Multiple sclerosis (MS) is a chronic disease of the central

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1 Orphn nucler receptor NRA expressed in T cells from multiple sclerosis medites production of inflmmtory cytokines Yoshimitsu Doi*, Shinji Oki*, Tomoko Ozw*, Hirohiko Hohjoh, Schiko Miyke*, nd Tkshi Ymmur* Deprtments of *Immunology nd Moleculr Genetics, Ntionl Institute of Neuroscience, Ntionl Center of Neurology nd Psychitry, -- Ogwhigshi, Kodir, Tokyo 87-8, Jpn Communicted y Tdmitsu Kishimoto, Osk University, Osk, Jpn, April, 8 received for review Mrch, 8 Multiple sclerosis MS is n utoimmune disese of the centrl nervous system CNS medited y Th7 nd Th cells. DNA microrry nlysis previously showed tht NRA, n orphn nucler receptor, is strongly up-regulted in the peripherl lood T cells of MS. Here, we report tht NRA plys pivotl role for mediting cytokine production from pthogenic T cells. In experimentl utoimmune encephlomyelitis EAE, n niml model of MS, NRA, ws selectively up-regulted in the T cells isolted from the CNS. Strikingly, forced expression of NRA ugmented promoter ctivities of IL-7 nd IFN- genes, leding to n excessive production of these cytokines. Conversely, tretment with for NRA, resulted in significnt reduction in the production of IL-7 nd IFN-. Furthermore, tretment with NRA reduced the ility of encephlitogenic T cells to trnsfer EAE in recipient mice. Thus, NRA is n essentil trnscription fctor for triggering the inflmmtory cscde of MS/EAE nd my serve s therpeutic trget. IL-7 interferon- EAE Th7 Multiple sclerosis MS is chronic disese of the centrl nervous system CNS, ccompnying multiple foci of inflmmtory lesions. MS is thought to hve n utoimmune pthogenesis, involving utoimmune T cells rective to myelin ntigens. Development of the CNS inflmmtion is triggered y proinflmmtory cytokines produced y the utoimmune T cells, which penetrte into the CNS prenchym fter eing ctivted in the periphery,. Although the precise mechnism for the peripherl T cell ctivtion remins oscure, studies indicted possile roles for cross-rective peptides, cytokines, or superntigen. Experimentl utoimmune encephlomyelitis EAE is prototype utoimmune disese model tht cn e induced in lortory nimls y ctive immuniztion with myelin ntigens mag or y pssive trnsfer of mag-specific T cells. Becuse Th cell clones rective to mag re cple of inducing clinicl nd pthologicl mnifesttions of EAE in nive mice, it hs long een elieved tht Th cells producing IFN- ply centrl role in the pthogenesis of EAE nd MS. This postulte is lso supported y the pst experience tht clinicl ppliction of IFN- tretment for MS turned out to worsen the disese 6. Furthermore, tretment with peptide nlogue of myelin sic protein MBP resulted in disese excertion long with n expnsion of MBP-rective Th cells 7. These results hve een repetedly mentioned to support the Th-medited pthogenesis of MS. However, this dogm hs recently een chllenged. Nmely, despite n ovious reduction of Th cells, mice deficient for IFN- or IFN- receptor 8 or for IL- signling were susceptile to EAE 9,. Susequent studies hve clrified tht IL- rther thn IL- is essentil for EAE induction. Ltely, the IL--dependent pthogenic T cells were identified s Th7 cells, novel helper T cells producing IL-7,. Currently, it is widely pprecited tht Th7 cells re crucil in the development of utoimmune diseses either independently or collortively with Th cells. DNA microrry nlysis reveled n up-regultion of IL-7 in the rin lesions of MS. More recently, pthologicl study hs demonstrted tht IL-7 secreting T cells re present in ctive lesions of MS. Gene expression profiling provided numer of potentil cndidte molecules tht might e pproprite s therpeutic trget, 6. We recently chrcterized gene signture of peripherl lood T cells from Jpnese MS ptients nd found tht nucler orphn receptor NRA is most significntly overexpressed in MS 7. NRA muttions re reported to cuse fmilil Prkinson s disese, reflecting its essentil role in the development nd survivl of sustnti nigr neurons 8. In contrst, much less ttention hs een pid onto its role in T cells. NRA fmily memers NRA nd - were shown to medite poptotic processes of mture 9, nd immture T cells,. However, these studies do not give insights into n overexpressed NRA in MS. Here, we report tht NRA is trnscription fctor regulting the expression of key cytokines in the pthogenesis of MS, including IL-7. Furthermore, we reveled tht silencing NRA expression y specific effectively prevents the production of the cytokines, therey inhiiting their pthogenic potentils to medite EAE. Results Up-Regultion of NRA in Peripherl Blood T Cells of MS. We nlyzed gene expression profiles of peripherl lood T cells from MS nd control sujects 7,. Comprison of the ptients nd helthy donors hs reveled tht 86 of,6 genes re differentilly expressed etween MS nd controls. Among genes up-regulted in MS, NRA ws most significntly overexpressed in MS in sttisticl P vlues nd n increse rtio.6-fold. To consolidte the overexpression of NRA in MS, we performed quntittive RT-PCR for NRA expression, using the sme smples previously nlyzed. Expression of NRA in T cells from MS incresed -fold on verge compred with helthy donors Fig. ; P.. T Cell Expression of NRA in EAE. NRA is trnscription fctor of steroid/thyroid receptor fmily implicted in vrious cellulr responses such s steroidogenesis, neuronl development, therogenesis, nd cell cycle regultion. However, its role in Author contriutions: Y.D., S.O., H.H., S.M., nd T.Y. designed reserch; Y.D., S.O., nd T.O. performed reserch; Y.D., S.O., S.M., nd T.Y. nlyzed dt; nd S.O. nd T.Y. wrote the pper. The uthors declre no conflict of interest. Freely ville online through the PNAS open ccess option. To whom correspondence my e ddressed. E-mil: soki@ncnp.go.jp or ymmur@ ncnp.go.jp. This rticle contins supporting informtion online t 8/DCSupplementl. 8 y The Ntionl Acdemy of Sciences of the USA MEDICAL SCIENCES cgi doi.7 pns.8 PNAS June 7, 8 vol. no

2 NRA SPL CNS NRA of mrna level HS MS. %.7 % Isotype control Anti-IL-7 ntiody IL-7-PE. **.6 % 8. % Fig.. Quntittive nlysis of NRA trnscription etween MS nd controls. CD T cells were isolted from PBMC of 7 MS ptients nd of 9 helthy donors, nd totl RNA ws extrcted. cdna ws synthesized nd the expression levels of NRA trnscript were nlyzed y quntittive RT-PCR. Ech smple ws normlized to GAPDH to djust for vritions. Open circles, MS ptients; filled circles, helthy controls. Brs indicte men vlues of ech group. The sttisticl difference ws determined y two-sided Student t test **, P.. EAE score EAE score 7 8 dys 7 dys ofnra mrna level ofnra mrna level.6... d NRA d d7 dd d8 NRA d9 d d SPL DLN PBMC SPL DLN CNS Fig.. Kinetic nlysis of NRA expression in the disese course of EAE. Left EAE ws induced in B6 mice y immuniztion with MOG - in CFA. Mice were killed on dys 7,, nd 8 fter immuniztion, nd T cells were isolted from dln, SPL, or PBMC, using nti-cd mgnetic eds. Right Totl RNAs were isolted from the T cell popultions, nd the expression levels of NRA were determined y quntittive RT-PCR. One representtive dt from three independent experiments is shown, nd dt re expressed s men SEM n for ech. EAE induced in B6 mice with MOG -. Clinicl scores were expressed s men SEM n. Here, we determined NRA expression in CD T cells isolted y using EPICS ALTRA cell sorter. The lymphoid cells SPL, dln, nd CNS were pooled from four mice on dys, 9,, nd nd used for cell sorting nd RT-PCR nlysis. The purity of the CNS-derived CD T cells ws 9%. Isotype control Anti-IFNγ ntiody IFNγ-PE Fig.. Accumultion of IL-7 or IFN- -producing inflmmtory T cells in the CNS. Mononucler cells were isolted from spleen or CNS on dy 7 fter immuniztion nd stimulted with PMA ng/ml nd ionomycin g/ml in the presence of mm monensin for h. Production of IL-7 nd IFN- ws nlyzed for the gted CD T cell popultion y intrcellulr cytokine stining. Blck line represents smples stined with either nti-il-7 or nti- IFN- A, nd the filled histogrm represents smples stined with isotype control. Given vlues show the percentge of cytokine producing-t cells present in ech pnel. T cell-medited utoimmune diseses is unknown. Therefore, we explored the functionl involvement of NRA in EAE induced in C7BL/6 B6 mice y immuniztion with MOG. CD T cells were isolted from SPL, dln, nd PBMC fter EAE induction nd the expression levels of NRA gene were mesured y quntittive RT-PCR Fig. Right. NRA expression ws detectle in PBMC-T cells on dys,, nd 8, showing mximum vlue on dy, which ws well correlted with the clinicl severity of EAE Fig. Left. NRA expression in SPL-T cells nd dln-t cells ws lso correlted with the severity of EAE, ut only mrginlly. In the course of EAE, mag-primed T cells would ccumulte into the CNS nd produce inflmmtory cytokines, leding to the formtion of inflmmtory lesions. We next exmined kinetic chnge of NRA in the T cells infiltrting into the CNS. As ssessed y quntittive RT-PCR, remrkle expression of NRA ws oserved in the CNS-T cells on dy 9, when n erly EAE sign ecme evident Fig.. The expression level decresed grdully therefter, ut ws still significnt until dy. These results suggest tht the CNS-T cells lso express NRA, ut the expression kinetics significntly differed from tht of PBMC-T cells. Accumultion of IL-7- nd IFN- -Producing T Cells in the CNS of EAE. Th cells specific for mag hve long een thought to induce EAE through their production of IFN-. However, recent studies indicte tht Th7 rther thn Th cells my ply centrl role. To mke this point cler in our experimentl setting, we exmined the ility of the CNS-T cells to produce IFN- nd IL-7. Mononucler cells were recovered from the CNS nd SPL on dy 7, nd stimulted with PMA nd ionomycin P/I. After immunostining, expression of IL-7 or IFN- in the CD T cells ws nlyzed y flow cytometry. Mjor proportions of the CNS-T cells were found to produce IL-7.7% of the cells or IFN- 8.% fter stimultion Fig.. In contrst, spleen cells contined lower numer of cells producing these cytokines. Trnscriptionl Up-Regultion of IL-7 nd IFN- After Introduction of NRA. The concomitnt expression of inflmmtory cytokines nd NRA hs guided us to investigte whether NRA directly ffects cytokine gene expression s trnscription fctor, using luciferse reporter plsmids contining the promoter frgment of IL-7, IFN-, or IL-. NRA gene trnsduction would result in twofold ugmenttion of IL-7 promoter ctivity nd, for IFN-, n even higher -fold induction Fig.. A significnt induction of IL- promoter ctivity ws lso noted. Intriguingly, n introduction of NRA plsmid 88 cgi doi.7 pns.8 Doi et l.

3 luciferse ctivity Normlized luciferse ctivity Normlized IL-7 IFN-γ IL P/I NRA LcZ NRA LcZ NRA LcZ 7 6 IL Plsmid conc. µg NRA LcZ CD -NRA -NRA GFP -NRA -NRA LTR IRES egfp LTR R R R R R:. % R: 8. % LTR NRA IRES egfp LTR R:. % R:. % IL-7-PE Fig.. Promoter ctivities of cytokine genes in the presence of NRA. The effect of NRA expression on IL-7, IFN-, nd IL- promoter ctivity. A reporter plsmid contining promoter of cytokine gene g nd Renill luciferse plsmid ng were introduced into EL cells y electroportion, together with pcdna-nra or pcdna-lcz g. Cells were stimulted for 8 h with P/I. Luciferse ctivity ws determined for ech cell lyste fter normliztion to the Renill luciferse ctivity. One representtive dt from three independent experiments is shown. Dt re expressed s men SD. The effect of NRA expression on sl promoter ctivity of IL-7 gene. EL cells trnsfected with pcdna-nra or pcdna-lcz together with IL-7 reporter plsmid nd Renill luciferse plsmid s desried in were cultured for 8 h without stimultion. One representtive dt from three independent experiments is shown. Dt re expressed s men SD. without P/I stimultion lso ugmented sl promoter ctivity of IL-7 genes in dose dependent mnner Fig.. Similrly, sl promoter ctivity of IFN- ws promoted dt not shown. Retrovirl Trnsduction of NRA Gene Enhnces Expression of Inflmmtory Cytokine in Primry T Cells. The results otined in EL lymphom cells need to e verified in more physiologicl settings. Next, we exmined whether forced expression of NRA my ffect the expression of cytokines in primry rodent T cells. Bicistronic retrovirl vector contining NRA gene frgment -NRA or empty vector were used for production of retroviruses Fig.. We infected the B6 T cells with either of the retroviruses s descried in ref. 6 nd compred the cytokine production etween GFP-positive infected nd GFP-negtive uninfected CD T cells y intrcellulr cytokine stining Fig. Top. CD T cells infected with - NRA-introduced retrovirus showed twofold enhncement of IL-7 expression 8.% compred with those infected with control retrovirus.% fter stimultion with P/I. In contrst, IL-7 production y uninfected T cells in either pnel ws lmost equivlent Fig. Middle. Furthermore, one-third of the CD T cells infected with -NRA-introduced retrovirus showed mssive IFN- expression.% compred with control retrovirus.% Fig. Bottom. Silencing of NRA Gene Expression Results in Reduced Production of IL-7 nd IFN-. Reporter gene nlysis nd retrovirl trnsduction experiments demonstrted tht T cell production of IL-7 nd IFN- is controlled y NRA Figs. nd. We further explored whether silencing of NRA gene my ffect the production of inflmmtory cytokines y CD T cells. An NRA-specific ws selected from three s sed on the inhiitory efficcy. The trgeting sequence of the NRA R: 7. % R:. % R R Isotype control R:. % R:. % IFN-γ-PE Fig.. The effect of retrovirlly trnsduced NRA on cytokine production y primry murine CD T cells. DNA frgments encoding wild-type NRA were cloned into the W icistronic retrovirl vector. LTR, long terminl repet; IRES, internl riosome entry site; egfp, enhnced green fluorescence protein. Splenic CD T cells were infected with retrovirus encoding NRA or control retrovirus, nd CD GFP T cells nd CD GFP T cells were gted s R nd R, respectively. Forced expression of NRA incresed the numer of CD T cells producing IL-7 or IFN-. The histogrm shows intrcellulr cytokine stining on the gted cells R or R. Blck line represents cells in R gte GFP stined with either nti-il-7 or nti-ifn- A, nd the filled histogrm represents cells in R gte GFP stined with isotype control. Given vlues show the percentge of cytokine producing-t cells present. is completely conserved etween mice nd humn. Therefore, we could pply it to humn T cells nd study whether NRA could e therpeutic trget in humn MS. In preprtory experiment, using FITC-leled, the trnsfection efficiency ws found to e 9%. We purified CD T cells from humn PBMC nd trnsfected them with the NRA or, using nucleofector II. The cells were stimulted with immoilized nti-cd A. As shown in Fig. 6, silencing NRA gene expression resulted in % reduction of IL-7 nd IFN- production. However, production of TNF-, IL-, or IL- ws not chnged significntly fter tretment Fig. 6. Intriguingly, the tretment lso induced modest reduction of IL- production. The moleculr mechnism of this inhiition is not clrified yet. Becuse silencing of NRA expression rther selectively inhiited the expression of inflmmtory cytokines, it is rgule tht NRA my e good trget for therpeutic intervention of MS. In this line, we next exmined whether the NRA is effective for inhiiting production of inflmmtory cytokines in MS. For this im, CD T cells were isolted from pirs of n MS ptient nd n ge- nd sex-mtched helthy donor nd were stimulted with nti-cd A fter eing trnsfected with the NRA or. We found tht the tretment significntly reduced the production of IL-7 nd IFN- y T cells from MS or helthy donors [supporting informtion SI Fig. S]. Agin we oserved some reduction of IL- fter tretment. However, the showed little effect on MEDICAL SCIENCES Doi et l. PNAS June 7, 8 vol. no. 88

4 l i i IL-7 conc. 7 Anti-CD ma IL I FN- γ conc. pg/m l IFN-γ prolifertion 7 ** ** * N.D. N.D. - + H-thymidine uptke x ccpm - + c l s c o r e n E A E c ******* 7 8 dys T NF- α conc. TNF-α IL- conc. IL- ** Fig. 6. The effect of NRA gene silencing on T cell cytokine production. Specific inhiition of T cell production of IL-7 nd IFN- y tretment. Humn CD T cells derived from PBMC were trnsfected with or nd stimulted y immoilized nti-cd A for 8 h. Cytokine levels in the culture superntnt were determined y ELISA or CBA humn Th/ cytokine kit. Prolifertion rte ws mesured y H-TdR uptke. Effect of tretment for T cell production of TNF-, IL-, IL-, nd IL- fter stimultion with immoilized nti-cd A. The dt re expressed s men SD *, P.; **, P.; Mnn Whitney U test. IL- conc. IL- IL- conc. IL- LFB stin HE stin production of TNF-, IL-, nd IL- from T cells used for ssys Tle S. Ameliortion of EAE y Silencing of NRA. Finlly, we investigted the therpeutic impliction of the experiments in model of pssively induced EAE, induced y doptive trnsfer of mag-ctivted LN cells. We prepred lymphoid cells from dln of SJL/J mice dys fter immuniztion with PLP 9. The dln cells were trnsfected with the NRA or control RNA nd stimulted with PLP 9 in vitro. Three dys lter, the cultured cells enriched in lympholsts were trnsferred to irrdited nïve SJL/J mice. In ddition to evluting clinicl mnifesttions, histology ws ssessed y hemtoxylin-eosin HE nd luxol fst lue LFB stining of prffin-emedded spinl cord sections. Notly, severity of clinicl Fig. 7 nd histologicl EAE on dy Fig. 7 ws significntly prevented in -treted group compred with -treted group Fig. 7. These results suggest tht modultion of NRA expression y specific s or other chemicl compounds might e promising tretment for ctive MS tht re hroring potent encephlitogenic T cells. Discussion Although mag-specific T cell clones isolted from the peripherl lood hs een widely used to gin insights into the pthogenesis of MS 7, nlysis of polyclonl T cells hs een undervlued for long time. However, it ws recently demonstrted tht peripherl T cells from MS nd helthy sujects significntly differ in surfce phenotype or gene expression profiling 7,, 8. Using cdna microrry, we hve identified NRA s gene most significntly up-regulted in the peripherl T cells of MS 7. We conducted the present study to clrify the impliction of this interesting oservtion. Inspired y the recent discovery tht retinoid-relted orphn receptor t ROR t is essentil for Th7 cell differentition 9 nd tht retinoic cids ply regultory role in Th7 cell differentition, we hve focused our efforts to explore the possile role of NRA in cytokine regultion. Reporter gene nlysis nd retrovirl trnsduction of NRA clerly demonstrted tht T cell production Fig. 7. The effect of T cell silencing of NRA expression on pssive EAE. Inguinl nd poplitel LNs cells were collected from femle SJL/J mice dys fter immuniztion with PLP 9-, nd were trnsfected with for NRA or, using HVJ-E vector kit. The cells were cultured in complete medi for 8 h. Then the medi were replced with fresh complete medi contining g/ml PLP 9-, nd the cells were stimulted for nother dys. After expnsion, cells were hrvested nd trnsferred i.p. 6 cells per mouse into Gy-irrdited nïve SJL/J mice n followed y i.p. injection of PT. Men SEM clinicl scores were indicted. *, P. y Mnn Whitney U test. Histologicl nlysis of spinl cords removed on dy fter doptive trnsfer of PLP 9- -rective T cells. Sections otined from cervicl cord regions were stined with HE or LFB. Infiltrtion of mononucler cells nd demyelintion of the cervicl cord regions were nlyzed for mice injected with PLP 9- -rective T cells pretreted with or for NRA. of inflmmtory cytokines, including IL-7 nd IFN-, is regulted y NRA, wheres silencing of NRA y specific prevents expression of these cytokines. Furthermore, tretment with the reduced the ility of pthogenic T cells to doptively trnsfer EAE. These results hve identified previously unchrcterized role for NRA in the regultion of T cell production of inflmmtory cytokines. NRA is memer of the orphn nucler NRA sufmily tht consists of NRA lso referred to s Nur77, NRA Nurr, nd NRA NOR-. The NRA memers shre highly conserved zing finger DNA inding domin nd less conserved puttive lignd-inding domin. All these memers ind to the DNA sequence NBRE AAAGGTCA or NurRE to ctivte trget gene expression. NRA nd NRA cn lso heterodimerize with retinoic X receptor RXR nd ctivte gene expression through DR. They exert pleiotropic functions nd re clssified s immedite erly genes induced y physiologicl nd physicl stimuli. Studies of gene-trgeted mice hve shown tht NRA nd NRA ply criticl role in T cell poptosis during the thymocyte development,. In contrst, developing thymocytes in NRA deficient mice p cgi doi.7 pns.8 Doi et l.

5 per to e norml,, which distinguishes NRA from other NRA memers. Involvement of orphn nucler receptor in T cell differentition hs recently ttrcted rod ttention, ecuse ROR t, splice vrint of ROR, ws found to ply n essentil role in the development of Th7 cells 9. ROR /ROR t were reported to ply n essentil function in survivl of CD CD8 thymocytes, nd in the genertion of fetl lymphoid tissue inducer LTi cells. It is prticulrly intriguing tht the consensus inding sequence for ROR [A/T AGGTCA] overlps with tht for NRA NBRE; AAAGGTCA, which hs encourged us to explore the functionl role of NRA in the production of IL-7 nd IFN-. Although the moleculr mechnism of cytokine production through the induced expression of NRA is not cler yet, NRA nd ROR t my hve n overlpping role in regulting the development nd effector functions of Th7 cells. NRA expression in the CNS-infiltrting T cells showed pek vlue t very erly phse of EAE dy 9 Fig.. We speculte tht this proly coincides with the entry of encephlitogenic cells into the CNS,. Consistently, similr kinetic chnge ws found in expression of T-et nd ROR t inthe CNS-T cells dt not shown. In contrst, up-regultion of NRA in peripherl lood T cells ws significntly delyed. This is likely to result from lte ctivtion of peripherl T cells fter peripherl recruitment of ntigen presenting cells engulfing myelin nd/or peripherl dispersion of myelin protein or its frgments. By pplying specific, we showed tht locking NRA expression is effective for inhiiting production of IL-7 nd IFN- from T cells from helthy donors nd MS ptients. Therpeutic impliction ws further demonstrted y using n doptive trnsfer EAE model. Becuse Th7 cells were identified s mjor plyer in utoimmunity,, it is sometimes rgued tht Th7 cells would e sole potent inducer of utoimmune inflmmtion. However, T-et-deficient mice nd Stt-deficient mice tht oviously lck Th cells would resist ginst induction of EAE, lthough they mintin lrge numer of Th7 cells 6, 7. This suggests tht oth Th nd Th7 cells re required for induction of full-lown EAE 8. In this context, the ility of the NRA to inhiit production of oth IL-7 nd IFN- suggests the dvntge of NRA trgeting therpy in controlling utoimmune inflmmtion. Mterils nd Methods EAE Induction. Active EAE ws induced with myelin oligodendrocyte glycoprotein MOG mino cids MOG - ; MEVGWYRSPFSRVVHLYRNGK s descried in ref. 9. Femle B6 mice were immunized s.c. with g of MOG mixed with mg of het-killed Mycocterium tuerculosis H7RA emulsified in Freund s djuvnt CFA. Pertussis toxin PT ng ws injected i.p. on dys nd fter immuniztion. Clinicl signs were scored dily s follows:, no clinicl signs;, loss of til tonicity;, flccid til;, prtil hind lim prlysis;, totl hind lim prlysis; nd, fore nd hind lim prlysis. Quntittive RT-PCR. DNse-treted totl RNAs were processed for cdna synthesis, using rndom hexmer primers nd SuperScript II reverse trnscriptse Invitrogen. cdnas were mplified y PCR on Light Cycler ST Roche Dignostics y using Light Cycler-FstStrt DNA Mster SYBR Green I kit Roche. Vlues for ech gene were normlized to those of housekeeping gene GAPDH to djust for vritions etween different smples. Forwrd primer for mplifying humn NRA gene ws -CGACATTTCTGCCTTCTCC- nd reverse primer -GGTAAAGTGTCCAGGAAAAG-. Mouse NRA forwrd primer ws designed s -GCATACAGGTCCAACCCAGT- nd reverse primer -AATGCAGGAGAAGGCAGAAA-. To evlute silencing efficcy of NRA-specific s, expression of NRA gene ws quntified y RT-PCR, using the primers to flnk the trget sequence forwrd, - TGCCACCACTTCTCTCCCCA- ; reverse, -GCGGCATCATCTCCTCAGAC-. Luciferse Assys. Ten million of EL thymom cells suspended in l of cold PBS nd trnsfected with g of pcdna-nra or pcdna-lcz in the presence of g of reporter plsmid, ng of Renill luciferse plsmid, nd g of DEAE-DEXTRAN Sigm y electroportion V, 97 F, time constnt ms with GenePulser electroportor II Bio-Rd. Six hours lter, cells were stimulted with ng/ml PMA nd g/ml ionomycin for h, followed y nlysis for luciferse ctivity. The dt were normlized for internl controls of Renill luciferse ctivity. Retrovirl Infection. Mouse CD T cells purified y AutoMACS using mouse CD T isoltion kit Miltenyi Biotec were stimulted with immoilized nti- CD A nd solule nti-cd8 A in complete medium supplemented with IL- units/ml for 8 h efore infection. The primed CD T cells were infected twice with retroviruses produced y 9T cells cotrnsfected with retrovirl vector nd pcl-eco pckging vector. The T cells were cultured in the presence of units/ml of IL- for dys nd were then sujected to intrcellulr cytokine stining. Silencing Effects of NRA on Pssive EAE. To evlute n effect of NRA, n doptive trnsfer EAE model in SJL/J mice ws pplied, ecuse consistent disese could e induced reltively esily. Femle SJL/J mice 8 weeks old Chrles River Lortories were immunized s.c. with g of proteolipid protein PLP mino cids 9 PLP 9- ; HSLGKWLGHPDKF nd mg of het-killed M. tuerculosis H7RA in CFA. Inguinl nd poplitel LNs hrvested dys fter immuniztion were trnsfected with s, using hemggultinting Virus of Jpn envelope HVJ-E vector kit GENOMEONE; Ishihr Sngyo. Eight hours lter, the cells were stimulted with PLP 9- peptide g/ml. After dys, collected cells were injected i.p. 6 cells per ody into irrdited mice Gy/ody with intrpelitonel injection of PT. For conventionl histologicl nlysis of EAE, prffin-emedded spinl cords were stined with either HE or LFB. Sttistics. For sttisticl nlysis, nonprmetric Mnn Whitney U test or Student t test ws used. P. ws considered sttisticlly significnt. Supporting Informtion. For further detils, see SI Mterils nd Methods. ACKNOWLEDGMENTS. We thnk Myumi Fujit for EAE induction, Miho Mizuno, Chihru Tomi, nd Yuki Kiki for excellent technicl ssistnce. This work ws supported y grnts from the Ministry of Helth, Lour nd Welfre of Jpn. MEDICAL SCIENCES. Sospedr M, Mrtin R Immunology of multiple sclerosis. Annu Rev Immunol : Hickey WF, Hsu BL, Kimur H 99 T-lymphocyte entry into the centrl nervous system. J Neurosci Res 8: 6.. Kwkmi N, et l. Live imging of effector cell trfficking nd utontigen recognition within the unfolding utoimmune encephlomyelitis lesion. J Exp Med :8 8.. Hur EM, et l. 7 Osteopontin-induced relpse nd progression of utoimmune rin disese through enhnced survivl of ctivted T cells. Nt Immunol 8:7 8.. Gold R, Linington C, Lssmnn H 6 Understnding pthogenesis nd therpy of multiple sclerosis vi niml models: 7 yers of merits nd culprits in experimentl utoimmune encephlomyelitis reserch. 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