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Symposium on Isoantigens
and Cell Interactions
Philadelphia, Pa.
March 17, 1965
.
MAY 5 1969
CONF-6.50314-1
The Immunogenetic Basis of Hybrid Resistance
To Parental Marrow Grafts
1:2**
Gustavo Cudkowicz
Biology Division, Oak Ridge National Laboratory
Oak Ridge, Tennessee
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Suatu, por lo conmiuston, nor say person scung on behalf of the Commission:
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primitoly owed rigua; or
B. Aanuman may liabiliu, nu respect to the use of, or for damcı rorWut from the
un of any laformalioa, apparaten, method, or procesu disclosed la ws report.
As und la tbe above, "persoa aclag on behall of the Complishm" Incluckeo way em.
ployee or contractor of the Commission, or employee of such contractor, to the extent that
Iwc soploys or coatractor of the Commission, or omployee of nich coal actor prepares,
diuvainiles, or provides accor. lo. any information purnant to be employmuat or coolract
with the Commission, or he employment milk sucd coolractor.

Running Title: Hybrid Resistance to Parental Marrow Grafts
Number of Tables: 8
Number of Figures: 6
Send proof to:
Dr. Gustavo Cudkowicz
Biology Division
Oak Ridge National Laboratory
P. O. Box Y
Oak Ridge, Tennessee 37831
INTRODUCTION
According to the predictions of the genetic laws of transplantation,
F, hybrid mice from crosses between two inbred strains should be considered
fully susceptible to tissue transplants of either parent, since the genetic
factors of hisiocompatibility are thought to be inherited as codominant
genes in mice (1). No exceptions have been noticed when grafts of normal
as
parental skin tissue have been placed on F, hybrids; however, in 1958
Snell reported the first apparent departure from the predicted pattern of
growth when transplantable lymphoma cells of parental origin were grafted
into F, hybrids (2). The lymphomas grew less and/or slower in the F,
hybrid mice than in mice of the susceptible parental strain. From the
time of this observation, an increasing number of communications have been
published which established that F, hybrid mice of certain genetic constitutions
are not able to support as well, mice of the native strains the growth of
transplantable lymphomas (3-6), sarcomas (7,8), carcinomas (7), and of
transplantable normal cells, such as antibody-forming (9,10) and hematopoietic
(11-13) cells.
It is not possible to say at the present time whether or not the deficient
growth on transplantation into F, heterozygotes of such a variety of cell
types of homozygous origin is due to similar mechanisms. For example, the
existence of genetic factors could only be determined for each graft type
by tests in segregating generations of mice or, alternatively, in F, progeny
from congenic-resistant parents, if the deficient growth of the grafts were
determined by histocompatibility factors. Results of genetic analysis are
available, however, only for a few of the aforementioned transplantable tissues;
such results suggest that deficient growth in F, wybrid mice of C57BL
2
lymphomas (2,3) and of normal parental hematopoietic cells (12,13) are
under genetic control and that in either case heterozygostity of the recipient
animals at I loci is responsible for the anomalous homozygous graft
behaviour, Furthermore, by studying marrow grafts taken from selected donor
strains, it was possible to recognize two types of hybrid resistance,
similar with respect to their general properties, but determined by different
genes. In fact, one case of hybrid resistance is controlled by a locus
which lies very close to,or within H-2 in linkage group IX (12,14,15), whereas
the other case appears to be related to a sex-associated gene, or to an
autosomal gene expressing itself in male hybrids only (13).
In this communication, I wish to describe the properties of the H-2-
associated hybrid resistance to parental marrow grafts and to summarize the
'results of an immunogenetic analysis. It 18 hopeful that similar studies on the
sex-associated hybrid resistance and on hybrid resistance toward transplantable
tissues other than marrow will indicate whether one is dealing with a peculiar
exception to the genetic laws of transplantation, relevant only for
hematopoietic grafts, or with the prototype of a noncodominant pattern of
inheritance of histocompatibility genes, affecting the survival of certain
tissue grafts, but not of skin grafts.
MATERIALS AND METHODS
Mice. Several inbred strains of mice, their congenic-resistant lines,
and F, hybrids were used; their origin and genotype are described elsewhere
(2,3,16,17). Pedigreed breeding pairs were supplied by G. D. Snell in
1961-1964 and by J. H. Stimpfling in 19614; each line was maintained thereafter
by strict single-line brother X sister matings in the animal colony of the
Biology Division, Oak Ridge National Laboratory. The B10 designation stands
for C57BL/10ScSn. The mice were used when 12-15 weeks old. C3H/Anf X C57BL
F, mice were purchased from Cumberland View Farms, Clinton, Tennessee. F
hybrids are designated by listing first the female and then the male strain.
Assay for proliferative activity of transplanted marrow. An appropriate
. number of femoral marrow cells suspended in Tyrode's solution was injected
intravenously into X irradiated recipient mice. Marrow contains a class of ,
cells capable of seeding the depleted hematopoietic sites of irradiated host
animals and of repopulating them through extensive proliferation and
differentiation into hemic cells. Five days after transplantation, 0.5uc of
5-Iodo-2'-deoxyuridine-1341 (13IVAR), a specific DNA precursor, was injected
into the peritoneal cavity of each animal to label the cells engaged in synthesis
of DNA. Unincorporated 1541 radioactivity is excreted from the regenerating
hematopoietic tissues within 12 hours. Therefore, the mice were killed 18
hours after injection of the label and their spleens were removed and placed in
glass test tubes. Retained radioactivity was measured in a well-type crystal
scintillation counter and expressed as percentage of the radioactivity injected
into the animals, above the percentage of splenic retention of radioactivity in
radiation control mice not injected with narrow. Under certain conditions,
uptake of 13-IUAR in the spleens of marrow recipients 18 a linear function of
the number of marrow cells transplanted (12); for this reason the uptake of
ISAIUAR estimates the extent to which the grafted marrow is capable of
producing DNA synthesizing (that is, dividing) hematopoietic cells and 18
referred to in the context of this paper as 'growth' or 'proliferative activity'.
In certain experiments, it was necessary to estimate the growth of
grafted marrow in the bone marrow cavities of the recipients. Both femurs
were removed from the labelled animals and processed like the spleens.
However, owing to the small mass of tissue in the femurs, it was desirable
to increase incorporation of 13 IUNR Into DNA of hematopoietic cells. This
was accomplished by injecting intraperitoneally 100% moles of 5-fluoro-2'-
deoxyuridine (FUAR) one hour prior to the injection of "SIUR. FUAR acts
by inhibiting the endogenous formation of thymi. ne precursors which would
'compete with 15-IUDR for incorporation into DNA.
Preparation of cell suspensions. Marrow cells were obtained from femurs
and tibias by flushing the bones with Tyrode's solution. Spleen cells were
suspended by teasing the capsule of the organ with a needle and gently
shaking the resulting spleen fragments in Tyrode's solution until most of the
cells become free.
The cell suspensions were filtered through a 200 mesh stainless steel
screen, and nucleated cells were counted in an electronic particle counter,
after red blood cells were lysed.
Irradiation. Recipient mice were irradiated with 300 kv (peak) x rays,
IN
HVL 0.5 mm Cu, at an exposure rate of approximately 70 R per minute. During
the exposure, the mice were housed in partitioned circular revolving Lucite
cages. Exposure measurements were made under comparable conditions with a
Victoreen R meter.
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RESULTS
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Description of Hybrid Resistance
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In a large series of experiments the growth of 10% to 10° grafted
marrow cells in the spleens of Irradiated isogenic recipients has not
varied considerably from strain to strain of nice nor has it varied as
the result of grading the recipient's exposure to radiation in the range
of 700 to 900 R of X rays (12,16,18). Such growth was characterized (1)
by exponential expansion of the DNA synthesizing cell population in the
recipient spleens from three to fire or six days after transplantation,
with a doubling time of apporximately twelve hours; and (11) by linear
relationship between grafted marrow cell dose and splenic repopulation five
days after transplantation. Figures 1 and 2 illustrate the growth of (F-1-2
10° grafted C57BL marrow cells in the spleen of isogenic and of C3H/Anf x
C57BL F, hybrid mice. Although the grafted cells were competent to proliferate
in isogenic recipients, they failed to do so in the F, hybrids exposed to
700 R of X rays throughout the 12 day duration of the experiment (Fig. 1).
Acute radiation injury in these F, mice is sublethal and, consequently,
spontaneous regeneration of host hematopoiesis occurs in the survivors. Hence,
the duration of the experiment could not be prolonged beyond 12 days owing to
the difficulty of distinguishing 3-IUDR incorporation promoted by regenerated
host cells as opposed to incorporation promoted by descendants of the grafted
donor cells. Host hematopoiesis does not regenerate so promptly, however, in
hybrids exposed to 900 R of X rays; under the latter condition growth of
parental donor cells in the hybrids was dectable, but it occurred considerably
later than 10 isogenic recipients (Fig. 1) and it was not adequate to prevent
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death of the F, mice exposed to the lethal dose of radiation. In order to
measure adequately the growth of C57BL marrow in F, recipients it was
necessary to inject an increased number of cells, about ten times as many .
as in isogenic recipients when the hybrids were born from C3H/Anf and
C57BL parents (Fig. 2). Thus, hybrid resistance manifests itself by the
deficient growth in the recipient's spleen of grafted parental marrow cells.
Its strength is measured by the magnitude of the overdose of donor cells
necessary to override it at a given exposure to radiation, for hybrid
resistance is weakened by increasing doses of x rays (Fig. 1, refs. 12, 16).
The strength of hybrid resistance varies considerably in hybrids of different
parentage (16,19). However, the range of values found 18 of the same order:
of magnitude as the range of values found for the resistance of inbred strains
of mice toward allogeneic marrow grafts (16). Similarly, the weakening effect
of whole-body X irradiation on the resistance to grafted C57BL marrow 18
not substantially different for C3H/Anf X C57BL F, hybrids than for allogeneic
DBA/2 mice (16).
To establish whether or not the spleen plays a role in the manifestation
of hybrid resistance, 100 C57BL marrow cells were grafted into intact and into
splenectomized mice exposed to 800 R of X rays. Splenectomy was performed
at ten weeks of age, one month prior to the experiment., At varying intervals
after marrow grafting, the production of hematopoietic cells was estimated
in the bone marrow cavities, instead of in the recipient spleens, by
measuring the uptake of 13 IUAR in two femur8. Isogenic recipients were able
to support the growth of the grafted marrow in their fimurs, as indicated in
Fig. 3. Expansion of the pool of DNA synthesizing cells was progressive
between day five and eleven in the femurs of intact C57BL mice, but had
already reached a plateau by day live in the femurs of splenectomized C57BL
mice, presumably, because in the absence of the spleen a larger proportion
of injected hematopoietic stem cells settled in the bones.' In contrast,
the femurs of C3H/Anf X C57BL F, mice were not detectably repopulated by
grafted C57BL marrow within 11 days after transplantation, irrespective
of the presence or absence of the spleen (Fig. 3). This indicates that
parental marrow grafts grow deficiently in both sites of hematopoiesis of
resistant F, recipients, and furthermore, that manifestation of resistance
is not dependent on the presence of the spleen at the time of parental
marrow grafting.
To determine at which age F, hybrids become resistant to parental marrow
grafts, infant BlO B10.D2 F, mice of either sex, 14 to 25 days old, were
exposed to 700 R of X rays and infused by tail vein injection with 5 X 105
B10 marrow cells harvested from aduit donors. In a previous experiment
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hybrids between the two congenic lines B10 and B10.D2 were found to be strongly
132
resistant to B10 marrow grafts (19). Recipient mice of different ages were
injected with 0.5 uc of 15-IVAR, irrespective of their body weight. It was
expected, therefore, that the uptake values of radioactivity in spleens of
mice of different weight would vary in relation with the different dilution
of the label in the body fluids, although each recipient mouse was given the
same number of marrow cells. Consequently, the spenic uptake values of 'S-IUAR
measured in F, mice of each age group were compared with the uptake values
measured in genetically susceptible Blo recipient mice of comparable size
and age, grafted with Bl0 cells of the same preparation. B10 x B10.D2 F1
mice are fully suceptible to grafted parental Bl0 marrow cells at the age of
14, 16, and 17 days, as shown in Fig. 4. At 20 days of age, the hybrids begin
to support less well than BlO mice the growth of B10 cells, but only at 24 days
of age do the hybrids become fully resistant.
20
The reported data show that minimal numbers (10° or less) of viable
parental marrow cells grafted into resistant hybrids exposed to 700 R of
X rays do not produce detectable numbers of dividing descendent cells
or more
within a period of five days. This does not rule out, however, that
grafted parental stem cells, although prevented from expanding and
differentiating, survive in the F, recipient spleens. To establish whether.
or not such F, spleens contain viable hematopoietic stem cells of Blo origin,
capable of repopulating susceptible irradiated mice, a measurement was
undertaken by a method of periodic sampling, as reported in the following.
Two million nucleated BlO marrow cells were injected into irradiated (700 R)
B10, B10 x B10.D2 Fy, and BlO.D2 recipient mice. One hour later, and at
daily intervals thereafter, four cimera mice of each group were killed to
remove their spleens. The cells of individual chimera spleens were dispersed
to form a suspension in 1 to 1.5 ml of Tyrode's solution, each of which was
assayed for its content of stem cells by injecting the whole suspension into
one irradiated (700 R) test-recipient mouse. The latter mice were of the
B10 strain and were, therefore, isogenic with the original marrow donors;
the test-recipients were preimmunized against 1soantigens of the B10.D2 strain
by two intraperitoneal injections of B10.D2 spleen cells given at weekly
intervals, to prevent viable F, hybrid cells eventually present in the chimera
spleens from proliferating upon transplantation into 'irradiated Bló test-mice.
It has been shown previously that admixed BIO X BLO.D2 F, cells do not affect
the hematopoietic competence of B10 cells (19). The extent to which the
chimera spleens are capable of promoting on transplantation the uptake of
1S-IUR in the spleens of B10 test-recipients estimates the fraction of stem
cells of the original BlO marrow inoculum which settled, survived, and
subsequently expanded in the spleens of the primary Blo, BLO X BLO.D2 and
B10.D2 recipients.
13
The results obtained are shown in Table 1. One hour after
transplantation the spleens of B10, and BlO.D2 chimera mice are equally
effective in promoting the uptake of 13-IUR in test-recipients, indicating
that similar fractions of infused stem cells settled and survived in such
spleens. One day after transplantation the hematopoietic competence of
the spleens decreased in all types of chimeras. A similar observation has
been reported earlier (20) and has been ascribed to non uniform efficiency
of extraction of dispersal of stem cells from the irradiated spleens as a
function of time. Two days after transplantation of the BlO marrow cells,
the hematopoietic competence of isogenic chimera spleens recovered and
during the following two days it increased steadily. In contrast, the
'hematopoietic competence of B10 x B10.D2 F, and B10.D2 chimera spleens was
negligible two days after transplantation and remained so on the third and
fourth day, indicating that viable BlO stem cells that settled in the spleens
of resistant F, and B10.D2 recipients during the first hour after transplantation
lost their ability to proliferate 24 to 48 hours afterwards.
Genetics of Hybrid Resistance
Genetic studies of hybrid resistance were made with segregating backcross
progeny mice (12), with F, hybrids from outcrosses between several inbred strains
of mice (16,18), and with F, hybrids from crosses between congenic-resistant?
mice differing et single H loci or at regions of the H-2 locus (14-16,
18,19). The manifestation of resistance in hybrids appeared to be dependent
on heterozygosity at the complex H-2 locus or at :D region, but not on
heterozygosity at one or more other I loci, or at the C and K regions of H-.
On the other hand, all the inbred strains of mice which have been found
to possess marrow cells growing deficiently on transplantation into H-2
C57BL,
heterozygotes shared the H-2° allele and were C57BL/6, C57BL/10. C57L,
129 and LP. (18). Mice carrying alleles other than H-2, namely H-2",
H-24, H-2+, H-2k, H-29, and H-2®, possessed marrow cells which grew without
appareat impairment on transplantation into the F, heterozygotes which were
resistant to grafted H-2° parental marrow (18). Furthermore, the marrow
cells of segregating C57BL X 101 F, mice grew deficiently in F, recipients
if they were H-2'homozygotes (21), but not if they were H-2°/H-25 or
H-2*/H-2* in phenotype. Lines of mice congenic with the strains A, C3H, and
DBA/1 acquired the trait of deficient marrow growth if the H-2° allele was
transferred into thej.r genome, but not when other H-2 alleles were transferred
(18).
Sneli (2,3) and Stimpfling (17) have produced a relatively large number
0:r mouse lines congenic with the B10 strain except for an allelic substitution
at a single H locus. These mouse lines and their F, hybrids have been
employed in experiments reported in the following to further demonstrate the
close association with 1-2 or with one of its regions of the genetic factor
or factors involved in hybrid resistance. Mice of congenic-resistant lines
differing from the BlO strain (H-2°/H-2°) at an H locus other than K-2 have
been mated with mice of strain B1O.D2 (H-2°/H-24) to provide F, hybrids
H-2°/H-2° in phenotype. Mice of the congenic-resistant lines characterized
by an allelic substitution at the H-2 locus were mated with mice of strain
B10 to also provide H-2 heterozygous F, hybrids. Adult hybrids were exposed
to 700 R of X rays and grafted with a standard dose of 10° nucleated marrow cells
harvested from parental donorks of the strain congenic with Blo. Depending on
whether the grafted marrow proliferated or not within five days in the spleens
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of the recipients, the growth pattern of the donor marrow was clasvified as
"optimal" or "deficient". Details on the experimental design and on the
con'rolled variables were reported elsewhere (12). The results .
Table 2) reconfirmed that allelic substitutions at the H-1, H-3, and .
H-4 loci of strain Blo did not modify the deficient growth pattern of
B10 marrow grafted into H-2 heterozygotes, whereas, substitution of the H-2
allele of strain B10 was invariably associated with loss of the trait and
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with optimal pattern of marrow growth on transplantation.
Jo In the course of studying the genetic structure of H-2, Stimpfling
identified several mouse lines carrying H-2 variant alleles, presumably
derived from crossing-overs with H-2 (17). The exceptional alleles, which
derived from exchanges between H-2 and H-2", resembled the H-2” and H-21
alleles and were transferred, by repeated backcrossing, onto a genetic
background congenic with B10 (17). Marrow cells of the variant lines were
grafted into irradiated BlO X B10.A F, hybrids because the phenotype of the
latter mice is H-2°/H-2" and should possess all the known codominant H-2
components of the variant lines. If the variant H-2 alleles maintained the
chromosome segment of H-2 responsible for deficient marrow growth in heterozygotes,
the BIO X B10.A hybrids should be adequate for demonstrating the trait. In
fact, such hybrids are resistant to Blo marrow (14) and are heterozygotes with
respect to components of H-2 in the D, C, and K regions. Marrow cells of
variant mouse lines which possess components of H-2° in the D region, but not iu
the C and K regions possess also the genetic determinants for deficient growth
(Table 3). In contrast, marrow of mice which possess components of H-2° in the
K region but not in the D and C regions are capable of optimal growth in
B10 x B10.A F, mice. Thus, the genetic factor which controls hybrid resistance
lies within or very closely adjacent to the D end of the H-2 locus.
14
It has been noticed before that H-2 heterozygous hybrids of different
parentage differ sometimes with respect to the strength of their resistance
to parental marrow grafts, as measured by the number of cells necessary to
---
override it, although they may share the same H-2 alleles (16). To investigate
whether the genetic background of the parental mice entering the F, crosses
influences the strength of the hybrid's resistance, the following F, mice
were produced from congenic-resistant pairs as prospective recipients of
marrow grafts from their H-2 parents: B10 x B10.D2; C3H.SW X C3H; A X A.BY;
and DBA/1 X D1.LP. Each of these hybrids is heterozygous at a chromosome
segment of linkage group IX containing the complex H-2 locus, but should be
homozygous for most of the genes contributed by the B10, C3H, A, and DBA/1
genomes, respectively. The hybrids were exposed to 700 R of x rays and
injected a few hours later with graded doses of nucleated parental strain
marrow cells. Donor cells of the same pool were also transplanted into
similarly irradiated isogenic recipient mice to assay the competence of the
cells to proliferate in the absence of histoincompatibilities. To avoid
sex-associated resistance, the donor and recipient mice of all strain combinations
were females. The promotion of splenic uptake of 15IUAR by grafted marrow in
isogenic recipients was comparable for cells of strains Blo, C3H.SW, A.BY, and
D1.LP, and was, furthermore, a linear function of the dose of grafted cells
(Fig. 5). Splenic uptake of 13-IUAR was also a linear function of donor
cell dose in resistant F, hybrid recipients; however, it was necessary to
graft larger numbers of cells than into isogenic mice to colonize the
recipient spleens, ab 16 indicated by the displacement to the right of the
dose-response lines (Fig. 5).
The number of donor cells required to overcome resistance varied
markedly in the four hybrids studied. Spleen repopulation in B10 x B10.D2
F, mice required 15-to-20 times as many cells as spleen repopulation in
isogenic recipients, whereas lower multiples of the cell doses inoculated
into isogenic mice were adequate for repopulating the spleens of c3H.SW X
C3H F, and A X A.BY F, mice. It is noteworthy that DBA/1 x D1.LP F, hybrids
(H-2°/H-2") were fully susceptible to D1.LP marrow grafts (Fig. 5), although
the grafts were donated by H-2° homozygous mice. To further investigate
exceptional
the absence or weakness of resistance in H-2 heterozygous hybrids, one million
nucleated marrow cells of strain A.BY and D1. LP donors were transplanted into
a series of irradiated (700 R) F, recipients which had in common one or
both H-2 alleles, contributed by parental mice of different strains. For
example, A X A.BY F, and Blo.A X A.BY F, mice are identical at the H-2 locus
(H-2/H-2°) since B10.A 18 a line congenic with strain B20 except for the
H-2e allele derived from strain A (17); however, the former hybrid does not
possess any trait of strain B10 and its resistance to A.BY marrow grafts is
welak (Fig. 5 and Table 4). In contrast, BlO.A X A.BY hybrids possess a
single dose of Bló genes and their resistance to A.BY marrow is strong
(Table 4). In a similar way, the strength of resistance toward D1. LP marrow
grafts varies considerably in the three H-2*/H-2 hybrids C3H x B10 F7,
c3H X LP F7, and C3H x D1.LP F(Table 4), in the face of similarity or
identity at the H-2 locus, since the H-2° allele of the D1. LP line was derived
from strain LP (2). The failure of DBA/1 X D1. LP F, and of c3H x D1.LP F,
hybrids to develop resistance toward parental marrow grafts may be related to
the failure of DBA/1 mice, congenic with 01. LP nice, to resist the transplantation
of a number of otherwise strasn specific tumors indigeneous to inbred lines
1
unrelated to DBA/1 (23). From these data it appears that the strength of

(hybrid resistance depends on determinants contributed by the genome of the
parental strains (other than H-2), which may interact with the H-2 associated
alleles primarily responsible for hybrid resistance.
C57BL marrow cells maintain their deficient growth pattern in F,
.
hybrids even after one year of residence in heavily irradiated resistant
F, hosts (21). To increase the probability of observing adaptive changes and
selection of variant cell lines capable of optimal or suboptimal growth in
H-2 heterozygous hybrids, Blo marrow cells were serially transplanted at
20-day intervals in C3H/Anf X C57BL F, hybrids esxposed to 900 R of x rays.
20 x 10° nucleated cells were grafted initially and an equal number of cells
were regrafted on each passage for a total of three transplant generations.
Sixty days after the initial cell transplantation, the chimeras were
sacrificed and 10° nucleated cells of their femoral marrow were assayed for
growth into irradiated (700 R) BlO mice, isogenic with the serially passaged
B10 X
marrow, and into c3H/Anf X C57BL F, and B10.A F, resistant hybrids. The
B10 and B10 x B10.A F, test-mice were preimmunized against isoantigens of
C3H/Anf X C57BL F, mice (two intraperitoneal injections of 10' spleen cells
at weekly intervals, three and two weeks before the experiment) to prevent
viable host cells of the B10 + F, chimeras from proliferating on transplantation.
The marrow of five individual chimeras of the first and of the second transplant
generations, and of severi chimeras of the third transplant generation was
tested (Table 5) and in no instance was it found to contain variant Bl0 marrow 1-3
cells capable of optimal growth in resistant hybrids.
Imunological Studies
Hybrid resistance can be abrogated in adult F, mice by multiple
injections of viable spleen cells gathered from H-2° parental strain donors
17
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but not by injections of spleen cells from parental strain mice of
different H-2 phenotype (19). Abrogation of the resistance has been
accomplished consistently when l-to-2 X 10' viable spleen cells were
given intraperitoneally with each injection, over a period of three to
four weeks. A single administration, or two injections of viable spleen
cells at weekly intervals do not affect the hybrid resistance. F, mice
which become susceptible to parental marrow grafts following the prolonged
treatement react normally to allogeneic marrow grafts and are not cellular
However,
chimeras with respect to dividing hematopoietic cells (19). , C3H/Anf x
C57BL F, worids made susceptible to C57BL marrow grafts,
susceptible also to marrow grafts from other H-2 homozygous donors, such
as A.BY, C3H.SW, D1.LP, LP, and 129 mice (18). Pretreatment of certain
resistant hybrids with multiple injections of 1.5 x 10' parental strain
marrow cells instead of spleen cells enhances slightly the strength of their
resistance to H-2 marrow grafts, without affecting, however, their
... are
resistance to allogeneic grafts (19). A list of resistant bybrids which
become susceptible upon treatment with parental strain spleen cells is given
in Table 6. Also listed are the hybrids whose resistance has been found to
increase or to remain unchanged upon treatment with parental strain marrow
cells.
Abrogation and enhancement of hybrid resistance, as described above,
are reminiscent of acquired unresponsiveness and acquired immunity, in that
they are effected by the parental cells toward which the hybrid 18 resistant
but not by the parental cells histocompatible with the hybrids. Furthermore,
once established, the new condition of the bybrids 18 specific for H-2°
.
18
homozygous marrow grafts Pentidai in this respect with the cells used for
the treatment. It was of interest to establish, therefore, whether the
injection of parental strain hematopoietic cells into newborn F, mice
would also abrogate or delay the appearance of hybrid resistance.
BLO X'BlO.D2 F, nice were injected intraperitoneally, within 12 hours from
birth, with 5 x 10° or 10' nucleated marrow cells from adult Bl0 female
donors. A few mice of each litter were not injected with cells or were .
injected with BlO.D2 marrow cells to serve as controls. In addition, one
litter of Bl0 newborn mice were injected with B10 cells from one of the
preparations used to inject the F7, mice. At 35 to 40 days of age, the
surviving animals were tested for resistance to 5 X 10° grafted BlO .
marrow cells following exposure to 700 R whole body X irradiation. Results
of preliminary experiments are reported in Table 7; uninjected F, mice and
F, mice given BlO.D2 cells were fully resistant, whereas six F, mice
neonatally injected with 10' B10 cells and two F, mice injected with 5 x 10°
B10 cells failed to develop resistance. Two of the four F, mice of the
latter group were found to be resistant. It seems, therefore, th at the
neonatal administration of parental hematopoietic cells to appropriate F,
hybrids may delay the development of resistance or induce immunological
tolerance to parental marrow grafts.
Hybrid resistance to parental marrow grafts has been transferred
adoptively by F, marrow or spleen cells to irradiated mice that do not
possess isoantigens associated with the H-2° homozygous phenotype, namely
to C3H, A and C3H x B10.BY mice (16). However, resistance was not transferred
to irradiated H-2 mice, even if the latter were bearing functional F,
hybrid hematopoietic grafts (16). Probably, F, hematopoietic cells and/or
F, immunocytes became paralyzed or specifically unresponsive upon transplantation
into H-2° homozygous mice, owing to the excess of parental 1soantigens in such
recipients. To test this interpretation, the experiments were extended as to .
ascertain whether F, cells from resistant donors that failed to transfer
hybrid resistance upon infusion into irradiated parental H-2° mice, would still
be capable of conferring resistance on retransplantation into secondary
recipient mice lacking the isoantigens associated with the homozygous H-2
phenotype. If the F, cells were paralyzed or specifically unresponsive to
parental 1soantigens when residing in irradiated H-2° mice, one would predict
that upon removal from such recipients the F, cells would regain their
reactivity toward the isoantigens of H-2° homozygous parental marrow grafts.
The design of the experiment is shown in Fig. 6. The first passage
of marrow was performed with 4 x 10' nucleated B10 X A F, cells injected
into the tail veins of irradiated (850 R) BIO recipients. After 122 days,
the peripheral blood hemoglobin of all the chimeras, analyzed by the method
of Popp and Cosgrove (24) was found to be of donor type. At this time,
six chimeras were reexposed to 700 R of X rays and tested for hybrid resistance
by grafting 10° nucleated Bl0 marrow cells. Regenerated marrow from the
remaining F, + parent chimeras was harvested and 2 x 10' nucleated cells were
retransplanted into each of several groups of irradiated recipients. The
experimental groups were strain A mice exposed to 800'R of x rays and strain
B1O X A F, mice exposed to 900 R of x rays. The different exposure levels
correspond to the LD, 100/30 of each strain. Control groups (not listed in
Fig. 6) were strain BlO mice injected with marrow of the primary F, + BLO
chimeras and strain B10 and A mice injected, respectively, with 18ogenic,
serially transplanted marrow cells. Forty days after the second marrow
.
20
passage, the secondary chimeras were tested as before for resistance to
B10 marrow grafts.
'
The results of this experiment are presented in Table 8. BlO marrow
test-grafts promoted the uptake of 134IUAR in the spleens of B10 isogenic
chimeras and, to a lesser extent, in the spleens of A strain isogenic chimeras.
This indicated that the chimeric.state, as it was produced here, did not alter
per se the ability of recipients to support the growth of B10 marrow grafts.
B10 marrow cells were also able to promote splenic uptake of 15-IUAR in
primary F, + Bl0 and in secondary Fi + B10 + B10 chimeras, as if these animals
were fully susceptible. In contrast, Blo marrow cells were not able to
grow in F, + B10 + A and in F, + B10 + F, secondary chimeras, as if these animals
were fully resistant.
Although this experiment is not yet complete with respect to the number
of transplant generations and to the variety of precipient strains contemplated
it cleary shows that the F, cells which failed to transfer hybrid resistance
to Blo recipients, were competengt to transfer resistance upon retransplantation
into strain A and B10 X A F, mice. The findings strengthen, therefore, the
interpretation given to earlier results (16) that the competent cells of
resistant F, mice become paralyzed or specifically unresponsive when exposed to
an excess of parental 1soantigen( 8)
*
..
.
-
.
.
.-
DISCUSSION
Inbred mouse strains homozygous for the H-2 allele possess in
the D region of the complex H-2 locus, or in close association with it,
a genetic determinant controliing a requirement for the optimal growth of
transplanted marrow cells in the hematopoietic sites of irradiated
.
.
.
.
recipients. Since F, hybrid mice heterozygous for this genetic determinant do
not support the growth of minimal numbers of grafted homozygous cells, it is,
concluded that this genetic determinant is not expressed in H-2°
heterozygotes. Allogeneic strains of mice homozygous for H-2 alleles other
than H-2 may, however, support the growth of H-2 /H-2° marrow grafts (11, 12,
16). Therefore, the resistance of F, heterozygotes toward grafts of
homozygous parental origin has been attributed to interallelic genetic
interactions rather than to heterozygosis for a recessive gene.
Infant F, hybrid mice are not resistant to grafted parental marrow
cells until they reach 20 days of age. However, once established the hybrid
resistance appears to be relatively strong: the proliferative integrity of .
parental marrow cells grafted into F, mice is inactivated within 24-48 hours and
the weakening, effect of whole-body X irradiation on hybrid resistance is
rather low (16). In general, the strength of hybrid resistance, as measured
by these two parameters and by the overdose of parental donor cells
necessary to override it, appears to be of the same' order of magnitude as
the strength of the resistance displayed by allogeneic inbred mice toward
H-2 homozygous marrow grafts. Studies with a varient) of H-2 heterozygous
hybrids have provided evidence that the strength of resistance may vary
22
..
..
.. ..
...."
L
I YO
1Y:
'.*
*
considerably among hybrids of different parentage, although the hybrids
may be identical with respect to their H-2 constitution. It appears
as if one or more independent allelomorphic genes may act as modifiers,
since the resistance of hybrids was strong when one or both parents
contributed the genetic background of strain C57BL, or of one of its
sublines (16), but decreased in strength in the following order when
the parents contributed the genetic background of strains C3H, A, and
DBA/1. An example of gene interaction modifying the expression of
red blood cell 1soantigens of the H-29 phenotype in DBA/1 mice has been
reported by Stimpfling and Snell (25).
Thus, hybrid resistance, or conversely, deficient growth of
parental marrow grafts in F, hybrids, can be ascribed to genetic
interaction in the inheritance of a histocompatibility gene affecting
specifically the fate of hematopoietic cell grafts. It can be speculated
that interallelic interaction leads to suppression in F, heterozygotes of
one or more isoantigens associated with the H-2 phenotype. The following
events can then be postulated: (1) hematopoietic tissues of H-2°
homozygous mice should be isoantigenic to H-2 heterozygous F, mice and
capable of inducing, under appropriate conditions, a state of specific
unresponsiveness and/or immunity in resistant F, mice; (2) skin
tissue of H-2° homozygotes should not share with hematopoietic tissue the
parental isoantigens; (3) hybrid resistance should þe transferred
adoptively by F, hematopoietic cells to mice lacking the parental
1soantigens. In the presence of large amounts of such 1soantigens, as
in H-2°/H-2 mice, transferred F, cells should rather become unresponsive.
The results of a series of experiments described here and
still
elsewhere (16, 19), some of which in a preliminary phase, are consistent
with the predictions, except for the fact that the induction of immunity
in resistant hybrids,(i.e., an induced state in which parental marrow
grafts become unacceptable to F, recipients which otherwise would accept
them) has so far been unsuccessful in most of the employed hybrids. In
a few F, strains, resistance was specifically increased by pretreatment
with parental hematopoietic cells, but the increase was relatively small
(19). It is conceivable that hybrid resistance, which develops relatively
after birth
late at about weaning age, but manifests itself suddenly (Fig. 4),
resulted from sensitization toward parental-like isoantigens during the .
neonatal period of life. It has been shown by Tiu et al. (26) that
isoantigenic variant cell lines are produced frequently by serial
transplantation of fetal liver cells but not of adult marrow cells.
If i soantigenic variation were to occur also spontaneous ly in the
hematopoietic cell population of H-2° heterozygous near-term embryos or
newborn mice, variant clones of cells may arise with isoantigens
controlled by the D region of H-2 similar to the isoantigens of parental
H-2° homozygotes. Such clones .would induce a homograft response which
tends' to eliminate them and to generate at the same time resistance toward
parental homozygous cells.
The hybrid resistance phenomenon, as described herein, differs
with respect to several properties from the 'syngeneic preference'
phenomenon, i.e., the deficient growth of certain transplantable parental
tumors in F, hybrids (5-7, 27). The major differences seem to reside in
24
the effect of radiation (5, 7) and of pretreatment with parental cells
(27) on the two phenomena; in the occurrence of adaptive changes on
serial transplantation of lymphoma cells (6), but not of marrow cells; and
in the different ability of the two types of parental cells to withstand
the presence of foreign isoantigens (11, 12, 19, 27). Nevertheless,
both phenomena way be part of the homeostatic mechanisms maintaining
homogeneous the phenotype of heterozygous somatic cells by eliminating
variant clones which lack certain isoantigens.
The search for isoantigenic
.
variants in hematopoietic cell populations of F, hybrid fetuses and of
infant F, mice before the development of hybrid resistance may provide
the means of verifying the validity of such a hypothesis.
25
SUMMARY
The growth of parental marrow grafts in the hematopoietic sites of
X irradiated F, hybrids is deficient if the donor mice are homozygotes for
the D region of the H-2° allele and if the recipient mice are heterozygotes
at the same locus. However, the extent to which F, heterozygotes resist
the growth of parental marrow grafts depends also on other genetic determinants,
presumably one or more modifier genes contributed by the genomes of the
parental strains entering the F, crosses.
Hybrid resistance develops relatively late in life, at about weaning
age. It is moderately radiosensitive, but can be abrogated by multiple
injections (ínto adult hybrids of viable parental strain spleen cellsVover a
period of three to four weeks. Forty-day-old hybrids injected during
neonatal life with parental strain marrow cells fail to develop resistance.
Hybrid resistance can be slightly enhanced by multiple injections into
adult animals of viable parental strain marrow cells, but only in some hybrid
strains. Hybrid resistance is transferred adoptively by F, marrow or spleen
cells tovirradiated recipient mice. (genetically susceptible
These findings are consistent with the interpretation that genetic
interaction lead to suppression of one or more parental isoantigen an diesel
between alleles in or near the D region of the complex H-2 locuska Such
isoantigens are relevant for the fate of hematopoietic cell grafts, but not
for the fate of 102mal skin grafts.

.
:
.
.
,..
-
!,'
,
26
.
..
FOOTNOTES
.... Research sponsored by the U. S. Atomic Energy Commission under
contract with the Union Carbide Corporation.
2. Congenic-resistant strains of mice share a given genetic background,
but differ with respect to a chromosome segment carrying an allele
at a single I locus. Such a difference results in resistance toward
tissue grafts exchanged between members of the congenic pair.
"
.
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....
.
.
.
.
.
.
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7
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'T'.
Literature Cited
1. Little, c. C1. 1941 The Genetics of Tumor Transplantation. In
Biology of the Laboratory Mouse, Ed. by G. D. Snell, Blakiston Co.,
Philadelphia. 279-309.
2. Snell, G. D. 1958 Histocompatibility Genes of the Mouse. II. Introduction
and Analysis of Isogenic-Resistant Lines. J. Nat. Cancer Inst. 21:843-877.
3. Snell, G. D., and L. Co Stevens 1961 Histocompatibility Genes of the
Mouse. III. H-1 and H-4, Two Histocompatibility Loci in the First
Linkage Group. Immunology 4:366-379.
4. Gorer, P. A., A. M. Tuffrey, and J. R. Batchelor 1962 Serological
Studies on the X Antigens. Ann. N. Y. Acad. Sci. 101:5-11.
2
5. Hellström, K. E. 1963 Differential Behaviour of Transplanted Mouse
Lymphoma Lines in Genetically compatible Homozygous and F,
Hybrid Mice. Nature 199:614-615.
6. Huemer, R. P. 1965 Repression of Growth, and subsequent Adaptation, of
a Parental Strain Tumour in Genetically compatible F, Hybrid Mice.
Nature 205:48-50.
22
'
"
.
28
7. Hellström, K. E. 1964 Growth Inhibition of Sarcoma and Carcinoma Cells
of Homozygous Origin, Science 143:477–478.
8. Oth, D., J. Robert, C. Michaud, and M. Crestin 1964 Comportement
anormal d'une tumeur 1sologue de Souris C3H transplantée chez des
hybrides F. C. R. Soc. Biol. 158:841-844.
9. Boyse, E. A. 1959 The Fate of Mouse Spleen Cells Transplanted into
Homologous and F, Hybrid Hosts. Immunology 2:170-181.
10. Celada, F., and W. J. Welshons 1962 Demonstration of F, Hybrid
Anti-parent Immunological Reaction. Proc. Nat. Acad. Sci.
U.S. 48:326–331.
11. McCulloch, E. A., and J. E. Till 1963 Repression of Colony-forming
Ability of C57BL Hematopoietic Cells Transplanted into Non-isologous
Hosts. J. Cell. Comp. Physiol. 61:301-308.
R
12. Cudkowicz, G., and J. H. Stimpfling 1964 Deficient Growth of C57BL
Marrow cells Transplanted in F, Hybrid Mice. Association with
the Histocompatibility-2 Locus. Immunology 1:291-306.
13. Cudkowicz, G. 1965 Sex-associated Hybrid Resistance to Parental
Marrow Grafts. (Abstract) Fed. Proc. 24:637.
USE
W
..c
'e':-.
.
:
n
.
..",..
.....
.
.
.
.
.
...
.
.
.
.
.
.
.
.
.
..
.
14. Cudkowicz, G., and J. H. Stimpfling 1964 Hybrid Resistance to
Parental Marrow Grafts: Association with the K Region of
H-2. Science 144:1339-1340.
15.
Cudkowicz, G., and J. H. Stimpfling 1965 Hybrid Resistance
Controlled by H-2 Region: Correction of Data. Science 147:1056.
16. Cudkowicz, G. 1965 Hybrid Resistance to Parental Hematopoietic Cell
Grafts: Implications for Marrow Chimeras. In La greffe des
cellules hematopoietique allogeniques. Ed. by G. Mathé, J. L.
Amiel and L. Schwarzenberg. C.N.R.s., Paris, in press.
17. Stimpfling, J. H., and A. Richardson
1965
of the Mouse
Recombination within the Histocompatibility-2 Locus Genetics,
in press.
18. Cudkowicz, G., and J. H. Stimpfling
1965 Lack of Expression of
Parental Isoantigen(s) in F, Hybrid Mice. Proc. Xth Congr.
int. Soc. Blood Transf., S. Karger, Basel, in press.
19. Cudkowicz, G., and J. H. Stimpfling 1964 Induction of Immunity and
of Unresponsiveness to Parental Marrow Grafts in Adult F,
Hybrid Mice. Nature 204:450-453.
30
20. McCulloch, E. A., and J. E. Till
1964 Proliferation of Hemopoietic
Colony-forming Cells Transplanted into Irradiated Mice. Rad.
Res. 22:383-397.
.21. Popp, R. A., and G. Cudkowicz 1965 Independence of Deficient Early
Growth and Later Regression of (C57BL X 101)F, Marrow Grafts
in (C57BL X 101)F, Hybrid Mice. Transplantation 3:
22. Cudkowicz, G., and J. H. Stimpifing 1965 In Preparation
23. Snell, G. D., E. Russell, E. Fekete, and P. Smith 1954 Resistance
of Various Inbred Strains of Mice to Tumor Homoiotransplants, and
its Relation to the H-2 Allele which Each Carries. J. Nat. Cancer
Inst. 14:485-491.
24. Popp, R. A., and G. E. Cosgrove 1959 Solubility of Hemoglobin as
Red Cell Marker in Irradiated Mouse Chimeras. Proc. Soc. Exper.
Biol. Med. (N.X.) 101:754-758.
25. Stimplring, J. H., and G.' D. önell 1962 Histocompatility Genes and
Some Immunogenetic Problems. In International Symposium on Tissue
Transplantation. Ed. by A. P. Crtistoffanini, and G. Hoecker,
Universidad de Chile, Santiago, 37-54.
31
26. Till, J. E., E. A. McCulloch, and L. Siminovitch
1964 Isolation
of Variant Cell Lines During Serial Transplantation of
Hematopoietic Cells Derived from Fetal Liver. J. Nat.
Cancer Inst. 33:707-720.
27. Hellström, K. E. 1965 Studies on the Syngeneic Preference
Phenomenon.
This Symposium.
♡
Table 1. Measurement of the Hematopoietic Cell Content of Recipient . Spleens Following Irradiation (700 R) and
Transplantation of 2 x 100 B10 Marrow cells:
Days after Transplantation
Splenic uptake of IVAR (%) in individual test-mice promoted by grafted
spleen cells of the following chimeras*
BlO 4 B10 10 B10 x B10.02 F, B20 — → B10.02
1/24
.45
.25 .26 .35 .39
.09 .11 .15 .18
.20 .27 .31 .33
.39 .47 .55 .59
.95 1.031.05 1.17
.27 .30 .33 .47
.07.07.11.12
.01 .02 .02 .04
.02 .02 .03 .03
.01 .02 .02 .03
.20 .30 .36
.01 .02 .03
.01 .01 .02
.01 .01 .01
.01 .01 .02
.03
.02
.03
*The cells of a whole spleen were infused into irradiated (700 R) B10 test-recipient mice preimmunized against
isoantigens of strain B10.D2.
Table 2 - The Effect of Allelic Substitutions at I Loci of B10 Mice on the Growth Pattern of
Parental Marrow Grafts in H-2 Heterozygous F, Hybridst
Allelic substitution
Strain designation
Growth pattern
of marrow
None
BLO#
· Deficient
H-12
H-2°
B10.BY
B10.129(5M)
Deficient
A-36
B10.LP
Deficient
Deficient
H-46
1-2 8-2 8-2d
-
28-29
B10.129(21M)
B10.A B10.BR
B20.02
Optimal
B10.M
B10.Y
*Data from Cudkowicz and Stimpfling (18,22)
*The relevant genotype of strain B10 is H-1°, H-2, H-3°, -4*.
.
.
34
"able 3 - The Growth Pattern of Marrow Cells from Mice Carrying Variant 1-2 Alleles (resulting from
crossing-over within H-2) Grafted into B10 x B10.A F, Hybrids*
Straio
H-2 Allele
H-2 Allele
H-2 Serotype
Growth Pattern
of marrow
B10
Deficient
H-2H-288
DM- C- H- K-
D- M- C+ 8+ K+
[+ M+ C+ H+ K-
D+ M+ C+ A+ K+
H-21-288
Deficient
Optimal
Optimal
B10.A
*Data of Cudkowicz and Stimpfling (22) - The hybrid recipients are heterozygotes for the five
H-2 components listed in the table.
-
.
in
Table 4 - The Strength of Resistance to H-2°/-2° Marrow Grafts in Hybrids of Different Parentage*
Donor Strain
Classification
. Recipients
Strain
H-2
Mean splenic uptake of 15-IVAR
(% + Stand. error)t.
A.BY
AXA.BY
Weakly resistant
B10.A X A.BY
0.25 = .03
0.03 = .001
0.59 • .04
Strongly resistant
Susceptible
B10 X A.BY
D1.LP
DBA/1 X DI.LP
a/b
Susceptible' ;
V
k/b
C3H x D.LP
СЗН x IP
0.77 • .05
0.74 + .07
0.15** .02
0.02 + .001
0.83 = .05
Susceptible 21
Weakly resistant
k/b
C3H X Blo
k/b
Strongly resistant
B10 X Dl.LP
b/b
Susceptible
* Data from Cudkowicz and Stimpfling (22).
+ 5 days after irradiation (700 R) and grafting of 10° nucleated cells. Ten to fifteen mice per group.
Tables
Table 5 - Proliferative Capacity of B10 Marrow Cells Serially Transplanted in C3H/Anf X C57BL F, Recipients
ng
Marrow donors
No. of marrow
donors tested
Splenic uptake of 131IUAR (%) in recipients of the following strains:
B10 x B10.A C3H/Anf X C57BL .
Blob
Primary B20-F, chimeras
5
Secondary BlO-Fchimeras
5
0.60
10.45 - 0.69)
0.37
(0.28 - 0.55)
0.36
(0.22 - 0.42)
0.01
(0.01 - 0.03)..
0.03
(0.02 - 0.09)
0.02
(0.01 - 0.08
0.03
(0.02 - 0.05)
0.02
(0.02 - 0.04)
0.03
(0.01 - 0.07)
Terfilary B10 -> F, chimeras
& 5 days after transplantation of 10° nucleated chimera marrow cells. Average value is given; lowest and highest
values in parenthesės.
Immunized against isoantigens of C3H/Anf X C57BL F, spleen cells. ·
--..:.
- --
-
.-... --
-..
-
-.-
Table 6 -
The Effect of Pretreatment with Parental Spleen or Marrow Cells on Hybrid Resistance
.
37
-
---
Resistant bybrids which become
susceptible following multiple
injections of parental spleen
cells
Hybrids whose resistance
increases following multiple
injections of parental marrow
Hybrids whose resistance
remains unchanged following
multiple injections of parental
** marrow cells
cells
C3H/Anf X C57BL
C3H/Anf X C57BL
СЗН x Bio
B10 x B10.02
C3H.SW X C38
c3H x 129
СЗН x Bio
C3H X 129
Α.ΒΥΧΑ
C3H.SW X C3H
B10 ΧΑ
A.BY X A
BIO X FU
Blo, x 4
DBA/1 X DI.LP
B10 x B10.D2
C57BL/6 X DBA/2
B1O X FU
C57BL/6 X DBA/2
Table 7
-
he effect of neonatal exposure to parental marrow cells on the development of hybrid resistance,
38
Recipient Strain
Treatment
Splenic Uptake of 131IUAR (%) Promoted by Test Grafts
In Individual Recipient Mice
B10 X B10.D2
None
.01
.01
.14
.08
.10
.06
.05
.01
.01
.14
.08
.18
10² B10.D2 Marrow cells
10' B10 Marrow cells
5 x 106 B10 Marrow cells
10'B10 Marrow cells
.01
.01
.16
.19
.20
.03
.02
.22
.02
.01
.19
26
.28
..05
.05
.23
BIO
.28
.30
8 5 X 10marrow cells given after exposures of 35-40 day old recipients to 700 R of X rays
* mention that
ñ
Table 8 -
Serial Transfer of Hybrid Resistance by B1O X A F, Marrow Cells
Chimeras to be Tested
, 1st Host / 2nd Host
Splenic Uptake of 131IUAR (% + Stand. Error)
Promoted by Test Grafts
(Donor
B10 , B10 / Blo
Α / Α Α
B10 x A | Blo / None
BLO XA , BLO | BLO
ві0 xA | Bio І ві0 xA
BLO XA , BLONA
0.58 1.07 1736
0.297.03 (5)
0.51 + .03 (6)
0.47+ .05 (7)
0.05 = .02 (7)
0.06+ .01 (5)
10° Blo marrow cells grafted after exposure of the chimeras to 700 R of X rays
'In parentheses - number of chimeras tested.
-
..
**
.
FIGURE LEGENDS
Figure 1 - Splenic uptake of SHIUR in isogenic and F, hybrid mice given
106 C57BL marrow cells, as a function of time after transplantation...
Five mice per point. .
O C57BL recipients exposed to 800 R
O 038/Anf X C57BL F, recipients exposed to 900 R
c3H/Anf X C57BL F, recipients exposed to 700 R
Figure 2 - Splenic uptake of 13-IUAR in isogenic and in F, hybrid mice given
C57BL marrow, as a function of the number of transplanted cells.
Five to seven nice per point.
C57BL recipients exposed to 800 R
O c3H/Anf X C57BL F, recipients exposed to 900 R
Figure 3 - Uptake of 13-IVAR in two femurs of irradiated (800 R) isogenic and
F, hybrid mice given 10° C57BL marrow cells, as a function of time after
transplantation. Splenectomy was performed one month prior to the
experiment. Recipient mice were injected with FUAR before being labeled
with 131 IUAR.
C57BL recipients
O c3H/Anf X C57BL F, recipients
Figure 4 - Uptake of 13AIUAR in the spleens of irradiated (700 R) isogenic and
F, hybrid infant mice given 5 x 10' B10 marrow cells at different ages.
B10 recipients
O B1O X B10.D2 F, recipients
Figure 5 - Splenic uptake of 15-IUAR in relation to the number of parental
marrow cells injected into irradiated (700 R) 18ogenic and F, hybrid
recipient mice (five mice per point). .
NII Parental strain donor cells. 'Isogenic recipients
B10 cells.
B10 x B10.D2 F,
O C3H.SW cells
C3H.SW X C3H F1
A A.BY cells
· A X A.BY F1
D1. LP cells
DBA/1 X D1.LP F
-
Figure 6 - Schematic outline of the procedure used for serial transfer of
-
-
B10 X AF, marrow cells and of hybrid resistance.
+
-
-:
:-
.
.
.
-
-
-
-
------
-
14,13.2
106 C57BL MARROW CELLS GRAFTED INTO:
O C57BL-800 R
O C3H/A-EX C57BL Fi - 900 R
O C3H/AC-X032 - F-700R
2.00-
90
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MEAN SPLENIC UPTAKE OF 13110R

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TIME AFTER
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ROW TAVSPLANTION (days;
14,131
2.007 0 C57BL-800 R
o C3H/Aní x C57BL F-900R
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MEAN SILENIC UPTAKE OF 3*, GR (%)


LAS
0.125 0.5 Ź ģ
NO. OF TRANSPLANTED MARROW
· CELLS X 106
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..........................
14,133
CO7SL-000 R
0 C3H/A > C575L F,- 800 R
0.
47 INTACT MICE
со
OC
OOO O
O
Occo o
0
CcO000
0000
000 00
UPTAKE OF 131 UdR IN 2 TEMUKSI
SPLENECTOMIZED MICE
C
o
000 00
co
0000
00 0000
000000
o 2000
0 19
NSPLANTION
-
.
.
.
.
now.romeni.
...
mmmon ..
0 OCD?
o oo
IV
O
000000
a comme-- .134
0
0.0.
24
O
000
o B10 x B10.D2 Fe MICE
o BIO MICE
00 0000
000
22
20
ACE OF RECIPIENTS (days)
0
0
0
0
0
L
0
0
0
0
0
0
0
9!
:!
sammen mengaman
0.27
SPLENIC UPTAKE OF 13? UUR (%)
DONOR
www RECIPIENT STRAINS
111
810
A
O
O
PARENTAL - ISOGENIC PARENTAL
- 1840 X 810.02)F,
A.BY - (A X A.BY)
01. LP - (DBA/1 X 01. LP) FR
C3H.SW - (C3H.SW X C3H)F,

MEAN SPLENIC UPTAKE OF 1311 UdR (%)

////////
-'..
.-.Nom
..
ini
en
m
. .;** ta
...
.
W ."
A
O
!
!
*
3
- 104
(567891 2 3 4 5 6 7 8 9 2 § 4 5 6 7 8 1
105
106
NO. OF TRANSPLANTED MARROW CELLS
-
10%
E
-'
'
14,162
-T
THE PROCEDURE USED FOR SERIAL TRANSPLANTATION OF BIO XA F, MARROW CELLS AND FOR SERIAL TRANSFER OF
HYBRID RESISTANCE
..
...
B10 X A F, MARROW CELLS
comme....

"
.
...
.
REGENERATED MARROW

.
IST PASSAGE IN IRRADIATED
2ND PASSAGE IN IRRADIATED
BIO HOSTS
A HOSTS
122 DAYS
40 DAYS
2ND PASSAGE IN IRRADIATED
BIO XA F, HOSTS
40 DAYS
TESTED FOR HYBRID
TESTED FOR HYBRID RESISTANCE
AND FOR HEMOGLOBIN TYPE
RESISTANCE

19.
.
.
.
: .
{
.
!
PL
NTCA
,
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T
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:
.
2
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.
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AVT
w
41
L
!
AR
DATE FILMED
6 / 29 /65
+
S
T
I
ly
.
..
- LEGAL NOTICE
This report was prepared as an account of Government sporisored work. Neither the United
States, nor the Commission, nor any person acting on behalf of the Commission:
A. Makes any warranty or representation, expressed or implied, with respect to the accu-
racy, completeness, or usefulness of the information contained in this report, or that the use
of any information, apparatus, method, or process disclosed in this report may not infringe
privately owned rights; or
B. Assumes any liabilities with respect to the use of, or for damages resulting from the
use of any information, apparatus, method, or process disclosed in this report.
As used in the above, "person acting on behalf of the Commission” includes any em-
ployee or contractor of the Commission, or employee of such contractor', to the extent that
such employee or contractor of the Commission, or employee of such contractor prepares,
disseminates, or provides access to, any information pursuant to his employment or contract
with the Commission, or his employment with such contractor.
END