Intersection of population variation and autoimmunity genetics in human activation Chun Jimmie Ye et al. Science 345, (2014); DOI: 10.1126/science.1254665

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a cohort of 348 healthy human volunteers RESEARCH ARTICLE SUMMARY representing three different ancestries (Af- rican, Asian, and European). Responses to IMMUNOGENETICS activation were evaluated by expres- sion profiling, focused on the transcripts that best represent the response, its vari- Intersection of population variation ability, and its functional consequences. Re- lating these data to dense single-nucleotide and autoimmunity genetics in human polymorphism genotypes from the partici- pants, we identified the genetic contribu- tions to this variation by using heritability T cell activation analysis and fine-mapped previously un- Chun Jimmie Ye, Ting Feng, Ho-Keun Kwon, Towfique Raj, Michael Wilson, known expression quantitative trait loci Natasha Asinovski, Cristin McCabe, Michelle H. Lee, Irene Frohlich, Hyun-il Paik, (eQTLs) that control these responses by us- Noah Zaitlen, Nir Hacohen, Barbara Stranger, Philip De Jager, Diane Mathis, ing trans-ethnic meta-analysis. Aviv Regev,* Christophe Benoist* RESULTS: We observed a high degree of interindividual variability, much of which was reproducible for a given subject. This variability followed complex patterns and

did not reduce simply to dominant TH1, 2, or 17 types. We identified 39 loci associated in cis with gene activation in T cells. These explained on average 25% of the repeatable ImmVar variation, but a major element could not be cohort ascribed to simple genetic effects, instead reflecting environmental influences, immu- nologic history, or complex integration of Activation network regulation. Of activation-induced , cytokines showed the most variabil- ity, but with little or no cis genetic control, in contrast to cytokine receptors, which CD4+ T cells were less variable but for which several eQTLs were detected. Ancestry of the do- nors markedly influenced T cell responses,

Analysis with stronger TH17 be- a G G ON OUR WEB SITE ing associated with African descent. We a a G Read the full article at http://dx.doi fine-mapped and vali- .org/10.1126/ dated experimentally a

Expression science.1254665 single-base variant that aa aG GG modulates binding of the transcription factor YY1 and hence the Characterizing the variation in T cell response. A total of 348 individuals from the ImmVar activity of an enhancer element controlling cohort donated blood for CD4+ T cell isolation and genotyping. The T cells were subsequently the autoimmune-associated IL2RA gene, af- activated to mimic the recognition of the cognate antigen. The variability in adaptive immune fecting its activity in activated but not regu- response, which may have genetic and nongenetic components, was characterized by profiling latory T cells. the response of activated T cells. A computational analysis uncovered genetic drivers for this variation and pinpointed molecular mechanisms by which they may act. CONCLUSION: Echoing the linked ImmVar studies, we find that the relevant cell type INTRODUCTION: The activation of CD4+ individual variation in the responsiveness and context are essential for discovering T lymphocytes by antigen initiates adap- of human CD4+ T cells and how genetic the genetic drivers of cell-specific responses tive immune responses, amplifying rare variation affects the tone and intensity of T and of connected autoinflammatory dis- antigen-specific clones and leading to func- cell responses in human individuals. eases. Our study lays the groundwork for tional specialization of effector T cells, in further explorations into the relative con- particular through the spectrum of cyto- RATIONALE: As the third arm of the tributions of genes and their environment kines they produce. Pathogens have exerted ImmVar project, we performed a rigorously on immunological processes, which should selective pressure during recent human controlled analysis of the responses of hu- aid in our understanding of autoimmune evolution and migrations, but selection for man blood CD4+ T cells to activation in un- disease and its genetic underpinnings. ■ a type of response that is optimal against biased conditions or in a culture regimen one microbe class could carry a price in that promotes differentiation to the T helper The list of author affiliations is available in the full article online. less-effective responses to other microbes 17 cell (T 17) phenotype. To permit an ap- *Corresponding author. E-mail: [email protected] H (A.R.); [email protected] (C.B.) or in impaired self-tolerance and autoim- preciation of the breadth of variation across Cite this article as C. J. Ye et al., Science 345, 1254665 mune disease. Little is known about inter- humans, we investigated these responses in (2014). DOI: 10.1126/science.1254665

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◥ T cells. Are some individuals inherently more RESEARCH ARTICLE responsive to T cell activation or more prone to mount TH1- or TH17-biased responses? Genetic variants in regulatory elements that affect tran- IMMUNOGENETICS script abundance can be mapped as expression quantitative trait loci (eQTLs) that associate in- dividual genetic variation with resulting varia- Intersection of population variation tion in gene expression (15, 16). Although these can, in principle, facilitate the interpretation of disease-associated variants, most eQTL studies and autoimmunity genetics in have been performed in cell types or states that are not directly relevant to pathogenic processes human T cell activation involving T lymphocytes.

Chun Jimmie Ye,1 Ting Feng,2 Ho-Keun Kwon,2 Towfique Raj,1,3 Michael T. Wilson,2 An experimental and analytic strategy Natasha Asinovski,2 Cristin McCabe,1,3 Michelle H. Lee,3 Irene Frohlich,3 Hyun-il Paik,2 to dissect interindividual variation Noah Zaitlen,4 Nir Hacohen,2 Barbara Stranger,5 Philip De Jager,1,3 Diane Mathis,1,2 We followed a stepwise approach to dissect the Aviv Regev,1,6* Christophe Benoist1,2* variationintheresponsesofprimaryTcellsin 348 healthy participants of African (91), Asian T lymphocyte activation by antigen conditions adaptive immune responses and (74), or European (183) ancestry from the Boston immunopathologies, but we know little about its variation in humans and its genetic or PhenoGenetic Project, which were also analyzed environmental roots. We analyzed gene expression in CD4+ T cells during unbiased in other arms of the ImmVar study (17, 18)(Fig. 1A and table S1). By design, we limited the age activation or in T helper 17 (TH17) conditions from 348 healthy participants representing European, Asian, and African ancestries. We observed interindividual variability, most range (18 to 56 years) to constrain the effects of marked for cytokine transcripts, with clear biases on the basis of ancestry, and following age on immune function and sampled the parti- cipants in a manner that minimizes variation re- patterns more complex than simple TH1/2/17 partitions. We identified 39 genetic loci specifically associated in cis with activated gene expression. We further fine-mapped lated to circadian or hormonal secretion rhythms and validated a single-base variant that modulates YY1 binding and the activity of an enhancer or food consumption (fig. S1). First, we estab- element controlling the autoimmune-associated IL2RA gene, affecting its activity in lished a rigorously standardized protocol to pu- activated but not regulatory T cells. Thus, interindividual variability affects the rify and activate, in a highly parallel mode, fresh + fundamental immunologic process of T helper activation, with important connections CD4 T cells from human blood. Cells were ac- to autoimmune disease. tivated via the T cell receptor (anti-CD3+CD28 beads—hereafter “a3+28”) alone or in biasing ctivationofTlymphocytesoccursthrough B cells to produce high-affinity immunoglobu- conditions that favor differentiation to a TH17 the recognition of the cognate antigen lins. Choices between effector phenotypes are phenotype (supplementation with TGF-b,IL-6, presented by molecules of the major histo- themselves modulated by the cytokine network, IL-23, IL-1B, anti–IL-4, and anti-IFN-g—hereafter “ ” A compatibility complex (MHC) and is ar- such as the reinforcement of the TH17 identity a3+28/TH17 ) or with addition of IFN-b. These guably the linchpin of the adaptive immune through IL-23. These pathways also drive major conditions allowed us to map interindividual var- system. This process initiates the amplification immunoinflammatory diseases. Pathogenic TH1 iation in T cell transcriptional responses that mimic of rare T cells specific for a given antigen within or TH17 cells have been implicated in rheumatoid activation through the antigen-specific receptor the naïve repertoire, which rapidly builds the arthritis, multiple sclerosis (MS), and inflamma- in an unbiased setting or when coerced toward cell cohorts required for an effective response. T tory bowel disease (IBD), and TH2-type responses the TH17 phenotype. Genome-wide expression pro- cell amplification also coincides with pheno- in asthma and other atopic diseases (1). filing established the time points to sample (with typic differentiation into various “flavors” of Pathogens exert strong selective pressures cells from a pool of donors) and the extent of effector CD4+ T cells, each of which is beneficial during human evolution, including the migra- interindividual variability in these responses (with in distinct contexts of microbial challenge (1): tions of the last tens of millennia (2). Optimal cells from a set of 15 donors). From these data, we Thelper1(TH1) cells producing interferon-g (IFN-g) fitness, however, must balance pathogen clear- selectedthefivemostinformativeconditions(stim- and tumor necrosis factor–a (TNF-a)aremost ance with the cost to the organism that may re- ulus time point pairs) and a set of 236 transcripts effective against viruses and intracellular mi- sult from collateral tissue damage or autoimmune (tableS2)thatbestcapturetheresponsesandtheir crobes; TH2 cells producing interleukin-5 (IL-5) deviation (3, 4). Akin to the diversification of variance across donors and activation states, as and IL-4/13 are most effective in controlling par- MHC alleles, it is plausible that selection for re- previously described (19), complemented with asites; TH17 cells and the IL-17 family cytokines sponses to different types of pathogens has di- transcripts of known importance, including 16 partake in antifungal and antibacterial defenses versified the overall efficacy and/or functional key defining cytokines. We then used direct mol- at mucosal interfaces; and follicular TH cells help bias of T cell activation in humans. However, ecule counting (NanoString Technologies, Seat- selection for a TH phenotype, advantageous in tle, WA) to measure the 236-transcript gene set one environment, might lead to subpar responses in CD4+ T cells from all 348 donors in each of 1 Broad Institute of Massachusetts Institute of Technology to dominant pathogens in other geographic lo- thesefiveconditions.Weestimatedthestable (MIT) and Harvard, Cambridge, MA 02142, USA. 2Division of Immunology, Department of Microbiology and Immunobiology, cales, or carry a penalty in terms of self/nonself interindividual variation from a subset of eight Harvard Medical School, Boston, MA 02115, USA. 3Program discrimination and autoimmune diseases. Indeed, donors who were sampled twice and distinguished in Translational NeuroPsychiatric Genomics, Institute for the there is evidence that genetic variants that in- components attributable to cis genetic or to non- Neurosciences, Departments of Neurology and Psychiatry, fluence T cell activation can enhance susceptibility genetic effects (demographic, physical, and envi- Brigham and Women’s Hospital, Boston, MA 02115, USA. 4 5–8 Department of Medicine Lung Biology Center, University of to immunologic diseases ( ), such as the var- ronmental) from the 183 donors of European California, San Francisco, San Francisco, CA 94158, USA. iants in the IL2RA locus, which encodes a key ancestry. Leveraging the enhanced resolution 5Section of Genetic Medicine, University of Chicago, Chicago, trophic cytokine receptor, associated with MS offered by meta-analysis across population groups 6 IL 60637, USA. Howard Hughes Medical Institute, and type 1 diabetes (T1D) (9–14). (20), we fine-mapped eQTLs that control these Department of Biology, MIT, Cambridge, MA 02139, USA. *Corresponding author. E-mail: [email protected] (A.R.); Little is known, however, about interindividual responses. On the basis of these results, we val- + [email protected] (C.B.) variation in the responsiveness of human CD4 idated and dissected molecular interactions around

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A 1. High-throughput isolation 2. Genome-wide 3. Targetted 4. Identifying PhenoGenetic 5. Test and activation of mRNA profiling mRNA profiling sources of causal prediction cohort primary human CD4+ (n = 58) (n = 1927, variation 1788 post filtering) Peripheral blood cis-(r)eQTL mapping Time course PSI Fine mapping /DNA binding (45 min - 72 h) gene set Enhancer assay African selection Inter vs. intra variation Mutagenesis American TGF Covariates male CD28 Individual responses Caucasian African (n = 15) female American T 17 signature (16) female CD3 CD4+ H Asian IL6R male Control (7) Asian Best Caucasian female IFN R IL-6, TGF activation IFN male 48 h proxies signature (121) 4 h 48 h IFN 4 h (47)

Activation classics (45)

IFN 236 targeted genes Activated Activated T 17 CD4+ CD4+ Stimulated H

Magnetic repurification

B 3+28 / Naïve C D IL1A IL1B 500 800 2 456 Genes Persistent IL10RA repressed 1 IL11RA 50 40 0 IL17RA 1 IL7R IL2 IL3 1000 10,000 Early 2 induced 1 240 Genes 100 50 IL18RAP 0 FOS IL4 IL6 1 Intermediate 5000 800 induced I 2 320 Genes 1 IL2RA 50 30 0 IL17A IL21 IL9 IL10 1 IL22 5000 1000 BATF Intermediate 2 induced II 50 50 658 Genes 1 IL9 IL17A IL17F 0 IL12RB2 5000 6000

MINA Counts 1 FOXP3 50 50 2 Late IL21 IL22 induced 1 532 Genes 5000 5000 Cell cycle 0

1 50 50

2 IL24 IFN 3+28+IFN / 3+28 270 Genes IFN 1 MX1 300 50,000 induced CXCL10 0 IFITM3 ISG20 50 50 1 TNF TGF 1 3000 10,000 3+28+T 17 / 3+28 289 Genes H 1 T 17 IL23R H IL17F induced 0 IFNG 50 500 4 h 4 h 48 h 48 h 4 h 4 h 48 h 48 h CCL20 Unst. Unst. 1 3+28 IFN 3+28 T 17 3+28 IFN 3+28 T 17 RORA H H 0.75 2 4 10 24 72 00.752 4 102472 Individuals Row Time (h) Row Min Max Time (h)

Fig. 1. A systematic approach to characterize the variation in T cell ac- relative to unbiased a3+28; middle) or to TH17 polarization cytokines (fold tivation and response. (A) Overview of study, from (1) cell purification change relative to unbiased a3+28; bottom). Each row (gene) is normalized and activation in four conditions, (2) genome-wide or (3) signature expres- to its mean fold change. (C) Profiles of gene expression for seven gene clus- sion profiling, (4) computational analysis to decompose expression variance ters, for conditions outlined and color-coded as in (A). A sample of represent- and identify eQTLs, and (5) functional validation of potential causal variants. ative genes in each cluster is listed. (D) Expression of 16 cytokines across (B) Time course profiles of gene expression in unbiased a3+28 cells (fold 348 individuals and 5 conditions [black, unstimulated; other color codes as change relative to 0-hour baseline; top), and response to IFN-b (fold change in (A)].

1254665-2 12 SEPTEMBER 2014 • VOL 345 ISSUE 6202 sciencemag.org SCIENCE RESEARCH | RESEARCH ARTICLE a single-nucleotide polymorphism (SNP) that con- 4hoursfora3+28+IFN-b,and48hoursfor lation, despite a slight positive correlation be- trols IL2RA expression upon T cell activation. a3+28/TH17 activations, and unstimulated con- tween IL17F and IFNG responses at 48 hours trols harvested at 4 hours. and the proportion of CD62Llo cells (Pearson r = Interindividual variation in T cell We determined the extent of interindividual 0.07 and 0.12; fig. S3). responses, co-regulated cytokine clusters variation in T cell responses across 348 indi- The intensity of initial T cell activation is an Genome-wide expression profiling determined viduals, with NanoString profiling of the 236- important determinant of the outcome of a re- the signatures of dynamic T cell activation and transcript set. For cytokine transcripts, there sponse to viral pathogens (25). To assess differ- differentiation responses to different stimuli in was a generally consistent pattern of responses ences in overall responsiveness, that is, whether a pool of donors of different genders and ances- across individuals [Fig. 1D; note the surprisingly individuals differ in the general intensity of T tries (Fig. 1, B and C). After a3+28 activation, we early response of many cytokine genes (for ex- cell responses, we calculated a “response index” observed successive waves of induction consist- ample, IL4, IL17A, IL22,andIFNG)andthetem- by averaging the centered expression values of ent with patterns of responses to T cell activation poral disconnect between IL17A and IL17F]. There 51 transcripts induced (fold change >1.68, table in vivo (21, 22) (Fig. 1, B and C), affecting 1750 was, however, a spectrum in the extent of inter- S4) at 48 hours of a3+28 activation. This index induced and 456 repressed genes (fold change individual variability (Fig. 1D): large for IL3 and spanned a somewhat narrow range (1.69-fold >1.68; table S3), including proliferation and ef- IL17A (9.3- and 5.3-fold, respectively) and tight across the 348 donors; fig. S1A) and was not or fector genes (23, 24). In TH17-biased conditions, for IL2 and TNF (2.4-fold; for comparison, the only minimally affected by participant gender or few transcripts (289) were affected that were not range for ACTB was 1.07). Indeed, a set of 14 tran- age (fig. S1, B to D). The response index corre- already induced in unbiased a3+28 conditions scripts, enriched for cytokines and chemokines lated strongly with a similar index computed (we did observe the expected overinduction of (13 of 14, P <1×10−7), showed an increase in from cytokine transcripts, which ranged over the IL17 family and, more surprisingly, of IFNG, variance relative to unstimulated baseline, con- 3.4-fold (fig. S1A). This variability in the activa- the prototypical TH1 cytokine). The 270 genes trarytothebulkofresponsivegeneswhosevar- tion response is significantly correlated [princi- induced in response to IFN-b peaked between 2 iance tended to shrink upon activation (fig. S2). pal component 2 (PC2): Pearson r = −0.52, P < and 4 hours, including canonical IFN-responsive The range of variability in cytokine responses 1×10−16;PC3:Pearsonr =0.36,P <1×10−12] targets. From these data, we selected represent- was largely independent of the proportion of with principal components computed over the ative time points at 4 and 48 hours for a3+28, CD62Llo “memory” cells in the starting popu- entire expression matrix (fig. S1, E and F).

A 4 h 3+28 48 h 3+28 B 48 h 3+28

12 African Race Asian European 10 Age (y) 18 56 Sex Female Male IL17F 8 TGFB1 TNF 6 IL2 IL24 IL21 810121416 810121416 IL1B IFN IFN IL6 12 IFNG IL17F 10 IL17A IL22

IL17F IL10 8 IL4 IL9 6 IL1A IL3 7 8 9 10 11 12 7 8 9 10 11 12 348 individuals –3 3 IL4 IL4

C 4 h 3+28 48 h 3+28 48 h 3+28 1 A B B1 F A 1 2 4 0 4 B 1 3 4 9 2 6 2 6 4 3 9 2 3 IL 1 A IL IL1A IL IL IL2 IL IL10 IL1 TNF TGF IL2 IL2 IL IL17 IFNG IL17 IL21 IL24 TGF IL IFNG IL17F IL1 IL IL22 TNF IL1B IL17A IL9 IL IL IL6 IL1B IL1A IL10 IL2 IL4 IL IL17 TGFB1 TNF IL IFNG IL IL17F IL22 IL2 1 1 IL6 TNF TNF IL1B IL2 IL2 IL1A IL24 IL21 IL24 TGFB1 IL1B TGFB1 IL1B IFNG IL10 IL6 IL6 IL4 IFNG IL17F IL9 IL21 IL17A TNF IL17F IL22 IL2 IL17A TGFB1 IFNG IL22 IL10 IL3 IL1A IL4 IL17F IL10 IL9 IL17A IL9 IL24 IL22 IL4 IL1A IL21 IL3 IL3 –0.7 0.7 –0.5 0.5 Covariate-adjusted data PCA-adjusted data Fig. 2. Interindividual variation in T cell response.(A) Relation between ex- in 48-hour a3+28 with demographic covariates race, age, and sex. (C)Pairwise pression values (log2 scale) for pairs of TH defining cytokines (IL-4, IL-17F, and IFN-g) correlation between 16 cytokines in covariate-adjusted data during early activation in unbiased 4-hour a3+28 and 48-hour a3+28 conditions; each point is an individual (left), late activation (middle), and late activation with principal components anal- sample. (B) Heatmap of 16 cytokines clustered by expression across 348 individuals ysis (PCA)–adjusted data to account for overall responsiveness (right).

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We asked whether some individuals could be were sampled on two different dates and stim- individuals of African ancestry), and IFNG,which distinguished by “response types,” skewed toward ulatedinthesameconditions(Fig.3A).Theaverage showed an opposite pattern. We are cognizant the cytokine signature of a particular TH subtype. repeatability (27) (linear mixed model bootstrap that such differences can be artifactually gener- No simple “THX-type” individuals emerged from R T SER, table S5, medium fill in Fig. 3A) was ated by systematic bias in sampling, batch, or the expression of signature cytokines: The levels highest in late response (45 T 2% in either 48-hour lifestyle confounders, but the differences observed of IL17F and IL4 were largely independent a3+28 or 48-hour TH17)andlowerinearlyre- likely have some genetic underpinning because (Pearson r = 0.07, P = 0.16), whereas the levels sponse (20 T 3% in 4-hour a3+28). Although there sampling was from the same geographical area, of IL17F and IFNG were positively correlated at was no general trend between repeatability and overthesametimeperiod,atthesamelocation, 48 hours (Pearson r =0.6,P <1×10−15)(Fig.2A). the coefficient of variation, some of the most var- under a controlled protocol, and samples from This complexity in individual responses is ex- iable genes, including IL17A/F, IL4, IFNG,andIL9, different ancestries were randomly distributed emplified by a cluster of donors characterized were also among the most repeatable (fig. S6). across experimental batches. After adjusting the by higher IL4 and IL9 expression, but which in- Next, we estimated the proportion of variance data for the first eight principal components to cluded some high IL17F or IFNG responders as attributable to known covariates or genetic com- account for unknown confounders, the pattern well (Fig. 2B). Some of the high IFNG responders ponents in samples only from the 183 donors of was weakened but still present for many genes had low IL4 responses, as would be predicted European ancestry. The proportion of expres- (ancestry explaining a median of 2.4 and 2.7% of from classic TH1/TH2 opposition, but this was sion variance explained by physiological covar- expression variation in the 48-hour conditions; certainlynotthenorm.Theseresponse-defining iates together (for example, age, gender, height, Fig.3Dandfig.S7).Eventhemoststringentes- cytokines are highly repeatable between repli- weight, and blood pressure) was generally low timates of population differentiation are higher than r2 T 2 17 30–33 cate draws from the same donors (fig. S4). (2.4 to 3.6%, adjusted fixed SErfixed,lightfillin previous estimates ( , ), which may par- Some groups of cytokines are co-regulated in Fig. 3, A and B), with sex [10 genes, t-test false tially be due to enrichment of immune-related genes mouse T cells in response to particular pathogen discovery rate (FDR) < 0.05] and age (53 genes, in our gene set, consistent with previous obser- challenges. We asked whether similar covaria- F-testFDR<0.05)theonlycovariatessignificantly vations (17, 30–33). Further, although the trend tion patterns of cytokine transcripts exist across correlated with gene expression in 48-hour a3+28 was present in unstimulated samples, it was am- healthy humans, as affected by genetic and envi- (table S6). plified after 48-hour stimulation, indicating that ronmental variation. We calculated, for each con- The average proportion of variance explained by the population differentiation required activation 2 dition, the pairwise correlation between 16 cytokine cis heritability (28, 29)(permutationh T SE 2 , to be manifest. cis hcis transcripts. Early in a3+28 activation, many classic dark fill in Fig. 3A) was higher in 48-hour activated In addition to genes with known popula- T cell cytokines tended to be correlated, with only conditions (9.2 T 0.9%) than in unstimulated T tion differentiation resulting from population- limited differentiation of TH17 cytokines and a cells (5.8%) and 4-hour activated conditions (~6.6%). specific genetic polymorphisms [UTS2 and IFITM3 well-individualized cluster of inflammatory cyto- Whereas the latter are consistent with previous es- (18, 34)], we detected population differentiation kines (IL1A/B and IL6; Fig. 2C, left). Clusters timates in whole tissue with identity by descent in transcripts encoding cytokines, chemokines, became more refined after 48 hours (Fig. 2C, mid- (28), the higher 48-hour estimates may reflect in- or their receptors (table S6 and Fig. 3C). Notably, dle), some of which were expected (IL17A/F and creased power to detect activation-specific eQTLs. IL2RA was one of the most statistically differen- IL22), and others more surprising (for example, Repeatable variance attributable to common tially expressed genes (t test, P <1×10−16 in both IFNG with IL21,orIL4 with IL9, IL10,andIL3). cis genetic and nongenetic factors varied widely 48-hour activation conditions, P =0.03afterPCA Expression of most cytokines still exhibited some between genes (Fig. 3, A and B). Cytokine tran- adjustment): On average, activated T cells from degree of positive correlation, perhaps indicative scripts were repeatable in their expression upon Europeans expressed ~15% less IL2RA mRNA of general responsiveness. Indeed, the distinc- activation for the eight individuals tested, but than individuals of African ancestry. Together, tions between cytokines became more apparent common genetic variation did not contribute these results suggest that there is strong popula- when the data were adjusted for eight principal substantially to their variability across the full tion differentiation in T cell responses even after components (Fig. 2C, right). data set of 348 individuals (Fig. 3B). In contrast, stringent removal of the general response and To test whether variation at early times may repeatable variation in cytokine receptors, in par- may include a genetic component. be predictive of later outcomes, we computed ticular IL23R and IL2RA, was significantly driven pairwise correlations between genes across time by cis genetic factors (~50%) in activated condi- Genetic variants associated with points (fig. S5) and found a positive correla- tions. Overall, our results suggest that although expression variation during T cell tion between the expression of the IL17 family at the T cell response is generally reproducible, activation and TH17 polarization 4hoursandIL17F expression at 48 hours and common cis genetic effects account for ~25% of To identify the genetic variants underpinning between early IFNG and later IFNG production, repeatable variability; physiological covariates ac- expression differences, we associated ~10 mil- indicating that interindividual variation sets the count for very little (~4%); and the rest may be lion imputed genotypes [1000 Genomes Project type of response. This early bias was not an am- due to established environmental effects, immu- (35), minor allele frequency (MAF) > 0.05] from plification of a predetermined state, because there nological history, or trans-genetic regulation. our cohort of 348 donors with gene expression was no correlation with the same in unstimulated levels in each condition. Almost half the genes samples. Other baseline transcripts were corre- Population differentiation in in our gene set (112 of 222, excluding X- and lated to responsiveness (26), including the abun- T cell response Y-encoded genes; Fig. 4A and table S7) had a dance of IFN-I–responsive transcripts (DDX58, When examining differences in T cell responses significant cis-eQTL within 1 Mb around the gene, IFIH1,andSTAT2)predictinglaterIL17F induc- across the cohort of 348 donors with a linear in at least one state (permutation FDR < 0.01, tion, suggesting an interesting connection be- model, ancestry differentiated the expression of tableS8).Somecis-eQTLsweredetectedinall tween tonic IFN-I signals and the ability to mount 94 of 229 (41%) genes in the two 48-hour con- conditions (Fig. 4B), but 38 were state-specific IL-17 responses (26). ditions (linear regression F test, FDR < 0.01, table (12 late, 2 early, 11 pan activation, and 13 other S6), explaining a median 7% of the expression patterns; table S7). We did not detect any cis-eQTLs Contributions of genetics variance. We observed ordering in the mean ex- specific to TH17 polarizing or IFN-b conditions and environment to repeatable pression of response genes, with a stronger trend beyond those detected in unbiased activation variation in T cell response of overexpression for donors of African ancestry, alone, although a few eQTLs (eight in TH17, five To estimate the relative contributions of genetic lower for European ancestry, and a mixed pattern in IFN-b) had larger effect sizes (table S7). Some and environmental factors, we first estimated for Asian ancestry (Fig. 3C). Differentially respon- of the condition-specific cis-eQTLs were related for each gene its intraindividual repeatability sive genes include key indicators of TH pheno- to transcript levels (Fig. 4, C and D), but this was with data from eight individuals whose cells types, IL17 family cytokines (overinduced in not the case for many (Fig. 4, E and F)—IL2RA

1254665-4 12 SEPTEMBER 2014 • VOL 345 ISSUE 6202 sciencemag.org SCIENCE RESEARCH | RESEARCH ARTICLE was expressed before stimulation, but it only We identified only six trans-eQTLs at genome- cis effects, we identified six other significant sig- had a significant cis-eQTL in the late activation wide significance (permutation FDR < 0.2), includ- nals, such as the association of the IL23R cis- conditions (rs12251836, meta P <1×10−16). The ing rs6498114, a variant in the CIITA locus, a gene eQTLs (rs2064689 and rs2863204) in the a3+28/ low-expression variant at rs12251836 was rare in known to control MHC class II expression, which TH17 condition with the change in expression of African (MAF = 0.13) and Asian (MAF = 0.03) but fittingly correlated with HLA-DRA expression IL21 between 48-hour a3+28/TH17 and unbiased common in European genomes (MAF = 0.41) (table S9). When restricting to test for trans im- stimulation (table S10), explaining 11 T 8.7% of (Fig. 4G). pact only those SNPs identified as having local the variance (permutation FDR <0.2; mediation

A C Unst. 4 h 3+28 4 h IFN 48 h 3+28 48 h TH17 European Asian African LYZ FCER2 ICOS IL7R POLR2A ICAM1 GZMB ITGAX HLA.DQA1 TRIM5 OASL DHX58 TIMP1 BST2 LIF APOBEC3G SCARNA4 SAMD3 TXN IL2RA CCL4 PML IFI44L

PIK3IP1 Genes GSTM1 SEMA7A LY6E HLA.DQB1 IFIT2 GLB1L2 CD69 IER3 IL17A STAT2 HDGFRP3 FOXP3 IL17F IL17F CCL20 MALT1 CTLA4 FLJ45825 BATF IL1RN CCL22 GN LY IFNG IL12RA JHDM1D UTS2 CXCL9 NKG7 MXRA7 FADS2 00.5 00.5 00.5 GSTM1 FGL2 XCL2 Population mean P value 0 10 1 10 10 1 Variance B D 48 h 3+28 Unst. 4 h 3+28 4 h IFN 48 h 3+28 48 h TH17 TGFB1 European / African Asian / African TNF IFNG FADS2 UTS2 IL24 IL22 IFIT2 PHEX IL21 DHX58 IL17F NAPSA DHX58 IL17A IL2RA FCER2 CXCL10 IL10 PLEK SOCS1 IL9 IL17A CXCL9 IL6 MXRA7 MXRA7 CCL22 IL4 Effect size IFITM3 IL6 IL6 IL3 CXCL10 HDGFRP3 IL2 CXCL9 CCL22 IL1B (covariate adjusted data) type IL17F Fixed IL1A Fixed Fixed IL17F 0 1 01010101 Variance Effect size (fully PCA-corrected data) P value Physiological variance Repeatable variance Cis-genetic heritability Fig. 3. Sources of variation across T cell activation conditions. (A and B) known covariates including gender, age, weight, height, BMI, and blood pressure Top 56 non-cytokine genes (A) or top 16 key cytokines (B) ranked by max- estimated from multiple regression. Error bars indicate bootstrap SEs around imum repeatability across all conditions estimated from eight recalled indi- each estimate. (C) Percent difference of average population expression (median) viduals. For each gene, repeatability (medium shading) and contributions of from overall average (median) that shows population differentiation in expres- cis heritability (dark shading) and of physiological covariates [age, gender, sion. (D) Fold change (effect size) in normalized expression for donors of body mass index (BMI), weight, height, diastolic, systolic, season, and month; European (left, n =183)andAsian(right,n = 74) ancestry relative to donors of light shading] to repeatable variation are estimated (European cohort only). African ancestry (n = 91), comparing 48-hour a3+28 data adjusted for all PCs (x Cis heritability is defined as the proportion of phenotypic variance explained axis) or for experimental and physiological covariates only ( y axis). Darker by variants within 1 Mb of the gene estimated using linear mixed models (29). shading indicates more statistically significant population differentiated genes as Physiological variance explained is the proportion of variance explained by all determined by an omnibus F test.

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A B Lnc RNA FLJ45825 (rs1757180)

Unst. 4 h α3+28 4 h IFN 48 h α3+28 48 h TH17 27.0 70 Unst. 0.6 10 51.2 23.6 25.9 0.61 0.48 0.37 0.32 40 0.4 8 20 0.2 0 6 99.6 0.97 CC tCtt CC tC tt CC tC tt CC tC tt CC tC tt 70 4 h α3+28 0.6 C FGL2 (rs2075761) 40 10 0.4 4.16 0.34 0.15 0.04 20 0.2 8 0 6 43.8 114 101 4 h IFN 0.97 1.53 1.45

70 0.6 Log

R CC tCtt CC tC tt CC tC tt CC tC tt CC tC tt value) 40 0.4 2 2 P

FADS2 (no ( D (rs61896141) 20 0.2 10 9 24.0 8.27 5.43 r

0.51 0.39 mali 0 0.81 -log 8 z

7 ed counts) 70 48 h α3+28 0.6 6 149 120 40 0.4 1.40 1.68 AA Accc AA Ac cc AA Accc AA Ac cc AA Ac cc 20 0.2 E IL23R (rs2863204) 0 9 0.21 2.24 2.37 0.02 0.14 0.15 8 70 48 h TH17 0.6 7 40 0.4 20 6 32.6 37.9 0.2 0.33 0.24 0 GG aG aa GG aG aa GG aG aa GG aG aa GG aG aa F IL2RA (rs12251836)

T5 12

I

LTA 1.46 TXN 2.31 CD9

F MX2 UTS2 IL23R

IFIT2 I BST2 IL2RA ADS2 FGL2 HMMR IFITM3 F 0.09 THBS1 NLRP2 0.13 TRGV3 NAPSA MXRA7 8 ENTPD1 POLR2A TAS2R43 SLC27A2 FLJ45825 HDGFRP3 HLA-DQA1 HLA-DQB1 Gene 11 7 0.32 16.0 21.0 G IL2RA 0.04 0.16 0.16 (rs12251836) 6 10 TT Taaa TT Ta aa TT Ta aa TT Ta aa TT Ta aa 48 h α3+28 12 African Fig. 4. Common cis variation associated with gene expression during T cell activation

) Asian

2 and differentiation. (A) Manhattan plot of the significance of cis (within 1 Mb of gene) European associations for the 236 genes in each condition. Permutation significant (FDR < 0.01) associations are colored. (B to F) Individual plots of expression per genotype for illustrative

11 SNPs; numbers in each panel indicate the significance [−log10(P value), upper number] and effect size [beta, lower number] of association. (G) Per-genotype plots of IL2RA ex- pression in 48-hour a3+28, distinguished by population. The width of boxes representing

Expression (log each genotype is scaled by its frequency in the population.

10 TT Ta aa analysis by conditioning on IL23R transcript S11), two key cytokine receptors implicated in a rs12095335 appeared as a secondary association to levels excluded that the effect might be due to number of autoimmune diseases. IL23R mRNA levels (meta P =2×10−4;Fig.5C), the nearby H3Q codon change), consistent with IL23R encodes the receptor for IL-23, partakes suggesting that there may be at least two inde- the amplification of IL-21 production by IL-23 in establishing stable TH17 differentiation (38), pendent effects on IL23R expressionintheregion. signals during TH17 differentiation (36, 37). and is strongly associated with IBD (39, 40). Here, The trans-association between these IL23R var- rs2863204 was the variant most significantly asso- iants and IL21 expression in the TH17 condition Overlap of genetically variable ciated with IL23R expression in 48-hour a3+28/ further implies functional consequences for the T cell responses with genome-wide TH17 (Fig. 5A). It is not on the same haplotype region with regard to TH17 differentiation. association study loci identifies as the IBD-associated nonsynonymous variant Perhaps most intriguing was the discovery of potential disease-relevant variants (rs11209026, R381Q) (40). However, meta-analysis SNPs associated with IL2RA expression. IL2RA To assess the relevance of activation eQTLs to dis- of ulcerative colitis and Crohn’s disease separately encodes CD25, the high-affinity IL-2 receptor, and ease, we intersected our eQTLs with the genome- (40) revealed allelic heterogeneity in IL23R where has intricately balanced functions, reflecting the wide association study (GWAS) catalog and an additional variant, tagged by rs12095335, is divergent roles of IL-2 as a trophic factor for identified intriguing disease-associated variants, associated with Crohn’sdiseasebutnotulcerative inhibitory T regulatory (Treg) cells and in aiding notably near IL23R and IL2RA (Fig. 5 and table colitis (Fig. 5B). When conditioned on rs2863204, the expansion of proinflammatory effector T cells

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Fig. 5. Trans-ancestry r2 tigated the functional effects of the top five can- fine mapping identi- 0.8 didate SNPs located in the 5′ flanking and first fies activation- 0.6 intron of IL2RA (Fig. 6A). We first tested for en- 0.4 specific cis-eQTLs in 0.2 hancer activity by introducing ~200– GWAS regions. (A)In IL23R IL2RA A D (bp)fragmentscenteredaroundeachSNP(Fig. 48-hour a3+28/TH17, 6A) into a luciferase reporter vector, driven by SV40 rs2863204 rs12722495 rs2863204 (MAF = 12 or IL2RA minimal promoters, and transfecting

0.53, European) is 48 h them into Jurkat T cells [+/− phorbol 12-myristate 40 10 Type 1 diabetes best associated with 13-acetate (PMA) + ionomycin activation]. Four IL23R 8 expression and 30 rs11209026 3+28/T of the segments were essentially inactive (or even is independent from 6 had slight inhibitory effects, fig. S8), but fragment rs11209026 (R381Q). 20 IV, which encompasses rs12251836, showed en- H

17 4 (B)AtIL23R, a coding hancer activity (“836” for short in Fig. 6B). The variant (rs11209026, 10 2 A allele, associated with lower IL2RA expression, R381Q, MAF = 0.06, had lower enhancer activity than the T allele 0 0 European) and a regula- 0 (Fig. 6B). The higher enhancer function of the T B E tory variant (rs12095335) allele was dependent on activation of the Jurkat are associated with 150 cells (Fig. 6C), suggesting that enhancer activity rs11209026 rs12251836 Crohn’sdisease.(C) Crohn’s disease 15 is boosted upon cell activation by interactions Conditioning on 48 h affectedbythesinglebasechangeatrs12251836. 100 rs12722495 rs2863204 recovers rs12095335 10 We examined whether the rs12251836 SNP af- rs12095335 (secondary 3+28 fects the binding of transcription factors (TFs). ’ Crohn s disease associ- 50 After incubation of nuclear extracts from primary 5 ation) as a significant human CD4+ T cells, we detected protein binding to secondary association. a 25-bp oligonucleotide encompassing rs12251836T, (D)AttheIL2RA locus, 0 0 but not to the rs12251836A counterpart (Fig. 6D). the best T1D association C F Furthermore, the formation of this DNA-protein is a regulatory variant, 8 Conditioned on rs28632048 Conditioned on rs12251836 complex in primary T cells depended on their rs12722495 rs12722495. (E)In 48 h stimulation (Fig. 6E), paralleling enhancer activity

48-hour a3+28, 6 6 48 h in the region. Searching for TF-binding motifs rs12095335 T rs12251836 (MAF = 0.41, 3+28/T showed that the rs12251836 sequence has putative −5

European) is best 3+28 P 4 4 ( <1×10 ) binding sites for YY1 and RUNX1 associated with IL2RA straddling the SNP, with a FOX-binding site im- rs2863204 H expression and is 17 mediately downstream (Fig. 6F) (46). The 836A 2 independent from 2 allele maintains the FOX-binding motif, but the pre- rs12722495. (F) dicted RUNX and YY1 sites are lost. To test these Conditioning on 0 0 predicted interactions, we expressed epitope-tagged rs12251836 recovers SLC35D1 IL23R IL15RA RBM17 versions of these three TFs and assayed for binding rs12722495 (primary to oligonucleotides spanning this segment. FOXP3 C1orf141 IL2RA PFKFB3 T1D association). x axis andRUNX1boundthers12251836oligonucleo- defines genomic inter- 67.5 67.6 67.7 6 6.05 6.1 6.15 tides equally for both alleles, but binding of YY1 vals local to each gene, Position on chr1 (Mb) Position on chr10 (Mb) was restricted largely to rs12251836T allele. The and y axis shows the allele-specific binding of YY1 was also confirmed by –log10 (P value) of association in GWAS (Crohn’s disease, T1D) and eQTL analysis (48-hour a3+28 or electrophoretic mobility shift assay (EMSA) (fig. 48-hour a3+28/TH17). S8). Furthermore, transfection of a YY1 expression vector boosted the enhancer activity of fragment IV, or natural killer cells (41). Complementary sus- in the IL2RA locus, which may have balancing but only with the plasmid carrying the rs12251836T ceptible and protective haplotypes in MS and T1D effects. We surmise that the associated variant variant (Fig. 6G). These results show that this var- have been demonstrated (9, 12, 42). These map rs12251836 is mainly active in acutely triggered iant is nested in an activation-augmented enhancer to the promoter region or first intron of IL2RA Teffectorcells,butnotinTreg cells, because IL2RA region and mediates differential IL2RA expres- and have been diversely associated with titers of mRNA in unstimulated CD4+ T cells would pre- sion by disrupting a functional YY1-binding site. hi soluble IL-2 receptor, expression of CD25 at the dominantly come from CD25 Treg cells, and this cell surface, or IL-2 signaling efficacy (12, 43, 44). association was not apparent in unstimulated cells. Discussion Three independent haplotypes have been reported On the other hand, the rs12722495 variant, weaker The exploration of how natural variation affects to be associated with T1D. The strongest, a pro- herebutdominantinfreshCD4+ blood T cells gene expression during human T lymphocyte tective haplotype tagged by rs12722495 (Fig. 5D), (43), might preferentially operate in chronically activation and differentiation identified complex leads to higher expression of IL2RA in steady- triggered cells such as Treg cells. On balance, these patterns of variability between individuals, more − state CD45RA memory and Treg cells (43–45). two variants would then control the effectiveness complexthansimpleTH paradigms. Ancestry had Here, rs12251836, the most significant variant as- of IL-2 signals in cells of opposite outcome in a a surprisingly profound effect on these response sociated with IL2RA expression in 48-hour a3+28 disease context. The divergent GWAS results for patterns. activated cells, was unlinked to rs12722495, which T1D and MS may also result from this interplay. By focusing on primary cells and profiling them showed little signal (Fig. 5E). However, by con- in the activated cellular state that has the most ditioning on rs12251836, we detected in our data a A disease-related SNP affecting likelihood to reveal regulatory variation related to secondary association between rs12722495 and YY1 binding and activity of an autoimmune pathogenesis, we estimated higher − IL2RA IL2RA expression in activated cells (P =5.3×10 7, enhancer motif in cis heritability and identified novel genetic asso- Fig. 5F). Together, these results point to functional Given the overlap between the IL2RA eQTL and ciations. This work represents the third arm of effects of at least two independent associations autoimmunity-associated GWAS SNPs, we inves- the ImmVar project, in which the same donors

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A

10 kb 6,11 Intron 1 6,09 Chr10 (Mb) IL2RA

DNase sensitivity

Conservation I II III IV V rs35285258 rs12253981 rs2476491 rs12251836 rs11256497

B C D Enhancer assay EMSA 836T 836A 836T/A TTTAAA 60 60 Competitor TA CD4+ T extract ++ ++ *** *** Relative enhancer activityRelative Relative enhancer activityRelative

0 0 CT AT GA TA CT Activation : –+ Fragment :- IIIIIIIVV

E F 836T 836A G Runx1 EMSA TGGTCCTCTATGGTTGGATGA TGGTCCTCTAAGGTTGGATGA T Foxp3 Foxp3 836 YY1 ZEB1, Pdx1 A 836T/A TTT TTT 836 Competitor ++ CD4+ T extract ++ ++ Cell activation -Flag ++ 60 GFP YY1-HA GFP Foxp3-FlagGFP Runx1 40 Input

20 836T Oligo pull down A Relative intensity 836 0 836T ++ IB : YY1 Foxp3 Runx1 836A ++ enhancer activityRelative (α HA) (α Flag) 0 YY1 +++ YY1 transfection : –+ Fig. 6. Validation of rs12251836, a causal variant in the IL2RA locus. (A) times, and apparent sizes should not be compared. (E) Specific binding of Genomic locations of five top associated SNPs in the IL2RA locus, together YY1 to the rs12251836T allele in nuclear extract of primary CD4+ T cells, with with mammalian sequence conservation and deoxyribonuclease (DNase) or without prior 6-hour activation with a3+28. (F)Top:PredictedTF-binding hypersensitivity sites (in TH1 cells). The ~200-bp fragments centered around sites around the two alleles of rs12251836. Bottom: Pull-down assay of the each of the variants used in enhancer assays are marked (I to V). (B)En- three main predicted TFs [transfected into human embryonic kidney 293 hancer activity in activated Jurkat cells for both alleles (as marked) of the five cells, hemagglutinin (HA)– or Flag-tagged] with oligonucleotides encompass- selected fragments cloned into a minimal promoter vector (firefly luciferase, ing both rs12251836 alleles or an irrelevant control (EBNA) and detected by all values normalized to cotransfected Renilla). (C) Enhancer activity of frag- immunoblotting for the relevant tags. (G) Trans-activating activity of overex- ment IV, as in (B), with or without PMA + ionomycin stimulation. (D)Protein pressed YY1 in Jurkat cells also transfected with the enhancer reporter binding to the two alleles of rs12251836 tested by EMSA with nuclear extract plasmids used in (B) (with either allele at rs12251836). Data are representa- from Jurkat or human primary CD4+ T cells—these gels were run for different tive of three or more independent experiments; error bars indicate SD. were profiled with respect to fresh immunocytes of 15 overlapped with those detected in IFN-treated triggering, cytokine transcripts showed the stron- at baseline (17) and to activation responses in dendritic cells (18), reflecting the sharing of IFN-b gest change in interindividual variance. This diver- dendritic cells (18). Sixty-nine of the 112 eQTLs response pathways. A comprehensive elucidation sification did not generally track along simple discovered here were not detected in baseline of the genetic control of immune responses will TH1/2/17 models but adopted more-complex pat- CD4+ T cells of Raj et al. (17). These eQTLs were require similar analyses in an array of relevant cell terns. This observation is of importance for the discovered for genes of direct importance in anti- types under relevant conditions of stimulation. relationship between T cell differentiation pro- infectious responses and autoimmunity (IL23R and AresomeindividualsmorepronetomountTH- files and immune diseases and for the definition IL2RA). Of the eQTLs for IFN-responsive genes, 11 biased responses? Of the changes elicited by T cell of biomarkers: Rather than correlating disease

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