Regulatory T cells prevent catastrophic autoimmunity throughout the lifespan of mice

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1 Regultory T ells prevent tstrophi utoimmunity throughout the lifespn of mie Jeong M Kim 1, Jeffrey P Rsmussen 1 & Alexnder Y Rudensky 1,2 Mie lking the trnsription ftor ( ) lk regultory T (T reg ) ells nd develop ftl utoimmune pthology. In mie, mny tivted effetor T ells express self-retive T ell reeptors tht re expressed in T reg ells in wild-type mie. Thus, in wild-type mie, most self-retive thymoytes esping negtive seletion re diverted into the T reg linege, nd whether T reg ells re ritil in self-tolerne in wild-type mie remins unknown. Here, ute in vivo ltion of T reg ells demonstrted vitl funtion for T reg ells in neontl nd dult mie. We suggest tht self-retive T ells re ontinuously suppressed y T reg ells nd tht when suppression is relieved, self-retive T ells eome tivted nd filitte elerted mturtion of dendriti ells. The trnsription ftor is speifilly expressed in regultory T ells (T reg ells) nd is required for their development 1 3. Loss-of-funtion muttions in the gene enoding in mie nd humns result in lk of T reg ells nd in ftl utoimmune pthology eginning t very erly ge nd ffeting multiple orgns 4 7. In ontrst, depletion of T reg ells y neontl thymetomy, y doptive trnsfer of nive T ell smples depleted of T reg ells into lymphopeni hosts or y tretment of mie with ntiodies speifi for CD25, whih is highly expressed on most T reg ells, results in muh milder nd more slowly progressing disese One potentil explntion for tht disrepny is provided y the oservtion tht protetive T reg ells, whih onstitute 1 15% of peripherl CD4 + T ells, express self-retive T ell reeptors (TCRs) 12. In -defiient mie, mny tivted T ells express TCRs normlly found in T reg ells in wild-type mie 13. Those dt suggest tht mny ells expressing T reg ell ssoited TCRs re not deleted in the sene of nd insted enter the pool of self-retive T ells, whih ontriute to disese progression. An extreme version of tht line of resoning is tht most self-retive T ells tht espe negtive seletion re diverted into the T reg ell linege nd tht diversion itself is minly responsile for preventing T ell medited utoimmunity. An dditionl, non mutully exlusive possiility is tht T reg ells re needed to estlish tolerne exlusively during erly postntl development, when self-retive reent thymi emigrnts might undergo preferentil homeostti prolifertion euse of moderte lymphopeni 14. During tht period, deletionl entrl tolerne might e reltively relxed euse of n immture thymi medullry omprtment in whih most negtive seletion egins 15. In ddition, T ells of the erly wve in neontl mie disply TCRs with inresed ross-retivity nd, therefore, self-retive potentil, euse of developmentl dely in expression of the terminl deoxynuleotidyl trnsferse enzyme 16,17. Finlly, the presene of T reg ells erly in life my filitte the differentition of T ells hving suppressive properties ut lking expression 18,19. Thus, the reltive importne of T reg ell medited suppression versus -independent suppressive mehnisms, inluding infetious tolerne nd immunosuppression medited y indued regultory T ells (T r 1 ells) nd T helper type 3 ells, remins unknown. Similrly, whether T reg ells re differentilly required for protetion ginst utoimmunity in neontl versus dult mie is lso not ler. To ddress those prinipl outstnding issues, we generted knok-in mie ontining the oding sequene of toxin reeptor inserted into the 3! untrnslted region of. Using those mie, we utely lted the entire T reg ell popultion y injeting toxin for vrying periods of time nd nlyzed the funtionl onsequenes of T reg ell ltion in neontl nd dult mie. In neworn nd dult mie, purging of T reg ells resulted in n utoimmune disese similr in severity to tht of -defiient mie. Our results emphsize the importne of T reg ell medited suppression throughout the lifespn of mie nd the indequy of other forms of peripherl tolerne estlished in dult mie. Furthermore, we identified other immune ells tht were tivted fter T reg ell ltion. Our dt suggest tht T ells, proly expressing self-retive TCRs, re trgets of ontinuous T reg ell medited suppression. After T reg ell ltion, those T ells eme tivted, produed sereted nd memrne-ound ytokines nd filitted dendriti ell (DC) mturtion. Those events proly led to the reruitment of dditionl self-retive T ell lones nd therey estlished viious yle of utoimmune hyper-retivity. RESULTS Induile T reg ell ltion in mie To generte mie llowing speifi elimintion of T reg ells in vivo, we designed trgeting onstrut in whih we inserted DNA enoding the 1 Deprtment of Immunology nd 2 Howrd Hughes Medil Institute, University of Wshington, Settle, Wshington 98195, USA. Correspondene should e ddressed to A.Y.R. (ruden@u.wshington.edu). Reeived 26 Otoer; epted 21 Novemer; pulished online 3 Novemer 26; orreted online 24 Deemer 26 (detils online); doi:1.138/ni1428 NATURE IMMUNOLOGY VOLUME 8 NUMBER 2 FEBRUARY

2 CD4 (%) Unt 13.5 DTR GFP / µg/kg 5. µg/kg 5 µg/kg LN Spl Thy humn diphtheri toxin reeptor (DTR) fused to sequenes enoding green fluoresent protein (GFP) nd equipped with n internl riosome entry site (IRES) into the 3! untrnslted region of to produe (Supplementry Fig. 1 online). We deteted GFP fluoresene nd DTR expression exlusively in T ells, not in CD4 + T ells (Fig. 1). If the DTR-GFP knok-in llele did not pertur the norml expression pttern of, whih is n X hromosome linked gene, hlf of the ells in /+ femle mie would e expeted to express GFP euse of rndom X-hromosome intivtion. Aordingly, pproximtely 5% of the peripherl T reg ells in /+ femle mie expressed GFP. These dt indited tht insertion of the IRES-DTR-GFP ssette in the lous did not lter the ompetitive fitness of T reg ells expressing the knok-in llele. Furthermore, unmnipulted hemizygous mle or homozygous femle mie showed no overt signs of utoimmunity or immune dysfuntion up to 5 months of ge, the mximum period monitored (dt not shown). To identify the dose of diphtheri toxin tht indued T reg ell elimintion, we treted mie nd wild-type ( ) littermtes with dily intrperitonel injetions of 5, 5. or.5 "g diphtheri toxin per kg ody weight for 7 onseutive dys. Tht tretment regimen ws sed on n study showing tht 7-dy injetion regimen is required for effiient elimintion of CD4 + T ells 2. Diphtheri toxin did not ffet the frequeny of T reg ells in ontrol littermtes t ny dose tested, ut it resulted in dose-dependent derese in T reg ell numers in the lymph nodes, peripherl lood, thymuses nd spleens of mie Figure 1 Chrteriztion of induile T reg ell ltion. () Flow ytometry of DTR expression (top row) nd GFP fluoresene (ottom row) y CD4 + (lue) nd CD4 + (green) lymph node ells (mouse identifition, ove histogrms). Colors in histogrms orrespond to gted popultions in the dot plot (left); red lines, isotype ontrol (DTR plots) or CD4 + ells from mie (GFP plots). () Flow ytometry of expression in CD4 + lymph node ells from untreted mie (Unt; fr left) or from mie (lue lines) nd mie (red lines) treted with diphtheri toxin (right three; dose, ove histogrm). Numers ove rketed lines indite perent ells mong CD4 + ells. () Flow ytometry of the reovery of CD4 + ells from the lymph nodes (LN), spleens (Spl) nd thymuses (Thy) of 8- to 1-week-old nd mie given two injetions of diphtheri toxin (5 "g/kg) on onseutive dys. Eh dt point (± s.e.m.) represents three to four mie. Dt re representtive of two to five experiments. (Fig. 1 nd dt not shown). Complete elimintion of T reg ells ws hieved fter 7 d of tretment with 5 "g diphtheri toxin per kg ody weight (5 "g/kg), the highest dose tested. After 2 d of diphtheri toxin injetions,.4% of CD4 + T ells expressed ; tht indited over 97% depletion within 48 h ompred with T ells in untreted mie or diphtheri toxin treted ontrol mie (Fig. 1). The extent of T reg ell elimintion remined onstnt throughout the time ourse nd ws not further ugmented t lter time points (Fig. 1 nd dt not shown). To determine the rte of T reg ell reovery fter ute T reg ell depletion, we treted mie with diphtheri toxin t h nd 24 h nd monitored the susequent pperne of ells in the thymus, lymph nodes nd spleen. Administrtion of diphtheri toxin did not ffet T reg ell numers in littermtes, ut ner-omplete elimintion of T reg ells ws hieved in the thymus, lymph nodes nd spleen 2 d fter the initil injetion (Fig. 1). On dy 4, thymi T reg ells reounded to 47% of their numers efore tretment, wheres peripherl T reg ells were present t 8% nd 6% of pretretment numers in lymph node nd spleen, respetively. The T ell suset in the thymus ws fully reovered y dy 1, nd pretretment numers of T reg ells in the lymph nodes nd spleen were rehed etween dys 1 nd 15 (Fig. 1). Although those tissue-speifi reovery kinetis n e onsidered s evidene supporting the ide tht the thymus is entrl to the genertion of T reg ells, further studies re needed to ddress tht importnt issue. Autoimmunity fter neontl T reg ell ltion Next we ompred the utoimmune pthology developing in mie sujeted to T reg ell elimintion t irth with tht in mie with germline deletion of. We injeted litters with 5 "g/kg of diphtheri toxin eginning 12 h fter irth nd ontinuing every other dy therefter. By 24 d fter irth, pproximtely 4% of mie hd moriund phenotype, inluding filure to thrive, lk of moility, ventrl skin lesions, hunhed posture nd onjuntivitis; the remining mie eme moriund within 27 d of irth (Fig. 2 nd dt not shown). In ddition, untreted mie nd diphtheri toxin treted mie developed similr lymphoprolifertive syndromes, s shown y similr degree of lymphdenopthy, whih ws not deteted in ontrol mie (Fig. 2). The severe nd rpid pthology of diphtheri toxin treted mie ws reminisent of tht of mie 1,21. One notle differene in diphtheri toxin treted versus mie ws the presene of severe til sling only in mie (dt not shown). Both diphtheri toxin treted mie nd untreted mie showed severe tissue pthology mnifested y mssive lymphoyti nd mononuler infiltrtes in liver sinusoids, lung interstitium nd epidermis of the skin nd other orgns (Fig. 2 e nd dt not shown). In ontrst, we found no tissue pthology in diphtheri toxin treted littermtes of mie or in untreted littermtes of mie. 192 VOLUME 8 NUMBER 2 FEBRUARY 27 NATURE IMMUNOLOGY

3 d e Figure 2 Consequenes of hroni T reg ell elimintion in neontes. () Survivl of progeny of nd /+ mie injeted with diphtheri toxin from irth every other dy. Mie were monitored for signs of terminl utoimmune disese nd were killed one moriund. () Splenomegly nd lymphdenopthy in 3-week-old (n = 1) nd (n = 6) littermtes treted with diphtheri toxin (+DT) s desried in, nd in untreted ( DT) (n = 5) nd (n = 4) littermtes. ( e) Hemtoxylin nd eosin stining of setions from livers, lungs nd skin of 3-week-old mie () or mie (d) treted with diphtheri toxin s desried in, nd of ge-mthed, untreted mie (e). Originl mgnifition, 25. Representtive dt re from two independent experiments. Beuse T reg ells suppress the prolifertion nd tivtion of nonregultory T ells, we next determined whether CD4 + T ells were hypertivted in mie tht were hronilly exposed to diphtheri toxin from irth. We ompred the tivtion sttus of CD4 + T ells from diphtheri toxin treted mie with tht of ells from diphther i toxin treted littermtes. CD4 + T ells from diphtheri toxin treted mie hd higher expression of T ell tivtion mrkers, inluding CD25, CTLA-4 nd ICOS, nd lower expression of CD62L (inditive of n tivted sttus) thn did ells from diphtheri toxin treted littermtes (Fig. 3). The extent of CD25 CD62L CTLA-4 ICOS Ki T ell tivtion of CD4 + T ells ws similr to tht of CD4 + T ells derived from ge-mthed -defiient mie (Fig. 3). Ativted CD4 + T ells from diphtheri toxin treted mie nd -defiient mie lso expressed Ki-67, mrker inditive of prolifertion (Fig. 3). CD4 + T ells from diphtheri toxin treted nd untreted ontrol mie hd low expression of those tivtion mrkers, ruling out the possiility of nonspeifi T ell tivtion due to potentil ontmintion of diphtheri toxin preprtions with teril produts. These results olletively showed tht elimintion of T reg ells in neontes indued n utoimmune syndrome similr to tht of mie, with the exeption of the differene in the severity of gross skin lesions. These dt indited tht self-retive T ells tht filed to ommit to the T reg ell linege were not the only use of the ftl utoimmune lesions in mie; in wild-type mie, self-retive T ells present in the non-t reg T ell suset must e ontrolled y T reg ells to prevent tstrophi lympho prolifertive disese. T reg ells nd self-tolerne in dult mie To ddress the forementioned possiility of requirement for T reg ells in setting up nd enforing -independent tolerne mehnisms exlusively during neontl development, we exmined the onsequenes of hroni ltion of T reg ells in 3-month-old mie. Like neontes, diphtheri toxin treted dult mie developed pthology mnifested y severe lymphdenopthy nd splenomegly, wsting disese hrterized y weight loss, filure to thrive nd redued moility, nd severe onjuntivitis (Fig. 4 nd dt not shown). Notly, T reg ell elimintion in dult nonlymphopeni mie resulted in n even more rpid development of terminl utoimmune disese thn in neontes. As erly s 1 d fter the first injetion of diphtheri toxin, some mie eme moriund, wheres ll mie suumed to terminl disese y 3 weeks of T reg ell ltion (Fig. 4). Therefore, -independent reessive nd dominnt tolerne mehnisms estlished in dult mie re not suffiient to protet mie from ftl utoimmunity fter T reg ell elimintion. Next we determined the kinetis of CD4 + T ell tivtion fter T reg ell ltion. At 2 d fter the initil diphtheri toxin tretment of mie, we deteted n inrese of out 2% in the proportion of CD4 + ells expressing the T ell tivtion mrkers CTLA-4 nd CD25 ompred with tht of ontrol mie (Fig. 5,). This result suggested tht in norml mie, mny non T reg CD4 + T ells were kept in hek y T reg ells. The reltive nd solute numers of CD4 + T ells with n tivted phenotype, inluding higher expression of CTLA-4 or CD25 nd lower expression of CD62L, grdully rehed plteu y 6 d fter diphtheri toxin tretment (Fig. 5,d). At tht time, the extent of CD4 + T ell tivtion ws similr to tht in 3-week-old mie sujeted to T reg ltion from irth (Fig. 3) Figure 3 T ell tivtion in nd mie depleted of T reg ells from irth. () Expression of tivtion mrkers on spleni CD4 + T ells from 3-week-old mie (lue lines) nd mie (red lines) injeted with diphtheri toxin from irth every other dy. () Expression of tivtion mrkers on spleni CD4 + T ells from ge-mthed mie (red lines) nd mie (lue lines). Numers ove rketed lines indite perent ells positive for mrker (elow histogrm). Dt re representtive of two independent experiments. NATURE IMMUNOLOGY VOLUME 8 NUMBER 2 FEBRUARY

4 1 Survivl (%) Figure 4 Consequenes of hroni T reg ell elimintion in dult mie. () Lymphdenopthy nd splenomegly of 3- to 5-month-old mie (n = 8) nd mie (n = 7) injeted with diphtheri toxin every other dy. () Survivl of nd mie treted with diphtheri toxin s desried in. Mie were exmined for linil signs of disese nd were killed one moriund. Representtive dt re from two independent experiments. Ativtion of DCs fter T reg ell ltion Next we ssessed the sttus of other immune ell types fter T reg ell ltion. At 7 d fter diphtheri toxin dministrtion, we noted inreses in the numers of CD19 + B22 + B ells, F4/8 + CD11 mrophges, GR- 1 + CD11 + grnuloytes nd nturl killer ells in the spleen nd lymph nodes of mie (Tle 1). We lso noted n inrese in the frequeny nd n inrese of nerly tenfold in the solute numers of CD11 hi DCs expressing the mjor histoomptiility lss II moleule I-A in the lymph nodes of dult mie (Fig. 6 nd Tle 1). The perentges nd solute numers of spleni CD11 hi I-A + ells inresed 2.5- nd 5-fold, respetively (Fig. 6 nd Tle 1). In ddition, 3-weekold mie treted with diphtheri toxin from irth hd fivefold CTLA-4 DTR higher frequeny of nd higher solute numers of CD11 hi I-A + lymph node ells thn their littermtes (Fig. 6 nd dt not shown). We deteted similr degree of DC popultion expnsion in ge-mthed mie, suggesting tht DC dysregultion is generl phenomenon resulting from the sene of T reg ells (Fig. 6). Among CD11 + ells, the proportion of myeloid CD11 + DCs inresed, wheres the proportion of DCs positive for the plsmytoid DC mrker PDCA-1 deresed fter T reg ell elimintion (Fig. 6). Nevertheless, oth DC susets inresed in solute numers (dt not shown). Furthermore, there ws moderte ut reproduile inrese in the expression of DC mturtion mrkers, inluding CD8 nd CD4, on CD11 + ut not PDCA-1 + DCs (Fig. 6d). These results olletively suggested tht T reg ells normlly restrin CD11 + DC mturtion. Next we sought to determine whether the inreses in DC numers were used y enhned DC survivl or prolifertion. To test whether T reg ell elimintion diminished the poptosis of CD11 + CD11 + DCs, we treted spleni nd lymph node ells from mie with diphtheri toxin for 7 onseutive dys, stined them with fluoresene-tgged nnexin V or z-vad-fmk nd nlyzed them y flow ytometry. Insted of showing less poptosis, CD11 + CD11 + DCs from diphtheri toxin treted mie showed slightly more poptosis thn DCs from diphther i toxin treted ontrol mie (dt not shown). Furthermore, CD11 + CD11 + DCs from mie did not express the prolifertion mrker Ki-67, regrdless of whether T reg ells were lted (dt not shown). Beuse the lk of Ki-67 stining suggested tht DCs were mostly nonprolifertive fter T reg ell elimintion, we were le to mesure the kinetis of the genertion of CD11 + CD11 + DCs through ontinuous leling CD Ativted CD4 + (%) CD25 + CD62L lo CTLA-4 + d Ativted CD4 + T ells ( 1 7 ) CD25 + CD62L lo CTLA Figure 5 Kinetis of T ell tivtion in response to ute elimintion of T reg ells from dult mie. (,) Flow ytometry of intrellulr CTLA-4 () nd surfe CD25 () expression on lymph node ells isolted from 4- to 5-week-old nd mie injeted with diphtheri toxin t h nd 24 h, ssessed 2 d fter the initil injetion. Red numers indite perent CD4 + T ells expressing CTLA-4 () or CD25 (); lk numers indite perent of totl ells in eh qudrnt. (,d) Perentges () nd solute numers (d) of CD4 + T ells with CD25 +, CD62L lo or CTLA-4 + phenotype in nd mie injeted dily with diphtheri toxin s desried in,. Dt re representtive of two to three experiments. 194 VOLUME 8 NUMBER 2 FEBRUARY 27 NATURE IMMUNOLOGY

5 LN DCs/mouse ( 1 6 ) CD I-A DT DT with 5-romodeoxyuridine (BrdU). In greement with pulished reports, in ontrol mie, pproximtely 5% of CD11 + DCs inorported BrdU fter 3 d of leling 22 (Supplementry Fig. 2 online). Between the dys 2 nd 3 of diphtheri toxin tretment, we deteted 2% inrese in the perentge of BrdU + CD11 + DCs in mie. The frequeny of BrdU + CD11 + DCs progressively inresed throughout the time ourse, Tle 1 Anlysis of expnded ell susets in response to T reg ell elimintion Lymph node Fold hnge Spleen Fold hnge CD19 + B ± ± ± ± TCR# + CD ± ± ± ± TCR# + CD ± ± ± ± CD11 hi IA ± ± ± ± GR-1 hi CD ± ± ± ± F4/8 + CD ± (n = 4) ± (n = 3) DT DT ± (n = 4) CD11 CD ± (n = 3) NK ± ± ± ± Lymph node ells nd splenoytes were isolted from nd mie fter 7 d of T reg ell elimintion y digestion with ollgense D. Men ± s.d. vlues re sed on mie (n = 6) nd mie (n = 5) unless otherwise noted. d e PDCA CD I-A 3.5 suggesting more effiient genertion of CD11 + DCs from preursors fter ltion of T reg ells. In tissue ulture studies, T reg ells hve een shown to inhiit the mturtion nd to lter the ytokine prodution of DCs Alterntively, T reg ells my indiretly ffet DC mturtion y suppressing self-retive CD4 + T ells tht themselves indue the mturtion of DCs. Consistent with the CD SMARTA Rg2 +/ SMARTA Rg2 / Figure 6 Inrese in DC numers fter T reg ell elimintion. ( ) Anlysis of splenoytes nd lymph node ells from nd mie 4 8 weeks of ge injeted dily with diphtheri toxin. () Flow ytometry of splenoytes (top) nd lymph node ells (ottom) isolted nd stined on dy 7 with ntiodies speifi for CD11 nd I-A. Numers ove oxed res indite perent ells in outlined gte. () Asolute numers of CD11 hi I-A + ells in lymph nodes of 3-week-old nd mie treted with diphtheri toxin from irth (+DT), nd in gemthed, untreted ( DT) nd littermtes. () Expression of CD11 nd PDCA-1 on CD11 + CD3 lymph node ells. (d) Expression of CD4 nd CD8 on CD11 hi I-A + CD11 + lymph node ells from mie (lue lines) nd mie (red lines). Blk lines, isotype ontrol. (e) CD11 nd I-A expression on splenoytes (top) nd lymph node ells (ottom) from SMARTA Rg2 +/ nd SMARTA Rg2 / mie (two to three mie per group). Numers ove oxed res indite perent ells in outlined gte. Representtive dt re from two independent experiments. ltter possiility, tivted T ells generted fter T reg ell elimintion expressed GM-CSF, interleukin 13, RANKL nd CD4L, ell surfe moleules tht promote DC development nd mturtion (Supplementry Fig. 3 online). In ddition, we deteted no DC tivtion in SMARTA TCRtrnsgeni mie on the Rg2 / (reomintion-tivting gene 2 defiient) geneti kground (Fig. 6e). As these mie lk T reg ells nd ll T ells express single TCR speifi for the lymphoyti horiomeningitis virus glyoprotein 33 epitope, the sene of T reg ells itself ws not suffiient to trigger DC tivtion. DISCUSSION The identifition of muttions in the gene enoding s the use of ggressive utoimmunity in humn ptients with IPEX syndrome (immunodysregultion, polyendorinopthy, enteropthy, X-linked syndrome) nd in the mutnt mouse strin surfy nd the susequent disovery of the essentil funtion of in the development of T reg ells hve provided geneti foundtion for the phenomenon of T reg ell medited dominnt tolerne 1 7. Those results suggest tht T reg ells re vitl in preventing utoimmunity. However, T reg ells express self-retive TCRs, nd TCR self lignd intertions in NATURE IMMUNOLOGY VOLUME 8 NUMBER 2 FEBRUARY

6 ertin rnge of inresed ffinity filitte T reg ell development in the thymus 12,27 3. Furthermore, mny T ells with tivted ut not nive phenotypes in mie use TCRs expressed y T reg ells in wild-type mie 13. Those results rise the possiility tht ommitment of self-retive thymoytes esping negtive seletion to the T reg ell linege neutrlizes most potentilly pthogeni self-retive T ells. Tht senrio indites tht in wild-type mie, T reg ells ontrol reltively few T ells tht re retive to self or environmentl ntigens nd tht elimintion of T reg ells would result in slowly progressing utoimmunity ompnied y mild lesions, in ontrst to the severe nd rpid utoimmunity in mie. Aording to tht ide, utoimmunity indued fter ltion of T reg ells in wild-type mie would e similr to tht in thymetomized mie t dy 3 or in lymphopeni reipients of CD4 + T ells. However, our nlysis of T reg ell ltion in mie showed tht the ontinuous presene of T reg ells in norml mie in physiologil onditions ws needed to prevent the devstting lymphoprolifertive disese resemling tht of mie. Notly, the disese indued fter the elimintion of T reg ells in dult mie ws even more ggressive thn tht in neontes sujeted to T reg ell ltion. We propose tht this is t lest prtilly due to the ft tht overll, the T ell omprtment, inluding self-retive T ells, is muh lrger in dult mie thn in neontes. Those results exlude the possiility tht T reg ells, y limiting the tivtion nd popultion expnsion of self-retive T ells filitted y trnsient lymphopeni inherent to neontes, re ritil erly in life ut not during dulthood 14. Furthermore, our findings emphsize the inility of reessive tolerne mehnisms estlished in unmnipulted dult mie (suh s entrl nd peripherl nergy) to restrin tstrophi utoimmunity eginning fter T reg ells re eliminted. In ddition to T reg ells, severl T ell susets, inluding interleukin 1 produing suppressive T ells, re reported to effiiently inhiit T ell responses to foreign nd self ntigens 31,32. It hs lso een proposed tht CD4 + CD25 + T reg ells medite suppression y generting t lest some of those suppressive T ells 33. A orollry to tht model is tht ltion of T reg ells in dults should result in less ggressive nd severe disese thn tht in neontes. However, the opposite trend oserved indites the inility of T reg ells to restrin utoimmunity on their own. Erly studies indited tht the thymus is entrl to T reg ell differentition 34,35. Susequently, it hs een proposed tht nonregultory T ells speifi to self or environmentl ntigens n lso upregulte nd quire regultory properties in the periphery However, our oservtion of very fst regenertion of thymi suset fter trnsient ltion of oth the thymi nd peripherl T reg omprtments, followed, with onsiderle dely, y the peripherl T reg suset, indites tht the thymus serves s hief soure of T reg ell development even when the peripherl T reg ell nihe is emptied fter ute T reg ell ltion. An extremely fst pe of deteriortion of mie sujeted to T reg ell ltion indites tht very high proportion of norml CD4 + T ell repertoire is ontrolled y T reg ells. Tht ide is supported y the finding of douling of the proportion of peripherl T ells expressing tivtion mrkers fter less thn 48 h of dministrtion of T reg ell ltion regimen. In ddition to T ell tivtion, notle onsequene of T reg ell ltion ws the inrese in DC numers, demonstrting tht T reg ells ontrol DC ell numers in vivo. Reports hve suggested tht T reg ells kill immture myeloid DC in grnzyme-dependent wy in vitro; however, we filed to find evidene to support tht ide, s neither DC poptosis nor survivl ws ltered fter T reg ell ltion 4. Insted, in vivo BrdU leling experiments showed tht the rte of CD11 + CD11 + DC genertion ws sustntilly elerted fter ltion of T reg ells. We lso noted n inrese in the expression of tivtion mrkers on DCs. Tht result is in pprent greement with in vitro studies proposing tht T reg ells ontrol T ell tivtion y suppression of DC tivtion In ddition, imging studies hve suggested tht T reg ells diminish the ility of DCs to form stle ontts with self-retive T ells nd therey diminish their tivtion 41,42. Our dt hve not definitively indited tht T ells or DCs or oth of those ell susets re primry trgets of T reg ells in vivo. Nevertheless, we propose tht the DC mturtion is proly the result of very erly tivtion of CD4 + T ells retive to self nd environmentl ntigens indued fter the ltion of T reg ells. The ide of dependene of DC tivtion nd popultion expnsion in the presene of self-retive T ells is onsistent with our oservtion tht DCs filed to expnd their popultions nd did not hve n tivted phenotype in TCR-trnsgeni Rg2-defiient mie lking T reg ells. In greement with those results, diphtheri toxin indued ltion of T reg ells omined with ntiody-medited depletion of totl CD4 T ell suset prevented the expnsion of DC numers (J.M.K. nd A.Y.R., unpulished oservtions). Furthermore, T reg ell elimintion triggered the prodution of ytokines suh s GM-CSF, whih is known to promote DC differentition, y CD4 + T ells. Although the mehnism of T reg ell medited suppression of DC tivtion remins unler nd its eluidtion requires further investigtion, we propose tht T reg ells my indiretly influene DC dynmis y suppressing self-retive CD4 + T ells. METHODS Mie. Mie with germline deletion of on C57BL/6 kground ( mie) hve een desried 1. C57BL/6 mie ( ) were purhsed from Jkson Lortories. All mie were red nd mintined in speifi pthogen free onditions in the niml fility t University of Wshington in ordne with the institutionl guidelines. The trgeting onstrut for mie ws generted y suloning of 7.9-kilose XI frgment of ontining exons 6 11 from 3.8-kilose osmid ontining the omplete gene 1 into pbluesript vetor ontining PGK-DTA negtive-seletion ssette. A SlI restrition site ws engineered in ple of the BeI site in the 3! untrnslted region of upstrem of the polydenyltion signl. The trgeting onstrut ws generted y first loning of the DTR-GFP DNA from the CD11-DTR-GFP plsmid y PCR mplifition into the IRES-MCS-BGHpA-FRT-NEO-FRT shuttle vetor. The IRES- DTR-GFP frgment ws then loned into the SlI site inserted into. The linerized trgeting onstrut ws introdued y eletroportion into R1 emryoni stem ells nd neomyin-resistnt lones were sreened y PCR ross the 3! rm for evidene of homologous reomintion. BspHIdigested genomi DNA of positive lones were then sreened y Southern lot. Emryoni stem ell lones ering the orretly trgeted lous were injeted into C57BL/6 lstoysts, nd himeri mle offspring were mted to FLP-deleter mie for exision of the PGK-Neo ssette. Trnsmission of the trgeted llele ws onfirmed y PCR. Cell isoltion. Single-ell suspensions from lymph nodes (superfiil ervil, mndiulr, xillry, lterl xillry, superfiil inguinl nd mesenteri), spleen nd thymus were generted y mehnil disruption. For DC studies, tissue smples were mined in 5% (volume/volume) FCS in RPMI medium ontining 2 mg/ml of ollgense D (Rohe) nd 2 "g/ml of DNseI (Rohe). Collgense digestion ws done for 3 min t 37 C nd single-ell suspensions were prepred y pssge of ells through filter with pore size of 1 "m (BD Flon). Regents nd flow ytometry. Diphtheri toxin ws purhsed from Sigm nd ws reonstituted ording to the mnufturer s protool. Frozen diphtheri toxin stoks were frozen nd thwed one nd 5 "g/kg of diphtheri toxin ws injeted intrperitonelly unless otherwise noted. Phyoerythrin-onjugted ntiody to CD25 (nti-cd25; PC61), llophyoynin nti- (FJK-16S), llophyoynin nti-cd11 (N418), phyoerythrin nti-cd8 (16-1A1), phyoerythrin nti-cd4l (MR1), nti-rankl (IK22/5), llophyoynin nti-cd19 (MB19-1) nd llophyoynin nti-f4/8 (BM8) were purhsed from ebiosiene; phyoerythrin, FITC or iotin nti-cd11 (M1/7), phyoerythrin or 196 VOLUME 8 NUMBER 2 FEBRUARY 27 NATURE IMMUNOLOGY

7 iotin nti-cd11 (HL3), peridinine hlorophyll protein nti-cd4 (L3T4), phyoerythrin nti-cd8$ (53-6.7), phyoerythrin or FITC nti-i-a (AF6-12), FITC nti-cd4 (HM 4.3), phyoerythrin or FITC nti-tcr# (H57-597), phyoerythrin nti-ctla4 (UC1-4F1-11), phyoerythrin nti-icos (7E.17G9), phyoerythrin nti-cd62l (MEL14), peridinine hlorophyll protein CD3 (2C11), llophyoynin nnexin V, nti-brdu, phyoerythrin nti-ki-67, phyoerythrin or FITC nti-cd45r (B22) nd FITC nti-gr-1 were purhsed from BD Phrmingen; iotin nti- HB-EGF (nti-dtr; BAF259) ws from R&D Systems; FITC z-vad-fmk ws from Promeg; nd iotin nti-mpdca-1 ws from Miltenyi Biote. For nnexin V, z-vad-fmk nd intrellulr nti- stining, ells were stined ording to the mnufturer s instrutions. Cells were nlyzed on FACSCnto (BD Biosienes) with FloJo softwre (Tree Str). Cytokine nlysis. CD4 + ells were purified with nti-cd4 eds nd n utomacs (Miltenyi Bioteh). Sorted CD4 + ells (1 1 6 ) were stimulted for 14 h with 5 ng/ml of phorol 12-myristte 13-ette nd 2 ng/ml of ionomyin nd superntnts were nlyzed with ytokine rry (Allied Bioteh). Interleukin 13 ws mesured y enzyme-linked immunosorent ssy (R&D Systems). Note: Supplementry informtion is ville on the Nture Immunology wesite. ACKNOWLEDGMENTS We thnk D. Liggit for ssistne with the nlysis of histopthology; A. Gll, L. Krpik, T. Chu, K. Forush, P. deroos nd M. Shwrtz for ssistne; R. Setoguhi nd M. Bevn (University of Wshington) for SMARTA Rg2 / mie; nd memers of the Rudensky l for disussions. The CD11-DTR-GFP plsmid ws gift from R. Lng (Children s Hospitl, Cininnti, Ohio). Suported y the Howrd Hughes Medil Institute (A.Y.R.), Cner Reserh Institute (J.M.K.) nd US Ntionl Institutes of Helth (A.Y.R.). COMPETING INTERESTS STATEMENT The uthors delre tht they hve no ompeting finnil interests. Pulished online t Reprints nd permissions informtion is ville online t reprintsndpermissions/ 1. 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Regultory T ells inhiit stle ontts etween CD4 + T ells nd dendriti ells in vivo. J. Exp. Med. 23, (26). 42. Tng, Q. et l. Visulizing regultory T ell ontrol of utoimmune responses in nonoese dieti mie. Nt. Immunol. 7, (26). NATURE IMMUNOLOGY VOLUME 8 NUMBER 2 FEBRUARY