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1 IL-7 ts on DCs to suppress the T ell response nd utoimmunity y induing expression of the immunoregultory moleule CD9 Ivn D Msnfroni,, Ad Yeste,, Silvio M Vieir, Evn J Burns, Bonny Ptel, Ido Slom, Yn Wu, Lior Myo, Rotem Ben-Hmo, Sol Efroni, Vijy K Kuhroo, Simon C Roson & Frniso J Quintn npg Nture Ameri, In. All rights reserved. Dendriti ells (DCs) ontrol the lne etween effetor T ells nd regultory T ells in vivo. Hene, the study of DCs might identify mehnisms of disese pthogenesis nd guide new therpeuti pprohes for disorders medited y the immune system. We found tht interleukin 7 (IL-7) signling in mouse DCs limited the genertion of effetor ells of the T H nd T H 7 susets of helper T ells nd the development of experimentl utoimmune enephlomyelitis (EAE). The effets of IL-7 were medited t lest in prt through indution of the immunoregultory moleule CD9 in DCs. IL-7-indued CD9 deresed the extrellulr onentrtion of ATP nd downregulted nuleotide-dependent tivtion of the NLRP inflmmsome. Finlly, therpeuti vintion with IL-7-onditioned DCs suppressed estlished relpsing-remitting EAE. Thus, IL-7 signling in DCs limited pthogeni T ell responses nd the development of utoimmunity. The dysregulted tivity of effetor ells of the T H nd T H 7 susets of helper T ells results in the development of tissue inflmmtion nd utoimmunity. Myelin-speifi T H nd T H 7 ells, for exmple, ontriute to disese pthogenesis in multiple slerosis (MS) nd its niml model, experimentl utoimmune enephlomyelitis (EAE). During EAE, dendriti ells (DCs) ontrol the tivtion nd differentition of myelin-speifi effetor T ells nd regultory T ells (T reg ells),. Moreover, DCs isolted from ptients with MS produe lrge mounts of T H - nd T H 7-polrizing ytokines, whih suggests tht DCs promote the genertion of the pthogeni T ell response in MS. Indeed, DCs ontrol severl pthogeni mehnisms ssoited with the development of entrl nervous system (CNS) utoimmunity. DCs promote the entry of T ells into the CNS, the tivtion nd differentition of pthogeni T ells in the CNS nd the spreding of the utoimmune response to new CNS epitopes 7. Thus, it is importnt to study the pthwys tht regulte DC tivity during the ourse of utoimmunity, to identify mehnisms of disese pthogenesis nd lso to develop new pprohes for therpeuti intervention. IL-7 is ytokine struturlly relted to IL- nd is omposed of p suunit nd the produt of Epstein-Brr virusindued gene (Ei). IL-7 signls through reeptor omposed of the ommon IL- reeptor hin gp (whih is used y severl other memers of the IL- nd IL- fmilies) nd unique IL-7 reeptor α-hin (IL-7RA) tht is homologous to the IL-Rβ hin of the IL- reeptor. IL-7 is produed y DCs in response to tivtion vi Toll-like reeptors through mehnism tht involves the utorine effets of interferon-β (IFN-β) 9. Indeed, the therpeuti effets of IFN-β dministrtion on relpsing-remitting MS hve een linked to the indution of IL-7 prodution y DCs. On the sis of its struturl homology to IL- nd its ility to trigger IFN-γ prodution, IL-7 ws initilly thought to e proinflmmtory ytokine. However, it ws susequently disovered tht IL-7 suppresses T H, T H nd T H 7 responses nd limits CNS inflmmtion in severl experimentl models. In the EAE model, the dministrtion of IL-7 inhiits disese development. Conversely, the lk of funtionl IL-7 reeptor results in exerted T H 7 responses nd the worsening of EAE,. IL-7 ts diretly on T ells to inhiit the development of pthogeni T H 7 ells nd to promote the differentition of IL--produing type T reg ells (Tr ells) 7. Thus, the rrest of EAE y IL-7 is thought to reflet its diret effets on T ells. However, severl ell types eyond T ells express the IL-7 reeptor, ut the relevne of IL-7 signling in those ells for the ontrol of EAE nd utoimmunity is unknown. DCs express funtionl IL-7 reeptor ; however, lthough IL-7 hs een reported to interfere with the tivtion of DCs in vitro, the physiologil relevne of IL-7 signling in DCs nd its effets on the ontrol of the T ell response nd utoimmunity re unknown. In this work, we studied the effets of IL-7 signling in DCs on the differentition of effetor T ells nd T reg ells nd CNS utoimmunity. We found tht IL-7 signling in DCs upregulted expression of the immunoregultory moleule CD9 (enoded y Entpd) nd limited the development of EAE. CD9 expressed y onventionl DCs (DCs) redued the extrellulr onentrtion of ATP nd deresed ATP-triggered tivtion of the NLRP inflmmsome. Moreover, we Center for Neurologi Diseses, Brighm nd Women s Hospitl, Hrvrd Medil Shool, Boston, Msshusetts, USA. The Min nd Everrd Goodmn Fulty of Life Sienes, Br Iln University, Rmt Gn, Isrel. Division of Gstroenterology nd Heptology, Beth Isrel Deoness Medil Center, Hrvrd Medil Shool, Boston, Msshusetts, USA. These uthors ontriuted eqully to this work. Correspondene should e ddressed to F.J.Q. (fquintn@ris.wh.hrvrd.edu). Reeived Mrh; epted 9 July; pulished online Septemer ; doi:./ni.9 9// VOLUME NUMBER OCTOBER nture immunology

2 npg Nture Ameri, In. All rights reserved. found tht vintion of mie with IL-7-onditioned DCs suppressed estlished hroni relpsing-remitting EAE. Together our dt demonstrte tht IL-7 signling in DCs ontrols pthogeni T ell responses in n CD9-dependent mnner nd identify IL-7- onditioned DCs s possile pproh for the tretment of disorders medited y the immune system. RESULTS Higher IL-7RA expression in DCs thn in plsmytoid DCs To investigte the role of IL-7 signling in DCs on the regultion of utoimmunity, we first nlyzed the expression of IL-7RA in plsmytoid DCs (pdcs; F/ CD CD lo B MHC lss II lo Ly ) nd DCs (F/ CD CD B MHC lss II Ly ) isolted from nive mie y flow ytometry (Supplementry Fig. ). IL-7RA ws expressed minly in DCs, with only low or sent expression on pdcs (Fig. ). We otined similr results for IL-7RA expression y quntittive PCR nd immunolot nlysis of sorted pdcs nd DCs (Fig.,). Thus, the expression pttern of IL-7RA suggested tht IL-7 ontrols the tivity of DCs. IL-7 modultes the funtion of DCs After ntigen uptke in the presene of DC-mturing stimuli, DCs upregulte their expression of mjor histoomptiility omplex (MHC) lss II nd ostimultory moleules. To investigte the effets of IL-7 on DC tivtion, we pretreted spleni DCs from nive mie with vehile or IL-7 nd studied their response to tivtion with lipopolyshride from Esherihi oli (). Pretretment of DCs with IL-7 followed y tivtion with led to signifintly lower expression of MHC lss II nd the ostimultory moleules CD, CD nd CD thn tht of DCs tivted with without IL-7 pretretment (Fig. nd Supplementry Fig. ). DCs ontrol T ell differentition vi the seretion of polrizing ytokines. Pretretment of spleni DCs with IL-7 followed y tivtion with led to signifintly lower prodution of IL- nd of IL- nd IL- (whih promote the differentition of T H nd T H 7 ells, respetively) thn tht of DCs tivted with without IL-7 pretretment (Fig. ). Pretretment of DCs with IL-7 followed y tivtion with lso upregulted Il7 expression (Fig. ), whih suggested positive feedk loop for IL-7 prodution. Indeed, in those onditions we deteted inresed prodution of IFN-β (Fig. ), whih is reported to t in n utorine mnner to trigger IL-7 prodution 9. We lso deteted inresed prodution of IL- in those onditions nd inresed prodution of nd trnsforming growth ftor-β (TGF-β) only in response to tretment with IL-7 (Fig. ). Together these dt suggested tht IL-7 deresed the prodution of ytokines tht promote the differentition of effetor T H nd T H 7 ells, while it enhned the prodution of ntiinflmmtory ytokines y DCs. The effets of IL-7 on the expression of MHC lss II, ostimultory moleules nd ytokines suggested tht IL-7 ffets the ility of DCs to tivte nd polrize T ells into speifi susets. Thus, we pretreted DCs with IL-7 nd tivted them with, then extensively wshed them nd ssessed their ility to tivte nive D CD T ells in the presene of their ognte trget ntigen: n epitope of mino ids of myelin oligodendroyte glyoprotein (MOG()). Pretretment of DCs with IL-7 followed y tivtion with led to signifintly lower prolifertive response of nive D T ells to MOG() thn tht eliited y DCs treted with without IL-7 pretretment (Fig. d). Moreover, the DCs pretreted with IL-7 nd tivted with hd deresed ility to indue the prodution of IFN-γ nd IL-7 y T ells, s mesured y enzyme-linked immunosorent ssy nd intrellulr ytokine stining (Fig. e,f). Conversely, pretretment of DCs with IL-7 efore tivtion with oosted their ility to promote the differentition of IL- nd Foxp CD T ells (Fig. e,f). We oserved similr effets for one mrrowderived DCs pretreted with IL-7 nd tivted with (dt not shown). IL-7 is known to t diretly on T ells to suppress their differentition into effetor T ells,7. We found tht DCs pretreted IL-7 nd tivted with showed diminished ility to trigger the prodution of IFN-γ nd IL-7 y T ells in the presene of exogenously dded T H - nd T H 7-polrizing ytokines thn tht of DCs tivted with without IL-7 pretretment (Fig. g). Conversely, pretretment of DCs with IL-7 inresed IL- prodution nd expression of the trnsription ftor Foxp in T ells when we dded Tr-polrizing ytokines or ytokines tht polrize differentition into Foxp T reg ells to the oulture (Fig. g,h), whih suggested tht IL-7 signling in DCs modulted T ell differentition in vivo even in the ontext of inflmmtion or other physiologil onditions tht generte polrizing ytokine milieu. Together these dt demonstrted tht IL-7 signling ontrolled the ntigenpresenting funtion of DCs. IL-7RA in DCs limits EAE development IL-7 hs n importnt role in the ontrol of CNS inflmmtion during EAE,,. In greement with pulished reports, we found signifint worsening of EAE in IL-7RA-defiient (Il7r / ) mie, hrterized y n inrese in the frequeny of CNS-infiltrting IFN-γ nd IL-7 CD T ells nd lower frequeny of IL- CD T ells (Supplementry Fig.,). Il7r / mie lso showed n inresed rell response to MOG() nd n inresed frequeny of CD CD CDL hi IFNγ, IL-7 nd IFN-γ IL-7 CD T ells in the lymph nodes nd spleen, onomitnt with deresed frequeny of Foxp nd IL- CD T ells (Supplementry Fig.,d). The pulished effets of IL-7 on enephlitogeni nd T reg ells,,7 would suggest tht the worsening of EAE in the Il7r / mie resulted from the lk of IL-7 signling in T ells. However, the Il7r / mie hd nonell-speifi deletion of IL-7RA; thus, it remined possile tht IL-7 ted on dditionl ells eyond T ells to limit the development of EAE. To investigte the role of IL-7 signling in DCs during EAE, we isolted DCs from wild-type nd Il7r / mie d fter disese indution. We found tht DCs from Il7r / mie showed n inresed ility to tivte nive D T ells in the presene of MOG() (Supplementry Fig. e), Events (% of mx).. IL-7RA Il7r mrna (fold) DC pdc IL-7RA Atin WT Il7r / pdc DC pdc DC Figure IL-7RA expression in DCs. Flow ytometry (), quntittive PCR () nd immunolot nlysis () of IL-7RA expression in sorted DCs nd pdcs. Numers ove rketed line () indite perent IL-7RA DCs (red) nd pdcs (lue); dotted line, isotype-mthed ontrol ntiody. Atin serves s loding ontrol throughout. WT, wild-type. P <. (Student s t-test). Dt re representtive of more thn three independent experiments with similr results (error rs (), s.e.m.). 9// nture immunology VOLUME NUMBER OCTOBER

3 npg Nture Ameri, In. All rights reserved. MHCII (MFI) Il7 mrna ( fold) ND CD (MFI) 9 d (.p.m.) CD (MFI) eifn-γ (ng/ml) whih suggested tht defetive IL-7 signling in DCs ontriuted to the worsening of EAE in Il7r / mie. In vivo, DCs re influened y their intertions with T ells nd the ytokine milieu. Thus, the inresed ntigen-presenting funtion of DCs isolted from Il7r / mie might hve refleted the exposure of the DCs to more inflmmtory ytokine milieu nd not diret effets of IL-7 on DCs. To study the role of IL-7 signling in DCs during EAE, we used himer-sed pproh to generte mie lking IL-7RA expression in DCs (Supplementry Fig. ). For this, we reonstituted lethlly irrdited wild-type mie with one mrrow ells from mie tht express the diphtheri toxin reeptor (DTR) under the ontrol of the promoter of the gene enoding CD (Itgx; lled CD-DTR mie here). After reonstitution, these mie n e depleted of CD DCs y the dministrtion of diphtheri toxin (DTx). DTx nnot e hronilly dministered to CD-DTR mie euse of dverse side effets; however, no dverse effets re ssoited with the hroni dministrtion of DTx to himers generted y the reonstitution of wild-type mie with one mrrow from CD-DTR mie (CD- DTR WT). Thus, months fter reonstitution of wild-type mie with CD-DTR one mrrow, we depleted those CD-DTR WT himers of DTR DCs y hroni dministrtion of DTx nd reonstituted their DC omprtment with DC preursors from wildtype mie (to generte mie) or from Il7r / mie (to generte DC(IL-7RA-KO) mie) (Supplementry Fig. ). We dministered DTx to nd DC(IL-7RA-KO) mie one every other dy until the ompletion of the experiment nd deteted no ntiodies to DTx fter months of DTx dministrtion (Supplementry Fig. ). DC(IL-7RA-KO) mie hd signifintly lower IL-7RA expression in DCs ut not in other ntigen-presenting ell (APC) popultions thn did DC (WT) mie (Fig. nd Supplementry Fig. d). IL-7 (ng/ml)..... IL-7,., Figure IL-7 modultes the ntigen-presenting funtion of DCs. () Flow ytometry of wild-type DCs left untreted () or treted with IL-7 ( ng/ml) or ( ng/ml) lone or sequentilly, presented s men fluoresene intensity (MFI). MHCII, MHC lss II. () Enzyme-linked immunosorent ssy of ytokines in ulture superntnts of DCs treted s in. () Quntittive CD (MFI) IL- (ng/ml)... IL- (ng/ml) IL-7. g T H IFN-γ (ng/ml) IL- (ng/ml)... TGF-β (ng/ml) We deteted no differene etween nd DC(IL-7RA-KO) mie in the frequeny or solute numer of DCs (Supplementry Fig. e). Tretment with MOG() resulted in fster development of EAE in DC(IL-7RA-KO) mie thn in mie; DC(IL- 7RA-KO) mie lso rehed signifintly higher disese sores thn did their ounterprts (Fig. ). The worsening of EAE in DC(IL-7RA-KO) mie ws ssoited with greter frequeny of T H nd T H 7 ells in the CNS nd signifintly fewer IL- T ells thn tht in mie (Fig. ). Moreover, nlysis of the CD CD CDL hi spleni T ell omprtment reveled higher rell prolifertive response to MOG() nd higher frequeny of IFN-γ nd IL-7 CD ells in DC(IL-7RA-KO) mie thn in mie, onomitnt with lower frequeny of Foxp T reg ells nd IL- CD ells in DC(IL-7RA-KO) mie thn in mie (Fig. d,e). We otined similr results when we indued EAE y trnsferring MOG()-retive T H or T H 7 ells (Supplementry Fig. fh). To study the effets of IL-7 signling in DCs during EAE, we isolted DCs from nd DC(IL-7RA-KO) mie d fter EAE indution. DCs from DC(IL-7RA-KO) mie hd higher expression of the proinflmmtory ytokines IL-, IL- nd IL-, onomitnt with lower expression of IL- nd IL-7, thn tht of mie (Fig. f). We onfirmed those results y dditionl quntittive profiling nlyses, whih deteted pronouned proinflmmtory trnsription profile in DCs isolted from DC(IL-7RA-KO) mie during EAE (Fig. g). Moreover, DCs from DC(IL-7RA-KO) mie showed n greter ility to tivte the prolifertion of D CD T ell nd promoted the prodution of inresed mounts of IFN-γ nd IL-7 nd deresed quntities of IL- nd TGF-β thn did their ounterprts (Fig. h,i). Together these dt showed tht IL-7 ted on DCs in vivo to limit the development of enephlitogeni T ells nd EAE ND ND T H 7 IL- (ng/ml)..... f (%) ells CDIFN-γ PCR nlysis of Il7 mrna in DCs treted s in, presented reltive to tht of the ontrol gene Gpdh. ND, not deteted. (df) (d), ytokines in ulture superntnts (e) nd frequeny of CD IFN-γ, IL-7, IL- nd Foxp ells (f) mong nive D CD T ells stimulted with MOG() plus DCs treted s in. (g,h) Cytokine seretion (g) nd frequeny of Foxp CD T ells (h) mong nive D CD T ells stimulted with MOG() plus DCs treted s in, IFN-β (ng/ml)..... CD IL-7 ells (%) h Foxp Foxp IL- (ng/ml)..... CD IL- ells (%) TGF-β ( ng/ml) IL CD IL-7. in the presene of exogenous ytokines to promote the differentition of T H, T H 7 nd Tr ells (g) or Foxp T ells (h). Numers in outlined res (h, left) indite perent Foxp CD T ells. P <. nd P <. (one-wy nlysis of vrine (ANOVA)). Dt re from three independent experiments (g,h, right; men nd s.e.m.) or re representtive of three independent experiments (h, left). IL-7 (ng/ml) IL- (ng/ml)..... Tr TGF-β (ng/ml) CD Foxp ells (%) CD Foxp ells (%) // VOLUME NUMBER OCTOBER nture immunology

4 npg Nture Ameri, In. All rights reserved. Events (% of mx) CNS CD T ells (%)..7 IL-7RA EAE linil sore DC (WT) DC (IL-7RA-KO) IFN-γ IL-7 IL- Foxp DC(IL-7RA-KO) Time (d) d (.p.m.) EAE linil sore Time (d) DC(IL-7RA-KO) MOG (µg/ml) DC(IL-7RA-KO)....9 Trnsriptionl effets of IL-7 on DCs To investigte the mehnisms tht medite the effets of IL-7 on DCs, we nlyzed y mirorry the expression profiles of primry spleni DCs isolted from nive wild-type mie nd treted in vitro with IL-7. Study of the expression dt y Ingenuity pthwy nlysis identified signifintly lower expression of genes enoding proinflmmtory moleules ssoited with the NF-κB nd Tolllike reeptor signling pthwys in IL-7-treted DCs thn in untreted DCs (P =. nd P =. 7, respetively; Supplementry Fig.,). Conversely, tretment with IL-7 led to signifint inrese over time in the expression of Tnip nd Tnfip, whih enode moleules known to inhiit NF-κB tivtion (Supplementry Fig. ). We lso found signifint upregultion over time of Ido, Ido, Il7, Il nd Entpd, whih enode nti-inflmmtory moleules (Supplementry Fig. ). We used the NetGenertor lgorithm to nlyze the trnsriptionl response of DCs to IL-7. This lgorithm integrtes expression profiles in model with prior knowledge of the genes under investigtion nd their onnetions. The resulting model suggested tht IL-7 ontrolled the expression of the nti-inflmmtory moleules desried ove in mnner dependent on the trnsription ftors STAT nd STAT (Supplementry Fig. d). Together these dt showed tht IL-7 limited the inflmmtory response of DCs nd triggered the expression of tolerogeni moleules. CD9 medites the inhiitory effets of IL-7 on DCs Our trnsriptionl profiling studies identified severl ndidte moleules tht proly medite the effets of IL-7 signling in DCs e IL-7 IL- Foxp.. IFN-γ CD.9. CD IL-7 IFN-γ Figure IL-7RA signling in DCs ontrols T ell differentition nd EAE development. () Flow ytometry of IL-7RA in spleni DCs sorted from nive or DC(IL-7RA-KO) mie. Numers ove rketed line indite perent IL-7RA DCs (lk) or DC(IL-7RA-KO) DCs (red); dotted line, isotype-mthed ontrol ntiody. () Development of EAE in nd DC(IL-7RA-KO) mie, presented s linil sore (left) nd liner-regression urves (right; thinner lines indite 9% onfidene intervl). () Frequeny of IFN-γ, IL-7, IL- nd Foxp ells mong CNS-infiltrting CD T ells, nlyzed y flow ytometry. (d) Rell response to MOG() (MOG) y splenoytes isolted from nd DC(IL-7RA-KO) mie d fter EAE indution. (e) Frequeny of CD CD CDL hi spleni IFN-γ, IL-7, IFN-γ IL-7 (DP), IL- nd Foxp CD T ells in nd DC(IL-7RA-KO) CD IL- ells (%) CD Foxp ells (%) on the tivtion of T ells. To identify the mehnisms tht medite the effets of IL-7 on the ntigen-presenting funtion of DCs, we used loking ntiodies to IL-7, IL-, IFN-β or TGF-β, or the indolemine,-deoxygense (IDO)-speifi inhiitor -methyl-dtryptophn (-D-MT). We pretreted spleni DCs with IL-7 nd then treted them with, extensively wshed the ells nd used them to tivte nive CD T ells with ntiody to CD (nti-cd) in the presene of the loking ntiodies to ytokines or -D-MT. The suppressive effet of pretreting DCs with IL-7 ws not loked y the ntiodies or -D-MT (Fig.,), whih suggested tht neither IDO nor the ytokines IL-7, IL-, IFN-β or TGF-β medited the suppressive effets of DCs pretreted with IL-7. Expression of PD-L (CD7), the lignd for the T ellinhiitory reeptor PD-, is reported to e upregulted following the tretment of mouse pdcs or humn monoytederived DCs 9 with IL-7. We did not detet sustntil upregultion of PD-L expression fter treting DC with IL-7 (Supplementry Fig. ). Moreover, we lso oserved the suppressive effets of IL-7-treted DCs on the tivtion of T ells when we used PD-L- or IL--defiient DCs (Supplementry Fig. ). Together these dt suggested tht the suppressive effets of IL-7 on the ntigen-presenting funtion of DCs were independent of PD-L or IL-. CD9 hs een linked to the suppressive tivity of mouse nd humn T reg ells,. Entpd (whih enodes CD9) ws signifintly upregulted in DCs in response to tretment with IL-7 in vitro (P =.; Supplementry Fig. ). Hene, we investigted the role of CD9 in the effets of IL-7 on DCs. Entpd defiieny olished the suppressive effets of the pretretment of DCs with IL-7 CD ells (%) CD ells (%) g CCL CCL CCL7 CD CD CD CSF CXCL CXCL CXCL Cd GS IL- IL- IL- IRF IRF IRF IRF IRF7 IRF Im Nod Nod NOS NQO NRF PARP RIG.I ReI Rnf STAT STAT STAT TGFβ TLR TLR TLR TLR9 DP f Il7r mrna (fold) Il7 mrna (fold) WT KO Expression (fold)..... h Events (% of mx) i IFN-γ (ng/ml) IL- (ng/ml) Il mrna (fold) Ifn mrna (fold) CFSE IL-7 (ng/ml) TGF-β (ng/ml) DC(IL-7RA-KO) Il mrna (fold) Il mrna (fold) ND index Il mrna (fold) Tgf mrna (fold) DC(IL-7RA-KO). DC (IL-7RA-KO) mie d fter EAE indution. Numers in qudrnts or djent to outlined res indite perent ells in eh throughout. (f) Expression of Il7r, Il, Il, Il, Il7, Ifn, Il nd Tgf mrna in DCs sorted from nd DC(IL-7RA-KO) mie d fter EAE indution, presented reltive to tht of Gpdh. (g) Quntittive expression profiling of DCs isolted from nd DC(IL-7RA-KO) mie d fter EAE indution, presented reltive to tht of endogenous ontrol genes. (h,i) (h) nd ytokine seretion (i) of nive D CD T ells leled with the division-trking dye CFSE nd stimulted with MOG() plus DCs sorted from nd DC(IL-7RA-KO) mie d fter EAE indution. Numers ove plots (h, left) indite perent CFSE (proliferted) ells; green line (h), unproliferted ells. P <., P <. nd P <., ompred with (Student s t-test). Dt re from one experiment representtive of three experiments with five or more mie per group (error rs (f,h,i), s.e.m.). 9// nture immunology VOLUME NUMBER OCTOBER 7

5 (.p.m.) IL-7 Bloking A f p-stat MFI/STAT MFI IC IL-7 IL- IFN-β TGF-β IL-7 (min) p-stat MFI/STAT MFI (.p.m.) IL-7 (min) g -D-MT Entpd (CD9), SRE- (.p.m.) IL-7 SRE- IRF- SRE: STAT-inding site STAT-STATinding site d Entpd mrna (fold) Strt of trnsription Events (% of mx), IRF-: STAT-inding site h STAT enrihment CD9 DC(IL-7RA-KO).. SRE- STAT enrihment SRE- e IL-7 (min): STAT enrihment p-stat STAT p-stat STAT Site STAT-STAT IL-7 IL-7 npg Nture Ameri, In. All rights reserved. Figure CD9 is required for the inhiitory effets of IL-7 on DCs. (,) of nive CD T ells stimulted with nti-cd plus wild-type DCs treted with lone ( ) or pretreted with IL-7 nd treted with (), in the presene of isotype-mthed ontrol ntiody (IC) or loking ntiody (A) to IL-7, IL-, IFN-β or TGF-β () or in the presene () or sene ( ) of -D-MT (). () of T ells stimulted with nti-cd plus DC(CD9-KO) DCs treted with lone or pretreted with IL-7 nd treted with. (d) Quntittive PCR on the tivtion nd polriztion of T ells. Pretretment of Entpd- defiient DCs with IL-7 did not signifintly derese the prolifertive response nd the prodution of IFN-γ nd IL-7, nd hd no signifint effet on IL- prodution nd Foxp expression y D T ells (P =.; Fig. nd Supplementry Fig.,). Thus, these dt suggested tht CD9 medited the effets of IL-7 signling in DCs on T ell tivtion. To further hrterize the role of CD9 on the effets of IL-7 in DCs, we studied the regultion of CD9 expression y IL-7. Freshly isolted DCs from DC(IL-7RA-KO) mie showed signifintly lower expression of oth Entpd mrna nd CD9 protein, refleted s signifint derese in CD9 DCs, thn did their ounterprts (Fig. d). Thus, IL-7 ontrolled Entpd expression in DCs in vitro nd in vivo. STAT nd STAT medite the response to IL-7 in T ells 7. We deteted signifint phosphoryltion of STAT in DCs min fter tretment with IL-7 nd modest ut signifint inrese in the phosphoryltion of STAT (Fig. e,f). Bioinformtis nlysis identified puttive STAT-inding element (IRF-), two STAT-inding elements (SRE- nd SRE-) nd ommon STAT-STATinding element upstrem of the trnsription strt site in the Entpd promoter (Fig. g). Chromtin-immunopreipittion (ChIP) ssys deteted inding of STAT to SRE-, SRE- nd the STAT-STAT inding element in the Entpd promoter in response to IL-7 (Fig. h). Binding of STAT to the Entpd promoter, however, ws unresponsive to IL-7 nd ws triggered y tivtion with insted (Fig. i). To investigte the funtionl onsequenes of the intertion of STAT nd STAT with the Entpd promoter, we did reporter ssys. Cotrnsfetion of HEK9 humn emryoni kidney ells with reporter onstrut ontining the gene enoding firefly luiferse under the ontrol of the Entpd promoter together with onstrut enoding onstitutively tivted STAT or STAT led to signifintly more in luiferse tivity thn did trnsfetion of ontrol ells with empty vetor, ut this effet ws signifintly stronger in response to onstitutively tivted STAT (Fig. j). These dt were in greement with the reported effets of STAT on CD9 expression in T ells 7 nd together suggested tht IL-7 ontrolled CD9 expression in DCs vi STAT. i STAT enrihment IRF- STAT enrihment Site STAT-STAT IL-7 IL-7 CD9 ontrols tivtion of the NLRP inflmmsome CD9 is n etonuleotidse tht tlyzes the degrdtion of extrellulr ATP nd ADP. Extrellulr ATP triggers tivtion of the NLRP inflmmsome 9, proess shown to ontrol the differentition of enephlitogeni T H nd T H 7 ells during EAE. Given the requirement for CD9 expression in the effets of IL-7 signling in DCs on the tivtion nd differentition of T ells (Fig. nd Supplementry Fig. ), we investigted the effets of IL-7 on the extrellulr onentrtion of ATP nd tivtion of the NLRP inflmmsome. Pretretment with IL-7 nd tretment with led to signifint derese the extrellulr onentrtion of ATP deteted fter tivtion of wild-type DCs with (Fig. ). However, pretretment with IL-7 hd no effet on the extrellulr onentrtion of ATP mesured in the superntnts of j Luiferse tivity Control STAT STAT STAT STAT nlyiss of Entpd mrna (left) nd flow ytometry of CD9 (right) in DCs sorted from nive nd DC(IL-7RA-KO) mie; mrna results re reltive to tht of Gpdh. Numers ove rketed lines (right) indite perent CD9 ells; dotted line, isotype-mthed ontrol ntiody. (e,f) Immunolot nlysis (e) nd flow ytometry (f) of phosphorylted (p-) nd totl STAT nd STAT in spleni DCs exposed for vrious times to IL-7 ( ng/ml). (g) STAT-inding site (green; IRF-), STAT-inding sites (lue; SRE- nd SRE-) nd STAT-STATinding site (green-lue) in the Entpd promoter. (h,i) Chromtin-immunopreipittion nlysis of the intertion of STAT (h) or STAT (i) with vrious inding sites of the Entpd promoter s in g (ove grphs) in DCs left untreted () or treted with IL-7 or lone or sequentilly. (j) Luiferse tivity in HEK9 ells trnsfeted with CD9 luiferse reporter lone (Control) or together with onstrut enoding onstitutively tivted STAT (STAT) or STAT (STAT) seprtely or together (STAT STAT). P <. nd P <. (one-wy ANOVA). Dt re representtive of more thn three independent experiments with similr results (error rs (d,f,hj), s.e.m.). 9// VOLUME NUMBER OCTOBER nture immunology

6 npg Nture Ameri, In. All rights reserved. Figure IL-7-indued CD9 ontrols extrellulr ATP nd tivtion of the NLRP inflmmsome. () Extrellulr ATP onentrtion in ulture superntnts of wild-type (WT), IL-7RA-defiient (Il7r / ) or CD9-defiient (Entpd / ) DCs treted with IL-7 or lone or sequentilly. () Residul extrellulr ATP in ulture superntnts of DCs treted with in the presene () or sene ( ) of LPS fter inution with µm exogenous ATP. () Thin-lyer hromtogrphy ssy of the enzymti tivity of CD9 in DCs s in. (d) Quntifition of AMP nd intensity, presented in ritrry units (AU) reltive to tht of ADP in CD9-defiient DCs treted s in. (e) Immunolot nlysis (left) nd densitometry (right) of spse- nd IL-β in DCs s in. (f) Quntifition of IL-β in ulture superntnts of DCs s in. P <., P <. nd P <. (one-wy ANOVA). Dt re representtive of two independent experiments with similr results (error rs (,,df), s.e.m.). IL-7RA- or CD9-defiient DCs treted with (Fig. ). Moreover, defiieny Atin in IL-7RA or CD9 in DCs led to signifintly higher extrellulr onentrtion of ATP deteted fter tivtion with LPS thn tht of their wild-type ounterprts (Fig. ). To further investigte the mehnisms linked to the redued mount of extrellulr ATP deteted in tissue ulture superntnts of wild-type DCs treted with IL-7, we dded exogenous ATP ( µm) to DCs treted with LPS, vehile or IL-7 nd quntified the residul extrellulr ATP in the superntnts fter h of inution. We found signifintly lower onentrtions of extrellulr ATP remining in superntnts of wild-type DCs treted with nd IL-7 thn in those of wild-type DCs treted with (Fig. ). However, IL-7 did not ffet the mount of residul extrellulr ATP in IL-7RA- or CD9-defiient ells (Fig. ), whih suggested tht the lower undne of ATP in superntnts of wildtype DCs treted with IL-7 resulted from its inresed tolism y CD9. In greement with tht interprettion, tretment with IL-7 signifintly inresed nuleoside triphosphte diphosphohydrolse tivity in wild-type DCs ut not in IL-7RA -or CD9-defiient DCs (Fig.,d). Moreover, we deteted less nuleoside triphosphte diphosphohydrolse tivity (mnifested s greter residul mounts of ATP) in IL-7RA-defiient DCs thn in wild-type DCs (Fig.,d), whih suggested tht the utorine effets of IL-7 ontrolled the ility of DCs to degrde extrellulr ATP. ATP tivtes the NLRP inflmmsome in ertin APCs suh s mrophges 9. Ativtion of the NLRP inflmmsome results in the genertion of tive spse, whih leds to the mturtion nd relese of IL- nd IL-β. We found tht pretretment with IL-7 signifintly suppressed tivtion of the NLRP inflmmsome in wild-type DCs tivted with, s shown y lower undne of tivted spse- nd mture IL-β (Fig. e) nd the seretion of signifintly lower mounts of IL-β into the ulture medium (Fig. f). In ordne with our dt on the extrellulr onentrtions of ATP, we did not detet the inhiitory effets of IL-7 on the tivtion of the NLRP inflmmsome nd the relese of IL- β in -treted IL-7RA- or CD9-defiient DCs (Fig. e,f)..... IL-7 ATP (µm) e Pro-spse- WT Il7r / Entpd / IL-7 Cspse- Pro-IL-β IL-β IL-7 WT Il7r / Entpd / Indeed, defiieny in IL-7RA or CD9 resulted in signifintly more relese of IL-β thn tht of wild-type DCs fter tretment with (Fig. f). Together these dt suggested tht the upregultion of CD9 expression triggered y IL-7 limited extrellulr ATP onentrtions nd, onsequently, tivtion of the NLRP inflmmsome in DCs. CD9 in DCs limits EAE development To study the role of CD9 in DCs during EAE, we reonstituted CD-DTR WT himers with CD9-defiient DC preursors to generte mie lking Entpd expression in DCs (DC(CD9-KO) mie). We used mie s ontrols. We found signifintly lower expression of CD9 protein in DCs from DC(CD9-KO) mie thn in DCs from mie (Fig. ), ut did not otin this result for other APC popultions (Supplementry Fig. d). We deteted no differene etween nd DC(CD9-KO) mie in the frequeny or solute numer of DCs (Supplementry Fig. e). The indution of EAE led to n erlier onset of EAE in DC(CD9- KO) mie thn in mie, nd the EAE lso rehed signifintly higher sores in the former (Fig. ). The worsening of EAE in DC(CD9-KO) mie ws orrelted with n inresed frequeny of T H nd T H 7 ells in the CNS (Fig. ). In greement with the worsening of EAE ssoited with CD9 defiieny in DCs, DC(CD9- KO) spleni T ells hd signifintly enhned prolifertive rell response to MOG(), nd DC(CD9-KO) mie hd signifintly greter frequeny of CD CD CDL hi spleni IFN-γ nd IL-7 CD T ells (Fig. d,e). We oserved no differene etween nd DC(CD9-KO) mie in the frequeny of Foxp T reg ells or IL- CD T ells (Fig. d,e). Anlysis of DCs isolted d fter the indution of EAE reveled signifintly lower CD9 expression, onomitnt with higher expression of mrna enoding the proinflmmtory ytokines IL-, IL- nd IL-, in DC(CD9-KO) mie thn in ells (Fig. f). We found no signifint differene etween nd DC(CD9-KO) 7 ATP (µm) WT Il7r / Entpd / (KD) Cspse-/tin (AU) IL-7 IL-β/tin (AU) WT Il7r / Entpd / famp/adp (AU) d IL-7 IL-7 IL-β (pg/ml) WT Il7r / Entpd / IL-7 Time (min): AMP ADP WT Il7r / Entpd / WT Il7r / Entpd / 9// nture immunology VOLUME NUMBER OCTOBER 9

7 npg Nture Ameri, In. All rights reserved. Events (% of mx) CNS CD T ell (%) CD EAE linil sore DC(CD9-KO) IFN-γ IL-7 IL- Foxp DC(CD9-KO) Time (d) Figure CD9 in DCs ontrols T ell differentition nd EAE development. () Flow ytometry of CD9 in spleni DC sorted from nive or DC(CD9-KO) mie. Numers ove rketed line () indite perent CD9 DCs (lk) or DC(CD9-KO) DCs (red); dotted line, isotype-mthed ontrol ntiody. () Development of EAE in nd DC(CD9-KO) mie (presented s in Fig. ). () Frequeny of IFN-γ, IL-7, IL- nd Foxp ells mong CNS-infiltrting CD T ells, nlyzed y flow ytometry. (d) Rell response to MOG() in splenoytes isolted from nd DC(CD9-KO) mie d fter EAE indution. (e) Frequeny of CD CD CDL hi spleni IFN-γ, IL-7, IFN-γ IL-7 (DP), IL- nd Foxp CD T ells in nd DC(CD9-KO) mie d fter EAE indution. (f) Expression of Entpd, Il, Il, Il, Il7, Ifn, Il nd Tgf in DCs sorted from nd mie in the expression of mrna enoding IL-, TGF-β, IFN-β or IL-7 (Fig. f). In vitro, DCs from DC(CD9-KO) mie showed n inresed ility to trigger the prolifertion of nive D CD T ells in the presene of MOG() nd indue the prodution of IL-7 nd IFN-γ, t the expense of deresed seretion of IL- nd TGF-β (Fig. g,h). Thus, expression of CD9 in DCs limited the enephlitogeni T H nd T H 7 T ell response nd the development of EAE. Vintion with IL-7-onditioned DCs suppresses EAE The tolerogeni effets of IL-7 signling in T ells nd DCs support the proposl of its therpeuti potentil in disorders medited y the immune system. However, IL-7 is reported to t on T ells to oost ytotoxi CD T ell responses, whih suggests tht diret dministrtion of IL-7 ould potentilly hve detrimentl side effets in immunologilly medited disorders. Vintion with DCs indues immunity to tumors nd pthogens, ut vintion with tolerogeni DCs hs een shown to indue ntigen-speifi tolerne. Thus, given the tolerogeni effets of IL-7 signling in DCs, nd to void the potentil pthogeni effets of IL-7 dministrtion, we investigted the therpeuti effets of vintion with IL-7- onditioned DCs on CNS utoimmunity. First we studied the effets of vintion with IL-7-onditioned one mrrowderived DCs on the model of relpsing-remitting EAE indued in SJL mie y immuniztion with peptide of mino ids 9 of proteolipid protein (PLP(9)). On dy fter EAE d (.p.m.) EAE linil sore Time (d) MOG (µg/ml) f Entpd mrna (fold) Il7 mrna (fold) DC(CD9-KO) Il mrna (fold) Ifn mrna (fold)... indution, during the remission phse of the disese, we rndomly lloted the mie into four groups nd treted them s follows: group, sline only; group, DCs loded with PLP(9); group, DCs treted with IL-7; group, DCs treted with IL-7 nd loded with PLP(9). An dditionl ontrol group reeived DCs not loded with peptide or pretreted with ytokine. We repeted the tretment three dditionl times, one every d. DCs not loded with peptide or pretreted with ytokine hd no signifint effet on disese development (P =.7; Supplementry Fig. 7). However, the dministrtion of IL-7-onditioned DCs loded with PLP(9) led to signifint redution in EAE nd signifint redution in the prolifertive rell response (mesured s the prodution of IFN-γ nd IL-7) to PLP(9), onomitnt with inresed prodution of IL- nd TGF-β, reltive to results otined for untreted mie (Fig. 7). In this model, the hroni phse of EAE is hrterized y spreding of the T ell response to the PLP epitope of mino ids 79 (PLP(79)) 7. Thus, we nlyzed the rell response to PLP(7 9) in DC-vinted mie. We found tht the dministrtion of IL-7-treted DCs loded with PLP(9) led to signifint suppression of the prolifertive response nd prodution of IFN-γ nd IL-7 triggered y PLP(79), onomitnt with inresed prodution of IL- nd TGF-β, reltive to the results otined for untreted mie (Fig. 7d,e). Thus, vintion with IL-7-treted DCs lso seemed to redue epitope spreding in this model of EAE. Il mrna (fold) e IL- IL-7 Foxp DC(CD9-KO) Il mrna (fold) IFN-γ CD DC(CD9-KO) CD Il mrna (fold) Tgf mrna (fold)..... IFN-γ (ng/ml).7 7. CD ells (%) CD IL- ells (%) CD Foxp ells (%) CFSE.. IL-7 (ng/ml)... IL-7 IFN-γ DC(CD9-KO)... CD ells (%) DC(CD9-KO).. DC(CD9-KO) mie d fter EAE indution, presented reltive to tht of Gpdh. (g,h) (g) nd ytokine seretion (h) of nive CFSEleled D CD T ells stimulted with MOG() plus DCs sorted from nd DC(CD9-KO) mie d fter immuniztion (results in g presented s in Fig. h). P <. nd P <., ompred with (Student s t-test). Dt re from one experiment representtive of three experiments with five or more mie per group (error rs (h), s.e.m.). g Events (% of mx) h IL- (ng/ml) index TGF-β (ng/ml).. DP 9// VOLUME NUMBER OCTOBER nture immunology

8 npg Nture Ameri, In. All rights reserved. EAE linil sore d DC PLP PLP DC IL-7 DC IL-7 PLP (9) DC vintion..... Time (d) Time (d) PLP e. (79) (.p.m.) IFN-γ (ng/ml) EAE linil sore To further investigte the effets of IL-7-onditioned DCs on the immune response, we used myelin ntigen rrys, whih n detet epitope spreding in EAE nd MS,. The mirorrys onsisted of olletion of CNS-relted utontigens, inluding tissue lystes, reominnt proteins, nd peptide lirries spnning the entire sequene of myelin proteins nd lipids found in the entrl nd peripherl nervous system (Supplementry Tle ). The therpeuti effet of vintion with DCs on EAE in SJL mie ws ompnied y lower serum onentrtion of immunogloulin G ntiodies to myelin ntigens thn tht in untreted mie (Fig. 7f). We then studied the mehnisms involved in the suppression of EAE with IL-7-onditioned DCs; for this we used the C7BL/ mouse model of EAE indued with MOG(). We initited the dministrtion of IL-7-onditioned DCs d fter the indution of EAE. Vintion with IL-7-onditioned wild-type DCs loded with MOG() led to signifintly diminished EAE reltive to tht of untreted mie (Supplementry Fig. 7d). We oserved similr suppression of EAE fter the dministrtion of MOG()-loded, IL-7-onditioned DCs defiient in IL- or PD-L (Supplementry Fig. 7d), whih suggested tht these moleules did not hve sustntil role in regulting the enephlitogeni response y IL-7 signling in DCs. Conversely, MOG()-loded, IL-7- onditioned DCs defiient in IL-7RA or CD9 did not hve signifint effet on EAE (P =.7979, P = 7 nd P =., respetively; Supplementry Fig. 7d). Together these dt demonstrted tht vintion with IL-7-treted DCs ontrolled the enephlitogeni response nd estlished relpsing-remitting EAE in therpeuti prdigm in n CD9-dependent mnner. DISCUSSION IL-7 limits tissue inflmmtion nd utoimmunity in vrious senrios. Indeed, defiieny in the reeptor for IL-7 is linked to the IL-7 (ng/ml) (.p.m.) IL- (ng/ml) IFN-γ (ng/ml) TGF-β (ng/ml).. development of exerted inflmmtion in niml experimentl models, nd polymorphisms in IL-7 re ssoited with humn inflmmtory disorders. The immunoregultory tions of IL-7 re usully ttriuted to its diret effets on T ells, wherey it rrests the development of T H 7 ells nd promotes the differentition of Tr ells through mehnisms tht involve the trnsription ftor AhR,. In this work we identified n importnt role for IL-7 signling in DCs in the ontrol of the T ell response nd CNS utoimmunity. We found tht CD9 ws required for modulting the ntigenpresenting funtion of DCs y IL-7 in vitro nd in vivo. However, IL-7 lso upregulted the expression of dditionl immunoregultory moleules, suh s IL-7 itself, IFN-β, TGF-β nd IDO. Although those moleules did not hve sustntil role under the experimentl onditions in whih we tested them here, they might ontriute to the suppressive effets of IL-7 on DCs in lterntive senrios. IL-, known to tivte STAT signling, hs lso een shown to indue tolerogeni phenotype in DCs 7. Conversely, STAT defiieny restrited to DCs results in the spontneous development of inflmmtion. Suh dt support the proposl of tolerogeni role for STAT in DCs nd suggest tht other ytokines tht tivte STAT signling might trigger CD9-dependent regultory pthwys similr to those triggered y IL-7. Moreover, mirogli nd tissue mrophges express CD9, nd their funtion is lso regulted y ytokines tht tivte STAT signling 9,. Thus, it is possile tht the indution of CD9 expression onstitutes ommon immunoregultory mehnism triggered y STAT-tivting ytokines in ells of the innte immune system. CD9 medites the suppressive tivity of mouse nd humn T reg ells, proly through the genertion of denosine,. The mehnisms y whih CD9 in APCs regultes dptive immunity re less ler, ut pulished dt suggest tht these effets involve ontrolling tivtion of the NLRP inflmmsome. Extrellulr ATP triggers IL-7 (ng/ml).. IL- (ng/ml) DC IL-7 DC PLP DC IL-7 PLP Figure 7 Vintion with IL-7-onditioned DCs suppresses EAE. () Course of EAE indued y no tretment () or immuniztion of nive SJL mie with PLP() lone (DC PLP) or IL-7 lone (DC IL-7) or oth (DC IL-7 PLP), followed y intrvenous dministrtion of DCs (downwrd rrows) four times one every d strting t dy, presented s linil sores for the entire oservtion period (left) nd s liner-regression urves from dy until the termintion of the experiment (right). (e) Rell prolifertive response (,d) nd ytokine response (,e) to PLP( ) (,) or PLP(79) (d,e) in splenoytes otined from DC-treted mie d fter EAE indution s in. (f) Het mp of the ntiody response to myelin ntigens (right mrgin) on dy fter EAE indution s in (ssessed y ntigen mirorry); eh olumn represents the men serum retivity of immunogloulin G (IgG) to eh tretment ondition (key, elow). P <., P <. nd P <., versus untreted ontrol mie (one-wy ANOVA). Dt re representtive of t lest three independent experiments (men nd s.e.m. of five mie per group in ; error rs (e), s.e.m.).... f TGF-β (ng/ml) DCIL-7PLP DCIL-7 DCPLP Antiody retivity DC IL-7 DC PLP DC IL-7 PLP MBP() Bovine MBP gpmbp CNPse(9) Frontl loe MBP() HSP HSP7 MBP(9) CNPse() MOG() MOG() PLP(7) CNPse(9) PLP() MBP(79) MOBP() MBP() MBP() mmbp PLP(9) MBP(9) CNPse(9) Humn -- Tetrsilo GQ CNPse() ABPF() SAP-β Thlmus Corpus llosum Gnglioside mixture 9// nture immunology VOLUME NUMBER OCTOBER

9 npg Nture Ameri, In. All rights reserved. suh tivtion. Initil support for the proposl of role of ATPdependent NLRP tivtion in the ontrol of T ell immunity ws provided y the oservtion of ugmented ontt hypersensitivity in Entpd-defiient mie. Conversely, Nlrp defiieny results in deresed ontt hypersensitivity. Follow-up studies hve determined tht tivtion of the NLRP inflmmsome in APCs ontrols T H, T H nd T H 7 responses,. Indeed, tivtion of NLRP inflmmsome is required for the differentition of enephlitogeni T H nd T H 7 ells nd the development of EAE, proly s result of its effets on the seretion of IL-β nd IL-. In our studies, the indution of CD9 expression in DCs y IL-7 led to signifint redution in the extrellulr onentrtion of ATP, onomitnt with signifint redution in tivtion of the NLRP inflmmsome nd redued differentition of T H nd T H 7 ells. These dt re omptile with model in whih, through the upregultion of CD9 expression, IL-7 limits ATP-dependent tivtion of the NLRP inflmmsome in DCs nd their ility to promote the differentition of pthogeni T H nd T H 7 ells. It is possile, however, tht in ddition to its effets on CD9 expression, IL-7- triggered signling diretly interferes with the expression of omponents of the NLRP inflmmsome nd their tivtion in DCs. The immunoregultory properties of IL-7 support the proposl of its use for the tretment of humn utoimmune diseses. However, IL-7 n oost ytotoxi CD T-ell responses, whih suggests tht the dministrtion of IL-7 ould potentilly worsen disorders medited y the immune system. Hene, to explore the therpeuti potentil of IL-7 nd void its potentil deleterious side effets, we studied the effets of vintion with IL-7-onditioned DCs. We found tht vintion with IL-7-onditioned DCs suppressed the enephlitogeni T H nd T H 7 response nd hlted estlished hroni EAE. Vintion with DCs hs een pproved for the tretment of prostte ner ; thus, vintion with IL-7-onditioned tolerogeni DCs might provide new venue for the tretment of utoimmune diseses. Alterntively, nnoprtiles hve een developed for the simultneous tivtion of tolerogeni pthwys nd the delivery of ntigens to APCs in vivo to indue ntigen-speifi tolerne. Thus, it is oneivle tht nnoprtiles engineered to tivte IL-7 signling nd deliver myelin ntigens to DCs would indue tolerogeni DCs in vivo nd rrest pthogeni T ell responses. Tht pproh would exploit the immunoregultory properties of IL-7 nd overome the limittions ssoited with the implementtion of ell-sed therpies in linil setting. In summry, we hve reported here tht IL-7 signling in DCs limited the differentition of pthogeni T H nd T H 7 ells nd the development of CNS utoimmunity through mehnism tht ws t lest prtilly dependent on CD9. This immunoregultory pthwy might provide new therpeuti venues for MS nd other utoimmune disorders. Methods Methods nd ny ssoited referenes re ville in the online version of the pper. Aession odes. GEO: mirorry dt, GSE9. Note: Any Supplementry Informtion nd Soure Dt files re ville in the online version of the pper. Aknowledgments We thnk A. Shrpe (Hrvrd Medil Shool) for PD-L defiient mie; D.J. Pinsky (University of Mihign Helth Systems) for the Entpd promoter reporter; nd D. Frnk (Dn-Frer Cner Institute, Boston) for vetors enoding onstitutively tive STAT nd STAT. Supported y the US Ntionl Institutes of Helth (AI7 nd AI99 to F.J.Q.) nd the Ntionl Multiple Slerosis Soiety (RGA to F.J.Q.). AUTHOR CONTRIBUTIONS I.D.M., A.Y., S.M.V., E.J.B., Y.W. nd L.M. did in vitro nd in vivo experiments; B.P., I.S., R.B.-H. nd S.E. did ioinformtis nlysis; V.K.K. nd S.C.R. provided Il7r / nd Entpd / mie; I.D.M. nd F.J.Q. wrote the mnusript; nd F.J.Q. supervised the study nd edited the mnusript. COMPETING FINANCIAL INTERESTS The uthors delre no ompeting finnil interests. Reprints nd permissions informtion is ville online t reprints/index.html.. Nylnder, A. & Hfler, D.A. Multiple slerosis. J. Clin. Invest., ().. Pierson, E., Simmons, S.B., Cstelli, L. & Govermn, J.M. Mehnisms regulting regionl loliztion of inflmmtion during CNS utoimmunity. Immunol. Rev., ().. Biley, S.L., Shreiner, B., Mmhon, E.J. & Miller, S.D. CNS myeloid DCs presenting endogenous myelin peptides preferentilly polrize CD TH-7 ells in relpsing EAE. Nt. Immunol., 7 (7).. Yogev, N. et l. Dendriti ells meliorte utoimmunity in the CNS y ontrolling the homeostsis of PD- reeptor regultory T ells. Immunity 7, 7 ().. Comell, M., Montln, X., Münz, C. & Lünemnn, J.D. Trgeting dendriti ells to tret multiple slerosis. Nt. Rev. Nephrol., 997 ().. Greter, M. et l. Dendriti ells permit immune invsion of the CNS in n niml model of multiple slerosis. Nt. Med., (). 7. MMhon, E.J., Biley, S.L., Cstend, C.V., Wldner, H. & Miller, S.D. Epitope spreding initites in the CNS in two mouse models of multiple slerosis. Nt. Med., 9 ().. Kstelein, R.A., Hunter, C.A. & Cu, D.J. Disovery nd iology of IL- nd IL-7: relted ut funtionlly distint regultors of inflmmtion. Annu. Rev. Immunol., (7). 9. Molle, C., Goldmn, M. & Goriely, S. Critil role of the IFN-stimulted gene ftor omplex in TLR-medited IL-7p gene expression reveling two-step tivtion proess. J. Immunol., 779 ().. Mitsdoerffer, M. & Kuhroo, V. New piees in the puzzle: how does interferon-β relly work in multiple slerosis? Ann. Neurol., 7 (9).. Hunter, C.A. & Kstelein, R. Interleukin-7: lning protetive nd pthologil immunity. Immunity 7, 999 ().. Fitzgerld, D.C. et l. Suppressive effet of IL-7 on enephlitogeni Th7 ells nd the effetor phse of experimentl utoimmune enephlomyelitis. J. Immunol. 79, 7 (7).. Btten, M. et l. Interleukin 7 limits utoimmune enephlomyelitis y suppressing the development of interleukin 7-produing T ells. Nt. Immunol. 7, 999 ().. Awsthi, A. et l. A dominnt funtion for interleukin 7 in generting interleukin -produing nti-inflmmtory T ells. Nt. Immunol., 9 (7).. Fitzgerld, D.C. et l. Suppression of utoimmune inflmmtion of the entrl nervous system y interleukin sereted y interleukin 7-stimulted T ells. Nt. Immunol., 779 (7).. Stumhofer, J.S. et l. Interleukin 7 negtively regultes the development of interleukin 7-produing T helper ells during hroni inflmmtion of the entrl nervous system. Nt. Immunol. 7, 979 (). 7. Stumhofer, J.S. et l. Interleukins 7 nd indue STAT-medited T ell prodution of interleukin. Nt. Immunol., 7 (7).. Wng, S., Miyzki, Y., Shinozki, Y. & Yoshid, H. Augmenttion of ntigenpresenting nd Th-promoting funtions of dendriti ells y WSX-(IL-7R) defiieny. J. Immunol. 79, (7). 9. Krkhnov, S., Bedke, T., Enk, A.H. & Mhnke, K. IL-7 renders DC immunosuppressive y indution of B7H. J. Leuko. Biol. 9, 7 ().. Mtt, B.M., Rimondi, G., Rosorough, B.R., Sumpter, T.L. & Thomson, A.W. IL-7 prodution nd STAT-dependent upregultion of B7H medite immune regultory funtions of liver plsmytoid dendriti ells. J. Immunol., 77 ().. Guermonprez, P., Vlldeu, J., Zitvogel, L., Thery, C. & Amigoren, S. Antigen presenttion nd T ell stimultion y dendriti ells. Annu. Rev. Immunol., 7 ().. Jung, S. et l. In vivo depletion of CD dendriti ells rogtes priming of CD T ells y exogenous ell-ssoited ntigens. Immunity 7, ().. M, A. & Mlynn, B.A. A: linking omplex regultor of uiquityltion to immunity nd humn disese. Nt. Rev. Immunol., 777 (). 9// VOLUME NUMBER OCTOBER nture immunology

10 npg Nture Ameri, In. All rights reserved.. Weer, M. et l. Inferene of dynmil gene-regultory networks sed on timeresolved multi-stimuli multi-experiment dt pplying NetGenertor V.. BMC Syst. Biol. 7, ().. Deglio, S. et l. Adenosine genertion tlyzed y CD9 nd CD7 expressed on regultory T ells medites immune suppression. J. Exp. Med., 7 (7).. Gndhi, R. et l. Ativtion of the ryl hydroron reeptor indues humn type regultory T elllike nd Foxp regultory T ells. Nt. Immunol., (). 7. Chlmin, F. et l. Stt nd Gfi- trnsription ftors ontrol Th7 ell immunosuppressive tivity vi the regultion of etonuleotidse expression. Immunity, 7 ().. Eltzshig, H.K., Sitkovsky, M.V. & Roson, S.C. Purinergi signling during inflmmtion. N. Engl. J. Med. 7, (). 9. Mrithsn, S. et l. Cryopyrin tivtes the inflmmsome in response to toxins nd ATP. Nture, ().. Gris, D. et l. NLRP plys ritil role in the development of experimentl utoimmune enephlomyelitis y mediting Th nd Th7 responses. J. Immunol., 979 ().. Mrtinon, F., Myor, A. & Tshopp, J. The inflmmsomes: gurdins of the ody. Annu. Rev. Immunol. 7, 9 (9).. Tken, P.J., de Vries, I.J., Torensm, R. & Figdor, C.G. Dendriti-ell immunotherpy: from ex vivo loding to in vivo trgeting. Nt. Rev. Immunol. 7, 79 (7).. Dhodpkr, M.V., Steinmn, R.M., Krsovsky, J., Munz, C. & Bhrdwj, N. Antigenspeifi inhiition of effetor T ell funtion in humns fter injetion of immture dendriti ells. J. Exp. Med. 9, ().. Quintn, F.J. et l. Antigen mirorrys identify unique serum utontiody signtures in linil nd pthologi sutypes of multiple slerosis. Pro. Ntl. Ad. Si. USA, 99 ().. Roinson, W.H. et l. Protein mirorrys guide tolerizing DNA vine tretment of utoimmune enephlomyelitis. Nt. Biotehnol., 9 ().. Apetoh, L. et l. The ryl hydroron reeptor interts with -Mf to promote the differentition of type regultory T ells indued y IL-7. Nt. Immunol., (). 7. Moore, K.W., de Wl Mlefyt, R., Coffmn, R.L. & O Grr, A. Interleukin- nd the interleukin- reeptor. Annu. Rev. Immunol. 9, 7 ().. Melillo, J.A. et l. Dendriti ell (DC)-speifi trgeting revels Stt s negtive regultor of DC funtion. J. Immunol., (). 9. Bker, B.J., Prk, K.W., Qin, H., M, X. & Benveniste, E.N. IL-7 inhiits OSMmedited TNF-α nd inos gene expression in mirogli. Gli, 9 ().. Färer, K. et l. The etonuleotidse d9/entpdse modultes purinergimedited miroglil migrtion. Gli, ().. Mizumoto, N. et l. CD9 is the dominnt Lngerhns ell-ssoited eto-ntpdse: modultory roles in inflmmtion nd immune responsiveness. Nt. Med., ().. Sutterwl, F.S. et l. Critil role for NALP/CIAS/Cryopyrin in innte nd dptive immunity through its regultion of spse-. Immunity, 77 ().. Eisenrth, S.C., Colegio, O.R., O Connor, W., Sutterwl, F.S. & Flvell, R.A. Cruil role for the Nlp inflmmsome in the immunostimultory properties of luminium djuvnts. Nture, ().. Meng, G., Zhng, F., Fuss, I., Kitni, A. & Stroer, W. A muttion in the Nlrp gene using inflmmsome hypertivtion potentites Th7 ell-dominnt immune responses. Immunity, 7 (9).. Jähnish, H. et l. Dendriti ell-sed immunotherpy for prostte ner. Clin. Dev. Immunol., 79 ().. Yeste, A., Ndeu, M., Burns, E.J., Weiner, H.L. & Quintn, F.J. Nnoprtilemedited odelivery of myelin ntigen nd tolerogeni smll moleule suppresses experimentl utoimmune enephlomyelitis. Pro. Ntl. Ad. Si. USA 9, 77 (). 9// nture immunology VOLUME NUMBER OCTOBER

11 npg Nture Ameri, In. All rights reserved. ONLINE METHODS Animls. IL-7RA- nd CD9-defiient mie hve een desried,. SJL, D, C7BL/, CD-Cre:DTR nd IL- defiient mie were from the Jkson Lortories. PD-L defiient mie were gift from A. Shrpe (Hrvrd Medil Shool). For onditionl ltion of DCs (CD-DTR WT), one mrrow himers were inoulted intrperitonelly every seond dy for weeks with ng DTx per grm ody weight. For the genertion of one mrrow himers, reipient mie were lethlly irrdited with dose of 9. Gy nd d lter were given intrvenous injetion of one mrrow ells isolted from donor femor nd tiie. Reipients of one mrrow were then llowed to rest for weeks efore use. Four to five rndomly ssigned mie were used per experimentl group per experiment. Mie were kept in onventionl, pthogen-free fility t the Hrvrd Institutes of Mediine. All experiments were rried out in ordne with guidelines of the Institutionl Animl Cre nd Use Committee t Hrvrd Medil Shool. Isoltion of spleni DCs nd CNS infiltrtes. Spleens were inuted for min with mg/ml ollgense D nd then were mshed through 7- µm ell striner. DCs were sorted s desried into F/ CD CD lo B MHCII lo LyC pdcs nd F/ CD CD B MHCII LyC DCs (ebiosiene). Cs were ultured for h with IL-7 ( ng/ml) or ( ng/ml; E. oli strin :B; Sigm). Prllel ultures mintined without stimuli were used s ontrols. IL-7 ws prepred ording to the mnufturer s protool (ebiosiene). CNS infiltrtes were isolted s desried 7. Mie were perfused with ie-old PBS. The rin nd spinl ord were removed nd inuted in PBS ontining ollgense type III ( mg/ml; Worthington) nd DNse ( units/ml; Sigm-Aldrih). Tissues were then homogenized nd loded on %-7%-7% Peroll grdient for enrihment of CNS infiltrtes. Flow ytometry stining nd quisition. The following ntiodies were used for flow ytometry: peridinin hlorophyll proteinonjugted nti- LYC (HK.) nd llophyoynin-onjugted nti-cd (M/7; oth from BioLegend); phyoerythrin-onjugted ntiody to MHC lss II (M/..), fluoresein isothioynteonjugted nti-f/ (BM) nd Alex Fluor 7nti-CD9 (DMS; ll from ebiosiene); llophyoynin-indotriroynineonjugted nti-cd (HL) nd phyoerythrinonjugted nti-b (RA-B; ll from BD Phrmingen); nd fluoresein isothioynteonjugted nti-il-7ra (; R&D Systems). For nlysis of intrellulr Foxp, ell preprtions were stined for ell surfe mrkers with llophyoynin-indotriroynineonjugted nti- CD (RM-; BioLegend), then were fixed nd mde permele with fixtionpermeiliztion uffers (ebiosiene) nd were stined with peridinin hlorophyll protein-ynine.onjugted nti-foxp (FJK-s; ebiosiene). For intrellulr ytokine stining, ells were stimulted for h with phorol -myristte -ette ( ng/ml) nd ionomyin ( ng/ml) in the presene of GolgiPlug (BD Phrmingen). Then, ells were stined for surfe moleules (ntiodies identified ove), fixed nd mde permele with Cytofix/ Cytoperm Plus kit (BD Biosiene) nd stined with the following ntiodies: phyoerythrin-indotriroynineonjugted nti-ifn-γ (XMG.; BioLegend), phyoerythrin-onjugted nti-il-7 (ebio7b7; ebiosiene) nd llophyoynin-onjugted nti-il- (JES-E; BD Phrmingen). Dt were olleted with LSR II or FACSAri (BD Biosienes), then were nlyzed with FlowJo softwre (Treestr). in the presene of PLP(9) or PLP(79). The ells were pulsed with [ H]thymidine ( µci/well) for the finl h of the inution period. The frequeny of T ells produing IL-7 (ebio7b7; ebiosiene), IFN-γ (XMG.; BioLegend) or IL- (JES-E; BD Phrmingen) nd Foxp T ells (FJK-s; ebiosiene) ws ssessed y flow ytometry. For CFSE-sed prolifertion ssy, D CD T ells were leled with µm CFSE (roxyfluoresein diette suinimidyl ester; Moleulr Proes). Dt were quired on n LSR II (BD Biosienes) nd were nlyzed with FlowJo softwre (TreeStr). Mesurement of ytokines. Sereted ytokines were mesured fter h y enzyme-linked immunosorent ssy s desried 9. Quntittive PCR nlysis. RNA ws extrted with RNAesy olumns (Qigen, USA), then DNA ws prepred ording to the mnufturer s instrutions (Applied Biosystems) nd ws used s templte for rel-time PCR. All primers nd proes were provided y Applied Biosystems nd were used on the ViiA 7 Rel-Time PCR System (Applied Biosystems). Expression ws normlized to the expression of the housekeeping gene Gpdh. Primers-proe mixtures were s follows (from Applied Biosystems; identifiers in prentheses): Il (Mm9_m), Il (Mm_m), Il (Mm_m), Il (Mm9_m), Il7 (Mm_ m), Il7r (Mm979_m), Entpd (Mm7_m), Tgf (Mm7_m), Ifn (Mm9_s), Ido (Mm7_m), Ido (Mm7_m), Tnip (Mm_m), Tnfip (Mm7_ m), Rmp (Mm_m), Esr (Mm_m) nd Gpdh (Mm999999_g). Gene-expression nlysis. Trnsriptomes were nlyzed y Affymetrix mirorry MoGene st of smples otined, nd h fter stimultion with LPS or IL-7 nd LPS. Dt were normlized with the roust multirry verge lgorithm. Network tivity ws inferred with the NetGenertor lgorithm nd softwre of the R projet for sttistil omputing (version.-) using s input the hnge of gene expression (fold) over time points. The network grph ws produed with DOT plin text grph desription lnguge through the open softwre olletion Grphviz. For ncounter nlysis of gene expression, multiplexed trget profiling of inflmmtion- nd immune systemrelted trnsripts ws ustomized nd used in ordne with the mnufturer s protool (Nnostring). This omintion of genes nd their differenes in expression in vivo in DCs llowed investigtion of immune systemrelted pthwys during EAE with the EXPANDER tool ( expression nlyzer nd displyer ). Immunolot nlysis. For immunolot nlysis, ells were lysed with rdioimmunopreipittion uffer supplemented with protese inhiitor oktil (Sigm). Totl lystes of DCs ( µg) were resolved y eletrophoresis through % Bis-Tris Nupge gels (Invitrogen, USA) nd were trnsferred onto PVDF memrnes (Millipore). The following primry ntiodies were used: nti-il-7ra (MAB9; R&D Systems); ntiody to phosphorylted STAT (9), ntiody to phosphorylted STAT (97), nti-stat (9), nti- STAT (97) nd nti-gapdh (; ll from Cell Signling Tehnology); nti-spse- (7), nti-il-β (97) nd nti-β-tin (7; ll from Am). Immunolot nlysis ws done s desried 7 nd lots were developed with SuperSignl West Femto Mximum Sensitivity Sustrte s suggested y the mnufturer (Piere). ssys. For in vitro experiments, DCs pretreted with IL-7 ( ng/ml) nd tivted for h with ( ng/ml; E. oli strin :B; Sigm) were used (t rtio of :) to stimulte nive T ells from wild-type or D mie. Polrizing onditions hve een desried : IL- ( ng/ml) ws used to generte T H ells, or IL- ( ng/ml); TGF-β ( ng/ml) ws used to generte T H 7 ells; nd TGF-β ( ng/ml) nd IL-7 ( ng/ml) were used to generte T reg ells nd Tr ells, respetively. Mouse IL-, IL-, IL- nd TGF-β were ll from R&D Systems. For in vivo experiments, spleni ells were otined from wild type, IL7RA- or CD9-defiient mie t dy fter immuniztion with MOG() nd were restimulted in vitro for d in the presene of MOG(). SJL mie were immunized with PLP(9), nd t the end of the experiment, spleni ells were olleted nd restimulted in vitro for d Chromtin immunopreipittion. DNA-protein omplexes in ells were rosslinked with % prformldehyde nd lysed with. ml lysis uffer (% SDS, mm EDTA nd mm Tris-HCl, ph.) ontining protese inhiitor oktil (Rohe Moleulr Biohemils). Chromtin ws shered y sonition nd superntnts olleted fter entrifugtion were diluted in uffer (% Triton X-, mm EDTA, mm NCl nd mm Tris-HCl, ph.). µg ntiody ws preound for minimum of h to protein A nd protein G Dynl mgneti eds (Invitrogen) nd smples were wshed three times with ie-old PBS ontining % BSA, nd then were dded to the diluted hromtin, followed y immunopreipittion overnight. The mgneti edhromtin omplexes were then wshed three times in rdioimmunopreipittion uffer ( mm HEPES, ph 7., mm EDTA,.7% nture immunology 9// doi:./ni.9

12 N deoxyholte, % NP- nd. M LiCl), followed y two wshes with Tris- EDTA uffer. Immunopreipitted hromtin ws then extrted with solution of % SDS nd. M NHCO nd ws heted t C for t lest h for reversl of the prformldehyde ross-linking. DNA frgments were purified with QIAquik DNA purifition Kit (Qigen) nd were nlyzed y SYBR Green rel-time PCR (Tkr Bio). The following ntiodies were used for hromtin immunopreipittion: nti-stat (9; Cell Signling Tehnology) nd nti-stat (97; Cell Signling Tehnology). The following primer pirs were used: Stt (SRE-), forwrd, -GCTGGGCTTTAGAGACTTGTGG GC-, nd reverse, -ACCCATGCAAATGGTTTGGGCA- ; Stt (SRE-), forwrd, -TGAGGGCCAGCCCACACTTCA-, nd rev: -GCTCACT GGGTACCTCTTGCCA- ; Stt (IRF-), forwrd, -GGAACAAAAATATA GAGAGAACTTGGGA-, nd reverse, -GTAGTTTGACCTAAGTGGACAT AGG- ; Stt- Stt, forwrd, -AGGCTCTTGTATCCTTGCCACCTCT-, nd reverse, -TGATGGTGGAGTGCTGTGTGCTG -. Plsmids. The Entpd promoter reporter ws provided y D.J. Pinsky, nd vetors enoding onstitutively tive STAT nd STAT were provided y D. Frnk. in omplete Freund s djuvnt, nd drining lymph nodes nd spleens were olleted 7 d fter immuniztion nd then were ultured for h with MOG() ( µg/ml) nd rrier free reominnt IL- ( ng/ml; R&D Systems) or IL- ( ng/ml; R&D Systems). Susequently, ells were trnsferred intrvenously into or DC(IL-7RA-KO) mie. Clinil signs of EAE were ssessed y investigtors linded to tretment onditions, ording to the following sore:, no disese;, loss of til tone;., poor righting ility;, hind-lim wekness;, hind-lim prlysis;, qudrepresis; nd, moriund. For the genertion of one mrrowderived DCs, one mrrow ells isolted from the femurs of nive mie were ultured for 7 d in the presene of the ytokine GM-CSF ( ng/ml; Peproteh). On dy 7, ells were purified with CD mgneti eds (Miltenyi), then were ultured for h with IL-7 ( ng/ml) nd, for the finl h efore dministrtion, were loded with µg MOG() or PLP(9). DCs ( ells per mouse) were then extensively wshed nd dministered intrvenously four times, one every d. All experiments were rried out in ordne with guidelines presried y the Institutionl Animl Cre nd Use Committee t Hrvrd Medil Shool. npg Nture Ameri, In. All rights reserved. Trnsfetion nd luiferse ssys. HEK9 ells were grown in DMEM supplemented with % FBS nd were trnsfeted with FuGENE HD trnsfetion regent nd µg of eh plsmids ording the mnufturer s instrutions (Rohe). Firefly nd renill luiferse tivity ws nlyzed h fter trnsfetion with Dul Luiferse Assy kit (Promeg). Free ATP mesurement. DCs were ultured for h with IL-7 or LPS s desried ove (Isoltion of spleni DCs nd CNS infiltrtes). Cells were then wshed twie with phenol redfree RPMI- medium (Gio) nd were deprived of serum for h. Cell-free medium ws then nlyzed for endogenous ATP with ENTILEN rluiferse/luiferin regent (Promeg). Bioluminesent tivity ws mesured with n Infinite Pro luminometer (Ten). Etonuleotidse enzymti tivity nlysis. Thin-lyer hromtogrphy ws done s desried with slight modifitions. DCs were treted for h with IL-7 or LPS, then were inuted with mci/ml [ C]ADP (GE Helthre Life Sienes) in mm C nd mm Mg. Hydrolysis produts of [ C]ADP were ssessed y TLC in -µl liquots olleted t., nd min nd were pplied onto sili gel mtrix pltes (Sigm-Aldrih). [ C]ADP nd its rdioleled derivtives were seprted with the pproprite solvent mixture s desried,. Adenosine uptke nd demintion ws loked with µm dipyridmole. [ C]ADP, [ C]AMP nd [ C]ADO inuted in PBS served s stndrds. EAE indution nd vintion with DCs. EAE ws indued y suutneous immuniztion of mie with µg MOG() or µg PLP(9) (ANASPEC) s desried 9. Adoptive trnsfer EAE ws indued s desried with some modifitions. D mie were immunized with µg MOG() Antigen mirorry. Antigens (Supplementry Tle ) were spotted onto Epoxy slides (TeleChem) s desried. Nonspeifi inding on mirorrys ws loked with % ovine serum lumin, followed y inution with test serum (: dilution in loking uffer). Arrys were then wshed nd inuted with indoroynine-onjugted got ntiody to mouse immunogloulin G (detetion ntiody; --7; Jkson ImmunoReserh Ls). Antigen retivity ws defined y the men intensity of inding to the replites of tht ntigen on the mirorry. Rw dt were normlized nd nlyzed with GeneSpring softwre (Silion Genetis). Sttistil nlysis. Prism softwre ws used for sttistil nlysis. Sttistil nlysis ws done ording to the reommendtions of Nture for reporting life sienes reserh. For omprison of two groups, liner regression with 9% onfidene intervl, nd unpired, two-tiled Student s t-test were used. One-wy ANOVA for pired dt ws used to determine the signifine of the time-response urves. P vlues of <. were onsidered sttistilly signifint. For djustment of the signifine vlue for multiple omprisons, Bonferroni orretion ws pplied with orreted signifine vlue of Strossom, S.C. et l. Gletin- detivtes lssilly tivted mirogli nd protets from inflmmtion-indued neurodegenertion. Immunity 7, 9 ().. Quintn, F.J. et l. Aiolos promotes T H 7 differentition y diretly silening Il expression. Nt. Immunol., (). 9. Quintn, F.J. et l. Control of T reg nd T H 7 ell differentition y the ryl hydroron reeptor. Nture, 7 ().. Sun, X. et l. CD9/ENTPD expression y CD Foxp regultory T ells promotes hepti metstti tumor growth in mie. Gstroenterology 9, (). doi:./ni.9 9// nture immunology

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