LETTER. Control of T H 17 cells occurs in the small intestine

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1 doi:1.138/nture18 of T H 17 cells occurs in the smll intestine Enric Esplugues 1,,3 *, Smuel Huer 1, *, Nicol Gglini, Anj E. Huser, Terrence Town,7, Yisong Y. Wn 8, Willim O Connor Jr 1, Anthony Rongvux 1, Nico Vn Rooijen 9, Ann M. Hermn 1, Yoichiro Iwkur 11, Vijy K. Kuchroo 1, Jy K. Kolls 13, Jeffrey A. Bluestone 1, Kevn C. Herold 1 & Richrd A. Flvell 1,1 Interleukin (IL)-17-producing T helper cells (T H 17) re recently identified 1 T cell suset distinct from T helper type 1 (T H 1) nd T helper type (T H ) cells 1.T H 17 cells cn drive ntigenspecific utoimmune diseses nd re considered the min popultion of pthogenic T cells driving experimentl utoimmune encephlomyelitis (EAE), the mouse model for multiple sclerosis. The fctors tht re needed for the genertion of T H 17 cells hve een well chrcterized 3. However, where nd how the immune system controls T H 17 cells in vivo remins uncler. Here, y using model of tolernce induced y CD3-specific ntiody, model of sepsis nd influenz A virl infection (H1N1), we show tht proinflmmtory T H 17 cells cn e redirected to nd controlled in the smll intestine. T H 17-specific secretion induced expression of the chemokine CCL in the smll intestine, fcilitting the migrtion of these cells specificlly to the smll intestine vi the CCR/CCL xis. Moreover, we found tht T H 17 cells re controlled y two different mechnisms in the smll intestine: first, they re eliminted vi the intestinl lumen; second, proinflmmtory T H 17 cells simultneously cquire regultory phenotype with in vitro nd in vivo immune-suppressive properties (rt H 17). These results identify mechnisms limiting T H 17 cell pthogenicity nd implicte the gstrointestinl trct s site for control of T H 17 cells. T H 17 cells hve een ssocited with the pthogenesis of severl chronic inflmmtory disorders, including rheumtoid rthritis nd multiple sclerosis,7. To study the cellulr nd moleculr mechnisms tht control pthogenicity medited y T H 17 cells we first used the CD3-specific ntiody tretment model. It is known tht CD3-specific ntiody tretment induces cytokine storm nd locl inflmmtion minly in the smll intestine 8. Despite this it hs een vlidted s n in vivo model of toleriztion 9 nd is now under study in humn clinicl trils 1. By mimicking ntigen, CD3-specific ntiody tretment leds to ctivtion-induced cell deth (AICD) of T cells 11,1 nd consequently systemic upregultion of IL- (ref. 9) nd trnsforming growth fctor- (TGF-1) induced y phgocyte engulfment of poptotic T cells 13. In line with these pulictions, we found tht CD3-specific ntiody tretment induced n immunoregultory environment mrked y simultneous expression of TGF-1 nd IL- (Fig. 1). The comintion of these cytokines is importnt for the development of T H 17 cells in vitro nd in vivo s it hs een previously clerly estlished 3,. Accordingly, we found elevted levels of in plsm of CD3-specific ntiody-treted nimls compred to controls (Fig. 1). First, we imed to investigte the source of. It hs een reported tht few hours fter injection of CD3-specific ntiody, there is rpid disppernce of the mjority of T cells from the Concentrtion (ng ml 1 ) c 1 TGF-β1 IL Time (h) fter nti-cd3 Time (h) fter nti-cd3 Time (h) fter nti-cd DAPI egfp Merged Wild type egfp Wild type egfp 38. Concentrtion (ng ml 1 ) Concentrtion (ng ml 1 ) Duodenum Jejunum Ileum Colon Figure 1 Accumultion of T H 17 cells in the smll intestine fter CD3- specific ntiody tretment. Mice were injected with CD3-specific ntiody., Plsm levels of TGF-1, IL- nd. Men s.e.m.; n., Flow cytometric nlysis of egfp expression (gted on 1 TCR 1 events); numers in qudrnts indicte percent cells in ech. c, Immunofluorescence stining of frozen sections of the smll intestine fter CD3-specific ntiody tretment (egfp, green;, red; cell nuclei, DAPI). Scle r, mm. Dt re representtive of t lest three independent experiments. 1 Deprtment of Immunoiology, Yle University School of Medicine, New Hven, Connecticut, USA. Germn Rheumtism Reserch Center (DRFZ), A Leiniz Institute, Berlin 1117, Germny. 3 Cluster of Excellence NeuroCure, Chrite-Universitätsmedizin Berlin, Berlin 1117, Germny. I. Medizinische Klinik, Universitätsklinikum Hmurg-Eppendorf, Hmurg, Germny. Sn Rffele Dietes Reserch Institute (HSR-DRI), Miln 13, Itly. Deprtments of Biomedicl Sciences nd Neurosurgery, Cedrs-Sini Medicl Center, Los Angeles, Cliforni 98, USA. 7 Deprtment of Medicine, Dvid Geffen School of Medicine, University of Cliforni, Los Angeles, Cliforni 98, USA. 8 Lineerger Comprehensive Cncer Center, Deprtment of Microiology nd Immunology, The University of North Crolin, School of Medicine, Chpel Hill, North Crolin 799, USA. 9 Deprtment of Moleculr Cell Biology, Fculty of Medicine, Vrije Universiteit, Amsterdm, 181 BT, The Netherlnds. 1 Deprtment of Lortory Medicine, Yle University School of Medicine, New Hven, Connecticut, USA. 11 Center for Experimentl Medicine, Institute of Medicl Science, University of Tokyo, Tokyo , Jpn. 1 Center for Neurologic Diseses, Brighm nd Women s Hospitl, Hrvrd Medicl School, Boston, Msschusetts 11, USA. 13 Deprtment of Genetics, LSU Helth Sciences Center, New Orlens, Louisin 711, USA. 1 Dietes Center t the University of Cliforni Sn Frncisco, Sn Frncisco, Cliforni 913, USA. 1 Howrd Hughes Medicl Institute, New Hven, *These uthors contriuted eqully to this work. 1 NATURE VOL 7 8 JULY Mcmilln Pulishers Limited. 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2 RESEARCH circultion 13,1. Surprisingly, in prllel with the disppernce of T cells from the periphery, we found concomitnt increse in the percentge nd the numer of totl T cells in the smll intestine, in prticulr in the duodenum (Supplementry Fig. 1 c). In newly generted egfp knock-in mouse (enhnced green fluorescent protein ws inserted in the Il17 locus; Methods nd Supplementry Fig. d nd 3 c) injected with CD3-specific ntiody, 8% of the 1 TCR 1 T cells locted in the duodenum were expressing (Fig. 1 nd Supplementry Fig. 1d, e). The percentge nd numer of T H 17 cells in the intestine decresed from the duodenum to the colon in grdient-like fshion (Fig. 1). Detection of 1 egfp 1 T cells y immunofluorescence nd two-photon-lserscnning microscopy confirmed the high frequency of T H 17 cells in the smll intestine in situ (Fig. 1c nd Supplementry Fig. c). Importntly, we lso found T H 17 cell infiltrtion in the duodenum when nimls were injected with therpeutic non-fcr-inding CD3-specific ntiody 1, lthough the frequency nd numers of the T H 17 cells were lower compred to the FcR-inding ntiody (Supplementry Fig. ). Similr results were oserved fter ntigenspecific stimultion when solule myelin oligodendrocyte glycoprotein ntigen (MOG) ws dministered to MOG-TCR trnsgenic mice (D mice) 1 (Supplementry Fig. ). Tken together these dt suggest tht the genertion nd the ccumultion of T H 17 cells in the smll intestine ws not restricted to the CD3-specific ntiody tretment, ut ws generl mechnism following strong T-cell receptor (TCR) stimultion. We next wnted to identify the moleculr signls importnt for the genertion of T H 17 cells in vivo fter CD3-specific ntiody tretment. Becuse IL- is known to e importnt for T H 17 cell genertion, we evluted the importnce of this cytokine. Il / nd wild-type mice were treted with CD3-specific ntiody. In the Il / mice, only very smll popultion of T H 17 cells (out %) could e found y flow cytometry in the smll intestine (Supplementry Fig. ) nd ws undetectle in the plsm (dt not shown). To study the cellulr source of IL-, we treted mice with clodronte-loded liposomes, which elimintes most mcrophges nd significnt proportion of dendritic cells compred to PBS-loded liposomes 13 (Supplementry Fig. c). IL- plsm levels were gretly reduced in mice treted with clodronte-loded liposomes compred to control mice fter CD3- specific ntiody injection (Supplementry Fig. d) nd profound reduction in T H 17 cells ws oserved (Supplementry Fig., c). Tken together, these dt support the notion tht IL- secreted y ntigen-presenting cells (APCs) is criticl for the genertion of T H 17 cells during CD3-specific ntiody tretment. We next nlysed the mechnism leding to the specific ccumultion of T H 17 cells in the smll intestine, predominntly in the duodenum. T H 17 cells re known to express the chemokine receptor CCR (ref. 17). Wheres CCR is relevnt in different utoimmune disese models 7,18, the role of the CCR/CCL xis in immune cell migrtion to the intestine during tolernce induction hs not yet een evluted. To study tht, we nlysed the expression of CCR on 1 egfp positive nd negtive cells (Fig. ) nd Ccl mrna expression (Fig. ) in the spleen nd the gut. CCR ws minly expressed in T H 17 cells from the spleen nd the gut h fter CD3-specific ntiody injection (Fig. ). Strikingly, when we performed time course to mesure the mrna levels of Ccl in different prts of the intestine during CD3-specific ntiody tretment, we oserved tht Ccl ws expressed t the highest level in the duodenum in stedy stte conditions nd ws selectively further upregulted fter CD3-specific ntiody tretment (Fig., c nd Supplementry Fig. 7). To test the importnce of the CCR/CCL xis for the migrtion of T H 17 cells from the periphery to the duodenum, we treted Ccr / nd control mice with CD3-specific ntiody. T H 17 cell numer (Fig. e) nd frequency (Fig. d) were strongly reduced in the intestine of the Ccr / compred to wild-type mice. In generl, we did not oserve signs of intestinl inflmmtion in the Ccr / mice s we did Ccl mrna c Ccl mrna Smll intestine GFP GFP + GFP GFP + S.I CCR CCR e Liver Duodenum Jejunum Ileum Colon Brin Kidney Lung Time (h) fter nti-cd3 g 1. Ccl mrna in wild-type controls fter CD3-specific ntiody tretment (dt not shown). Interestingly, we detected higher numer of T H 17 cells in the spleen nd lymph nodes of Ccr / mice when compred to control nimls (Fig. e). This increse ws ccompnied y splenomegly nd enlrgement of lymph nodes (dt not shown), indicting tht CCR does not hve mjor role in the genertion nd expnsion of T H 17 cells. In conclusion, CCR seems to e essentil for the migrtion of T H 17 cells to the smll intestine fter CD3-specific ntiody tretment, nd the intestinl inflmmtion is dependent on this migrtion. Thus our dt indicte tht T H 17 cell migrte to the smll intestine leding to intestinl inflmmtion nd dmge. However, we cnnot exclude tht prolifertion of gut resident T H 17 cells lso contriutes to the oserved phenomenon. To evlute the contriution of nd IL-17F (T H 17 signture cytokines) in the induction of CCL expression in the duodenum, we treted Il17 / or Il17r / mice with CD3-specific ntiody. We found decresed levels of CCL in the Il17 / nd the Il17r / mice versus the controls fter CD3-specific ntiody tretment (Fig. f), indicting tht IL-17 signlling hs mjor role in the induction of CCL in the duodenum. We next studied the cellulr source of CCL. Ccl mrna ws only detectle in the intestinl epithelil cells in untreted mice. Tretment with CD3-specific ntiody led to f Ccl mrna d Numer of T H 17 cells ( 1 3 ) 1 1 Wild type Ccr / LN MLN Colon Ileum Jejunum Duodenum WT Il17A / Il17r / CD8 + + CD11 + EC CD8 CD11 8 WT Ccr / EC Figure The xis CCR/CCL is essentil for the recruitment of T H 17 cells to the smll intestine., CCR expression h fter nti-cd3 tretment., c, Ccl mrna expression (men s.e.m.; n ). S.I., smll intestine. d, IL- 17A expression (gted on 1 TCR1 events) s mesured y intrcellulr cytokine stining. e,t H 17 cell numers in different orgns (men s.d.; n ). LN, lymph node; MLN, mesenteric lymph node. f, Ccl mrna expression in duodenum of wild-type (WT), Il17 / nd Il17r / mice (men s.e.m.;n ). g, Ccl mrna levels of epithelil nd hemtopoietic cells isolted from the smll intestine. EC, epithelil cells. Pnels, d g show results 1 h fter the first nti-cd3 injection. Dt re representtive of t lest three independent experiments Mcmilln Pulishers Limited. All rights reserved 8 JULY 11 VOL 7 NATURE 1

3 RESEARCH LETTER further upregultion of Ccl mrna y the epithelil cells. Additionlly, the 1 T cells present in the smll intestine fter CD3-specific ntiody tretment, most of which were T H 17 cells, expressed high levels of Ccl mrna (Fig. g). In conclusion, T H 17 cells vi nd IL-17F production directly upregulte CCL production y the intestinl epithelil cells, which then leds to the susequent recruitment of CCR 1 T H 17 cells, which lso produce CCL. Of note, the intestinl inflmmtion fter CD3-specific ntiody tretment ws trnsient nd 1% of the mice recovered. To understnd etter the mechnisms underlying this process, we first ssessed poptosis of T H 17 cells in the smll intestine ut we did not detect significnt numer of poptotic cells (dt not shown). When we studied the in vivo prolifertion cpcity of 1 TCR 1 T cells from the CD3-specific ntiody-treted nimls, we found tht T H 17 cells from the duodenum were ctively proliferting (Supplementry Figs 8 nd 9, ). Using IL17A egfp 3 FoxP3 mrfp doule reporter mice (monomeric red fluorescent protein ws inserted in the foxp3 locus) we determined tht 1 1 T cells were proliferting t higher rte thn 1 T cells in the duodenum (Supplementry Figs 8 nd 9). Using two-photon lser-scnning microscopy, we found tht the T H 17 cells in the duodenum did not show the typicl ehviour of n poptotic T cell, conversely, they ehved like ctivted T cells in terms of their pttern of speed nd direction of migrtion (Supplementry Video). Tken together these dt indicte tht T H 17 cells do not die in the smll intestine, ut re rther ctively proliferting. In line with previous pulictions 8 we found tht CD3-specific ntiody tretment cused dirrhoe, oedem, inflmmtion nd tissue destruction in the smll intestine (Supplementry Fig. 1, ), which correlted with the recruitment of T H 17 cells. However, the intestinl pthology ws only trnsient nd mice fully recovered. We therefore egn to investigte the fte of T H 17 cells in the smll intestine. Interestingly, we found frction of T H 17 cells in the intestinl lumen egfp T H 17 (EAE) T H 17 (nti-cd3) Effector T reg T H 17 CFSE Il-17A egfp TNFα IL CTLA IL17F IL17A CCL IL1 Smll intestine Fold induction T H 17 (EAE) vs T H 17 (nti-cd3) IL1 IL17F CCL CCR1 CCR CCL AHR IL IL17A BATF3 IL1 IL3R IFNγ RORC RORA ILRA CCL9 CXCL9 CXCL1 TNF-α CCR CXCR3 IL Figure 3 Functionl nd moleculr chrcteriztion of rt H 17 cells., Suppression ssy ws performed using egfp mrfp 1 (Effector), egfp mrfp 1 1 (Tregs) or egfp 1 mrfp 1 (T H 17) cells sorted from spleen or smll intestine. (Br represents undivided CFSE-lelled 1 CD responder T cells). Dt re representtive of six independent experiments., Gene expression nlysis compring T H 17 cells (egfp 1 mrfp 1 ) from centrl nervous system t dy 17 fter EAE induction versus T H 17 cells isolted from the smll intestine of nti-cd3 treted egfp 3 Foxp3 mrfp doule reporter mice. of the CD3-specific ntiody-treted mice (Supplementry Fig. 1c, d). Given the severe inflmmtion, dirrhoe nd tissue dmge (Supplementry Fig. 1, ), it is most likely tht these cells were pssively wshed out, lthough n ctive mechnism cnnot e excluded. Considering tht the remining T H 17 cells in the duodenum were ctively proliferting, ut the intestinl pthology ws only trnsient, we were curious out the functionl cpilities of these cells. Surprisingly, we found tht the remining T H 17 cells in the duodenum were le to suppress prolifertion of responder T cells in vitro (Fig. 3). To study the moleculr properties of these suppressive T H 17 cells (which we refer from now on s rt H 17 cells), we performed genome-wide trnscriptionl profiling ssy (Fig. 3). We compred the gene expression pttern of rt H 17 cells from CD3-specific ntiody-treted mice nd genes expressed y pro-inflmmtory T H 17 cells tht were hrvested from the centrl nervous system of EAE-induced mice. The signture genes of T H 17 cells, like Rorc, Ror, Il17, Il or Il3r, were similrly expressed etween oth types of T H 17 cells. Also the ctivtion sttus of these cells seemed to e similr, ecuse ctivtion mrkers such s CD9, CD nd were eqully expressed. However, we found tht the rt H 17 cells from the CD3-specific ntiody-treted mice showed non-inflmmtory gene expression profile compred to pro-inflmmtory T H 17 cells isolted from the centrl nervous system of EAE-induced mice. Notly, the expression levels of Tnf- nd Il-, two cytokines with cler pro-inflmmtory roles 19,, were gretly reduced in the rt H 17 cells from the smll intestine. In contrst, these cells expressed high levels of IL-1, cytokine with potent nti-inflmmtory ctivities 1 (Supplementry Fig. 11 nd Fig. 3). These dt re supported y previous report showing tht in-vitro-generted non-pthogenic T H 17 cells re le to express IL-1 (ref. ). To evlute the moleculr mechnisms involved in the suppressive function of the rt H 17 cells, different molecules were locked in n in vitro suppression ssy using monoclonl ntiodies (Supplementry Fig. 11). The suppressive cpcity of the rt H 17 cells ws prtilly dependent on IL-1, CTLA- nd TGF-. Blocking ll three pthwys resulted in lck of suppression y the rt H 17 cells. T H 17 cells isolted from the spleen showed n intermedite phenotype. They exhiited limited cpcity to suppress the prolifertion of T cells in vitro (Fig. 3), nd lso produced more TNF- nd IL-, ut less IL-1 compred to T H 17 cells isolted from the smll intestine (Supplementry Fig. 11). However, ecuse some of the T H 17 cells in the smll intestine downregulted CCR (Fig. ), it is possile tht some rt H 17 might hve migrted ck from the smll intestine to the spleen. If development of the suppressive cpility occurred in the smll intestine, then preventing the migrtion of the T H 17 cells to tht site should prevent the development of these tolerogenic cells. To test this hypothesis, we nlysed T H 17 cells isolted from the spleen of Ccr / mice, ecuse we showed lredy tht these T H 17 cells re unle to migrte to the smll intestine. Consistent with the hypothesis, Ccr / T H 17 cells in the spleen showed high TNF- production, filed to suppress T-cell prolifertion in vitro, nd were even proinflmmtory, cusing inflmmtory owel disese in vivo upon trnsfer into lymphopenic host (Supplementry Fig. 1 d). These dt indicte tht proinflmmtory T H 17 cells do indeed cquire their suppressive phenotype in the smll intestine. To confirm our findings in n niml disese model, EAE-induced mice were treted with CD3-specific ntiody. In line with previous puliction 3 we oserved protective effect when the tretment ws dministered during the course of the disese (Supplementry Fig. 13). More importntly, we demonstrted tht T H 17 cells were recruited to the duodenum of the CD3-specific ntiody treted nimls nd these mice hd strongly reduced numers of T H 17 cells in the centrl nervous system (dt not shown). Using MOG-specific tetrmer, we determined tht significnt percentge of T H 17 cells in the duodenum were ntigen-specific (Supplementry Fig. 13), demonstrting tht MOG-specific T H 17 cells were recruited to the duodenum following CD3-specific ntiody tretment. In contrst the frequency 1 NATURE VOL 7 8 JULY Mcmilln Pulishers Limited. All rights reserved

4 RESEARCH of MOG-tetrmer-positive T H 17 cells ws much lower in other orgns of EAE-induced mice, which hd not een treted with CD3-specific ntiody (dt not shown). This is evidence ginst generl increse in MOG-specific T H 17. Therefore our results show tht ntigenspecific T H 17 cells, with proinflmmtory properties, generted in the periphery cn e redirected to the smll intestine. To confirm tht rt H 17 cells isolted from the smll intestine of CD3-specific ntiodytreted mice re indeed in vivo immune-suppressive we tested their suppressive cpcity in n EAE trnsfer model. We co-trnsferred MOG-specific in-vitro-differentited T H 17 either lone or together with MOG-specific rt H 17 cells isolted from the smll intestine of CD3-specific ntiody-treted D trnsgenic mice. Strikingly, we found tht rt H 17 cells were le to completely suppress the development of EAE in these trnsfer experiments (Supplementry Fig. 13c, d), indicting tht the rt H 17 cells re indeed stle in terms of their immune suppressive function. As mentioned ove CD3-specific ntiody tretment is lredy used in clinicl trils 9,1, nd we therefore imed to confirm our results using teplizum (hokt3c1(al-al)), one CD3-specific ntiody used in these trils. To tht end we used humnized mouse system: we reconstituted Bl/c Rg- / cc / doule knockout mice with humn peripherl lood mononucler cells. Two weeks fter the trnsfer we treted these mice with either OKT-3, n FcR-inding S. ureus Totl Vβ8 Vβ8 + TSST-1 SEB Smll intestine CCR PE CCR PE d Cell numer CFSE Vβ8 Vβ Percentge of mx. c 37.3 Ccl mrna Smll intestine Vβ8 Vβ Foxp3 + T reg s rt H 17. SEB Figure T H 17 cells re recruited to the smll intestine during sepsis.,, egfp expression is shown (gted on 1 TCR 1 events). Mice were injected with Stphylococcus ureus () or SEB nd TSST-1 (). c, CCR expression h fter the first SEB injection (top). Ccl mrna levels in the smll intestine 1 h fter the first injection (men s.e.m.; n ) (ottom). d, In vitro suppression ssy using 1 egfp 1 cells from the smll intestine or 1 Foxp3 mrfp 1 cells from the spleen of SEB-treted mice s suppressor cells. Results re representtive of t lest two independent experiments CD3-specific ntiody used in the first humn studies, or teplizum, n FcR non-inding CD3-specific ntiody. Strikingly, we found humn T cells in the smll intestine fter tretment with oth of these CD3-specific ntiodies (Supplementry Fig. 1, ). The presence of humn -, IL-1- nd CCL-producing cells in the smll intestine in OKT-3- nd teplizum-treted mice ws confirmed y reltime PCR (Supplementry Fig. 1c). Tken together our results otined in the CD3-specific ntiody model suggest tht T H 17 cells, y upregulting CCL expression in the duodenum vi IL-17 signlling, hve developed n elegnt mechnism to limit the pthogenicity in order to void life-thretening immune response. This predicted in turn tht this mechnism should e generl to most strong immune responses tht result in T H 17 cells. T H 17 cells hve crucil role in controlling different microorgnisms in vivo. We next investigted whether this mechnism of T H 17 cell control lso functions during strong immune response elicited y pthogenic microorgnisms. We first used murine model of sepsis. We injected Stphylococcus ureus, which is one of the most frequent orgnisms responsile for sepsis in humns,, intrvenously into egfp reporter mice. Mice were nlysed 3 dys fter the injection, t time when they displyed severe clinicl symptoms of sepsis (weight loss, dehydrtion, lethrgy). Strikingly, we found the highest frequency nd numer of T H 17 in the smll intestine (Fig. ). Interestingly, most T H 17 ppered to e TCR V8 1. The injection of the superntigen SEB (Stphylococcus ureus enterotoxin B), which is produced y the cteri used in these experiments nd inds to V8 1 T cells, ws sufficient to induce the ccumultion of T H 17 in the smll intestine just s in the nti-cd3 studies. As control we injected mice with TSST-1 (toxic shock syndrome toxin 1), superntigen tht does not ind to V8 nd is not produced y the cteri we used. Of note, we oserved tht the dministrtion of TSST-1 ws less effective t inducing the ccumultion of T H 17 cells in the smll intestine (Fig. ). Finlly, we could confirm tht the T H 17 cells, induced y SEB tretment, expressed CCR nd tht CCL is specificlly upregulted in the smll intestine following SEB tretment (Fig. c). Furthermore, while supopultion of the T H 17 cells ws found in the intestinl lumen (Supplementry Fig. 1), the remining T H 17 cells demonstrted n immune-suppressive phenotype (Fig. d nd Supplementry Fig. 1), gin comprle to our results otined in the CD3-specific ntiody tretment. Interestingly, it is known tht SEB cn induce tolernce 7, which is in line with our results tht SEB leds to the genertion of rt H 17 cells. Accordingly, we found tht SEB nd, to lesser extent, TSST-1 tretment of EAE-induced mice led to the meliortion of disese (dt not shown), which is in line with one previous puliction 8. In ddition to nti-cteril immunity, viruses re the next key clss of pthogens to which we must respond, yet contin excessive immunopthology which is commonly the cuse of moridity nd mortlity 9. To ddress such n immune response, we nlysed influenz, virl infection tht hs devstted humn popultions. Notly, we gin found incresed T H 17 cell frequencies in the smll intestine in mice infected with influenz A (H1N1) (Supplementry Fig. 17). In conclusion, we propose generl mechnism tht could explin how pro-inflmmtory T H 17 immune response, which is eneficil in clering infection, ut immunopthogenic in excess, cn e controlled y the mechnisms we descrie here: nmely y cquisition of n immune-suppressive phenotype or elimintion into the intestinl lumen (Supplementry Fig. 18). These findings nd further studies iming to identify the underlying mechnism of the conversion of pro-inflmmtory T H 17 cells into rt H 17 cells my help in designing new strtegies to control uto-rective T H 17 cells in utoimmune diseses like multiple sclerosis. METHODS SUMMARY, SEB, TSST-1 tretment nd Stphylococcus ureus infection. Mice were injected intrperitonelly three times with either CD3-specific ntiody 11 Mcmilln Pulishers Limited. All rights reserved 8 JULY 11 VOL 7 NATURE 17

5 RESEARCH LETTER (clone C11, mg per mouse) SEB ( mg per mouse) or TSST-1 ( mg per mouse) t, 8 nd 9 h. Mice were nlysed 1 h fter the first injection, if not otherwise specified. Stphylococcus ureus ws injected intrvenously ( colony-forming units per mouse) in order to induce sepsis. Mice were killed 3 dys fter the injection. Flow cytometric nlysis. Cells were isolted from the orgn s indicted. IL- 17A egfp nd CCR expression ws ssessed directly fter isoltion. When indicted cells were restimulted nd intrcellulr cytokine stining for ws performed. Numers in dot-plot qudrnts indicte percent cells in ech. Cells were gted on 1 TCR 1 events. Rel-time PCR. Ccl mrna expression ws mesured in different tissues s indicted using rel time PCR with reverse trnscription. In vitro suppression ssy. Different suppressor cells were co-cultured with croxyfluorescein dicette succinimidyl ester (CFSE)-lelled 1 CD responder T cells, which were isolted from the spleen of.1 congenic mice. Br represents undivided CFSE-lelled responder T cells. Full Methods nd ny ssocited references re ville in the online version of the pper t Received 1 My 1; ccepted 19 My 11. Pulished online 17 July Korn, T., Bettelli, E., Oukk, M. & Kuchroo, V. K. IL-17 nd Th17 cells. Annu. Rev. Immunol. 7, 8 17 (9).. Lngrish, C. L. et l. IL-3 drives pthogenic T cell popultion tht induces utoimmune inflmmtion. J. Exp. Med. 1, 33 (). 3. Bettelli, E. et l. Reciprocl developmentl pthwys for the genertion of pthogenic effector T H 17 nd regultory T cells. Nture 1, 3 38 ().. Mngn, P. R. et l. Trnsforming growth fctor- induces development of the T H 17 linege. Nture 1, 31 3 ().. Mnel, N., Unutmz, D. & Littmn, D. R. The differentition of humn T H -17 cells requires trnsforming growth fctor- nd induction of the nucler receptor RORct. Nture Immunol. 9, 1 9 (8).. Yng, L. et l. IL-1 nd TGF- re required for differentition of humn T H 17 cells. Nture, 3 3 (8). 7. Hirot, K. et l. Preferentil recruitment of CCR-expressing Th17 cells to inflmed joints vi CCL in rheumtoid rthritis nd its niml model. J. Exp. Med., (7). 8. Merger, M. et l. Defining the roles of perforin, Fs/FsL, nd tumour necrosis fctor in T cell induced mucosl dmge in the mouse intestine. Gut 1, 1 13 (). 9. Chtenoud, L. & Bluestone, J. A. CD3-specific ntiodies: portl to the tretment of utoimmunity. Nture Rev. Immunol. 7, 3 (7). 1. Herold, K. C. et l. Tretment of ptients with new onset type 1 dietes with single course of nti-cd3 ma teplizum preserves insulin production for up to yers. Clin. Immunol. 13, (9). 11. Crpenter, P. A. et l. Non-Fc receptor-inding humnized nti-cd3 ntiodies induce poptosis of ctivted humn T cells. J. Immunol. 1, 13 (). 1. Smith, C. A., Willims, G. T., Kingston, R., Jenkinson, E. J. & Owen, J. J. Antiodies to CD3/T-cell receptor complex induce deth y poptosis in immture T cells in thymic cultures. Nture 337, (1989). 13. Perruche, S. et l. CD3-specific ntiody-induced immune tolernce involves trnsforming growth fctor- from phgocytes digesting poptotic T cells. Nture Med. 1, 8 3 (8). 1. Chtenoud, L., Primo, J. & Bch, J. F. CD3 ntiody-induced dominnt self tolernce in overtly dietic NOD mice. J. Immunol. 18, 97 9 (1997). 1. Alegre, M. L. et l. An nti-murine CD3 monoclonl ntiody with low ffinity for Fc gmm receptors suppresses trnsplnttion responses while minimizing cute toxicity nd immunogenicity. J. Immunol. 1, 1 1 (199). 1. Bettelli, E. et l. Myelin oligodendrocyte glycoprotein-specific T cell receptor trnsgenic mice develop spontneous utoimmune optic neuritis. J. Exp. Med. 197, (3). 17. Annunzito, F. et l. PhenotypicndfunctionlfeturesofhumnTh17 cells. J. Exp. Med., (7). 18. Reoldi, A. et l. C-C chemokine receptor -regulted entry of T H -17 cells into the CNS through the choroid plexus is required for the initition of EAE. Nture Immunol. 1, 1 3 (9). 19. Brown, S. J. & Myer, L. The immune response in inflmmtory owel disese. Am. J. Gstroenterol. 1, 8 9 (7).. Petitto, J.M., Streit, W.J.,Hung,Z., Butfiloski, E.& Schiffenuer,J.Interleukin-gene deletion produces roust reduction in susceptiilitytoexperimentl utoimmune encephlomyelitis in C7BL/ mice. Neurosci. Lett. 8, 7 (). 1. Pestk, S. et l. Interleukin-1 nd relted cytokines nd receptors. Annu. Rev. Immunol., ().. McGechy, M. J. et l. TGF- nd IL- drive the production of IL-17 nd IL-1 y T cells nd restrin T H -17 cell-medited pthology. Nture Immunol. 8, (7). 3. Kohm, A. P. et l. Tretment with nonmitogenic nti-cd3 monoclonl ntiody induces 1 T cell unresponsiveness nd functionl reversl of estlished experimentl utoimmune encephlomyelitis. J. Immunol. 17, 3 ().. Khder, S. A., Gffen, S. L. & Kolls, J. K. Th17 cells t the crossrods of innte nd dptive immunity ginst infectiousdiseses t the mucos. Mucosl Immunol., 3 11 (9).. Rodriguez-Creixems, M. et l. Bloodstrem infections: evolution nd trends in the microiology worklod, incidence, nd etiology, 198. Medicine (Bltimore) 87, 3 9 (8).. Mrtin, G. S., Mnnino, D. M., Eton, S. & Moss, M. The epidemiology of sepsis in the United Sttes from 1979 through. N. Engl. J. Med. 38, 1 1 (3). 7. Ochi, A., Yuh, K. & Migit, K. Not every superntigen induces tolernce in vivo. Semin. Immunol., 7 3 (1993). 8. Soos, J. M., Schiffenuer, J. & Johnson, H. M. Tretment of PL/J mice with the superntigen, stphylococcl enterotoxin B, prevents development of experimentl llergic encephlomyelitis. J. Neuroimmunol. 3, 39 3 (1993). 9. Chowell, G. et l. Severe respirtory disese concurrent with the circultion of H1N1 influenz. N. Engl. J. Med. 31, 7 79 (9). Supplementry Informtion is linked to the online version of the pper t Acknowledgements The uthorswould like tothnkf. Mnzo for expert dministrtive ssistnce, L. Evngelisti nd C. Hughes for generting emryonic stem cells nd chimeric mice, respectively, J. Stein for initil screening of knock-in mice, T. Ferrndino for ssistnce with the mouse colony, E. Eynon nd J. Aldermn for mnging the mouse progrm nd A. Lin for ssistnce with Gene Arry nlysis. We lso thnk T. Tylor nd G. Tokmoulin for expert help with the FACS sorting nd D. Gonzlez for help with the multiphoton microscopy. We would like to thnk J. P. Allison for providing the nti-ctla- ntiody, F. Wldron-Lynch nd J. S. Poerfor providing peripherl lood mononucler cells, nd the NIH Tetrmer core fcility for providing the tetrmers. W.O. ws supported y fellowship from the Ntionl Multiple Sclerosis Society. S.H. ws supported y the DFG (HU 171/1-1) nd y Jmes Hudson Brown Alexnder B. Coxe Fellowship. E.E. ws supported y the Spnish Ministry of Science postdoctorl fellowship nd y Jmes Hudson Brown Alexnder B. Coxe Fellowship. R.A.F. is ninvestigtor ofthe HowrdHughesMediclInstitute. The genertion ofmice for thiswork wssupported ythe TrnsgenicCore ofthe YleDERC DK73 nd some of the work supported y Pilot project from DK73. This work ws lso supported y the JDRF. Author Contriutions E.E., S.H. nd R.A.F. designed the study nd wrote the mnuscript; N.G. did the in vitro suppression ssys nd the flow nlysis for IL-1 expression; A.E.H. nd A.M.H did the two-photon lser-scnning microscopy experiments; T.T. did the immunohistochemistry nlysis; W.O. supported the work with key suggestions nd y editing the mnuscript; E.E. nd S.H. did ll other in vitro nd in vivo experimentl work; Y.Y.W. provided Foxp3 mrfp mice; A.R. did the virl infection experiments; N.V.R. provided clodronte-loded liposomes, V.K.K provided D mice nd feedck on the mnuscript; Y.I. provided Il17 / mice nd feedck on the mnuscript; J.K.K. provided the Il17r / mice nd feedck on the mnuscript nd J.A.B. provided CD3-specific ntiodies nd feedck on the mnuscript; K.C.H. provided teplizum nd key suggestions. E.E. nd R.A.F. co-directed the project. Author Informtion Reprints nd permissions informtion is ville t The uthors declre no competing finncil interests. Reders re welcome to comment on the online version of this rticle t Correspondence nd requests for mterils should e ddressed to E.E. (enric.esplugues@yle.edu) or R.A.F. (richrd.flvell@yle.edu). 18 NATURE VOL 7 8 JULY Mcmilln Pulishers Limited. All rights reserved

6 RESEARCH METHODS Mice. BALB/c mice (lstocyst donors), CD1 mice (foster mothers), Tet-Cre trnsgenic mice ( deletor mice, C7BL/ ckground), C7BL/ mice (B), C7BL/.Ly.1 mice (.1 1 ), IL / mice nd CCR / mice were purchsed from The Jckson Lortories. MOG-trnsgenic mice (D mice, C7BL/ ckground) 3 nd Foxp3 reporter mice (FIR mice, C7BL/ ckground) 31 were intercrossed with the egfp reporter mice. We lso used Il17 /, Il17r / nd IL-1 egfp mice (Tiger mice) 3 3. All mice were kept under specific pthogenfree conditions in the niml cre fcility t Yle University. The mice were studied t 1 week of ge. All the experiments were pproved y the Institutionl Animl Cre nd Use Committee of Yle University. Genertion of IRES egfp reporter mice. A BAC clone consisting of Il17 genomic DNA derived from C7BL/ mice ws purchsed from BcPc (Oklnd, CA). An 8-k BmHI MluI frgment comprising exons 1, nd 3 for the Il17 gene ws cloned into pesy-flox vector djcent to the thymidine kinse selection mrker. The internl riosome entry site (IRES) egfp cssette ws linked to LoxP-flnked neomycin (Neo) selection mrker to otin the IRES egfp Neo cssette. The trgeting construct ws generted y cloning the IRES egfp Neo cssette into ScII site etween the trnsltion stop codon (UGA) nd the polydenyltion signl (AUA3) of the Il17 gene. The trgeting construct ws linerized y ClI clevge nd susequently electroported into Bruce C7BL/ emryonic stem (ES) cells. Trnsfected ES cells were selected in the presence of 3 mgml 1 G18 nd 1 mm gnciclovir. Drug-resistnt ES cell clones were screened for homologous recomintion y PCR. To otin chimeric mice, correctly trgeted ES clones were injected into BALB/c lstocysts, which were then implnted into CD1 pseudopregnnt foster mothers. Mle chimers were red with C7BL/ to screen for germ-line trnsmitted offspring. Germline trnsmitted mice were red with germline Cre trnsgenic mice (Tet-Cre mice) to remove the neomycin gene. Mice ering the trgeted Il17 llele were screened y PCR (Il17 knock-in (KI) sense: 9-CACCAGCGCTGTGTCAAT-39, Il17 KI nti-sense: 9-ACAAACACGAAGCAGTTTGG-39 nd Il17 IRES: 9-ACCGGCCTTATTCCAAGC-39). Antiodies, tetrmers nd intrcellulr cytokine stining. Anti- (L3T), nti-cdl (MEL-1), nti- (IM7), nti-.1 (A) nd nti-. (1), nti-tcr, nti-il-, nti-, nti-tnf, nti-ki-7 nd nti-brdu (9-romo--deoxyuridine) were purchsed from Becton Dickinson Phrmingen. For intrcellulr cytokine stining, the cells were restimulted with phorol 1-myristte 13-cette (PMA) (Sigm, ng ml 1 ) nd ionomycin (Sigm,. mgml 1 ) for h. Golgistop (BD Bioscience) ws dded during the lst 3 h of restimultion. After restimultion, the cells were wshed nd Ficoll grdient ws performed. The cells were fixed with 1% prformldehyde (electron microscopy grde) for 1 min on ice. After two wshes, cells were incuted with FITCconjugted nti-gfp ntiody (Rocklnd) nd phycoerythrin (PE)-conjugted nti- (BD Bioscience) in wsh/perm solution (BD Bioscience) for 3 min on ice. Cells were wshed twice nd resuspended in PBS. Acquisitions were mde with LSRII cytometer (BD Bioscience). For ex vivo-stining with MOG 38-9 /I-A()-tetrmer-llophycocynin (APC)- lelled (mouse myelin oligodendrocyte glycoprotein 38-9, GWYRSPFSRWH, NIH Tetrmer Fcility), single-cell suspensions were incuted t density of 1 7 cells ml 1 with neurminidse (.7 muml 1, neurminidse type X from Clostridium perfringens, Sigm) in serum-free DMEM t 37 uc/1% CO for min efore incution with the I-A() multimers (3 mgml 1 ) in DMEM supplemented with % FCS (ph 8.) t room temperture for h. After wshing, cells were stined for 7-AAD (Moleculr Proes), (RM-) nd TCR. hclip/i- A()-tetrmer-APC-lelled ws used s control ( PVSKMRMATPLLMQA, NIH Tetrmer Fcility). The percentge of tetrmer cells ws determined in the /TCR gte of live (7-AAD ) cells. Stined cells were nlysed on LSRII cytometer (BD Bioscience) nd dt were nlysed with FlowJo softwre (Treestr). Flow cytometry nd FACS sorting. Collected lymphocytes were treted with mmonium chloride lysis uffer (BioSource Interntionl) to remove red lood cells nd wshed with RPMI contining 1% FBS (Gemini Biologicl Products). Cells were then stined with 1: dilution of the indicted ntiodies together with 1 mgml 1 nti-fc-receptor locking ntiody (.G, Americn Type Culture Collection) in PBS contining % FBS nd then wshed twice with PBS. For isolting T cells, 1 T cells were first enriched y mgnetic-ctivted cell sorting eds (Miltenyi Biotec) nd then stined with the indicted ntiodies. The Becton Dickinson FACSVntge system nd MoFlo sorter (DAKO Cytomtion) were used for fluorescence detection nd cell sorting. T H 17 differentition in vitro. Splenocytes from IRES egfp mice nd C7BL/ mice were incuted with -microeds nd then positively selected through LS columns (Miltenyi Biotec). After enrichment, nive cells ( 1 CD CDL hi low ) were sorted y FACS s mentioned ove. 1 nive T cells were grown for dys t 1 cells ml 1 with plte-ound nti-cd3 ( mg ml 1 ) nd solule nti-cd8 ( mgml 1 ) in medium (Bruff s medium supplemented with 1% FCS, L-glutmine, penicillin nd streptomycin) under T H 17 conditions (TGF-, IL-, IL-3, nti-ifn-c, nti-il). (egfp) expression ws determined y flow cytometry. Multiphoton imging. The smll intestine (duodenum) from n egfp 3 Foxp3 mrfp doule reporter mouse treted with CD3-specific ntiody ws mounted on glss slide in chmer consisting of silicone isoltor ( mm dimeter 3. mm, Electron Microscopy Sciences). The tissue ws immersed in PBS nd covered y glss coverslip. An Olympus BXWI microscope equipped with 3x, numericl perture.9 Olympus ojective nd LVision TriMScope Multiphoton System controlled y Imspector Softwre (LVision Biotec) ws used to collect imges. For excittion, Coherent Chmeleon Ti:Spphire lser ws tuned to 9 nm. Imges of mm size were recorded t resolution of 1, 3 1, pixels with 1-mm z-spcing. Emitted light ws collected with nondescnned detectors fter hving pssed 3/9, / nd 1/1 nm ndpss filters. Volocity softwre (Improvision) ws used to crete three-dimensionl imge stcks, nd QuickTime Pro ws used to generte imge sequences. In vivo T-cell stimultion nd intestinl lymphocyte isoltion. Different mice in C7BL/ ckground were injected with nti-cd3 ( mg, 1 C11) 3,3 intrperitonelly 1 3 times t n intervl of dys etween injections nd killed h fter the finl injection. For the controls, isotype control or PBS ws injected. The intrepithelil lymphocytes (IEL) nd lmin propri lymphocytes (LPL) were collected s descried with some modifictions 3. In rief, smll or lrge intestines were removed nd Peyer s ptches were dissected. The first cm of the smll intestine were considered s duodenum. Intestines were opened longitudinlly nd then were cut into strips 1 cm in length. Tissues were wshed with Hnk s uffered sline nd incuted in the presence of mm of EDTA t 37 uc for 3 min. The relesed cells were loded onto Percoll grdient nd centrifuged. The cells etween % nd 1% Percoll were collected nd used s intestinl epithelil lymphocytes. LPL were collected y digesting gut tissue, which ws removed for IEL isoltion s descried ove. The tissue ws digested with collgense IV (1 U, Sigm) t 37 uc for 1 h nd loded onto Percoll grdient nd centrifuged. The cells etween % nd 1% Percoll were collected nd used s LPL. For the lumen content isoltion nd nlysis, mice were nesthetized using isoflurne. One ligtion ws mde fter the pylorus nd second one out cm distl from the first ligtion. A smll incision without reching the vessel proximl to the second ligtion ws mde nd 1 ml of pre-wrmed PBS ( ml min 1 ) ws injected using syringe nd 7G1/ needle. The fluids were collected in Petri dish plced under the incision proximl to the second ligtion. The collected fluids were incuted for 1 min in HBSS/ EDTA nd then filtered through 7 mm cell striner efore FACS nlysis. Adoptive trnsfer of 1 T cells. 1 FoxP3 1 nd the 1 FoxP3 T cells from the thymus or from the periphery of IL17A egfp 3 Foxp3 mrfp doule reporter mice were collected nd purified y mgnetic-ctivted cell sorting (MACS; Miltenyi Biotec). After MACS enrichment, totl 1 Foxp3 1 nd 1 FoxP3 T cells were FACS-sorted nd 3 1 T cells were doptively trnsferred (intrvenously) into su-lethlly irrdited, sex-mtched wild-type.1 1 recipient mice. Four weeks fter trnsfer, nimls were injected with CD3-specific ntiody ( mg) nd the smll intestines were recovered nd exmined for egfp nd mrfp y FACS. Immunofluorescence microscopy. Smll intestines were removed from egfp reporter mice nd wild-type littermtes fter CD3-specific ntiody tretment in vivo. Smll intestines were fixed in % prformldehyde for 1 h. After two wshes with PBS % sucrose solution ws dded. The % sucrose solution ws replced 1 h lter with 3% sucrose solution. On the next dy, the smples were wshed nd then snp-frozen in OCT nd stored t 8 uc. Cryosections were cut t 1 mm on Leic model CM18 freezing microtome, pplied to Superfrost Plus Gold slides (Fisher Scientific), ir-dried, nd PAP pen pplied (Zymed Lortories). Sections were locked for 3 min t mient temperture with serum-free protein lock (Dko Cytomtion) nd were stined with PE nti- (BD) nd Alex88 nti-gfp (Invitrogen) overnight t uc. Smples were wshed three times y immersing in PBS for min nd then mounted with Prolong gold mounting medi with DAPI (Invitrogen). Sections were oserved under drk field in independent fluorescence chnnels using n utomted Olympus BX-1 microscope. Experimentl utoimmune encephlomyelitis. Mice were immunized sucutneously with mg of MOG 3 (Yle Keck fcility) emulsified in CFA (BD Difco). Mice received ng pertussis toxin (PTx, List Biologicl Lortories) intrperitonelly t the time of immuniztion nd 8 h lter. Mice were checked for clinicl symptoms dily, nd signs were trnslted into clinicl score s follows:, no detectle signs of EAE;., til wekness; 1, complete til prlysis;, prtil hind lim prlysis;., unilterl complete hind lim prlysis; 3, complete 11 Mcmilln Pulishers Limited. All rights reserved

7 RESEARCH LETTER ilterl hind lim prlysis; 3., complete hind lim prlysis nd prtil forelim prlysis;, totl prlysis of forelims nd hind lims (mice with score ove 3. to e killed);, deth. All niml experiments were conducted ccording to the IACUC policies. Cytokine ssys. Cytokines were quntified in plsm y ELISA (TGF-1, Promeg) or y Cytometric Bed Arry (IL- nd, BD Bioscience) following the mnufcturer s instructions. The plsm ws otined y centrifugtion of lood collected on EDTA-coted tues fter crdic puncture. Gene expression nlysis. Totl RNA extrcted (1 ng; RNesy, Qigen) from intestinl rt H 17 cells (from CD3-specific ntiody-treted nimls) or from proinflmmtory T H 17 cells (from EAE-induced mice) were used to perform genomewide trnscriptionl profiling ssy (GeneChip Mouse 1. ST Arry, Affymetrix). Dt ws nlysed with GeneSpring GX 1 (Agilent Technologies). Reltive gene expression nlysis. RNA from cells/tissues ws isolted with the RNesy/QIAshredder purifiction system (Qigen) in ccordnce with the mnufcturer s protocol. RNA ws sujected to reverse trnscriptse with Superscript II (Invitrogen) with oligo(dt) primer in ccordnce with the mnufcturer s protocol. cdna ws semi-quntified using commercilly ville primer/proe sets (Applied Biosystems) nd nlysed with the DDC t (chnge in cycle threshold) method. All results were normlized to Hprt quntified in prllel mplifiction rections during ech PCR quntifiction. Suppression ssys. CFSE ( mm)-lelled 1 CD T cells (responder cells) were cultured in 9-well round ottom pltes t 3 1 cells per well with 1 irrdited APCs (spleenocytes MACS-depleted for 1 nd CD8 1 T cells) s feeder cells in the presence of 3 1 cells per well of FACS-sorted 1 IL- 17A 1 Foxp3 or 1 IL 17A Foxp3 1 T cells. Cell cultures were stimulted with mgml 1 of nti-cd3 ntiody (C11) in the presence or not of nti-tgf- (1D11), nti-ctla- (9H1) nd nti-il1r. After dys, cells were collected, stined nd the CFSE signl ws nlysed y flow cytometry. Sepsis induced y infection nd superntigen tretment: Stphylococcus ureus (ATCC 18, SEB 1 TSST-1 ) ws injected intrvenously into egfp reporter mice (1 8 colony-forming units per mouse). Mice were killed 3 dys fter the injection, t time when they displyed severe clinicl symptoms of sepsis (weight loss, dehydrtion, lethrgy) nd the presence of 1 IL17A 1 T cells ws tested in different orgns (spleen, lymph node, smll intestine) using FACS nlysis. Similr experiments were done injecting the superntigens SEB nd TSST-1. All of them were purchsed from Toxin Technology. All superntigens were dministered three times (, 8, 9 h) intrperitonelly t mg per mouse. For the influenz A infection, mice were infected with plque-forming units of influenz A/PR8 (H1N1) virus vi the intrnsl route. Infection ws performed y the intrnsl ppliction of ml virus stock diluted in PBS (or n equl volume of PBS s control) to mice tht hd een deeply nesthetized with nfne (Ivesco). Lungs nd smll intestines were hrvested 3 nd dys fter infection for flow cytometry nlysis. Peripherl lood mononucler cells isoltion nd dministrtion. Humn leukocytes were collected y leukpheresis of dult volunteer donors under protocol pproved y the Yle Humn Investigtions Committee. The peripherl lood mononucler cells were isolted using Lymphocyte Seprtion Medium (Cppel) ccording to the mnufcturer s instructions. The cells were stored in 1% DMSO/9% FBS t 19 uc nd were thwed nd wshed efore use. Rg / 3 cc / doule knockout mice were reconstituted with humn peripherl lood mononucler cells y intrperitonelly inocultion weeks efore nti-cd3 specific ntiody tretment. The numer of humn T cells ( 1 1 ) in the smll intestine ws evluted y flow cytometry. Animls demonstrted no signs of grft-vs-host disese. Rre nimls tht filed to reconstitute with humn T cells were, y prior design, excluded from nlysis. Endoscopic procedure. Colonoscopy ws performed in linded fshion for colitis scoring using the Coloview system (Krl Storz, Germny). Briefly: colitis scoring ws sed on grnulrity of mucosl surfce, stool consistence, vsculr pttern, trnslucency of the colon nd firin visile ( 3 points for ech). Sttisticl nlysis. Where indicted, the Student t test for non-pired dt nd the Mnn Whitney U test were used to clculte sttisticl significnce for differences in prticulr mesurement etween different groups. A P-vlue of less thn. ws considered significnt. 3. Bettelli, E. et l. Myelin oligodendrocyte glycoprotein-specific T cell receptor trnsgenic mice develop spontneous utoimmune optic neuritis. J. Exp. Med. 197, (3). 31. Wn, Y. Y. & Flvell, R. A. Identifying Foxp3-expressing suppressor T cells with icistronic reporter. Proc. Ntl Acd. Sci. USA 1, (). 3. Nke, S. et l. Antigen-specific T cell sensitiztion is impired in IL-17-deficient mice, cusing suppression of llergic cellulr nd humorl responses. Immunity 17, (). 33. Ye, P. et l. Requirement of interleukin 17 receptor signling for lung CXC chemokine nd grnulocyte colony-stimulting fctor expression, neutrophil recruitment, nd host defense. J. Exp. Med. 19, 19 8 (1). 3. Kmnk, M. et l. Expression of interleukin-1 in intestinl lymphocytes detected y n interleukin-1 reporter knockin tiger mouse. Immunity, 91 9 (). 3. Alegre, M. L. et l. An nti-murine CD3 monoclonl ntiody with low ffinity for Fc gmm receptors suppresses trnsplnttion responses while minimizing cute toxicity nd immunogenicity. J. Immunol. 1, 1 1 (199). 11 Mcmilln Pulishers Limited. All rights reserved