Selection of Foxp3 + regulatory T cells specific for self antigen expressed and presented by Aire + medullary thymic epithelial cells

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1 Selection of Foxp3 + regultory T cells specific for self ntigen expressed nd presented y Aire + medullry thymic epithelil cells Kthrin Aschenrenner 1, Louise M D Cruz 1,3, Eliseth H Vollmnn 1,3, Mri Hintererger 1, Jn Emmerich 1, Lee Kim Swee 2, Antonius Rolink 2 & Ludger Klein 1 The prmeters specifying whether utorective CD4 + thymocytes re deleted (recessive tolernce) or differentite into regultory T cells (dominnt tolernce) remin unresolved. Dendritic cells directly delete thymocytes, prtly through crosspresenttion of peripherl ntigens promiscuously expressed in medullry thymic epithelil cells (mtecs) positive for the utoimmune regultor Aire. It is uncler if nd how mtecs themselves ct s ntigen-presenting cells during tolernce induction. Here we found tht n sence of mjor histocomptiility clss II molecules on mtecs resulted in fewer polyclonl regultory T cells. Furthermore, trgeting of model ntigen to Aire + mtecs led to the genertion of specific regultory T cells independently of ntigen trnsfer to dendritic cells. Thus, routing of mtec-derived self ntigens my determine whether specific thymocytes re deleted or enter the regultory T cell linege. The ide tht the thymic epithelium is criticl for the induction of dominnt tolernce dtes ck to the 198s 1. The cell type mediting tolernce in those studies remined elusive; however, follow-up studies suggested suset of CD4 + T cells 2. It is now widely ccepted tht nturl Foxp3 + + regultory T cells (T reg cells), the est chrcterized meditors of dominnt tolernce so fr, originte in the thymus 3 5. Erly studies suggesting thymic origin of T reg cells 6,7 hve een complemented y T cell receptor (TCR) sequence nlyses demonstrting considerle overlp etween thymic nd peripherl T reg cell repertoires 8,9. Findings in mice expressing trnsgenes encoding oth TCR nd cognte ntigen hve olstered this ide y estlishing tht expression of self ntigen in the thymic epithelium cn e sufficient for the genertion of ntigen-specific T reg cells. Those findings were often (incorrectly) interpreted to men tht recognition of the respective ntigen in the context of mjor histocomptiility complex (MHC) clss II on epithelil cells induces T reg cell differentition. The ntigen-presenting cell (APC) tht ultimtely displyed thymic epithelium derived ntigen ws not identified in ny of the models. Furthermore, most of those studies did not llow for distinction etween corticl thymic epithelil cells (ctecs) nd medullry thymic epithelil cells (mtecs) s source of ntigen 1 15, nd only one report suggested involvement of exclusively mtec-derived ntigen 16. Open issues pertining to the exct nture of the ntigen-expressing nd/or ntigen-presenting cells involved in T reg cell development re intimtely relted to the issue of whether specifiction of T reg cells is n erly (corticl) or lte (medullry) event during thymocyte development. Studies of polyclonl T cell repertoires hve yielded conflicting results, some fvoring corticl specifiction 17 nd others fvoring medullry specifiction 18 of T reg cell fte. A seprte line of experiments hs firmly estlished tht mtecs contriute to T cell tolernce y promiscuously expressing wide rnge of otherwise tissue-specific ntigens 19,2. Muttions in the gene encoding the utoimmune regultor Aire, which in epithelil comprtments of the thymus is expressed exclusively in the medull, pertur promiscuous expression of tissue-specific ntigens nd cuse utoimmune polyendocrine syndrome in humns nd generl utoimmunity in mice 21. Evidence suggests tht tissue-specific ntigens expressed in mtecs contriute to centrl tolernce in n essentil wy y inducing recessive tolernce (such s clonl deletion of utorective thymocytes) 22,23, wheres their eventul function in the induction of dominnt tolernce remins specultive. Very limited informtion is ville out the type of APC tht ultimtely presents mtecderived ntigens to trigger deletion of ntigen-specific thymocytes. The importnce of TEC-derived self ntigens for the induction of oth dominnt nd recessive modes of centrl tolernce is undisputed However, it remins uncler whether thymocyte deletion or the genertion of T reg cells resulting from ntigen expression in mtecs or TECs in generl is triggered in TEC-utonomous wy. This issue is of prticulr importnce ecuse intercellulr trnsfer 1 Reserch Institute of Moleculr Pthology, 13 Vienn, Austri. 2 Deprtment of Clinicl nd Biologicl Sciences, Center for Biomedicine, University of Bsel, CH-451 Bsel, Switzerlnd. 3 Present ddresses: Division of Biologicl Sciences, University of Cliforni, Sn Diego, L Joll, Cliforni 9293, USA (L.M.D.) nd The CBR Institute for Biomedicl Reserch, Deprtment of Pthology, Brighm nd Women s Hospitl, Hrvrd Medicl School, Boston, Msschusetts 2115, USA (E.H.V.). Correspondence should e ddressed to L.K. (klein@imp.univie.c.t). Received 27 Novemer 26; ccepted 26 Jnury 27; pulished online 25 Ferury 27; doi:1.138/ni1444 NATURE IMMUNOLOGY VOLUME 8 NUMBER 4 APRIL

2 CD4 RTOC Host RTOC Host WT WT WT H2-A1 / 11.9 ± ± 13.4 CD8 Gted on CD4 + SP thymocytes Foxp3 3.2 ± ±.9 B6 WT thymus 12.9 ± ±.3 of self ntigens from epithelil to hemtopoietic cells in the thymic microenvironment is well documented phenomenon 27,28. In fct, two reports hve suggested centrl function for thymic dendritic cells (DCs) in the estlishment of oth recessive nd dominnt CD4 + T cell tolernce. First, deletion of TCR-trnsgenic CD4 + thymocytes in response to model ntigen expressed in mtecs requires ntigen trnsfer to hemtopoietic cells, most likely DCs 29. Second, in vitro evidence suggests tht thymic DCs conditioned with thymic stroml lymphopoietin my promote the genertion of T reg cells in the humn thymus 3. Those findings collectively sprked the ide tht thymic DCs my serve dul, possily mndtory, function in centrl CD4 + T cell tolernce y inducing negtive selection s well s T reg cell genertion; in contrst TECs my merely serve s n ntigen reservoir. The ensuing mode of tolernce might then e dictted y the mturtion stte of the DCs 31. In mechnistic terms, this model my offer missing link etween thymic induction of nturl T reg cells nd peripherl, new genertion of induced T reg cells through suimmunogenic delivery of cognte ntigen 32,33. An lterntive view would hold tht quntittive nd/or qulittive fetures of intrthymic ntigen recognition in the context of DCs versus TECs would fvor deletion versus T reg cell differentition, respectively. We egn our study here to clrify the functions of epithelil versus hemtopoietic ntigen presenttion during the genertion of T reg cells in mouse thymus. Furthermore, we imed to explore the potentil contriution of mtecs to T reg cell development, with prticulr emphsis on their potentil functions s ntigen reservoirs nd/or s true APCs. RESULTS Selection of polyclonl T reg cells y mtecs To test whether MHC clss II expressed exclusively on TECs ws sufficient for the selection of polyclonl T reg cells, we generted chimeric mice in which only TECs expressed MHC clss II. To do this, we mde use of the fct tht reggregte thymic orgn cultures (RTOCs) 34 form norml thymic microenvironment fter engrftment under the kidney cpsule 35. Experiments ddressing this with fetl thymic loes t emryonic dy 1 (E1) or treted with deoxygunosine hve een clled into question ecuse of the forml cvet tht minute mounts of MHC clss II positive hemtopoietic cells my hve persisted in the grft 31.Torigorouslypreventcontmintion with crryover hemtopoietic cells, we prepred RTOCs from cell suspensions of MHC clss II positive E14 thymi fter stringently depleting them of CD45 + hemtopoietic cells nd trnsplnted the RTOCs into MHC clss II sufficient C57BL/6 recipient mice (WT-WT) or MHC clss II deficient C57BL/6 recipient mice Figure 1 Selection of polyclonl Foxp3 + + T reg cells y MHC clss II expressed exclusively on thymic epithelium. RTOCs were generted from CD45 thymic stroml cells prepred from E14 MHC clss II positive (wild-type) donors. After 48 h of in vitro culture, RTOCs were grfted under the kidney cpsules of wild-type or MHC clss II deficient (H2-A1 / ) recipients; 8 1 weeks lter, single-cell suspensions of grfts were stined with ntiodies specific for CD4, CD8, nd intrcellulr Foxp3. () Thymocyte susets in grfts (donors nd hosts, ove plots) nd in thymi of C57BL/6 mice (B6). Wild-type host, n ¼ 3; H2-A1 / host, n ¼ 6; C57BL/6 mice, n ¼ 5. () Expression of surfce nd intrcellulr Foxp3 on gted CD4 + SP thymocytes. Numers ove outlined res indicte percent (± s.d.) CD4 + SP cells () orfoxp3 + + cells (). Dt re representtive of two independent experiments. (WT-H2-A1 / ). Anlysis of thymocyte susets in the grfts fter 8 1 weeks showed tht selection of Foxp3 + + T reg cells occurred efficiently in WT-H2-A1 / nd WT-WT chimers (Fig. 1). As these grfts did not differ in their overll cellulrity, the somewht lower frequency of Foxp3 + + T regs mong CD4 + single-positive (SP) cells in WT-H2-A1 / grfts ws more thn compensted for in terms of solute numers y the much greter frequency of CD4 + SP cells in the sence of MHC clss II on hemtopoietic cells (Fig. 1). The extent to which CD4 + SP numers were higher ws in good greement with estimtes tht normlly out 6% of CD4 + SP cells re negtively selected fter encounter with self ntigen on thymic DCs 36. Next we sought to delinete the contriution of corticl versus medullry TECs to the genertion of polyclonl Foxp3 + T reg cells. We hypothesized tht elimintion of MHC clss II expression on medullry TECs would demonstrte involvement of mtecs in T reg cell selection. To test this ide, we gin grfted RTOCs into H2-A1 / mice. This time, the RTOCs contined 1:1 mixture of CD45 cells from wild-type nd H2-A1 / E14 thymi. Flow cytometry of these mixed RTOCs t 8 1 weeks fter grfting confirmed tht ulk T cell development ws efficiently supported nd histologicl exmintion confirmed tht the grfts were properly comprtmentlized into corticl nd medullry regions (dt not shown). Notly, wheres the cortex contined rndom mixture of MHC clss II positive nd MHC clss II negtive input cells, some medullry islnds formed tht, ecuse medullry regions rise clonlly from individul precursors 35, were composed of either MHC clss II positive or MHC clss II negtive cells (Fig. 2 nd Supplementry Fig. 1 online). An MTS1 MHC clss II MTS1 Foxp3 MHC + medull MHC medull Foxp3 + cells/1 6 µm 3 P = MHC + MHC Figure 2 In situ detection of Foxp3 + cells in MHC clss II positive or MHC clss II negtive medullry islnds in mixed RTOCs. () Stining of seril cryosections through entire grfts for medullry res (MTS1) nd MHC clss II to identify MHC clss II positive (MHC + ) or MHC clss II negtive (MHC ) medullry regions. Top row, MTS1 (green) nd MHC clss II (red); ottom row, MTS1 (green) nd Foxp3 (red) in n djcent section. Scle rs, 5 mm. () Quntifiction of Foxp3 + cells in medullry islnds ; two to four exclusively MHC clss II positive or MHC clss II negtive medullry regions could e identified in ech of totl of three mixed RTOCs. 352 VOLUME 8 NUMBER 4 APRIL 27 NATURE IMMUNOLOGY

3 HA-GFP Aire Act Ly51 Gted on CD45 ctec EpCAM mtec DC Mφ mtec ctec HA-GFP experimentl limittion of our pproch ws tht the clonlly derived spheres expnded over time nd thus ultimtely ecme confluent; considerle frction of medulle consisted of ptchwork of MHC clss II positive nd MHC clss II negtive regions (dt not shown). Therefore, we used seril sectioning to identify medullry regions tht were composed exclusively of either MHC clss II positive or MHC clss II negtive cells. We then quntified Foxp3 + cells per volume unit in MHC clss II positive or MHC clss II negtive medulle (Fig. 2) nd found significntly more Foxp3 + T reg cells in MHC clss II positive medullry regions. These results collectively supported the ide tht interctions of developing thymocytes with MHC clss II on mtecs re criticl for the development of polyclonl T reg cells. Antigen-specific differentition of T reg cells The function of specific ntigen remins uncler when studying polyclonl T reg cells. To ddress the function of mtec-derived self ntigen in the genertion of ntigen-specific T reg cells, we exploited the specific expression pttern of Aire to produce model ntigen exclusively in mtecs. We generted cteril rtificil chromosome trnsgenic construct contining 5 kiloses of 5 region nd 122 kiloses of 3 region flnking Aire, in which we replced the Aire strt codon with n open reding frme encoding fusion protein of influenz hemgglutinin (HA) nd green fluorescent protein (GFP). Preliminry chrcteriztion of two trnsgenic founder lines indicted they hd identicl phenotypes, nd we chose one founder line (clled AIRE-HA here) for ll further nlyses. We nlyzed the expression pttern of the trnsgene in highly purified thymic stroml cell B7.1 c Gted on mtecs Aire Act MHC clss II B7.1 hi B7.1 lo Figure 3 Medull-specific expression of HA-GFP with cteril rtificil chromosome trnsgenesis. () Sorting gtes (outlined ovls) used for the isoltion of ctecs (CD45 EpCAM + Ly51 + ) nd mtecs (CD45 EpCAM + Ly51 ; left) s well s MHC clss II high B7.1 hi nd MHC clss II low B7.1 lo mtecs (right). (,c) RT-PCR nlysis of the expression of trnsgenic HA- GFP nd endogenous Aire in thymic stroml cell susets (ove lnes). Seril fivefold dilutions (wedges) of cdna prepred from sorted cells were normlized to Act trnscripts (encoding -ctin) efore PCR mplifiction of HA-GFP nd Aire trnscripts. Mf, mcrophge. Dt re representtive of t lest two experiments. popultions y RT-PCR (Fig. 3). Expression of the HA-GFP cssette fithfully reflected tht of endogenous Aire, with sustntil quntities in mtecs nd fint signls in DCs. Neither the trnsgene nor endogenous Aire ws expressed in ctecs (Fig. 3). In sufrctionted MHC clss II high B7.1 hi nd MHC clss II low B7.1 lo mtecs 37, trnsgene expression gin closely mimicked tht of endogenous Aire, with sustntilly more in MHC clss II high B7.1 hi mtecs (Fig. 3c). Attempts to directly exploit GFP fluorescence for nlyses of expression y histology or flow cytometry were hmpered y insufficient expression of the trnsgene (dt not shown). To visulize the fte of hemgglutinin-specific CD4 + T cells developing in AIRE-HA thymi, we crossed AIRE-HA mice with mice expressing trnsgenic TCR tht recognizes peptide of mino cids of influenz hemgglutinin (HA(17 119)) in the context of I-E d (ref. 38). The thymic cellulrity of AIRE-HA mice ws not significntly different from tht of mice expressing only the trnsgene ( ± (n ¼ 11) versus ± (n ¼ 8); P ¼.58), nd the frequency of CD4 + SP cells ws essentilly identicl (Fig. 4). In the CD4 + SP popultion, the frequency of cells expressing the clonotype ws lower in AIRE-HA mice thn in control mice ( ± versus ± ). Aout hlf of those CD4 + SP + cells expressed, suggesting efficient differentition into the T reg cell linege (Fig. 4). In ccordnce with tht oservtion, 23.9% ± 6.8% ( ± )ofcd4 + + cells in AIRE-HA mice expressed Foxp3, wheres the frequency of CD4 + + Foxp3 + cells in TCR- HA mice ws negligile (corresponding to ±5 1 2 cells; Fig. 4c). In vitro suppression ssys showed tht sorted CD4 + SP TCR- HA + + thymocytes from AIRE-HA mice efficiently inhiited the prolifertion of nive CD4 + + T cells otined CD4 CD8 AIRE-HA c d Gted on CD4 + SP thymocytes 14.3 ± ± ± ± 4.4 Gted on CD4 + SP + thymocytes.4 ± ± 6.8 Foxp3 1 3 c.p.m nive Co-culture (1:1) + Figure 4 Genertion of Foxp3 + + T reg cells in AIRE-HA mice. () Frequency of CD4 + SP thymocytes in mice (n ¼ 8) nd AIRE-HA mice (n ¼ 11). () Frequency of + cells in + CD4 + SP popultions. (c) Expression of Foxp3 in + cells, ssessed y intrcellulr stining. Numers ove outlined res (,c) nd in qudrnts () indicte the verge frequency (± s.d.) of CD4 + SP thymocytes (), of + + cells mong CD4 + SP cells () oroffoxp3 + + cells in the + popultion (c). Mouse strins, ove top plots. (d) Prolifertion of nive peripherl + CD4 + T cells from mice deficient in recomintion-ctivting gene nd + + CD4 + SP thymocytes from AIRE-HA mice; cells were cultured lone or together in the presence of syngeneic splenocytes nd HA(17 119) nd prolifertion ws ssessed y scintilltion counting fter pulse with [ 3 H]thymidine for the lst 2 h of 96-hour incution period. Dt re representtive of 8 mice nd 11 AIREHA mice ( c) or re representtive of three independent experiments (d). NATURE IMMUNOLOGY VOLUME 8 NUMBER 4 APRIL

4 from mice deficient in recomintion-ctivting gene 2 (Fig. 4d). The peripherl phenotype of AIRE-HA mice reflected the sitution in the thymus; distinct + + suset ws present mong CD4 + T cells (Supplementry Fig. 2 online). To formlly demonstrte tht mtec-derived ntigen ws cusing T reg WT AIRE-HA cell differentition in this mouse model system, we grfted deoxygunosine-treted thymi from E14 AIRE-HA fetuses into mice. This experiment fithfully recpitulted the phenotype of AIRE-HA mice (Fig. 5). In contrst, + CD4 + SP cells in AIRE-HA -WT one mrrow chimers hd phenotype identicl to tht of control -WT chimers, indicting tht Aire locus driven expression of HA in hemtopoietic cells, if existent, ws too low to mesurly ffect T cell development (Fig. 5). The system hs een used in mny settings to study T reg cell development 11,12,14,15. Therefore, we ddressed whether T reg cell differentition fter intrthymic ntigen encounter might possily reflect n inherent predisposition of + thymocytes. Trgeting of HA to DCs with trnsgenic construct driven y the promoter of the gene encoding CD11c 39 resulted in complete deletion of TCR- HA + thymocytes, indicting tht + thymocytes cn e deleted in the thymus when pproprite conditions re met (Supplementry Fig. 3 online). We next ddressed whether our findings in the AIRE-HA model could e reproduced with second model ntigen nd nother TCR trnsgene. We used fusion protein of HA nd ovlumin (OVA) contining the OVA epitope (mino cids ) recognized y the DO11.1 TCR nd trgeted the fusion protein to mtecs under control of the Aire locus with cteril rtificil chromosome trnsgenic pproch similr to tht descried ove. The T cell phenotype of the resultnt AIRE-OVA DO11.1 mice strongly resemled tht of AIRE-HA mice; we detected Foxp3 + + T reg cells expressing the DO11.1 TCR in the thymus nd periphery (Supplementry Fig. 4 online). These dt AIRE-HA BALB/c BALB/c Figure 5 Expression of HA in AIRE-HA thymic epithelium ut not expression in hemtopoietic cells recpitultes the phenotype of AIRE-HA mice. () Phenotype of CD4 + SP thymocytes in wild-type or AIRE-HA thymi grfted under the kidney cpsules of recipients, nlyzed 8 12 weeks fter trnsplnttion of deoxygunosine-treted E14 thymi. () Phenotype of CD4 + SP thymocytes in BALB/c wild-type recipients of or AIRE-HA one mrrow. Numers in qudrnts indicte percent + + cells (top right) or + cells (ottom right) mong CD4 + SP cells. Dt re representtive of two independent experiments with t lest seven grfts ech () or ten chimers ech in two independent experiments () GFP + cells (%) collectively indicted tht self ntigen expressed in mtecs cn efficiently induce the differentition of Foxp3 + + T reg cells. DCs nd mtecs present mtec-derived ntigen Our experiments so fr hd estlished tht expression of HA exclusively in mtecs ws sufficient for the differentition of TCR- HA + thymocytes into T reg cells. The issue remined open, however, of whether this confined expression pttern coincided with similrly restricted pttern of HA(17 119) presenttion exclusively y mtecs or whether mtec-derived HA ws eventully tken up, processed nd presented y thymic DCs. To directly mesure presenttion of HA(17 119) y vrious thymic stroml cell popultions from AIRE-HA mice, we used the A5 T cell hyridom, which expresses the clonotypic nd crries GFP reporter trnsgene driven y elements controlling the gene encoding interleukin 2 (ref. 4). Titrtion of HA(17 119) into cocultures of A5 cells nd wild-type thymic DCs showed dynmic rnge of GFP expression crossing three orders of mgnitude nd strting from detection limit of pproximtely 1 ng/ml (Fig. 6). The dose-response curve otined with wild-type mtecs s APCs ws essentilly superimposle (Fig. 6). Antigen presenttion ssys with ctecs, mtecs nd DCs isolted ex vivo from AIRE-HA mice showed strong stimultion of A5 cells y mtecs nd wek yet significnt stimultion y DCs, ut no significnt stimultion y ctecs (Fig. 6). To confirm tht presenttion of HA(17 119) y DCs ws indeed due to cpture of mtec-derived ntigen rther thn to processing of endogenously expressed ntigen, we generted series of one mrrow chimers. Indeed, thymic DCs isolted from WT-AIRE-HA chimers stimulted A5 cells s efficiently s did those from control AIRE-HA-AIRE-HA chimers; in contrst, DCs otined from AIRE-HA-WT chimers did not stimulte A5 cells (Supplementry Fig. 5 online). Presenttion of HA(17 119) y MHC clss II high B7.1 hi nd MHC clss II low B7.1 lo mtecs isolted from AIRE-HA mice reflected in more pronounced wy the difference etween these mture nd immture mtecs in terms of mrna quntities (Fig. 3c). Although MHC clss II high B7.1 hi mtecs strongly stimulted A5 cells, presenttion y MHC clss II low B7.1 lo mtec ws rely mesurle (Fig. 6c). These results collectively indicted tht ntigen exclusively expressed y mtecs cn e presented concomitntly y mtecs themselves s well s y DCs. 1 HA (17 119) (µg/ml) 1 mtec DC GFP + cells (%) NS P =.8 ctec mtec DC c GFP + cells (%) P =.24 B7.1 hi B7.1 lo Figure 6 Disply of HA(17 119) y isolted mtecs nd DCs ut not y ctecs from AIRE-HA thymi. () Dose-response curve of ctivity of the GFP reporter construct in A5 T hyridom cells stimulted y wild-type mtecs or DCs (key) plus HA(17 119) (concentrtion, horizontl xis). The frequency of GFP + cells fter 17 h of coculture ws determined y flow cytometry. Dt re representtive of two experiments. () Stimultion of A5 cells fter 17 h of culture together with ctecs, mtecs or DCs from AIRE-HA thymi (drk gry) or wild-type thymi (light gry). NS, not significnt. Dt re representtive of more thn five independent experiments. Stimultion of A5 cells fter 17 h of culture together with MHC clss II-high B7.1 hi nd MHC clss II low B7.1 lo mtecs from AIRE-HA thymi (drk gry) or wildtype thymi (light gry). Dt re representtive of two independent experiments. 354 VOLUME 8 NUMBER 4 APRIL 27 NATURE IMMUNOLOGY

5 CD4 WT H-2 d (nude ) AIRE-HA H-2 d (nude ) CD8 Gted on CD4 + SP thymocytes WT H-2 (nude ) AIRE-HA H-2 (nude ) 27.4 ± ± ± ± 6.2 Gted on CD4 + SP thymocytes Gted on CD4 + SP + thymocytes Gted on CD4 + SP + thymocytes.1 ± ± ± ± 6.1 Foxp3.3 ± ±.9.2 ± ± 1.2 T reg cell selection independent of hemtopoietic cells The detection of mtec-derived ntigen presented on thymic DCs rised the issue of whether the genertion of T reg cells in AIRE-HA mice ws orchestrted utonomously y mtecs or y DCs tht cptured nd presented mtec-derived ntigen. If the ltter were true, experimentl interference with the cpcity of DCs to present ntigen would olish T reg cell differentition. The fct tht mice with trgeted muttion of the gene encoding I-E d, to which the is restricted, hve not een generted rendered strightforwrd pproch with I-E d -deficient one mrrow impossile. Therefore, s n lterntive strtegy, we mde use of the fct tht + T cells neither llorect to C57BL/6 (H-2 ) APCs nor recognize cognte HA peptide in the context of I-A (dt not shown). We generted one mrrow chimers with thymic mice y reconstituting irrdited C57BL/6 nude mice (H-2 ) with H-2 one mrrow (clled H-2 (nude-) here) or BALB/c nude mice (H-2 d ) with H-2 d one mrrow (clled H-2 d (nude-) here). Susequently, we engrfted those chimers with deoxygunosine-treted AIRE-HA thymi (AIRE- HA-H-2 (nude-) nd AIRE-HA-H-2 d (nude-tcr- HA)) or wild-type thymi (WT-H-2 (nude-) nd WT- H-2 d (nude-)) otined from BALB/c (H-2 d ) E14 donors. Positive selection of CD4 + + cells would in ll situtions e medited y H-2 d thymic epithelium. The fte of + cells in AIRE-HA-H-2 d (nude-) grfts ws expected to emulte tht of AIRE-HA mice. In AIRE-HA-H-2 (nude-) grfts, however, T reg cell development, depending on its requirement for the presenttion of mtec-derived ntigen y hemtopoietic APCs, would or would not occur. c Thymus WT TG WT TG Bone mrrow H-2 d H-2 H-2 d (nude ) recipient I-A d ± 3.3 I-A.5 ±.3 H-2 (nude ) recipient 1.2 ± ± 4.1 H-2 (nude ) recipient We nlyzed grfts 6 8 weeks fter trnsplnttion. The size of the CD4 + SP comprtment ws not very different mong the experimentl groups (Fig. 7). Somewht unexpectedly, we consistently detected fewer + cells mong CD4 + SP cells for WT-H-2 (nude- ) thymi thn for WT-H-2 d (nude-) thymi (in the sence of cognte ntigen; Fig. 7). We deem it likely tht this ws pssive, indirect effect due to less-stringent DC-imposed censorship of thymocytes expressing endogenously rerrnged TCRs (such s CD4 + SP cells) in thymi with MHC-mismtched (H-2 ) hemtopoietic comprtment. Given tht, the phenotypes of AIRE- HA-H-2 d (nude-) thymi nd AIRE-HA-H-2 (nude- ) thymi were very similr. The frequency of + cells mong CD4 + SP cells ws in oth cses lower thn tht in control thymi (Fig. 7). + CD4 + SP popultions in oth experimentl groups tht received AIRE-HA thymi contined similrly high frequencies of Foxp3 + + cells, wheres only minute popultions of cells with tht phenotype were detectle in the sence of cognte ntigen (Fig. 7,). To verify expression of the expected MHC hplotypes y hemtopoietic APCs, we digested one third of ech grft with collgense nd nlyzed the resulting cell suspensions y flow cytometry. Most DCs from H-2 (nude-) recipients expressed I-A, wheres cells expressing I-A d were essentilly undetectle (Fig. 7c). These dt confirmed efficient seeding of thymic grfts with hostderived hemtopoietic APCs. In summry, our findings indicte tht T reg differentition of + thymocytes resulting from expression of HA exclusively in mtecs did not require crosspresenttion of HA y thymic DCs ut insted ws utonomously medited y mtecs. Foxp3 + + in + (%) Thymus WT TG WT TG Bone mrrow H-2 d H-2 Figure 7 Efficient genertion of Foxp3 + + T reg cells independently of cross-presenttion y hemtopoietic cells. () Cellulr composition of deoxygunosine-treted AIRE-HA or wild-type thymi (H-2 d ) t 6 1 weeks fter eing grfted into H-2 d (nude-) or H-2 (nude-) recipients. Numers ove outlined res or in top right qudrnts indicte the frequency (± s.d.) of CD4 + SP cells (top row), of + + cells in the CD4 + SP popultion (middle row) or of Foxp3 + + cells mong gted CD4 + SP + cells (ottom row). All plots show dt fter gting on CD cells to identify thymocytes (nude mice nd thymus grfts were CD ). Dt were clculted from three independent experiments (WT-H-2 d (nude- ), n ¼ 8 chimers; AIRE-HA-H-2 d (nude-), n ¼ 8 chimers; WT-H-2 (nude-), n ¼ 7 chimers; AIRE-HA-H-2 (nude-tcr- HA), n ¼ 19 chimers). The cellulrity of grfts vried considerly within experimentl groups, ut ws not sustntilly different etween groups (dt not shown). () Summry of the dt in. Left, percent + cells mong CD4 + SP cells; right, percent Foxp3 + + cells mong gted CD4 + SP + cells. TG, AIRE-HA trnsgenic. (c) Crryover of donor-derived CD11c + DCs in deoxygunosine-treted grfts. Expression of I-A d nd I-A on gted CD45 + CD11c + cells in H-2 d (nude-) or H-2 (nude-) recipients ws nlyzed in thymic cell suspensions prepred y enzymtic digestion. Numers in qudrnts indicte percent cells (± s.d.) in ech. Dt re representtive of t lest four grfts ech. + in CD4SP (%) Isotype contr. 1. ±.3 I-A 64.7 ± 3.5 NATURE IMMUNOLOGY VOLUME 8 NUMBER 4 APRIL

6 DISCUSSION Using two complementry pproches, we hve provided evidence here for n utonomous function of mtecs, which ct concomitntly s ntigen-expressing nd ntigen-presenting cells in T reg cell genertion. Our initil experiments with RTOCs from mteril rigorously purged of hemtopoietic cells provided unmiguous evidence in fvor of the ide tht TECs re sufficient to support polyclonl T reg cell development. Our oservtions support model wherey TECthymocyte interctions shpe the T reg cell comprtment nd dominnt tolernce, wheres hemtopoietic APCs (minly thymic DCs) medite deletionl tolernce. A study of the selection of superntigen-rective T reg cells in the sence or presence of MHC clss II on hemtopoietic cells reched similr conclusions in terms of the generl function of the thymic epithelium during T reg cell development 41. However, delinetion of the respective contriution of medullry versus corticl TECs remined experimentlly chllenging. When MHC clss II expression is restored in MHC clss II deficient mice with purportedly ctec-specific trnsgene, functionl + CD4 + T cell pool of norml size is selected 17. Those results suggest tht interctions of developing thymocytes with MHC clss II on ctecs re sufficient to support T reg cell differentition. However, in tht system it remins uncler whether mtecs truly lck MHC clss II; this cvet nd concern is prticulrly vlid, given the notorious propensity of mtecs to ectopiclly express trnsgenes 26. Tking dvntge of the clonl origin of medullry islnds, we generted chimeric RTOCs in which individul medulle either expressed or lcked MHC clss II (ref. 35). The significntly fewer Foxp3 + T reg cells in medulle lcking MHC clss II supported the ide of involvement of mtec-thymocyte interctions in generting the polyclonl T reg cell pool. Severl scenrios might ccount for the residul Foxp3 + cells in MHC clss II deficient medulle. First, smll clones of MHC clss II positive mtec positioned djcent to MHC clss II-negtive regions my hve disppered eventully, wheres their footprint in the locl T reg cell comprtment my hve persisted for some time. This dynmic nture of medull morphology over time hs een reported efore 42,43. Second, the possiility of lterl movement of Foxp3 + cells generted in more distnt MHC clss II positive medulle cnnot e formlly excluded. Finlly, initilly stochstic (ntigen-independent) specifiction of T reg cell fte my initilly occur in the cortex. After migrtion of thymocytes from the cortex to the medull, interctions with cognte ntigen on mtecs my then serve to selectively induce the survivl nd/or popultion expnsion of otherwise short-lived precursors. The phenotype of mice deficient in tumor necrosis fctor receptor ssocited fctor 6 (TRAF6) supports the ide tht mtec-thymocyte interctions re criticl for T reg cell genertion 44. Asence of TRAF6 from the thymic epithelium is ssocited with generl utoimmunity, most likely s result of disorgnized medullry comprtment lcking mture AIRE + mtecs, the exct cell type to which HA ws trgeted in our model. The CD4 + SP comprtment of TRAF6- deficient mice ws mostly devoid of + T reg cells. A complementry study of the involvement of TRAF6 in T cells hs shown tht this lck of T reg cells is not due to T cell intrinsic requirement for TRAF6, s functionl T reg cells rise when TRAF6 is lted specificlly nd only in T cells 45. Our findings my explin why the utoimmunity cused y TRAF6 deficiency in thymic epithelium seems more severe thn tht in AIRE-deficient mice 2. AIRE deficiency leds to lower expression of peripherl ntigens in otherwise pprently norml mture mtecs 23. TRAF6 deficiency more profoundly results in complete sence of such cells nd thus lso ffects ny tolerogenic function of mtecs eyond their function s reservoir of tissuespecific ntigens. A chief limittion inherent to nlysis of the polyclonl T reg cell repertoire is tht the function of specific ntigen during T reg cell selection remins mostly oscure. Therefore, we complemented our oservtions with studies in systems involving defined ntigen specificity. Trgeting of HA s well s OVA to mtecs efficiently directed TCR-trnsgenic CD4 + T cells of the cognte TCR specificity into the T reg cell linege, wheres DC-specific expression of HA led to the deletion of + thymocytes. Those dt suggested tht the APC type specifies the outcome of the recognition of self ntigen, with the cvet tht expression of self ntigen in mtecs cnnot necessrily e interpreted to men tht the ensuing mode of tolernce is in fct result of ntigen presenttion y mtecs 29. Indeed, direct ssessment of ntigen presenttion in the AIRE-HA model demonstrted severl slient fetures of ntigen routing in this system. First, oth mtecs nd DCs, ut not ctecs, presented sustntil mounts of HA(17 119). Second, presenttion of HA(17 119) y DCs ws the result of cquisition nd processing of mtec-derived ntigen. Third, presenttion y mtecs ws most likely due to endogenous MHC clss II loding rther thn ntigen trnsfer in the mtec comprtment. Regrdless of their limited ility to process exogenous ntigens, mtecs presented HA(17 119) fr more efficiently thn did DCs, which re rguly etter indictors of freely ville ntigen 46. Using chimers with MHC-mismtched thymi nd one mrrow, we ruled out the possiility tht trnsfer of mtec-derived ntigen to DCs ws essentil for the genertion of + T reg cells. This finding contrsts with the strict requirement for trnsfer of mtecderived ntigen to hemtopoietic cells during deletion of OT-II CD4 + thymocyes in the RIP-mOVA model 29. These findings collectively support the ide tht the modlities of routing of mtec-derived self ntigens re indeed criticl prmeter in specifying recessive or dominnt mechnisms of tolernce. We deem it likely tht such considertions might likewise pply to different epitopes derived from given self ntigen. How likely is it tht the nture of the stroml cell presenting mtecderived ntigen (mtecs themselves versus cross-presenting DCs) is the sole determinnt of whether recessive or dominnt tolernce is induced? Differentition of + cells into the T reg cell linege in AIRE-HA mice coincided with much lower solute numers of these cells thn in control mice. According to strictly qulittive model, this my hve occurred through deletion of certin frction of + cells fter ntigen recognition on DCs concomitnt with T reg cell selection y mtecs. However, elimintion of the cpcity of DCs to present mtec-derived ntigen did not restore the frequency of + cells. Thus, it seems tht even in conditions in which HA is presented exclusively y mtecs, deletion nd T reg cell selection occur side y side. Severl mutully nonexclusive explntions exist. First, it is possile tht the strength of gonistic signls delivered y mtecs in our model is close to threshold tht my distinguish deletion from T reg cell selection; hence, smll fluctutions in ntigen expression in mtecs my tip the lnce in fvor of one or the other mode of tolernce. Second, functionl heterogeneity in the mtec comprtment (for exmple, vrying mounts of costimultory molecules) my influence the outcome of deletion versus T reg cell fte specifiction. Evidence suggests tht CD28-B7 interctions my e involved Finlly, the possiility of stochstic heterogeneity mong thymocytes, even those expressing n identicl TCR, regrding the predisposition to 356 VOLUME 8 NUMBER 4 APRIL 27 NATURE IMMUNOLOGY

7 differentilly respond to ntigenic stimuli delivered y mtecs cnnot e ruled out. Our conclusion tht interctions with AIRE + mtecs shpe the T reg cell repertoire is not incomptile with the fct tht the utoimmunity in AIRE-deficient mice hs een scried minly to defect in deletionl rther thn dominnt tolernce 23. Muttions in Aire my chnge the profile of peptide MHC clss II complexes displyed on mtecs in very sutle wy. At the sme time, Aire-deficient mtecs cn still e expected to present plethor of self ntigens nd thus my contriute to the genertion of sufficiently diverse T reg cell repertoire. Consistent with tht ide, even mice expressing single MHC clss II ound peptide hve T reg cell repertoire of considerle diversity 9.It remins uncler in how fr ntigen-specific holes in the T reg cell repertoire my eventully predispose to utoimmunity. We propose tht mtecs provide criticl niche tht fosters T reg cell development. Bsed on considertions pertining to T reg cell ontogeny nd positioning in the thymic microenvironment, the existence of such niche hs een postulted efore, lthough its exct nture (hemtopoietic or epithelil) hs remined uncler 18. Wht is the function of this niche? At first, the enormous increse in the numer of ntigen-specific T reg cells triggered y the presenttion of endogenously expressed ntigen y mtecs my suggest de novo induction of T reg cell phenotype. However, our oservtions re lso comptile with stochstic component of T reg cell development 51.Thus,selection nd/or popultion expnsion of stochsticlly primed T reg cell precursors my require, in ddition to gonistic ntigen, externl cues specificlly provided y mtecs. Deciphering the moleculr signture of this mtec niche in terms of criticl surfce molecules nd/or solule fctors should provide dditionl insights into T reg cell development. Signling vi CD28 (ref. 49) s well s through the thymic stroml lymphopoietin receptor 52 hs een shown to directly induce Foxp3 expression in thymocytes in vitro. Consistent with our proposition, their lignds (B7 nd thymic stroml lymphopoietin, respectively) re undntly provided y mtecs. METHODS Mice. Animls were red in the niml fcility of the Reserch Institute of Moleculr Pthology in individully ventilted cges. C57BL/6 nude nd BALB/c nude mice were from Tconic; other strins were from in-house reeding colonies. All niml studies were pproved y locl uthorities (MA58) nd were done ccording to Austrin regultions. The genertion of trnsgenic mice is descried in the Supplementry Methods online. Antigen presenttion ssy. Thymic stroml cells (2 1 4 ) from wild type or AIRE-HA mice were cultured together with A5 T hyridom cells (2 1 4 )in 2 ml Iscove s modified Dulecco s medium (IMDM) supplemented with 1% (vol/vol) FCS in 96-well round-ottomed pltes. Then, 17 h lter, stimultion ws mesured y flow cytometry of GFP reporter expression on gted + CD4 + cells. Equl input numers of APCs (except in experiments with B7.1 hi nd B7.1 lo mtec susets) were ensured y verifiction tht similr stimultion ws chieved with sturting mounts (1 mg/ml) of exogenous HA(17 119). Suppression ssys. Sorted + + CD4 + SP T cells (2 1 4 )from AIRE-HA thymi nd/or nive + CD4 + T cells (2 1 4 ) enriched y mgnetic-ctivted cell sorting from spleen nd lymph nodes of mice deficient in recomintion-ctivting gene were cultured together with irrdited (3, rds) BALB/c splenocytes (2 1 5 ) in the presence of 1 mg/ml of HA(17 119). Prolifertion ws mesured y scintilltion counting fter cells were pulsed with 1 mci [ 3 H]thymidine per well for the lst 2 h of 96-hour incution period. Preprtion of thymic strom. Thymi from 2-week-old mice were cut into smll pieces nd were digested t 37 1C in IMDM contining.2 mg/ml of collgense (Roche),.2 mg/ml of dispse I (Roche), 2% (vol/vol) FCS, 25 mm HEPES, ph 7.2, nd 25 mg/ml of DNse I, followed y incution for 5 min in 5 mm EDTA. Cells were wshed nd were resuspended in Percoll (r, 1.115; GE Helthcre). A discontinuous grdient ws then generted y the ddition of lyer of Percoll (r, 1.5) followed y lyer of PBS on top of this cell suspension. Grdients were spun for 3 min t 1,35g t 4 1C, nd low-density cells were collected from the upper interfce, wshed nd stined for sorting y flow cytometry. RTOC. Single-cell suspensions of E14 fetl loes were prepred y enzymtic digestion (descried ove). Smples were depleted of CD45 + cells with iotinylted CD45-specific monoclonl ntiody nd streptvidin MACS eds (Miltenyi Biotech) ccording to stndrd procedures. Cells were spun down nd were resuspended t density of pproximtely cells/ml. Drops of.2.4 ml were deposited onto.45-mm nylon memrnes (Millipore) supported y Gelfom sponges (Phrmci & Upjohn) in six-well pltes contining 3 ml IMDM nd 1% (vol/vol) FCS. RTOCs were incuted for 48 h efore trnsplnttion. Deoxygunosine tretment. E14 thymic loes were plced on.45-mm memrne filters (Millipore) supported y Gelfom (Phrmci&Upjohn) nd were incuted for 5 d with 1% (vol/vol) IMDM supplemented with 1.35 mm 2-deoxygunosine efore trnsplnttion. Immunofluorescence. Frozen sections 11 mm in thickness were fixed in cold cetone, were wshed nd were locked for 2 min with 1% (vol/vol) FCS in PBS, followed y incution overnight with the pproprite primry ntiodies: Foxp3-specific polyclonl rit serum, MTS1 superntnt (rt immunogloulin M) nd/or iotinylted ntiody to MHC clss II (nti MHC clss II; P7/7). Sections were incuted with secondry ntiodies for 2 h t 21 1C fter eing wshed three times for 15 min ech in.1% (vol/vol) Tween 2 in PBS. Secondry regents were Alex Fluor 488 conjugted nti-rt immunoloulin M (Moleculr Proes), indocrocynine-conjugted ntirit (Jckson ImunoLortories) nd/or indocrocynine-conjugted streptvidin (Jckson ImmunoLortories). MHC clss II positive or MHC clss II negtive medulle were identified in consecutive sections through entire grfts for verifiction of the sence of MHC clss II positive cells in three dimensions. The volume of the respective medullry segments ws determined s follows: re of the MTS1 + medullry region (in mm 2 ) thickness of the section (in mm). The numer of Foxp3 + cells per unit volume in MHC clss II positive nd MHC clss II negtive medullry regions ws determined nd the men vlue ws clculted. Sttisticl nlysis. Sttisticl significnce ws ssessed y the two-tiled Student s t-test. Note: Supplementry informtion is ville on the Nture Immunology wesite. ACKNOWLEDGMENTS We thnk M.S. Anderson, B. Kyewski nd J. Derinski for comments on the mnuscript. K. Krjlinen (Nnyung Technologicl University) provided the A5 hyridom; A. Rudensky (University of Wshington) provided Foxp3-specific polyclonl rit serum; R. Boyd (Monsh Medicl School) provided MTS1 superntnt; nd B. Kyewski (Germn Cncer Reserch Center) provided iotinylted ntiody to MHC clss II. Supported y Boehringer Ingelheim (Reserch Institute of Moleculr Pthology), the Europen Union (Euro-Thymide FP6 Integrted Project; LSHB-CT ) nd the Austrin Ntionl Science Fund (Z58-B1 nd Sonderforschungsereich F23). AUTHOR CONTRIBUTIONS K.A. generted nd nlyzed AIRE-HA mice; L.M.D. did the RTOC experiments; E.H.V. generted nd nlyzed AIRE-OVA DO11.1 mice together with J.E.; M.H. contriuted to the genertion nd nlysis of nude chimers; L.K.S. nd A.R. generted nd nlyzed CD11c-HA mice (Supplementry Fig. 3); nd L.K. prepred the mnuscript. COMPETING INTERESTS STATEMENT The uthors declre no competing finncil interests. NATURE IMMUNOLOGY VOLUME 8 NUMBER 4 APRIL

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Numer of T reg cells tht differentite does not increse upon encounter of gonist lignd on thymic epithelil cells. J. Exp. Med. 2, (24). 52. Jing, Q., Su, H., Knudsen, G., Helms, W. & Su, L. Delyed functionl mturtion of nturl regultory T cells in the medull of postntl thymus: role of TSLP. BMC Immunol. 7, 6 (26). 358 VOLUME 8 NUMBER 4 APRIL 27 NATURE IMMUNOLOGY