Trnscriptionl reprogrmming of mture + helper T cells genertes distinct MHC clss II restricted cytotoxic T lymphocytes npg 213 Nture Americ, Inc. All rights reserved. Dniel Mucid 1,8,9, Mohmmd Mushtq Husin 1,9, Swko Muroi 2,9, Femke vn Wijk 1,8,9, Ryo Shinnksu 1,8, Yoshinori Noe 2,8, Bernrdo Sgri Reis 3, Yujun Hung 1, Florence Lmolez 1, Michel Docherty 1,8, Antoine Attinger 1,8, Jr-Wen Shui 1, Gisen Kim 1, Christopher J Len 1, Shiny Skguchi 4, Chizuko Miymoto 2, Peng Wng 5,8, Koji Atrshi 6,8, Yunji Prk 1,8, Toshinori Nkym 7, Keny Hond 6,8, Wilfried Ellmeier 4, Mitchell Kronenerg 1, Ichiro Tniuchi 2 & Hilde Cheroutre 1 TCR thymocytes differentite into either CD8 + cytotoxic T lymphocytes or + helper T cells. This functionl dichotomy is controlled y key trnscription fctors, including the helper T cell mster regultor ThPOK, which suppresses the cytolytic progrm in mjor histocomptiility complex (MHC) clss II restricted + thymocytes. ThPOK continues to repress genes of the CD8 linege in mture + T cells, even s they differentite into effector helper T cell susets. Here we found tht the helper T cell fte ws not fixed nd tht mture, ntigen-stimulted + T cells terminted expression of the gene encoding ThPOK nd rectivted genes of the CD8 linege. This unexpected plsticity resulted in the post-thymic termintion of the helper T cell progrm nd the functionl differentition of distinct MHC clss II restricted + cytotoxic T lymphocytes. + T cells re commonly clssified s helper T cells on the sis of their roles in providing help to promote or dmpen cellulr nd humorl immune responses. In contrst, β + cytotoxic T lymphocytes (CTLs) provide direct protective immunity y killing infected or trnsformed cells. The helper T cell progrm is initilly induced during thymic development, during which thymocytes expressing mjor histocomptiility complex (MHC) clss II rective T cell ntigen receptor (TCR) develop into the + helper T cell linege, wheres thymocytes with specificity for MHC clss I differentite into the CD8 + CTL linege. The functionl progrmming, which coincides with ut does not depend on the MHC restriction or expression of the coreceptor or β, is controlled y the ction nd counterction of key trnscription fctors. Together with Tox nd GATA-3, the helper T cell trnscription fctor ThPOK (ckrox; encoded y Zt7 (clled Thpok here)) first induces the + helper T cell fte nd prevents thymocytes from differentiting into CD8 + CTLs 1 6. Runx3, memer of the Runx fmily of trnscription fctors, hs the opposite effect nd termintes expression while promoting differentition into the CTL linege 7,8. The CD8- linege dichotomy persists in the periphery for mture T cells, in which ThPOK continues to suppress the cytotoxic fte of MHC clss II restricted + T cells even s they differentite into effector helper T cell susets controlled y dditionl trnscription fctors 6. Tht linege seprtion, however, is not ll encompssing, nd reports hve repetedly indicted the presence of + T cells with cytolytic functions in vrious species, including humns nd rodents 9 12. At stedy stte, popultions of the effector cells tht reside in the intestine s intrepithelil lymphocytes (IELs) show enrichment for cytotoxic T cells, including + T cells 13 15, wheres under inflmmtory conditions, including virl infections, utoimmune disorders nd in response to tumor ntigens, mny cytolytic + T cell popultions expnd in the lood nd peripherl tissues 9 12,16,17. Their widespred undnce nd prticiption in vrious eneficil s well s pthogenic dptive immune responses 9 12,16,17 underscore the physiologicl relevnce of cytolytic + effector cells. However, the genertion of convincing evidence directly linking them to specific 1 Division of Developmentl Immunology, L Joll Institute for Allergy & Immunology, L Joll, Cliforni, USA. 2 Lortory for Trnscriptionl Regultion, RIKEN Reserch Center for Allergy nd Immunology, Yokohm, Kngw, Jpn. 3 Lortory of Mucosl Immunology, The Rockefeller University, New York, New York, USA. 4 Institute of Immunology, Center for Physiology, Pthophysiology nd Immunology, Medicl University of Vienn, Vienn, Austri. 5 Division of Vccine Discovery, L Joll Institute for Allergy & Immunology, L Joll, Cliforni, USA. 6 Deprtment of Immunology, Grdute School of Medicine, University of Tokyo, Tokyo, Jpn. 7 Deprtment of Immunology, Grdute School of Medicine, Chi University, Chi, Jpn. 8 Present ddresses: Lortory of Mucosl Immunology, The Rockefeller University, New York, New York, USA (D.M.), Deprtment of Peditric Immunology, University Medicl Center Utrecht, Wilhelmin Children s Hospitl, Utrecht, The Netherlnds (F.v.W.), RIKEN Reserch Center for Allergy nd Immunology, Yokohm, Kngw, Jpn (R.S., K.A. nd K.H.), Section of Immunology, Deprtment of Mechnism of Aging, Ntionl Center for Geritrics nd Gerontology, Jpn (Y.N.), Deprtment of Gstroenterology, University of Cliforni Sn Diego Medicl Center, Sn Diego, Cliforni, USA (M.D.), Merck-Serono, Versoix, Switzerlnd (A.A.), The Center for Nnomedicine, Shnghi Advnced Reserch Institute, Chinese Acdemy of Science, Shnghi, Chin (P.W.) nd Division of Integrtive Bioscience nd Biotechnology, Pohng University of Science nd Technology, Pohng, Kore (Y.P.). 9 These uthors contriuted eqully to this work. Correspondence should e ddressed to H.C. (hilde@lii.org) or I.T. (tniuchi@rci.riken.jp). Received 19 Septemer 212; ccepted 14 Decemer 212; pulished online 2 Jnury 213; doi:1.138/ni.2523 nture immunology VOLUME 14 NUMBER 3 MARCH 213 281
npg 213 Nture Americ, Inc. All rights reserved. spects of the dptive immune response hs een difficult, s they hve een viewed merely s functionl vrints of the well-defined T helper type 1 (T H 1) sutype 12,18. The lck of defined gene signture hs lso gretly impired progress in elucidting the iology of cytolytic + T cells nd in designing clinicl strtegies tht specificlly trget these cells in vrious diseses. Here we provide proof indicting tht cytotoxic + T cells represent distinct suset of effector cells tht cn e defined y the sence of the mster regultor ThPOK, which mintins the helper T cell fte in ll susets of clssicl + helper T cells y continuously suppressing the CD8 + CTL linege progrm 6. Nevertheless, we lso found tht the ThPOK + CTLs originted from ThPOK-expressing progenitor cells tht initilly committed to the ThPOK-controlled helper T cell linege during thymic selection. Our findings therefore chllenge the view tht the helper T cell progrm in + T cells is fixed nd show tht mture + T cells cn lose ThPOK expression post-thymiclly nd cn e functionlly reprogrmmed to ecome MHC clss II restricted CTLs. We lso identify the Thpok silencer s the trnscriptionl switch tht terminted Thpok trnscription nd y defult drove the derepression of the CTL progrm in mture + effector cells. At stedy stte, + CTLs remined immunologiclly quiescent even in the continuous presence of their cognte ntigens. However, in response to restimultion in the context of interleukin 15 (IL-15), + CTLs gretly incresed their inflmmtory nd cytolytic functions nd differentited into potent killer effector cells. Overll, our dt demonstrte tht + CTLs re not simple vrint of clssicl ThPOK-controlled T H 1 cells ut insted re distinct functionl MHC clss II restricted effector cells tht cn e chrcterized y the loss of ThPOK expression nd the derepression of spects of the gene-expression progrm of the CD8 + CTL linege. Spleen f (reltive MFI) 12 1 8 6 4 2 + mln mln 99.5 99.8 + spleen + IEL + + IEL α + IEL + β + + + α + g CD17 29.1 + siel 47.5.3.8 + IEL 7.6 36.2 27.1 c + siel 59.7.3 21.4 CD8β + β + IEL 51.4 RESULTS Not ll mture + T cells express ThPOK The reported cytolytic ctivity of mture + T cells is inconsistent with the ide tht ThPOK continuously suppresses the CTL progrm in ll mture, MHC clss II restricted + T cells 6 nd suggests tht these cells might not e under the negtive control of ThPOK. To investigte this, we nlyzed ThPOK expression in mture T cells isolted from reporter mice with sequence encoding green fluorescent protein (GFP) knocked in to the ThPOK locus (Thpok-GFP mice) 19. As expected, + Thpok-GFP lymphocytes isolted from the spleen or mesenteric lymph nodes (mlns), which re mostly nive T cells, were GFP + (Fig. 1), which indicted tht they ll expressed ThPOK, s is typicl of mture cells of the + helper T cell linege. Conversely, ll cells in the CD8 + frction were GFP (Fig. 1), consistent with the sence of ThPOK expression in cells of the CTL linege. Unexpectedly, mny of the Thpok-GFP + effector T cells tht ccumulted in the intestine t stedy stte were GFP (Fig. 1,c), which indicted tht like their CD8 + counterprts, they did not express ThPOK. Notly, most of the GFP + cells were in the suset of doule-positive (DP) IELs tht coexpressed nd without CD8β 2 (Fig. 1 d). Consistent with the lck of ThPOK-medited suppression, these + + CD8β cells lso hd functionl fetures similr to those of mture CD8 + CTLs, including undnt expression of grnzyme (Fig. 1e,f) nd sustntil expression of the ctivtion-induced degrnultion mrker CD17 (LAMP-1), glycoprotein present in the memrne of cytotoxic grnules nd exposed on the surfce of ctivted cytolytic cells 21 (Fig. 1g,h). The induction of CD17 y the DP suset ws similr to tht of typicl TCRαβ + CD8 + CTLs, wheres ctivted + single-positive (SP) IELs or helper T cells from the spleen did not induce this cytolytic mrker 18.6 d 58.8 2.6 CD17 + cells (%) 38.5 CD8β h 8 + siel 6 4 2 + spleen. TCRγδ IEL e α IEL Spleen mln siel.6.4.1 2.3 55.7 β IEL + IEL + + IEL.1 i Cytotoxicity (%) 8 6 4 2 + spleen + IEL + + IEL 21.7 β + IEL Figure 1 Some mture + T cells do not mintin ThPOK expression in the periphery. () Frequency of GFP + cells (numers ove rcketed lines) mong gted 5 + TCRβ + lymphocytes isolted from the spleen nd mlns of nive Thpok-GFP reporter mice (n = 4 or 5). () Frequency of GFP + cells (numers ove rcketed lines) mong gted 5 + TCRβ + IELs isolted from the smll intestine (siel) of nive Thpok-GFP reporter mice (n = 4 or 5). (c) Cell surfce stining for nd GFP on gted 5 + TCRβ + CD8β + IELs s in. Numers in qudrnts indicte percent cells in ech throughout. (d) Cell surfce stining for nd CD8β on gted 5 + TCRβ + + IELs s in. (e) Cell surfce stining for nd intrcellulr for stining grnzyme B (Gzm) on or in gted 5 + TCRβ + CD8β + lymphocytes isolted from the spleen nd mlns nd IELs from the smll intestine of nive wild-type C57BL/6 mice (n = 4 or 5). (f) Men fluorescence intensity (MFI) of grnzyme B in +, + + or α + T cells mong gted 5 + TCRβ + CD8β lymphocytes from the spleen nd mlns nd IELs of nive wild type C57BL/6 mice (n = 4 or 5); results re presented reltive to those of the + + suset, set s 1%. P <.5 (nlysis of vrince (ANOVA) with Bonferroni s post-test). (g) Frequency of CD17 + cells (numer in outlined re t right) mong totl gted 5 + TCRβ + CD8β + nd 5 + TCRβ + β + IELs fter stimultion with ntiody to TCRβ. (h) Frequency of CD17 + cells mong + splenocytes or TCRγδ +, α +, β +, + nd + + IEL susets fter stimultion with ntiody to TCRβ (n = 4 replictes). P =.2 (nonprmetric two-tiled Mnn-Whitney test). (i) Cytotoxicity of lymphocyte susets, ssessed y lctte dehydrogense relese ssy. Dt re representtive of two (,), three (c f) or four (g i) independent experiments (error rs (f,h,i), s.e.m.). 282 VOLUME 14 NUMBER 3 MARCH 213 nture immunology
CD8 linege S TH P TH Endogenous Thpok locus E 4 P 4 ThPOK-Cre trnsgene S TH Cre IRES CD2 PA c pln 31.5 Ros26-YFP 32.3 + + + Ros26-YFP x ThPOK-Cre 38.1 36.6 ThPOK linege S TH P TH E 4 P 4 S TH Cre IRES CD2 PA 63.5 18.6 59.8 23.4 Inctive stte Active stte IEL 8.4 4.1 6 1.2 Thymus 1 2 3 4 5.4 18 44.1 5.8 49.5 YFP YFP hcd2 3.5 6 1 2 3 4 5 hcd2.2 6.1 33.8 44.7 48.8 Ros26-YFP x ThPOK-Cre Ros26-YFP npg 213 Nture Americ, Inc. All rights reserved. YFP (Fig. 1g,h). Moreover, ctivted DP cells lso effectively killed trget cells in vitro, s mesured y the relese of lctte dehydrogense fter lysis of trget cells (Fig. 1i nd Supplementry Fig. 1). In sum, these dt demonstrted tht in norml mice, not ll + effector cells expressed ThPOK nd, furthermore, tht those ThPOK + lymphocytes expressed (ut not CD8β) nd hd cytolytic ctivity tht closely resemled tht of mture CD8 + CTLs. Mture ThPOK + T cells derive from ThPOK + thymocytes ThPOK is the mster regultor of the helper T cell linege nd is first expressed in the thymus, where it countercts Runx3 nd suppresses the CTL fte of MHC clss II restricted thymocytes 4 6. The sence of ThPOK expression ssocited with cytotoxicity in mture + T cells could suggest tht they might hve originted from ThPOK progenitors. To investigte this, we designed fte-mpping mouse model in which we trcked previous ThPOK expression in mture T cell susets (Fig. 2). Inctivtion of Thpok trnscription in MHC clss I specific thymocytes of the CD8 + CTL linege is medited y repressive fctors, such s Runx proteins, tht ind to the Thpok silencer DNA element 22,23. In MHC clss II restricted cells, however, ThPOK itself inds to the Thpok silencer nd prevents Runx-medited silencing, which results in positive feedck loop with continuous expression of Thpok (Fig. 2). A fte mrker tht reports ny previous ctivity of the Thpok silencer in thymocytes is therefore n ccurte reporter for the linege origin nd initil commitment of mture + T cells. On the sis of tht rtionle, we designed mouse strin with trnsgenic expression of Cre recominse, ThPOK-Cre, in which Thpok silencer inserted into Cd4 enhncer-promoter locus controls expression of trnsgene encoding Cre (Fig. 2). YFP Figure 2 Mture ThPOK + T cells re the progeny of ThPOK-expressing thymocytes. () Endogenous Thpok (left) nd the trnsgene encoding ThPOK-Cre (right) in thymocytes of the CD8 nd lineges. S TH, Thpok silencer; P TH, Thpok promoter; E 4 P 4, Cd4 enhncer-promoter; IRES, internl riosoml entry site; CD2, humn gene encoding CD2; PA, poly(a) til. () Stining for nd (middle) nd for humn CD2 (hcd2) nd YFP (left nd right) in thymocytes gted on expression of nd (gtes 1 6 (middle), where 1 represents immture DP thymocytes nd 5 nd 6 represent mture + nd CD8 + SP thymocytes, respectively), from Ros26-YFP mice nd Ros26-YFP mice expressing ThPOK-Cre (with the trnsgene in ; Ros26-YFP ThPOK-Cre). Numers ove rcketed lines (left nd right) indicte percent cells positive for humn CD2 (ThPOK-Cre + ; top rows) or YFP + cells (ottom rows). (c) Stining for nd (contour plots) nd YFP (histogrms) in cells from the peripherl lymph nodes (pln) nd IELs from the smll intestine of mice s in, gted on TCRβ + + lymphocytes. Numers djcent to outlined res indicte percent cells in ech. Dt re representtive of t lest three independent experiments. In contrst to the widely used strin of mice tht express Cre under the control of the Cd4 enhncer-promoter strting t the + β + thymocyte stge, in ThPOK-Cre mice, the Thpok silencer prevents Cd4 enhncer-promoter ctivity in immture thymocytes nd cells of the CD8 linege, which results in Cre expression exclusively in mture + SP thymocytes committed to the helper T cell linege (Fig. 2). Consistent with tht, in progeny of cross of ThPOK-Cre mice with reporter mice expressing yellow fluorescent protein (YFP) from the uiquitous Ros26 promoter (Ros26-YFP), we found tht only cells derived from thymocytes tht were committed to the + helper T cell linege were mrked y the expression of the YFP reporter fter Cre-medited recomintion (Fig. 2,c). In those progeny of the Ros26-YFP ThPOK-Cre cross, CD8 + SP thymocytes nd mture peripherl CD8 + T cells, including CD8 + mucosl T cells, did not express YFP, wheres, s expected, most + SP lymph node T cells nd IELs were YFP +. Notly, -expressing + IELs were lso YFP +, to n extent similr to tht of conventionl + SP cells (Fig. 2,c), which indicted tht they hd induced ThPOKdependent Cre expression nd therefore tht the mture ThPOK + T cells must hve een derived from progenitor cells tht expressed ThPOK t n erlier stge. ThPOK + + helper T cells lose ThPOK expression The ide tht the mture ThPOK + cells previously expressed ThPOK, together with the oservtion tht those cells minly ccumulted mong effector cells, suggested tht the loss of ThPOK expression might hve een the result of post-thymic ctivtion or mturtion process tht mture T cells undergo in the periphery. To ssess this, we doptively trnsferred highly purified nture immunology VOLUME 14 NUMBER 3 MARCH 213 283
siel 31.1.3 2.8 liel 2.6 2.3 2.1 IL-17 Spleen mln 2.1 96 1.7 97 slpl llpl 9.5 89.4 4.1 95 siel liel 32.6 65 15.8 83 c Spleen + siel + liel 94 ± 2 mln + + siel + + liel 94 ± 1.8 64 ± 8 9.3 ±.2 86 ± 3 23 ± 2 d GFP + TCRβ + + cells (%) 1 8 6 4 2 Spleen mln + + + siel + + + liel npg 213 Nture Americ, Inc. All rights reserved. e 1.4 ±.3 mln IEL 25.6 ± 3.2 f Cd8 Cd8 IV III II I V Figure 3 ThPOK + effector cells lost ThPOK s mture cells in the periphery. () Stining of nd intrcellulr IL-17 in gted 5 + TCRβ + + IELs from the smll intestine (siel) nd lrge intestine (liel) of Rg1 / recipient mice 8 weeks fter doptive trnsfer of nive 5RB hi TCRβ + CD25 + spleen T cells. () Stining of nd in gted 5 + TCRβ + + T cells from the spleen nd mlns, lymphocytes from the lmin propri of the smll intestine (slpl) nd lrge intestine (llpl) nd IELs from the smll intestine nd lge intestine of Rg1 / recipient mice 8 weeks fter doptive trnsfer of nive 5RB hi TCRβ + CD25 spleen T cells. (c) Expression of Thpok-GFP in gted 5 + TCRβ + + (SP) or + + CD8β (DP) lymphocytes isolted from vrious tissues (s in,) of Rg1 / recipient mice 8 weeks fter trnsfer of sorted 5RB hi TCRβ + CD25 + spleen T cells from Thpok-GFP donor mice. Numers ove rcketed lines indicte percent GFP + cells (men ± s.e.m.). (d) Frequency of GFP + cells mong gted 5 + TCRβ + + (SP) or + + CD8β (DP) lymphocytes from spleen or mlns or IELs of Rg1 / recipients fter trnsfer of sorted nive 5RB hi TCRβ + GFP + CD25 + spleen T cells from Thpok-GFP donor mice. P <.1 (ANOVA nd Bonferroni s post-test). (e) Surfce stining for on retrnsferred sorted (>99.7% purity) 5 + TCRβ + + donor IELs isolted from mlns (pooled from three recipient mice) nd IELs of the smll intestine nother Rg1 / recipient 8 weeks fter the second trnsfer. Numers djcent to outlined res indicte percent + cells (men ± s.e.m.). (f) ChIP with tiling rry of + SP thymocytes from FH-ThPOK mice. I V, Cd8 enhncers; green rrowhed indictes inding of ThPOK to the E8 I region. (g) Frequency of + cells (numers djcent to outlined res) mong 5 + TCRβ + CD8β + IELs isolted from wild-type mouse () nd n E8 I -deficient mouse (E8 I -KO). (h) Frequency of + + IELs from Rg1 / recipient mice 8 weeks fter trnsfer of wild type or E8 I -deficient 5RB hi TCRβ + CD25 + spleen T cells. Ech symol represents n individul mouse; smll horizontl lines indicte the men. P <.1 (ANOVA nd Bonferroni s post-test). Dt re representtive of three independent experiments ( d; error rs (d), s.e.m.), two independent experiments (e,f,h) or three independent experiments with four or five mice per genotype (g). g 48.2 7.3 E8 I -KO h + + (%) 35 25 15 5 E8 I -KO siel E8 I -KO liel ThPOK-expressing GFP + nive Thpok-GFP + T cells into recipient mice deficient in recomintion-ctivting gene 1 (Rg1 / mice). The lymphopenic conditions in this model induced strong prolifertion nd differentition of the donor cells, which ccumulted minly s cells of the T H 17 suset of helper T cells (chrcterized y the expression of the trnscription fctor RORγt nd the cytokine IL-17) in the lrge intestine, nd notly, lso s DP effector cells, especilly in the smll intestine of the Rg1 / hosts (Fig. 3,). Donor cells in the spleen nd mlns were GFP + (Fig. 3c), which indicted tht they continued to express ThPOK. In contrst, mny of the Thpok- GFP + T cells tht ccumulted s effector cells in the intestine did not hve detectle expression of GFP (Fig. 3c), which indicted tht they hd sustntilly diminished or complete loss of ThPOK expression. Consistent with the oservtions otined with immunologiclly replete mice, the loss of GFP expression ws gin gretest in those + cells tht lso reinduced expression, lthough smll frction of + SP IELs were lso GFP (Fig. 3c,d). Seril trnsfer of those SP donor cells into second set of Rg1 / recipients generted mny more DP cells (Fig. 3e), which indicted tht the reexpression of on mture + T cells followed the loss of ThPOK expression nd further indicted tht the Cd8 locus in conventionl + helper T cells might e constitutively suppressed y ThPOK. To nlyze this, we used chromtin immunoprecipittion (ChIP) comined with tiling rrys of cells from geneticlly engineered mice tht express Flg-hemgglutinin epitope tgged ThPOK (FH-ThPOK) from the Thpok locus 19. FH-ThPOK ssocited with the E8 I enhncer element 24 in the Cd8 locus in SP + thymocytes (Fig. 3f). We lso confirmed tht result in mture + SP T cells y ChIP ssy (Supplementry Fig. 2), which suggested tht in mture + thymocytes nd lymphocytes, ThPOK prevented expression y direct suppression of the E8 I enhncer element in the Cd8 locus, nd lso tht the expression of without CD8β in mture ThPOK + T cells ws driven y derepression of the E8 I enhncer. In support of tht finding, oth the frequency of -expressing + IELs nd CD8 expression itself were much lower in E8 I -deficient mice thn in wild-type mice (Fig. 3g), finding lso true for the progeny of E8 I -deficient cells versus wild type donor cells in Rg1 / recipients of n equl numer of oth genotypes of donor cells (Fig. 3h nd Supplementry Fig. 2). Overll the dt indicted tht nive ThPOK + + helper T cell cells might hve lost expression of ThPOK in the periphery nd consequently regined expression of ThPOK-suppressed genes such s Cd8. 284 VOLUME 14 NUMBER 3 MARCH 213 nture immunology
IL-17 d + IEL.3 4 24.5 71.3 4 2 2 4 Expression (log 2 ) + + + + + ThPOK + ThPOK + + Il17e Il22 Ccr6 Il17re Cxcl3 Ccl2 Rorc e Thpok Ror t 8 6 4 2 2 15 1 5 Ccr6 Ccr7 Klf4 Il17 Il22 Il1 Cd81 Tnfsf8 Rorc Cd28 Ad Bcl6 Cd27 Mzr 2B4 Lt2 Swp7 Ccl3 Ifng Tx2l Eomes Cd13 Cd11c Prf1 Crtm + + ThPOK + ThPOK + + Il17 Il22 4 3 2 1 2 15 1 5 3 +3 Expression (fold) f Thpok Ror t 2 15 1 5 4 3 2 1 Il17 Il22 2, 1,5 1, 5 75 5 25 c + + ThPOK + ThPOK + + gcd8 Crtm 8 6 4 2 32 24 16 8 Ccr6 Ccr7 Klf4 Il17 Il22 Il1 Cd81 Tnfsf8 Rorc Cd28 Ad Bcl6 Cd27 Mzr 2B4 Lt2 Swp7 Ccl3 Ifng Tx2l Eomes Cd13 Cd11c Prf1 Crtm Ifn (fold 1 3 ) 2 15 1 5 8 6 4 2 3 +3 Expression (fold) h Cd8 (fold 1 3 ) Crtm 4 3 2 1 24 18 12 6 Ifn (fold 1 3 ) 24 18 12 6 2.4 1.8 1.2.6 npg 213 Nture Americ, Inc. All rights reserved. i ThPOK- GFP 2B4 + + ThPOK + ThPOK + + ThPOK loss coincides with CTL differentition Although susets of polrized + effector cells re controlled y unique trnscription fctors, such s T-et for T H 1, GATA-3 for T H 2 nd RORγt for T H 17 cells, ThPOK continues to function s mster regultor of the helper T cell linege nd continues to suppress the CTL progrm. Consistent with tht, nive Thpok-GFP donor spleen cells tht differentited into T H 17 effector cells in the intestine of the Rg1 / recipient mice remined GFP + (Fig. 4), which indicted tht their gene-expression profile ws still regulted y the ThPOK. To exmine the effect of the loss of ThPOK expression on the gene-expression pttern of + effector cells, we nlyzed gene microrrys generted from RNA isolted from sorted ThPOK + or ThPOK SP nd DP effector cells from norml unmnipulted Thpok-GFP reporter mice 19 (Fig. 4) nd from Rg1 / recipients of nive Thpok-GFP + SP donor cells (Fig. 4c nd Supplementry Fig. 3). Notly, we found tht DP nd SP ThPOK + T cells hd unique ut similr gene-expression pttern tht differed from tht of SP ThPOK + + cells, lthough in the trnsfer experiments, the donor cells originted from the sme pool of nive lymphocytes nd differentited in the sme host environment (Fig. 4,c nd Supplementry Fig. 3). As expected, mny of the ThPOK + + SP cells isolted from the intestine hd distinct T H 17 gene-expression pttern, wheres expression of those genes ws rely detectle in the ThPOK nd DP susets (Fig. 4 d). Furthermore, RT-PCR nlysis CD8 (fold 1 3 ) 3.5 2.5 1.5.5 (fold 1 4 ) 5 4 3 2 1 Crtm 45 3 15 Thpok 15 1 5 j GFP GFP + 36.8 of genes chrcteristic of T H 17 cells, including those encoding the cytokines IL-17A, IL-17F nd IL-22, the cytokine receptor IL-23R nd the T H 17 hllmrk nucler trnscription fctor RORγt, confirmed tht ThPOK + effector cells were distinct from T H 17 cells (Fig. 4e,f nd Supplementry Fig. 3). Moreover, nlysis of genes chrcteristic of other + helper T cell types, such s T H 1 nd T H 2 cells, demonstrted tht the gene-expression pttern of + SP lymphocytes nd DP lymphocytes tht hd lost ThPOK expression did not resemle the ptterns of the known helper T cell effector susets (Supplementry Fig. 3c). In contrst, in ddition to reexpressing, ThPOK + effector cells lso expressed mny other genes typiclly ssocited with the CD8 + CTL progrm. This included the expression of vrious genes encoding cytolytic proteins, such s severl grnzymes nd perforin, s well s interferon-γ (IFN-γ) nd severl receptors expressed y nturl killer cells nd mture CD8 + CTLs (Fig. 4,c,g,h nd Supplementry Fig. 3d,e). Of prticulr note ws their expression of the cytotoxicityrelted, MHC clss I restricted, T cell ssocited molecule CRTAM nd the CD2 fmily memer CD244 (2B4), oth known to promote the cytolytic function nd IFN-γ production of CD8 + T cells 25 27. The reciprocl expression in mture + effector cells of either ThPOK or CTL signture genes (Fig. 4i) confirmed the hypothesis tht ThPOK continuously suppressed the CTL progrm in conventionl helper T cell cells ut lso demonstrted tht the differentition 1 2B4 GFP + + GFP + GFP + + Figure 4 Activted + helper T cells tht lose ThPOK expression differentite into CTLs. () Intrcellulr stining for IL-17 nd GFP in gted 5 + TCRβ + + IELs from Rg1 / recipients of sorted nive 5RB hi TCRβ + GFP + CD25 + spleen T cells from Thpok-GFP donor mice, ssessed 8 weeks fter cell trnsfer. () Gene-expression microrry nlysis of mrna from sorted 5 + TCRβ + ThPOK + +, ThPOK + nd ThPOK + + IELs from nive Thpok-GFP reporter mice; results re reltive to the chnge in expression in the ThPOK + + suset. (c) Gene-expression microrry nlysis of mrna from sorted 5 + TCRβ + ThPOK + +, ThPOK + nd ThPOK + + IELs from Rg1 / recipients of sorted nive TCRβ + GFP + 5RB hi CD25 + spleen T cells from Thpok-GFP donor mice (presented s in ). (d) Het mp of normlized expression of T H 17 signture genes in donor + T cells s in c, determined y microrry nlysis s in. (e,f) Quntittive rel-time PCR nlysis of mrna from T H 17-ssocited genes in sorted T cell susets s in c (e) or d (f); results re presented reltive to those for Rpl32. (g,h) Quntittive rel-time PCR nlysis of mrna from CTL-ssocited genes in sorted T cell susets s in c (g) or d (h), presented s in e,f. (i) Expression of nd 2B4 in gted 5 + TCRβ + + IELs s in (left), nd quntittive rel-time PCR nlysis of mrna encoding, grnzyme B, CRTAM nd ThPOK in IELs sorted for (5 + TCRβ + + ) GFP +, GFP nd GFP + susets isolted from Rg1 / recipients s in (n = 4; right). (j) Stining for nd (left nd middle) nd 2B4 (right) in gted TCRβ + + IELs from Rg1 / recipients (n = 3) of untrnsfected (GFP ) or ThPOK-trnsfected (GFP + ) TCRβ + 5RB hi CD25 + donor spleen T cells. Dt re representtive of three (,e j) or two ( d) independent experiments (men nd s.e.m. in e i). nture immunology VOLUME 14 NUMBER 3 MARCH 213 285
npg 213 Nture Americ, Inc. All rights reserved. 69.2.7 18.3 11.8 4.2 44.1 2.1 B2m / 49.5 Thpok S /S B2m / 1.6 2.8 Figure 5 The Thpok silencer forms the switch tht termintes Thpok expression in mture + T cells. () Stining for nd on gted TCRβ + + IELs isolted from the smll intestine of nive wild type, B2m / nd Thpok S /S B2m / mice (left; n = 2 or 3 mice per genotype). Right, frequency of + + cells mong those TCRβ + + IELs (ech symol represents n individul mouse; smll horizontl lines indicte the men). P <.1 (two-tiled unpired t-test). () Stining s in of IELs isolted from the smll intestine of Rg1 / recipients 4 5 weeks fter doptive trnsfer of Thpok Sfl/Sfl TCRβ + + cells trnsfected with n empty retrovirl vector encoding GFP lone (EV) or retrovirl vector encoding Cre-GFP (Cre), with gting on GFP + or GFP cells (left). Right, frequency of + + cells mong those TCRβ + + IELs (presented s in ). (c) Stining for nd GFP on IELs isolted from the smll intestine of Rg1 / recipients fter trnsfer of sorted of + CTL effector cells coincided with the post-thymic loss of ThPOK. In greement with tht, fter enforced expression of ThPOK vi retrovirl trnsduction, + donor cells no longer differentited into CTLs in vivo (Fig. 4j), wheres trnsfection of construct encoding Thpok with spontneous muttion found in the helper-deficient mouse strin 4 did not prevent the differentition of helper T cells into + CTLs (Supplementry Fig. 3f). These dt indicted tht the loss of ThPOK coincided with derepression of the CTL phenotype in mture + effector cells. Thpok silencer derepression termintes Thpok expression In thymocytes committed to the CD8 + CTL linege, ThPOK expression is switched off t the trnscriptionl level y the Thpok silencer 22. Consequently, in mice with germline deletion of this silencer (Thpok S /S mice), MHC clss I restricted thymocytes mture s -expressing T cells in the periphery 22. To determine if the Thpok silencer engges in similr role to terminte Thpok expression in mture MHC clss II restricted precursors of + CTLs, we nlyzed the peripherl mturtion of + T cells in Thpok S /S mice. To eliminte the MHC clss I restricted -expressing cells in these mice, we crossed the Thpok S /S mice with MHC clss I deficient (β 2 -microgloulin-deficient (B2m / )) mice, which rogted the development of MHC clss I restricted + CD8 + T cells. Notly, we found considerle enhncement of ll + popultions, including DP IELs, in B2m / mice, in the sence of mture CD8 + T cells (Fig. 5); however, Thpok S /S B2m / mice hd considerly fewer mture MHC clss II restricted -expressing + T cells (Fig. 5). These oservtions indicted tht the Thpok silencer ws criticl genomic switch in the process of the differentition of + CTLs tht turned off Thpok trnscription in mture + T cells. We further confirmed tht ide with + T cells in which loxp-flnked Thpok silencer (Thpok Sfl/Sfl ) ws deleted t the mture stge y trnsfection of the cells with retrovirl construct contining sequence encoding Cre linked to GFP. The trnsfer of trnsfected Thpok Sfl/Sfl + T cells (which expressed Cre (GFP + ) nd consequently hd deletion of the Thpok silencer) into Rg1 / recipient mice resulted 2.7 93 + + cells (%) 5 4 3 2 1 B2m / Thpok S /S B2m / c 44.8 5.7 9.3 Thpok Sfl/Sfl GFP GFP + MAZR-KO 9.7 19 nive TCRβ + GFP + 5RB hi CD25 + spleen T cells from wild-type donor mice () or MAZR-deficient Thpok-GFP donor mice (MAZR-KO), gted s in. (d) Frequency of + GFP + IELs s in c (presented s in ). P =.3 (two-tiled unpired t-test). Dt re representtive of three (,c,d) or two () independent experiments. EV Cre 28.2 18.9 4.2 3.7 36.8 2. in impired ccumultion of -expressing + CTLs in the intestine of the recipients (Fig. 5), which further emphsized the criticl role of the Thpok silencer in terminting Thpok expression s prt of the CTL-differentition process of + effector cells. In contrst to ThPOK, which suppresses the repressive ctivity of the Thpok silencer, the zinc-finger trnscription fctor MAZR is known to ctivte the Thpok silencer 28, which results in MAZR-induced negtive regultion of Thpok trnscription. Consistent with the prticiption of MAZR in rectivtion of the Thpok silencer in mture T cells, MAZR-deficient + donor cells trnsferred into Rg1 / recipient mice formed fewer -expressing + CTLs thn did their wild-type counterprts in the intestine of the recipients (Fig. 5c,d). These results indicted tht trnscriptionl regultion of Thpok ws key for control of the helper T cell phenotype of mture + T cells nd tht rectivtion of its silencer led to termintion of the helper T cell progrm nd, conversely, to the functionl differentition of MHC clss II restricted + CTLs. + CTL differentition is driven in vivo y ntigen The loss of ThPOK oserved in progeny of nive ThPOK + donor cells suggested tht induction of the CTL progrm in mture + T cells coincided with n ctivtion or mturtion process. The reexpression of s well s the cytolytic functionl differentition of mture + T cells ws reminiscent of the cytotoxic-linege differentition of positively selected CD8 + SP thymocytes medited y IL-7 (ref. 29). Despite tht, however, we found significntly more -expressing + CTLs in mice deficient in the receptor for IL-7 thn in wildtype mice (Fig. 6), which indicted tht the in vivo differentition of mture + CTLs ws not n IL-7-driven process. Similr to other effector cells in the intestine, DP cells were not present in germ-free mice (Supplementry Fig. 4) nd seemed to e present in norml numers in germ-free mice reconstituted with specific pthogen free microorgnisms (Supplementry Fig. 4), which indicted tht some microil fctors directly or indirectly promoted the ccumultion of + CTLs in the intestine. In contrst, unlike clssic + T H 17 effector cells, they did not increse in numer in the intestine of 67.6 + + cells (%) 5 4 3 2 1 d + GFP cells (%) EV GFP GFP + 1 75 5 25 Cre GFP GFP + MAZR-KO 286 VOLUME 14 NUMBER 3 MARCH 213 nture immunology
c d e ThPOK + ThPOK + + + + cells (%) 9 75 6 45 3 15 Il-7r / + + + BrdU Ki67 CD69 IEL 37.8.4 2 microns 9 LPL.8.4 3 Foxp3 IEL 5.2 1.3 2.9 27.6 mlns.7.2 5.3 93.6 Thpok-GFP CD17 + cells (%) f CD17 + cells (%) 5 4 3 2 1 1 8 6 4 2 5 5 4 4 3 3 2 2 1 1 3 3 3 3 3 3 IFN-γ + cells (%) IFN-γ + cells (%) TNF + cells (%) + + + 1 1 8 8 6 6 4 4 2 2 3 3 3 3 3 3 TNF + cells (%) npg 213 Nture Americ, Inc. All rights reserved. Figure 6 The ThPOK loss nd reprogrmming of + CTLs is n ntigen-driven process in vivo. () Frequency of + + T cells mong gted 5 + TCRβ + + CD8β IELs from wild-type mice or mice deficient in the receptor for IL-7 (Il7r / ). Ech symol represents n individul mouse; smll horizontl lines indicte the men. P =.3 (nonprmetric two-tiled Mnn-Whitney test). () BrdU stining of IELs from the smll intestine of nive wild type mice fter intrperitonel injection of 1 mg BrdU, followed y 6 d of BrdU (.8 mg/ml) in the drinking wter (left), nd stining for Ki67 (middle) nd CD69 (right) of + or gted 5 + TCRβ + CD8β + IELs from nive wild-type mice. (c) Stining for nd Foxp3 in gted 5 + TCRβ + CD8β + IELs (top) nd lymphocytes from the lmi propri (ottom) of OT-II Rg1 / mice (n = 4) fter 4 weeks on n OVA-contining diet. (d) Frequency of + nd GFP + cells mong 5 + TCRβ + + OT-II Thpok-GFP IELs isolted from Rg1 / recipient mice fter 4 weeks on n OVA-contining diet. (e) Frequency of CD17 +, IFN-γ + nd TNF + cells mong OT-II Thpok-GFP ThPOK or ThPOK + 5 + TCRβ + + IELs s in d, nlyzed t dy without IL-15 exposure () or fter 3 d of in vitro culture with recominnt IL-15 (3) efore restimultion with OVA(323 339). (f) Frequency of CD17 +, IFN-γ + nd TNF + cells mong wild-type + nd 5 + TCRβ + CD8β + polyclonl IELs cultured without or with IL-15 s in e efore restimultion with ntiody to TCR-β. Dt re representtive of two ( c), five (d) or three (e,f) independent experiments. germ-free mice monocolonized with segmented filmentous cteri lone 3 (Supplementry Fig. 4) or in response to n infection with Citrocter rodentium, pthogen known to induce strong T H 17 response in the lrge intestine (Supplementry Fig. 4c), wheres they were present in norml numers in the intestine of mice deficient in the dptor MyD88. These oservtions indicted tht the microil conditions required for the stedy-stte ccumultion of + CTLs in the intestine were proly not estlished y single microorgnism nd did not depend on IL-1 signls or signls induced y Toll-like receptors (Supplementry Fig. 4d). Pulished reports hve indicted tht + CTLs re ntigen-experienced cells tht differentite in response to repeted ctivtion signls 1,12. In greement with tht, DP cells with CTL phenotype isolted from Rg1 / recipients of nive spleen + SP T cells lso showed evidence of strong or repeted ctivtion nd considerle uptke of the thymidine nlog BrdU ut only wek stining for the ctive cell-cycle mrker Ki67 (Fig. 6), which indicted tht they were resting cells tht hd previously een ctivted nd hd previously intensely proliferted. In ddition, + CTLs lso hd higher expression of the ctivtion mrker CD69 thn did + SP effector helper T cells (Fig. 6), which suggested tht the ctivtion process tht coincided with the loss of ThPOK expression nd derepression of the CTL progrm lso coincided with those + effector cells tht received strong or repeted ctivtion signls. To directly investigte tht, we nlyzed the effector differentition of monoclonl OT-II + T cells (which hve trnsgenic expression of TCR specific for ovlumin mino cids 323 339 (OVA(323 339)) in response to continuous ctivtion in vivo with their cognte peptide OVA(323 339) presented y I-A. T cells migrte s effector cells only to peripherl tissues, nd in greement with tht, in the sence of OVA ntigen, very few OT-II + T cells ccumulted in the intestine of OT-II mice. Similrly, fter trnsfer of nive OT-II + T cells into Rg1 / recipient mice, only limited numer of the donor cells migrted to the intestine of the recipient mice in the sence of OVA ntigen (dt not shown). In contrst, fter mice were fed n OVAcontining diet for t lest 4 weeks, lrge numer of ctivted OT-II + cells ccumulted in the intestine of the OT-II mice nd the Rg1 / recipients, nd, notly, mny reexpressed (Fig. 6c), wheres those tht remined SP hd tendency to express the trnscription fctor Foxp3 (Fig. 6c). To determine if the derepression of expression on the responder OT-II + T cells coincided with the loss of ThPOK nd their differentition into CTLs, we isolted cells from OT-II Thpok-GFP reporter mice, trnsferred those nive donor cells into OVA-fed Rg1 / recipient mice nd nlyzed the phenotype nd function of the OVA-responding cells tht ccumulted s effector cells in the intestine of the hosts. As expected, mny OT-II Thpok-GFP DP cells ccumulted in the smll intestine ut, notly, they were ll GFP (Fig. 6d), which indicted tht they hd lost ThPOK expression. Furthermore, in ddition to the reexpression of, OT-II GFP effector cells lso newly induced the expression of typicl cytolytic mrkers, such s 2B4 nd grnzyme B (Supplementry Fig. 5). Notly, they hd n ntigen-specific cytolytic response when restimulted in vitro with the OT-II TCR specific peptide OVA(323 339) (Fig. 6e) ut not when restimulted with the MHC clss I restricted OVA peptide SIINFEKL (mino cids 257 264; Supplementry Fig. 5). Despite their functionl potentil, OT-II ThPOK + CTLs remined immunologiclly quiescent in vivo even in the continuous presence of their cognte ntigen in the diet. Tht finding ws similr to results otined with MHC clss I restricted β + CTLs, which lso seem to e inctive under stedy-stte conditions 31. After chllenge with excess mounts of IL-15, however, s in ctive celic disese 18,32, CD8 + CTLs ecome pthogenic killer cells 33,34 tht lso considerly upregulte secretion of the inflmmtory cytokines IFN-γ nd tumor-necrosis fctor (TNF) 18,35,36,. To determine if + CTLs might e s responsive to IL-15 s CD8 + CTLs, we restimulted the diet-induced OT-II ThPOK + CTLs in vitro in the context of IL-15. As expected, the ddition of IL-15 resulted in higher expression 37 ; however, IL-15 lone or together with the irrelevnt MHC clss I restricted peptide SIINFEKL hd no effect on the functionl differentition or mturtion of the MHC clss II restricted OT-II + CTLs (Supplementry Fig. 5). In contrst, the ddition of IL-15 resulted in considerly enhnced immune responses of OVA(323 339)-stimulted OT-II ThPOK + CTLs, s mesured y the sustntl upregultion of CD17 expression nd the nture immunology VOLUME 14 NUMBER 3 MARCH 213 287
npg 213 Nture Americ, Inc. All rights reserved. much greter production of IFN-γ nd TNF (Fig. 6e). These dt therefore demonstrted tht ntigen-induced + CTLs generted in the sence of inflmmtion were poised to exert potent effector functions when rectivted y their cognte ntigen in the context of the inflmmtory cytokine IL-15. We confirmed those dt with polyclonl ThPOK + CTLs isolted from the intestine of norml lymphocyte-sufficient mice nd showed tht unmnipulted + CTLs nlyzed ex vivo lso hd the potentil in response to polyclonl ctivtion nd to increse their cytolytic nd inflmmtory immune responses when exposed to IL-15. Although IL-15 lone supported short-term survivl of wild-type + CTLs nd β + CTLs, this cytokine lone did not induce cytolytic effector functions in these polyclonl cells (Supplementry Fig. 5c). In contrst, similr to results otined with the dietry ntigen induced OT-II ThPOK + CTLs or norml β + CTLs (Supplementry Fig. 5d), the ddition of IL-15 lso resulted in considerly enhnced cytolytic nd inflmmtory functions of wild-type polyclonl TCR-stimulted + CTLs, wheres it hd only negligile effect on ThPOK + + helper T cells (Fig. 6f). These dt emphsizes the likely pthogenic potentil of ntigen-induced ThPOK + CTLs, which were poised to kill under conditions in which IL-15 ws undnt. DISCUSSION The results reported here hve indicted n unexpectedly lrge degree of plsticity for + helper T cells nd hve demonstrted tht in response to chronic or strong stimultion with ntigen, mture + cells were le to terminte their expression of the helper T cell mster regultor ThPOK (the signture trnscription fctor of the linege) nd differentite into functionl CTLs. The identifiction of dditionl functionl + T lymphocyte susets defined y signture trnscription fctors, such s T H 17 cells nd Foxp3 + regultory T cells, hs een n importnt dvnce in immunology reserch. Although cytotoxic ctivity of + T cells hs long een known, here we hve shown tht this ws not simply function of T H 1-like cells. Insted, here we identified previously unknown type of mture, ntigenexperienced + T cell chrcterized y the ctivtion-induced loss of ThPOK. These cells hd the concomitnt ppernce of cellsurfce nd functionl phenotype tht ws distinct from tht of conventionl helper T cell susets ut resemled tht of CD8 + effector CTLs. Our dt therefore indicte tht the ThPOK-driven commitment to the + helper T cell linege is not fixed nd tht plsticity controlled t the level of ThPOK expression my led to the functionl differentition of defined ThPOK MHC clss II restricted + CTLs, despite their initil ThPOK expression nd commitment to the helper T cell linege or the MHC clss II restriction of their TCRs. The differentition of + T lymphocytes into CTLs insted of their suppression nd prevention from ecoming inflmmtory T H 1 or T H 17 cells mkes teleologicl sense t epithelil interfces, where rpid elimintion of infected cells is importnt not only for resistnce to the initil invsion of intrcellulr pthogens ut lso for limiting or preventing excessive infiltrtion of systemiclly ctivted cytokine secreting cells tht could jeoprdize the integrity of the mucosl rrier 38. In ddition, the cytotoxic function of the + CTLs my lso contriute to diminishing infection nd inflmmtion y eliminting infected migrtory dendritic cells tht could further prime nive cells or medite systemic spred of the pthogen. The MHC clss II restriction of + CTLs might render these lymphocytes le to restrin virl infections tropic for MHC clss II positive trget cells, including infected MHC clss II positive epithelil cells 39, Epstein-Brr virus trnsformed B cells 4 or humn immunodeficiency virus infected humn + T cells 41. Such T cells might lso e criticl for protection ginst viruses, including humn immunodeficiency virus nd cytomeglovirus, tht hve developed mechnisms to escpe surveillnce y MHC clss I restricted CD8 + CTLs 4,42 or under conditions in which the control response of CD8 + CTLs ecomes exhusted, s is the cse in mny chronic infections. In ddition, s with β + CTLs, the responsiveness of + CTLs to IL-15 might provide mens for promoting their ntigenspecific or ystnder protective ility ginst vriety of pthogens or for enhncing the efficcy of ntitumor therpies involving doptive cells. It is lso possile, however, tht in inflmmtory disorders tht involve IL-15, such s celic disese or virus-induced inflmmtory conditions 32,43, + CTLs my incresingly ecome pthogenic effector cells tht hrmfully destroy their trget tissue nd recruit other cell types of the immune system, therey contriuting to inflmmtory pthogenesis. In the context of celic disese, it hs een difficult to connect the known pthogenic fctors, including dietry ntigens, ssocition with MHC clss II, IL-15 nd TCR-dependent nd TCR-independent killer cells 18,36. Our finding tht dietry ntigen induced, MHC clss II restricted + CTLs ecme potent cytotoxic effector cells, producing lrge mounts of IFN-γ nd TNF, when stimulted y their cognte ntigen in the presence of IL-15 my form the long-sought missing link tht ties together the genetic nd environmentl fctors involved in the pthogenesis of celic disese. In summry, the results presented here hve identified + CTLs s distinct + effector cells defined y their post-thymic loss of ThPOK nd the reciprocl functionl differentition of these MHC clss II restricted T lymphocytes into CTLs. Our dt hve lso identified the Thpok silencer s the moleculr switch tht drives the unique post-thymic reprogrmming of these + effector cells. The unexpected plsticity of mture + T cells in differentiting into CTLs expnds the functionl ility of MHC clss II restricted + effector cells to lso include direct protective functions nd provides the immune system with n lterntive protective mechnism, lthough the findings here hve lso demonstrted tht these + CTLs my e centrl in driving the immunologicl pthology ssocited with inflmmtory MHC clss II restricted T cells. + effector cells with cytolytic functions hve een known for long time, nd they hve frequently een ssocited with eneficil s well s pthologicl immune responses. Nevertheless, progress in directly linking such cells to those events or trgeting them s prt of n immunologicl therpy hs een extremely difficult minly ecuse they could not e seprted from clssic effector helper T cells. Therefore, the new insights reported here defining + CTLs s distinct suset of effector cells nd identifying the mechnism tht leds to the unique differentition of these cells provide informtion to finlly move forwrd the field of + CTLs nd to specificlly trget these cells to enhnce protective immunity or to prevent or tret inflmmtory diseses nd immunologicl pthology. Methods Methods nd ny ssocited references re ville in the online version of the pper. Accession codes. GEO: microrry dt, GSE41257 nd GSE42277. Note: Supplementry informtion is ville in the online version of the pper. Acknowledgments We thnk A. Lrnge nd I. Vicente-Surez for discussions nd criticl reding of the mnuscript; M. Cheroutre for contriutions in conceiving of the project; 288 VOLUME 14 NUMBER 3 MARCH 213 nture immunology
npg 213 Nture Americ, Inc. All rights reserved. Y. Wng-Zhu for prepring tetrmers of the thymic leukemi ntigen nd reeding Rg1 / mice; C. Kim nd K. Vn Gunst for cell sorting; D. Littmn (New York University) for E8 I -deficient mice; nd D. Kppes (Fox Chse Cncer Center) for Thpok vectors. Supported y the US Ntionl Institutes of Helth (R1 AI5265 nd DP1 OD6433 to H.C.; F32 DK82249 to J.-W.S.; nd P1 DK46763 to M.K. nd H.C.), the Reserch Center for Allergy nd Immunology (H.C. nd I.T.), the Jpn Society for the Promotion of Science (I.T.), Ter Meulen fund (F.v.W.) nd the Crohn s & Colitis Foundtion of Americ (D.M.). This is mnuscript 1263 from the L Joll Institute for Allergy nd Immunology. AUTHOR CONTRIBUTIONS H.C., I.T., M.K., D.M. nd M.M.H. conceived of the project; M.M.H., D.M. nd F.v.W. generted the phenotypic nd functionl dt; I.T., S.M., Y.N. nd C.M. generted the dt on fte mpping nd deletion of the Thpok silencer nd did the ChIP ssys; R.S. trnsfected cells; Y.H. provided the dt on IL-7R-deficient mice; B.S.R., M.D. nd A.A. generted the gene rrys; G.K., F.L. nd C.J.L. trnsferred cells nd nlyzed mice; J.-W.S. nd D.M. infected mice with citrocter; K.A. nd K.H. reconstituted germ-free mice; S.S. generted the dt on the role of MAZR; Y.P. nlyzed Myd88 / mice; P.W., D.M., F.v.W., B.S.R. nd H.C. nlyzed the generry dt; T.N. nd W.E. provided expertise; M.K. provided conceptul dvice nd helped with dt nlysis nd writing of the mnuscript; I.T. nd H.C. generted concepts, designed experiments, nlyzed dt nd wrote the mnuscript; nd ll uthors contriuted to the writing of the mnuscript nd provided dvice. COMPETING FINANCIAL INTERESTS The uthors declre no competing finncil interests. Pulished online t http://www.nture.com/doifinder/1.138/ni.2523. Reprints nd permissions informtion is ville online t http://www.nture.com/ reprints/index.html. 1. Alihmd, P., Kdvllore, A., de l Torre, B., Kppes, D. & Kye, J. TOX is required for development of the T cell linege gene progrm. J. Immunol. 187, 5931 594 (211). 2. Hernández-Hoyos, G., Anderson, M.K., Wng, C., Rothenerg, E.V. & Alerol-Il, J. GATA-3 expression is controlled y TCR signls nd regultes /CD8 differentition. Immunity 19, 83 94 (23). 3. Pi, S.Y. et l. Criticl roles for trnscription fctor GATA-3 in thymocyte development. Immunity 19, 863 875 (23). 4. He, X. et l. The zinc finger trnscription fctor Th-POK regultes versus CD8 T-cell linege commitment. Nture 433, 826 833 (25). 5. Sun, G. et l. The zinc finger protein ckrox directs linege differentition during intrthymic T cell positive selection. Nt. Immunol. 6, 373 381 (25). 6. Wng, L. et l. The zinc finger trnscription fctor Zt7 represses CD8-linege gene expression in peripherl + T cells. Immunity 29, 876 887 (28). 7. Tniuchi, I. et l. Differentil requirements for Runx proteins in repression nd epigenetic silencing during T lymphocyte development. Cell 111, 621 633 (22). 8. Woolf, E. et l. Runx3 nd Runx1 re required for CD8 T cell development during thymopoiesis. Proc. Ntl. Acd. Sci. USA 1, 7731 7736 (23). 9. Appy, V. et l. Chrcteriztion of + CTLs ex vivo. J. Immunol. 168, 5954 5958 (22). 1. Appy, V. The physiologicl role of cytotoxic + T-cells: the holy gril? Clin. Exp. Immunol. 138, 1 13 (24). 11. Brown, D.M. Cytolytic cells: Direct meditors in infectious disese nd mlignncy. Cell. Immunol. 262, 89 95 (21). 12. Mrshll, N.B. & Swin, S.L. Cytotoxic T cells in ntivirl immunity. J. Biomed. Biotechnol. 1.1155/211/95462 (211). 13. Guy-Grnd, D., Mlssis-Seris, M., Briottet, C. & Vsslli, P. Cytotoxic differentition of mouse gut thymodependent nd independent intrepithelil T lymphocytes is induced loclly. Correltion etween functionl ssys, presence of perforin nd grnzyme trnscripts, nd cytoplsmic grnules. J. Exp. Med. 173, 1549 1552 (1991). 14. Sydor, B.C. et l. Intestinl intrepithelil lymphocytes re ctivted nd cytolytic ut do not proliferte s well s other T cells in response to mitogenic signls. J. Immunol. 15, 2179 2191 (1993). 15. Sshr, T., Tmuchi, H., Ikewki, N. & Kuot, K. Unique properties of cytotoxic + CD8 + intrepithelil T-cell line estlished from the mouse intestinl epithelium. Microiol. Immunol. 38, 191 199 (1994). 16. Nscimeni, M., Shin, E.C., Chiriog, L., Kleiner, D.E. & Rehermnn, B. Peripherl + CD8 + T cells re differentited effector memory cells with ntivirl functions. Blood 14, 478 486 (24). 17. Strutt, T.M., McKinstry, K.K. & Swin, S.L. Functionlly diverse susets in T cell responses ginst influenz. J. Clin. Immunol. 29, 145 15 (29). 18. DePolo, R.W. et l. Co-djuvnt effects of retinoic cid nd IL-15 induce inflmmtory immunity to dietry ntigens. Nture 471, 22 224 (211). 19. Muroi, S. et l. Cscding suppression of trnscriptionl silencers y ThPOK sels helper T cell fte. Nt. Immunol. 9, 1113 1121 (28). 2. Cheroutre, H. Strting t the eginning: new perspectives on the iology of mucosl T cells. Annu. Rev. Immunol. 22, 217 246 (24). 21. Burkett, M.W., Shfer-Wever, K.A., Strol, S., Bseler, M. & Mlyguine, A. A novel flow cytometric ssy for evluting cell-medited cytotoxicity. J. Immunother. 28, 396 42 (25). 22. Setoguchi, R. et l. Repression of the trnscription fctor Th-POK y Runx complexes in cytotoxic T cell development. Science 319, 822 825 (28). 23. He, X., Prk, K. & Kppes, D.J. The role of ThPOK in control of /CD8 linege commitment. Annu. Rev. Immunol. 28, 295 32 (21). 24. Ellmeier, W., Sunshine, M.J., Losos, K., Htm, F. & Littmn, D.R. An enhncer tht directs linege-specific expression of CD8 in positively selected thymocytes nd mture T cells. Immunity 7, 537 547 (1997). 25. Kennedy, J. et l. A moleculr nlysis of NKT cells: identifiction of clss-i restricted T cell-ssocited molecule (CRTAM). J. Leukoc. Biol. 67, 725 734 (2). 26. Boles, K.S., Brchet, W., Dicovo, T., Cell, M. & Colonn, M. The tumor suppressor TSLC1/NECL-2 triggers NK-cell nd CD8 + T-cell responses through the cell-surfce receptor CRTAM. Blood 16, 779 786 (25). 27. Boles, K.S., Stepp, S.E., Bennett, M., Kumr, V. & Mthew, P.A. 2B4 (CD244) nd CS1: novel memers of the CD2 suset of the immunogloulin superfmily molecules expressed on nturl killer cells nd other leukocytes. Immunol. Rev. 181, 234 249 (21). 28. Skguchi, S. et l. The zinc-finger protein MAZR is prt of the trnscription fctor network tht controls the versus CD8 linege fte of doule-positive thymocytes. Nt. Immunol. 11, 442 448 (21). 29. Prk, J.H. et l. Signling y intrthymic cytokines, not T cell ntigen receptors, specifies CD8 linege choice nd promotes the differentition of cytotoxic-linege T cells. Nt. Immunol. 11, 257 264 (21). 3. Ivnov, I.I. et l. Induction of intestinl Th17 cells y segmented filmentous cteri. Cell 139, 485 498 (29). 31. Cheroutre, H. & Lmolez, F. Douting the TCR coreceptor function of α. Immunity 28, 149 159 (28). 32. Mention, J.J. et l. Interleukin 15: key to disrupted intrepithelil lymphocyte homeostsis nd lymphomgenesis in celic disese. Gstroenterology 125, 73 745 (23). 33. Eert, E.C. Interleukin 15 is potent stimulnt of intrepithelil lymphocytes. Gstroenterology 115, 1439 1445 (1998). 34. Hirose, K. et l. Interleukin-15 my e responsile for erly ctivtion of intestinl intrepithelil lymphocytes fter orl infection with Listeri monocytogenes in rts. Infect. Immun. 66, 5677 5683 (1998). 35. Ye, W., Young, J.D. & Liu, C.C. Interleukin-15 induces the expression of mrnas of cytolytic meditors nd ugments cytotoxic ctivities in primry murine lymphocytes. Cell. Immunol. 174, 54 62 (1996). 36. Adie, V., Discepolo, V. & Jri, B. Intrepithelil lymphocytes in celic disese immunopthology. Semin. Immunopthol. 34, 551 566 (212). 37. Gngdhrn, D. et l. Identifiction of pre- nd postselection TCRαβ + intrepithelil lymphocyte precursors in the thymus. Immunity 25, 631 641 (26). 38. Cheroutre, H., Lmolez, F. & Mucid, D. The light nd drk sides of intestinl intrepithelil lymphocytes. Nt. Rev. Immunol. 11, 445 456 (211). 39. Hersherg, R.M. et l. Highly polrized HLA clss II ntigen processing nd presenttion y humn intestinl epithelil cells. J. Clin. Invest. 12, 792 83 (1998). 4. Khnn, R. et l. Clss I processing-defective Burkitt s lymphom cells re recognized efficiently y + EBV-specific CTLs. J. Immunol. 158, 3619 3625 (1997). 41. Ko, H.S., Fu, S.M., Winchester, R.J., Yu, D.T. & Kunkel, H.G. I determinnts on stimulted humn T lymphocytes. Occurrence on mitogen- nd ntigen-ctivted T cells. J. Exp. Med. 15, 246 255 (1979). 42. Alcmi, A. & Koszinowski, U.H. Virl mechnisms of immune evsion. Immunol. Tody 21, 447 455 (2). 43. McInnes, I.B. & Grcie, J.A. Interleukin-15: new cytokine trget for the tretment of inflmmtory diseses. Curr. Opin. Phrmcol. 4, 392 397 (24). nture immunology VOLUME 14 NUMBER 3 MARCH 213 289