The ion channel TRPV1 regulates the activation and proinflammatory properties of CD4 + T cells

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1 A rt i l s Th ion hnnl TRPV rgults th tivtion n proinflmmtory proprtis of CD + T lls Smul Brtin, Yukri Aoki-Nonk,, Ptrus Ruolf Jong, Lilin L Nohr,8, Hongjin Xu,8, Shwn R Stnwoo, Sonl Sriknth, Jihyung L, Kith To, Lior Armson, Timothy Yu, Tiffny Hn, Rnim Toum 5, Xingli Li, José M Gonzálz-Nvjs, Sott Hrmn, Mript Corr, Guo Fu,7, Hui Dong, Yousng Gwk, Alssnr Frno 5, Wilfr A Jffris & Eyl Rz npg Ntur Amri, In. All rights rsrv. TRPV is C + -prml hnnl stui mostly s pin rptor in snsory nurons. Howvr, its rol in othr ll typs is poorly unrstoo. Hr w foun tht TRPV ws funtionlly xprss in CD + T lls, whr it t s non stor-oprt C + hnnl n ontriut to T ll ntign rptor (TCR)-inu C + influx, TCR signling n T ll tivtion. In mols of T ll mit olitis, TRPV promot olitogni T ll rsponss n intstinl inflmmtion. Furthrmor, gnti n phrmologil inhiition of TRPV in humn CD + T lls rpitult th phnotyp of mous Trpv / CD + T lls. Our finings suggst tht inhiition of TRPV oul rprsnt nw thrputi strtgy for rstrining proinflmmtory T ll rsponss. Following nggmnt of th T ll ntign rptor (TCR) n formtion of th immunologil synps, lium (C + ) hnnls mit th C + influx nssry for th tivtion of T lls. C + influx from th xtrllulr miliu into th ytosol is mntory for th tivtion of ownstrm TCR signling pthwys n ky trnsription ftors, inluing NFAT n NF-κB. Ligtion of th TCR triggrs phosphoryltion vnts tht l to tivtion of th C + rls tivt C + hnnl omplx n to stor-oprt C + ntry. Howvr, this nonil shm of C + influx os not ount for th involvmnt of othr plsm-mmrn C + hnnls. Stuis hv oumnt th importnt ontriutions of voltggt C + hnnls,5 n mmrs of th trnsint rptor potntil (TRP) fmily of ion hnnls,7 to T ll tivtion. Th mmmlin TRP fmily of ion hnnls onsists of 8 mmrs tgoriz into six sufmilis: nonil (TRPC), with svn mmrs; vnilloi (TRPV), with six mmrs; mlsttin (TRPM), with ight mmrs; nkyrin (TRPA), with on mmr; polyystin (TRPP), with thr mmrs; n muolipin (TRPML), with two mmrs 8. Although physiologil rols of TRP hnnls r wll hrtriz in th nrvous systm, thir rol in th tivtion n funtion of T lls is still lusiv,5,. Th founing mmr of th TRPV sufmily, TRPV, is highly prml to C + (with prmility to lium rltiv to prmility to soium (P C/ P N ) of ~) 9 n ws initilly intifi s th rptor for psiin, th pungnt ingrint in hili ppprs. In this stuy, w foun tht mous n humn primry CD + T lls xprss funtionl TRPV hnnl (ll TRPV CD hr). W foun tht TRPV CD ontriut to TCR-inu C + influx n ws rquir for propr ownstrm TCR signling. Our t init ll-intrinsi rol for th TRPV hnnl in th tivtion of n quisition of proinflmmtory proprtis y CD + T lls. RESULTS Exprssion of funtionl TRPV y CD + T lls W first ompr th xprssion of mrna noing vrious TRP hnnls in primry CD + T lls isolt from th spln of C57BL/ (wil-typ) mi. Among mrna noing th ight iffrnt TRP hnnls nlyz, Trpv trnsripts h th highst xprssion (Fig. ). W us CD + T lls isolt from Trpv / mi s ontrol (Fig. ). W onfirm xprssion of TRPV protin in mous spln CD + T lls y flow ytomtry (Fig. ) s wll s in humn primry CD + T lls n in th Jurkt humn T lymphoyt ll lin y immunolot nlysis (Supplmntry Fig. ). Confol mirosopy show xprssion of TRPV prominntly t th plsm mmrn of rsting CD + T lls (Fig. n Supplmntry Fig.,). In, th fluorsn signls of TRPV n CD wr lrgly ololiz (Prson s orrltion offiint,.9) (Fig. ). Furthrmor, ll-surf iotinyltion ssys onfirm loliztion of TRPV t th plsm mmrn of Jurkt T lls (Supplmntry Fig. ). W nxt vlut th funtionlity of th TRPV hnnl in CD + T lls y th whol-ll pth-lmp thniqu. W us th prototypil TRPV gonist psiin n ror psiin-inu Dprtmnt of Miin, Univrsity of Cliforni, Sn Digo, L Joll, Cliforni, USA. Division of Orl Sin for Hlth Promotion, Niigt Univrsity Grut Shool of Mil n Dntl Sins, Niigt, Jpn. Mihl Smith Lortoris, Cntr for Bloo Rsrh, Th Brin Rsrh Cntr, Dprtmnt of Mil Gntis, Dprtmnt of Miroiology n Immunology n Dprtmnt of Zoology, Univrsity of British Columi, Vnouvr, Cn. Dprtmnt of Physiology, Dvi Gffn Shool of Miin t Univrsity of Cliforni, Los Angls, Los Angls, Cliforni, USA. 5 Dprtmnt of Pitris Univrsity of Cliforni, Sn Digo, L Joll, Cliforni, USA. Dprtmnt of Immunology n Miroil Sin, Th Sripps Rsrh Institut, L Joll, Cliforni, USA. 7 Stt Ky Lortory of Cllulr Strss Biology, Innovtion Cntr for Cll Biology, Shool of Lif Sins, Ximn Univrsity, Fujin, Chin. 8 Ths uthors ontriut qully to this work. Corrsponn shoul rss to W.A.J. (wilf@msl.u.) or E.R. (rz@us.u). Riv 8 August; pt Sptmr; pulish onlin 5 Otor ; oi:.8/ni.9 ntur immunology VOLUME 5 NUMBER NOVEMBER 55

2 Artils Tr pm Tr p Tr p Tr v p Tr v p Tr v pv Tr p Tr v5 pv Clls (%) W T Tr pv / mrna (rltiv) Figur TRPV is xprss in CD+ T lls. () Quntittiv IgG (trl) 8 α-trpv PCR nlysis of trnsripts noing TRP protins in splni CD + α-trpv + T lls isolt from C57BL/ (wil-typ) mi; rsults normliz to ppti thos for th houskping gn Gph (noing GAPDH) n prsnt GMF (p) rltiv to thos for Trpm (known to xprss in CD+ T lls7), 5. Trpv 88 st s., not signifint; P <.5, P <. n P <.. C (on-wy nlysis of vrin (ANOVA) with post-ho Dunntt s tst)..8 () Eltrophorsis of th prouts of quntittiv PCR nlysis of splni Gph 5 CD+TCRβ+ T lls isolt from wil-typ () n Trpv / mi n TRPV sort y flow ytomtry (purity, >98%): th Trpv-spifi prout (top) DAPI TRPV CD Mrg migrts t th xpt siz of 88 s pirs (p); C (noing CDδ) srvs s T ll mrkr; Gph (noing glyrlhy phospht hyrogns) srvs s loing ontrol. () Flow ytomtry of splnoyts isolt from wil-typ mi, prinut with loking ppti orrsponing to TRPV (α-trpv + ppti) or not (α-trpv) CD n thn stin for CD n TCRβ (gt s CD+TCRβ+ T lls) n with nti-trpv, or stin with th isotyp-mth ontrol ntioy immunogloulin G lon (IgG) (trl); numrs in plot init gomtri mn fluorsn (GMF) of th stining intnsity. () Confol mirosopy of th sullulr loliztion of TRPV n CD in splni CD + T lls stin with th DNA-ining y DAPI (fr lft), with got nti-trpv (primry) follow y Alx Fluor 5 onjugt nti-got (sonry) (mil lft) or with Alx Fluor 88 onjugt nti-cd (mil); mil right, mrg img (yllow, TRPV-CD ololiztion); fr right, nlrgmnt (5.5 ) of r outlin t lft. Sl r, 5 µm. () Cololiztion of TRPV n CD, ssss with Vloity softwr (Prson s orrltion offiint,.9). Dt r rprsnttiv of thr or mor inpnnt xprimnts (mn n s..m. of fiv mi in ). CAP / Trpv CAP Trpv /.5 + SB pa s Currnt CAP Currnt nsity (pa/pf) osrv in wil-typ CD+ T lls ws in TRPV urrnt, w msur psiin-inu urrnts in th prsn of SB79, spifi TRPV ntgonist. SB79 inhiit pproximtly 8% of th inwr urrnt osrv in wil-typ CD+ T lls in rspons to psiin (untrt ontrol,. ±. pa/pf (mn ± s..m.), n = lls; SB79 trt,. ±. pa/pf (mn ± s..m.), n = lls) (Fig.,). To ttr hrtriz th psiin-inu urrnt in CD+ T lls, w us rmp-puls protool to msur th urrnt-voltg rltionship ftr th ition of psiin (Fig. ). Th urrntvoltg rltionship of th psiin-gt urrnt onfirm th outwr-rtifition hrtristi of th TRPV urrnt rport for rt n humn TRPV hnnls,. To furthr vlut funtionlity of th TRPV hnnl in CD+ T lls, w prform singl-ll rtiomtri C+ imging n flow Currnt nsity (pa/pf) urrnts in wil-typ n Trpv / CD+ T lls hl t 85 mv (Fig. ). In wil-typ CD+ T lls, th pplition of psiin rpily inu smll inwr urrnt (.7 ±. pa/pf (mn ± s..m.); n = 9 lls) tht ws snsitiz with prolong xposur to th gonist (Fig.,). This snsitiztion ws proly us y th prsn of C+ ions in th xtrnl solution, s monstrt for in rorings of rt TRPV urrnts in HEK9 humn mryoni kiny lls trnsft to xprss TRPV (rfs.,). In Trpv / CD+ T lls, w ror only n insignifint mount of inwr urrnt (. ±. pa/pf (mn ± s..m.); n = 8 lls) in rspons to psiin (Fig.,). Aitionlly, with th sm roring onitions, w routinly ror - to -na psiin-gt urrnts from trnsform humn kiny (tsa) lls with trnsint xprssion of rt TRPV (t not shown). To furthr onfirm tht th urrnt..5. Figur TRPV is funtionl C+ hnnl in CD T lls. () Inwr urrnts ror on wil-typ, Voltg (mv) Trpv / n SB79-trt wil-typ ( + SB) SB + CD+ T lls trt with µm psiin (CAP; gry rs, C + xposur urtion) n hl t 85 mv; ott lins, Pk C CD+ T lls. CAP zro urrnt. (,) Currnt nsity of wil-typ CD T lls (n = 9) n Trpv / CD+ T lls (n = 8) () or. of wil-typ CD+ T lls lft untrt (SB ) (n = ) (kd). Trpv / or trt with µm SB79 (SB +) (n = ) (), Trpv / TRPV - 9 Trpv-TG Trpv-TG. ssss ftr trtmnt with psiin, lult y -. β-tin normliztion of pk urrnt ror t 85 mv to th TRPV/β-tin. pitn of h ll. () Currnt-voltg rltionship µm µm of th psiin-inu urrnt in CD+ T lls trt Tim (s) CAP with µm psiin n with voltg rmp ( 7 mv to +7 mv) livr ovr ms; rsults lult y sutrtion of urrnt for th ition of psiin from urrnt ftr tht ition n normliztion rltiv to th urrnt t 7 mv ( 9. ±. pa). () [C+]i in wil-typ, Trpv / n Trpv-TG splni CD+CD5 (niv) T lls lo with th lium initor Fur- AM n trt with µm psiin (gry r) in th prsn of mm CCl ( C; lk r), monitor y onfol imging n prsnt s th rtio of Fur- mission t n xittion of nm to tht t 8 nm (/8). (f) Quntifition of C+influx profils otin s in with or µm psiin. (g) Immunolot nlysis of TRPV n β-tin (loing ontrol throughout) in totl lysts of wil-typ n Trpv-TG splni CD+ T lls; oults t ~95 kiloltons (kd) n 5 kd orrspon to th nonglyosylt n glyosylt forms of th TRPV hnnl. Blow lns, nsitomtry nlysis (n intnsity rltiv to tht of β-tin). P <.5, P <., P <. n P <. (two-til Stunt t-tst (,) or on-wy ANOVA with th post-ho Bonfrroni tst (f)). Dt r rprsnttiv of thr or mor inpnnt xprimnts (mn n s..m. in,; vrg of four lls in ; mn n s..m. of 5 lls in f). 5 g VOLUME 5 NUMBER NOVEMBER pv W T - Fur- (/8) TG f Tr Fur- (/8) npg Ntur Amri, In. All rights rsrv. TRPV ntur immunology

3 A rt i l s Fur- (/8) Fur- (/8) TPSG Pk C + C α-cd Iono 5 Tim (s) Trpv / Trpv-TG f Trpv / Trpv-TG Fur- (/8) α-cd Trpv / α-cd Pk C + Sustin C + α-cd Fur- (/8) 5 s Ionomyin Pk C + 5 pa g Currnt nsity (pa/pf) Trpv / Trpv-TG 8 Trpv / Ino- (5/5) h Ino- (5/5).8.. C or C α-cd + α-cd Tim (s) C 5 C α-cd + α-cd8 Tim (s) Trpv / BCTC (µm) (vh). Fur- (/8).... i Ino- (5/5) 9 Tim (s) C TPSG Pk C +. BCTC (µm) C Ino- rtio/tim Slop C Trpv / Trpv-TG. BCTC (µm) npg Ntur Amri, In. All rights rsrv. Figur TRPV CD ts s non stor-oprt C + hnnl n ontriuts to TCR-inu C + influx. () [C + ] i in wil-typ, Trpv / n Trpv- TG splni CD + CD5 (niv) T lls lo with Fur- AM n stimult y rosslinking with nti-cd (α-cd) in th prsn of mm CCl ( C), with µm ionomyin (Iono) t th n of th quisition (monitor n prsnt s in Fig. ). () Quntifition of th C + -influx profils in. () [C + ] i in wil-typ n Trpv / spln CD + T lls lo with th fluorsnt C + initor y Ino- AM n stimult with solul nti-cd ( µg/ml) n nti-cd8 ( µg/ml) (α-cd + α-cd8) in C + -fr mium, ssss for ( C) n ftr ( or C) th ition of CCl ( mm (wil-typ) or mm (Trpv / )) to th xtrllulr mium, monitor y flow ytomtry; rsults r prsnt s th rtio of Ino- mission t 5 nm to tht t 5 nm (5/5). () Stor-oprt C + ntry in wil-typ, Trpv / n Trpv-TG splni CD + CD5 (niv) T lls lo with Fur- AM, ssss for ( C) n ftr ( C) th ition of mm CCl n ftr pssiv pltion of intrllulr stors with µm thpsigrgin (TPSG). () Quntifition of th C + -influx profils in. (f) Inwr urrnts in wil-typ n Trpv / CD + T lls tivt y rosslinking with nti-cd n strptviin (r ov lso inits urtion of strptviin pplition), n hl t 85 mv. (g) TCR-inu urrnts in wil-typ CD + T lls (n = 7) n Trpv / CD + T lls (n = 9), lult y normliztion of pk urrnt to pitn of h ll. (h) [C + ] i in wil-typ CD + T lls lo with Ino- AM n prtrt for 5 min with th TRPV ntgonist BCTC (. or µm) or vhil lon (vh;.% imthyl sulfoxi (DMSO)), thn stimult with nti-cd plus nti-cd8 in C + -fr mium, with 5 mm CCl (5 C) to th xtrllulr mium uring th quisition (prsnt s in ). (i) Quntifition of th C + -influx profils in h. P <.5; P <. n P <. (two-til Stunt t-tst (g) or on-wy ANOVA with th post-ho Bonfrroni tst (,,i)). Dt r rprsnttiv of thr or mor inpnnt xprimnts (mn n s..m. of 5 lls in,; mn n s..m. in g,i). ytomtry s C + flux msurmnts. Cpsiin inrs th intrllulr lium onntrtion ([C + ] i ) in onntrtionpnnt wy in wil-typ niv CD + T lls ut not in thir Trpv / ountrprts (Fig.,f). In ition, w gnrt mi with trnsgni xprssion of TRPV spifilly in CD + T lls (Trpv-TG mi) y rossing mi xprssing Trpv from th uiquitous Ros lous with mi xprssing Cr romins from th T ll spifi C promotr. W foun tht psiin inu sustntil C + influx in Trpv-TG CD + T lls (Fig.,f) tht ovrxprss TRPV (Fig. g). Cpsiin lso inu sustntil C + influx in Jurkt T lls, whih ws lmost ompltly olish ftr knokown of TRPV mit y short hirpin RNA (Supplmntry Fig. ). Colltivly, ths finings init tht th TRPV hnnl ws funtionlly xprss on th plsm mmrn of CD + T lls (TRPV CD ). TRPV CD ontriuts to TCR-inu C + influx W nxt invstigt th physiologil rol of TRPV CD y ompring hngs in [C + ] i ftr stimultion of th TCR in wiltyp, Trpv / n Trpv-TG CD + T lls. C + influx inu y rosslinking with ntioy to th invrint signling protin CD (nti-cd) ws signifintly lowr in Trpv / CD + T lls thn in wil-typ lls (Fig.,). W osrv this ft in Trpv / CD + T lls inpnntly of th xtrllulr lium onntrtion tst or of th C + flux monitoring protool us (for xmpl, TCR stimultion for th ition of CCl or vi vrs; Supplmntry Fig. ). In ition, C + -titrtion xprimnts rvl tht th TCR-inu C + influx ft in Trpv / CD + T lls ws rsu y 5% inrs in th xtrllulr lium onntrtion in th ultur mium (Fig. ). In orn with th loliztion of TRPV CD t th plsm mmrn (Fig., n Supplmntry Fig. ), th C + fflux from intrllulr stors ws similr in wil-typ n Trpv / CD + T lls ftr stimultion of th TCR (Supplmntry Fig. ). Th TCR-inu C + influx ws signifintly mor sustin in Trpv-TG CD + T lls thn in wil-typ lls (Fig., n Supplmntry Fig. f). Howvr, w osrv no iffrn mong wil-typ, Trpv / n Trpv-TG CD + T lls in C + influx following stimultion with th C + ionophor ionomyin (Fig., n Supplmntry Fig. g,h) or with th sroplsmi rtiulum C + -ATPs pump inhiitor thpsigrgin (Fig., n Supplmntry Fig. i), whih ypss proximl TCR signling n inu tivtion of C + rls tivt C + hnnls n stor-oprt C + ntry. Sin TRPV shrs 5% homology with TRPV n n form htromultimrs with tht hnnl 9,5, w lso nlyz th C + -influx profil of spln CD + T lls isolt from Trpv / mi. Howvr, in ontrst to C + influx in Trpv / CD + T lls, w foun norml C + influx in ths lls upon stimultion of th TCR (Supplmntry Fig. j). W furthr onfirm th spifi ontriution of TRPV to TCR-inu C + urrnts y whol-ll pth-lmp xprimnts. Crosslinking of TCRs prou signifintly smllr inwr urrnts in Trpv / CD + T lls hl t 85 mv (. ±. pa/ pf (mn ± s..m.); n = 9 lls) thn in wil-typ lls unr th sm xprimntl onitions (. ±. pa/pf (mn ± s..m.), n = 7 lls) (Fig. f,g). Finlly, prtrtmnt of wil-typ CD + T lls with ithr of two iffrnt TRPV ntgonists, BCTC (Fig. h,i) or I-RTX 7 (Supplmntry Fig. k), rs th C + influx inu y TCR stimultion to lvl similr to tht in Trpv / CD + T lls n i so in onntrtion-pnnt wy. Colltivly, ths rsults suggst tht TRPV CD t s non stor-oprt C + hnnl n ontriut to TCRinu C + influx. ntur immunology VOLUME 5 NUMBER NOVEMBER 57

4 A rt i l s npg Ntur Amri, In. All rights rsrv. Figur TRPV CD prtiipts in TCR signling. () Confol mirosopy of wil-typ spln CD + T lls for (α-cd ) n ftr (α-cd +) rosslinking with nti-cd, in th prsn (+) or sn ( ) of th Sr-fmily kins inhiitor PP ( µm); rrowhs init rruitmnt of TRPV to TCR lustrs; yllow (mrg imgs) inits lustring of Lk n TRPV. Sl r, µm. () Immunossy of Jurkt E. lls (wil-typ) n J.CM. lls (Lk / ) lft unstimult ( ) or stimult (+) for 5 min with solul ntioy to humn CD (.5 µg/ml) n ntioy to humn CD8 ( µg/ml), follow y immunopripittion (IP) with nti-trpv n immunolot nlysis (IB) of immunopripitts (top lot) or totl ll lysts (mil n ottom lots) with ntioy to phosphorylt tyrosin (p-tyr), nti-trpv n nti-lk (lft mrgin). () [C + ] i in wil-typ n Lk / Jurkt T lls lo with Fur- AM n trt with µm psiin in th prsn of mm CCl ( C), with th ition of µm ionomyin (Iono) t th n of th quisition (positiv ontrol) (monitor n prsnt s in Fig. ). Right, quntifition of C + -influx pk t lft. P <. (two-til Stunt t-tst). () Immunolot nlysis of phosphorylt (p-) Zp7 n Lt in th ytosoli frtion of wil-typ n Trpv / spln CD + T lls lft unstimult () or stimult for 5 min (ov lns) with solul nti-cd (5 µg/ml) n nti-cd8 ( µg/ml). Right mrgin, molulr siz (in kd). () Intrllulr stining of phosphorylt PLC-γ in gt CD + TCRβ + wil-typ n Trpv / splni CD + T lls lft unstimult (US) or stimult for min with solul f PP + α-cd + + DAPI TRPV Lk Mrg Trpv / α-cd + α-cd8 (min) 5 5 (kd) p-zp7-7 p-lt β-tin - - p-zp7/β-tin p-lt/β-tin Trpv / α-cd + 5 α-cd8 (min) 5 p-erk/ Erk/ p-erk//erk/ p-p8 p8 p-p8/8 p-jnk Jnk p-jnk/jnk nti-cd (5 µg/ml) n nti-cd8 ( µg/ml). Numrs ov n low rkt lins init prnt PLC-γ + lls (olors mth ky). (f) Immunolot nlysis of phosphorylt n totl Erk/, p8 n Jnk in th ytosoli frtion of lls stimult s in. (g) Immunolot nlysis of NFAT- in th nulr frtion of lls stimult s in. (h) Eltrophorti moility-shift ssy of NF-κB in th nulr frtion of lls stimult s in ;, nonspifi low-molulr wight n (loing ontrol). Blow lns (,f h), nsitomtry nlysis. Dt r rprsnttiv of thr or mor inpnnt xprimnts (mn n s..m. of 5 lls in ). (kd) g h Fur- (/8) Lk / α-cd + α-cd8 + + IP: TRPV IB: p-tyr IB: TRPV Cll ounts IB: Lk. CAP. Lk /. Tim (s) p-plcγ β-tin p-trpv TRPV Lk NFAT-/β-tin Trpv / α-cd + α-cd8 (min) 5 5 NF-κB C Iono Pk C + (/8) Trpv CAP Trpv / α-cd + α-cd8 (min) 5 5 (kd) NFAT- - 9 NF-κB/ Lk / IgG (trl) US α-cd + α-cd8 ( min) - TRPV CD is prt of th TCR signling s W thn invstigt how TRPV CD ws tivt upon stimultion of th TCR n ssss its fft on TCR signling. W first ompr th sullulr loliztion of TRPV in rsting n tivt CD + T lls y onfol mirosopy. W foun tht unr rsting onitions, TRPV loliz togthr with omponnts of th TCR omplx, suh s th orptor CD (Fig.,) n th Sr-fmily tyrosin kins Lk (Fig. ). In ition, Lk n TRPV wr rpily rruit to TCR lustrs ftr ligtion of th TCR (Fig. ). Sin formtion of th p strutur is known to pnnt on tyrosin phosphoryltion 8,9, w invstigt whthr TRPV lustring ws pnnt on similr mhnism. In, w foun tht PP, n inhiitor of th Sr fmily of tyrosin kinss, inhiit pping of Lk n TRPV (Fig. ). Bus th phosphoryltion of TRP hnnls rgults thir hnnl tivity, w invstigt whthr nggmnt of th TCR inu tyrosin phosphoryltion of TRPV CD. To rss th possil rol of Lk in this systm, w xmin th tyrosin-phosphoryltion sttus of nognous TRPV immunopripitt from wil-typ Jurkt T lls (lon E.) n rivtiv mutnt of Jurkt T lls tht lk Lk xprssion (J.CM.). Stimultion of th TCR inu tyrosin-phosphoryltion of TRPV in th prntl wil-typ E. ll lin ut not in th mutnt, Lk-fiint J.CM. lls (Fig. ). In orn with pulish stuis tht hv monstrt n ssntil rol for tyrosin kinss of th Sr fmily in th tivtion of TRPV hnnls, w foun tht TRPV CD -mit C + influx ws lmost ompltly olish in th sn of Lk (Fig. ). Colltivly, ths rsults suggst tht TRPV is prt of th proximl TCR signling s n tht tyrosin phosphoryltion y Lk is possil gting mhnism for TRPV CD ftr stimultion of th TCR. W nxt vlut th fft of TRPV fiiny on TCR signling. W foun no iffrn in phosphoryltion of th TCR-proximl signling omponnts Zp7, Lt (Fig. ) n PLC-γ (Fig. ), slight iffrn in phosphoryltion of th kinss Erk/ n p8, n lss tivtion of th kins Jnk (Fig. f) in Trpv / CD + T lls rltiv to tht in wil-typ CD + T lls. Consistnt with thir rs TCR-inu C + influx, Trpv / CD + T lls fil to sustin loliztion of NFAT- to th nulus (Fig. g) n isply ru tivtion of NF-κB (Fig. h) upon stimultion of th TCR. 58 VOLUME 5 NUMBER NOVEMBER ntur immunology

5 A rt i l s Cytokin (pg/ml) IFN-γ (pg/ml) 8 α-cd + α-cd8 IFN-γ IL-7A Vh. BCTC (µm) IL- (pg/ml) IL- IL- IL- TNF,, 8 Vh. 8,,,, BCTC (µm) PMA + iono IFN-γ IL- AnnV + lls (%) 5 IL- (pg/ml) CCl (mm) 5 Vh. BCTC (µm) vh + BCTC Trpv / f IFN-γ (ng/ml) IFN-γ (ng/ml) OVA BCTC 5 OVA IL-7A (ng/ml) IL-7A (ng/ml) OVA.5..5 OVA BCTC IL- (ng/ml) IL- (ng/ml) 8 OVA 5 5 OVA BCTC Trpv +/+ OT-II Trpv / OT-II OT-II + vh OT-II + BCTC npg Ntur Amri, In. All rights rsrv. Figur 5 Gnti ltion or phrmologil inhiition of TRPV CD rss TCR-inu proution of ytokins. () Enzym-link immunosornt ssy (ELISA) of ytokins (horizontl xs) in wil-typ n Trpv / splni CD + T lls stimult for h (IFN-γ, IL-7A, IL- n TNF) or 8 h (IL- n IL-) with plt-oun nti-cd ( µg/ml) n solul nti-cd8 ( µg/ml) (lft; n = mi pr group) or with PMA (5 ng/ml) n ionomyin (5 nm) (right; n = mi pr group). () IL- in suprntnts of wil-typ n Trpv / CD + T lls (n = mi pr group) stimult for h with nti-cd plus nti-cd8 in RPMI mium supplmnt with vrious onntrtions (horizontl xis) of CCl. () IFN-γ, IL-7A n IL- in suprntnts of CD + T lls rovr from oulturs of wil-typ splni CD + DCs lo with OVA (OVA +) or not (OVA ) n inut for 5 with OVA-spifi Trpv +/+ or Trpv / OT-II splni CD + T lls (n = mi pr group); qul numrs of th CD + T lls rovr wr rstimult for h with nti-cd plus nti-cd8 for nlysis. () ELISA of IFN-γ n IL- in suprntnts of wiltyp splni CD + T lls (n = mi pr group) prinut for min with vrious onntrtions of BCTC (TRPV inhiitor) or vhil (Vh;.% DMSO) n stimult for h with nti-cd plus nti-cd8. () Apoptosis of CD + T lls mong lls rovr in, stin for CD, nnxin V (AnnV) n th DNA-intrlting y 7-AAD, nlyz y flow ytomtry. (f) Cytokin proution y splni OT-II CD + T lls (n = mi pr group) prtrt for min with µm BCTC (+) or vhil (.% DMSO) ( ), thn wsh n ultur for 5 togthr with wil-typ splni DCs lo with OVA or not, follow y prossing of CD + T lls s in. P <.5, P <., P <. n P <. (two-til Stunt t-tst () or on-wy ANOVA with th post-ho Bonfrroni (,f) or Dunntt s (,,) tst). Dt r rprsnttiv of fiv (, lft), thr (,f) or two (, right, ) inpnnt xprimnts (rror rs, s..m.). Thus, TRPV CD sm to ispnsl for proximl TCR signling ut ws rquir for th propr trnsution of istl TCR signling vnts. TRPV CD ontriuts to TCR-inu ytokin proution W rson tht th ltr TCR signling in Trpv / CD + T lls woul fft thir susqunt ytokin proution. W osrv tht Trpv / CD + T lls prou lowr onntrtions of vrious ytokins (intrfron-γ (IFN-γ), intrlukin 7A (IL-7A), IL-, IL-, IL- n tumor-nrosis ftor (TNF)) ftr stimultion with nti-cd n ntioy to th orptor CD8 (nti-cd8) thn i wil-typ lls (Fig. 5). W osrv th opposit phnotyp for Trpv-TG CD + T lls (Supplmntry Fig. ). Th iminish ytokin proution y Trpv / CD + T lls ws not xplin y inrs poptosis or lowr prolifrtiv rspons of ths lls (Supplmntry Fig. ). Morovr, TCR-inpnnt tivtion of Trpv / n wil-typ CD + T lls with th phorol str PMA n ionomyin rsult in similr srtion of IFN-γ n IL- (Fig. 5). Using IL- s rout, w foun tht its iminish proution y Trpv / CD + T lls ws rsu y n inrs in th xtrllulr lium onntrtion in th ultur mium (Fig. 5). Ths t suggst tht th iminish ytokin proution in Trpv / CD + T lls ws proly u to th rs TCR-inu C + influx osrv in ths lls (Fig. n Supplmntry Fig. ). In ition, in n ntign-spifi mol (OT-II lls, whih hv trnsgni xprssion of n ovlumin (OVA)-spifi TCR), w foun tht Trpv / OT-II CD + T lls stimult with OVA-lo wil-typ nriti lls (DCs) h lowr proution of IFN-γ, IL-7A n IL- thn i ontrol (Trpv +/+ ) OT-II lls unr th sm stimultory onitions (Fig. 5 n Supplmntry Fig. ). To furthr onfirm th ontriution of TRPV CD -inu signling to ytokin proution, w trt wil-typ CD + T lls with th TRPV ntgonist BCTC. BCTC rs th rls of ytokins from CD + T lls in onntrtion-pnnt wy upon stimultion with nti-cd plus nti-cd8 (Fig. 5). Th osrv rs in ytokin proution ws not u to n inrs in th poptosis of CD + T lls (Fig. 5). BCTC lso signifintly iminish ytokin proution y OT-II CD + T lls upon stimultion with OVA-lo DCs (Fig. 5f). Togthr ths rsults suggst ll-intrinsi rol for TRPV CD in promoting TCR-inu ytokin proution. TRPV inhiition rss humn CD + T ll tivtion To xplor th rlvn of th t gnrt in th mous to th humn systm, w trmin th fft of phrmologil inhiition of TRPV on th tivtion profil of primry CD + T lls nrih from th priphrl loo of hlthy onors. W stimult th lls with nti-cd plus nti-cd8 in th prsn or sn of th TRPV ntgonist SB79. W nlyz uprgultion of th surf xprssion of tivtion mrkrs (i.., CD5 n HLA-DR) 8 h ltr n foun tht SB79 rs thir xprssion in onntrtion-pnnt wy (Fig.,). Consistnt with rsults otin with th mous ntur immunology VOLUME 5 NUMBER NOVEMBER 59

6 A rt i l s npg Ntur Amri, In. All rights rsrv. CD5 HLA-DR US 5.5 M 5 CD 5. M 5 CD α-cd + α-cd8 + vh. 8. α-cd + α-cd8 + SB79 (µm). 7.7 systm, inhiition of TRPV lso rs IL- proution (Fig. ). Notly, w osrv this fft vn t vry low onntrtions of th ntgonist SB79 (. µm), n it ws not ssoit with inrs poptosis of CD + T lls (Fig. ). To onfirm thos finings, w knok own TRPV in primry humn CD + T lls through th us of smll intrfring RNA (sirna). Similr to phrmologil inhiition of TRPV, knokown of TRPV rs th xprssion of CD5 n HLA-DR (Fig.,f) n th proution of IL- (Fig. g,h) upon stimultion with nti-cd plus nti-cd8. Colltivly, ths t init th TRPV hnnl ontriut to th tivtion of humn CD + T lls. TRPV CD rgults proinflmmtory CD + T ll rsponss To nlyz th ontriution of TRPV-inu signling to CD + T ll rsponss in vivo, w ppli two iffrnt mols of CD + T ll mit olitis. W first us th Il / mol 5 n ompr th svrity of olitis in Il / mi n Il / Trpv / mi. Il / mi lost mor wight thn Il / Trpv / mi i ftr th inution n synhroniztion of olitis y orl trtmnt with th nonsltiv ylooxygns inhiitor piroxim (Fig. 7). Histologil nlysis of th olon onfirm tht Il / mi vlop svr inflmmtion with pithlil hyprprolifrtion, rypt loss n llulr infiltrtion in th muos n sumuos, whrs w osrv signifintly lowr inflmmtory rspons in Il / Trpv / mi (Fig. 7,). In ition, Il / mi trt with th TRPV.7..9 Figur Gnti n phrmologil inhiition of TRPV CD rs th tivtion of humn primry CD + T lls. (,) Flow ytomtry (lft) of priphrl loo mononulr lls isolt from hlthy humn onors n lft unstimult (US) or stimult for 8 h with nti-cd plus nti-cd8 (oth µg/ml; solul) in th prsn of vrious onntrtions (ov plots) of th TRPV ntgonist SB79 or its vhil (vh;.% DMSO) n stin for CD5 n CD () or HLA-DR n CD (). Numrs in top right qurnts init prnt CD5 + CD + lls () or HLA-DR + CD + lls (). Right, quntifition of th rsults t lft. () IL- in suprntnts of lls trt s in,. () Apoptosis of CD + T lls in, stin for CD n nnxin V n nlyz y flow ytomtry. () TRPV xprssion in frshly isolt priphrl loo mononulr lls trnsft y nuloftion with TRPV-spifi sirna or nontrgting ontrol (Ctrl) sirna n stimult with nti-cd plus nti-cd8 h ltr, ssss y flow ytomtry.9 CD5 HLA-DR..9 CD + CD5 + lls (%) CD + HLA-DR + lls (%) CD US Vh.. US Vh.. SB79 (µm) SB79 (µm) 5 CD + AnnV + lls (%) f sirna Ctrl TRPV α-cd + α-cd US Vh.. Vh.. ntgonist SB79 show ttnut oloni inflmmtion n T ll riv proution of inflmmtory ytokins ompr with tht of Il / mi trt with vhil (Supplmntry Fig. 5). To onfirm th ll-intrinsi rol of TRPV CD in intstinl inflmmtion, w trnsfrr wil-typ or Trpv / niv (CD + CD5RB hi CD5 ) T lls into ripint mi fiint in romintion-tivting gn (Rg / mi) n ssss thir ility to liit olitis 5. W us Rg / ripints of wil-typ niv CD + T lls trnsfrr togthr with rgultory T lls (T rg lls) (CD + CD5RB lo CD5 + ) s ontrol. Th optiv trnsfr of wiltyp niv CD + T lls lon inu svr olitis in th ripints, s rflt y signifint oy wight loss, high iss tivity inx n histologil signs of svr olitis (Fig. 7 g n Supplmntry Fig.,). W i not osrv this ftr th trnsfr of Trpv / niv CD + T lls (Fig. 7 g n Supplmntry Fig.,). Mssiv llulr infiltrtion oinint with svr rypt loss ws vint in th olons of Rg / mi givn trnsfr of wil-typ niv CD + T lls, ut not in th olons of thos givn trnsfr of Trpv / niv CD + T lls (Supplmntry Fig. ). To quntify th inflmmtory ytokins prou in th oloni muos of ths mi, w ultur oloni xplnts x vivo. Th oloni xplnts of Rg / mi givn trnsfr of wil-typ niv CD + T lls rls mor IFN-γ, IL- 7A n TNF thn i thos from ripints of Trpv / niv CD + T lls (Fig. 7h). Th unn of mrna noing svrl proinflmmtory ytokins (Ifng, Il7, Il, Tnf n Il) n hmokins (Cxl, Cxl9, Cxl, Cl n Cl) ws lso lowr in olon SB79 (µm) g h IL- (pg/ml) SB79 (µm) Clls (%) sirna: TRPV β-tin TRPV/ β-tin 5 5 TRPV IgG (trl) TRPV sirna Ctrl sirna (numrs in plot s in Fig. ). (f) Exprssion of th surf tivtion mrkrs CD5 n HLA-DR on gt CD + T lls sort from lls s in t 8 h ftr trnsftion ( h ftr stimultion), n in untrnsft untrt lls (lft). Numrs in outlin rs init prnt CD5 + CD + lls (top row) or HLA-DR + CD + lls (ottom row). (g,h) Immunolot nlysis of TRPV (g) n ELISA of IL- (h) in humn CD + T ll lon 8 h ftr trnsftion n h ftr stimultion s in. P <.5, P <., P <. n P <. (on-wy ANOVA with post-ho Dunntt s tst). Dt r rprsnttiv of four inpnnt xprimnts ( ; mn n s..m. of two or thr onors) or thr inpnnt xprimnts (with on onor ( h)). IL- (pg/ml).5 Ctrl TRPV GMF 8, 9,95 (kd) 9 Ctrl TRPV sirna VOLUME 5 NUMBER NOVEMBER ntur immunology

7 A rt i l s npg Ntur Amri, In. All rights rsrv. Boy wight (%) DAI Il / (no PXC) Il / Trpv / (+ PXC) Il / (+ PXC) 9 8 PXC 7 Tim () 8 T Trpv / T T + T rg f T Trpv / T + PXC Il / (no PXC) Il / Il / Trpv / T + T rg Figur 7 TRPV CD rgults T ll mit olitis. () Wsting iss in Il / n Il / Trpv / mi ftr th inution n synhroniztion of olitis y piroxim (PXC; ox (ottom lft), urtion), prsnt s oy wight rltiv to initil oy wight, st s %. Sttistil nlysis omprs Il / vrsus Il / Trpv /. () Miroopy of olon stions from mi s in, stin with hmtoxylin n osin. Sl rs, µm (min imgs; ojtiv) or mm (insts; ojtiv). () Colitis sors of th mi in. () Boy wight of Rg / ripint mi wks ftr optiv trnsfr of 5 niv homognts from ripints of Trpv / CD + T lls thn in thos of ripints of wil-typ CD + T lls (Supplmntry Fig. ). Th trnsfrr Trpv / CD + T lls srt signifintly lowr mounts of proinflmmtory ytokins (IFN-γ, IL-7A n TNF) thn i th trnsfrr wil-typ CD + T lls (Fig. 7i). Morovr, th numr of CD + T lls prouing IFN-γ, IL-7A or IL- ws lso muh lowr in th spln, msntri lymph nos n lmin propri of th ripints of Trpv / niv CD + T lls thn in thos of th ripints of wil-typ niv CD + T lls (Supplmntry Fig. 7). This suggst tht th rs olitogniity of Trpv / niv CD + T lls ws possily u to ftiv tivtion n iffrntition in situ. In lin with this hypothsis, w foun tht Trpv / OT-II niv CD + T lls wr lss l to iffrntit into fftor lls of th T H, T H n T H 7 susts of hlpr T lls in vitro thn wr ontrol (Trpv +/+ ) OT-II niv lls (Supplmntry Fig. 8). To nlyz th potntil ontriution of TRPV to othr ll typs in th ripints, w ompr th olitis inu y th trnsfr of wiltyp niv CD + T lls into Rg / mi with tht inu in Rg / Trpv / ripints. W foun tht Rg / n Rg / Trpv / ripints vlop similr oy wight loss n oloni inflmmtion n isply similr proinflmmtory CD + T ll rsponss (Fig. 8). Finlly, in lin with thir nhn tivtion profil in vitro (Supplmntry Fig. ), Trpv-TG niv CD + T lls inu xrt olitis ftr trnsfr into Rg / ripints (Supplmntry Fig. 9). Togthr ths rsults monstrt ll-intrinsi rol of TRPV CD in promoting th tivtion n inflmmtory rsponss of T lls (propos mol for th rgultion of T ll tivtion y TRPV, Supplmntry Fig. ). g Colitis sor 8 Colitis sor PXC + + Il / / / Trpv +/+ +/+ / h IFN-γ (ng/mg) T Trpv / T T + T rg..5 DISCUSSION Whil C + rls tivt C + hnnls hv n sri s th mjor sour for th ntry of C + into T lls,, svrl itionl fmilis of hnnls xprss on th plsm mmrn of T lls my hv importnt rols in this pross, inluing voltg-gt C + hnnls n TRP hnnls,5,. In prtiulr, th ontriution of TRP hnnls to this pross is not wll fin. TRP hnnls my rs C + influx in T lls (for xmpl, TRPM) 7 or inrs suh influx (for xmpl, TRPC) 7,8, n it is s yt unlr whthr thy irtly fft TCR signling n T ll tivtion. In th prsnt stuy, w foun tht th TRPV hnnl ws funtionlly xprss in CD + T lls (TRPV CD ) n rport prviously unknown funtion for TRPV yon its wll-rogniz rol s pin rptor,9. Our rsults init n ssntil rol for TRPV CD in th tivtion n th quisition of inflmmtory proprtis y CD + T lls. TRPV xprssion hs n rport in priphrl loo lymphoyts in rts n in humns 7,, ut th funtionlity of th TRPV hnnl ws not ssss n it rmin unknown whthr TRPV hs rol in th tivtion n funtion of T lls. In this stuy, w monstrt tht TRPV ws onstitutivly xprss in mous n humn CD + T lls s wll s in th Jurkt humn lukmi T ll lin. By whol-ll pth lmp, w monstrt th funtionlity of th TRPV hnnl t th plsm mmrn n ror psiin-inu urrnts in wil-typ CD + T lls ut not in Trpv / CD + T lls. Th smllr TRPV urrnt osrv in CD + T lls thn tht rport for in snsory nurons or in lls with htrologous ovrxprssion of TRPV might ount for th following ll-intrinsi ftors: lowr Boy wight (%) IL-7A (ng/mg) 9 CE 5..5 i SP CD + T lls 5 IFN-γ (ng/ml) IL-7A (ng/ml) T Trpv / T 8 7 Tim () TNF (ng/ml).8... ND TNF (ng/mg) T + T rg T Trpv / T T + T rg (CD + CD5RB hi CD5 ) T lls (sort y flow ytomtry) from wil-typ mi ( T) or Trpv / mi (Trpv / T) or mixtur of 5 wil-typ niv CD + T lls plus.5 5 wil-typ (CD + CD5RB lo CD5 + ) T rg lls ( T + T rg ), prsnt s in ; sttistil nlyss ompr T vrsus Trpv / T. () Diss tivity inx (DAI) of th mi in. (f) Miroopy of olon stions from th mi in, stin with hmtoxylin n osin. Sl rs, µm (min imgs; ojtiv) or mm (insts; ojtiv). (g) Colitis sors of th mi in. (h) Cytokin onntrtions in oloni xplnts (CE) from th mi in ftr h of ultur. (i) Cytokin proution y splni CD + T lls isolt from th mi in n rstimult for h with nti-cd ( µg/ml, plt-oun) n nti-cd8 ( µg/ml, solul). P <.5, P <., P <. n P <. (on-wy (,,g i) or two-wy (,) ANOVA with th post-ho Bonfrroni tst). Dt r from on xprimnt rprsnttiv of two xprimnts (mn n s..m. of n = 7 ( ), 8 ( g) or (h,i) mi pr group). ntur immunology VOLUME 5 NUMBER NOVEMBER

8 A rt i l s npg Ntur Amri, In. All rights rsrv. Figur 8 TRPV xprssion in non-cd + T lls os not fft olitis svrity in n optiv-trnsfr mol. () Boy wight of Rg / or Rg / Trpv / ripint mi wks ftr trnsfr of 5 wil-typ or Trpv / niv (CD + CD5RB hi CD5 ) T lls (sort y flow ytomtry), prsnt rltiv to initil oy wight, st s %. () Colon wight of th mi in, prsnt s rtio to olon lngth. () Miroopy of olon stions from mi s in, stin with hmtoxylin n osin. Sl rs, µm (min imgs; ojtiv) or mm (insts; ojtiv). () Colitis sors of th mi in. () ELISA of ytokins in pool splni (SP) n msntri lymph no (MLN) CD + T lls t h ftr rstimultion with nti-cd ( µg/ml plt-oun) n nti-cd8 ( µg/ml solul). P <.5 n P <. (on-wy ANOVA with th post-ho Bonfrroni tst). Dt r rprsnttiv of two inpnnt xprimnts (mn n s..m. of four mi pr group). TRPV xprssion in CD + T lls thn in lls with htrologous ovrxprssion of TRPV; iffrnt post-trnsltionl moifitions (for xmpl, phosphoryltion n glyosyltion) tht oul inrs th TRPV-tivtion thrshol n rsult in lowr snsitivity to psiin; n/or th prsn of iffrnt rgultory protins ssoit with TRPV CD (rfs.,5,). Nonthlss, it is wll known tht vn smll urrnts n prou physiologilly importnt inrss in [C + ] i n hv profoun onsquns on ll physiology 7. Aoringly, y C + imging n flow ytomtry s C + - monitoring thniqus, w intifi TRPV CD s funtionl C + hnnl n show tht it ontriut to TCR-inu C + influx in mnnr inpnnt of stor-oprt C + ntry. W lso onfirm th ontriution of TRPV to TCR-inu C + urrnts y whol-ll pth lmp n foun tht rosslinking of TCRs prou signifintly smllr inwr urrnts in Trpv / CD + T lls thn in wil-typ lls. Our rsults suggst tht TRPV is omponnt of th TCR signling omplx, sin it is rpily rruit to TCR lustrs upon TCR stimultion in Sr-pnnt mnnr. Through th us of Jurkt ll lon tht lks xprssion of th Sr-fmily kins Lk, w show tht Lk rpily tyrosin-phosphorylt TRPV ftr stimultion vi th TCR n rgult TRPV CD hnnl tivity. Tyrosin phosphoryltion y Sr kins hs n shown to ssntil for th tivtion of TRPV hnnls in othr ll typs. In, psiin-inu urrnts in orsl root gnglion nurons r lok y th Sr-fmily kins inhiitor PP n r nhn y th tyrosin phosphts inhiitor soium orthovnt. PP lso olishs urrnts in HEK9 lls trnsft to xprss rt TRPV, whrs otrnsftion of lls to xprss TRPV with n tivt Sr kins (v-sr) rsults in fivfol inrs in psiin-inu urrnts. Finlly, in lls trnsft to xprss ominnt-ngtiv non-rptor (-Sr) tyrosin kins, psiin-inu urrnts r onsirly iminish,. This mo of tivtion y Sr-fmily kinss is not rstrit to TRPV n hs lso n shown for othr mmrs of th TRP fmily. Our finings r thrfor onsistnt with th rport protin-protin intrtion of TRPV n Sr n mphsiz th rol of Sr-fmily kinss in rgulting th tivity of TRPV hnnls. Tyrosin-phosphoryltion y Lk is thrfor possil gting mhnism for TRPV CD ftr stimultion of th TCR. Anlysis of TCR signling in wil-typ n Trpv / CD + T lls rvl iminish tivtion of th p8 n Jnk pthwys n lss trnslotion of NF-κB to th nulus in Trpv / CD + T lls. In lin with thir rs TCR-inu C + influx, Trpv / CD + T lls lso fil to mintin loliztion of NFAT- to th nulus Boy wight (%) IFN-γ (ng/ml) Rg / + T Rg / Trpv / + T Rg / + Trpv / T 9 8 Rg / + T 5 5 Colon wight/lngth (g/m) SP ftr stimultion vi th TCR. Consquntly, Trpv / CD + T lls isply signifintly iminish ytokin-proution profil ftr ithr ntign-spifi stimultion (OVA-lo DCs) or nonspifi stimultion (with nti-cd plus nti-cd8). Colltivly, ths t init tht TRPV ws nssry for propr TCR-inu signling n ytokin proution ut ws ispnsl for CD + T ll prolifrtion unr our xprimntl onitions. Consistnt with our in vitro t, w foun rol for TRPV CD in T ll inflmmtory rsponss in vivo in two iffrnt mols of inflmmtory owl iss. Il / Trpv / mi n Il / mi trt with TRPV ntgonist h lss oloni inflmmtion thn i untrt Il / ontrol mi. In ition, Trpv / niv CD + T lls h n impir ility to liit olitis whn trnsfrr into Rg / ripints. As th tivtion of TRPV in snsory ffrnts ontriuts to nurogni inflmmtion, whih might fft th svrity of olitis 8, w ompr th olitis inu y th trnsfr of wil-typ niv CD + T lls into Rg / mi with tht inu in Rg / Trpv / ripint mi. Gnti ltion of TRPV in onor CD + T lls trmin th svrity of oloni inflmmtion in this mol, ut gnti ltion of TRPV in othr lls of th ripints i not. Th T ll intrinsi rol of TRPV ws furthr monstrt y th nhn tivtion profil n proinflmmtory proprtis of Trpv-TG CD + T lls. Finlly, our rsults init tht inhiition of TRPV CD in mous n humn CD + T lls with TRPV ntgonists or with TRPV- spifi sirna rpitult th phnotyp of Trpv / CD + T lls. As TRPV funtions s noiptor (i.., pin rptor) in snsory nurons 9, TRPV ntgonists wr vlop s nlgsi gnts 9. Th immunomoultory proprtis of TRPV ntgonists intifi in our stuy suggst tht thir pplition might nfiil in ptints with CD + T ll mit immunopthologis suh s inflmmtory owl iss. Mthos Mthos n ny ssoit rfrns r vill in th onlin vrsion of th ppr Rg / + T Rg / Trpv / + T Rg / + Trpv / T SP MLN IL-7A (ng/ml) Rg / Trpv / + T Rg / + Trpv / T MLN TNF (ng/ml) Rg / + T Rg / Trpv / + T Rg / + Trpv / T Colitis sor SP 7 5 MLN VOLUME 5 NUMBER NOVEMBER ntur immunology

9 A rt i l s npg Ntur Amri, In. All rights rsrv. Not: Any Supplmntry Informtion n Sour Dt fils r vill in th onlin vrsion of th ppr. Aknowlgmnts W thnk C. Conh n K. Sur for hlp with msurmnts of C + ; A. Ptpoutin (Th Sripps Rsrh Institut) for Chins hmstr ovry lls trnsft to xprss TRPV; E. Gri n T. Snuth for ss to ltrophysiologil quipmnt; J. L n C. Quinly for isussions; M. Sholl for niml ring; T. Rmlo for ll sorting; S. Shnou for tissu prossing; N. Vrki n L. Ekmnn for hlp with histologil vlutions; n J. Sntini for thnil ssistn with onfol imging t th Nurosin mirosopy shr fility of th Univrsity of Cliforni Sn Digo (support y th Ntionl Institut of Nurologil Disorrs n Strok of th US Ntionl Instituts of Hlth (P 7)). Support y th US Ntionl Instituts of Hlth (U AI95 n P DK58 to E.R., n AI8 to Y.G.), th Cnin Instituts of Hlth Rsrh (MOP-98 to W.A.J.), th Bro Fountion (IBD-R to E.R.), th Crohn s & Colitis Fountion of Amri (SRA 8 to E.R., RFA 57 to S.B., n RFA 97 to P.R..J.), th Europn Molulr Biology Orgniztion (ALTF 88-9 to S.B.), th Fulright Assoition (S.B.), th Philipp Fountion (S.B.) n th Jpn Soity for th Promotion of Sin (Y.A.-N.). AUTHOR CONTRIBUTIO S.B., P.R..J., A.F. n E.R. sign th stuy; S.B., Y.A.-N., P.R..J., K.T., L.A., T.Y., T.H., X.L. n J.M.G.-N. prform most of th in vitro n in vivo xprimnts; S.B. msur th C + flux y flow ytomtry, with th thnil ssistn of X.L. n G.F.; S.S. n Y.G. prform singl-ll C + imging in mous CD + T lls; S.B. n J.L., with th hlp of H.D., prform singl-ll C + imging in Jurkt lls; L.L.N., H.X., S.R.S. n W.A.J. plnn n sign th ltrophysiologil ssys, L.L.N., H.X. n S.R.S. prform ths ssys, n L.L.N., H.X., S.R.S. n W.A.J. wrot th orrsponing stions of th mnusript; R.T. n A.F. prform th humn T ll xprimnts with TRPV ntgonists; S.H. n M.C. took r of th mous olony n gnotyp th mi; L.L.N., H.X., S.R.S., W.A.J., S.B., Y.A.-N., P.R..J., T.Y., K.T., L.A., A.F. n E.R. nlyz n intrprt th t; S.B., E.R. n W.A.J. rvis th mnusript for pulition; n S.B. n E.R. wrot th mnusript. COMPETING FINANCIAL INTERESTS Th uthors lr ompting finnil intrsts: tils r vill in th onlin vrsion of th ppr. Rprints n prmissions informtion is vill onlin t rprints/inx.html.. Oh-hor, M. & Ro, A. Clium signling in lymphoyts. Curr. Opin. Immunol., 5 58 (8).. Gllo, E.M., Cnt-Brrtt, K. & Crtr, G.R. Lymphoyt lium signling from mmrn to nulus. Nt. Immunol. 7, 5 ().. Hogn, P.G., Lwis, R.S. & Ro, A. Molulr sis of lium signling in lymphoyts: STIM n ORAI. Annu. Rv. Immunol. 8, 9 5 ().. Omilusik, K. t l. Th C(v). lium hnnl is ritil rgultor of T ll rptor signling n niv T ll homostsis. Immunity 5, 9 (). 5. Omilusik, K.D., Nohr, L.L., Stnwoo, S. & Jffris, W.A. Wft, wrp, n wv: th intrit tpstry of lium hnnls rgulting T lymphoyt funtion. Front. Immunol., ().. Shwrz, E.C. t l. TRP hnnls in lymphoyts. Hn. Exp. Phrmol. 79, 5 5 (7). 7. Wnning, A.S. t l. TRP xprssion pttrn n th funtionl importn of TRPC in primry humn T-lls. Biohim. Biophys. At 8, (). 8. Vnkthlm, K. & Montll, C. TRP hnnls. Annu. Rv. Biohm. 7, 87 7 (7). 9. Owsinik, G., Tlvr, K., Vots, T. & Nilius, B. Prmtion n sltivity of TRP hnnls. Annu. Rv. Physiol. 8, ().. Ctrin, M.J. t l. Th psiin rptor: ht-tivt ion hnnl in th pin pthwy. Ntur 89, 8 8 (997).. Tousk, F., Mrskov, L., Tisingr, J. & Vlhov, V. A ut snsitiztion of TRPV. Curr. Phrm. Biothnol., 9 ().. Gunthorp, M.J. t l. Intifition n hrtristion of SB-79, potnt n sltiv vnilloi rptor (VR/TRPV) ntgonist. Nurophrmology, 9 ().. Arnkil, B.R., Klin, M.E., Dvison, I.G., Ktz, L.C. & Ehlrs, M.D. Gnti ontrol of nuronl tivity in mi onitionlly xprssing TRPV. Nt. Mthos 5, 99 (8).. Prkh, A.B. & Pnnr, R. Stor pltion n lium influx. Physiol. Rv. 77, 9 9 (997). 5. Smith, G.D. t l. TRPV is tmprtur-snsitiv vnilloi rptor-lik protin. Ntur 8, 8 9 ().. Vlnzno, K.J. t l. N-(-trtiryutylphnyl)--(-hloropyriin--yl)ttrhyropyrzin-(H)-rox-mi (BCTC), novl, orlly fftiv vnilloi rptor ntgonist with nlgsi proprtis: I. in vitro hrtriztion n phrmokinti proprtis. J. Phrmol. Exp. Thr., 77 8 (). 7. Whl, P., Fog, C., Tullin, S. & Thomsn, C. Ioo-rsinifrtoxin, nw potnt vnilloi rptor ntgonist. Mol. Phrmol. 59, 9 5 (). 8. Brr, V.A., Brnot, K.M., Shffr, M.H., Burkhrt, J.K. & Smlson, L.E. Formtion of STIM n Ori omplxs: punt n istl ps. Immunol. Rv., 8 59 (9). 9. Oh-hor, M. Clium signling in th vlopmnt n funtion of T-ling lls. Immunol. Rv., (9).. Hnk, J.H. t l. Disovry of novl, potnt, n Sr fmily-sltiv tyrosin kins inhiitor. Stuy of Lk- n FynT-pnnt T ll tivtion. J. Biol. Chm. 7, 95 7 (99).. Jin, X. t l. Moultion of TRPV y nonrptor tyrosin kins, -Sr kins. Am. J. Physiol. Cll Physiol. 87, C558 C5 ().. Yo, X., Kwn, H.Y. & Hung, Y. Rgultion of TRP hnnls y phosphoryltion. Nurosignls, 7 8 (5).. Zhng, X., Hung, J. & MNughton, P.A. NGF rpily inrss mmrn xprssion of TRPV ht-gt ion hnnls. EMBO J., (5).. Strus, D.B. & Wiss, A. Gnti vin for th involvmnt of th lk tyrosin kins in signl trnsution through th T ll ntign rptor. Cll 7, (99). 5. Wirtz, S. & Nurth, M.F. Mous mols of inflmmtory owl iss. Av. Drug Dliv. Rv. 59, 7 8 (7).. Brg, D.J. t l. Rpi vlopmnt of olitis in AID-trt IL--fiint mi. Gstrontrology, 57 5 (). 7. Luny, P. t l. TRPM rgults lium osilltions ftr T ll tivtion. Sin, 7 77 (). 8. Philipp, S. t l. TRPC mits T-ll rptor-pnnt lium ntry in humn T-lymphoyts. J. Biol. Chm. 78, 9 8 (). 9. Ctrin, M.J. t l. Impir noiption n pin snstion in mi lking th psiin rptor. Sin 88, ().. Shumhr, M.A., Moff, I., Sungunt, S.P. & Lvin, J.D. Molulr loning of n N-trminl spli vrint of th psiin rptor. Loss of N-trminl omin suggsts funtionl ivrgn mong psiin rptor sutyps. J. Biol. Chm. 75, 75 7 ().. Sunrs, C.I., Kun, D.A., Crwfor, A. & Grghty, D.P. Exprssion of trnsint rptor potntil vnilloi (TRPV) n (TRPV) in humn priphrl loo. Mol. Immunol., 9 5 (7).. Englr, A. t l. Exprssion of trnsint rptor potntil vnilloi (TRPV) in synovil firolsts from ptints with ostorthritis n rhumtoi rthritis. Biohm. Biophys. Rs. Commun. 59, (7).. Spinsnti, G. t l. Quntittiv rl-tim PCR ttion of TRPV gn xprssion in humn lukoyts from hlthy n hyposnsitiv sujts. Mol. Pin, 5 (8).. Bhioo, V. t l. Lymphoyt TRPV gn xprssion n MIF loo lvls in young girl linilly ignos with HSAN IV. Clin. J. Pin 7, (). 5. Shin, J.S. t l. Diffrns in snsitivity of vnilloi rptor trnsft to humn mryoni kiny lls n psiin-tivt hnnls in ultur rt orsl root gnglion nurons to psiin rptor gonists. Nurosi. Ltt. 99, 5 9 ().. Voolstr, O. & Hur, A. Post-trnsltionl moifitions of TRP hnnls. Clls, (). 7. Armstrong, D.L., Erxln, C. & Whit, J.A. Pth lmp mthos for stuying lium hnnls. Mthos Cll Biol. 99, 8 97 (). 8. G, M., Prsn, A.E., Kristnsn, N.N., Frnnz C, F. & Clsson, M.H. Blokg of th nurokinin rptor n psiin-inu ltion of th ntri ffrnt nrvs prott SCID mi ginst T-ll-inu hroni olitis. Inflmm. Bowl Dis. 5, 7 8 (9). 9. Morn, M.M., MAlxnr, M.A., Biro, T. & Szllsi, A. Trnsint rptor potntil hnnls s thrputi trgts. Nt. Rv. Drug Disov., ().. Ki, N. t l. Anlysis of th ntiv qutrnry strutur of vnilloi rptor. J. Biol. Chm. 7, 8 89 (). ntur immunology VOLUME 5 NUMBER NOVEMBER

10 npg Ntur Amri, In. All rights rsrv. ONLINE METHODS Rgnts n ntiois. For immunolot nlysis, w us th following ntiois: ntioy to phosphorylt Erk/ (D..E), ntioy to phosphorylt p8 (DF9), ntioy to phosphorylt Jnk (8E), nti- Erk/ (7F5), nti-p8 (9), nti-jnk (95), ntioy to Zp7 phosphorylt t Tyr9 (7) n ntioy to PLC-γ phosphorylt t Tyr78 (8; ll from Cll Signling Thnology); ntioy to Lt phosphorylt t Tyr9 (7-78) n ntioy to phosphorylt tyrosin (G-; oth from Millipor); nti-cdε (M-; s-7), nti-lk (; s-) n nti-trpv (P-9; s-98; ll from Snt Cruz Biothnology); nti-trpv (ACC-; Alomon); monolonl nti-nfat (7; Am); n nti-β-tin (AC-7; Sigm). For immunofluorsn stuis, w us nti-trpv (s-98; Snt Cruz Biothnology), nti-lk (s-; Snt Cruz Biothnology), nti-cd (GK.5; Biosin), nti-cd5 (-F; BD Biosins) n nti-luin- (RB-95; Thrmo Sintifi). For T ll stimultion, w us monolonl nti mous CDε (5-) n nti mous CD8 (PV-; oth from BioXll), n monolonl nti humn CDε (UCHT) n nti humn CD8 (CD8.; oth from Biosin). Othr rgnts inlu th following: PMA (phorol -myristt -tt), ionomyin, thpsigrgin n phytohmgglutinin (ll from Sigm); PP (Cymn Chmil); BCTC (N-(-trtiryutylphnyl)--(-hloropyriin--yl)ttrhyropyrzin- (H)-rox-mi) n I-RTX (ioo-rsinifrtoxin) (Toris); SB79 (Enzo Lif Sins); n nnxin-v n 7-mino-tinomyin D (7-AAD) poptosis-ttion kit (BD Biosins); n CFSE (roxyfluorsin itt suinimiyl str), Ino- AM, n Fur- AM (Invitrogn). Isoltion of mrna n quntittiv PCR. RNA ws isolt with n RNsy Mini Kit oring to th mnufturr s protool (Qign). µg of RNA smpl ws us for rvrs trnsription n synthsis of DNA with qsript DNA suprmix (Qunt Biosins). Quntittiv rl-tim PCR ws prform on n AB7 (Appli Bisosytms) with PrfCT SYBR Grn FstMix (Qunt Biosins). Primrs for spifi trgt gns wr sign on th sis of thir rport squns n wr synthsiz y IDT Thnologis (squns, Supplmntry Tls n ). Th xprssion of gns noing svrl TRP hnnls in mous CD + T lls ws ompr ftr vrifition tht mplifition ffiinis for th iffrnt trgt gns wr similr. For nlysis of TRPV xprssion, quntittiv PCR prouts wr sprt y ltrophorsis through % gros gl, follow y stining with SYBR Sf DNA (Invitrogn) for furthr onfirmtion of th spifiity of th primrs us. Mi. Six- to tn-wk-ol mi wr us for ll xprimntl prours. Spifi pthogn fr C57BL/ (B) mi wr from Hrln Sprgu Dwly or wr r in th niml fility of th Stin Clinil Rsrh uiling t th Univrsity of Cliforni, Sn Digo. Trpv / mi 9, Trpv / mi, mi xprssing Trpv from th Ros lous, mi xprssing Cr from th C promotr (C-Cr), Rg / mi n OT-II mi on th B kgroun (ll from th Jkson Lortoris) wr r in th niml fility of th Stin Clinil Rsrh uiling. For th gnrtion of Trpv / OT-II mi, Il / Trpv / mi or Rg / Trpv / mi, Trpv / mi wr intrross with OT-II mi, Il / mi or Rg / mi rsptivly. Mi with C- Cr rivn xprssion of trnsgn noing TRPV (Trpv-TG) wr gnrt y rossing of mi with llls for ROSA-stop flox -TRPV-IRES-ECFP (whih xprss Trpv from th Ros lous) n C-Cr in th niml fility of th Stin Clinil Rsrh uiling. Mi wr r for mor thn months n wr gnotyp for thy wr us in ny xprimnts. All xprimntl prours wr onut in orn with institutionl guilins for niml r n us of th Univrsity of Cliforni, Sn Digo. Isoltion n stimultion of CD + T lls. Mous CD + T lls wr isolt from th spln or msntri lymph nos with CD + T ll ngtiv sltion kit (975; StmCll). Purity of th nrih popultions ws ontroll y stining with nti-cd (GK.5; Biosin) n nti- TCRβ (H57-597; Biosin) y flow ytomtry n ws typilly >9% for CD + T lls riv from th spln n >97% for CD + T lls riv from th msntri lymph nos. For ELISA, CD + T lls wr stimult with µg/ml plt-oun nti-cd (5-; BioXll) n µg/ml solul nti-cd8 (PV-; BioXll) in RPMI- mium supplmnt with % ht-intivt FCS, mm l-glutmin, U/ml pniillin n µg/ml strptomyin. Cultur suprntnts wr ollt t h n 8 h for nlysis of ytokin proution (ELISA kits; Biosin). For signling xprimnts, CD + T lls wr stimult for vrious tims (for xmpl,, 5, or min) with 5 µg/ml nti-cd n µg/ml nti-cd8 (oth solul; ntiois intifi ov). Splni CD + T lls from OT-II or Trpv / OT-II mi wr isolt s sri ov. Bon mrrow riv DCs wr ultur n hrvst s sri. CD + on mrrow riv DCs or splni DCs wr isolt with CD + positiv sltion kit (8758; StmCll) n thn wr lo for h with µg/ml of I-A -rstrit OVA ppti (mino is -9 (ISQAVHAAHAEINEAGR); PptioGni Rsrh) for th ition of wil-typ or Trpv / OT-II CD + T lls to th ultur (DC/T ll rtio, :). Aftr 5 of oultur, CD + T lls wr rovr n thn wr rstimult for h with nti-cd plus nti-cd8 (intifi ov). Suprntnts wr thn ollt n ytokins wr msur y ELISA. For xprimnts with primry humn CD + T lls, priphrl loo mononulr lls (PBMCs) wr isolt from th loo of hlthy onors y Fioll nsity-grint thniqu. For som xprimnts, CD + T lls h n isolt from th PBMCs n thir popultions xpn with phytohmgglutinin n llogni irrit fr lls ( 5 lls pr wll) s sri. Th CD + T ll lons or th PBMCs wr lft untrt or wr trt with th TRPV ntgonist SB79 n wr stimult for 8 h with ntioy to humn CD (UCHT; Biosin) n ntioy to humn CD8 (CD8.; Biosin) (oth µg/ml; solul) in RPMI- mium. For nlysis of phosphoryltion of TRPV y immunolot, Jurkt T lls (lons E. n J.Cm.; Amrin Typ Cultur Colltion) wr grown in omplt RPMI- mium (supplmnt with 5% ht-intivt FCS, mm l-glutmin, U/ml pniillin, n µg/ml strptomyin) n wr stimult for th pproprit tim with solul ntioy to humn CD (.5 µg/ml; OKT; BioLgn) n ntioy to humn CD8 ( µg/ml; CD8.; Biosin). In vitro T ll iffrntition. CD + CD5 (niv) T lls wr isolt from th spln of wil-typ OT-II or Trpv / OT-II mi, n on mrrow riv DCs wr lo for h t 7 C with OVA ppti ( µg/ml; mino is 9) n wr γ-irrit ( Gy from 7 Cs sour). Thos lls wr ultur togthr (t nsity of T lls pr wll n 5 5 DCs pr wll) in flt-ottom -wll plts in omplt RPMI mium (unlss init othrwis) in th prsn of th following: for T H iffrntition, rominnt mous IL- ( ng/ml; Biosin) n nutrlizing nti-il- ( µg/ml; BVD-G; BioXll); for T H iffrntition, rominnt mous IL- ( ng/ml), nutrlizing nti-ifn-γ ( µg/ml; XMG.; BioXll) n nutrlizing nti-il- ( µg/ml; R-5D9; BioXll); for T H 7 iffrntition, rominnt mous IL- ( ng/ml) n TGF-β ( ng/ml; oth from Biosin), plus nutrlizing nti-ifn-γ ( µg/ml; XMG.; BioXll) n nti-il- ( µg/ml; BVD-G; BioXll) in omplt IMDM; n for T rg ll iffrntition, rominnt mous TGF-β ( ng/ml; Biosin) n IL- ( ng/ml; Biosin). At y, rominnt mous IL- ( ng/ml) ws to th T H n T H ulturs. Aftr, CD + T lls wr rovr n thn wr rstimult for 5 h or not with µg/ml plt-oun nti-cd n µg/ml solul nti-cd8 (ntiois intifi ov) in th prsn of GolgiStop (BD Biosins). Intrllulr ytokins wr msur with nti- IFN-γ (XMG.), nti-il- (B), nti-il-7a (Bio7B7) n nti-il- (JES5-E) oring to th mnufturr s instrutions (Biosin). Knokown of TRPV. Primry humn CD + T ll lons or frshly isolt PBMCs wr trnsft with nm TRPV-spifi sirna ( pool of thr iffrnt sirna uplxs; s-8; Snt Cruz Biothnology) or ontrol sirna (Non-Trgting sirna #; Dhrmon) t nsity of 5 lls pr µl humn T ll nuloftor solution (VPA-; Lonz) with th Amx Nuloftor II vi (progrm U-; Lonz). Aftr nuloftion, lls wr immitly trnsfrr into prwrm omplt RPMI- n wr ultur in -wll plt t 7 C in 5% CO humiifi inutor. Six hours ftr trnsftion, lls wr stimult for furthr h with ntioy to humn CD n ntioy to humn CD8 (intifi ov; oth µg/ml, solul). TRPV-knokown ffiiny in lls ws thn nlyz (y flow ntur immunology oi:.8/ni.9