Polymorphic estrogen receptor binding site causes CD2-dependent sex bias in the susceptibility to autoimmune diseases

Polymorphic estrogen receptor binding site causes CD2-dependent sex bias in the susceptibility to autoimmune diseases | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Help Center Sign In Submit a Preprint Cite Share Download PDF Article Polymorphic estrogen receptor binding site causes CD2-dependent sex bias in the susceptibility to autoimmune diseases Gonzalo Fernandez Lahore, Michael Förster, Martina Johannesson, and 7 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-337166/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 22 Sep, 2021 Read the published version in Nature Communications → Version 1 posted You are reading this latest preprint version Abstract Complex autoimmune diseases are sexually dimorphic. An interplay between predisposing genetics and sex-related factors likely determines the sex discrepancy in the immune response, but conclusive evidence is lacking regarding the underlying molecular mechanisms. Using forward genetics, we positionally identified a polymorphic estrogen receptor binding site that regulates CD2 expression, leading to female-specific differences in mouse models of T cell-dependent autoimmunity. Female mice with reduced CD2 levels displayed diminished expansion of autoreactive T cells. Mechanistically, CD2 affected T cell activation by inhibiting LAG-3 expression. Our findings explain the sexual dimorphism in human autoimmunity, as CD2 associated with rheumatoid arthritis and its regulation through 17-β-estradiol was conserved in human T cells. Hormonal regulation of CD2 has implications for CD2-targeted therapy. Indeed, anti-CD2 treatment was more potent in female mice. In conclusion, our results demonstrate the relevance of sex-genotype interactions and provide strong evidence for CD2 as a sex-sensitive predisposing factor in autoimmunity. Immunology Molecular Genetics Immunogenetics Gene regulation autoimmune diseases Polymorphic estrogen receptor CD2 Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction Women mount a more vigorous immune response and are more susceptible to most autoimmune diseases 1 , 2 . These diseases have a strong but complex genetic component, and it has been difficult to identify the underlying polymorphisms 3 – 5 . The female preponderance in autoimmunity is sex hormone related 6 but could also be genetically dependent 7 . Not only through sex chromosomes but also through distinct sex hormone regulated expression of autosomal genes. However, conclusive evidence is still lacking, as it is difficult to positionally identify the underlying polymorphisms controlling complex traits in a sex-dependent manner. Analysis of genetically segregated inbred animal strains dramatically enhances the power to isolate polymorphisms underlying complex diseases. Compared with association studies of human cohorts, studies in mice reduce environmental variability and allow for proof-of-concept experiments in biologically relevant systems, making it possible to conclusively identify genes underlying complex traits. In the context of previous such work to identify genetic loci that regulate autoimmune arthritis 8 – 10 , we have identified a locus on mouse chromosome 3 (Cia21) that affects expression of the T cell activation marker CD2 and regulates arthritis severity in females, but not in males 9 . We herein find the cause of the effect to be a polymorphic estrogen receptor binding site (ERBS) within Cia21 that recapitulates the phenotypic properties of its parent locus. This polymorphic ERBS orchestrates expression of surrounding genes in a sex-specific manner, including CD2. We isolated these polymorphisms in a congenic mouse line (D3-31) and used these mice to study the consequences of estrogen-mediated regulation of CD2 for T cell-dependent autoimmunity. In addition, we found estrogen regulation of CD2 expression to be a conserved mechanism in humans that likely contributes to the sexual dimorphism in T cell-mediated autoimmune diseases. Results We have set out to identify major genetic polymorphisms underlying the development of autoimmune arthritis, using animal models. As part of these efforts we previously described a major quantitative trait locus (QTL) on chromosome 3 qF2.2, which we termed Cia21 9 . Cia21 was identified from an inter-cross between the collagen-induced arthritis (CIA)-susceptible C57BL/10.RIII (BR) and the CIA-resistant RIIIS/J (R3) mouse strains 11 . Cia21 contains several differentially expressed genes, including CD2 and PTPN22 9 . Both CD2 and PTPN22 play a key role in T cell activation and were proposed as strong candidate genes. The aim of the present study is to identify the polymorphisms underlying the Cia21 QTL. A minimal non-coding genetic interval proximal to CD2 recapitulates the arthritis-regulating properties of Cia21 To dissect the Cia21 QTL, we bred heterozygous Cia21 mice and evaluated the resulting recombinant mice (shown in Fig. 1 a) using CIA (Fig. 1 b). Out of all the evaluated recombinants, only two, numbers 1 and 5, recapitulated the protective arthritis phenotype previously observed in Cia21 mice 9 . Thus, the Cia21 QTL results from individual contributions of these two sub-QTLs. Importantly, the phenotype driving recombinant regions 1 and 5 mapped to the previously proposed 9 candidate genes CD2 and PTPN22 , respectively. Recombinant fragment 1 (proximal to CD2 ), however, was significantly smaller than fragment 5 providing better conditions for the positional identification of underlying polymorphisms. Therefore, we focused our efforts on the former. Recombinant fragment 1 stretched from markers D3KV1 to MF31 (Fig. 1 a, ca. 0.2 Mbp), but could be further redefined to the significantly smaller D3KV1-MF96 interval (ca. 0.02 Mbp) through a recombination assisted breeding strategy. Although recombinant fragments 1, 2, and 3 overlapped significantly, only fragment 1 regulated arthritis. Thus, we concluded that the causative polymorphisms must be positioned between markers D3KV1 and MF96 (Fig. 1 a, highlighted yellow). D3KV1-MF96 is a non-coding 0.02 Mbp region proximal to CD2 , located in-between the genes ATP1A1 and IGSF3 (Fig. 1 c). We isolated the D3KV1-MF31 recombinant fragment (termed D3-31) in a congenic mouse line for further investigations. D3-31 congenic mice carry the parental R3 allele of D3-31 on an otherwise BR background. For simplicity, we hereon refer to the congenic line as D3-31 and to wild type littermates as BR. D3-31 congenic mice are protected from several T cell-dependent models of autoimmunity in a sex-specific manner In accordance with our previous data on Cia21, the R3 allele of D3-31 protected congenic mice in T cell-dependent 12 – 14 autoimmune inflammatory models, including collagen induced arthritis (CIA), experimental autoimmune encephalomyelitis (EAE), and delayed type hypersensitivity (DTH) (Fig. 2 a-f). We also investigated the T cell-independent 15 collagen antibody-induced arthritis (CAIA) model, but observed no phenotypic differences (supplementary fig. S1). As the DTH model does not depend on B cells 12 , these results indicated a critical role for T cells. Interestingly, and as previously described for Cia21 9 , only female D3-31 mice were protected from T cell mediated autoimmunity (Figs. 2 a-f). Thus, we concluded that D3-31 regulates T cell dependent autoimmune phenotypes, and likely T cells, in a sex-specific manner. Female sex hormones are required for the protective phenotype in D3-31 mice To discriminate between influence of sex chromosomes versus hormones, we performed CIA and EAE experiments in castrated female mice (Fig. 3 a-c). Castration of female mice depletes gonadal production of 17-β-estradiol (E2) 16 , which constitutes the major circulating estrogenic compound in females. Castration reverted the protective effect of the D3-31 fragment both in CIA and EAE (Fig. 3 a-c), which demonstrated the crucial contribution of female sex hormones, most likely E2, to the protective phenotype in female D3-31 mice. We next defined the genetic mechanisms underlying this sexually dimorphic immune phenotype by sequencing the D3-31 fragment. Polymorphisms in an estrogen receptor binding site (ERBS) affect E2-mediated transcriptional activity DNA sequencing of the D3-31 BR and R3 alleles revealed four single nucleotide polymorphisms (SNPs) in the critical D3KV1-MF96 interval (Fig. 4 a and b). None of the variants affected the coding region of known genes, indicating distal (cis) regulation of gene expression, likely by interfering with regulatory elements. Given our previous observations, we speculated that the identified polymorphisms could be located within an ERBS, interfering with sex-dependent regulation of gene expression. Estrogen receptors (ERα and ERβ) are nuclear hormone receptors that translate E2-mediated signalling. Both ERα and ERβ are expressed in immune cells 17 , and act as transcription factors regulating the expression of proximal and distant genes 18 , 19 . To test our hypothesis, we screened publicly available ChIP-seq data for ERα binding sites overlapping with one or more of the sequenced SNPs within D3KV1-MF96 interval. Indeed, one of the SNPs, AC > GG on chr3:101310478-479 (termed SNP478), clearly overlapped with an ERα binding site (Fig. 4 c). In fact, bioinformatic analysis also revealed an estrogen response element (i.e. an ER core binding motif) in close proximity to SNP478. We sought to verify this finding, and confirmed binding of ERα to SNP478 in spleen cells using ChIP-qPCR (Fig. 4 d). Comparison of SNP478 between mouse inbred strains revealed that this SNP is in fact part of a highly polymorphic AC/GT simple repeat (supplementary fig. S2, extracted from 20 ). To address whether SNP478 had functional consequences for E2-mediated transcriptional activity (i.e. interfered with the binding of ERα to the DNA), we cloned the candidate D3KV1 ERBS (± 100 bp) in its two variant forms (AC and GG) into luciferase reporter constructs. We assessed transcriptional activity of these constructs in transfected ERα + MCF-7 cells treated with increasing concentrations of E2 (Fig. 4 e). In the context of the reporter construct, an increased occupancy of the ERBS by ERα (as a function of increasing E2) resulted in suppression of transcriptional activity. Although surprising, similar observations have been reported elsewhere 21 . Given the stronger transcriptional inhibition when using the BR derived construct, we concluded that ERα has a higher affinity for the BR allele than for the D3-31 allele. Importantly, these data demonstrate that SNP478 has functional consequences for E2-mediated transcriptional activity. Polymorphism in an ERBS leads to female-specific changes in CD2 expression Next, we tested the biological relevance of our findings by comparing the gene expression profile in lymphoid tissue from male and female D3-31 and BR mice. We observed female-specific changes in the expression of three genes adjacent to the polymorphic ERBS, namely CD2 , IGSF3 and MAB21L3 (Fig. 5 a). We also investigated the expression of ATP1A1 as well as more distal genes ( CD101 and SLC22A15 ) previously implicated in the non-obese diabetic (NOD) mouse model of type 1 diabetes 22 , but found no changes in their expression level. Notably, the female-specific reduction of CD2 expression in D3-31 mice was also evident at protein level (Fig. 5 b), and correlated with our previously reported gene expression results 9 . Out of the differentially expressed genes, CD2 was the only gene predominantly expressed in lymphoid tissue (Fig. 5 c), particularly in activated CD4 + T cells (Fig. 5 d). IGSF3 and MAB21L3 regulate neural 23 and ocular 24 development, whereas CD2 has been involved in immune function 25 and associated with human autoimmune conditions 4 , 26 . Indeed, treatment of lymph node cells with anti-CD2 mAb inhibited T cell activation as demonstrated by reduced secretion of pro-inflammatory cytokines (Fig. 5 e). Considering these data and normal development of D3-31 mice, we concluded that CD2 is driving the T cell-dependent immune phenotype observed in D3-31 mice. Given the sex-specific differences in gene expression, we next investigated the relation between E2 and CD2 expression. T cells cultured in the presence of E2 up-regulated CD2 in a dose-dependent manner (Fig. 5 f). Conversely, use of E2 depleted medium (achieved by using charcoal-stripped serum) reduced the expression of CD2, and, more importantly, neutralized the observed differences in CD2 expression between BR and D3-31 mice. Additionally, differences in CD2 expression could be re-established by reintroducing E2 to the medium (Fig. 5 g). This not only demonstrates direct regulation of E2 on CD2 expression, but also proves that the identified polymorphisms interfere with this regulation. Consequently, we speculated that E2-mediated regulation of CD2 was contributing to sex-specific differences in the T cell responses. A sex-dependent reduction of CD2 expression in female D3-31 mice could likely limit the T cell responses. E2-dependent regulation of CD2 expression leads to sex-specific differences in autoreactive T cell activation To investigate the impact of sex hormone-dependent alterations in CD2 expression on the T cell responses, we compared the activation of T cells between BR and D3-31 female mice. In a first set of in vitro experiments, we found an impaired response in D3-31 T cells to TCR stimulation, as evidenced by reduced proliferation and IL-2 production (Fig. 6 a). Importantly, the difference in T cell proliferation between BR and D3-31 mice could be enhanced in a dose-dependent manner by E2 (Fig. 6 b), much like the E2-dependent expression differences observed for CD2 (Fig. 5 g). A diminished T cell response in D3-31 mice was also evident in vivo . Compared to BR mice, D3-31 mice showed a lower level of antigen specific T cells responses 10 days after immunization with CIA antigen bovine collagen type II, as demonstrated by reduced secretion of proinflammatory cytokines in lymph node cultures recalled with antigen (Fig. 6 c). Flow cytometry analysis of D3-31 draining lymph nodes revealed lower numbers of antigen experienced CD40L + CD4 + T cells (Fig. 6 d), which expressed reduced levels of CD2 and L-17A after ex vivo restimulation with PMA (Fig. 6 d and e). Differences in T cell activation status were also evident by lower numbers of induced regulatory T cells after immunization (Fig. 6 f). Importantly, the observed differences in T cell activation were strictly sex-specific (Fig. 6 d-f), mirroring sex-specific differences in CD2 expression. Treatment with anti-CD2 strongly reduced the expression of IL-17 in naïve and autoreactive CD4 + T cells (Fig. 6 g and e, respectively), as well as FOXP3 in naïve T cells (Fig. 6 g), demonstrating a key role for CD2 in the differentiation of Th17 and Treg cells. Consequently, we concluded that reduced CD2 expression in female D3-31 mice limits T cell activation in a sex-specific manner. Although CD2 can regulate TCR signalling by increasing the stability of the immune synapse, we thought it plausible that persistent differences in CD2 signalling could elicit more profound phenotypic changes. Proteomic and flow cytometric analysis of CD4 + T cells treated with an anti-CD2 mAb resulted in a selective up-regulation of the immune inhibitory marker LAG-3 (Fig. 6 h). Thus, our data suggests that CD2 signalling modulates T cell activation not only by stabilizing the immune-synapse, but also by regulating the expression of the inhibitory marker LAG-3. CD2 associates with rheumatoid arthritis (RA) and is regulated by E2 in humans Our results in mice suggested a regulatory role for CD2 on T cell-dependent autoimmunity, which is genetically determined in a sex-linked manner. We therefore explored the relevance of our findings in humans in the context of RA. In a genetic association study, we found a significant association between CD2 polymorphisms and RA (Fig. 7 a). While this association was more often found in females than in males, this was likely due to higher prevalence of RA in females (female to male ratio 3:1). Interestingly, several of the SNPs associated with RA (p < 0.05) can enhance expression of CD2 (Fig. 7 b), as we determined from the GTEx database 27 . Further analysis of available microarray datasets 28 revealed a mild yet significant correlation between CD2 expression in RA synovia and disease activity (Fig. 7 c). Moreover, CD2 is strongly up-regulated in the synovial tissue from RA patients when compared to osteoarthritis or healthy synovium (Fig. 7 d). Thus, it is likely that CD2 is involved in joint inflammation, and that CD2 polymorphisms affecting its expression contribute to the development or perpetuation of joint autoimmunity. Importantly, women expressed higher levels of CD2 than men, both in RA synovium and healthy PBMCs (Fig. 7 c and 7 e, respectively), suggesting the E2-mediated regulation of CD2 observed in mice is conserved in humans as well. To corroborate our findings, we stimulated CD4 + T cells from healthy human donors with increasing amounts of E2. Firstly, we noticed a strong up-regulation of CD2 in antigen experienced CD45RO + T cells compared to their naïve CD45RA + counterparts (Fig. 7 f). But more importantly, expression of CD2 could be enhanced in CD45RO + T cells by incubation with E2 in a concentration-dependent manner (Fig. 7 g). Indeed, analysis of available ChIP-seq data 29 revealed that ERα robustly binds the human CD2 gene locus (Fig. 7 h). Thus, these data demonstrate the evolutionary conserved nature of E2-mediated regulation of CD2. We reasoned that hormonal regulation of CD2 expression could have implications for anti-CD2-mediated therapy, as previous research suggests that anti-CD2 (Alefacept) preferentially targets CD2 hi T cells 30 . To test this, we compared the in vivo effects of anti-CD2 mAb administration on circulating T cells from male and female mice (Fig. 7 i). Anti-CD2 mAb treatment partially depleted circulating T cells, and resulted in the relative enrichment of remaining effector CD44 + T cells, skewing the naïve CD62L + /effector CD44 + T cell ratio. This effect was significantly more pronounced in females, which, like in humans, expressed higher levels of CD2 in circulating T cells. Taken together, these data demonstrate that sex-dependent differences in CD2 expression determine the response to anti-CD2 mAb. Discussion Using forward genetics, we have positionally identified a polymorphic ERBS regulating T cell-dependent autoimmunity. This site orchestrates expression of surrounding genes in a sex-specific manner, including expression of the T cell co-stimulatory molecule, CD2. We find that E2-mediated regulation of CD2 is a conserved mechanism that influences T cell activation in a sex-specific manner, contributing to the sexual dimorphism in autoimmune diseases. Understanding the sexual dimorphic immune responses is fundamental for personalized medicine but is methodologically challenging. Common approaches to study this phenomenon rely on intricate manipulation of gonadal or hormonal systems 31 , which has yielded valuable insights but with limited physiological relevance. Our study provides a more physiological perspective by the identification of a naturally occurring polymorphism in an ERBS, which enables studies on sex-associated differences in T cell-mediated autoimmunity. Since we used a hypothesis-free approach, our findings strongly suggest E2-mediated regulation of CD2 as a key physiological mechanism contributing to sex differences in the T cell responses and susceptibility to autoimmunity. Our results also highlight the importance of genotype-sex interactions for the sexual dimorphism in autoimmune diseases. While much attention has been devoted to the contribution of sex chromosomes, epigenetic mechanisms or direct actions of hormones to the sexually dimorphic immune responses, the interactions between sex and predisposing autosomal polymorphisms have remained elusive. Isolated studies have demonstrated sex-dependency of expression (e) QTLs 32 and sex differences in the genetic associations to inflammatory diseases 33 – 35 , but evidence is limited. Using a hypothesis-free approach, we for the first time conclusively identify a sex biased QTL with direct consequences for the development of autoimmunity. Polymorphisms in the identified ERBS modulate E2-driven CD2 expression, leading to sex-specific differences in T cell autoimmunity. Our results demonstrate not only that genetic polymorphisms influence hormonal regulation of gene expression, but also that genotype-sex interactions shape the sexually dimorphic immune response. Independent of our sex-related findings, this study provides valuable insights into CD2 immunobiology. Polymorphisms in the CD2 locus have been previously associated with several autoimmune diseases 4 , 26 , but not much attention was given to the mechanism of action of these polymorphisms. Similarly, CD2 has been explored as a therapeutic target, but its mechanism of action beyond depletion of circulating T cell is poorly characterised 36 , and complex adverse effects (including malignancies 37 ) warrant further research. CD2 in isolation affects the formation of the immune synapse 3839 and T cell activation 4041 , but the relevance of these findings in vivo are less clear. For example, targeting CD2 in mice does not seem to affect immune system development 42 or thymic T cells 43 , unless TCR transgenic systems are used 4445 . Thus, there is a need to study this therapeutically promising pathway in a physiologically relevant context. The D3-31 mice used in this study exhibit discrete changes in CD2 expression mediated by E2, thus enabling us to study the effect of CD2 on T cell mediated autoimmunity in a physiological setting. We show for the first time that changes in CD2 expression, caused by natural polymorphisms, affect the T cell responses. Reduced CD2 expression protected mice from T cell-dependent inflammation and autoimmunity by reducing the activation and proliferation of antigen-specific T cells. This is consistent with studies demonstrating that CD2 membrane density is proportional to TCR signalling strength 38 , and that peptide-based blocking of CD2 signalling reduces CIA severity 46 . Our results also implicate CD2 in the generation of Th17 and Treg- type T cell responses. Mice with reduced CD2 expression had a diminished T cell response characterized by a reduced expansion of Th17 and Treg cells. Accordingly, blocking CD2 resulted in the suppression of both cell types in vitro . Indeed, CD2 has been linked to Treg 47 , 48 and Th17 phenotypes 39 before, and targeting CD2 is effective in the treatment of Th17-mediated inflammatory diseases like psoriatic arthritis 49 . In summary, this suggests a key role for CD2-mediated activation in the induction of Th17 and Treg cells. Mechanistically, CD2 seems to play a role in T cell activation beyond its ability to stabilize the immune synapse, as blocking CD2 results in selective up-regulation of the exhaustion marker, LAG-3. This finding is supported by studies showing an inverse correlation between CD2 expression and exhaustion of T cells 38 50 , and up-regulation of LAG-3 in human CD8 T cells after treatment with Alefacept (anti-CD2) 51 . Thus, together with other studies, our data suggests that CD2 signalling maintains T cell autoreactivity by reducing the expression of inhibitory LAG-3 molecule. Our findings in mice are likely relevant to the sexual dimorphism observed in human autoimmune conditions. CD2 associates with RA and E2 regulation of CD2 expression is highly conserved in human T cells. Women, who are generally more prone to autoimmunity, express higher levels of CD2 than men. In mice, we demonstrate that these type of discrete and sex-specific differences in CD2 expression result in sexually dimorphic T cell responses and autoimmune phenotypes. Thus, subtle, physiological changes in CD2 expression caused by natural polymorphisms likely modify the risk of T cell-dependent autoimmunity in humans. E2-mediated regulation of CD2 probably contributes to sex differences in the immune responses, both in homeostasis as well as autoimmune conditions. Sex-dependent differences in CD2 expression have implications for several sexually dimorphic immune processes involving T or other CD2 expressing cells. Hormonal regulation of CD2 could contribute to more vigorous humoral immune responses in women 2 , helping to protect their off-spring from infections 52 at the cost of an enhanced risk to autoimmunity post-partum 53 . Alternatively, an enhanced CD2 expression in women might facilitate the induction of regulatory T cell phenotypes (as we observed in mice) to facilitate foetal-maternal immune tolerance. A hormonal regulation of CD2 expression could have wide ranging implications for the personalized therapy of T cell-mediated inflammatory diseases, as Alefacept was shown to preferentially target CD2 hi T cells 30 . Indeed, we demonstrate strong effects of anti-CD2 mAb administration on the naïve/effector T cell ratio in female, but not male mice. As such, sex-specific differences in T cell CD2 expression may offer a useful biomarker for stratification of patients in the context of CD2 targeted therapies. In conclusion, our results highlight the importance of genotype-sex interactions for the sexual dimorphism in autoimmunity, demonstrating that sex can determine the penetrance of predisposing genetic factors. Our findings show that CD2 is a sex-sensitive regulator of T cell-mediated autoimmunity. Hormonal regulation of CD2 is a conserved mechanism that has implications for the sexual dimorphism in the susceptibility to -and treatment of- autoimmune diseases like RA. Materials And Methods Animals . The BR.Cia21.D3-31 congenic founder mice were obtained from a partial advanced intercross (PAI) described elsewhere and they were subsequently back crossed for four additional generations 9 . In order to ensure strain purity, BR.Cia21.D3-31 mice were screened with a custom designed 8k Illumina chip at genome wide level 54 and the mice were found to be devoid of any contaminating RIIIS/J alleles. No SNPs were present between the congenic and the B10.RIII background strain. Mice were kept under specific pathogen free (SPF) conditions in the animal house of the Section for Medical Inflammation Research, Karolinska Institute in Stockholm. Animals were housed in individually ventilated cages containing wood shavings in a climate-controlled environment with a 14 h light-dark cycle, fed with standard chow and water ad libitum . All the experiments were performed with age-, sex- and cage-matched mice and all the genetic experiments were performed with littermate controls. All the experimental procedures were approved by the ethical committees in Stockholm, Sweden with ethical permit numbers; 12923/18 and N134/13 (genotyping and serotyping), N35/16 (CIA) and N83/13 (EAE). Preparation of mouse single cell suspensions . Briefly, spleen or lymph nodes were harvested and mechanically dissociated on a 40 µM cell strainer (Falcon) using a 1ml syringe plunger (Codan). Cells were counted on a Sysmex KX-21 cell counter. All centrifugation steps throughout the study were carried out a 350 x g for 5 min at RT. For spleen samples, red blood cells were lysed in RBC buffer (155 mM NH4Cl, 12 mM NaHCO3, 0.1 mM EDTA) before counting. Preparation of human peripheral blood mononuclear cells (PBMC). Human PBMCs were prepared from 8 ml whole blood of healthy donors using SepMate (Stemcell Technologies) tubes and Ficoll density gradient medium (Sigma) according to the manufacturer. Ethical permit number: Dnr 2020–05001. Cell culture . 10 6 splenocytes, 5×10 5 lymph node cells, or 10 5 PBMCs were cultured in 200 µl of complete RPMI per well in Nunclon U-shaped bottom 96-well plates (Thermo Scientific). Cells were incubated at 37°C and 5% CO 2 . Complete RPMI: RPMI 1640 with GlutaMAX™ (Thermo Scientific); 10% heat inactivated FBS (Thermo Scientific); 10 µM HEPES (Sigma); 50 µg/ml streptomycin sulfate (Sigma); 60 µg/ml penicillin C (Sigma); 50 µM β-Mercaptoethanol (Thermo Scientific). FBS was heat-inactivated for 30 min at 56°C. To assess the effect of 17-β-estradiol (Sigma) on CD2 expression, the medium was supplemented with charcoal-stripped FBS instead (Thermo Scientific). 17-β-estradiol was solved in ethanol. ELISA . 10 6 lymph node cells from CIA mice were plated per well and stimulated with 100 µg/ml bovine collagen type II (bCII) in complete RPMI for 48 h as described in cell culture . Supernatants were used for cytokine analysis. Flat 96-well plates (Maxisorp, Nunc) were coated overnight at 4°C with the capture antibody (Ab, listed below) in PBS. After removing the coating solution, supernatant from cell cultures were added. Plates were incubated for 3 h at RT before washing (0.05% Tween PBS) and adding the biotinylated detection Ab (listed below) in PBS (1 h at RT). Plates were washed and incubated 30 min at RT with Eu-labelled streptavidin (PerkinElmer, 1:1000) in 50 mM Tris-HCl, 0.9% (w/v) NaCl, 0.5% (w/v) BSA and 0.1% Tween 20, 20 µM EDTA. After washing, DELFIA Enhancement Solution (PerkinElmer) was added and fluorescence read at 620 nm (Synergy 2, BioTek). Monoclonal antibodies (mAbs) to IL-2 (capture Ab 5 µg/ml JES6-IA12; detection Ab 2 µg/ml biotinylated-JES6-5H4, in-house produced), IL-17A (capture Ab 5 µg/ml TC11-18H10.1; detection Ab 2,5 µg/ml TC11-8H4, Biolgend), IFN-γ (capture Ab 5 µg/ml AN18; detection Ab 2,5 µg/ml biotinylated R46A2, in-house produced). Analysis of mRNA expression . 10 6 lymph node cells per well were stimulated for 24 h using mAb LEAF hamster anti-mouse CD3 (1 µg/ml, 500A2, BD Pharmingen) and LEAF hamster anti-mouse CD28 (1 µg/ml, 37.51, BD Pharmingen) as described in cell culture . Cells were washed in PBS and RNA was extracted using Qiagen RNeasy columns according to the manufacturer without DNAse digestion. RNA concentration was determined using a NanoDrop 2000 (Thermo Scientific). Sample concentrations were normalized before proceeding with reverse transcription. Samples were stored at -20°C for short-term storage. cDNA synthesis was carried out using the iSrcipt cDNA synthesis kit (Bio-Rad) according to the manufacturer. qRT-PCR primers covered an exon-exon junction to minimize amplification of genomic DNA and were used at a final concentration of 300 nM. The qPCR reaction was carried out using the iQSYBR Green Mix (Bio-Rad) in white 96-well plates (Bio-Rad) using a CFX96 real-time PCR detection system (Bio-Rad). ACTB or GAPDH were used as an internal control. Primer sequences are listed in supplementary table 2. Data were analysed according to the ∆∆Ct method 55 , assuming equal efficiency for all the primer pairs. ChIP-qPCR. 10x10 6 spleen cells/ml were fixed for 10 min in 1% formaldehyde PBS at RT. The reaction was stopped by adding 125 mM glycine and cells were washed twice in ice-cold PBS. Complete protease inhibitor cocktail (Roche) was added in all the following steps. 2x10 6 cells were lysed in 1 ml cell lysis buffer 56 on ice for 15 min, and the extracted nuclei lysed in 1 ml nuclear lysis buffer 56 on ice for 15 min. Lysates were sonicated for 15 cycles (on high settings, 30’’ON-30’’OFF) using a Diagenode Bioruptor. The water bath was cooled to 4°C before beginning sonication. Average DNA length after sonication was 500 bp. 450 µl of the lysates were incubated with 10 µg/ml rabbit anti-mouse ERα Ab (clone E115, Abcam) or polyclonal rabbit IgG isotype control (Abcam) on a shaker at 4°C over night. Next day, DNA-Ab complexes were precipitated using protein G magnetic beads (Thermos Scientific). Beads were washed twice for 5 min at RT in buffers of increasing salt concentration according to 56 . DNA was eluted by incubating beads in 100 µl elution buffer 56 at 65°C for 30 min with occasional vortex. Beads were pelleted and fixation was reversed by incubation of supernatants for 8 h at 65°C in the presence of 0.3 M NaCl in 96-well plates. On the third day, 10 µg/ml RNAse A (Thermo Scientific) was added for 30 min (37°C) before incubation with 10 µg/ml of Proteinase K (Thermo Scientific) at 55°C for 30 min. DNA was purified using GeneJET PCR purification kit (Thermo Scientific) and used for qPCR. Primers used for amplification of recovered DNA are listed in supplementary table 3. Data was analysed according to 56 , but briefly, results are presented as fold change over their respective mock IP controls. Flow cytometry . 10 6 cells were blocked in 20 µl of PBS containing 5 µg in-house produced 2.4G2 in 96-well plates for 10 min at RT. Samples were washed with 150 µl of PBS and subsequently stained with the indicated antibodies in 20µl of PBS diluted 1:100 or 1:200 at 4°C for 20 min in the dark (Ab list follows). Cells were washed once, fixed and permeabilized for intracellular staining using BD Cytofix/Cytoperm™ (BD) according to the manufacturer. Cells were stained intracellularly with 20 µl of permeabilization buffer (BD), using the antibodies at a 1:100 final dilution, for 20 min at RT. FOXP3 staining required nuclear permeabilization and was carried out using Bioscience™ FOXP3/Transcription Factor Staining Buffer. For intracellular cytokine staining, cells were stimulated in vitro with phorbol 12-myristate 13-acetate (PMA) 10 ng/ml, ionomycin 1 µg/ml, and BFA 10 µg/ml for 4–6 h at 37°C prior to fixation, permeabilization and staining. Flow cytometry anti-mouse antibodies (BD Pharmingen): CD3 (clone: 145-2C11); TCRB (H57-597); CD4 (RM4-5); CD8 (53 − 6.7); CD19 (1D3, 6D5); CD11B (M1/70); CD11C (HL3, N418); FOXP3 (FJK-16s); CD25 (7D4); CD44 (IM7); CD62L (MEL-14); CD2 (RM2-5); LY6C (AL-21); LAG-3 (C9B7W); CD40L (MR1); IFN-γ (R46A2); IL-17A (TC11-18H10.1). CD16/CD32 (2.4G2, in house). Flow cytometry anti-human antibodies (BD Pharmingen): CD45 (clone: HI30); CD2 (RPA-2,10); TCRB (IP26); CD4 (OKT4); CD45RA (Hl100); CD45RO (UCHL1). Proliferation assay . 10 7 lymph node cells were labelled using CellTrace™ Violet Cell Proliferation Kit (ThermoFisher Scientific) according to the manufacturer. 5x10 5 naïve lymph node cells were cultured per well in U 96-well plates as described under cell culture in the presence of hamster anti-mouse CD3 (1 µg/ml, 500A2, BD Pharmingen) and hamster anti-mouse CD28 (1 µg/ml, 37.51, BD) for 72–96 h. Proliferation by dilution of CTV was assessed using flow cytometry. Complementary antibody staining was done as described under flow cytometry . Proliferation parameters were analysed and calculated using FlowJo 8.8.7. Collagen-induced arthritis (CIA) . 12-week-old mice were immunized with 100 µg of bovine collagen type II (bCII) in 100 µl of a 1:1 emulsion with CFA (BD ) and PBS intradermally at the base of the tail. Mice were challenged at day 35 with 50 µg of bCII in 50 µl of IFA (BD) emulsion. Mice were monitored for arthritis development as described in 57 . In short, each visibly inflamed (i.e. swollen and red) ankle or wrist was given 5 points, whereas each inflamed knuckle and toe joint was given 1 point each, resulting in a total of 60 possible points per mouse and day. Collagen antibody-induced arthritis (CAIA) . CII-specific antibodies (M2139, CIIC1, CIIC2 and UL1) were generated and purified as previously described 15 . The sterile cocktail of M2139, CIIC1, CIIC2 and UL1 mAbs (4 mg per mouse) was injected intravenously. On day 7, lipopolysaccharide (O55:B5 LPS from Merck; 25 µg in 200 µl per mouse) was injected intraperitoneally to all mice to increase severity of the disease. Mice were scored as described for CIA. Experimental induced autoimmune encephalomyelitis (EAE) . 12-week-old mice were immunized with a 100 µl emulsion of 250 µg myelin basic protein peptide (MBP) 89–101 peptide in PBS and 50 µl IFA (incomplete Freud’s adjuvant) containing 50 µg Mycobacterium tuberculosis H37RA (BD). Animals were boosted with 200 ng of Bordetella pertussis toxin (Sigma Aldrich, St. Louis, MO, USA) i.p. on day 0 and 48 h post initial immunization. EAE severity was evaluated as described in 58 . Briefly, mice were scored as follows: 0, no clinical signs of disease; 1, tail weakness; 2, tail paralysis; 3, tail paralysis and mild waddle; 4, tail paralysis and severe waddle; 5, tail paralysis and paralysis of one limb; 6, tail paralysis and paralysis of two limbs; 7, tetraparesis; 8, moribund or deceased. Delayed type hypersensitivity (DTH) . Hypersensitivity reaction was elicited by initially immunizing mice with 100 µg bCII emulsified in 50 µl CFA (Difco, Detroit, MI, USA). Ten days later mice were challenged with an injection of 10 µg bCII in 10 mM acetic acid into the dorsal part of the right ear and vehicle control in the left one. Ear swelling was assessed 48 and 72 h later using a calliper. Ovariectomy . In brief, ovaries of female mice were removed after a single incision through the back skin and bilateral flank incision through the peritoneum. Thereafter, mice were rested for a minimum of 14 days prior to immunization for EAE or CIA as described elsewhere. Luciferase reporter assay . 2x10 4 MCF-7 cells were seeded into flat 96-well flat bottom plates (Thermo Scientific) and left to adhere overnight. Then cells were transfected with pGL4.17 (Promega) luciferase reporter construct containing the BR or R3 allele of the candidate ERBS (pGL4.17.BR and pGL4.17.R3, respectively). ERBS cloning primers 5’-3’, Fw: AGATCTCGAGGGGGAAAGCTCTGACTTGGG; Rv: GTCAAGCTTGAGAAAGAATTTTGCTTATTTAGTCC. Cells were transfected in OPTIMEM medium (Thermo Scientific) using lipofectamine 3000 (Thermo Scientific) according to the manufacturer. The transfection mix (per well) contained 400 ng plasmid, 0.3 µl lipofectamine, and 0.2 µl P3000 reagent. Respective stimuli (20 ng/ml PMA, 10–100 nM E2) were added after 24 h, and cells were further incubated overnight before lysis. Luciferase activity was measured using Pierce Firefly Luc One-Step Glow Assay Kit (Thermo Scientific) in a Synergy-2 plate reader (BioTek). Genetic association study . Data for genetic variations within CD2-CD58 locus was extracted from previous Immunochip data published elsewhere (PMID: 23143596). After filtering these data correspond to 263 SNPs in 1940 healthy controls (M/F 524/1416) and 2762 RA patients (M/F 817/1945) from the Swedish EIRA study. Association was analysed by PLINK separately for female and male individuals. Analysis of public microarray expression data . Microarray data was extracted from NCBI GEO Database 28 and analysed using Shiny GEO 59 . GEO accession number is cited wherever NCBI GEO data has been used. Statistical analysis . Statistical analysis was performed using GraphPad Prism v6.0 or higher. Statistical comparison of two unpaired groups was carried out using Mann-Whitney U non-parametric test. CIA and EAE disease curves were compared using two-way ANOVA multiple comparisons test. P-values under 0.05 were considered statistically significant and are denoted with the symbol *. P-values under 0.01 are denoted **. Proteomic analysis of enriched CD4 + T cells . CD4 + T cells were enriched from naïve spleens using untouched CD4 + T cell mouse kit (Dynabeads, Life Technologies). 96-well U bottom plates were pre-coated with 1 µg/ml of anti-CD3 and 1 µg/ml of anti-CD2 in PBS for 3 h at 37°C. 2.5x10 5 CD4 + T cells were plated on the pre-coated plates and cultured for 48 h. Cell pellets were lysed in a buffer consisting of 1% SDS, 8 M urea and 20 mM EPPS pH 8.5 and sonicated using a Branson probe sonicator (3 s on, 3 s off pulses, 45 s, 30% amplitude). Protein concentration was measured using BCA assay and subsequently 50 µg of protein from each sample were reduced with 5 mM DTT at RT for 45 min followed by alkylation with 15 mM IAA in the dark at RT for 45 min. The reaction was quenched by adding 10 mM DTT and the samples were precipitated using methanol-chloroform mixture. Dried protein pellets were dissolved into 8 M urea, 20 mM EPPS pH 8.5. EPPS (20 mM, pH 8.5) was added to lower the urea concentration to 4 M and LysC digestion was done at a 1:100 ratio (LysC/protein, w/w) overnight at RT. Then urea concentration was lowered to 1 M and trypsin digestion was conducted at a 1:100 ratio (Trypsin/protein, w/w) at RT for 5 h. TMTpro plex (Thermo Fischer Scientific) reagents were dissolved into dry acetonitrile (ACN) to a concentration of 20 µg/µl and 200 µg were added to each sample. The ACN concentration in the samples was adjusted to 20% and the labelling was conducted at RT for 2 h and quenched with 0.5% hydroxylamine (ThermoFischer Scientific) for 15 min at RT. The samples were then combined and dried using Speedvac to eliminate the ACN. Then samples were acidified to pH < 3 using TFA and desalted using SepPack (Waters). Lastly, peptide samples were dissolved into 20 mM NH4OH and 150 µg of each sample was used for off-line fractionation. Samples were fractionated off-line in a high-pH reversed-phase manner using an UltimateTM 3000 RSLCnano System (Dionex) equipped with a XBridge Peptide BEH 25 cm column of 2.1 mm internal diameter, packed with 3.5 µm C18 beads having 300 Å pores (Waters). The mobile phase consisted of buffer A (20 mM NH 4 OH) and buffer B (100% ACN). The gradient started from 1% B to 23.5% in 42 min, then to 54% B in 9 min, 63% B in 2 min and stayed at 63% B for 5 min and finally back to 1% B and stayed at 1% B for 7 min. This resulted in 96 fractions that were concatenated into 24 fractions. Samples were then dried using Speedvac and re-suspended into 2% ACN and 0.1% FA prior to LC-MS/MS analysis. Peptides were separated on a 50 cm EASY-spray column, with a 75 µm internal diameter, packed with 2 µm PepMap C18 beads, having 100 Å pores (Thermo Fischer Scientific). An UltiMate™ 3000 RSLCnano System (Thermo Fischer Scientific) was used that was programmed to a 91 min optimized LC gradient. The two mobile phases consisted of buffer A (98% milliQ water, 2% ACN and 0.1% FA) and buffer B (98% ACN, 2% milliQ water and 0.1% FA). The gradient was started with 4% B for 5 min and increased to 26% B in 91 min, 95% B in 9 min, stayed at 95% B for 4 min and finally decreased to 4% B in 3 min and stayed at 4% B for 8 more min. The injection was set to 5 µL corresponding to approximately 1 µg of peptides. Mass spectra were acquired on a Q Exactive HF mass spectrometer (Thermo Fischer Scientific). The Q Exactive HF acquisition was performed in a data dependent manner with automatic switching between MS and MS/MS modes using a top-17 method. MS spectra were acquired at a resolution of 120,000 with a target value of 3.10 6 or maximum integration time of 100 ms. The m/z range was from 375 to 1500. Peptide fragmentation was performed using higher-energy collision dissociation (HCD), and the normalized collision energy was set at 33. The MS/MS spectra were acquired at a resolution of 60,000 with the target value of 2.10 5 ions and a maximum integration time of 120 ms. The isolation window and first fixed mass were set at 1.6 m/z units and m/z 100, respectively. TMT-10 labelling quantification Protein identification and quantification were performed with MaxQuant software (version 1.6.2.3). MS2 was selected as the quantification mode and masses of TMTpro labels were added manually and selected as peptide modification. Acetylation of N-terminal, oxidation of methionine and deamidation of asparagine and glutamine were selected as variable modifications while carbamidomethylation of the cysteine was selected as fixed modification. The Andromeda search engine was using the UP000000589_Mus musculus database (22129 entries) with the precursor mass tolerance for the first searches and the main search set to 20 and 4.5 ppm, respectively. Trypsin was selected as the enzyme, with up to two missed cleavages allowed; the peptide minimal length was set to seven amino acid. Default parameters were used for the instrument settings. The FDR was set to 0.01 for peptides and proteins. “Match between runs” option was selected with a time window of 0.7 min and an alignment time window of 20 min. Declarations Acknowledgments We would like to thank Dr Leonid Padyukov and the EIRA study group for providing the genetic data. Funding This work was supported by grants from the Knut and Alice Wallenberg Foundation, the Swedish Association against Rheumatism, the Swedish Medical Research Council, the Swedish Foundation for Strategic Research and Karolinska Institute-KID. Author contributions G.F.L. wrote the manuscript with the help of M.F. and R.H. G.F.L. designed, performed, analysed, and interpreted most experiments. M.F. and M.J. designed, performed, and analysed all the experiments shown in figs. 1b, 2 and 3. R.Z. and P.S. performed and analysed experiments requiring mass spectrometry (fig. 6h). K.S.N. helped secure funding and reviewed the manuscript. E.L., M.A., and Y.H. helped with data collection, analysis and interpretation. All authors revised and approved the manuscript. R.H. initiated, designed, and supervised the project and takes overall responsibility for the data. 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Perez-Riverol, Y. et al. The PRIDE database and related tools and resources in 2019: Improving support for quantification data. Nucleic Acids Res. (2019) doi: 10.1093/nar/gky1106 . Zheng, R. et al. Cistrome Data Browser: Expanded datasets and new tools for gene regulatory analysis. Nucleic Acids Res. (2019) doi: 10.1093/nar/gky1094 . Fornes, O. et al. JASPAR 2020: Update of the open-Access database of transcription factor binding profiles. Nucleic Acids Res. (2020) doi: 10.1093/nar/gkz1001 . Additional Declarations There is NO Competing Interest. Supplementary Files NCOMMS2110552.pdf Reporting Summary SupplementaryMaterials.docx Cite Share Download PDF Status: Published Journal Publication published 22 Sep, 2021 Read the published version in Nature Communications → Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-337166","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":19446676,"identity":"7c69fe4a-f5d6-45a7-82fd-5db7cbc84f2a","order_by":0,"name":"Gonzalo Fernandez Lahore","email":"","orcid":"https://orcid.org/0000-0002-4291-0251","institution":"Karolinska Institute","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Gonzalo","middleName":"Fernandez","lastName":"Lahore","suffix":""},{"id":19446677,"identity":"db0a2903-c7d9-48b7-83de-a1b8812ca6cf","order_by":1,"name":"Michael Förster","email":"","orcid":"","institution":"Karolinska Institute","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Michael","middleName":"","lastName":"Förster","suffix":""},{"id":19446678,"identity":"4a7a9a5b-8e9a-4bc2-b1b3-3fde3961a5ff","order_by":2,"name":"Martina Johannesson","email":"","orcid":"https://orcid.org/0000-0002-4353-7583","institution":"Karolinska Institutet","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Martina","middleName":"","lastName":"Johannesson","suffix":""},{"id":19446679,"identity":"4ebd6cc8-a319-4ead-9ee6-f30ff8a5ca94","order_by":3,"name":"Pierre Sabatier","email":"","orcid":"https://orcid.org/0000-0002-2734-1791","institution":"Karolinska Institute","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Pierre","middleName":"","lastName":"Sabatier","suffix":""},{"id":19446680,"identity":"8c502750-00e7-48aa-a9a4-6a8212db70f2","order_by":4,"name":"Erik Lönnblom","email":"","orcid":"https://orcid.org/0000-0002-1311-2541","institution":"Division of Medical Inflammation Research","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Erik","middleName":"","lastName":"Lönnblom","suffix":""},{"id":19446681,"identity":"275f220a-0c76-42f9-abf7-fef5248e0c0b","order_by":5,"name":"Mike Aoun","email":"","orcid":"","institution":"Karolinska Institute","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Mike","middleName":"","lastName":"Aoun","suffix":""},{"id":19446682,"identity":"0333e0e3-1eab-4eca-be81-16279d1b13bf","order_by":6,"name":"Yibo He","email":"","orcid":"","institution":"Karolinska Insitutet","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Yibo","middleName":"","lastName":"He","suffix":""},{"id":19446683,"identity":"9ba0e13c-c3c6-4335-aeea-e9fa9b0afd9c","order_by":7,"name":"Kutty Nandakumar","email":"","orcid":"https://orcid.org/0000-0001-7790-8197","institution":"Southern Medical University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Kutty","middleName":"","lastName":"Nandakumar","suffix":""},{"id":19446684,"identity":"8553cd41-115f-4aac-96f7-b6656b724cd0","order_by":8,"name":"Roman Zubarev","email":"","orcid":"https://orcid.org/0000-0001-9839-2089","institution":"Karolinska Institute","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Roman","middleName":"","lastName":"Zubarev","suffix":""},{"id":19446685,"identity":"c6cd244f-f254-4b22-ad6b-b013e16874e7","order_by":9,"name":"Rikard Holmdahl","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA6ElEQVRIiWNgGAWjYDACZjYgwcaQwMDOwCDB2GAhJ8FMtBZmsBYJY8JaGNC0JM4gpMG8nS3xc0UZQx4/M3fijZ87JNJntjMwf/iAR4vMYbbDkmfOMRRLNvNutuw9I5E7m5mBTRKfVRLM7A2SjW0MiRsO826T4G2TyJ0H1MLMg19L80+YFsm/bRLpcswMzJ//4NXCdgxuizTQlgRpYDhI4/M+UEuaZcM5icSZQL9Yy7ZJGM5sZmyT7MGnhf+Y8c2GMpvEfvbejTffttnIS5w/fPjDD3zWQHUicxgbCGsYBaNgFIyCUYAXAAC5dUILxAE8eQAAAABJRU5ErkJggg==","orcid":"https://orcid.org/0000-0002-4969-2576","institution":"Karolinska Institute","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Rikard","middleName":"","lastName":"Holmdahl","suffix":""}],"badges":[],"createdAt":"2021-03-17 13:16:30","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-337166/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-337166/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41467-021-25828-5","type":"published","date":"2021-09-22T04:00:00+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":7616238,"identity":"f98d0f1c-1c80-44c2-8cc9-816b0ac5cc11","added_by":"auto","created_at":"2021-04-02 16:47:52","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":255113,"visible":true,"origin":"","legend":"Schematic representation of Cia21 and phenotype driving D3KV1-MF31 (D3-31) recombinant fragment. The Cia21 QTL resulted from an inter-cross between the CIA susceptible C57BL/10.RII (BR) and the CIA resistant RIIIS/J (R3) strains. Cia21 is present on chromosome 3 qF2.2 and is 3 Mbp in size. a) Schematic representation of the Cia21 QTL and recombinant mice derived by intercrossing of Cia21 heterozygotes. Important genetic markers and genes are indicated on the left. The critical D3KV1-MF96 interval is highlighted in yellow. Uncertainty borders are dashed. b) Collagen-induced arthritis in female recombinant mice from (a) compared to BR littermate controls. Incidence and total number of mice are indicated in parenthesis on the respective graphs. Data are summarized as mean (SEM). c) Detailed view of D3KV1-MF31 (fragment 1) and close-by genes. The critical D3KV1-MF96 interval is highlighted in yellow. Coordinates according to mouse NCBI37/mm9 build. n.s., not significant; *, p \u003c 0.05; **, p \u003c 0.01.","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-337166/v1/ac02632f0bf896193494a379.png"},{"id":7616122,"identity":"9a9e477c-8672-4e54-a358-4282f7e3586b","added_by":"auto","created_at":"2021-04-02 16:44:52","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":128657,"visible":true,"origin":"","legend":"D3-31 mice are protected from several models of T cell-dependent autoimmunity in a sex-specific manner. Collagen-induced arthritis (CIA) in a) female and b) male BR and D3-31 mice. Delayed-type hypersensitivity (DTH) reaction in c) female and d) male BR and D3-31 mice. MBP89-101-induced experimental autoimmune encephalomyelitis (EAE) in e) female and f) male BR and D3-31 mice. Incidence and total number of mice used are indicated in parenthesis. Data are summarized as mean (SEM). *, p \u003c 0.05; **, p \u003c 0.01.","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-337166/v1/ed3ed28f6cf81ab6ba11da95.png"},{"id":7616126,"identity":"15e1246d-3f46-4e9c-b938-7b2ba84d3925","added_by":"auto","created_at":"2021-04-02 16:44:52","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":272377,"visible":true,"origin":"","legend":"Female sex hormones are required for the protective phenotype in D3-31 mice. a) CIA severity and incidence (in parenthesis) in ovariectomized D3-31 and BR mice. b) Incidence of EAE in ovariectomized (OVX) and sham operated (SHAM) D3-31 and BR mice. c) Table comparing incidence, maximal score and accumulated severity of EAE experiment shown in (b). Data are summarized as mean (SEM). *, p \u003c 0.05.","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-337166/v1/f78dbe23bb472e68cca06700.png"},{"id":7616125,"identity":"b9eb5ee1-cb11-4a3b-b60e-ead1664a9d54","added_by":"auto","created_at":"2021-04-02 16:44:52","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":174295,"visible":true,"origin":"","legend":"Polymorphism in D3-31 estrogen receptor binding site affects E2-mediated transcriptional activity. a) Sequencing results showing genetic variants within critical D3KV1-MF96 interval. b) Detailed schematic overview of polymorphisms (denoted by red lines) in the D3KV1-MF96 interval. SNP478 denotes a AC \u003e GG substitution on chr3:101310478-79. c) ChIP-seq data from mouse uterus (extracted from GSM894054 61) showing ERα binding intensity to polymorphic regions listed in (a). Consensus ER binding motif (UN0308.1 62) and SNP478 are highlighted in blue and red, respectively. Coordinates according to mouse NCBI37/mm9 build. d) Rabbit anti-mouse ERα ChIP-qPCR data confirming binding of ERα to SNP478 in spleen cells. A gene dessert was used as negative control (-ctrl) and a known ERα binding site (CSF2RA 29) as positive control (+ ctrl). Values are expressed as fold enrichment over rabbit IgG mock IP (n=5/group). e) Effect of SNP478 on the transcriptional activity of the D3KV1 ERα binding site shown in (c). The candidate ERα binding site (chr3:101310478 ± 100 bp to each side) was cloned in its two variant forms (AC and GG) into luciferase reporter constructs. The constructs were transfected into MCF7 cells to evaluate transcriptional activity (n=5/group). Data are summarized as mean (SEM). *, p \u003c 0.05; **, p \u003c 0.01.","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-337166/v1/3cbc27e614f4907350d9b387.png"},{"id":7616237,"identity":"214f961f-a672-4e09-9111-2e445e7413e6","added_by":"auto","created_at":"2021-04-02 16:47:52","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":214220,"visible":true,"origin":"","legend":"D3-31 mice show sex-specific differences in CD2 expression. a) Expression of genes surrounding the D3-31 congenic fragment in lymph nodes cells. Dotted lines indicate fragment borders. Expression in female and male mice is shown in red and blue, respectively. b) CD2 protein expression in lymph node CD4+ T cells from female and male D3-31 and BR mice (flow cytometry). c) Expression of CD2 and other surrounding genes in lymph nodes from BR mice. d) CD2 protein expression in blood circulating T cells, B cells, and monocytes (flow cytometry). e) Secretion of IL-17A and IFN-ƴ in T cells stimulated with anti-CD3 mAb only, or anti-CD3 and anti-CD2 mAb. f) CD2 expression in lymph node T cells after in vitro culturing with increasing concentrations of 17-β-estradiol (E2). g) Comparison of CD2 expression in T cells from D3-31 and BR mice cultured in normal medium (ctrl), medium (charcoal) stripped of E2 (-E2), or -E2 medium supplemented with 10 nM E2. Data are summarized as mean (SEM) from n=5 mice per group. *, p \u003c 0.05; **, p \u003c 0.01. ","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-337166/v1/2ab1876579a63864682eb3ab.png"},{"id":7616123,"identity":"f5fd9bc3-4e25-4c96-9772-624b605495c2","added_by":"auto","created_at":"2021-04-02 16:44:52","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":276184,"visible":true,"origin":"","legend":"Sex-specific differences in CD2 expression limit the T cell responses in female D3-31 mice. a) Proliferation and IL-2 secretion of CD4+ lymph node T cells after stimulation with anti-CD3/anti-CD28 mAbs. b) Proliferation of BR and D3-31 CD4+ T cells as in (a) in the presence of increasing concentrations of E2 (10-100 nM). c) Antigen recall assay showing proinflammatory cytokine secretion by lymph node cell cultures from CIA mice after recall with bovine collagen type II (bCII). Lymph nodes were harvested 10 days after immunization with bCII (day 10). d) Quantification of antigen experienced CD40L+CD4+ T cells in lymph nodes from CIA mice (day 10), and expression of CD2 in these cells. e) Gating and quantification of IL-17A+CD40L+ T cells in lymph nodes from CIA mice (day 10). Cells were restimulated ex vivo with PMA in the presence or absence of anti-CD2 mAb before staining for flow cytometry. f) Gating and quantification of CD25+FOXP3+Tregs in lymph nodes from CIA mice (day 10). g) Expression of IL-17A and FOXP3 in CD4+ naïve T cells stimulated with PMA in the absence or presence of anti-CD2 mAb. h) Volcano plot comparing the proteomic profile of CD4+ T cells stimulated with anti-CD3 mAb in the presence and absence of anti-CD2 mAb (left). Flow cytometry data showing LAG-3 expression in CD4+ T cells after culture with anti-CD2 mAb (right). Data are summarized as mean (SEM) from n=5 mice per group. *, p \u003c 0.05; **, p \u003c 0.01. ","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-337166/v1/ea7a4201f19349a3ecc0b205.png"},{"id":7615837,"identity":"8802888a-1a89-483d-b529-df40fc20675c","added_by":"auto","created_at":"2021-04-02 16:41:52","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":183208,"visible":true,"origin":"","legend":"E2-mediated regulation of CD2 is conserved in humans. a) Genetic association data showing association between CD2 polymorphisms and rheumatoid arthritis (RA) in female (top) and male (bottom) patients (EIRA cohort, 1341 males and 3361 females). b) Effect of indicated SNPs on expression of CD2 in human spleen as determined using GTEx 32. c) CD2 expression in synovia from RA patients plotted against disease activity (DAS28-CRP). Data was extracted from GEO Dataset GSE45867. d) Expression of CD2 in synovial tissue from RA patients, osteoarthritis (OA) patients, or healthy controls (GEO GDS5401-3). Females are shown in red and males in blue. e) Expression of CD2 in PBMCs from healthy males and females (GEO GDS5363). f) CD2 expression on antigen experienced CD45RO+ or naïve CD45RA+ CD4+ T cells from blood of a healthy donor. g) CD2 expression in CD45RO+ T cells after 24h incubation with 10-100 nM E2 (n=3/group). h) Anti-ERα ChIP-seq data showing binding of ERα to the human CD2 locus in MCF7 cell line (extracted from 29, SRX1995230). i) Naïve BR male and female mice were injected i.p. with 50 µg anti-CD2 mAb (RM2-5). Circulating CD4+ T cells were analysed before (0 h) and 48 h after mAb injection. Ratio of naive (CD62L+) to effector (CD44+) CD4+ T cells (left), and CD2 expression in CD4+ T cells (right). Data are expressed as mean (SEM). *, p \u003c 0.05; **, p \u003c 0.01.","description":"","filename":"Fig7.png","url":"https://assets-eu.researchsquare.com/files/rs-337166/v1/5fc9ca7714034e6ffc9a96a4.png"},{"id":15779218,"identity":"e9663003-290b-40c7-97db-ffd9ca44d4d5","added_by":"auto","created_at":"2021-11-22 15:36:03","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2045672,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-337166/v1/c58c15f1-63d1-4cd3-abc7-f2429931447f.pdf"},{"id":7615833,"identity":"8eca42f0-39c2-42d4-b55e-e724d27cb161","added_by":"auto","created_at":"2021-04-02 16:41:52","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":372348,"visible":true,"origin":"","legend":"Reporting Summary","description":"","filename":"NCOMMS2110552.pdf","url":"https://assets-eu.researchsquare.com/files/rs-337166/v1/2d1e29896b0d6a2565816755.pdf"},{"id":7616120,"identity":"2a26ec03-6860-41a7-8647-0ee01cfecd7c","added_by":"auto","created_at":"2021-04-02 16:44:52","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":229100,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryMaterials.docx","url":"https://assets-eu.researchsquare.com/files/rs-337166/v1/7aed890507dd5ac4d5b1a7b4.docx"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"Polymorphic estrogen receptor binding site causes CD2-dependent sex bias in the susceptibility to autoimmune diseases","fulltext":[{"header":"Introduction","content":" \u003cp\u003eWomen mount a more vigorous immune response and are more susceptible to most autoimmune diseases \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e,\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. These diseases have a strong but complex genetic component, and it has been difficult to identify the underlying polymorphisms \u003csup\u003e\u003cspan additionalcitationids=\"CR4\" citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. The female preponderance in autoimmunity is sex hormone related \u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e but could also be genetically dependent \u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. Not only through sex chromosomes but also through distinct sex hormone regulated expression of autosomal genes. However, conclusive evidence is still lacking, as it is difficult to positionally identify the underlying polymorphisms controlling complex traits in a sex-dependent manner.\u003c/p\u003e \u003cp\u003eAnalysis of genetically segregated inbred animal strains dramatically enhances the power to isolate polymorphisms underlying complex diseases. Compared with association studies of human cohorts, studies in mice reduce environmental variability and allow for proof-of-concept experiments in biologically relevant systems, making it possible to conclusively identify genes underlying complex traits. In the context of previous such work to identify genetic loci that regulate autoimmune arthritis \u003csup\u003e\u003cspan additionalcitationids=\"CR9\" citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e, we have identified a locus on mouse chromosome 3 (Cia21) that affects expression of the T cell activation marker \u003cem\u003eCD2\u003c/em\u003e and regulates arthritis severity in females, but not in males \u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. We herein find the cause of the effect to be a polymorphic estrogen receptor binding site (ERBS) within Cia21 that recapitulates the phenotypic properties of its parent locus. This polymorphic ERBS orchestrates expression of surrounding genes in a sex-specific manner, including CD2. We isolated these polymorphisms in a congenic mouse line (D3-31) and used these mice to study the consequences of estrogen-mediated regulation of CD2 for T cell-dependent autoimmunity. In addition, we found estrogen regulation of CD2 expression to be a conserved mechanism in humans that likely contributes to the sexual dimorphism in T cell-mediated autoimmune diseases.\u003c/p\u003e "},{"header":"Results","content":"\u003cp\u003eWe have set out to identify major genetic polymorphisms underlying the development of autoimmune arthritis, using animal models. As part of these efforts we previously described a major quantitative trait locus (QTL) on chromosome 3 qF2.2, which we termed Cia21 \u003csup\u003e9\u003c/sup\u003e. Cia21 was identified from an inter-cross between the collagen-induced arthritis (CIA)-susceptible C57BL/10.RIII (BR) and the CIA-resistant RIIIS/J (R3) mouse strains \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. Cia21 contains several differentially expressed genes, including \u003cem\u003eCD2\u003c/em\u003e and \u003cem\u003ePTPN22\u003c/em\u003e \u003csup\u003e9\u003c/sup\u003e. Both CD2 and PTPN22 play a key role in T cell activation and were proposed as strong candidate genes. The aim of the present study is to identify the polymorphisms underlying the Cia21 QTL.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA minimal non-coding genetic interval proximal to\u003c/strong\u003e \u003cspan class=\"BoldItalic\"\u003eCD2\u003c/span\u003e \u003cstrong\u003erecapitulates the arthritis-regulating properties of Cia21\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo dissect the Cia21 QTL, we bred heterozygous Cia21 mice and evaluated the resulting recombinant mice (shown in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003ea) using CIA (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eb). Out of all the evaluated recombinants, only two, numbers 1 and 5, recapitulated the protective arthritis phenotype previously observed in Cia21 mice \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. Thus, the Cia21 QTL results from individual contributions of these two sub-QTLs. Importantly, the phenotype driving recombinant regions 1 and 5 mapped to the previously proposed \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e candidate genes \u003cem\u003eCD2\u003c/em\u003e and \u003cem\u003ePTPN22\u003c/em\u003e, respectively. Recombinant fragment 1 (proximal to \u003cem\u003eCD2\u003c/em\u003e), however, was significantly smaller than fragment 5 providing better conditions for the positional identification of underlying polymorphisms. Therefore, we focused our efforts on the former.\u003c/p\u003e\n\u003cp\u003eRecombinant fragment 1 stretched from markers D3KV1 to MF31 (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003ea, ca. 0.2 Mbp), but could be further redefined to the significantly smaller D3KV1-MF96 interval (ca. 0.02 Mbp) through a recombination assisted breeding strategy. Although recombinant fragments 1, 2, and 3 overlapped significantly, only fragment 1 regulated arthritis. Thus, we concluded that the causative polymorphisms must be positioned between markers D3KV1 and MF96 (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003ea, highlighted yellow). D3KV1-MF96 is a non-coding 0.02 Mbp region proximal to \u003cem\u003eCD2\u003c/em\u003e, located in-between the genes \u003cem\u003eATP1A1\u003c/em\u003e and \u003cem\u003eIGSF3\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003ec). We isolated the D3KV1-MF31 recombinant fragment (termed D3-31) in a congenic mouse line for further investigations. D3-31 congenic mice carry the parental R3 allele of D3-31 on an otherwise BR background. For simplicity, we hereon refer to the congenic line as D3-31 and to wild type littermates as BR.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eD3-31 congenic mice are protected from several T cell-dependent models of autoimmunity in a sex-specific manner\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn accordance with our previous data on Cia21, the R3 allele of D3-31 protected congenic mice in T cell-dependent \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e12\u003c/span\u003e\u0026ndash;\u003cspan class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e autoimmune inflammatory models, including collagen induced arthritis (CIA), experimental autoimmune encephalomyelitis (EAE), and delayed type hypersensitivity (DTH) (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003ea-f). We also investigated the T cell-independent \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e collagen antibody-induced arthritis (CAIA) model, but observed no phenotypic differences (supplementary fig. S1). As the DTH model does not depend on B cells \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e, these results indicated a critical role for T cells. Interestingly, and as previously described for Cia21 \u003csup\u003e9\u003c/sup\u003e, only female D3-31 mice were protected from T cell mediated autoimmunity (Figs.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003ea-f). Thus, we concluded that D3-31 regulates T cell dependent autoimmune phenotypes, and likely T cells, in a sex-specific manner.\u003c/p\u003e\n\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\n\u003ch2\u003eFemale sex hormones are required for the protective phenotype in D3-31 mice\u003c/h2\u003e\n\u003cp\u003eTo discriminate between influence of sex chromosomes versus hormones, we performed CIA and EAE experiments in castrated female mice (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003ea-c). Castration of female mice depletes gonadal production of 17-\u0026beta;-estradiol (E2) \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e, which constitutes the major circulating estrogenic compound in females. Castration reverted the protective effect of the D3-31 fragment both in CIA and EAE (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003ea-c), which demonstrated the crucial contribution of female sex hormones, most likely E2, to the protective phenotype in female D3-31 mice. We next defined the genetic mechanisms underlying this sexually dimorphic immune phenotype by sequencing the D3-31 fragment.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\n\u003ch2\u003ePolymorphisms in an estrogen receptor binding site (ERBS) affect E2-mediated transcriptional activity\u003c/h2\u003e\n\u003cp\u003eDNA sequencing of the D3-31 BR and R3 alleles revealed four single nucleotide polymorphisms (SNPs) in the critical D3KV1-MF96 interval (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003ea and b). None of the variants affected the coding region of known genes, indicating distal (cis) regulation of gene expression, likely by interfering with regulatory elements. Given our previous observations, we speculated that the identified polymorphisms could be located within an ERBS, interfering with sex-dependent regulation of gene expression.\u003c/p\u003e\n\u003cp\u003eEstrogen receptors (ER\u0026alpha; and ER\u0026beta;) are nuclear hormone receptors that translate E2-mediated signalling. Both ER\u0026alpha; and ER\u0026beta; are expressed in immune cells \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e, and act as transcription factors regulating the expression of proximal and distant genes \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e18\u003c/span\u003e,\u003cspan class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. To test our hypothesis, we screened publicly available ChIP-seq data for ER\u0026alpha; binding sites overlapping with one or more of the sequenced SNPs within D3KV1-MF96 interval. Indeed, one of the SNPs, AC\u0026thinsp;\u0026gt;\u0026thinsp;GG on chr3:101310478-479 (termed SNP478), clearly overlapped with an ER\u0026alpha; binding site (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003ec). In fact, bioinformatic analysis also revealed an estrogen response element (i.e. an ER core binding motif) in close proximity to SNP478. We sought to verify this finding, and confirmed binding of ER\u0026alpha; to SNP478 in spleen cells using ChIP-qPCR (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003ed). Comparison of SNP478 between mouse inbred strains revealed that this SNP is in fact part of a highly polymorphic AC/GT simple repeat (supplementary fig. S2, extracted from \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e).\u003c/p\u003e\n\u003cp\u003eTo address whether SNP478 had functional consequences for E2-mediated transcriptional activity (i.e. interfered with the binding of ER\u0026alpha; to the DNA), we cloned the candidate D3KV1 ERBS (\u0026plusmn;\u0026thinsp;100 bp) in its two variant forms (AC and GG) into luciferase reporter constructs. We assessed transcriptional activity of these constructs in transfected ER\u0026alpha;\u0026thinsp;+\u0026thinsp;MCF-7 cells treated with increasing concentrations of E2 (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003ee). In the context of the reporter construct, an increased occupancy of the ERBS by ER\u0026alpha; (as a function of increasing E2) resulted in suppression of transcriptional activity. Although surprising, similar observations have been reported elsewhere \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. Given the stronger transcriptional inhibition when using the BR derived construct, we concluded that ER\u0026alpha; has a higher affinity for the BR allele than for the D3-31 allele. Importantly, these data demonstrate that SNP478 has functional consequences for E2-mediated transcriptional activity.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePolymorphism in an ERBS leads to female-specific changes in\u003c/strong\u003e \u003cspan class=\"BoldItalic\"\u003eCD2\u003c/span\u003e \u003cstrong\u003eexpression\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNext, we tested the biological relevance of our findings by comparing the gene expression profile in lymphoid tissue from male and female D3-31 and BR mice. We observed female-specific changes in the expression of three genes adjacent to the polymorphic ERBS, namely \u003cem\u003eCD2\u003c/em\u003e, \u003cem\u003eIGSF3\u003c/em\u003e and \u003cem\u003eMAB21L3\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003ea). We also investigated the expression of \u003cem\u003eATP1A1\u003c/em\u003e as well as more distal genes (\u003cem\u003eCD101\u003c/em\u003e and \u003cem\u003eSLC22A15\u003c/em\u003e) previously implicated in the non-obese diabetic (NOD) mouse model of type 1 diabetes \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e, but found no changes in their expression level. Notably, the female-specific reduction of CD2 expression in D3-31 mice was also evident at protein level (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eb), and correlated with our previously reported gene expression results \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eOut of the differentially expressed genes, \u003cem\u003eCD2\u003c/em\u003e was the only gene predominantly expressed in lymphoid tissue (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003ec), particularly in activated CD4\u003csup\u003e+\u003c/sup\u003e T cells (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003ed). \u003cem\u003eIGSF3\u003c/em\u003e and \u003cem\u003eMAB21L3\u003c/em\u003e regulate neural \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e and ocular \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e development, whereas CD2 has been involved in immune function \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e and associated with human autoimmune conditions \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e4\u003c/span\u003e,\u003cspan class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e. Indeed, treatment of lymph node cells with anti-CD2 mAb inhibited T cell activation as demonstrated by reduced secretion of pro-inflammatory cytokines (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003ee). Considering these data and normal development of D3-31 mice, we concluded that CD2 is driving the T cell-dependent immune phenotype observed in D3-31 mice.\u003c/p\u003e\n\u003cp\u003eGiven the sex-specific differences in gene expression, we next investigated the relation between E2 and CD2 expression. T cells cultured in the presence of E2 up-regulated CD2 in a dose-dependent manner (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003ef). Conversely, use of E2 depleted medium (achieved by using charcoal-stripped serum) reduced the expression of CD2, and, more importantly, neutralized the observed differences in CD2 expression between BR and D3-31 mice. Additionally, differences in CD2 expression could be re-established by reintroducing E2 to the medium (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eg). This not only demonstrates direct regulation of E2 on CD2 expression, but also proves that the identified polymorphisms interfere with this regulation. Consequently, we speculated that E2-mediated regulation of CD2 was contributing to sex-specific differences in the T cell responses. A sex-dependent reduction of CD2 expression in female D3-31 mice could likely limit the T cell responses.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\n\u003ch2\u003eE2-dependent regulation of CD2 expression leads to sex-specific differences in autoreactive T cell activation\u003c/h2\u003e\n\u003cp\u003eTo investigate the impact of sex hormone-dependent alterations in CD2 expression on the T cell responses, we compared the activation of T cells between BR and D3-31 female mice. In a first set of \u003cem\u003ein vitro\u003c/em\u003e experiments, we found an impaired response in D3-31 T cells to TCR stimulation, as evidenced by reduced proliferation and IL-2 production (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003ea). Importantly, the difference in T cell proliferation between BR and D3-31 mice could be enhanced in a dose-dependent manner by E2 (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eb), much like the E2-dependent expression differences observed for CD2 (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eg).\u003c/p\u003e\n\u003cp\u003eA diminished T cell response in D3-31 mice was also evident \u003cem\u003ein vivo\u003c/em\u003e. Compared to BR mice, D3-31 mice showed a lower level of antigen specific T cells responses 10 days after immunization with CIA antigen bovine collagen type II, as demonstrated by reduced secretion of proinflammatory cytokines in lymph node cultures recalled with antigen (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003ec). Flow cytometry analysis of D3-31 draining lymph nodes revealed lower numbers of antigen experienced CD40L\u003csup\u003e+\u003c/sup\u003e CD4\u003csup\u003e+\u003c/sup\u003e T cells (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003ed), which expressed reduced levels of CD2 and L-17A after \u003cem\u003eex vivo\u003c/em\u003e restimulation with PMA (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003ed and e). Differences in T cell activation status were also evident by lower numbers of induced regulatory T cells after immunization (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003ef). Importantly, the observed differences in T cell activation were strictly sex-specific (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003ed-f), mirroring sex-specific differences in CD2 expression. Treatment with anti-CD2 strongly reduced the expression of IL-17 in na\u0026iuml;ve and autoreactive CD4\u003csup\u003e+\u003c/sup\u003e T cells (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eg and e, respectively), as well as FOXP3 in na\u0026iuml;ve T cells (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eg), demonstrating a key role for CD2 in the differentiation of Th17 and Treg cells. Consequently, we concluded that reduced CD2 expression in female D3-31 mice limits T cell activation in a sex-specific manner.\u003c/p\u003e\n\u003cp\u003eAlthough CD2 can regulate TCR signalling by increasing the stability of the immune synapse, we thought it plausible that persistent differences in CD2 signalling could elicit more profound phenotypic changes. Proteomic and flow cytometric analysis of CD4\u003csup\u003e+\u003c/sup\u003e T cells treated with an anti-CD2 mAb resulted in a selective up-regulation of the immune inhibitory marker LAG-3 (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eh). Thus, our data suggests that CD2 signalling modulates T cell activation not only by stabilizing the immune-synapse, but also by regulating the expression of the inhibitory marker LAG-3.\u003c/p\u003e\n\u003cp\u003e\u003cspan class=\"BoldItalic\"\u003eCD2\u003c/span\u003e \u003cstrong\u003eassociates with rheumatoid arthritis (RA) and is regulated by E2 in humans\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eOur results in mice suggested a regulatory role for \u003cem\u003eCD2\u003c/em\u003e on T cell-dependent autoimmunity, which is genetically determined in a sex-linked manner. We therefore explored the relevance of our findings in humans in the context of RA. In a genetic association study, we found a significant association between \u003cem\u003eCD2\u003c/em\u003e polymorphisms and RA (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003ea). While this association was more often found in females than in males, this was likely due to higher prevalence of RA in females (female to male ratio 3:1). Interestingly, several of the SNPs associated with RA (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) can enhance expression of \u003cem\u003eCD2\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003eb), as we determined from the GTEx database \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e. Further analysis of available microarray datasets \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e revealed a mild yet significant correlation between \u003cem\u003eCD2\u003c/em\u003e expression in RA synovia and disease activity (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003ec). Moreover, \u003cem\u003eCD2\u003c/em\u003e is strongly up-regulated in the synovial tissue from RA patients when compared to osteoarthritis or healthy synovium (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003ed). Thus, it is likely that CD2 is involved in joint inflammation, and that \u003cem\u003eCD2\u003c/em\u003e polymorphisms affecting its expression contribute to the development or perpetuation of joint autoimmunity.\u003c/p\u003e\n\u003cp\u003eImportantly, women expressed higher levels of \u003cem\u003eCD2\u003c/em\u003e than men, both in RA synovium and healthy PBMCs (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003ec and \u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003ee, respectively), suggesting the E2-mediated regulation of \u003cem\u003eCD2\u003c/em\u003e observed in mice is conserved in humans as well. To corroborate our findings, we stimulated CD4\u003csup\u003e+\u003c/sup\u003e T cells from healthy human donors with increasing amounts of E2. Firstly, we noticed a strong up-regulation of CD2 in antigen experienced CD45RO\u003csup\u003e+\u003c/sup\u003e T cells compared to their na\u0026iuml;ve CD45RA\u003csup\u003e+\u003c/sup\u003e counterparts (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003ef). But more importantly, expression of CD2 could be enhanced in CD45RO\u003csup\u003e+\u003c/sup\u003e T cells by incubation with E2 in a concentration-dependent manner (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003eg). Indeed, analysis of available ChIP-seq data \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e revealed that ER\u0026alpha; robustly binds the human \u003cem\u003eCD2\u003c/em\u003e gene locus (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003eh). Thus, these data demonstrate the evolutionary conserved nature of E2-mediated regulation of CD2.\u003c/p\u003e\n\u003cp\u003eWe reasoned that hormonal regulation of CD2 expression could have implications for anti-CD2-mediated therapy, as previous research suggests that anti-CD2 (Alefacept) preferentially targets CD2\u003csup\u003ehi\u003c/sup\u003e T cells \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e. To test this, we compared the \u003cem\u003ein vivo\u003c/em\u003e effects of anti-CD2 mAb administration on circulating T cells from male and female mice (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003ei). Anti-CD2 mAb treatment partially depleted circulating T cells, and resulted in the relative enrichment of remaining effector CD44\u003csup\u003e+\u003c/sup\u003e T cells, skewing the na\u0026iuml;ve CD62L\u003csup\u003e+\u003c/sup\u003e/effector CD44\u003csup\u003e+\u003c/sup\u003e T cell ratio. This effect was significantly more pronounced in females, which, like in humans, expressed higher levels of CD2 in circulating T cells. Taken together, these data demonstrate that sex-dependent differences in CD2 expression determine the response to anti-CD2 mAb.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"Discussion","content":" \u003cp\u003eUsing forward genetics, we have positionally identified a polymorphic ERBS regulating T cell-dependent autoimmunity. This site orchestrates expression of surrounding genes in a sex-specific manner, including expression of the T cell co-stimulatory molecule, CD2. We find that E2-mediated regulation of CD2 is a conserved mechanism that influences T cell activation in a sex-specific manner, contributing to the sexual dimorphism in autoimmune diseases.\u003c/p\u003e \u003cp\u003eUnderstanding the sexual dimorphic immune responses is fundamental for personalized medicine but is methodologically challenging. Common approaches to study this phenomenon rely on intricate manipulation of gonadal or hormonal systems \u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e, which has yielded valuable insights but with limited physiological relevance. Our study provides a more physiological perspective by the identification of a naturally occurring polymorphism in an ERBS, which enables studies on sex-associated differences in T cell-mediated autoimmunity. Since we used a hypothesis-free approach, our findings strongly suggest E2-mediated regulation of CD2 as a key physiological mechanism contributing to sex differences in the T cell responses and susceptibility to autoimmunity.\u003c/p\u003e \u003cp\u003eOur results also highlight the importance of genotype-sex interactions for the sexual dimorphism in autoimmune diseases. While much attention has been devoted to the contribution of sex chromosomes, epigenetic mechanisms or direct actions of hormones to the sexually dimorphic immune responses, the interactions between sex and predisposing autosomal polymorphisms have remained elusive. Isolated studies have demonstrated sex-dependency of expression (e) QTLs \u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e and sex differences in the genetic associations to inflammatory diseases \u003csup\u003e\u003cspan additionalcitationids=\"CR34\" citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e, but evidence is limited. Using a hypothesis-free approach, we for the first time conclusively identify a sex biased QTL with direct consequences for the development of autoimmunity. Polymorphisms in the identified ERBS modulate E2-driven CD2 expression, leading to sex-specific differences in T cell autoimmunity. Our results demonstrate not only that genetic polymorphisms influence hormonal regulation of gene expression, but also that genotype-sex interactions shape the sexually dimorphic immune response.\u003c/p\u003e \u003cp\u003eIndependent of our sex-related findings, this study provides valuable insights into CD2 immunobiology. Polymorphisms in the \u003cem\u003eCD2\u003c/em\u003e locus have been previously associated with several autoimmune diseases \u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e,\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e, but not much attention was given to the mechanism of action of these polymorphisms. Similarly, CD2 has been explored as a therapeutic target, but its mechanism of action beyond depletion of circulating T cell is poorly characterised \u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e, and complex adverse effects (including malignancies \u003csup\u003e\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e) warrant further research. CD2 in isolation affects the formation of the immune synapse \u003csup\u003e3839\u003c/sup\u003e and T cell activation \u003csup\u003e4041\u003c/sup\u003e, but the relevance of these findings \u003cem\u003ein vivo\u003c/em\u003e are less clear. For example, targeting CD2 in mice does not seem to affect immune system development \u003csup\u003e\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e or thymic T cells \u003csup\u003e\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/sup\u003e, unless TCR transgenic systems are used \u003csup\u003e4445\u003c/sup\u003e. Thus, there is a need to study this therapeutically promising pathway in a physiologically relevant context. The D3-31 mice used in this study exhibit discrete changes in CD2 expression mediated by E2, thus enabling us to study the effect of CD2 on T cell mediated autoimmunity in a physiological setting.\u003c/p\u003e \u003cp\u003eWe show for the first time that changes in CD2 expression, caused by natural polymorphisms, affect the T cell responses. Reduced CD2 expression protected mice from T cell-dependent inflammation and autoimmunity by reducing the activation and proliferation of antigen-specific T cells. This is consistent with studies demonstrating that CD2 membrane density is proportional to TCR signalling strength \u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e, and that peptide-based blocking of CD2 signalling reduces CIA severity \u003csup\u003e\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u003c/sup\u003e. Our results also implicate CD2 in the generation of Th17 and Treg- type T cell responses. Mice with reduced CD2 expression had a diminished T cell response characterized by a reduced expansion of Th17 and Treg cells. Accordingly, blocking CD2 resulted in the suppression of both cell types \u003cem\u003ein vitro\u003c/em\u003e. Indeed, CD2 has been linked to Treg \u003csup\u003e\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e,\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e\u003c/sup\u003e and Th17 phenotypes \u003csup\u003e\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e before, and targeting CD2 is effective in the treatment of Th17-mediated inflammatory diseases like psoriatic arthritis \u003csup\u003e\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e\u003c/sup\u003e. In summary, this suggests a key role for CD2-mediated activation in the induction of Th17 and Treg cells.\u003c/p\u003e \u003cp\u003eMechanistically, CD2 seems to play a role in T cell activation beyond its ability to stabilize the immune synapse, as blocking CD2 results in selective up-regulation of the exhaustion marker, LAG-3. This finding is supported by studies showing an inverse correlation between CD2 expression and exhaustion of T cells \u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e 50\u003c/sup\u003e, and up-regulation of LAG-3 in human CD8 T cells after treatment with Alefacept (anti-CD2) \u003csup\u003e\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e\u003c/sup\u003e. Thus, together with other studies, our data suggests that CD2 signalling maintains T cell autoreactivity by reducing the expression of inhibitory LAG-3 molecule.\u003c/p\u003e \u003cp\u003eOur findings in mice are likely relevant to the sexual dimorphism observed in human autoimmune conditions. \u003cem\u003eCD2\u003c/em\u003e associates with RA and E2 regulation of CD2 expression is highly conserved in human T cells. Women, who are generally more prone to autoimmunity, express higher levels of \u003cem\u003eCD2\u003c/em\u003e than men. In mice, we demonstrate that these type of discrete and sex-specific differences in CD2 expression result in sexually dimorphic T cell responses and autoimmune phenotypes. Thus, subtle, physiological changes in CD2 expression caused by natural polymorphisms likely modify the risk of T cell-dependent autoimmunity in humans. E2-mediated regulation of CD2 probably contributes to sex differences in the immune responses, both in homeostasis as well as autoimmune conditions.\u003c/p\u003e \u003cp\u003eSex-dependent differences in CD2 expression have implications for several sexually dimorphic immune processes involving T or other CD2 expressing cells. Hormonal regulation of CD2 could contribute to more vigorous humoral immune responses in women \u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e, helping to protect their off-spring from infections \u003csup\u003e\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e\u003c/sup\u003e at the cost of an enhanced risk to autoimmunity post-partum \u003csup\u003e\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e\u003c/sup\u003e. Alternatively, an enhanced CD2 expression in women might facilitate the induction of regulatory T cell phenotypes (as we observed in mice) to facilitate foetal-maternal immune tolerance. A hormonal regulation of CD2 expression could have wide ranging implications for the personalized therapy of T cell-mediated inflammatory diseases, as Alefacept was shown to preferentially target CD2\u003csup\u003ehi\u003c/sup\u003e T cells \u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e. Indeed, we demonstrate strong effects of anti-CD2 mAb administration on the na\u0026iuml;ve/effector T cell ratio in female, but not male mice. As such, sex-specific differences in T cell CD2 expression may offer a useful biomarker for stratification of patients in the context of CD2 targeted therapies.\u003c/p\u003e \u003cp\u003eIn conclusion, our results highlight the importance of genotype-sex interactions for the sexual dimorphism in autoimmunity, demonstrating that sex can determine the penetrance of predisposing genetic factors. Our findings show that CD2 is a sex-sensitive regulator of T cell-mediated autoimmunity. Hormonal regulation of CD2 is a conserved mechanism that has implications for the sexual dimorphism in the susceptibility to -and treatment of- autoimmune diseases like RA.\u003c/p\u003e "},{"header":"Materials And Methods","content":" \u003cp\u003e\u003cb\u003eAnimals\u003c/b\u003e. The BR.Cia21.D3-31 congenic founder mice were obtained from a partial advanced intercross (PAI) described elsewhere and they were subsequently back crossed for four additional generations \u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. In order to ensure strain purity, BR.Cia21.D3-31 mice were screened with a custom designed 8k Illumina chip at genome wide level \u003csup\u003e\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e\u003c/sup\u003e and the mice were found to be devoid of any contaminating RIIIS/J alleles. No SNPs were present between the congenic and the B10.RIII background strain. Mice were kept under specific pathogen free (SPF) conditions in the animal house of the Section for Medical Inflammation Research, Karolinska Institute in Stockholm. Animals were housed in individually ventilated cages containing wood shavings in a climate-controlled environment with a 14 h light-dark cycle, fed with standard chow and water \u003cem\u003ead libitum\u003c/em\u003e. All the experiments were performed with age-, sex- and cage-matched mice and all the genetic experiments were performed with littermate controls. All the experimental procedures were approved by the ethical committees in Stockholm, Sweden with ethical permit numbers; 12923/18 and N134/13 (genotyping and serotyping), N35/16 (CIA) and N83/13 (EAE).\u003c/p\u003e \u003cp\u003e \u003cb\u003ePreparation of mouse single cell suspensions\u003c/b\u003e. Briefly, spleen or lymph nodes were harvested and mechanically dissociated on a 40 \u0026micro;M cell strainer (Falcon) using a 1ml syringe plunger (Codan). Cells were counted on a Sysmex KX-21 cell counter. All centrifugation steps throughout the study were carried out a 350 x g for 5 min at RT. For spleen samples, red blood cells were lysed in RBC buffer (155 mM NH4Cl, 12 mM NaHCO3, 0.1 mM EDTA) before counting.\u003c/p\u003e \u003cp\u003e\u003cb\u003ePreparation of human peripheral blood mononuclear cells (PBMC).\u003c/b\u003e Human PBMCs were prepared from 8 ml whole blood of healthy donors using SepMate (Stemcell Technologies) tubes and Ficoll density gradient medium (Sigma) according to the manufacturer. Ethical permit number: Dnr 2020\u0026ndash;05001.\u003c/p\u003e \u003cp\u003e \u003cb\u003eCell culture\u003c/b\u003e. 10\u003csup\u003e6\u003c/sup\u003e splenocytes, 5\u0026times;10\u003csup\u003e5\u003c/sup\u003e lymph node cells, or 10\u003csup\u003e5\u003c/sup\u003e PBMCs were cultured in 200 \u0026micro;l of complete RPMI per well in Nunclon U-shaped bottom 96-well plates (Thermo Scientific). Cells were incubated at 37\u0026deg;C and 5% CO\u003csub\u003e2\u003c/sub\u003e. Complete RPMI: RPMI 1640 with GlutaMAX\u0026trade; (Thermo Scientific); 10% heat inactivated FBS (Thermo Scientific); 10 \u0026micro;M HEPES (Sigma); 50 \u0026micro;g/ml streptomycin sulfate (Sigma); 60 \u0026micro;g/ml penicillin C (Sigma); 50 \u0026micro;M β-Mercaptoethanol (Thermo Scientific). FBS was heat-inactivated for 30 min at 56\u0026deg;C. To assess the effect of 17-β-estradiol (Sigma) on CD2 expression, the medium was supplemented with charcoal-stripped FBS instead (Thermo Scientific). 17-β-estradiol was solved in ethanol.\u003c/p\u003e \u003cp\u003e \u003cb\u003eELISA\u003c/b\u003e. 10\u003csup\u003e6\u003c/sup\u003e lymph node cells from CIA mice were plated per well and stimulated with 100 \u0026micro;g/ml bovine collagen type II (bCII) in complete RPMI for 48 h as described in \u003cem\u003ecell culture\u003c/em\u003e. Supernatants were used for cytokine analysis. Flat 96-well plates (Maxisorp, Nunc) were coated overnight at 4\u0026deg;C with the capture antibody (Ab, listed below) in PBS. After removing the coating solution, supernatant from cell cultures were added. Plates were incubated for 3 h at RT before washing (0.05% Tween PBS) and adding the biotinylated detection Ab (listed below) in PBS (1 h at RT). Plates were washed and incubated 30 min at RT with Eu-labelled streptavidin (PerkinElmer, 1:1000) in 50 mM Tris-HCl, 0.9% (w/v) NaCl, 0.5% (w/v) BSA and 0.1% Tween 20, 20 \u0026micro;M EDTA. After washing, DELFIA Enhancement Solution (PerkinElmer) was added and fluorescence read at 620 nm (Synergy 2, BioTek). Monoclonal antibodies (mAbs) to IL-2 (capture Ab 5 \u0026micro;g/ml JES6-IA12; detection Ab 2 \u0026micro;g/ml biotinylated-JES6-5H4, in-house produced), IL-17A (capture Ab 5 \u0026micro;g/ml TC11-18H10.1; detection Ab 2,5 \u0026micro;g/ml TC11-8H4, Biolgend), IFN-γ (capture Ab 5 \u0026micro;g/ml AN18; detection Ab 2,5 \u0026micro;g/ml biotinylated R46A2, in-house produced).\u003c/p\u003e \u003cp\u003e\u003cb\u003eAnalysis of mRNA expression\u003c/b\u003e. 10\u003csup\u003e6\u003c/sup\u003e lymph node cells per well were stimulated for 24 h using mAb LEAF hamster anti-mouse CD3 (1 \u0026micro;g/ml, 500A2, BD Pharmingen) and LEAF hamster anti-mouse CD28 (1 \u0026micro;g/ml, 37.51, BD Pharmingen) as described in \u003cem\u003ecell culture\u003c/em\u003e. Cells were washed in PBS and RNA was extracted using Qiagen RNeasy columns according to the manufacturer without DNAse digestion. RNA concentration was determined using a NanoDrop 2000 (Thermo Scientific). Sample concentrations were normalized before proceeding with reverse transcription. Samples were stored at -20\u0026deg;C for short-term storage. cDNA synthesis was carried out using the iSrcipt cDNA synthesis kit (Bio-Rad) according to the manufacturer. qRT-PCR primers covered an exon-exon junction to minimize amplification of genomic DNA and were used at a final concentration of 300 nM. The qPCR reaction was carried out using the iQSYBR Green Mix (Bio-Rad) in white 96-well plates (Bio-Rad) using a CFX96 real-time PCR detection system (Bio-Rad). \u003cem\u003eACTB\u003c/em\u003e or \u003cem\u003eGAPDH\u003c/em\u003e were used as an internal control. Primer sequences are listed in supplementary table 2. Data were analysed according to the ∆∆Ct method \u003csup\u003e\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e\u003c/sup\u003e, assuming equal efficiency for all the primer pairs.\u003c/p\u003e \u003cp\u003e \u003cb\u003eChIP-qPCR.\u003c/b\u003e 10x10\u003csup\u003e6\u003c/sup\u003e spleen cells/ml were fixed for 10 min in 1% formaldehyde PBS at RT. The reaction was stopped by adding 125 mM glycine and cells were washed twice in ice-cold PBS. Complete protease inhibitor cocktail (Roche) was added in all the following steps. 2x10\u003csup\u003e6\u003c/sup\u003e cells were lysed in 1 ml cell lysis buffer \u003csup\u003e\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e\u003c/sup\u003e on ice for 15 min, and the extracted nuclei lysed in 1 ml nuclear lysis buffer \u003csup\u003e\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e\u003c/sup\u003e on ice for 15 min. Lysates were sonicated for 15 cycles (on high settings, 30\u0026rsquo;\u0026rsquo;ON-30\u0026rsquo;\u0026rsquo;OFF) using a Diagenode Bioruptor. The water bath was cooled to 4\u0026deg;C before beginning sonication. Average DNA length after sonication was 500 bp. 450 \u0026micro;l of the lysates were incubated with 10 \u0026micro;g/ml rabbit anti-mouse ERα Ab (clone E115, Abcam) or polyclonal rabbit IgG isotype control (Abcam) on a shaker at 4\u0026deg;C over night. Next day, DNA-Ab complexes were precipitated using protein G magnetic beads (Thermos Scientific). Beads were washed twice for 5 min at RT in buffers of increasing salt concentration according to \u003csup\u003e\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e\u003c/sup\u003e. DNA was eluted by incubating beads in 100 \u0026micro;l elution buffer \u003csup\u003e\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e\u003c/sup\u003e at 65\u0026deg;C for 30 min with occasional vortex. Beads were pelleted and fixation was reversed by incubation of supernatants for 8 h at 65\u0026deg;C in the presence of 0.3 M NaCl in 96-well plates. On the third day, 10 \u0026micro;g/ml RNAse A (Thermo Scientific) was added for 30 min (37\u0026deg;C) before incubation with 10 \u0026micro;g/ml of Proteinase K (Thermo Scientific) at 55\u0026deg;C for 30 min. DNA was purified using GeneJET PCR purification kit (Thermo Scientific) and used for qPCR. Primers used for amplification of recovered DNA are listed in supplementary table 3. Data was analysed according to \u003csup\u003e\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e\u003c/sup\u003e, but briefly, results are presented as fold change over their respective mock IP controls.\u003c/p\u003e \u003cp\u003e \u003cb\u003eFlow cytometry\u003c/b\u003e. 10\u003csup\u003e6\u003c/sup\u003e cells were blocked in 20 \u0026micro;l of PBS containing 5 \u0026micro;g in-house produced 2.4G2 in 96-well plates for 10 min at RT. Samples were washed with 150 \u0026micro;l of PBS and subsequently stained with the indicated antibodies in 20\u0026micro;l of PBS diluted 1:100 or 1:200 at 4\u0026deg;C for 20 min in the dark (Ab list follows). Cells were washed once, fixed and permeabilized for intracellular staining using BD Cytofix/Cytoperm\u0026trade; (BD) according to the manufacturer. Cells were stained intracellularly with 20 \u0026micro;l of permeabilization buffer (BD), using the antibodies at a 1:100 final dilution, for 20 min at RT. FOXP3 staining required nuclear permeabilization and was carried out using Bioscience\u0026trade; FOXP3/Transcription Factor Staining Buffer. For intracellular cytokine staining, cells were stimulated \u003cem\u003ein vitro\u003c/em\u003e with phorbol 12-myristate 13-acetate (PMA) 10 ng/ml, ionomycin 1 \u0026micro;g/ml, and BFA 10 \u0026micro;g/ml for 4\u0026ndash;6 h at 37\u0026deg;C prior to fixation, permeabilization and staining.\u003c/p\u003e \u003cp\u003eFlow cytometry anti-mouse antibodies (BD Pharmingen): CD3 (clone: 145-2C11); TCRB (H57-597); CD4 (RM4-5); CD8 (53\u0026thinsp;\u0026minus;\u0026thinsp;6.7); CD19 (1D3, 6D5); CD11B (M1/70); CD11C (HL3, N418); FOXP3 (FJK-16s); CD25 (7D4); CD44 (IM7); CD62L (MEL-14); CD2 (RM2-5); LY6C (AL-21); LAG-3 (C9B7W); CD40L (MR1); IFN-γ (R46A2); IL-17A (TC11-18H10.1). CD16/CD32 (2.4G2, in house).\u003c/p\u003e \u003cp\u003eFlow cytometry anti-human antibodies (BD Pharmingen): CD45 (clone: HI30); CD2 (RPA-2,10); TCRB (IP26); CD4 (OKT4); CD45RA (Hl100); CD45RO (UCHL1).\u003c/p\u003e \u003cp\u003e \u003cb\u003eProliferation assay\u003c/b\u003e. 10\u003csup\u003e7\u003c/sup\u003e lymph node cells were labelled using CellTrace\u0026trade; Violet Cell Proliferation Kit (ThermoFisher Scientific) according to the manufacturer. 5x10\u003csup\u003e5\u003c/sup\u003e na\u0026iuml;ve lymph node cells were cultured per well in U 96-well plates as described under \u003cem\u003ecell culture\u003c/em\u003e in the presence of hamster anti-mouse CD3 (1 \u0026micro;g/ml, 500A2, BD Pharmingen) and hamster anti-mouse CD28 (1 \u0026micro;g/ml, 37.51, BD) for 72\u0026ndash;96 h. Proliferation by dilution of CTV was assessed using flow cytometry. Complementary antibody staining was done as described under \u003cem\u003eflow cytometry\u003c/em\u003e. Proliferation parameters were analysed and calculated using FlowJo 8.8.7.\u003c/p\u003e \u003cp\u003e \u003cb\u003eCollagen-induced arthritis (CIA)\u003c/b\u003e. 12-week-old mice were immunized with 100 \u0026micro;g of bovine collagen type II (bCII) in 100 \u0026micro;l of a 1:1 emulsion with CFA (BD ) and PBS intradermally at the base of the tail. Mice were challenged at day 35 with 50 \u0026micro;g of bCII in 50 \u0026micro;l of IFA (BD) emulsion. Mice were monitored for arthritis development as described in \u003csup\u003e\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e\u003c/sup\u003e. In short, each visibly inflamed (i.e. swollen and red) ankle or wrist was given 5 points, whereas each inflamed knuckle and toe joint was given 1 point each, resulting in a total of 60 possible points per mouse and day.\u003c/p\u003e \u003cp\u003e \u003cb\u003eCollagen antibody-induced arthritis (CAIA)\u003c/b\u003e. CII-specific antibodies (M2139, CIIC1, CIIC2 and UL1) were generated and purified as previously described \u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. The sterile cocktail of M2139, CIIC1, CIIC2 and UL1 mAbs (4 mg per mouse) was injected intravenously. On day 7, lipopolysaccharide (O55:B5 LPS from Merck; 25 \u0026micro;g in 200 \u0026micro;l per mouse) was injected intraperitoneally to all mice to increase severity of the disease. Mice were scored as described for CIA.\u003c/p\u003e \u003cp\u003e \u003cb\u003eExperimental induced autoimmune encephalomyelitis (EAE)\u003c/b\u003e. 12-week-old mice were immunized with a 100 \u0026micro;l emulsion of 250 \u0026micro;g myelin basic protein peptide (MBP) 89\u0026ndash;101 peptide in PBS and 50 \u0026micro;l IFA (incomplete Freud\u0026rsquo;s adjuvant) containing 50 \u0026micro;g \u003cem\u003eMycobacterium tuberculosis\u003c/em\u003e H37RA (BD). Animals were boosted with 200 ng of \u003cem\u003eBordetella pertussis\u003c/em\u003e toxin (Sigma Aldrich, St. Louis, MO, USA) i.p. on day 0 and 48 h post initial immunization. EAE severity was evaluated as described in \u003csup\u003e\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e\u003c/sup\u003e. Briefly, mice were scored as follows: 0, no clinical signs of disease; 1, tail weakness; 2, tail paralysis; 3, tail paralysis and mild waddle; 4, tail paralysis and severe waddle; 5, tail paralysis and paralysis of one limb; 6, tail paralysis and paralysis of two limbs; 7, tetraparesis; 8, moribund or deceased.\u003c/p\u003e \u003cp\u003e \u003cb\u003eDelayed type hypersensitivity (DTH)\u003c/b\u003e. Hypersensitivity reaction was elicited by initially immunizing mice with 100 \u0026micro;g bCII emulsified in 50 \u0026micro;l CFA (Difco, Detroit, MI, USA). Ten days later mice were challenged with an injection of 10 \u0026micro;g bCII in 10 mM acetic acid into the dorsal part of the right ear and vehicle control in the left one. Ear swelling was assessed 48 and 72 h later using a calliper.\u003c/p\u003e \u003cp\u003e \u003cb\u003eOvariectomy\u003c/b\u003e. In brief, ovaries of female mice were removed after a single incision through the back skin and bilateral flank incision through the peritoneum. Thereafter, mice were rested for a minimum of 14 days prior to immunization for EAE or CIA as described elsewhere.\u003c/p\u003e \u003cp\u003e \u003cb\u003eLuciferase reporter assay\u003c/b\u003e. 2x10\u003csup\u003e4\u003c/sup\u003e MCF-7 cells were seeded into flat 96-well flat bottom plates (Thermo Scientific) and left to adhere overnight. Then cells were transfected with pGL4.17 (Promega) luciferase reporter construct containing the BR or R3 allele of the candidate ERBS (pGL4.17.BR and pGL4.17.R3, respectively). ERBS cloning primers 5\u0026rsquo;-3\u0026rsquo;, Fw: AGATCTCGAGGGGGAAAGCTCTGACTTGGG; Rv: GTCAAGCTTGAGAAAGAATTTTGCTTATTTAGTCC. Cells were transfected in OPTIMEM medium (Thermo Scientific) using lipofectamine 3000 (Thermo Scientific) according to the manufacturer. The transfection mix (per well) contained 400 ng plasmid, 0.3 \u0026micro;l lipofectamine, and 0.2 \u0026micro;l P3000 reagent. Respective stimuli (20 ng/ml PMA, 10\u0026ndash;100 nM E2) were added after 24 h, and cells were further incubated overnight before lysis. Luciferase activity was measured using Pierce Firefly Luc One-Step Glow Assay Kit (Thermo Scientific) in a Synergy-2 plate reader (BioTek).\u003c/p\u003e \u003cp\u003e \u003cb\u003eGenetic association study\u003c/b\u003e. Data for genetic variations within CD2-CD58 locus was extracted from previous Immunochip data published elsewhere (PMID: 23143596). After filtering these data correspond to 263 SNPs in 1940 healthy controls (M/F 524/1416) and 2762 RA patients (M/F 817/1945) from the Swedish EIRA study. Association was analysed by PLINK separately for female and male individuals.\u003c/p\u003e \u003cp\u003e \u003cb\u003eAnalysis of public microarray expression data\u003c/b\u003e. Microarray data was extracted from NCBI GEO Database \u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e and analysed using Shiny GEO \u003csup\u003e\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e\u003c/sup\u003e. GEO accession number is cited wherever NCBI GEO data has been used.\u003c/p\u003e \u003cp\u003e \u003cb\u003eStatistical analysis\u003c/b\u003e. Statistical analysis was performed using GraphPad Prism v6.0 or higher. Statistical comparison of two unpaired groups was carried out using Mann-Whitney U non-parametric test. CIA and EAE disease curves were compared using two-way ANOVA multiple comparisons test. P-values under 0.05 were considered statistically significant and are denoted with the symbol *. P-values under 0.01 are denoted **.\u003c/p\u003e \u003cp\u003e \u003cb\u003eProteomic analysis of enriched CD4\u003c/b\u003e \u003csup\u003e \u003cb\u003e+\u003c/b\u003e \u003c/sup\u003e \u003cb\u003eT cells\u003c/b\u003e. CD4\u003csup\u003e+\u003c/sup\u003e T cells were enriched from na\u0026iuml;ve spleens using untouched CD4\u003csup\u003e+\u003c/sup\u003e T cell mouse kit (Dynabeads, Life Technologies). 96-well U bottom plates were pre-coated with 1 \u0026micro;g/ml of anti-CD3 and 1 \u0026micro;g/ml of anti-CD2 in PBS for 3 h at 37\u0026deg;C. 2.5x10\u003csup\u003e5\u003c/sup\u003e CD4\u003csup\u003e+\u003c/sup\u003e T cells were plated on the pre-coated plates and cultured for 48 h.\u003c/p\u003e \u003cp\u003eCell pellets were lysed in a buffer consisting of 1% SDS, 8 M urea and 20 mM EPPS pH 8.5 and sonicated using a Branson probe sonicator (3 s on, 3 s off pulses, 45 s, 30% amplitude). Protein concentration was measured using BCA assay and subsequently 50 \u0026micro;g of protein from each sample were reduced with 5 mM DTT at RT for 45 min followed by alkylation with 15 mM IAA in the dark at RT for 45 min. The reaction was quenched by adding 10 mM DTT and the samples were precipitated using methanol-chloroform mixture. Dried protein pellets were dissolved into 8 M urea, 20 mM EPPS pH 8.5. EPPS (20 mM, pH 8.5) was added to lower the urea concentration to 4 M and LysC digestion was done at a 1:100 ratio (LysC/protein, w/w) overnight at RT. Then urea concentration was lowered to 1 M and trypsin digestion was conducted at a 1:100 ratio (Trypsin/protein, w/w) at RT for 5 h. TMTpro plex (Thermo Fischer Scientific) reagents were dissolved into dry acetonitrile (ACN) to a concentration of 20 \u0026micro;g/\u0026micro;l and 200 \u0026micro;g were added to each sample. The ACN concentration in the samples was adjusted to 20% and the labelling was conducted at RT for 2 h and quenched with 0.5% hydroxylamine (ThermoFischer Scientific) for 15 min at RT. The samples were then combined and dried using Speedvac to eliminate the ACN. Then samples were acidified to pH\u0026thinsp;\u0026lt;\u0026thinsp;3 using TFA and desalted using SepPack (Waters). Lastly, peptide samples were dissolved into 20 mM NH4OH and 150 \u0026micro;g of each sample was used for off-line fractionation.\u003c/p\u003e \u003cp\u003eSamples were fractionated off-line in a high-pH reversed-phase manner using an UltimateTM 3000 RSLCnano System (Dionex) equipped with a XBridge Peptide BEH 25 cm column of 2.1 mm internal diameter, packed with 3.5 \u0026micro;m C18 beads having 300 \u0026Aring; pores (Waters). The mobile phase consisted of buffer A (20 mM NH\u003csub\u003e4\u003c/sub\u003eOH) and buffer B (100% ACN). The gradient started from 1% B to 23.5% in 42 min, then to 54% B in 9 min, 63% B in 2 min and stayed at 63% B for 5 min and finally back to 1% B and stayed at 1% B for 7 min. This resulted in 96 fractions that were concatenated into 24 fractions. Samples were then dried using Speedvac and re-suspended into 2% ACN and 0.1% FA prior to LC-MS/MS analysis.\u003c/p\u003e \u003cp\u003ePeptides were separated on a 50 cm EASY-spray column, with a 75 \u0026micro;m internal diameter, packed with 2 \u0026micro;m PepMap C18 beads, having 100 \u0026Aring; pores (Thermo Fischer Scientific). An UltiMate\u0026trade; 3000 RSLCnano System (Thermo Fischer Scientific) was used that was programmed to a 91 min optimized LC gradient. The two mobile phases consisted of buffer A (98% milliQ water, 2% ACN and 0.1% FA) and buffer B (98% ACN, 2% milliQ water and 0.1% FA). The gradient was started with 4% B for 5 min and increased to 26% B in 91 min, 95% B in 9 min, stayed at 95% B for 4 min and finally decreased to 4% B in 3 min and stayed at 4% B for 8 more min. The injection was set to 5 \u0026micro;L corresponding to approximately 1 \u0026micro;g of peptides.\u003c/p\u003e \u003cp\u003eMass spectra were acquired on a Q Exactive HF mass spectrometer (Thermo Fischer Scientific). The Q Exactive HF acquisition was performed in a data dependent manner with automatic switching between MS and MS/MS modes using a top-17 method. MS spectra were acquired at a resolution of 120,000 with a target value of 3.10\u003csup\u003e6\u003c/sup\u003e or maximum integration time of 100 ms. The m/z range was from 375 to 1500. Peptide fragmentation was performed using higher-energy collision dissociation (HCD), and the normalized collision energy was set at 33. The MS/MS spectra were acquired at a resolution of 60,000 with the target value of 2.10\u003csup\u003e5\u003c/sup\u003e ions and a maximum integration time of 120 ms. The isolation window and first fixed mass were set at 1.6 m/z units and m/z 100, respectively.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eTMT-10 labelling quantification\u003c/h2\u003e \u003cp\u003eProtein identification and quantification were performed with MaxQuant software (version 1.6.2.3). MS2 was selected as the quantification mode and masses of TMTpro labels were added manually and selected as peptide modification. Acetylation of N-terminal, oxidation of methionine and deamidation of asparagine and glutamine were selected as variable modifications while carbamidomethylation of the cysteine was selected as fixed modification. The Andromeda search engine was using the UP000000589_Mus musculus database (22129 entries) with the precursor mass tolerance for the first searches and the main search set to 20 and 4.5 ppm, respectively. Trypsin was selected as the enzyme, with up to two missed cleavages allowed; the peptide minimal length was set to seven amino acid. Default parameters were used for the instrument settings. The FDR was set to 0.01 for peptides and proteins. \u0026ldquo;Match between runs\u0026rdquo; option was selected with a time window of 0.7 min and an alignment time window of 20 min.\u003c/p\u003e \u003c/div\u003e "},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe would like to thank Dr Leonid Padyukov and the EIRA study group for providing the genetic data.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by grants from the Knut and Alice Wallenberg Foundation, the Swedish Association against Rheumatism, the Swedish Medical Research Council, the Swedish Foundation for Strategic Research and Karolinska Institute-KID.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eG.F.L. wrote the manuscript with the help of M.F. and R.H. G.F.L. designed, performed, analysed, and interpreted most experiments. M.F. and M.J. designed, performed, and analysed all the experiments shown in figs. 1b, 2 and 3. R.Z. and P.S. performed and analysed experiments requiring mass spectrometry (fig. 6h). K.S.N. helped secure funding and reviewed the manuscript. E.L., M.A., and Y.H. helped with data collection, analysis and interpretation. All authors revised and approved the manuscript. R.H. initiated, designed, and supervised the project and takes overall responsibility for the data.\u0026nbsp;\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe mass spectrometry proteomics data were deposited to the ProteomeXchange Consortium via the PRIDE partner repository \u003csup\u003e60\u003c/sup\u003e with the dataset identifier PXD024126.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eBilli, A. C., Kahlenberg, J. M. \u0026amp; Gudjonsson, J. E. 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An interplay between predisposing genetics and sex-related factors likely determines the sex discrepancy in the immune response, but conclusive evidence is lacking regarding the underlying molecular mechanisms. Using forward genetics, we positionally identified a polymorphic estrogen receptor binding site that regulates \u003cem\u003eCD2\u003c/em\u003e expression, leading to female-specific differences in mouse models of T cell-dependent autoimmunity. Female mice with reduced CD2 levels displayed diminished expansion of autoreactive T cells. Mechanistically, CD2 affected T cell activation by inhibiting LAG-3 expression. Our findings explain the sexual dimorphism in human autoimmunity, as CD2 associated with rheumatoid arthritis and its regulation through 17-β-estradiol was conserved in human T cells. Hormonal regulation of CD2 has implications for CD2-targeted therapy. Indeed, anti-CD2 treatment was more potent in female mice. In conclusion, our results demonstrate the relevance of sex-genotype interactions and provide strong evidence for CD2 as a sex-sensitive predisposing factor in autoimmunity.\u003c/p\u003e","manuscriptTitle":"Polymorphic estrogen receptor binding site causes CD2-dependent sex bias in the susceptibility to autoimmune diseases","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2021-04-02 16:41:50","doi":"10.21203/rs.3.rs-337166/v1","editorialEvents":[],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"nature-communications","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"NCOMMS","sideBox":"Learn more about [Nature Communications](http://www.nature.com/ncomms/)","snPcode":"","submissionUrl":"https://mts-ncomms.nature.com/","title":"Nature Communications","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature Communications","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"d625dcb4-3060-40e0-b10f-afffa516fe73","owner":[],"postedDate":"April 2nd, 2021","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":3382905,"name":"Immunology"},{"id":3382906,"name":"Molecular Genetics"}],"tags":[],"updatedAt":"2021-11-22T15:33:49+00:00","versionOfRecord":{"articleIdentity":"rs-337166","link":"https://doi.org/10.1038/s41467-021-25828-5","journal":{"identity":"nature-communications","isVorOnly":false,"title":"Nature Communications"},"publishedOn":"2021-09-22 04:00:00","publishedOnDateReadable":"September 22nd, 2021"},"versionCreatedAt":"2021-04-02 16:41:50","video":"","vorDoi":"10.1038/s41467-021-25828-5","vorDoiUrl":"https://doi.org/10.1038/s41467-021-25828-5","workflowStages":[]},"version":"v1","identity":"rs-337166","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-337166","identity":"rs-337166","version":["v1"]},"buildId":"oE6Zbj460LM0Up2FdVbMZ","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}