key: cord-0807466-z1ljij2t
authors: Hsieh, Li-En; Grifoni, Alba; Dave, Hiral; Wang, Jasmine; Johnson, Diana; Zellner, Jennifer; Sidney, John; Chambers, Christina; Franco, Alessandra
title: SARS-CoV-2-specific T cell responses and immune regulation in infected pregnant women
date: 2021-12-11
journal: J Reprod Immunol
DOI: 10.1016/j.jri.2021.103464
sha: 85c0bf73fe558a0f37dc1571b9ef1610b33e57b1
doc_id: 807466
cord_uid: z1ljij2t

We studied the T cell response to SARS-CoV-2 spike and non-spike peptide epitopes in eight convalescent pregnant women together with the immune monitoring that included innate tolerogenic dendritic cell populations important to maintain the immunological mother/fetus interface to address a potential risk for the antiviral cellular response in the outcome of pregnancy. Four subjects had pre-existing chronic inflammatory conditions that could have potentially affected the SARS-CoV-2-specific T cell response. Seven of eight subjects responded to SARS-CoV-2 peptides with differences within CD4 + T helper (Th) and CD8+ cytotoxic T cells (CTL). SARS-CoV-2-specific inducible regulatory T cells (iTreg) were numerous in circulation. CD4 + T cell memory included central memory T cells (T(CM)) and effector memory (T(EM)). As far as the CD8+ memory repertoire, T(CM) and T(EM) were very low or absent in eight of eight subjects and only effector cells that revert to CD45RA+, defined as T(EMRA) were measurable in circulation. T cells were in the normal range in all subjects regardless of pre-existing inflammatory conditions. The immune phenotype indicated the expansion and activation of tolerogenic myeloid dendritic cells including CD14+ cDC2 and CD4+ ILT-4+ tmDC. In summary, SARS-CoV-2 infection induced a physiological anti-viral T cell response in pregnant women that included SARS-CoV-2-specific iTreg with no negative effects on the tolerogenic innate dendritic cell repertoire relevant to the immune homeostasis of the maternal-fetal interface. All eight subjects studied delivered full-term, healthy infants.

Early data suggest that the special population of pregnant women is more susceptible to severe disease when infected with SARS-CoV-2 (Delahoy et al. 2020 , du Fosse 2020 , Ellington et al. 2020 , Zambrano et al. 2020 , and is at increased risk for adverse outcomes such as preterm delivery (Woodworth et al. 2020 ). Immune function is uniquely modified during pregnancy to allow for tolerance of the fetal allograft.

The immune cells that reside at the interface between the placenta and the uterus are in fact enriched or excluded from the decidua depending upon their regulatory or inflammatory function (Red-Horse 2004 , Moffett 2006 , Mold 2012 , Erlebacher 2013 . Specifically, dendritic cells (DC), the antigen presenting cells (APC) that activate T cells via the presentation of peptides through the human leucocyte antigen (HLA), are very sparse in the decidua. The maternal adaptive immunity that responds to pathogens is "confined" to prevent bi-directional trafficking across the placenta. The immune cells that constitute the maternal-fetal interface are the immune cells that populate the decidua. Specific lineages of suppressive myeloid dendritic cells prevent miscarriage of the pregnancy via IL-10 secretion and by priming regulatory T cells (Treg): tolerogenic dendritic cells (tmDC) defined by the expression of CD4, CD14, the immunoglobulin-like transcript (ILT)-4 with the canonical markers CD11 c and CD11 b (Burns 2013). HLA-G expression on DC leads to the polarization of T cell lineages toward a regulatory phenotype via the ILT-4/HLA-G pathway (Gregori 2010 , Amodio 2015 . At the maternal-fetal interface, ILT-4+, HLA-G+ DC, named DC-10, are very important in orchestrating the early interaction with the trophoblast (Amodio 2013, Gregori 2015) . Within the adaptive immunity, maternal Treg play a critical role in the immune homeostasis at the maternal-fetal interface. Treg restore immune tolerance to self J o u r n a l P r e -p r o o f and allo-antigens in humans (Roncarolo 2007 , von Boehmer 2010 , Wing 2010 .

Immune response to infection with the novel SARS-CoV-2 virus in pregnancy has not been well characterized.

In a recent case report, it was suggested that an infected pregnant woman had a good antibody response and there was no evidence of fetal damage (du Fosse 2020). Here we sought to characterize SARS-CoV-2-specific CD4+ and CD8+ T cell responses and the innate and adaptive immune phenotype in a small sample of pregnant women convalescent from SARS-CoV-2 infection.

The study sample included eight pregnant women ranging in age from 26 to 40 years who were documented to be infected with SARS-CoV-2 at varying times in gestation. Four women were healthy prior to SARS-CoV-2 infection, one was autoimmune diagnosed with polyarthritis rheumatism, one was allergic with atopic dermatitis, and two had ulcerative colitis (Table 1) .

Eligible subjects were selected for this study from pregnant women residing in the U.S. who were enrolled in the MotherToBaby prospective cohort study conducted by the OTIS Research Center at UC San Diego (Chambers 2001 , Chambers et al. 2010 , Chambers et al. 2013 , Bandoli 2017 , Chambers 2018 , Chambers 2019 . All enrolled pregnant women provide detailed information about their demographics, health history, and status of SARS-CoV-2 infection through maternal interviews and medical record review. None of the subjects had a severe SARS-CoV-2 infection and hospitalization was not required. The subjects with pre-existing inflammatory conditions were not on steroids medications at the time of the study. The two subjects with ulcerative colitis were on mercaptopurine and anti-47 integrin receptor respectively during pregnancy. Each subject provided a blood sample through a Quest Laboratory closest to their residence, which was subsequently sent by overnight mail to UC San Diego for processing and analysis. The study was approved by the Human Research J o u r n a l P r e -p r o o f Protection Program at UC San Diego, and all women provided written informed consent to participate.

All the SARS-CoV-2 peptides were derived by the bioinformatics analyses and synthesized as crude material (T.C. laboratory, San Diego, CA), resuspended in DMSO, and pooled according to megapool (MP) composition followed by sequential lyophilization steps. SARS-CoV-2 CD4 and CD8 peptides MP have been designed and validated in acute and convalescent samples, as previously reported (Grifoni 2020a , Grifoni 2020b , Mateus 2020 , Weiskopf 2020 . The MP design was carried out on the Wuhan-Hu-1 reference isolated (GenBank Accession Number: MN908947). To ensure a comprehensive assessment of CD4-spike specific reactivity, the main target of vaccine candidates, overlapping 15mers by 10 spanning the entire protein have been synthetized and pooled separately (Spike; n=253).

The remainder of the SARS-CoV-2 proteome was filtered applying the "7-allele-method" CD4-T cell prediction with a cutoff of ≤ 20, aiming to predict promiscuous epitopes with the capability to bind across the most common HLA class II specificities (Non-spike; n=221). SARS-CoV-2 CD8 MP epitopes were predicted using the 12 most frequent HLA A & B alleles with broad worldwide coverage in human population (A*01:01, A*02:01, A*03:01, A*11:01, A*23:01, A*24:02, B*07:02, B*08:01, B*35:01, B*40:01, B*44:02, B*44:03) and applying the IEDB recommended NETMHCpan4.0 EL algorithm. The top 100 resulting predicted epitopes for each HLA allele ranked based on prediction were selected and clustered using the IEDB cluster 2.0 tool applying the clusterbreak method with 70% cut off for sequence identity (Grifoni 2020a , Grifoni 2020b , Mateus 2020 , Weiskopf 2020 ) for a total of 628 peptides divided in two CD8 MPs (A and B) composed by 314 peptides each.

Peripheral mononuclear cells (PBMC) were separated from heparinized whole blood from the subjects by Ficoll-Hypaque density gradient. 1x10 6 PBMC were stimulated in 96 wells U bottom plates with 1 μg/ml of peptide MPs. PBMC cultured with 0.1% DMSO, the same concentration of DMSO (solvent) in J o u r n a l P r e -p r o o f the MP-stimulated cultures, served as un-stimulated controls. 24 hours after cultures, T cell activation was determined by the activation-induced cell markers (AIM) assay by measuring the co-expression of 4-1BB and OX40 on CD4+ T cells, and 4-1BB and CD69 on CD8+ T cells . The memory phenotypes   of AIM+ T cells defined effector memory T cells (TEM, CD45RA− CCR7−), central memory T cells (TCM,   CD45RA− CCR7+) , and terminally differentiated effector T cells (TEMRA, CD45RA+ CCR7−). CD4+ CD25 high regulatory T cells (Treg) that responded to SARS-CoV-2 MPs were also studied. The monoclonal antibodies used for these assays included anti-CD3-AF700 (clone OKT3, mouse IgG2aκ, Biolegend), anti-CD4-BV605 (clone RPA-T4, mouse IgG1κ, BD Bioscience), anti-CD8-BV650 (RPA-T8, mouse IgG1κ, Biolegend), anti-4-1BB-Allophycocyanin (clone 4B4-1, mouse IgG1κ, Biolegend), anti-OX40-PE/Cy7 (clone Ber-ACT35, mouse IgG1κ, Biolegned), anti-CD69-PE (clone FN50, mouse IgG1κ, BD Bioscience), anti-CD45RA-BV421 (clone HI100, mouse IgG1κ, Biolegend), anti-CCR7-FITC (clone G043H7, mouse IgG2aκ, Biolegend), anti-CD25-BUV395 (clone M-A251, mouse IgG1κ, BD Bioscience). Data were recorded on LSRFortessa X-20 (BD Biosceince) and analyzed with FlowJo software version 10 (Tree Star).

Innate myeloid, plasmacytoid and uterus resident immune cells were defined from the same PBMC preparation used to determine SARS-CoV-2-specific T cell responses by surface markers by staining with monoclonal antibodies and analyzed by flow cytometry gating on specific populations: anti-human CD11c-Allophycocyanin, clone B-ly6, mouse IgG1κ; anti-human CD11b-Allophycocyanin/Cy7, clone ICRF44, mouse IgG1κ; anti-human CD14-PE/Cy7, clone M5E2, mouse IgG2aκ (BD Biosciences); antihuman CD123-BV421, clone 6H6, mouse IgG1κ; anti-human BDCA-1-PE/Dazzle594, clone L161, mouse IgG1κ (Biolegend); anti-human ILT-4-PerCp/eF710, clone 42D1, rat IgG2aκ (eBioscience); antihuman CD4-AF700, clone RPA-T4, mouse IgG1κ (BD Biosciences); anti-human HLA-G-PE, clone 87G, mouse IgG2aκ (Biolegend); anti-human CD56-PE/Cy7, clone CMSSB, mouse IgG1κ (eBioscience); J o u r n a l P r e -p r o o f anti-human-CD209-BV421, clone 9E9A8, mouse IgG2aκ (Biolegend). The activation/maturation of the innate immune cells that present antigen to T cells was defined by the expression of CD86 by using anti-human-CD86 FITC, clone FUN-1, mouse IgG1κ (BD Biosciences). Naïve and activated CD4+ and CD8+ T cells were defined with anti-human CD4-PerCp/Cy5.5, clone RPA-T4, mouse IgG1κ; antihuman CD45RA-Allophycocyanin, clone HI100, mouse IgG2bκ; anti-human IL-7Rα-FITC, clone eBioRDR5, mouse IgG1κ; and anti-human CD56-PE/Cy7, clone CMSSB, mouse IgG1κ to define NKT cells (eBioscience) and anti-human CD8-AF700, clone RPA-T8, mouse IgG1κ (BD Biosciences). Data were acquired on BD CANTO II and analyzed with FlowJo software version 10 (Tree Star).

PBMC from the eight subjects were cultured 24 hours with peptide pools to study T cell activation, antigen-specific Treg expansion and T cell memory in response to SARS-CoV-2.

The assay involved PBMC separation and 24-hour stimulation with SARS-CoV-2 peptides that included spike and non-spike epitopes for CD4+ T cell responses and spike and non-spike epitopes for CD8+ cytotoxic T cell (CTL) responses. The readout was an activation induced markers (AIM) assay based on flow cytometry to detect the early expression of molecules that define CD4+ and CD8+ T cell As shown in Figure 1A , eight of eight subjects showed a T cell response to SARS-CoV-2 with some differences within the Th and CTL repertoire as previously observed in convalescent adults (Grifoni J o u r n a l P r e -p r o o f 2020b). Within the four healthy women before infection, only subject #1, had expanded CD4+ T cells specific for spike and non-spike proteins and CD8+ T cells. In this subject, the CD8+ T cell expansion was very pronounced. Coordinated CD4+ and CD8+ T cell responses were also detectable in subject #2: this subject showed only non-spike-specific CD4+ T cells. Subject #4 did not show CD8+ T cell expansion but CD4+ T cells recognized spike and non-spike proteins, as subject #3, that had only CD4+ spike-specific T cells in circulation. Subject #5 with polyarthritis rheumatism and subject #6 with allergy showed only CD8+ T cell responses but not Th responses. In contrast, subjects #7 and #8 with ulcerative colitis had a very pronounced CD4+ T cell response to spike and non-spike proteins but not CD8+ T cell responses.

In pregnancy, regulatory T cells are of the most importance in the maternal-fetal interface. To address the antigen-specific SARS-CoV-2 adaptive regulation, we enumerated CD4+ CD25 high Treg within the AIM+ T cells ( Figure 1B ). Eight of eight subjects had numerous spike-specific and non-spike-specific inducible Treg (iTreg) ( Figure 1C and Supplementary Figure 1) , indicating a role in controlling the antiviral response.

The development of T cell memory plays an important role not only to insure protection for the mother from possible secondary SARS-CoV-2 infection, but also protection of the child during birth and possibly later through maternal immunological microchimera and milk transfer (Hsieh 2019 , Franco 2020 ). When we examined TEM and TCM by gating in AIM+ positive T cells, we found differences depending upon timing of infection ( Figure 1C ). The main differences were observed between spike-specific CD4+ TEM (numerous in six of eight subjects) and non-spike CD4+ TEM repertoire which occurred at a much lower rate ( Figure 1C ). Of note, none of the eight subjects showed a TEM or a TCM CD8+ T cell response irrespective of the timing of infection ( Figure 1C ). Within the CD8+ T cell memory populations, TEMRA were inversely numerous within CD8+ T cells but not CD4+ T cells ( Figure 1C ). T cell numbers were in the normal range and comparable between subjects with CD4+ T cells ranging from 29.6 to 58.7% (median 46.9%) and CD8+ T cells ranging from 9.4 to 24.6% (median 19.8%). CD4+ and CD8+ J o u r n a l P r e -p r o o f CD45RA+ naïve T cells were in the normal range, 54.8 to 88.9% (median 79.8%). Antigen experienced IL-7R+ T cells were comparable between subjects, 42.1 to 82.6% (median 67.8%) ( Figure 2 ).

Next, we explored the antigen presenting cell (APC) compartments for T cell presentation including myeloid cells important for the immune tolerance for the protection of the fetus from the same PBMC preparation tested for SARS-CoV-2 T cell responses. The most relevant regulatory innate cells in pregnancy include three myeloid lineages that abundantly secrete the suppressive lymphokine IL-10: CD14+ cDC2 and tmDC defined by the expression of ILT-4 and CD4 that can be found in circulation and tissue resident DC10, defined by the expression of ILT-4 and HLA-G. We included monocytes, macrophages, pro-inflammatory cDC1, CD14− cDC2 and plasmacytoid DC. The expression of CD86 served to define cell activation/maturation in these innate populations. The gating strategies for the analysis of the populations by flow cytometry can be found in Figure 3A . We also enumerated in circulation tissue resident DC-10, myeloid DC with suppressive function defined by the expression of the HLA-G and ILT-4 in addition to HLA-G+ regulatory decidual macrophages, and CD209+ decidual NK cells. These cells are important for the physiological formation of the villi and the protection of the fetal compartment from pro-inflammatory T cell responses that should not cross the myometrium that could be tracked in the periphery heading to tissues.

Tolerogenic innate cells CD14+ cDC2 and tmDC were numerous in seven of the eight subjects with the exception of subject #8 who had ulcerative colitis, although not statistically significant ( Figure 3B ). When we measured the CD86 expression, we found that CD14+ cDC2 and tmDC were fully activated and mature, suggesting that immune tolerance was the most relevant efferent arm of the antigen presentation irrespective of maternal health status before pregnancy and the timing in pregnancy of SARS-CoV-2 infection ( Figure 3B ).

Classical APC enumeration within the myeloid compartment included: monocytes, macrophages, cDC1, J o u r n a l P r e -p r o o f CD14− cDC2. The results pointed to few differences between subjects and a much lower activation of these cells compared with the tolerogenic counterpart ( Figure 3B ). cDC1 and CD14− cDC2, canonical APC for T cell responses, were found in the normal range. Levels of CD86 expression, a marker of activation/maturity, were high in the two subjects whose samples were collected three weeks after onset of the infection (#1 and #7, respectively), suggesting that the anti-viral T cell response was still ongoing. We also enumerated CD123+ plasmacytoid DC, relevant in the innate response to viruses.

These were found to be very low (lower than 6% in total PBMC) and again, not activated in the subjects whose samples were collected close to onset of the SARS-CoV-2 infection ( Figure 3C ).

Tolerogenic tissue resident DC-10 and decidual macrophages were measurable in circulation in all the subjects studied with no significant differences in women with inflammatory conditions ( Figure 4A ) as CD209+ decidual NK cells ( Figure 4B ).

Here we report the SARS-CoV-2-specific T cell response and immune phenotype in eight SARS-CoV- an open question about patterns of T cell recognition in infected pregnant women and the integrity of the mechanisms involved in protecting the fetus from pro-inflammatory T cell responses. The fetal HLA differs from maternal HLA and their physiology would suggest an immune response to "allogeneic" antigens. The delicate balance between the necessary pro-inflammatory systemic immune response and strong immune regulation within the placenta occurs through a very complex cross-talk between maternal immune cells and the placental trophoblast. Low numbers and/or functional defects within innate and adaptive cell lineages that regulate the immunity in the maternal-fetal interface contribute to several clinical complications including premature delivery. In physiological conditions, during a viral infection the maternal immune response clears the pathogen, avoiding fetal damage due to proinflammatory T cells in the placental interface. A mechanism that prevents T cell activation is the very J o u r n a l P r e -p r o o f low number of DC in the decidua that limits the ability to initiate adaptive immune responses in the draining lymph nodes not only to fetal antigens but also to antigens derived from viruses or pathogens (Erlebacher 2013) .

This work reports the T cell anti-viral response in eight SARS-CoV-2 infected pregnant women highlighting the CD4+ and CD8+ recognition of spike and non-spike peptides peptide epitopes. The SARS-CoV-2 proteome was probed using 1,102 peptides (9 to 15 amino acids in length) spanning the whole genome, ensuring the detection of HLA class II-restricted CD4+ T cell responses and HLA class I-restricted CD8+ T cell responses (Grifoni 2020a , Grifoni 2020b , Lipsitch 2020 , Mateus 2020 , Rydyznski Moderbacher 2020 ).

The results indicated that coordinated CD4+ Th cell and CD8+ CTL responses to SARS-CoV-2 were found in subjects closer to the onset of infection (three weeks), and showed greater activation of classical myeloid APC for T cell presentation as cDC1 and CD14− cDC2. This suggested that the T cell response to the virus is still ongoing in subjects infected months prior to the blood sample collection.

Of interest, only the subject with allergy (#6, atopic dermatitis) did not respond to any of the SARS-CoV-2 peptide pools, although the T cell enumeration has not been informative in capturing differences with the other subjects as the number of CD4+ T cells were comparable.

Memory T cells, TEM and TCM, have been found confined to the CD4+ compartment and less numerous in healthy women close to delivery, raising the question of a possible specific homing to the uterus and T cell transfer to the fetus. The absence of TEM and TCM memory CD8+ T cells in circulation but numerous TEMRA (these antigen-experienced effector T cells revert to a naïve CD45RA+ phenotype and are ready to expand upon encounter with the antigen), raises a question on the different timing within the T cell memory development between Th and CTL. This is an important point to better understand the risk of possible re-infection in convalescent subjects.

iTreg that recognized viral epitopes were numerous regardless of the timing of infection, timing of pregnancy or pre-existing conditions. We did not detect Treg in COVID-19 convalescent women (Hsieh J o u r n a l P r e -p r o o f 2021) but further investigation is required to drive final conclusions about the unique presence of SARS-CoV-2-specific iTreg in pregnancy. In pregnancy, Treg play a critical role in preventing allogeneic T cell responses and our data indicate that SARS-CoV-2 infection elicits not only a pro-inflammatory T cell response in pregnant women but also a regulatory T cell response. Of interest, two subjects with ulcerative colitis showed the most pronounced response to the virus and the larger number of Treg in circulation. The gut and reproductive system share several lymphoid stations suggesting that women with gut inflammation require further control of pro-inflammatory T cells that may affect the maternalfetal interface.

The predominant innate populations in circulation were tolerogenic in all the subjects studied: CD14+ cDC2 and tmDC were numerous and highly activated, indirectly suggesting that the immune homeostasis of the maternal-fetal interface is not compromised by SARS-CoV-2 infection. cDC1 and CD14− cDC2, classical APC for T cell presentation, were found in the normal range and activated in women studied close to the time of infection with no significant differences within the subjects.

In summary, SARS-CoV-2 infection does not jeopardize the physiology of the immunity at the maternalfetal interface. Further evidence is the enumeration of tolerogenic tissue resident innate cells as DC-10 and decidual macrofages that we found in circulation in comparable numbers between healthy women and women with pre-existing conditions before SARS-CoV-2 infection. This is supportive of the current recommendations regarding the safety of vaccination to SARS-CoV-2 any time during pregnancy. Furthermore, in support of this interpretaton, all the subjects studied delivered full-term, healthy infants.

Little is known about the placental infection in SARS-CoV-2-infected pregmant women. In two case reports viral load has ben found in the placenta associated either with pre-eclampsia at the third trimester (Hosier 2020) or neurological pathology in the baby (Vivanti et al. 2020) suggesting that healthy deliveries of healthy babies in SARS-CoV-2-infected women does not involve placental infection. More studies are needed to validate this statement: we do not have information in our cohort J o u r n a l P r e -p r o o f but the low numbers of pDC, critical innate cells in the anti-viral response and CD14-cDC2 would suggest lack of placental infection.

We acknowlege the strengths and limitations of this work. The sample size of women with inflammatory disorders was small to generalize on all autoimmune and allergic conditions. We first explored the virusspecific T cell repertoire and immune phenotype in SARS-CoV-2 infected pregnant women. However, we did not examine cord blood tissues and blood samples from infants born to the study subjects. This is a limitation in addressing the homing of the T cells, in particular memory T cells, their relation to fetal protection and possible T cell transfer to the baby as a mechanism of protection.

The authors declare no competing interests. LJI has filed for patent protection for various aspects of vaccine design and identification of specific epitopes. 

PBMC from eight pregnant women infected by SARS-CoV-2 were separated from heparinized whole blood using Histopaque. PBMC were stimulated with SARS-CoV-2 CD4 spike (253 epitopes), CD4 non-spike (221 epitopes) peptide MPs, or CD8 (MP A and MP B, 314 epitopes each) for 24 hours followed by the staining using a combination of monoclonal antibodies to study the T cell and iTreg activation in response to stimulation. The T cell memory phenotype of the antigen-specific AIM+ CD4+ and CD8+ T cells was also studied 24 hours after stimulation. T cell and iTreg activation is Table 1 studied three weeks after acute SARS-CoV-2 symptoms and at 12.4 weeks of gestation and one pregnancy subject with ulcerative colitis studied three weeks after acute SARS-CoV-2 symptoms and at 38.1 week of gestation, #7 in Table 1 

Characteristics and maternal and birth outcomes of hospitalized pregnant women with laboratory-confirmed covid-19 -covid-net, 13 states

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Hlas-g orchestrate the early interaction of human trophoblasts with maternal niche

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CD45RA− CCR7+) were enumerated in the antigen-specific, AIM+ CD4+ and CD8+ T cell populations. Treg were defined by the expression of CD4+ CD25

Supplementary Figure 2. Enumeration of AIM+ CD4+ and CD8+ T cells in response to the stimulation with anti-CD3/anti-CD28 agonistic antibodies. (A) Representative FACS plots of the

AIM+ T cell responses after anti-CD3/anti-CD28 stimulation from one healthy pregnancy subject

SARS-CoV-2 symptoms and at 12.4 weeks of gestation and one pregnancy subject with ulcerative colitis studied three weeks after acute SARS-CoV-2 symptoms and at 38.1 week of gestation

This work was partially supported by a grant from the National Institutes of Health (RO1AI43586) to AF, by a grant Resource-based Center for the study of the joint microenvironment in rheumatology;University of California San Diego, NIH Grant P30AR073761 to AF, a grant from the National