Origin of Triple hi and Triple lo T reg cells Triple hi and Triple lo T reg cells present in the thymus (Fig. 2a) could represent CD4 + GITR CD4 PD-1

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1 Affinity for self ntigen selets with distint funtionl properties Len Wyss 1,2, Brin D Stdinski 3, Crolyn G King 1, Sonj Shllenerg 4, Nihols I MCrthy, Jun Young Lee 6,7, Krsten Kretshmer 4,8, Luigi M Terrino 9, Grhm Anderson, Chrles D Surh 6,7,1, Eri S Husey 3 & Ed Plmer 1,2 216 Nture Ameri, In. All rights reserved. The mnner in whih regultory T ells () ontrol lymphoyte homeostsis is not fully understood. We identified two T reg ell popultions with differing degrees of self-retivity nd distint regultory funtions. We found tht hi hi CD2 hi (Triple hi ) were highly self-retive nd ontrolled lympho-prolifertion in peripherl lymph nodes. lo lo CD2 lo (Triple lo ) were less self-retive nd limited the development of olitis y promoting the onversion of CD4 + T onv ells into indued (i). Although Foxp3-defiient (Surfy) mie lked, they ontined Triple hi -like nd Triple lo -like CD4 + T ells with distint pthologil properties. Surfy Triple hi CD4 + T ells infiltrted the skin, wheres Surfy Triple lo CD4 + T ells indued olitis nd wsting disese. These findings indite tht the ffinity of the T ell ntigen reeptor for self ntigen drives the differentition of into distint susets with non-overlpping regultory tivities. The importne of CD4 + in mintining lymphoyte homeostsis is est ppreited in mie nd humns tht lk these ells. Foxp3-defiient (Surfy) mie 1 3 nd ptients with immunodysregultion polyendorinopthy enteropthy X-linked (IPEX) syndrome 4 suffer from exessive lymphoyte tivtion, lymphoyti infiltrtion into peripherl orgns nd olitis, leding to deth t n erly ge. In helthy individuls, ontrol homeostti prolifertion of onventionl T nd B ells nd prevent olitis 7. re omprised of thymi (tt reg ell) nd peripherlly indued (p or i), whih originte from different preursor ells nd develop in different lotions. t develop in the thymus nd their development requires stimultion of the T ell ntigen reeptor (TCR) with gonist peptide mjor histoomptiility omplex (MHC) lss II ntigens 8 1. In ontrst, i re generted in the periphery from nive, mture CD4 + onventionl T ells (T onv ells) during T ell tivtion in the presene of the ytokine TGF-β 11. Both popultions re suppressive nd their funtionl properties hve een exmined. Severl studies hve suggested tht t re required to ontrol immune homeostsis nd utoimmunity,12,13. On the other hnd, i hve speilized funtions depending on the type of inflmmtion, nd hve primry role in ontrolling muosl immunity nd fetl tolerne, However t y themselves re not suffiient to suppress hroni inflmmtion nd utoimmunity in the sene of i 1. re lso hrterized y their expression of surfe mrkers nd loliztion in different tissues On the sis of their expression of CD44 nd the lymph node homing reeptor CD62L, n e rodly divided into CD44 lo CD62L + entrl T reg (T reg ) nd CD44 hi CD62L lo/ effetor T reg (et reg ) ells 16. T reg ells re quiesent, primrily reside in seondry lymphoid tissues, express high levels of CD2 nd re interleukin-2 (IL-2) dependent. In ontrst, e, the dominnt T reg ell popultion in nonlymphoid tissues, re CD2 lo nd highly prolifertive, ut prone to poptosis. It hs een suggested tht et reg ell mintenne is driven y TCR nd o-stimultory signls, ut not y IL-2 (ref. 16). Severl studies hve demonstrted the importne of TCR stimultion in tivting to generte suppressive e 8,9. Furthermore, studies hve provided diret evidene tht TCR expression is indispensle for T reg ell survivl nd suppressive funtion 19,2. Although the T reg ell repertoire ontins oth self-retive 8,21,22 nd foreign-ntigen-retive 23 TCRs, the TCR ffinity of for self ntigen hs not yet een fully hrterized. Although it s generlly epted tht nd nive CD4 + T onv ells hve non-overlpping TCR repertoires, smll perentge of TCRs re found in oth CD4 + T ell popultions 24,2. Furthermore, the TCR repertories of t 1 Deprtment of Biomediine, University Hospitl Bsel nd University of Bsel, Bsel, Switzerlnd. 2 Deprtment of Nephrology, University Hospitl Bsel nd University of Bsel, Bsel, Switzerlnd. 3 Deprtment of Pthology, University of Msshusetts Medil Shool, Worester, Msshusetts, USA. 4 Moleulr nd Cellulr Immunology/Immune Regultion, DFG-Center for Regenertive Therpies Dresden (CRTD), Tehnishe Universität Dresden, Dresden, Germny. MRC Centre for Immune Regultion, Institute for Immunology nd Immunotherpy, University of Birminghm, Birminghm, UK. 6 Ademy of Immunology nd Miroiology, Institute for Bsi Siene, Pohng, Repuli of Kore. 7 Deprtment of Integrtive Biosienes nd Biotehnology, Pohng University of Siene nd Tehnology, Pohng, Repuli of Kore. 8 Pul Lngerhns Institute Dresden, Germn Center for Dietes Reserh (DZD), Dresden, Germny. 9 Institute of Pthology, Moleulr Pthology Division, University Hospitl of Bsel, Bsel, Switzerlnd. 1 Division of Developmentl Immunology, L Joll Institute for Allergy nd Immunology, L Joll, Cliforni, USA. Correspondene should e ddressed to E.P. (ed.plmer@unis.h). Reeived 1 April; epted 27 June; pulished online 1 August 216; doi:1.138/ni.322 nture immunology DVANCE ONLINE PUBLICATION

2 216 Nture Ameri, In. All rights reserved. nd i hve een shown to e distint 26,27. Although the tt reg ell TCR repertoire is ised towrd self-reognition, TCRs expressed in i n reognize foreign ntigens with high ffinity 24,26. Consistent with these results, tivted CD4 + T ells from TCRβ trnsgeni (TCRβ-tg) Surfy mie preferentilly used TCRs found in the T reg ell TCR repertoire of TCRβ-tg wild-type mie 21. Despite these findings, it s still not ler how T reg ell s ntigen speifiity influenes its regultory properties. We identified two funtionlly distint sugroups of t with distint TCR repertoires nd differing TCR ffinities for self ntigens. Triple lo expressed TCRs whose ffinities for self ntigens were lose to the negtive seletion threshold, wheres Triple hi expressed TCRs with ffinities well ove this threshold. Funtionlly, Triple lo ontrolled olitis y filitting onversion of CD4 T onv ells into i, wheres Triple hi mintined lymphoyte homeostsis with peripherl lymph nodes (LNs). Finlly, Foxp3-defiient (Surfy) mie ontined Triple hi - like nd Triple lo -like CD4 + T ells with distint pthologil properties. Our results provide evidene tht the degree of thymoyte self-retivity drives the genertion of distint sutypes, eh of whih ontrol different spets of lymphoyte homeostsis. RESULTS Distint T reg ell susets Foxp3 + hs ontinuum of expression of nd (Fig. 1). Given tht hi hi hd higher expression of CD2 thn tht of lo lo (Fig. 1), we refer to these popultions s Triple hi nd Triple lo, respetively. To ompre these T reg ell popultions with previously desried T reg ells susets 16 18, we exmined their expression of vrious homing nd hemokine reeptors (Fig. 1), s well s the trnsription ftor Helios nd the semphoring reeptor Nrp-1(Fig. 1). Bsed on CD4 + 1 their expression of these proteins, we onluded tht Triple hi nd Triple lo re 4 Figure 1 Chrteriztion of Triple hi nd Triple lo. () Flow ytometry nlyzing the expression of Foxp3,, nd CD2 in CD4 + LN ells from 6 12-week-old B6 mie. Numers djent to outlined res indite the perentge of Foxp3 + CD4 + ells (left; green) nd frequenies of Triple hi ( hi hi CD2 hi, middle, red) nd Triple lo ( lo lo CD2 lo, middle, rown) mong Foxp3 + CD4 + ells (n = 6 mie). Middle left, quntifition of these results. Histogrm (right) shows CD2 expression on Triple hi (red line) or Triple lo (rown, line) (n = 4 mie) nd quntifition of medin fluoresene intensity (MFI) of those results. (,) Flow ytometry nlyzing the expression of CD44, CD62L, CD13, CCR7 nd ICOS (), nd Helios nd Nrp-1 () y Foxp3 + CD4 + Triple hi (red lines nd rs) nd Triple lo (rown lines nd rs) from LNs of 6 12-week-old B6 mie (n = 4 mie) nd quntifition of those results. Eh symol represents n individul mouse; rs grphs indite the men (±s.e.m.). = not signifint (P >.), *P.1, P.1, *P.1 (unpired, two-tiled t test). Dt re from three () or two (,) independent experiments. Foxp3 1 3 distint from eh other nd distint from T reg nd e (Tle 1). This nlysis lso reveled tht T reg nd e re ontined in the Triple int gte (Supplementry Fig. 1). Origin of Triple hi nd Triple lo Triple hi nd Triple lo present in the thymus (Fig. 2) ould represent reirulting from the periphery s opposed to de novo generted t 28,29. To resolve this, we exmined thymi T reg ells in mie expressing Foxp3-RFP nd Rg-GFP reporters (Fig. 2). RFP + GFP + CD4 single-positive (SP) thymoytes represent de novo generted thymi, s they re still Rg-GFP +, wheres RFP + GFP CD4 SP ells in the thymus represent reirulting from the periphery 29. The frequeny of de novo generted (RFP + GFP + ) Triple hi nd Triple lo in the thymus ws similr to tht oserved mong LN. The ft tht oth Triple hi nd Triple lo developed in the thymus rgues ginst the ide tht either popultion onsists of i. To ddress the possiility tht Triple hi nd Triple lo might e indued y foreign ntigens or inflmmtion, we exmined in germ-free (GF) nd ntigen-free (AF) mie. AF mie re the offspring of GF mie wened onto nd rised on the elementl diet of gluose nd mino ids 3. As these nimls lk miroiome nd re not exposed to dietry ntigens, they ontin only self ntigens. GF nd AF mie ontined similr frequenies of LN s stndrd speifi-pthogen-free (SPF) nimls (Fig. 2). Notly, SPF, GF nd AF mie ontined similr frequenies of Triple hi nd Triple lo (Fig. 2), whih lso expressed similr levels of Nrp-1 nd Helios protein (Supplementry Fig. 2). These dt rule out the ide tht the Triple hi nd Triple lo phenotypes re response to inflmmtion. Furthermore, these results strongly suggest tht Triple hi nd Triple lo re exlusively generted through reognition of self ntigens in LN (%) CD4 CD2 CD44 + ells (%) Foxp CD44 CD62L CD13 + ells (%) * 4 * CD62L + ells (%) 9 * CD2 MFI (1 2 ) Triple hi Triple lo CD13 CCR7 ICOS Helios Helios MFI ( 1 2 ) * CCR7 MFI ( 1 2 ) Nrp-1 Nrp-1 MFI ( 1 2 ) 4 * ICOS MFI ( 1 2 ) * DVANCE ONLINE PUBLICATION nture immunology

3 Tle 1 Mrker expression y CD44 CD62L CD2 ICOS CCR7 CD13 Helios Nrp1 e High Low Low High Low High High (similr to ) High (similr to ) Triple hi High Low High High Int (similr to Triple lo ) ~7% neg High High Low High High Low High Low High (similr to etreg ells) High (similr to etreg ells) Triple lo Low High Low Low Int (similr to Triple hi ) ~9% neg Low Low Expression of surfe nd intrellulr mrkers y Triple hi, Triple lo, e nd. Int, intermedite. 216 Nture Ameri, In. All rights reserved. TCR repertoires expressed y Triple hi nd Triple lo To diretly ompre the TCR repertoires of Triple lo nd Triple hi T reg ells to CD4 + T onvs ells, we sorted ll three popultions expressing the vrile α-hin region 2 (V α 2) fmily (Fig. 3) from Rg +, single TCRβ hin strin (Ye62, Vβ8.2, TCRα +/KO, Foxp3 GFP-KI ; Supplementry Fig. 3) nd sujeted them to deep sequening. The most frequent lonotypes in eh group were nlyzed for their similrity (Fig. 3 d) nd diversity (Fig. 3e,f). Morisit-Horn nlysis, whih mesures sequene similrity, reveled tht the CD4 + T onv sequenes from three independent groups of mie (Online Methods) were similr to eh other, ut signifintly different from Triple lo nd Triple hi TCR sequenes otined from the sme mie (Fig. 3). Triple lo sequenes isolted from different groups of mie were similr to eh other s well, ut were different from CD4 + T onv nd Triple hi TCRs (Fig. 3). Triple hi TCR sequenes were not only different from CD4 + T onv nd Triple lo sequenes, ut they lso vried etween different groups of mie (Fig. 3d). Despite their signifint sequene differenes, the TCR repertoires of CD4 + T onv ells nd Triple lo were similrly diverse (Fig. 3e,f), wheres the Triple hi T reg ell TCR repertoire my e less diverse, t lest ording to Shnnon Entropy nlysis, whih is used to mesure sequene diversity. Tken together, deep sequening reveled tht Triple hi, Triple lo nd Foxp3 + CD4SP in thymus (%) Triple hi Triple lo Foxp3 + CD4SP 33.4 Figure 2 Anlysis of Triple hi nd Triple lo T reg ell origin. () Flow ytometry nlyzing the expression of nd on Foxp3 + CD4 SP thymoytes of 6 12-week-old B6 mie. Numers djent to outlined res (left) indite frequenies of Triple hi (red) nd Triple lo (rown) mong Foxp3 + CD4 SP thymoytes. Br grph (right) shows quntifition of those results (n = 1 mie). () Flow ytometry nlyzing expression of Rg-GFP reporter, nd in Foxp3 + CD4SP thymoytes from Foxp3-RFP Rg2-GFP dul-reporter mie. Numers ner rketed lines indite perent Rg-GFP (left) nd Rg-GPF + (right) mong Foxp3 + CD4 SP thymoytes. Numers djent to outlined res indite frequeny of Triple hi (red) nd Triple lo (rown) CD4 + T onv ells hve lerly distint TCR repertoires, implying tht TCR speifiity is importnt for seleting these T reg ell sutypes. Self-retivity of Triple hi nd Triple lo To diretly test whether Triple hi nd Triple lo T reg ell differ in their degree of self-retivity, we exmined CD nd Nur77 protein expression in eh suset (Fig. 4). The expression of these mrkers reflets T ell tivtion nd orreltes with TCR ffinity for its peptide-mhc (pmhc) lignd 31,32. We oserved higher expression of Nur77 nd CD y Triple hi, s ompred with Triple lo (Fig. 4), rguing tht Triple hi re more self-retive thn their Triple lo T reg ell ounterprts. To test this ide, we used in vivo BrdU leling nd oserved tht Triple hi proliferte more frequently in vivo thn Triple lo T reg ell nd CD4 + T onv ells (Fig. 4). Furthermore, when we ultured unsorted CD4 + T ells on syngenei one mrrow dendriti ells (BMDCs), we found tht Triple hi proliferted more extensively thn Triple lo nd CD4 + T onv ells (Fig. 4); this prolifertion required expression of MHCII self ntigens on ntigen-presenting ells (APCs). To exmine the influene of ntigen ffinity on the genertion of CD4SP thymoytes with Triple hi or Triple lo phenotype (Fig. 4d), we ultured B3K8TCR-tg Rg2 / thymoytes, whih express the Reirulting (Rg-GFP ) Rg-GFP SPF GF AF Foxp CD (%) Thymi (Rg-GFP + ).4 CD4 + Foxp3 + ells in LN (%) in LN (%) (%) 2 SPF SPF AF GF GF AF SPF Triple hi Triple lo Triple hi Triple lo mong reirulting (Rg-GPF, middle) nd de novo generted thymi (Rg-GPF +, right). Middle right nd fr right, quntifition of results t left. (n = 3 mie, dt tken from one experiment). () Flow ytometry nlyzing Foxp3, CD4, nd expression in LN ells from speifi pthogen free (SPF), germ free (GF) nd ntigen free (AF) B6 mie. Numers djent to outlined res indites frequenies of Foxp3 + CD4 + T ells (top row, gry) nd Triple hi (red) nd Triple lo (rown) in these mie (ottom row). Br grphs (right) show quntifition of those results (n = 2 mie per group). Eh symol represents n individul mouse; r grphs indite the men (±s.e.m.). = P >. (Kruskl-Wllis test). Dt re from five independent () or one (,) experiments AF GF nture immunology DVANCE ONLINE PUBLICATION

4 216 Nture Ameri, In. All rights reserved. Cell frequeny of V α 2 + CD4 + T ells (%) CD4 + T onv ells Triple hi Triple lo B3K8 TCR nd reognize the 3K, P2A nd P-1A peptides presented y I-A, on syngenei BMDCs in the presene of TGF-β nd IL-2. The ddition of P-1A peptide (threshold ffinity negtive seletor) indued development of Triple lo CD4SP thymoytes, wheres ddition of the P2A peptide (intermedite ffinity negtive seletor) indued intermedite (Triple int ) CD4 SP thymoytes; finlly, 3K peptide (high-ffinity negtive seletor) indued only Triple hi CD4 SP thymoytes (Fig. 4d). Foxp3 + were lso generted in these ultures, ut only in the presene of negtive seleting peptides (Fig. 4d nd Supplementry Fig. 4). Culturing B3K8TCR-tg Rg2 / thymoytes with the negtive seleting lignds P-1A, P2A nd 3K generted Foxp3 + expressing inresing mounts of (Fig. 4d), CD2 nd Helios protein (Supplementry Fig. 4). Tken together, these dt suggest tht threshold-, intermedite- nd high-ffinity negtive seleting ntigens indue Triple lo, Triple int nd Triple hi, respetively (Fig. 4d nd Supplementry Fig. 4). Tht the threshold negtive seletor indued weker TCR signls is supported y its deresed ility to indue pcd3ζ, pjun nd perk (Supplementry Fig. 4,d). We onfirmed these in vitro results using one mrrow himers, in whih OT-II thymoytes developed in RIP-OVA host expressing the ognte ntigen, ovlumin, in the thymus nd the pnres under the ontrol of the rt insulin promoter (Fig. 4e). These himeri mie ontined Triple hi nd Triple int, ut not Triple lo, in the thymus. Tken together, these dt imply tht Triple hi nd Triple lo T reg ells re likely generted y exposure to negtively seleting ntigens; moreover, the resulting phenotype is most likely determined y the ffinity of its TCR for self ntigen. Suppressive funtion of Triple hi To ompre the regultory properties of these two popultions, we trnsferred sorted B6 derived Triple hi or Triple lo, whih re unffeted y diphtheri toxin (DTx), to Foxp3 DTR mie (Supplementry Fig. ),. Endogenous from the Foxp3 DTR host were depleted y injeting DTx every other dy, eginning fter 3 d. LN lymphoytes were then exmined y flow ytometry 11 d fter the onset of T reg ell depletion (Supplementry Fig. ). Triple hi ontrolled the extensive prolifertion of T ells nd B ells in peripherl LNs of mie depleted of their endogenous (Fig. ), wheres Triple lo funtioned poorly in this respet. CD4 + T onv (MHI) 1.. * * CD4 + T onv TCRs Triple lo T reg TCRs Triple hi T reg TCRs * 1. * Triple lo T reg (MHI) e Shnnon entrophy (H). Triple lo T reg TCRs CD4 + T onv TCRs Triple hi T reg TCRs d Triple hi T reg (MHI) f Simpson diversity (D) As expeted, Triple hi limited the tivtion of CD4 + T onv ells (Fig.,). Tken together, these results show tht Triple hi regulte lymphoyte homeostsis in peripherl LNs. Suppressive funtion of Triple lo To exmine whether ny of these T reg ell susets ontrol olitis, we injeted CD3-defiient (Cd3e / ) mie with sorted nive CD4 + T onv ells (Supplementry Fig. 6), tretment tht resulted in weight loss (Fig. 6) nd olitis (Fig. 6), s previously desried 7. Co-trnsfer of Triple lo, ut not Triple hi, prevented weight loss nd limited lymphoyte infiltrtion of the oloni muos (Fig. 6,). Anlysis of LN ells from these mie reveled tht o-trnsferred Triple lo filitted the onversion of some CD4 + T onv ells into i (Fig. 6,d). Mie reeiving Triple lo hd the highest perentge of i (Fig. 6,d), very limited infiltrtion of the oloni muos (Fig. 6) nd mintined their weight (Fig. 6). To test whether i re required to ontrol olitis 1, we trnsferred CD4 + T onv ells isolted from Foxp3 DTR mie into Cd3e / mie (Supplementry Fig. 6). These nimls were lso treted with DTx every third dy to deplete ny i tht developed from trnsferred Foxp3 DTR CD4 + T onv ells. it reg ell depletion elerted weight loss nd development of olitis (Fig. 6,e). Co-trnsferred B6 Triple lo (whih re unffeted y DTx) were unle to ontrol the development of olitis when i were depleted (Fig. 6,,e). These dt support the ide tht Triple lo filitte onversion of some CD4 + T onv ells into Foxp3 + i (Fig. 6d), whih, in ggregte, limit development of olitis. Tken together, our dt (Figs. nd 6) suggest tht there re two popultions of : Triple hi, whih ontrol lymphoprolifertion in peripherl LNs, nd Triple lo, whih limit the development of olitis (t lest in lymphopeni setting). It should e noted tht the phenotypes of Triple hi were stle over the 11-d time ourse of the experiment (Fig. ), wheres Triple lo were stle over the 6-week time ourse of the olitis experiment (Fig. 6 nd Supplementry Fig. 6). Given tht Triple lo did not suppress lympho-prolifertion nd Triple hi did not suppress olitis, there ws no evidene for sustntil degree of trns-differentition etween the two susets during the time frme of these experiments. 1.. Triple hi T reg TCRs CD4 + T onv TCRs Triple lo T reg TCRs CD4 + T onv TCRs CD4 + T onv TCRs Figure 3 Sequene similrity nlysis etween Triple hi T reg, 8 Triple lo T reg TCRs * Triple lo T reg TCRs 6 Triple hi 1. Triple lo T reg nd CD4 + T onv TCRs. V α 2 + TCRα sequenes generted T reg TCRs Triple hi T reg TCRs from Triple hi T reg, Triple lo T reg nd CD4 + T onv LN ells from 4.8 Ye62 Vβ8.2 (single TCRβ hin), TCRα +/KO Foxp3-GFP KI mie. Sequenes from eh suset in eh group of mie were individully 2.6 ompred to ll susets from ll groups of mie. () Frequeny of V α 2-expressing ells mong CD4 + T onv ells (lue), Triple hi (red) nd Triple lo (rown). Evlution of TCR sequene similrity sed on the Morisit-Horn similrity index (MHI). ( d) MHI omprison of V α 2 + TCR sequenes. () CD4 + T onv lonotypes (lue) were ompred with eh other nd with Triple lo (rown) T reg nd Triple hi (red) T reg lonotypes. () Triple lo T reg lonotypes (rown) were ompred to eh other nd to CD4 + T onv (lue) nd Triple hi T reg (red) lonotypes nd (d) Triple hi T reg lonotypes (red) were ompred with eh other nd with CD4 T onv (lue) nd Triple lo T reg (rown) lonotypes (Online Methods). Evlution of TCR sequenes diversity sed on Shnnon entropy (e) nd Simpson diversity (f) sores (Online Methods). Eh symol represents the vlue from group of mie () or sequenes (e,f); in d, eh symol represents MHI omprison etween lonotypes from two individul groups; smll horizontl lines indite the men (±s.e.m.). = not signifint (P >.8), *P =.8, P.1, *P.1 (Mnn-Whittney U test, d; unpired, two-tiled t test, e,f). Dt re from one experiment with three independent groups of TCRβ hin mie (two mie per group). DVANCE ONLINE PUBLICATION nture immunology

5 216 Nture Ameri, In. All rights reserved. Figure 4 Self-retivity of Triple hi nd Triple lo. () Flow ytometry nlyzing Triple hi (red) nd Triple lo (rown) LN from 6 1-week-old B6 mie for CD (n = 4 mie) nd Nur77-GFP (n = 2 mie) expression nd quntifition of MFI of those results. () Prolifertion of LN-derived Triple hi T reg (red), Triple lo T reg (rown) nd T onv (lue) ells in vivo fter intrperitonel injetion of BrdU into B6 mie (n = 4 mie). Frequenies of BrdU-inorporting (BrdU + ) ells re shown. () Flow ytometry nlyzing in vitro prolifertion (CFSE dilution) of LN-derived Triple hi T reg (red) Triple lo T reg (rown) nd CD4 + T onv (lue) ells from purified CFSEleled CD4 + LN-derived T ells ultured on B6 or B6.MHCII KO BMDCs. Representtive histogrm (left) nd quntifition of those results (numers of proliferted ells, right) re shown (n = iologil replites). (d) Flow ytometry nlyzing,, Foxp3 nd CD4 expression y 3BK8TCR-tg CD4SP (top nd middle row) or nd expression y 3BK8TCR-tg Foxp3 + CD4SP thymoytes (ottom row) fter 48 h of ulture in the presene of P-1A, P2A, 3K or no peptide, mture B6 BMDCs s APCs, IL-2 nd TGF-β. (e) Flow ytometry nlyzing expression of Foxp3, CD4 on OTII CD4SP thymoytes (left) nd nd on Foxp3 + OTII CD4SP thymoytes otined from himers generted y reonstitution of RIP-OVA (top) or B6 (ottom) hosts with mixture of one mrrow ells from OTII Rg2 / nd B6 mie (n = 4 mie eh Triple hi nd Triple lo CD4 + T ells in Surfy mie Although Surfy mie nnot develop, s they lk funtionl Foxp3, they do rry out negtive seletion 21. For this reson, we wondered whether Surfy mie ontined Triple hi - like nd Triple lo - like Foxp3 CD4 + T ells. Flow ytometry nlysis reveled tht these mie ontined hi hi CD2 hi (Surfy Triple hi ) nd lo lo CD2 lo (Surfy Triple lo ) CD4 + T ells. Surfy d CD4 SP Foxp3 CD Nur No peptide Foxp3 + CD4 SP CD MFI ( 1 2 ) Nur77 MFI ( 1 2 ) CD4 6 * P-1A (1 µm) BrdU + ells (%) Triple hi Triple lo P2A (1 µm) Triple hi T reg ells Triple lo T reg ells CD4 + T onv ells 3K (1 µm) B6 BMDCs MHCII KO BMDCs e CFSE CD4 + T onv ells Triple lo Triple hi RIP-OVA host B6 host Triple hi CD4 + T ells resemled B6 Triple hi in terms of,, CD2, Helios, CD nd CD62L protein expression (Fig. 7). Given their lk of Foxp3 expression nd suppressive pity, Surfy Triple hi CD4 + T ells my e similr to previously reported T reg ell wnnes 21,33,34. Surfy Triple lo CD4 + T ells, on the other hnd, resemled CD4 + T onv ells with respet to their expression of these mrkers (Fig. 7). Foxp Proliferted ells ( 1 3 ) OTII CD4 SP CD4 Triple hi Triple lo * B MHCII KO 1 2 CD4 + Foxp group). Numers in qudrnts indite perent ells; numers djent to outlined res indite (d, middle) perent Foxp3 + mong 3BK8TCR-tg CD4SP nd (e, left) perent of Foxp3 + mong OTII CD4 SP. Eh symol represents n individul himer (,). Br grphs indite the men (±s.e.m.). = not signifint (P >.4), *P.4, P.1, *P.1 (unpired, two-tiled t test). Dt re from one (, Nur77), two (, CD;,) independent experiments or from one representtive experiment from totl of three (d) or two (e) independent experiments with similr results. Figure Triple hi, ut not Triple lo, suppress in vivo lymphoprolifertion. Anlysis of in vivo suppressive funtion of sorted Ly.2 Triple hi T reg, Ly.2 Triple lo or totl Ly.2 trnsferred into 6 1-week-old Ly.1 Foxp3 DTR mie treted every other dy with DTx. () Expnsion of endogenous CD4 + (left), CD8 + (middle) or B (right) ells of DTx-treted Ly.1 Foxp3 DTR mie, previously injeted (intrvenously) with Triple lo T reg (rown, n = 4 mie), Triple hi T reg (red, n = 6 mie) or totl T reg (green, n = 3 mie) ells ws nlyzed t d fter ell trnsfer. DTx-treted Ly.1 Foxp3 DTR mie reeiving no ells (lk, n = 1 mie) or DTx-treted CD CD4 + ells ( 1 6 ) No in B6 CD62L No in Foxp3 DRT 13 CD8 + ells ( 1 6 ) +Triple lo 6 * Triple hi 4 B ells ( 1 6 ) Totl 3 * Nive CD4 + T onv ells (%) No ells in FoxP3 DTR Triple lo Triple hi Totl No ells in B6 * No ells in B6 No ells in Foxp3 DTR Triple lo Triple hi Totl B6 mie (gry, n = 4 mie) were used s ontrols. () Representtive flow ytometry nlyzing CD44 nd CD62L expression on endogenous CD4 + T ells, isolted from LNs of DTx treted Ly.1 Foxp3 DTR or B6 mie (desried in ). () quntifition of endogenous nive (CD44 CD62L + ) CD4 + T onv ells of the results shown in. Numers in qudrnts indite perent ells in eh throughout. Br grphs indite the men (±s.e.m.). = not signifint (P >.), *P., P.1 (unpired, two-tiled t test). Dt re from 2 4 independent experiments (,). nture immunology DVANCE ONLINE PUBLICATION

6 1 T onv ells DTR T onv ells T onv ells T onv ells (+ Triple lo (+ Triple hi ) (+ Triple lo ) +DTx) DTR T onv ells (+DTx) Weight (%) 1 mln Nture Ameri, In. All rights reserved. 2 Tonv ells DTR T onv ells Time (weeks) CD4 + T onv ells CD4 + T onv ells + Triple lo CD4 + T onv ells + Triple hi No ells T onv ells + Triple lo DTR T onv ells + Triple lo T onv ells + Triple hi No ells LN d i (FoxP3 + T onv ells) (%) Foxp CD4 * mln T onv ells + Triple hi T onv ells T onv ells + Triple lo DTR T onv ells DTR T onv ells + Triple lo LN e Weight (%) Time (weeks) No ells CD4 + DTR T onv ells CD4 + DTR T onv ells + Triple lo Figure 6 Triple lo, ut not Triple hi, suppress olitis. Anlysis of in vivo suppressive funtion of sorted Ly.2 Triple hi T reg nd Ly.2 Triple lo o-trnsferred with sorted, nive (CD4 + CD2 ) CD4 + T onv ells from B6 Ly.1 (CD4 + B6 T onv ells, d) or Ly.1 Foxp3 DTR mie (CD4 + Foxp3 DTR T onv ells, e) into 6 1-week-old T-ell-defiient Cd3e / mie. () Weight hnge in Cd3e / mie following intrvenous doptive trnsfer of CD4 + T onv ells lone (lue, n = 9 mie), CD4 + B6 T onv + Triple lo (rown, n = 9 mie) or CD4 + B6 T onv + Triple hi T reg (red, n = 6 mie) ells. Cd3e / mie injeted with no ells (lk, n = ) were used s ontrols. () Hemtoxylin-nd-eosin stining of olon setions from Cd3e / mie doptively trnsferred with ell popultions indited in nd e. Sle r represents 1 µm. () Flow ytometry nlyzing of Foxp3 nd CD4 expression y CD4 + B6 T onv ells or CD4 + Foxp3 DTR T onv ells isolted from LNs nd mesenteri LNs (mlns) from mie desried in nd e 6 weeks fter trnsfer. (d) Numers djent to outlined res indite frequenies of Foxp3 + CD4 + (i) mong those ells nd quntifition of those results. (e) Weight hnge in Cd3e / mie following intrvenous doptive trnsfer of CD4 + FoxP3 DTR T onv ells lone (dshed lue, n = 3 mie) or CD4 + FoxP3 DTR T onv + Triple lo (dshed rown, n = 3 mie). Cd3e / mie not reeiving ells (lk, n = ) were used s ontrols. All mie were treted with DTx. = not signifint (P >.68), *P =.68, P., *P.1, P.1(unpired, two-tiled t test). Dt re from 2 4 independent experiments. Eh symol represents n individul mouse (d); men ± s.e.m. in,d,e. * To investigte their pthologil tivities, we sorted Surfy Triple lo nd Surfy Triple hi CD4 + T ells (Supplementry Fig. 7) nd seprtely trnsferred them into T-ell-defiient Cd3e / hosts (Supplementry Fig. 7). Trnsferred Surfy Triple lo CD4 + T ells promoted weight loss (Fig. 7) nd olitis (Fig. 7). Moreover, they umulted in mesenteri LNs (Supplementry Fig. 7), where ~3% of these ells expressed α 4 β 7, n integrin tht enles homing to the gut 3 (Supplementry Fig. 7d). In ontrst, trnsferred Surfy Triple hi CD4 + T ells did not use weight loss (Fig. 7) nd preferentilly umulted in peripherl, ut not mesenteri, LNs (Fig. 7d nd Supplementry Fig. 7). Moreover, Surfy Triple hi CD4 + T ells indued mssive inflmmtion in the skin, ut only miniml inflmmtion in the olon (Fig. 7 nd Supplementry Fig. 7e). Tken together, these results indite tht the sene of norml is not the sole use of Surfy disese; the tivity of dysregulted (T reg ell like) Surfy Triple hi CD4 + T ells ounts for some of the pthology oserved in these mie. DISCUSSION We exmined the funtionlity of susets with distint TCR repertoires nd differing ffinities for self ntigens. Our dt suggest tht Triple hi nd Triple lo re generted s n offshoot of negtive seletion. The high-ffinity self-retive TCRs expressed y Triple hi likely drive their seletion in the thymus nd their suppressive tivity in peripherl LNs 36. On the other hnd, thymi preursors expressing lower ffinity self-retive TCRs plusily differentite into Triple lo. Triple hi nd Triple lo re distint from entrl nd effetor T reg ell susets on the sis of their expression of CD2, CCR7, CD13, Helios nd Nrp-1 proteins 16. Foxp3-RFP Rg-GFP dul reporter mie lerly showed tht Triple hi nd Triple lo T reg ell were present mong de novo generted, Rg-GFP +, thymi. AF mie ontined virtully no foreign ntigens (they lked miroiome nd were fed n elementl diet), ut expressed norml frequenies of Triple hi nd Triple lo ; this demonstrtes tht their differentition is driven exlusively y self ntigens. Tken together, these dt demonstrte tht Triple hi nd Triple lo re generted in progrmmed fshion, sed on their ffinity for self ntigens. nd CD4 + T onv ells re differently seleted nd hve dissimilr TCR repertoires 24,2. A omprison of the TCR repertoires expressed in thymi nd peripherl (indued) is diffiult s DVANCE ONLINE PUBLICATION nture immunology

7 CD2 Surfy Triple hi CD4 + T ells Surfy Triple lo CD4 + T ells B6 Triple hi B6 T onv ells CD2 Weight (%) Skin (til) Colon B6 Time (weeks) Surfy Triple hi ells Cd3e / No ells Surfy Triple hi CD4 + T ells Surfy Triple lo CD4 + T ells Surfy Triple lo ells Cd3e / d Cell numer ( 1 6 ) Surfy Triple hi CD4 + T ells Surfy Triple lo CD4 + T ells * * LN mln 216 Nture Ameri, In. All rights reserved Helios CD CD62L Figure 7 Foxp3-defiient (Surfy) mie ontin B6 T reg -ell-like ells with distint pthogeniities. () Flow ytometry nlyzing the expression of nd CD2 on LN CD4 + T ells from Foxp3-defiient (Surfy) mie (top) nd,, CD2 (middle), Helios, CD nd CD62L (ottom) expression y Surfy Triple hi ( hi hi CD2 hi ; ornge solid line) CD4 + T ells, Surfy Triple lo ( neg neg CD2 neg ; purple solid line) CD4 + T ells, B6 CD4 + Triple hi (dotted red line) nd B6 CD4 + T onv ells (dotted lue line) from 2 3-week-old mie (n = 4 mie eh group). ( d) Anlysis of in vivo pthogeniity indued y sorted Surfy Triple hi nd Surfy Triple lo CD4 + T ells isolted from sik, 2 3-week-old FoxP3- defiient mie nd doptively trnsferred into 6 1-week-old Cd3e / mie. () Weight hnge over time following intrvenous doptive trnsfer of Surfy Triple hi (ornge) or Surfy Triple lo (purple) CD4 + T ells into 6 1-week-old Cd3e / mie (n = 14 mie eh group). Cd3e / mie reeiving no ells (lk, n = 7 mie) were used s ontrols. () Representtive hemtoxylin-nd-eosin stining of olon nd til skin setions from Cd3e / mie doptively trnsferred with ell popultions indited in nd B6 ontrol mie. Sle rs represent 1 µm. (d) Numers of Surfy Triple hi (ornge) nd Surfy Triple lo (purple) CD4 + T ells reovered from peripherl LNs nd mlns 6 weeks fter ell trnsfer (n = 1 mie eh group). = not signifint (P >.), *P., P.1 (unpired, two-tiled t test). Dt re from seven () or five (d) independent experiments or one experiment representtive of two () or five () independent experiments with similr results; men ± s.e.m. in. result of the sene of speifi mrkers for ell sorting 8,9,14,37,38. However, nlysis of peripherl (ssumed to e thymus derived) nd oloni (ssumed to e peripherlly indued) reveled different TCR repertories expressed in these two popultions 26. Deep sequening showed tht the TCR repertoires of Triple hi nd Triple lo T reg nd CD4 + T onv ells re distint; this is expeted if TCR speifiity is linked to T reg ell differentition. The deresed TCR diversity mong Triple hi my e result of oligolonl expnsion; this is onsistent with their inresed prolifertion in vivo. Bsed on CD nd Nur77-GFP reporter expression 31,32, the ffinity hierrhy for self-retivity is likely Triple hi > Triple lo > CD4 + T onv ells. Exposing MHCII-restrited TCR-tg thymoytes to threshold- (wek deleting), intermedite- (moderte deleting) or high-ffinity (strong deleting) ntigens genertes Triple lo, Triple int or Triple hi, respetively. The priniple tht thymoyte ffinity for self ntigen determines ell fte lso pplies to T reg ell development. Whether different T reg ell popultions suppress different spets of utoimmunity is not fully known 1. Aute T reg ell ltion in Foxp3 DTR mie leds to the tivtion of T ells speifi for ville ntigens, inluding genome-enoded self, environmentl nd food ntigens 39. We found tht the mssive expnsion of T onv nd B ells in T reg -ell-lted mie ws ontrolled y trnsferring Triple hi, ut not Triple lo. Triple hi my suppress lymphoprolifertion in peripherl LNs y either modifying dendriti ells (DCs) towrd tolerogeni phenotype 4 or y diretly interting with T onv ells 39,41,42. A numer of reports hve shown tht o-trnsfer of, prtiulrly miroiot-speifi, prevents the onset of or even ures mie of olitis 7,43,44. i re essentil for mintining immune homeostsis, espeilly t muosl interfes; in ddition, i ontriute to fetl tolerne,12,13. In the gut, nive CD4 + T ells re onverted into i following TCR stimultion in the presene of TGF-β nd IL-2; other ompounds suh s retinoi id or shorthin ftty ids from miroiot medite onversion s well 7. IL-1 is lso key plyer in mintining lymphoyte homeostsis in the gut, s IL-1-defiient mie suffer from spontneous olitis 7. Our results lerly show tht Triple lo suppressed olitis indution. Triple lo y themselves did not ontrol olitis indution, ut insted funtioned y promoting the genertion of i from CD4 + T onv ells. To the est of our knowledge, there is no study showing tht prtiulr T reg ell popultion n promote the onversion of CD4 + T onv ells to i in vivo. Reently, M2 mrophges were shown to promote supportive environment for i nd to diretly ontriute to immunologil homeostsis in the gut 4. Nevertheless, the mnner in whih Triple lo filitte the genertion of i is still n open question. T reg -ell-like wnne CD4 + T ells umulte in surfy mie 33,34. These T reg -ell-like Surfy CD4 + T ells re phenotypilly similr to on fide nd even express similr TCRs 21. Notly, trnsfer of T onv -like CD4 + T ells from Surfy mie results in olitis, ut not the other fetures of Surfy disese 33. We found tht Surfy Triple hi CD4 + T ells re similr to on fide Triple hi with respet to,, CD2 nd Helios expression. Trnsferred Surfy Triple hi CD4 + T ells proliferted extensively in peripherl LNs, infiltrted the skin nd used utneous lesions similr to those seen in Surfy mie. Notly, IL-2-defiient Surfy mie did not develop nture immunology DVANCE ONLINE PUBLICATION

8 216 Nture Ameri, In. All rights reserved. skin lesions, wheres IL-4-, IL-6-, IL-1-, Stt6- or CD13-defiient Surfy mie do 46, suggesting tht IL-2 ts s n importnt meditor of skin inflmmtion in Surfy mie. Surfy Triple hi CD4 + T ells likely require, ut do not produe, their own IL-2, s they express Helios, repressor of IL-2 trnsription 34. This might explin the umultion of Surfy Triple hi CD4 + T ells round IL-2-sereting, skin-resident DCs in the dermis 47. In ontrst, Surfy Triple lo CD4 + T ells indued severe olitis within 4 weeks of eing trnsferred to T-ell-defiient reipients. It s unler whether Surfy Triple lo CD4 + T ells re the Surfy equivlent of B6 Triple lo or of B6 CD4 + T onv ells. A portion of Surfy Triple lo CD4 + T ells re likely to e miroiot speifi, s GF surfy mie re less prone to develop olitis thn Surfy mie housed under SPF onditions 26,36. Tken together, these results indite tht Surfy disese is pleotropi. Although the sene of on fide is the mjor ontriutor to the surfy phenotype, the presene of dysregulted T reg -like ells very likely initites severl pthologil spets of this disese. In summry, we found tht the extent of self-retivity underlies the development of two distint popultions of regultory T ells. The highly self-retive Triple hi ontrolled the homeostti prolifertion of lymphoytes in peripherl LNs, wheres the less self-retive Triple lo filitted the genertion of i to mintin lymphoyte homeostsis in the olon. Surfy mie ontined dysregulted T reg -ell-like CD4 + T ells, whih ontriute to the pthology of Surfy disese. Methods Methods nd ny ssoited referenes re ville in the online version of the pper. Aession odes. Sequene Red Arhive: TCR sequene dt, PRJNA Note: Any Supplementry Informtion nd Soure Dt files re ville in the online version of the pper. Aknowledgments We thnk U. Shneider for niml husndry, E. Truneker nd T. Kres for ell sorting, nd G. DeLiero, L. Jeker nd O. Stepnek for reviewing the mnusript. This study ws funded y grnts /1 [SNF], Syill [EU FP7], nd TerrInognit [ERC] (E.P.); RO1-DK977, U19 AI1988 nd UMss DERC grnt DK322 (E.S.H.); T32 AI 7349 (B.D.S.); Federl Ministry of Edution nd Reserh grnt (BMBF), Germn Center for Dietes Reserh (grnt DZD e.v., FKZ1GI924) nd Center for Regenertive Therpies Dresden, Cluster of Exellene grnt FZT 111 (K.K.), Progrmme Grnt from MRC (G.A.); Projet IBS-R-D1 from the Inst. for Bsi Siene, Koren Ministry of Siene (C.D.S.) nd Onosuisse KFS-3169 (L.M.T.). AUTHOR CONTRIBUTIO L.W. nd E.P. oneived nd designed the experiments. L.W. performed ll of experiments exept for re-ggregted thymi orgn ultures, whih were rried out y C.G.K.; nlysis of in Foxp3.RFP/GFP mie, whih ws rried out y S.S. nd K.K.; deep sequening nd nlysis of TCR lonotypes, whih were rried y B.D.S. nd E.S.H.; nlysis of thymi in Foxp3-RFP/Rg-GFP dul reporter mie, whih ws rried out y N.I.M. nd G.A.; nlysis of in in GF, AF nd SPF mie, whih ws rried out y J.Y.L. nd C.D.S.; nd evlution of histologil setions, whih ws rried out y L.M.T. The mnusript ws written y L.W. nd E.P. All of the uthors red the mnusript. COMPETING FINANCIAL INTERESTS The uthors delre no ompeting finnil interests. Reprints nd permissions informtion is ville online t reprints/index.html. 1. Fontenot, J.D., Gvin, M.A. & Rudensky, A.Y. Foxp3 progrms the development nd funtion of CD4 + CD2 + regultory T ells. Nt. Immunol. 4, (23). 2. Khttri, R., Cox, T., Ysyko, S.A. & Rmsdell, F. An essentil role for Surfin in CD4 + CD2 + T regultory ells. Nt. Immunol. 4, (23). 3. Hori, S., Nomur, T. & Skguhi, S. Control of regultory T ell development y the trnsription ftor Foxp3. Siene 299, (23). 4. Bennett, C.L. et l. The immune dysregultion, polyendorinopthy, enteropthy, X-linked syndrome (IPEX) is used y muttions of FOXP3. Nt. Genet. 27, 2 21 (21).. Josefowiz, S.Z., Lu, L.F. & Rudensky, A.Y. Regultory T ells: mehnisms of differentition nd funtion. Annu. Rev. Immunol. 3, (212). 6. Ohkur, N., Kitgw, Y. & Skguhi, S. Development nd mintenne of regultory T ells. Immunity 38, (213). 7. Hrrison, O.J. & Powrie, F.M. Regultory T ells nd immune tolerne in the intestine. Cold Spring Hr. Perspet. Biol., (213). 8. Jordn, M.S. et l. Thymi seletion of CD4+CD2+ regultory T ells indued y n gonist self-peptide. Nt. Immunol. 2, (21). 9. Apostolou, I., Srukhn, A., Klein, L. & von Boehmer, H. Origin of regultory T ells with known speifiity for ntigen. Nt. Immunol. 3, (22). 1. Klein, L. & Jovnovi, K. Regultory T ell differentition: turning hrmful into useful. Immunity 37, (212). 11. Chen, W. et l. Conversion of peripherl CD4+CD2- nive T ells to CD4+CD2+ regultory T ells y TGF-et indution of trnsription ftor Foxp3. J. Exp. Med. 198, (23). 12. Zheng, Y. et l. Role of onserved non-oding DNA elements in the Foxp3 gene in regultory T-ell fte. Nture 463, (21). 13. Smstein, R.M., Josefowiz, S.Z., Arvey, A., Treuting, P.M. & Rudensky, A.Y. Extrthymi genertion of regultory T ells in plentl mmmls mitigtes mternl-fetl onflit. Cell 1, (212). 14. Ydv, M. et l. Neuropilin-1 distinguishes nturl nd induile regultory T ells mong regultory T ell susets. in vivo. J Exp Med 29, (212). 1. Hrihi, D. et l. A requisite role for indued regultory T ells in tolerne sed on expnding ntigen reeptor diversity. Immunity 3, (211). 16. Smigiel, K.S. et l. CCR7 provides lolized ess to IL-2 nd defines homeosttilly distint regultory T ell susets. J. Exp. Med. 211, (214). 17. Huehn, J. et l. Developmentl stge, phenotype, nd migrtion distinguish nivend effetor/memory-like CD4+ regultory T ells. J. Exp. Med. 199, (24). 18. Miyr, M. et l. Funtionl delinetion nd differentition dynmis of humn CD4+ T ells expressing the FoxP3 trnsription ftor. Immunity 3, (29). 19. Levine, A.G., Arvey, A., Jin, W. & Rudensky, A.Y. Continuous requirement for the TCR in regultory T ell funtion. Nt. Immunol. 1, (214). 2. Shmidt, A.M. et l. Regultory T ells require TCR signling for their suppressive funtion. J. Immunol. 194, (21). 21. Hsieh, C.S., Zheng, Y., Ling, Y., Fontenot, J.D. & Rudensky, A.Y. An intersetion etween the self-retive regultory nd nonregultory T ell reeptor repertoires. Nt. Immunol. 7, (26). 22. Lee, H.-M., Butist, J.L., Sott-Browne, J., Mohn, J.F. & Hsieh, C.-S. A rod rnge of self-retivity drives thymi regultory T ell seletion to limit responses to self. Immunity 37, (212). 23. Pholzyk, R. et l. Nonself-ntigens re the ognte speifiities of Foxp3+ regultory T ells. Immunity 27, (27). 24. Hsieh, C.S. et l. Reognition of the peripherl self y nturlly rising CD2+ CD4+ T ell reeptors. Immunity 21, (24). 2. Pholzyk, R., Igntowiz, H., Krj, P. & Igntowiz, L. Origin nd T ell reeptor diversity of Foxp3+CD4+CD2+ T ells. Immunity 2, (26). 26. Lthrop, S.K. et l. Peripherl edution of the immune system y oloni ommensl miroiot. Nture 478, 2 24 (211). 27. Ydv, M., Stephn, S. & Bluestone, J.A. Peripherlly indued Tregs: role in immune homeostsis nd utoimmunity. Front. Immunol. 4, 232 (213). 28. Yng, E., Zou, T., Leihner, T.M., Zhng, S.L. & Kmyshi, T. Both retention nd reirultion ontriute to long-lived regultory T-ell umultion in the thymus. Eur. J. Immunol. 44, (214). 29. Cown, J.E., MCrthy, N.I. & Anderson, G. CCR7 ontrols thymus reirultion, ut not prodution nd emigrtion, of Foxp3(+) T ells. Cell Rep. 14, (216). 3. Kim, K.S. et l. Dietry ntigens limit muosl immunity y induing regultory T ells in the smll intestine. Siene 31, (216). 31. Morn, A.E. et l. T ell reeptor signl strength in Treg nd inkt ell development demonstrted y novel fluoresent reporter mouse. J. Exp. Med. 28, (211). 32. Mndl, J.N., Monteiro, J.P., Vrisekoop, N. & Germin, R.N. T ell-positive seletion uses self-lignd inding strength to optimize repertoire reognition of foreign ntigens. Immunity 38, (213). 33. Kuzm, M. et l. Foxp3-defiient regultory T ells do not revert into onventionl effetor CD4+ T ells ut onstitute unique ell suset. J. Immunol. 183, (29). 34. Lin, W. et l. Regultory T ell development in the sene of funtionl Foxp3. Nt. Immunol. 8, (27). 3. Wgner, N. et l. Critil role for et7 integrins in formtion of the gut-ssoited lymphoid tissue. Nture 382, (1996). 36. Killerew, J.R. et l. A self-retive TCR drives the development of Foxp3+ regultory T ells tht prevent utoimmune disese. J. Immunol. 187, (211). DVANCE ONLINE PUBLICATION nture immunology

9 37. Thornton, A.M. et l. Expression of Helios, n Ikros trnsription ftor fmily memer, differentites thymi-derived from peripherlly indued Foxp3+ T regultory ells. J. Immunol. 184, (21). 38. Weiss, J.M. et l. Neuropilin 1 is expressed on thymus-derived nturl regultory T ells, ut not muos-generted indued Foxp3+. J Exp Med 29, (212). 39. Kim, J.M., Rsmussen, J.P. & Rudensky, A.Y. Regultory T ells prevent tstrophi utoimmunity throughout the lifespn of mie. Nt. Immunol. 8, (27). 4. Morelli, A.E. & Thomson, A.W. Tolerogeni dendriti ells nd the quest for trnsplnt tolerne. Nt. Rev. Immunol. 7, (27). 41. Liu, K. et l. In vivo nlysis of dendriti ell development nd homeostsis. Siene 324, (29). 42. Tdokoro, C.E. et l. Regultory T ells inhiit stle ontts etween CD4+ T ells nd dendriti ells in vivo. J. Exp. Med. 23, 11 (26). 43. Mottet, C., Uhlig, H.H. & Powrie, F. Cutting edge: ure of olitis y CD4+CD2+ regultory T ells. J. Immunol. 17, (23). 44. Round, J.L. & Mzmnin, S.K. Induile Foxp3+ regultory T-ell development y ommensl terium of the intestinl miroiot. Pro. Ntl. Ad. Si. USA 17, (21). 4. Hrihi, D. et l. Alterntively tivted mrophges oost indued regultory T nd Th17 ell responses during immunotherpy for olitis. J. Immunol. 196, (216). 46. Shrm, R., Sung, S.S., Gskin, F., Fu, S.M. & Ju, S.T. A novel funtion of IL-2: hemokine/hemottrtnt/retention reeptor genes indution in Th susets for skin nd lung inflmmtion. J. Autoimmun. 38, (212). 47. Zelnte, T., Fri, J., Wong, A.Y. & Riirdi-Cstgnoli, P. Interleukin-2 prodution y dendriti ells nd its immuno-regultory funtions. Front. Immunol. 3, 161 (212). 216 Nture Ameri, In. All rights reserved. nture immunology DVANCE ONLINE PUBLICATION

10 216 Nture Ameri, In. All rights reserved. ONLINE METHODS Mie. CD4.1 ongeni C7BL/6 (B6 Ly.1, strin:214), CD4.2 ongeni C7BL/6J (B6, strin:664), RIP-OVA mie expressing memrne ound form of Ov under the ontrol of the rt insulin promoter (RIP, strin: 433) 48,49 OTII TCR-tg mie reognizing IA /OVA (strin: 4194), B6.Nur77-GFP(strin: 18974) 31 nd Foxp3-defiient (Surfy, strin: 19933) mie 1 were ll otined from The Jkson Lortory. 3BK6 TCR-tg nd 3BK8TCR-tg mie reognizing IA /3K nd mie defiient for MHC lss II, invrint hin nd Rg2 gene (referred here s MHCII KO mie) were provided y P. Mrrk nd J. Kppler (Ntionl Jewish Helth) nd re desried elsewhere 2. Foxp3 DTR mie 39 were kindly provided y A. Rudensky (Memoril Slon Kettering Cner Center). Foxp3eGFP nd Cd3e / mie were kindly provided y T. Rolink (University of Bsel) nd single TCR-β hin (OT-I Vβ) trnsgeni mie kindly provided y D. Zehn (Tehnil University of Munih) nd re desried elsewhere 3. Mle Foxp3-defiient mie were used t 2 3 weeks of ge, for ll other strins, mle nd femle mie t the ge of 12 weeks were used for experiments. Mie were housed under speifi pthogenfree onditions nd red in our olony (University Hospitl Bsel) in ordne with Cntonl nd Federl lws of Switzerlnd. Animl protools were pproved y the Cntonl Veterinry Offie of Bselstdt, Switzerlnd. Mie expressing the YAe62TCRβ-hin 6,7 nd ll mouse su-lines were mintined in pthogen-free environment in ordne with institutionl guidelines in the Animl Cre Fility t the University of Msshusetts Medil Shool. Foxp3.RFP/GFP mie on the B6 kground were red nd mintined t the niml fility of the CRTD under speifi pthogen-free onditions; niml experiments were performed in ordne with the Germn lw on re nd use of lortory nimls nd pproved y the Regieriungspräsidium Dresden. Antigen free nd germ free B6 mie 3 were red nd mintined t the niml fility of the Pohng University of Siene nd Tehnology. This reserh ws pproved y the Institutionl Animl Cre nd Use Committees (IACUC) of the Pohng University of Siene nd Tehnology ( ). Mouse re nd experimentl proedures were performed in ordne with ll institutionl guidelines for the ethil use of non-humn nimls in reserh nd protools from IACUC of the Pohng University of Siene nd Tehnology. Foxp3-RFP Rg-GFP dul reporter mie 29 on the B6 kground were red nd mintined t the niml fility of the Biomedil Servies Unit t the University of Birminghm nd ll experiments were performed in ordne with lol nd ntionl Home Offie regultions. Flow ytometry nd ell sorting. Thymoytes nd lymphoytes were stined with LIVE/DEAD Fixle ner-ir stin Kit (Life Tehnologies, Invitrogen) nd surfe ntiodies ginst CD3 (14-2C11), CD4 (RM4-), CD (3-7.3), CD8 (3.8), CD19 (ID3), CD2 (PC61), CD44 (IM7), CD4.1 (A2), CD4.2 (14), CD4R (B22, RA3-6B2), CD62L (MEL-14), CD13 (2E7), CD197 (CCR7, 4B12), CD278 (ICOS, 7E.17G9), CD279 (, RMP 1-3), CD37 (, DAT-1/ Y76), Nrp-1 (polylonl), TCRβ (H7-97) nd α 4 β 7 (DATK32). Intrellulr stining for Foxp3 (FJK-16s/ 1D), Helios (22F6), pjun (D47G9), pcd3ζ (K ) nd perk (197G2) ws performed using the Foxp3 stining kit (ebiosiene). All ntiodies hve een vlidted y their suppliers nd referenes n e found on their wesite or on the online vlidtion dtses Antiodypedi nd 1DegreeBio. Antiody dilutions were 1:1 for surfe stinings nd 1: for intrellulr stinings. For BrdU experiments, mie were injeted with 1mg/d BrdU (-romodeoxyuridine, BD Biosiene) for 3 d nd ells were then stined for inorported BrdU using BrdU Flow Kit (BD Biosiene) followed y stining for intrellulr mrkers. All ntiodies were purhsed from BD Biosiene, BioLegend, ebiosiene or CellSignling Tehnology. For flow ytometri nlysis, FACS CntoII (BD Biosiene) nd FlowJo softwre (TreeStr) were used. For ell isoltion, CD4 + T ells were enrihed using Dyneds Untouhed Mouse CD4 Cells Kit (Life Tehnologies, Invitrogen) from ell suspensions from different soures (peripherl LN, mesenteri LN, spleen); supopultions of enrihed CD4 ells were further sorted on FACSAriIII or Influx ell sorter (BD Biosienes). Cell numers were determined using AuChek Counting Beds (Life Tehnologies, Invitrogen) ording to mnufturer s instrutions. All kits were used ording to mnufturer s instrutions. In vitro ssys. Bone mrrow derived DCs (BMDCs) were generted from one mrrow ells of 7-week-old B6 or B6.MHCII KO mie. Bone mrrow ells were ultured under mturtion onditions for 1 dys in full medium supplemented with GM-CSF (hyridom superntnt, LUTZ-GMCSF, kindly provided y V.Horejsi). Autologous mixed lymphoyte retions (uto-mlrs) were performed y o-ulturing 1 syngenei (B6 or MHCII KO) BMDCs with 3 1 CFSE leled (Life Tehnologies, Invitrogen) mgneti ed enrihed CD4 ells (Dyneds, Invitrogen) in 96-well-U-shped pltes for d. For in vitro, T reg ell development experiments, 1 thymoytes from 3BK8TCR-tg mie were o-ultured with 1 B6 BMDCs in the presene of IL-2 (2 U/ml, hyridom X63 superntnt) nd reominnt mouse TGF-β1 (1 ng/ml, R&D Systems) for 48 h with or without 1 µm 3K (FEAQKAKANKAV), P2A (FEAAKAKANKAVD) or P-1A (FAAQKAKANKAVD) peptides (ll otined from Eurogente). Re-ggregted thymi orgn ultures were performed s previously desried 8. In rief, re-ggregted thymi orgn ultures were estlished from B3K8TCR-tg, MHCII KO thymoytes nd thymi epithelil ells from B6 mie nd were ultured in presene of P-1A (2 µm), P2A (2 µm) or 3K (.2 µm) peptides for 7 d efore nlysis. All in vitro ssys were performed t 37 C in % CO 2 using omplete RPMI medium (GIBCO, Life Tehnologies). Genertion of one mrrow himeri mie. For generting one mrrow himeri mie, previously desried protool 49 ws dpted. Reipient mie (CD4.1 CD4.2) were lethlly irrdited with 9 rd (GmmCell, Best Thertronis). Bone mrrow ells from 8-week-old B6 mie (CD4.1) nd OT-II Rg2 / mie (CD4.2) were isolted nd depleted of mture CD4 + nd CD8 + T ells. A mixture of 9:1 of B6 nd OT-II Rg2 / one mrrow ells (4 1 6 totl ells) were injeted intrvenously (i.v.) into irrdited reipient mie. Mie were nlyzed weeks fter reonstitution nd treted with ntiiotis (Nopil, Meph Phrm AG) in the drinking wter until 2 weeks efore nlysis. The ongeni mrkers CD4.1 nd CD4.2 were used to identify T ells derived from different donor one mrrows s well s the host. In vivo suppression ssys. Foxp3 DTR mie were injeted intr-peritonel (i.p.) with DTx (Cliohem) every other dy for 1 12 d (first nd seond injetion µg/kg; susequent injetions 2 µg/kg). In some groups, 2. 1 sorted from pooled LNs were injeted i.v. 3 d efore first DTx injetion. Mie were nlyzed 1 d fter lst their DTx injetion. For olitis experiments, 6 1-week-old T-ell-defiient Cd3e / mie reeived (i.v.) sorted nive CD4 + T ells from pooled LNs of B6Ly.1 or Foxp3 DTR Ly.1 mie. In some groups,.8 1 sorted from pooled LN were o-trnsferred. Reipients of nive Foxp3 DTR CD4 + T ells (CD4 + GFP ) were injeted every third dy with DTx (1 µg/kg), i.p. For doptive trnsfer of Surfy CD4 + T ells, 6 1-week-old T-ell-defiient Cd3e / were reonstituted with 1 sorted CD4 + supopultions from pooled LNs of 2 3-week-old sik Foxp3- defiient (Surfy) mle mie. Reipient mie were weighed weekly t the sme time of dy nd srified when initil ody weight droped more thn 2% or t the ltest 6 weeks fter T ell trnsfer. The ongeni mrkers, Ly.1 nd Ly.2 were used to identify T ells from the different donors s well the host. Tissue smples were fixed in 4% prformldehyde, emedded in prfin, setioned nd stined with hemtoxylin nd eosin. Histology. Formlin-fixed tissues were proessed, stined with hemtoxylin nd eosin nd evluted lindly. Clonotype nlysis. Nive CD4 + (CD4 + CD2 Foxp3 ), Triple lo T reg (CD4 + CD2 lo Foxp3 + lo lo ) or Triple hi T reg (CD4 + CD2 hi Foxp3 + G ITR hi hi ) ell popultions were sorted from three replite groups (two mie per group) of single TCRβ-tg (B6.YAe62β tg + TCRα +/ ) mie were sorted to 98% purity (FACS Ari, BD Biosienes). RNA ws isolted using Trizol nd preipitted with RNse free glyogen (Invitrogen) following the mnuftures protool. DNA ws prepred using oligo-dts (Promeg) nd Omnisript RT kit (Qigen). DNA ws mplified with 2 rounds PCR with generi V α 2 primer ( -CCCTGGGGAAGGCCCTGCTCTCCTGATA-3 ) nd TCR Cα primer ( -GGTACACAGCAGGTTCTGGGTTCTGGATG-3 ). One-tenth volume of the first round PCR ws mplified with n dditionl 2 rounds of PCR using roded primers, for post sequene identifition of originting T ell popultion, ontining Illunim PE red primer nd P/7 regions, respetively. The resulting 3-p frgment ws gel purified nture immunology doi:1.138/ni.322