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1 Continuous requirement for the TCR in regultory T cell function Andrew G Levine 1,, Aron Arvey 1,,4, Wei Jin 1,,4 & Alexnder Y Rudensky Nture Americ, Inc. All rights reserved. Foxp3 + regultory T cells (T reg cells) mintin immunologicl tolernce, nd their deficiency results in ftl multiorgn utoimmunity. Although heightened signling vi the T cell ntigen receptor (TCR) is criticl for the differentition of T reg cells, the role of TCR signling in T reg cell function remins lrgely unknown. Here we demonstrted tht inducile ltion of the TCR resulted in T reg cell dysfunction tht could not e ttriuted to impired expression of the trnscription fctor Foxp3, decresed expression of T reg cell signture genes or ltered ility to sense nd consume interleukin (IL-). Insted, TCR signling ws required for mintining the expression of limited suset of genes comprising 5% of the ctivted T reg cell trnscriptionl signture. Our results revel criticl role for the TCR in the suppressor cpcity of T reg cells. Regultory CD4 + T cells tht express the trnscription fctor Foxp3 hve n essentil role in mintining immune tolernce 1. In the thymus, incresed ffinity for enggement of the T cell ntigen receptor (TCR) y immture CD4 + single-positive thymocytes is required for the initition of progrm for the differentition of regultory T cells (T reg cells) nd induction of Foxp3 expression. As consequence, T reg cells exported to the periphery exhiit TCR repertoire skewed towrd self-recognition 3,4. However, it remins uncler whether TCR signling is needed to medite the suppressive function of T reg cells in the periphery. T reg cells exhiit impired clcium flux, ctivtion of the kinse Akt nd phosphoryltion of the kinse Erk upon TCR stimultion reltive to tht of conventionl CD4 + T cells, nd Foxp3 is known to potently repress t lest some TCR-induced genes, s well s some genes encoding molecules involved in the TCR signling pthwy 5 8. At the sme time, Foxp3 + T reg cells hve high sl expression of severl cell surfce molecules tht re known to contriute to T reg cell function (such s CD5, CD39 nd CTLA-4), nd the expression of these molecules in conventionl CD4 + T cells is dependent upon TCR stimultion It is not known whether high-ffinity interctions of the TCR with complexes of self peptide nd mjor histocomptiility complex (MHC) clss II contriute to constitutive expression of these genes nd, consequently, to T reg cell function. T reg cells, despite their intrinsiclly dmpened response to TCR stimultion, cquire n ctivted phenotype nd expnd their popultions in response to their cognte ntigens in settings of ctivtion of the immune system, such s infection nd utoimmunity 15,16. These oservtions indicte tht recognition of self ntigen helps mintin T reg cells of prticulr specificities nd my potentite their suppressive ility during immunologicl chllenge 17. Nevertheless, strict relince on TCR expression for T reg cell ctivtion, rther thn preferentil ctivtion of ntigen-specific T reg cells, hs not een demonstrted, nor hs enggement of the TCR in vivo een shown to e required for T reg cell function in ny context. We used inducile genetic ltion of cell-surfce TCR complexes to directly ddress the requirement for TCR expression in the immunosuppressive ility of T reg cells. Notly, the TCR ws lrgely dispensle for Foxp3 expression, for linege stility nd for high expression of mny signture genes in T reg cells. Nevertheless, these fetures were not sufficient to preserve T reg cell function or to prevent ctivtion of the immune system. Loss of suppressor cpcity in the sence of the TCR ws not due to n impired ility of T reg cells to gin ccess to interleukin (IL-) nd, ccordingly, dministrtion of exogenous IL- filed to rescue systemic utoimmunity. Insted, TCR expression ws essentil for the ctivtion of T reg cells nd for T reg cells to mintin expression of limited set of genes found to e expressed lmost exclusively in ctivted T reg cells. Among those genes, expression of the trnscription fctor IRF4 contriuted to the optiml function nd homeostsis of T reg cells. Our results demonstrte n essentil role for the TCR in eliciting the suppressor function of differentited T reg cells. RESULTS Mintennce of T reg cell identity in the sence of the TCR To investigte the role of TCR signling in T reg cell function, we crossed Trc FL mice (which hve loxp-flnked llele encoding the TCR α-chin constnt region (C α or TCRα)) with Foxp3 egfp-cre-ert mice (with expression of enhnced green fluorescent protein (egfp) fused to Cre recominse estrogen-receptor-lignd-inding-domin protein from the 3 untrnslted region of Foxp3; clled Foxp3 Cre-ERT here) to chieve tmoxifen-inducile deletion of Trc specificlly in T reg cells 18,19. In this model, Cre-induced loss of the conditionl 1 Howrd Hughes Medicl Institute, Memoril Slon-Kettering Cncer Center, New York, New York, USA. Immunology Progrm, Memoril Slon-Kettering Cncer Center, New York, New York, USA. 3 Ludwig Center, Memoril Slon-Kettering Cncer Center, New York, New York, USA. 4 Present ddresses: Giled Sciences, Foster City, Cliforni, USA (A.A.) nd Tsinghu University School of Medicine, Beijing, Chin (W.J.). Correspondence should e ddressed to A.Y.R. (rudensk@mskcc.org). Received 4 August; ccepted 5 Septemer; pulished online 8 Septemer 14; doi:1.138/ni.34 nture immunology DVANCE ONLINE PUBLICATION

2 14 Nture Americ, Inc. All rights reserved. Figure 1 Mintennce of T reg cell identity in the sence of the TCR. (,) Expression of TCRβ () nd medin fluorescence intensity (MFI) of Foxp3 () in CD4 + Foxp3 + lymph node cells from 8- to 1-week-old Trc Foxp3 Cre-ERT mice (), Trc Foxp3 Cre-ERT mice () nd Trc Foxp3 Cre-ERT mice () treted with tmoxifen y gvge on dys nd 1 nd nlyzed on dy 9. Gry shding (), TCRβ stining on CD4 TCRβ cells. (c) Flow cytometry of Foxp3 + T cells mong CD4 + YFP + cells sorted to >99% purity from the spleens nd lymph nodes of Trc Foxp3 Cre-ERT Ros6 YFP mice on dy 13 following tretment with tmoxifen on dys nd 1 nd intrperitonel injection of IL--neutrlizing ntiody (Anti-IL-) or isotype-mtched control ntiody (Isotype) on dys 4 nd 8. Numers djcent to outlined res (left) indicte percent Foxp3 + cells mong cells (right) or TCRβ cells (left). Ech symol (,c (right)) represents n individul mouse; smll horizontl lines indicte the men. P <.5, P <.1 nd P <.1 (two-tiled unpired t-test). Dt re representtive of two independent experiments with four or more (,) or two or more (c) mice per group in ech. Trc llele upon tmoxifen dministrtion elimintes TCRα expression, which prevents formtion of heterodimers of TCRα nd TCRβ (TCRαβ) t the cell surfce. We dministered tmoxifen vi orl gvge on dys nd 1 nd nlyzed mice on dy 9. Allelic exclusion t the Trc locus in heterozygous Trc Foxp3 Cre-ERT mice yielded smll popultion (5%) of TCR-deficient (s ssessed y flow cytometry ( ; clled TCR here)) T reg cells (~5%), wheres in homozygous Trc Foxp3 Cre-ERT mice, the mjority of T reg cells (~6 7%) lcked cell surfce TCRβ (Fig. 1). Although we cnnot definitively exclude the possiility tht few residul TCR complexes were present in minute mounts (elow the detection limit of flow cytometric nlyses), functionl in vitro nlyses confirmed loss of TCR crosslinking dependent ctivtion of TCR T reg cells (Supplementry Fig. 1 d). Becuse inding sites for the trnscription fctors NFAT nd c-rel hve een identified in the Foxp3 locus, nd ecuse TCR enggement driven signling vi the trnscription fctor NF-κB is criticl for induction of Foxp3 expression, we speculted tht the TCR might e essentil for mintining Foxp3 expression. However, Foxp3 expression ws reduced only mrginlly in TCR T reg cells in Trc Foxp3 Cre-ERT mice nd ws not reduced t ll in TCR T reg cells in Trc Foxp3 Cre-ERT mice, reltive to its expression in TCRexpressing (s ssessed y flow cytometry ( ; clled TCR + here)) T reg cells in the sme mice nd in Trc Foxp3 Cre-ERT mice (Fig. 1). Similrly, the expression of genes encoding severl T reg cell signture molecules, including CD5, GITR, CD39 nd CD73, ws lrgely unffected in TCR T reg cells from oth Trc Foxp3 Cre-ERT nd Trc Foxp3 Cre-ERT mice (Supplementry Fig. 1e). These results indicted tht in the stedy stte, continuous TCR-medited recognition of self did not contriute sustntilly to Foxp3-dependent mintennce of expression of these genes 11,3. In contrst, CTLA-4 expression ws notly diminished in TCR T reg cells in Trc Foxp3 Cre-ERT mice, ut not in TCR T reg cells in Trc Foxp3 Cre-ERT mice, reltive to its expression in TCR + T reg cells in Trc Foxp3 Cre-ERT mice (Supplementry Fig. 1e). The frequency nd solute numer of Foxp3 + cells in the spleens nd lymph nodes of Trc Foxp3 Cre-ERT nd Trc Foxp3 Cre-ERT mice were unltered compred with tht in Trc Foxp3 Cre-ERT mice (Supplementry Fig. 1f). However, to ddress the possiility tht portion of T reg cells completely lost Foxp3 expression upon ltion of the TCR nd tht these former T reg cells were not ccounted for in this experimentl setup, we crossed Trc Foxp3 Cre-ERT mice with mice tht express the recomintion reporter Ros6 YFP (with sequence encoding yellow fluorescent protein (YFP) expressed form the uiquitous Ros6 locus). We ssessed the expression Events (% of mx) Foxp3 (MFI 1 3 ) of TCRβ nd Foxp3 in CD4 + YFP + cells sorted from the spleens nd lymph nodes of Trc Foxp3 Cre-ERT Ros6 YFP mice on dy 9 or 5 following tmoxifen dministrtion on two consecutive dys. YFP-expressing CD4 + TCRβ nd CD4 + cell susets contined similrly low frequencies of Foxp3 cells t oth time points (dt not shown). Furthermore, following in vivo neutrliztion of IL-, condition known to promote loss of Foxp3 expression 19, in Trc Foxp3 Cre-ERT Ros6 YFP mice, popultions of CD4 + YFP + TCRβ cells retined higher percentge of Foxp3 + cells thn did popultions of CD4 + YFP + cells (Fig. 1c). Together these dt indicted tht TCR signling ws dispensle for the mintennce of the T reg cell phenotype nd linege stility nd, moreover, tht TCR signling drove the loss of Foxp3 when IL- mounts were limiting. Requirement for the TCR in effector differentition of T reg cells Although the T reg cell phenotype ws lrgely preserved upon ltion of the TCR, we oserved reltive enrichment for nive-like CD44 lo CD6L hi cells mong TCR T reg cells in lymph nodes nd spleens of Trc Foxp3 Cre-ERT nd Trc Foxp3 Cre-ERT mice (Fig. nd Supplementry Fig. 1g). T reg cell prolifertion is restricted lmost exclusively to the CD44 hi suset, nd in prt the enrichment we oserved ppered to e consequence of severely impired prolifertive cpcity of CD44 hi T reg cells in the sence of the TCR 4 (Fig. ). The smll popultion of TCR T reg cells in Trc Foxp3 Cre-ERT mice ws predominntly nondividing: these cells showed miniml expression of the prolifertion mrker Ki67, filed to incorporte the thymidine nlog BrdU over 4-hour leling period nd contined the gretest frequency of CD44 lo CD6L hi cells mong ll TCR + or TCR T reg cell popultions in Trc Foxp3 Cre-ERT, Trc Foxp3 Cre-ERT nd Trc Foxp3 Cre-ERT mice (Fig., nd Supplementry Fig. 1h). In Trc Foxp3 Cre-ERT mice, however, TCR T reg cells exhiited considerle prolifertive ctivity, leit reduced reltive to tht of TCR + T reg cells present in the sme mouse (Fig. nd Supplementry Fig. 1h). Incresed Ki67 stining in CD44 hi T reg cells ws inversely correlted with lower frequency of CD44 lo CD6L hi cells mong ll TCR + or TCR T reg cell popultions in mice of ll three genotypes (Fig.,). To ddress the possiility tht continuous peripherl differentition of nive-like T reg cells into CD44 hi cells ws impeded in the sence of TCR expression nd tht such differentition lock contriuted to the TCRβ c Isotype Anti-IL- Foxp CD4 Foxp3 + cells (%) Isotype Anti-IL- DVANCE ONLINE PUBLICATION nture immunology

3 14 Nture Americ, Inc. All rights reserved. CD44 lo CD6L hi cells (%) predominntly CD44 lo CD6L hi phenotype of TCR T reg cells, we red Trc FL mice with Foxp3 YFP-Cre mice (which express fusion of Cre nd YFP from the 3 untrnslted region of Foxp3) to induce ltion of the TCR in newly generted, nive T reg cells 5,6. We resoned tht if the TCR were criticl for the effector differentition of T reg cells in the periphery, TCR T reg cells in these mice would retin CD44 lo CD6L hi nive-like phenotype. Immture HSA hi CD4 + Foxp3 + cells in the thymi of Trc Foxp3 YFP-Cre mice hd cell surfce expression of TCR complexes similr to tht in their wild-type counterprts; more mture HSA lo CD4 + Foxp3 + thymocytes showed only slightly reduced TCR expression (Supplementry Fig. ). Among Foxp3 + cells present in the spleens nd lymph nodes of Trc Foxp3 YFP-Cre mice, ~5 15% were TCRβ, wheres ~8% of Foxp3 + cells in Trc Foxp3 YFP-Cre mice lcked surfce TCRβ expression (Fig. e nd Supplementry Fig. ). We gin oserved slight decrese in the mount of Foxp3 protein in the TCR T reg cells in Trc Foxp3 YFP-Cre mice, ut not in Trc Foxp3 YFP-Cre mice, while expression of T reg cell signture genes ws vrily ffected in the TCR T reg cell popultions in oth mouse strins reltive to expression in TCR + T reg cells in Trc Foxp3 YFP-Cre, Trc Foxp3 YFP-Cre nd Trc Foxp3 YFP-Cre mice (Supplementry Fig. c,d). Despite their generlly intct T reg cell surfce phenotype, nerly ll TCR T reg cells in helthy Trc Foxp3 YFP-Cre mice hd nivelike CD6L hi CD44 lo phenotype nd lcked expression of ll T reg cell differentition mrkers tested, including KLRG1, CD13 nd CXCR3 (Fig. c nd dt not shown). Notly, we lso oserved this pttern under severe inflmmtory conditions in Trc Foxp3 YFP-Cre mice, which were moriund y 3 weeks of ge (Fig. c nd Supplementry Fig. e,f). The lck of CD44 hi cells mong TCR popultions in Trc Foxp3 YFP-Cre nd Trc Foxp3 YFP-Cre mice correlted with the decresed prolifertion nd mrkedly diminished frequency nd numer of TCR T reg cells reltive to tht of TCR + T reg cells in the sme mice nd in Trc Foxp3 YFP-Cre mice; this pttern ws evident in lymph nodes nd ws prticulrly pronounced in the spleen nd other tissues such s the liver nd lungs (Fig. d,e Figure Requirement for the TCR in the differentition nd popultion expnsion of T reg cells. (,) Expression of CD44 nd CD6L on or TCRβ CD4 + Foxp3 + lymph node cells () nd of Ki67 on CD44 hi CD4 + Foxp3 + lymph node cells () isolted on dy 9 from Trc Foxp3 Cre-ERT, Trc Foxp3 Cre-ERT nd Trc Foxp3 Cre-ERT mice treted Ki67 + cells (%) c CD CD6L with tmoxifen on dys nd 1. (c) Expression of CD44 nd CD6L on or TCRβ CD4 + Foxp3 + lymph node cells in.5-week-old Trc Foxp3 YFP-Cre, Trc Foxp3 YFP-Cre nd Trc Foxp3 YFP-Cre mice (left), nd expression of differentition mrkers in those of the Trc Foxp3 YFP-Cre mice. Numers djcent to outlined res (left) indicte percent CD44 hi CD6L lo cells; numers in qudrnts (right) indicte percent cells in ech throughout. (d,e) Ki67 expression in or TCRβ CD4 + Foxp3 + lymph node cells (d) nd frequency of or TCRβ CD4 + Foxp3 + cells mong totl CD4 + cells in the spleen nd lymph nodes () (e) of mice s in c. Ech symol (,,d,e (right)) represents n individul mouse. P <.1 nd P <.1 (two-tiled unpired t-test). Dt re representtive of two independent experiments with four or more mice per group in ech (,) or three independent experiments with six or more mice per group (c) or re pooled from three independent experiments with six or more mice per group (d,e) KLRG1 CD TCRβ e Foxp3 + cells (%) nd Supplementry Fig. g,h). Together these dt were consistent with n solute requirement for TCR expression, the loss of which could not e compensted for even in conditions of extreme ctivtion of the immune system, for the peripherl effector differentition of nive-like T reg cells nd cquisition of n ctivted CD44 hi phenotype. TCR-dependent effector function of mture T reg cells Foxp3 expression nd T reg cell popultion expnsion re fcilitted y signling vi the receptor for IL- (IL-R) 7 9. The increse in Foxp3 protein expression nd prolifertive ctivity of TCR T reg cells in Trc Foxp3 Cre-ERT mice compred with tht in such cells in Trc Foxp3 Cre-ERT mice led us to suspect tht ltion of TCR expression, even on mture T reg cells, might precipitte ctivtion of the immune system nd elevte the production of IL- nd other cytokines y ctivted CD4 + T cells. Indeed, nlysis of Trc Foxp3 Cre-ERT mice treted twice with tmoxifen nd nlyzed on dy 9 fter tretment reveled incresed percentges of CD44 hi T cells nd incresed numers of IL- producing CD4 + T cells compred with tht of tmoxifen-treted Trc Foxp3 Cre-ERT nd Trc Foxp3 Cre-ERT mice (dt not shown). To more rigorously investigte the role of TCR expression in mture T reg cell function, we dministered four doses of tmoxifen to mice (on dys, 3, 7 nd 1) to mximize Cre-ERT medited recomintion. On dy 13, we noted loss of TCR expression in ~75 8% of T reg cells in Trc Foxp3 Cre-ERT mice nd ~5 3% of T reg cells in Trc Foxp3 Cre-ERT mice (Fig. 3,). Despite the norml or even incresed frequency of totl Foxp3 + cells in the lymph nodes nd spleens of Trc Foxp3 Cre-ERT mice, we found elevted numers of CD4 + Foxp3 nd CD8 + T cells in the lymph nodes of these mice nd higher frequency of CD44 hi CD4 + nd CD8 + T cells in their lymph nodes nd spleens (Fig. 3c,d). CD8 + T cells from Trc Foxp3 Cre-ERT mice produced more interferon-γ (IFN-γ), nd CD4 + T cells from these mice produced more IFN-γ, IL-, IL-4, IL-13, IL-5 nd IL-17, thn did T cells from Trc Foxp3 Cre-ERT nd Trc Foxp3 Cre-ERT mice (Fig. 3e nd dt not shown). 3 1 d Ki67 + cells (%) nture immunology DVANCE ONLINE PUBLICATION

4 14 Nture Americ, Inc. All rights reserved. Figure 3 TCR-dependent effector function of mture T reg cells in dult mice. () Expression of Foxp3 nd TCRβ y CD4 + cells in the lymph nodes of Trc Foxp3 Cre-ERT, Trc Foxp3 Cre-ERT nd Trc Foxp3 Cre-ERT mice on dy 13 following tmoxifen tretment on dys, 3, 7 nd 1. Numers djcent to outlined res indicte percent Foxp3 + cells (right) or Foxp3 + TCRβ cells (left). () Frequency of Foxp3 + cells mong splenic nd lymph node CD4 + cells in Trc Foxp3 Cre-ERT, Trc Foxp3 Cre-ERT nd Trc Foxp3 Cre-ERT mice, nd of totl Foxp3 + cells mong CD4 + cells for ech genotype (Totl Foxp3 + ). (c e) Numer (c), CD44 expression (d) nd cytokine production (e) of CD4 + Foxp3 or CD8 + T cells in the spleen (c e) nd lymph nodes (c,d) of mice s in. Ech symol (c e) represents n individul mouse; smll horizontl lines indicte the men. NS, not significnt (P.5); P <.5 nd P.1 (two-tiled unpired t-test). Dt re representtive of three experiments with three mice or more per group in ech () or re pooled from three experiments with nine or more per group ( e; error rs (), men ± s.e.m.). The ctivtion of the immune system in Trc Foxp3 Cre-ERT mice ws milder thn tht resulting from complete depletion of T reg cells in Foxp3 DTR mice, which express the humn diphtheri toxin receptor (DTR) concomitntly with Foxp3 (ref. 3). Thus, it ws possile tht the lrge numer of TCR T reg cells in Trc Foxp3 Cre-ERT mice retined mesurle TCR-independent suppressor ility nd were still cple of immunoregultion. Alterntively, the smll popultion of remining TCR-sufficient T reg cells in these mice might hve limited, to some degree, the ctivtion of effector T cells nd the ssocited utoimmunity. To exmine these possiilities, we ttempted to reduce the proportion of T reg cells mong CD4 + cells in Foxp3 DTR mice to pproximte the frequency of residul TCR-sufficient T reg cells in Trc Foxp3 Cre-ERT mice following four doses of tmoxifen 31. We resoned tht if TCR T reg cells were cple of sustntil suppression, utoimmunity in Foxp3 DTR mice sujected to only prtil depletion of T reg cells would e more severe thn tht in Trc Foxp3 Cre-ERT mice. Injection of diphtheri toxin depletes mice of T reg cells within 4 h, wheres tmoxifen-induced, Cre-ERT medited recomintion progressively increses over 4-dy period (dt not shown). Therefore, we treted Foxp3 DTR, Trc Foxp3 Cre-ERT nd Trc Foxp3 Cre-ERT mice with diphtheri toxin 4 d fter their first dose of tmoxifen to ccount for the time needed for complete Cre-ERT medited deletion of Trc (we dministered oth tximofen nd diphtheri toxin to ll genotypes; Supplementry Fig. 3,). Prtil depletion of the T reg cell comprtment in Foxp3 DTR mice resulted in ctivtion nd cytokine production of CD4 + Foxp3 T cells grossly similr to tht oserved in Trc Foxp3 Cre-ERT mice with popultions of TCR-sufficient T reg cells of similr or even lrger size (Supplementry Fig. 3 d). Together these results demonstrted tht T reg cells required continuous TCR expression for the effective elortion of their suppressor function, nd suggested tht TCR T reg cells, which were undnt in Trc Foxp3 Cre-ERT mice, were lrgely devoid of detectle suppressor ility. TCR T reg cell dysfunction is not secondry to impired IL-R signling We considered tht the pprent loss of suppressive ility of T reg cells in the sence of the TCR might e n indirect consequence Foxp3 CD8 + T cells ( 1 6 ) CD4 + T cells ( 1 6 ) TCRβ NS NS CD44 hi CD8 + T cells (%) CD44 hi CD4 + T cells (%) c d e Foxp3 + cells (%) 4 of impired ility to loclize in TCR- nd ntigen-dependent mnner to sites of CD4 + T cell ctivtion nd to therey cquire IL-, cytokine known to e criticl for the function nd homeostsis of T reg cells. This would explin the decresed expression of Foxp3 nd miniml prolifertion of TCR T reg cells in helthy Trc Foxp3 Cre-ERT mice, in which IL- mounts were not elevted nd would not e le to prtilly remedy these defects 3. However, direct ex vivo nlysis of phosphoryltion of the trnscription fctor STAT5, which occurs downstrem of IL- signling in T reg cells, showed tht in the spleen nd lymph nodes of oth Trc Foxp3 Cre-ERT nd Trc Foxp3 Cre-ERT mice, the proportion of phosphorylted STAT5 in TCR T reg cells ws t lest equivlent to tht in TCR + T reg cells (Fig. 4 nd dt not shown). In Trc Foxp3 Cre-ERT mice, TCR T reg cells hd more phosphorylted STAT5 thn did TCR + T reg cells; this mirrored their expression of CD5 nd CD6L, which remined high on TCR cells ut ws decresed on the residul ctivted TCR + T reg cells present in these mice (Figs. nd 4 nd Supplementry Fig. 1e,g). These results were consistent with the oservtion tht T reg cells tht contin phosphorylted STAT5 re found minly in the CD6L hi CD44 lo (nd CD5 hi ) suset of T reg cells; in contrst to the ctivted CD44 hi CD6L lo (nd CD5 int ) T reg cell suset, this group of cells hs een reported to rely on IL-R signling rther thn enggement of costimultory receptors for their mintennce 4. In vitro nlysis confirmed tht lck of TCR expression did not sustntilly influence the phosphoryltion of STAT5 in response to IL-, nor did it impir the ility of T reg cells to cpture nd deplete IL- from culture medi (Supplementry Fig. 4,), which suggested tht in the sence of TCR expression, T reg cell medited deprivtion of IL- might not e n importnt mechnism of immunosuppression. Furthermore, tretment of Trc Foxp3 Cre-ERT, Trc Foxp3 Cre-ERT nd Trc Foxp3 Cre-ERT mice with neutrlizing ntiody to IL- (nti-il-) or isotype-mtched control ntiody hd similr effect on TCR + nd TCR T reg cells, reducing Foxp3 expression nd lowering the frequency of Foxp3 + cells mong totl CD4 + cells (Fig. 4). Together these dt suggested tht TCR T reg cells efficiently cptured IL- during ctivtion of the immune system nd t stedy stte. It remins to e determined wht signl(s) drove IL- + CD4 + T cells (%) IFN-γ + CD8 + T cells (%) IFN-γ + CD4 + T cells (%) IL-4 + CD4 + T cells (%) Totl Foxp DVANCE ONLINE PUBLICATION nture immunology

5 14 Nture Americ, Inc. All rights reserved. Figure 4 TCR expression y T reg cells is dispensle for IL-R signling in vivo. () Phosphoryltion of STAT5 t Tyr694 (p-stat5(y694)) in or TCRβ CD4 + Foxp3 + lymph node cells from Trc Foxp3 Cre-ERT, Trc Foxp3 Cre-ERT nd Trc Foxp3 Cre-ERT mice on dy 9 following tmoxifen dministrtion on dys nd 1. Numers djcent to outlined res indicte percent CD4 + Foxp3 + cells with phosphorylted STAT5. () Medin fluorescence intensity of Foxp3 in or TCRβ Foxp3 + cells (top) nd frequency of or TCRβ Foxp3 + cells mong CD4 + cells (ottom) in the lymph nodes of Trc Foxp3 Cre-ERT, Trc Foxp3 Cre-ERT nd Trc Foxp3 Cre-ERT mice on dy 13 following tmoxifen tretment on dys, 3, 7 nd 1 nd intrperitonel injection of IL--neutrlizing or isotype-mtched contol ntiody on dys 4 nd 8. Numers ove plots indicte comprison of results otined for mice treted with isotype-mtched control ntiody reltive to those for mice treted with nti-il-. Ech symol () represents n individul mouse; smll horizontl lines indicte the men. P <.5, P <.1 nd P <.1 (two-tiled unpired t-test). Dt re representtive of three experiments involving totl of three or more mice per group () or of two experiments with two or more mice per group in ech (). the prolifertion of TCR T reg cells selectively in disesed Trc Foxp3 Cre-ERT mice. However, we oserved incresed expression of the costimultory molecules CD8 nd CD86 on lymph node dendritic cells (DCs) in Trc Foxp3 Cre-ERT mice, nd ctivted DCs were le to induce limited prolifertion of TCR T reg cells in vitro (Supplementry Fig. 4c,d). Finlly, the dministrtion of complexes of IL- nd nti-il- to Trc Foxp3 Cre-ERT mice did not mesurly diminish the ctivtion or lymphoprolifertion of effector T cells cused y loss of TCR expression in T reg cells (Supplementry Fig. 5). Conversely, IL- depletion did not further excerte utoimmunity (dt not shown). Notly, this ws the cse despite 1.5-fold popultion expnsion of TCR T reg cells, ut not of TCR + T reg cells, following IL- dministrtion (proly consequence of higher CD5 expression nd heightened IL- responsiveness in TCR T reg cells) nd reduction of over twofold in TCR T reg cells following depletion of IL- (Fig. 4 nd Supplementry Fig. 5). These oservtions further confirmed tht TCR T reg cells, even when present in elevted numers, hd miniml suppressive ility. Together these results indicted tht neither TCR-dependent interctions with ntigen-presenting cells nor continuous TCR-medited locliztion within lymphoid orgns were required for T reg cells to cquire IL-, nd tht T reg cell dysfunction in the sence of the TCR could not e ttriuted to ltered IL-R signling. TCR expression promotes T reg cell dhesive properties in vitro The in vitro suppressive ility of T reg cells requires enggement of the TCR 33,34. This might involve pthwys independent of the ctlytic ctivity of the signling kinse Zp7, which is essentil for the effector function of conventionl T cells, ut dependent on memrne-proximl inside-out ctivtion of integrins nd susequent enhncement of the interction of T reg cells with ntigen-presenting cells 8. To ddress this possiility, we cultured TCR + or TCR T reg cells isolted from Trc Foxp3 Cre-ERT, Trc Foxp3 Cre-ERT or Trc Foxp3 Cre-ERT mice with DCs nd ssessed the formtion of DC T reg cell conjugtes. We did not detect ny difference in conjugte formtion etween DCs nd TCR + or TCR T reg cells following 3 min of incution (dt not shown). However, following overnight culture, TCR T reg cells isolted from Trc Foxp3 Cre-ERT or Trc Foxp3 Cre-ERT mice were less efficient thn were TCR + T reg cells t forming conjugtes with DCs (3.7% of TCR T reg cells compred with 7.5% of TCR + T reg cells; Supplementry Fig. 6). Conjugte formtion ws unffected y the presence or sence of MHC clss II p-stat5(y694) Isotype Anti-IL Foxp3 (MFI 1 3 ) Foxp3 + cells (%) CD molecules on DCs (Supplementry Fig. 6), which might indicte tht the greter dhesion of TCR + T reg cells thn of TCR T reg cells in this ssy ws not result of interctions etween TCR nd MHC clss II nd might hve een consequence of the overll heightened ctivtion sttus of TCR + T reg cells reltive to tht of TCR T reg cells. As expression of the integrin LFA-1 ws higher on CD44 hi T reg cells thn on CD44 lo CD6L hi T reg cells (Supplementry Fig. 6), it is possile tht greter conjugte formtion y TCR + T reg cells, which show greter enrichment for CD44 hi cells thn do TCR T reg cells, ws due t lest in prt to incresed expression of this integrin. Further work is needed to determine precisely how enggement of the TCR in vivo ffects signling pthwys to modulte the dhesive properties of T reg cells. However, our results indicted tht TCR expression contriuted to optiml contct-dependent interctions etween T reg cells nd ntigen-presenting cells, which might support TCR-dependent immunosuppressive function. TCR modultion of the effector T reg cell trnscriptionl signture To explore whether TCR signls, prt from influencing T reg cell dhesion, might drive trnscriptionl events to license suppressor function in vivo, we nlyzed the gene expression of TCR + nd TCR T reg cells. Flow cytometry showed tht loss of TCR expression hd stronger effect on effector-like CD44 hi CD6L lo T reg cells thn on nive-like CD44 lo CD6L hi T reg cells (dt not shown). This prompted us to investigte the gene-expression profiles of these two popultions seprtely in the TCR + or TCR T reg cell popultions isolted from helthy Trc Foxp3 Cre-ERT mice (to void confounding effects of ctivtion of the immune system). We found tht 155 genes were downregulted y t lest twofold nd only five genes were upregulted y tht mount in effector-like CD44 hi CD6L lo TCR T reg cells reltive to their expression in CD44 hi CD6L lo TCR + T reg cells (Fig. 5). 16 genes were downregulted in the nive-like CD44 lo CD6L hi TCR T reg cells reltive to their expression in CD44 lo CD6L hi TCR + T reg cells nture immunology DVANCE ONLINE PUBLICATION

6 14 Nture Americ, Inc. All rights reserved. Figure 5 TCR signling mintins the effector T reg cell trnscriptionl signture. () Genes expressed differently in CD44 hi CD6L lo versus CD44 lo CD6L hi T reg cells plotted ginst those expressed differently in CD44 hi CD6L lo TCRβ versus CD44 hi CD6L lo T reg cells (top) or CD44 lo CD6L hi TCRβ versus CD44 lo CD6L hi T reg cells (ottom) mong supopultions sorted y flow cytometry on dy 14 from Trc Foxp3 Cre-ERT mice treted with tmoxifen on dys, 1 nd 3; numers in plots indicte genes upregulted (lue) or downregulted (red) y twofold or more in the sence of the TCR (q <.1). () Genes downregulted twofold or more in CD44 hi CD6L lo TCRβ T reg cells reltive to their expression in CD44 hi CD6L lo T reg cells (TCR) mong those upregulted twofold or more in CD44 hi CD6L lo T reg cells reltive to their expression in CD44 lo CD6L hi T reg cells (Up) (left), nd genes upregulted twofold or more in CD44 hi CD6L lo T reg cells reltive to their expression in CD44 lo CD6L hi T reg cells (Up) mong those downregulted twofold or more in CD44 hi CD6L lo TCRβ T reg cells reltive to their expression in CD44 hi CD6L lo T reg cells (TCR) (right). (c) Cumultive distriution function plot of TCR-dependent genes plotted ginst ll genes expressed differently in Foxp3 GFP-KO T cells versus Foxp3 + CD4 + T cells. P < 1 (two-smple Kolmogorov-Smirnov test). (d) Expression ptterns of TCR-dependent genes encoding trnscription fctors, cell surfce molecules nd secreted molecules in T reg cells. Dt re representtive of one experiment with two or more replictes with five or more mice per replicte. (ll of these were lso downregulted in CD44 hi CD6L lo TCR vs. CD44 hi CD6L lo TCR + T reg cells), wheres one gene ws upregulted (Fig. 5). Some 535 genes showed higher expression (twofold or greter) in effector-like CD44 hi CD6L lo TCR + T reg cells thn in nivelike CD44 lo CD6L hi TCR + T reg cells, nd the expression of 136 of these (5%) ws TCR dependent (Fig. 5). Notly, 17 of the 155 genes (8%) downregulted in the sence of the TCR in CD44 hi CD6L lo T reg cells showed t lest twofold higher expression in effector-like T reg cells thn in nive-like T reg cells (Fig. 5). Foxp3 hs een proposed to solidify nd mplify trnscriptionl progrm initited in T reg cell precursors y enggement of the TCR 9,1,35,36. We compred the expression of the 155 genes identified ove s eing mintined y the TCR in T reg cells (clled TCRdependent genes here) to the expression of Foxp3-dependent genes, identified s eing upregulted in wild-type T reg cells reltive to their expression in egfp + T cells from Foxp3 GFP-KO mice, which express egfp from Foxp3-null llele 36. We found tht sustntilly more of the TCR-dependent genes thn ll genes were lso Foxp3 dependent (Fig. 5c). These oservtions suggested tht the TCR-driven trnscriptionl progrm in T reg cells ws enhnced y Foxp3 expression, ut tht Foxp3 lone ws not sufficient to mintin the full trnscriptionl signture of effector T reg cells. Exmintion of the TCR-dependent genes identified severl trnscription fctors tht were upregulted in TCR + CD44 hi CD6L lo T reg cells reltive to their expression in TCR CD44 hi CD6L lo, TCR + CD44 lo CD6L hi nd TCR CD44 lo CD6L hi T reg cells, including NFATc1, c-rel, Bcl-6 nd IRF4 (Fig. 5d); pulished dt hve shown tht Bcl-6 nd IRF4 re importnt for the differentition nd function of effector T reg cells Of the 155 TCR-dependent genes, we identified only one dhesion molecule encoding gene (Vcm1) whose expression ws upregulted in CD44 hi CD6L lo T reg cells reltive to its expression in CD44 lo CD6L hi T reg cells in mnner tht depended on TCR expression (Fig. 5d). Genes encoding severl other potentil effector molecules were lso upregulted in CD44 hi CD6L lo T reg cells reltive to their expression in CD44 lo CD6L hi T reg cells, in TCR-dependent mnner, including those encoding IL-1R ( decoy receptor for IL-1); the immunoinhiitory molecules CD83, CD nd LAG-3; IL-1; nd EBI3 ( suunit of the cytokines IL-7 nd IL-35) 6,4 45. In ddition, the chemokine-encoding genes Cxcl1 Expression (log fold) CD44 hi T reg vs CD6L hi T reg Expression (log fold) CD44 hi T reg vs CD6L hi T reg c Cumultive frction genes 5 genes Expression (log fold) CD44 hi TCR + vs TCR T reg genes 1 gene Expression (log fold) CD6L hi TCR + vs TCR T reg 1..5 All genes TCR dependent Expression (log fold) Foxp3 + vs Foxp3 GFP-KO Genes d nd Ccl1, s well s Ccr8 (which encodes the receptor for CCL1), were significntly downregulted in TCR T reg cells reltive to their expression in TCR + T reg cells, which suggested tht T reg cells might signl ech other through the expression of chemokines nd their corresponding receptors or might recruit into close proximity the trgets of their suppressive ctivity 46. Together these dt indicted tht under physiologicl conditions, sustntil portion of the effector T reg cell trnscriptionl progrm, ut not the nive-like T reg cell trnscriptionl progrm, chrcterized y elevted expression of severl potentil T reg cell effector molecules, ws mintined y continuous TCR signling. IRF4 expression promotes T reg cell function nd homeostsis To egin to ssess the importnce of the TCR-dependent trnscriptionl progrm for continuous T reg cell function in vivo, we focused on IRF4 s downstrem trget of the TCR signling pthwy in T reg cells. We confirmed tht elevted IRF4 expression in T reg cells ws restricted to CD44 hi cells nd ws reduced to sl expression upon ltion of the TCR in oth Trc Foxp3 Cre-ERT nd Trc Foxp3 Cre-ERT mice (Supplementry Fig. 7). Irf4 FL/ Foxp3 Cre-ERT mice, in which IRF4 is constitutively deleted in T reg cells, hve een shown to develop severe utoimmunity dominted y type helper T cell cytokines y 8 weeks of ge 37. T reg cells in Irf4 / mice hve een shown to hve n lmost exclusively nive-like phenotype 38, nd we similrly found tht T reg cells in Irf4 Foxp3 YFP-Cre mice were lrgely CD44 lo CD6L hi, even in the context of severe inflmmtion (dt not shown), which suggested impired differentition, survivl nd/or popultion expnsion of effector T reg cells. To determine whether fully differentited T reg cells require IRF4 expression downstrem of TCR enggement for their in vivo suppressive function, we dministered tmoxifen on dys, 3, 7 nd 1 to Irf4 Foxp3 Cre-ERT nd Irf4 Foxp3 Cre-ERT littermtes 47. On dy 13, we oserved slightly ut reproducily lower IRF4 protein expression in CD44 hi T reg lymph node cells from Irf4 Foxp3 Cre-ERT 6 4 5% Egr Egr1 N Nr41 Irf4 Rel Nftc1 Bcl6 Il1r Cd83 Pdcd1 CD Lg3 Ctl4 Vcm1 Ccr8 Tnfrsf9 Ei3 Il1 Cxcl1 Ccl1 TCR Up CD44 hi Genes TCR 8% Up CD6L hi TCR TCR + TCR TCR + Trnscription fctors Cell surfce proteins Secreted proteins Expression (log fold) DVANCE ONLINE PUBLICATION nture immunology

7 Events (% of mx) Events (% of mx) IRF Irf4 CD6L hi Irf4 CD44 hi Irf4 CD6L hi Irf4 CD44 hi Irf4 Irf IRF4 Irf4 (AU) Foxp3 + (%) Irf4 Irf4 Foxp3 Foxp3 + Trc Irf4 c CD4 + T cells ( 1 6 ) d IFN-γ + CD4 + T cells (%) Trc NS Irf4 CD44 hi CD4 + T cells (%) IL-4 + CD4 + T cells (%) Ki67 + CD4 + T cells (%) Trc Irf4 Trc Irf4 Trc Irf4 Trc Irf4 Trc Irf4 IL-13 + CD4 + T cells (%) Nture Americ, Inc. All rights reserved. Figure 6 IRF4 expression contriutes to optiml suppressive ility nd homeostsis of T reg cells. () IRF4 expression in CD44 hi CD6L lo (CD44 hi ) nd CD44 lo CD6L hi (CD6L hi ) CD4 + Foxp3 + lymph node cells from Irf4 Foxp3 Cre-ERT mice (Irf4 ) nd Irf4 Foxp3 Cre-ERT mice (Irf4 ) nlyzed on dy 13 following tmoxifen tretment on dys, 3, 7 nd 1 (left), quntittive PCR nlysis of Irf4 mrna in CD4 + egfp nd CD4 + egfp + cells sorted from pooled spleens nd lymph nodes of those mice. AU, ritrry units. () IRF4 expression in colonic lmin propri CD4 + Foxp3 + cells in Irf4 Foxp3 Cre-ERT nd Irf4 Foxp3 Cre-ERT mice (left), nd frequency of Foxp3 + cells mong CD4 + cells in the lrge intestine lmin propri in Trc Foxp3 Cre-ERT or Irf4 Foxp3 Cre-ERT mice (open circles) nd Trc Foxp3 Cre-ERT or Irf4 Foxp3 Cre-ERT mice (filled circles) nlyzed on dy 13 following tmoxifen tretment on dys, 3, 7 nd 1. Gry shded curve (left), CD4 + Foxp3 cells. (c,d) Numer of cells nd frequency of CD44 hi or Ki67 + cells (c) or cytokine-producing cells (d) mong CD4 + Foxp3 T cells in the lymph nodes (c) nd spleens (d) of Trc Foxp3 Cre-ERT mice (open circles), Irf4 Foxp3 Cre-ERT mice (open squres), Trc Foxp3 Cre-ERT mice (filled circles) nd Irf4 Foxp3 Cre-ERT mice (filled squres) nlyzed on dy 13 following tmoxifen tretment on dys, 3, 7 nd 1. Ech symol ( d) represents n individul mouse; smll horizontl lines indicte the men. P <.5, P <.1 nd P <.1 (two-tiled unpired t-test). Dt re representtive of two experiments with four or more mice per group in ech (,, left) or re pooled from two experiments with four or more mice per group in ech (,, right, c,d (error rs (), s.d.). mice thn in cells from Irf4 Foxp3 Cre-ERT mice (Fig. 6). We lso sw higher GFP expression in the T reg cells, mesured s sum of fluorescence from the Irf4-deletion GFP reporter (whose expression is switched on in the Irf4 locus y Cre-medited deletion of the Irf4 FL llele) nd from the GFP-Cre-ERT fusion protein expressed concomitntly with Foxp3 (Supplementry Fig. 7). Quntittive PCR nlysis indicted ~5% lower undnce of Irf4 mrna in T reg cells sorted from pooled spleens nd lymph nodes of tmoxifen-treted Irf4 Foxp3 Cre-ERT mice thn in those of Irf4 Foxp3 Cre-ERT mice (Fig. 6). We hypothesized tht the suoptiml ~5% reduction in mrna trnscripts nd the only slight reduction in IRF4 protein expression in the spleens nd lymph nodes of Irf4 Foxp3 Cre-ERT mice compred with tht of Irf4 Foxp3 Cre-ERT mice might hve resulted from competitive disdvntge of T reg cells tht lck IRF4 protein. Such disdvntge might led to preferentil popultion expnsion of the IRF4-sufficient T reg cells remining in the Irf4 Foxp3 Cre-ERT mice. This would e consistent with the pulished oservtion tht the survivl nd expnsion of strongly ntigen-stimulted CD8 + T cells is gretly impired in the sence of IRF4 (ref. 48). To determine whether IRF4 might similrly contriute to the mintennce of T reg cells tht hve een strongly ctivted, we ssessed the colonic lmin propri, in which nerly ll T reg cells were CD44 hi nd hd proly recently experienced enggement of the TCR, given their roust IL-1 production (dt not shown). Indeed, we oserved lower frequency of colonic lmin propri T reg cells in Irf4 Foxp3 Cre-ERT mice thn in Irf4 Foxp3 Cre-ERT mice (Fig. 6), similr to lower frequency of T reg cells in the colonic lmin propri of Trc Foxp3 Cre-ERT mice thn in Trc Foxp3 Cre-ERT mice noted ove. In contrst to spleen nd lymph node T reg cells, nd consistent with their lower frequency, colonic lmin propri T reg cells in Irf4 Foxp3 Cre-ERT mice hd much lower expression of IRF4 protein thn did those in Irf4 Foxp3 Cre-ERT mice (Fig. 6). We hypothesized tht the reportedly low influx of circulting T reg cells into the colonic lmin propri t stedy stte my hve precluded IRF4-sufficient cells from ecoming ctivted nd repopulting to wild-type frequency the T reg cell niche in this tissue 4. Despite the only slightly lower IRF4 expression in T reg cells isolted from the spleens nd lymph nodes of Irf4 Foxp3 Cre-ERT mice thn in their counterprts from Irf4 Foxp3 Cre-ERT mice, we were le to detect very mild, ut sttisticlly significnt, increse in the frequency of CD44 hi nd Ki67 + lymph node Foxp3 CD4 + T cells, s well s incresed production of IFN-γ, IL-4 nd IL-13 y splenic Foxp3 CD4 + T cells (Fig. 6c,d). This suggested tht IRF4 expression downstrem of TCR signling in T reg cells contriuted to the suppressive function of T reg cells. An increse in the production of type helper T cell cytokines ws consistent with the phenotype of mice with constitutive ltion of IRF4 in T reg cells, wheres the incresed IFN-γ ws proly consequence of the sustntil type 1 helper T cell is in C56B/L6 dult mice efore induced Irf4 deletion 37. As control, we confirmed tht in the sence of tmoxifen tretment, the expression of CD44, Ki67, IFN-γ, IL-4 nd IL-13 in CD4 + Foxp3 T cells from Irf4 Foxp3 Cre-ERT mice ws indistinguishle from tht in their counterprts from Irf4 Foxp3 Cre-ERT mice, s ws the frequency of T reg cells in the colonic lmin propri (dt not shown). This suggested tht the modest differences etween Irf4 Foxp3 Cre-ERT nd Irf4 Foxp3 Cre-ERT mice tht we oserved upon tmoxifen tretment were not consequence of the Irf4 FL llele itself. Together these dt indicted tht even prtil loss of IRF4 expression downstrem of TCR enggement in T reg cells interfered with optiml suppressive function of these cells. DISCUSSION Despite mjor progress in understnding the moleculr mechnisms of TCR enggement driven differentition of T reg cells, the role of nture immunology DVANCE ONLINE PUBLICATION

8 14 Nture Americ, Inc. All rights reserved. the TCR in T reg cell function in vivo hs remined uncler. Here we demonstrted tht TCR signling in differentited T reg cells ws dispensle for the mintennce of Foxp3 expression nd for the expression of mny chrcteristic mrkers of T reg cells. Although the ulk of the T reg cell specific gene signture ws lso preserved in the sence of the TCR, suppressor function ws criticlly dependent on the TCR. Given tht ntigen-ctivted CD4 + Foxp3 T cells in lymphoid orgns re thought to produce IL- in sptilly restricted mnner, we considered the possiility tht T reg cells might nlogously require their TCRs to correctly position themselves to gin preferentil ccess to IL-. This might elicit the suppressive function of T reg cells y stimulting IL-R. However, our in vivo nd in vitro dt suggested tht T reg cells cquired nd proly effectively depleted IL- in TCR-independent mnner. Thus, these cells my insted rely predominntly on expression of the chemokine receptor CCR7 to ensure sufficient IL- exposure, s hs een proposed 4. Our oservtion tht newly generted T reg cells in Trc Foxp3 YFP-Cre nd Trc Foxp3 YFP-Cre mice remined nive-like upon loss of the TCR nd did not populte nonlymphoid tissues suggested tht effector differentition nd popultion expnsion were processes dependent on the TCR nd proly dependent on ntigens. This finding my help explin the oservtion tht distinct TCR repertoires re displyed y T reg cell popultions in different lymphoid orgns nd tissues in dult mice 49,5. Furthermore, inducile ltion of the TCR resulted in fr greter chnge in gene expression in the effector-like T reg cell suset thn in the nive-like suset. This suggested tht continuous TCR signling might e selectively driving the homeostsis nd suppressor function of effector-like T reg cells. As inducile deletion of the TCR in differentited T reg cells precipitted utoimmunity, our dt my suggest tht ll or most of the suppressor function of T reg cells in vivo is medited y the CD44 hi effector-like T reg cell suset. We found tht prtil inducile ltion of Irf4, expressed downstrem of TCR enggement in CD44 hi T reg cells, resulted in very mild ut reproducile ctivtion of the immune system. This result suggested tht IRF4 expression ws importnt for TCR-dependent T reg cell function; however, given the suoptiml deletion of Irf4 nd very modest ctivtion of the immune system, it remins to e determined to wht extent nd in wht wy TCR-dependent induction of IRF4 in T reg cells contriutes to their ility to suppress spontneous utoimmunity. IRF4 my ct minly to control the mintennce of highly ctivted T reg cells, which ws prticulrly evident in the colonic lmin propri nd which, when ltered, my ffect the ility of the T reg cell pool to suppress. Alterntively, IRF4 my hve more direct role in promoting the suppressive ctivity of T reg cells, perhps y driving the expression of certin T reg cell effector molecules. Although we oserved decresed expression of the inducile costimultor ICOS on colonic T reg cells tht lcked IRF4 (dt not shown), the remining IRF4-sufficient T reg cells in lymphoid orgns of Irf4 Foxp3 Cre-ERT mice preclude more rigorous identifiction of IRF4 trgets. However, we note tht even though nive-like T reg cells did not express IRF4 nd were overwhelmingly unffected y TCR deletion on trnscriptionl level, this did not necessrily indicte tht these cells were nonfunctionl or were not experiencing TCR enggement. Indeed, severl genes, including Egr1, Egr nd Nr41, were downregulted in this T reg cell suset in the sence of the TCR. Additionl experiments re needed to elucidte the contriutions of other individul TCR-dependent genes in T reg cells to the mintennce of immunotolernce in the stedy stte nd to the restrint of immune responses directed ginst commensl microorgnisms, food nd environmentl ntigens nd pthogens. In connection with this, we note tht lthough IL-1 production y T reg cells hs een linked to the control of inflmmtory responses t environmentl interfces such s the gut, lungs nd skin, it hs lso een shown to e dispensle for T reg cell control over systemic utoimmunity 6. Likewise, we found tht wheres constitutive deletion of clcineurin B1 in Cn1 Foxp3 YFP-Cre mice (which eliminted clcineurin-dependent ctivtion of NFAT in T reg cells) resulted in lethl erly-onset utoimmunity, highly efficient inducile deletion in dult lymphoreplete Cn1 Foxp3 Cre-ERT mice hd no detectle dverse effects on T reg cell function (dt not shown). Together these findings suggest tht enggement of the TCR on T reg cells my drive focused trnscriptionl progrm, select spects of which re required in context-dependent mnner for medition of rod rnge of T reg cell immunosuppressive functions. Methods Methods nd ny ssocited references re ville in the online version of the pper. Accession codes. GEO: microrry dt, GSE6177. Note: Any Supplementry Informtion nd Source Dt files re ville in the online version of the pper. Acknowledgments We thnk M. Schmidt-Supprin (Technicl University Munich) nd K. Rjewsky (Mx Delrück Center) for Trc FL mice. Supported y the US Ntionl Institutes of Helth (R37AI346 to A.Y.R.), the Ludwig Cncer Center t Memoril Slon- Kettering Cncer Center (A.Y.R.) nd the Howrd Hughes Medicl Institute (A.Y.R.). AUTHOR CONTRIBUTIONS A.G.L. nd A.Y.R. designed the experiments; A.G.L. conducted experiments nd wrote the mnuscript; A.Y.R. supervised the reserch nd edited the mnuscript; nd W.J. prepred smples for microrry nlysis, nd A.A. nd A.G.L. conducted these nlyses. COMPETING FINANCIAL INTERESTS The uthors declre no competing finncil interests. Reprints nd permissions informtion is ville online t reprints/index.html. 1. Josefowicz, S.Z., Lu, L.F. & Rudensky, A.Y. Regultory T cells: mechnisms of differentition nd function. Annu. Rev. Immunol. 3, (1).. Lee, H.M., Butist, J.L., Scott-Browne, J., Mohn, J.F. & Hsieh, C.S. A rod rnge of self-rectivity drives thymic regultory T cell selection to limit responses to self. Immunity 37, (1). 3. Hsieh, C.S., Zheng, Y., Ling, Y., Fontenot, J.D. & Rudensky, A.Y. An intersection etween the self-rective regultory nd nonregultory T cell receptor repertoires. Nt. Immunol. 7, (6). 4. Hsieh, C.S. et l. Recognition of the peripherl self y nturlly rising CD5+ CD4+ T cell receptors. Immunity 1, (4). 5. Gvin, M.A., Clrke, S.R., Negrou, E., Gllegos, A. & Rudensky, A. Homeostsis nd nergy of CD4 + CD5 + suppressor T cells in vivo. Nt. Immunol. 3, (). 6. Ouyng, W. et l. Novel Foxo1-dependent trnscriptionl progrms control T(reg) cell function. Nture 491, (1). 7. Mrson, A. et l. Foxp3 occupncy nd regultion of key trget genes during T-cell stimultion. Nture 445, (7). 8. Au-Yeung, B.B. et l. A geneticlly selective inhiitor demonstrtes function for the kinse Zp7 in regultory T cells independent of its ctlytic ctivity. Nt. Immunol. 11, (1). 9. Gvin, M.A. et l. Foxp3-dependent progrmme of regultory T-cell differentition. Nture 445, (7). 1. Lin, W. et l. Regultory T cell development in the sence of functionl Foxp3. Nt. Immunol. 8, (7). 11. Willims, L.M. & Rudensky, A.Y. Mintennce of the Foxp3-dependent developmentl progrm in mture regultory T cells requires continued expression of Foxp3. Nt. Immunol. 8, (7). 1. Wing, K. et l. CTLA-4 control over Foxp3 + regultory T cell function. Science 3, (8). DVANCE ONLINE PUBLICATION nture immunology