Lean, but not obese, fat is enriched for a unique population of regulatory T cells that affect metabolic parameters

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1 Len, ut not oese, ft is enrihe for unique popultion of regultory T ells tht ffet metoli prmeters Mrkus Feuerer,5, Lur Herrero 2,5, Dniel Cipollett,4,5, Afi Nz 2, Jmie Wong,5, Ali Nyer 2, Jongsoon Lee 2, Allison B Golfine 3, Christophe Benoist,5, Steven Shoelson 2 & Dine Mthis,5 29 Nture Ameri, In. All rights reserve. Oesity is ompnie y hroni, low-gre inflmmtion of ipose tissue, whih promotes insulin resistne n type-2 ietes. These finings rise the question of how ft inflmmtion n espe the powerful rmmentrium of ells n moleules normlly responsile for guring ginst runwy immune response. CD4 + + T regultory (T reg ) ells with unique phenotype were highly enrihe in the ominl ft of norml mie, ut their numers were strikingly n speifilly reue t this site in insulin-resistnt moels of oesity. Loss-of-funtion n gin-of-funtion experiments revele tht these T reg ells influene the inflmmtory stte of ipose tissue n, thus, insulin resistne. Cytokines ifferentilly synthesize y ft-resient regultory n onventionl T ells iretly ffete the synthesis of inflmmtory meitors n gluose uptke y ulture ipoytes. These oservtions suggest tht hrnessing the nti-inflmmtory properties of T reg ells to inhiit elements of the metoli synrome my hve therpeuti potentil. Type 2 ietes n other elements of the metoli synrome hve inrese t n lrming rte over the pst severl ees. There hs een prllel rise in the iniene of oesity, now reognize to e mjor ontriutor to the insulin resistne unerlying this spetrum of metoli normlities. Just how oesity promotes insulin resistne is still unler, ut results from linil, epiemiologil n moleulr stuies hve onverge to highlight the role of inflmmtion. Prolonge nutrient overlo results in stte of hroni, low-gre inflmmtion in ipose tissue 2 n systemilly 3, prtiulrly in viserl ft epots 4. Viserl ft proues numer of inflmmtory ytokines n hemokines (suh s leptin, tumor nerosis ftor- (TNF-), mrophge hemottrtnt protein- n interleukin-6 (IL-6), mong others), whose proution n e pthologilly ysregulte in the oese stte, ontriuting in n importnt mnner to insulin resistne 5. At the ellulr level, mrophges hve key role. With inresing oesity, they umulte in viserl ft tissue, sometimes ontriuting s muh s hlf of the ellulrity 6,7. The inrese in numer is ompnie y n evolution in phenotype from the nti-inflmmtory (or lterntively tivte ) M2 form to the proinflmmtory (or lssilly tivte ) M form 8,9, whih orreltes with n inrese in insulin resistne (for exmple, see ref. ). In the oese stte, mrophges n ipoytes mke severl of the sme inflmmtory regultors n meitors, inluing TNF-, IL-6, mtrix metlloproteinses (MMPs), peroxisome prolifertor tivte reeptor-g n ftty i ining protein-4 (reviewe in refs.,). These finings, long with others, suggest tht ipoytes n ipose-tissue mrophges my ontriute to insulin resistne in onert, oth inhiiting n enhning eh other s tivities. Like ny inflmmtory stte, the hroni, low-gre inflmmtion ssoite with oesity shoul e sujet to the ontrol mehnisms tht normlly ur overtive immune responses. These mehnisms enompss numer of ell types, whih n operte through ell-ell ontt vi vriety of reeptors, or through iversity of solule meitors. Cells with potentilly regultory phenotype hve previously een ssoite with oesity (for exmple, nturl killer T ells) 2, n there hve een previous reports of nti-inflmmtory ytokines eing etete in ipose tissue (for exmple, IL- n trnsforming growth ftor- (TGF-)) 8,9. Yet, reserh on this topi hs een quite limite, n the influene or loss of influene of mjor ontrol mehnisms remins unresse. For exmple, the role of rguly the most potent regultory ell popultion, CD4 + + T reg ells, hs yet to e explore. T reg ells, smll suset of T lymphoytes, normlly onstituting only 5 2% of the CD4 + omprtment, re thought to e one of the oy s most ruil efenses ginst inpproprite immune responses, operting in ontexts of utoimmunity, llergy, inflmmtion, infetion n tumorigenesis 3,4. Typilly, they ontrol the ehvior of Immunology n Immunogenetis, 2 Cellulr n Moleulr Physiology n 3 Clinil Reserh, Joslin Dietes Center, Deprtment of Meiine, Brighm n Women s Hospitl, Hrvr Meil Shool, Boston, Msshusetts, USA. 4 Europen Shool of Moleulr Meiine, Nples, Itly. 5 Present resses: Deprtment of Pthology, Hrvr Meil Shool, Boston, Msshusetts, USA (M.F., D.C., C.B. n D.M.); Deprtment of Biohemistry n Moleulr Biology, Shool of Phrmy, University of Brelon, Brelon, Spin (L.H.); Alnylm Phrmeutils, Cmrige, Msshusetts, USA (J.W.). Corresponene shoul e resse to D.M. (m@hms.hrvr.eu). Reeive Septemer 28; epte 5 June 29; pulishe online 26 July 29; oi:.38/nm.22 NATURE MEDICINE ADVANCE ONLINE PUBLICATION

2 CD4: 52. ± ± ±.3 CD8: 8.3 ± SSC CD3 CD4 FSC SSC CD8 gte CD gte CD gte CD8 Isotype gte CD4 29 Nture Ameri, In. All rights reserve. + of CD4 + 7 Spl LN Lung Liver A. ft other T ell popultions, ut they n lso influene the tivities of ells of the innte immune system 5 7.T reg ells re hrterize y expression of the forkhe winge-helix trnsription ftor,. Defiienies in this ftor use the lymphoprolifertion n multiorgn utoimmunity foun in surfy mutnt mie n humns with immunoysregultion polyenorinopthy enteropthy X-linke synrome. Here we exmine the T reg ells resiing in mouse ipose tissue with regrs to oth their proportion n phenotype in viserl versus suutneous ft epots in len versus oese mie. We evlute their funtionl importne in loss-of-funtion, gin-of-funtion n in vitro experiments. Lstly, we sought n nlogous popultion in humn ipose tissue smples. Our oservtions ientify unique ft-resient T lymphoyte popultion whose prouts my hve pplitions in treting the metoli synrome. RESULTS Aipose tissue T reg ells Aipose tissue is ompose of multiple ell types. Most prominent re ipoytes, ut vsulr enothelil ells, mrophges 6,7 n lymphoytes 2,8 re lso foun in the stromovsulr frtion. Aoring to multiprmeter flow ytometry, out % of the ells in the stromovsulr frtion of ominl ft from B3-week-ol C57BL/6 (B6) mie fell within the lymphoyte gte (Fig. ). Close to hlf of the ells in this gte were of the CD3 + T linege, split 3: etween the + of CD4 + 7 Aominl ft S.. ft Age (weeks) Figure Aominl (epiiyml) n suutneous ft ps s well s spleen, lymph noe, lung n liver were isolte from retire-reeer B6 mle mie, n the stromovsulr frtion ws stine for, CD3, CD4, CD8 n. () Top, T ell istriution in stromovsulr frtion from ominl ft tissue. Numers on top inite the men ± s.. from six experiments for ells in the lymphoyte gte (left), frtion of CD3 + T ells mong lymphoytegte ells (enter) n istriution of CD4 + n CD8 + T ells (right). Bottom, perentge of + + T ells in ominl ft tissue gte on CD4 + (left) or CD8 + (enter) T ells. Orgns of five mie were poole. Representtive ot plots re shown. FSC, forwr stter; SSC, sie stter. Numers on the grphs inite the perentge of ells in tht gte for tht prtiulr experiment. () Frequeny of + TellsmongCD4 + ells in ifferent orgns. Mens ± s.. from t lest three inepenent experiments re shown, n orgns from four or five mie per experiment were poole. Spl, spleen; LN, lymph noe. () KinetisofT reg ell pperne in ominl n suutneous (s..) ft tissue s well s in spleen. Error rs represent men ± s.. () Immunohistology of ominl ipose tissue. Arrowhe inites stining. expression is restrite to the nuleus. Asterisks refer to e ipoyte resiue surroune y rown-like struture forme y immune ells. Control stining is with isotype ntioy. Originl mgnifition: top left, 4; ll others,. (Control) CD4 + n CD8 + omprtments, respetively (Fig. ). To our surprise, more thn hlf of the CD4 + T ells expresse (Fig. ), muh higher frtion thn tht normlly foun in lymphoi (for exmple, in the spleen or lymph noes) or nonlymphoi (for exmple, lung or liver) tissues (Fig. ), inluing in suutneous ft (Fig. ). Viserl n suutneous ipose tissue h similr low frtions of T reg ells t irth, with progressive umultion over time in the viserl, though not the suutneous, epot (Fig. ). This ihotomy in T reg ell umultion etween these two ft epots is potentilly ruil, given the ssoition of viserl, ut not suutneous, ft with insulin resistne 4,9. Immunohistologil exmintion revele + ells in the spes etween ipoytes, minly, ut not only, in regions where severl of them intersete (Fig. ). Ft tissue, espeilly from oese iniviuls, n host sustntil numers of mrophges, whih umulte in so-lle rown-like strutures tht re replete with e ipoyte resiues 6,7,2.WeoserveT reg ells in suh strutures, in lose proximity to mrophges n other leukoyte ggregtes (Fig. ). We estimte tht 5, 2, + ells resie in g of epiiyml ipose tissue in n B3-week-ol B6 mouse. Expression profiling of ft T reg ells revels unique phenotype We next resse whether the + + ells in ominl ipose tissue were of the typil T reg phenotype (Fig. 2). They funtione s effetively s nlogous ells isolte from the spleen 2 ADVANCE ONLINE PUBLICATION NATURE MEDICINE

3 29 Nture Ameri, In. All rights reserve. when introue into stnr in vitro suppression ssy (Fig. 2). Ft-resient T onventionl (T onv ) ells lso performe s expete; tht is, they h no suppressive tivity n h norml prolifertive response (Fig. 2). The lility n low reoverle numers of ftresient T reg ells hve so fr prevente us from ssying their tivities in in vivo suppressor ssys. So we turne to the well-estlishe trnsriptionl T reg ell signture, erive from the t of multiple groups 2 24,sninitorofin vivo funtion. The overll trnsript profile of the T reg ell popultion from viserl ft iffere from the ptterns of its spleen n lymph noe ounterprts more thn the ltter two i from eh other (Fig. 2). This oservtion lso hel for the T onv ell popultions t these sites, though not s notly so (Fig. 2). Fousing speifilly on the oumente T reg ell signture 2 24, we foun tht our t from the spleen showe n exellent repitultion of its mjor fetures; s ntiipte, most genes known to e upregulte in T reg ells esene to the right on the P vlue versus fol hnge volno plot, wheres most ownregulte loi roppe to the left (Fig. 2). Fully 93% of the signture ws present. In ontrst, eviene y their position t the volno summit, mny of the signture T reg ell genes were not signifintly up- or ownregulte in the orresponing popultion from viserl ft (for exmple, CD3 n G protein ouple reeptor-83) (Fig. 2). We onfirme the Figure 2 Funtionl omprison of T reg n T onv ells from ominl ipose tissue, lymph noe n spleen. CD4 + + T reg n T onv ells were isolte from retire-reeer B6 mie. () A stnr in vitro suppression ssy. erive CD4 + effetor T ells (responer ells) were inute t vrious rtios with ifferent T ell popultions (Ft: ipose tissue-erive T ells). ( e) Anlysis with Affymetrix mirorry hips. Normlize expression vlues for profiles iretly ompring T reg ells () in ft versus spleen (left), ft versus lymph noe (enter) n lymph noe versus spleen (right) or for profiles iretly ompring T onv ells () in ft versus spleen (left), ft versus lymph noe (enter) or lymph noe versus spleen (right). The numers in n inite the numer of genes whose expression iffere y more or less thn twofol. () Volno plots of gene expression t ompring P vlue versus fol hnge for proes from the onsensus T reg ell signture 2,24, plotte for spleen T reg ells versus T onv ells (left), ft T reg ells versus T onv ells (enter); ft T reg ells versus lymph noe T onv ells (right). The olor oing in the figures enotes genes.5-fol over- (re) or uner- (lue) expresse in T reg ells in ll four referene t sets. (e) Fol hnge versus fol hnge plots ompring T reg expression profiles etween ft T reg ells (x xis) n lymph noe T reg ells (y xis) (left) n spleen T reg ells (x xis) n lymph noe T reg ells (y xis) (right). Gene trnsripts uniquely up- or ownregulte in ft T reg ells re highlighte in re n lue, respetively. Spl T reg Spl T onv P vlue e LN T reg / LN T onv FsL..,98 IFN-γ Tet CCL5 CXCR3 t on CD3 y flow ytometri nlysis (Fig. 3). These oservtions on the T reg ell signture were true whether the omprtor ws T onv ells from the ft or the lymph noe (Fig. 2), rguing tht they reflet speil fetures of ipose tissue T reg ells. Nonetheless, the ftresient CD4 + + ells were lerly T reg ells, s muh (63%) of the signture ws intt, inluing overexpression of hllmrk trnsripts suh s those enoing, gluoortioi-inue tumor nerosis ftor reeptor (GITR), ytotoxi T lymphoyte ntigen-4 (CTLA-4), OX4 n killer ell letin-like reeptor G, in ition to itself (Figs. 2 n 3). We onfirme the elevte expression of severl of these signture genes in ft T reg ells y RT-PCR n flow ytometri LN T reg Ft T reg Ft T reg LN T reg 843 Prolifertion normlize,97, LN T onv Ft T onv Ft T onv LN T onv Gpr83 Gpr83 CD3 CTLA-4 Igfp4 Igfp4 OX4 Pe3 GITR Pe3 CD Ox4 Klrg 5 Gpr83 4 CD3 Pe3 Ox4 CTLA Igfp4 CTLA-4 Klrg 6 GITR Klrg 9... Spl T reg / spl T onv Ft T reg / ft T onv Ft T reg / LN T onv Consensus T reg signture : :.5 :.25 Responer to inhiitor rtio Ft T reg / ft T onv Ft T reg speifi genes ,98,2 CXCL IL- GM96 CCR2 CCR Itgv CCR Overexpresse LN T reg / LN T onv :. Overexpresse,869, Effetor T ells Spl T reg Spl T onv + Spl T reg + Ft T reg + Spl T onv + Ft T onv Effetor only Unerrepresente IFN-γ IL-... Spl T reg / spl T onv Unerrepresente NATURE MEDICINE ADVANCE ONLINE PUBLICATION 3

4 Aominl ft CD CTLA IL- Lung A. ft Nture Ameri, In. All rights reserve. f Aritrry unit 3, 2,,8 e 3.6 GITR CD3 CTLA-4 Ft T onv LN T onv Ft T reg LN T reg Ft T reg Ft T onv LN T reg LN T onv 66.5 Aominl ft 38 TNF-α Gte CD3 + CD CD Tr sequenes CTLA A. ft Lung Liver Fol hnge g Gte CD3 + CD4 + Gte CD3 + CD4 + IFN-γ Tet Ft T reg Ft T onv LN T reg LN T onv CD69 Ly6 A. ft Gte CD3 + CD4 + IL- IFN-γ IL-4 LN Liver Lung A. ft LN Liver Lung Figure 3 Phenotypi hrteriztion of T reg ells from ominl ft tissue, spleen, lung n liver. (,) Cells were isolte from retire-reeer B6 mie, n the stromovsulr frtion ws stine for, CD3, CD4, CD8,, gluoortioi-inue tumor nerosis ftor reeptor (GITR), CD3 n CTLA-4. Numers on the grph inite the perentge of ells in tht gte for tht prtiulr experiment. (,) Reltive RNA expression of selete genes from T reg n T onv ells from lymph noe n ft. Error rs represent mens ± s.. (,e) Cytokine expression profile from T reg n T onv ells from spleen, lung n ft tissue. Representtive ot plots of t lest three inepenent experiments re shown. Orgns from four to six mie were poole per experiment. Numers on the grph inite the perentge of ells in tht gte for tht prtiulr experiment. (f) Tr sequenes of ft-erive T reg n T onv ells. Aominl ft n lymph noe T reg n T onv ells were isolte from ge mle mie from the LTD mouse line. The frequeny of the CDR3 sequenes ws nlyze on single-ell sis. Depite is grphil isply of the TCR sequenes in het-mp formt from T reg n T onv ells. (g) Stining of CD3, CD4, CD8, n the tivtion mrkers CD69 n Ly6 in the stromovsulr frtion of ells isolte from ominl ipose tissue, lymph noe, liver n lung from retire-reeer B6 mie. Representtive ot plots re shown. Numers on the grph inite the perentge of ells in tht gte for tht prtiulr experiment IL- IFN-γ IL-4 quntifition (Fig. 3, n t not shown). The gene expression ifferenes oserve in T reg ells isolte from the ft versus from the spleen n lymph noe were not simple refletion of iffering tivtion sttuses, s we oserve lerly ivergent trnsription ptterns in iret omprison etween ft-erive n tivte T reg ells (Supplementry Fig. ). A lrge numer of genes were overexpresse, mny of them mrkely so, y the CD4 + + T ells resiing in ominl ipose tissue ut not y the orresponing popultion t other sites exmine (Fig. 2e; liste in Supplementry Tle ). Chief mongst these overexpresse genes were those enoing moleules involve in leukoyte migrtion n extrvstion: Gm96 (n IL- inuile CXCR2 lign 25 ), CCR, CCR2, CCR9, CCL6, integrin V, tivte leukoyte ell hesion moleule (Alm), CXCL2 n CXCL (Fig. 2e, Supplementry Tle n Supplementry Fig. 2). In ontrst, some moleules of similr funtion, for exmple CCL5 n CXCR3, were unerexpresse in the viserl ft T reg ells (Fig. 2e). Also noteworthy were the extremely high IL- trnsript levels in CD4 + + ominl ipose tissue ells (Fig. 2e n Supplementry Fig. 2). We estimte 36-fol ugmenttion of IL- trnsripts in ft versus lymph noe T reg ells from RT-PCR quntifition (Fig. 3); we oul lso etet the inrese y intrellulr stining for IL- protein in the T reg ells of ft versus spleen n lung (Fig. 3). Notly, pthwy nlysis suggeste tht the T reg ells not only proue lrge mounts of IL- ut lso seeme to e responing to it, s numer of genes ownstrem of the IL- reeptor were upregulte in ft T reg ells ompre with lymph noe T reg ells (Supplementry Fig. 3). Another set of genes ws upregulte speifilly in CD4 + T ells resiing in ipose tissue s ompre with their lymph noe ounterprts, ut not in spleen versus lymph noe (Fig. 2e; liste in Supplementry Tle ). Some of these loi lso oe for moleules implite in migrtion n extrvstion, inluing CXCR3 n CCL5. Ft-resient T onv ells seeme to e highly polrize to T helper type phenotype, s they expresse high levels of T-ox 2 (Tet) n interferon-g (IFN-g) trnsripts (Fig. 2e, Fig. 3 n 4 ADVANCE ONLINE PUBLICATION NATURE MEDICINE

5 29 Nture Ameri, In. All rights reserve. + of CD4 + g + of CD4 + Lep o/o Lep o/ Numer of + CD4 + ells per g ft ( 3 ) 3 n = 7 7 n = 4 WT Lep o/o Lep o/o n = 8 A y / e + of CD4 + P =.6 Lep o/wt Lep o/o NC n = 6 Lep o/+ Lep o/o Lep o/+ Lep o/o WT A y / NC S.. ft Supplementry Fig. 2), unnt intrellulr IFN-g n TNF- (Fig. 3,e), n little, if ny, intrellulr IL-4 (Fig. 3). Although the IL- effet oul lso e iserne with ft-resient T onv ells, it ws not s striking (Supplementry Fig. 3,). Ft-resient T reg ells hve speifi T ell reeptor repertoire The T ell reeptor (TCR) repertoire is nother useful prmeter for ssessing the egree of similrity of T ell popultions. For exmple, it hs een shown tht T reg n T onv ell popultions hve istint repertoires, with only limite overlp In ition, the TCR repertoire of T reg ells in the ominl ipose tissue might give n inition of whether their unne reflets n influx n/or retention of ells of prtiulr speifiity or lol ytokine-inue onversion 29. To rener the repertoire nlysis more mngele n interpretle, we exploite the Limite (LTD) mouse line, wherein TCR iversity is restrite to the omplementrity-etermining region 3 (CDR3) vi the omintion of trnsgeni Tr minilous n the Tr-knokout muttion 3. We etermine CDR3 sequenes from 98 iniviully sorte viserl ft CD4 + + ells tht lso expresse RNA, n we ompre their istriution with tht of CDR3 sequenes from ft T onv ells or lymph noe T reg n T onv ells. Unfortuntely, we were unle to otin enough T reg ells from suutneous ft to perform prllel TCR sequene nlysis on this epot. As expete, the het mps generte from these sequenes revele istint TCR repertoires for the lymph noe T reg n T onv ell popultions, with only limite overlp (Fig. 3f). Similrly, the ft-resient T reg n T onv ell popultions lso h ifferent repertoires (Fig. 3f), renering it very unlikely tht the umultion of + T reg ells in the ominl ipose tissue resulte from lol onversion of T onv ells. Notly, the ft-resient T reg ells h very restrite istriution of sequenes, representing istint suset of those normlly foun in their lymph noe T reg ell ounterprts (Fig. 3f n Supplementry Tle 2). The CDR3 sequenes hrteristi of ft-resient T reg ells were often inepenently generte y ifferent nuleotie sequenes; 5% of sequenes (three of six) foun more thn three times per iniviul mouse showe suh nuleotie vrition (Supplementry Tle 3). In ontrst, none of the sequenes (zero of ten) from ft-resient T onv ells i (Supplementry Tle 4), suggesting h + of CD4 + 3 f + of CD4 + A y / + of CD Age (weeks) 7 Lep o/+ WT NC A y / Lep o/o HOMA-IR i + of CD Lep o/o Lep o/+ Figure 4 Anlysis of ft T reg ells in three mouse moels of oesity: Lep o/o, A y / n. ( ) Aominl ipose tissue from Lep o/o n heterozygote Lep o/+ mie ws nlyze for T reg ell frequeny. () Representtive ot plots of CD4 + T ells from 3-week-ol mie. () Totl numer of T reg ells per grm of ft. Mens ± s.. re shown. P ¼.6. () Chnges of T reg ell representtion over time. Mens ± s.. re shown. () of T reg ells in ominl ipose tissue of 24-week-ol A y / or littermte (WT) mie. P ¼.8. (e) of ft T reg ells in mie fe for 29 weeks with or norml how (NC). P ¼.64. (f) Correltion of homeostsis moel ssessment of insulin resistne (HOMA-IR) n frtion of T reg ells. (g i) of T reg ells in the spleen n suutneous ft in Lep o/o (g), A y / (h) n -fe mie (i). the repete seletion of T reg ells with similr ntigen reeptors rther thn the prolifertion of single lone. The sequenes were reprouily frequent in thee ifferent mie, gin pointing to TCR-riven seletion (Supplementry Tle 3). These t inite tht the speifiity of the TCR my e instrumentl in generting the high frequeny of T reg ells in viserl ft, perhps through lol reognition of ognte ntigen. Inee, ft-resient T reg ells showe unusully high expression of the erly tivtion mrkers CD69 n lymphoyte ntigen 6 omplex, lous C (Ly6) (Fig. 3g), lthough it remins possile tht suh inreses inste, or lso, reflet ytokine influenes. Though TGF- is reily etetle in ipose tissue 3 n is known to promote T reg ell ifferentition n survivl 32 34, its effets re n unlikely explntion for the high representtion n tivtion stte of T reg ells in ft, euse we i not oserve the typil hnges in gene expression promote y this growth ftor in this popultion (Supplementry Fig. 4). For exmple, CD3 ws not upregulte (Fig. 2 n Fig. 3). This oservtion lso rgues ginst TGF- meite onversion of CD4 + ells to CD4 + + ells in viserl ft, s hs een oserve in few systems 29. Implition of ft-resient T reg ells in metoli ontrol To lern how this unique popultion of T reg ellsresponstoexess iposity, we exmine it in three mouse moels of oesity: leptinefiient mie (Lep o/o ; ommonly referre to s o/o) 35, mie heterozygous for the yellow spontneous muttion (A y /) 36 n mie hronilly fe high-ft iet () 3, ll on the B6 geneti kgroun n ll showing insulin resistne (Supplementry Fig. 5). Notly, the T reg ell popultion in ominl ft ws mrkely reue in ge o/o mie ompre with heterozygous littermtes, whether we quntifie the frtion of T reg ells in the CD4 + omprtment or the numer of T reg ells per grm of ft (Fig. 4,). Wheres 5-week-ol leptin-efiient mie h somewht higher (P ¼.2) numers of CD4 + + T ells in viserl ft (3%) thn i wiltype ge-mthe littermtes (%), this suset progressively eline in the mutnt mie n rose in the ontrol mie (Fig. 4)(P¼.). As ntiipte, o/o ft-resient T reg ells were reltively eplete mong IL- prouers (Supplementry Fig. 6). The norml proportions of T reg ells in the spleen n suutneous ft of o/o mie rgue tht the reltive srity of this suset in viserl ft ws not just refletion of the leptin efiieny; inee, the sene of this hormone ws reently reporte to foster the prolifertion of T reg ells 37.Numers of onventionl CD4 + T ells in the ominl ft of o/o ults were only milly lower, n the perentge of these ells mking IFN-g remine essentilly unhnge (Supplementry Fig. 6,). There were lso low proportions of CD4 + + ells in ominl ft, ut not t other sites, in the A y / mie n in -fe mie (Fig. 4 i). These low frequenies were not s striking s for o/o mie (Fig. 4), onsistent with less insulin resistne in the ltter two NATURE MEDICINE ADVANCE ONLINE PUBLICATION 5

6 moels (Supplementry Fig. 5). Inee, we sw goo orreltion etween insulin resistne n the frtion of T reg ells in ominl ft (Fig. 4f). The oserve orreltion etween oesity n insulin resistne on the one hn n erth of CD4 + + ells in ominl ipose tissue on the other hn suggests tht T reg ells might e implite in the reltionship etween the inflmmtory n metoli prmeters. To test this notion, we first ttempte loss-of-funtion experiments. Given tht it is not urrently fesile to lte T reg ells speifilly in ft, we use mie expressing the iphtheri toxin reeptor (DTR) uner the ontrol of trnsriptionl regultory elements, wherein ministrtion of iphtheri toxin results in rpi systemi epletion of T reg ells. We reently evelope line of teril rtifiil hromosome (BAC)-trnsgeni nonoese ieti (NOD) mie expressing DTR-eGFP fusion protein uner the ontrol of trnsriptionl regultory elements. Routinely, 85 9% of T reg ells re eliminte in the spleen n lymph noes 2 fter iphtheri toxin ministrtion to these mie (M.F., D. Littmn, C.B. n D.M., unpulishe t), similr to wht hs een esrie y nother group with their inepenently erive line 38. Cell eth inue y iphtheri toxin is poptoti n therefore oes not set off proinflmmtory immune response 39 42, prompting wiespre use of this pproh to proe iverse immunologil issues through speifi ltion of prtiulr ell types, inluing T reg 29 Nture Ameri, In. All rights reserve. e Fol hnge Fol hnge Gluose (mg l ) NC PBS IL-2 nti IL-2 TNF-α IL-6 A2 RANTES SAA-3 HOMA-IR 5 5 Insulin (ng ml ) NC PBS IL-2 nti IL-2.4 RANTES Epi ft DTR Insulin DTR + 6 pir Epi ft 4 Liver Epi ft 4 IR 2 2 pir Liver IR 2 Musle 2 pir IR Musle pir IR Insulin + + SAA-3 DTR DTR + Lung Epi ft f Gluose (mg l ).7 5 NS Gluose (mg l ) pir / IR (AU) h i j g PBS IL-2 nti IL NC gte CD4 + CD3 + NC : PBS : IL-2 nti IL Time (min) 7. + CD4 + + CD4 + AUC ( 4 ) Epi ft of + CD4 + Epi ft NC : PBS : IL-2 nti IL-2 CD CD4 NC PBS IL-2 nti IL-2 DTR DTR Epi ft Figure 5 Chnges in inflmmtory meitors n metoli prmeters in loss- n gin-of-funtion experiments. ( f) Loss-of-funtion experiment y epleting T reg ells expressing DTR. -week-ol mle mie, either DTR-positive or DTR-negtive, were trete every other y for 4 ( ) or9( f) with iphtheri toxin. () of T reg ells from spleen or the ominl ft (Epi ft) fter 4 of tretment. (,) Insulin signling in epiiyml white ipose tissue (WAT), liver musle n spleen, s inite y immunopreipittion n western lotting of insulin-inue insulin reeptor phosphoryltion (pir) () Blot t. () Quntifition of pir normlize y totl IR (n Z 4, P o.4, t test). AU, ritrry units. () of T reg ells from the ominl ft (top) or spleen (ottom) fter 9 of tretment, with representtive ot plot s n inset. (e) Top, expression of TNF-, IL-6, A2, RANTES n SAA-3 trnsripts in ominl ipose tissue. Three mie per group, one of two inepenent experiments is shown. Bottom, omprison of RANTES n SAA-3 trnsripts in spleen, lung n ominl ft (epi ft). (f) Fsting insulin (top) n fsting gluose (ottom) onentrtions fter 9 of tretment every other y. Six mie per group from two inepenent experiments. P ¼.26. Signifine ws etermine y the Mnn-Whitney U test. (g j) Gin-offuntion experiment. (g) Dot plots (left) n summrizing r grph (right) showing T reg ells from spleen n ominl ft tissue (epi ft) from mie fe norml how (NC) or with 5 weeks of. Mie were trete with IL-2 IL-2 speifi ma (nti IL-2) omplex or sline for 6 n nlyze on y 4 (n ¼ 6 for eh group). Numers on the grphs inite the perentge of ells in tht gte for tht prtiulr experiment. Bloo gluose (h) HOMA-IR (P ¼.78) (i) n n intrperitonel GTT (P ¼.25) (j) of the mie esrie in g re shown. (j) Right, lulte re uner the urve (AUC) from ll mie teste y GTT (n ¼ in eh group), inluing the t set esrie in g. P ¼.3, P ¼.36, P ¼.4. P vlues were lulte with the t test. Error rs in ll pnels represent the mens ± s.. 6 ADVANCE ONLINE PUBLICATION NATURE MEDICINE

7 29 Nture Ameri, In. All rights reserve. Reltive expression Reltive expression e / CD , IL-6 MMP-3 SAA-3 RANTES SAA-3 IL-6 Omentl /CD3 ells 39, However, euse T reg ell lte mie evelop multiorgn utoimmunity 2 weeks fter iphtheri toxin tretment 38,this strtegy require us to look t erly initors of potentil T reg ell funtion, nmely ltertions in ipose tissue RNAs enoing inflmmtory meitors or upstrem hnges in metoli signling pthwys; previous t suggeste tht 2 weeks my e too erly to see hnges in more ownstrem metoli prmeters, inluing performne in gluose tolerne tests (GTTs) 46. For one set of experiments, we trete -week-ol mle mie with iphtheri toxin every other y for 4, whih lowere the T reg ell level in ominl ft to out one-fourth the norml level (Fig. 5), wheres the spleen n lung popultions were t out one-thir the usul level (Fig. 5 n t not shown). The epletion of T reg ells ws ompnie y sustntil ereses in insulin-stimulte insulin reeptor tyrosine phosphoryltion in epiyiml ft n liver, ut not in musle n spleen (Fig. 5,). We otine prllel results on AKT protein kinse phosphoryltion (t not shown). At this erly time point, in vivo metoli hnges were mrginl (not shown), so we onute seon set of experiments in whih we trete mie with iphtheri toxin for longer times. Mie injete every other y for 9 h T reg ell frtion out 3% the usul size of tht in the ft, wheres the spleen, lung n lymph noe popultions h oune k to out 7% of norml (Fig. 5). We o not know why there is suh preferentil loss of T reg ells in ipose tissue with this protool, ut it is likely to reflet slower repopultion kinetis. Conomitntly, mny of the genes enoing inflmmtory meitors (for exmple, TNF-, IL-6, RANTES n serum myloi A-3 (SAA- 3)) were inue in the viserl ft epot (Fig. 5e) ut muh less so in the spleen n lung (Fig. 5e). Insulin mounts were signifintly higher in the T reg ell-eplete mie, initing insulin resistne s.. Control TNF IL- TNF + IL- RANTES 2 M TNF-α IFN-γ IL-β M TNF-α IFN-γ IL-β M TNF-α IFN-γ IL-β Reltive expression Glut4 # Reltive expression M TNF-α IFN-γ IL-β elt (om-s..) /CD IL- onentrtion (ng ml ) Glut4 (fol hnge) TNF-α Tregs TNF No TNF BMI R 2 =.29 Figure 6 Cytokine effets on ipoytes n humn orreltes. () Expression of IL-6, MMP-3, SAA-3 n RANTES, s mesure with quntittive PCR uner unmnipulte ulture onitions (ontrol); ipoytes were trete with TNF- (TNF), IL- (IL-) lone or TNF- n IL- (TNF + IL-). () Reltive expression of IL-6 in ifferentite ipoytes, ose response urve of IL-. TNF, ells trete with TNF- n vrious onentrtions of IL-. No TNF, ells trete only with IL-. Representtive experiments re shown. () Expression of SAA-3, RANTES, IL-6 n Glut4 in ifferentite ipoytes, unmnipulte ifferentite ipoytes (M) or ipoytes trete with TNF-, IFN-g n IL-. Representtive experiments re shown. () Expression of Glut4 in ifferentite ipoytes either unmnipulte or trete with TNF- in presene or sene of spleen T reg ells. Men ± s.. of three inepenent experiments re shown. P ¼.6. P vlue ws lulte with t test. (e) Pire humn omentl n suutneous ipose smples from mostly oese iniviuls (BMI rnge: , verge: 44.85). Expression of FOXP3 n CD3 ws mesure y quntittive PCR. Left, the rtios of FOXP3 versus CD3 for omentl n suutneous ipose tissue. Right, the erese in FOXP3/CD3 rtio in omentl (om) versus suutneous ipose tissue plotte ginst the BMI for eh iniviul onor from the left grph. The positive vlue of sujet 7 (42 s..from the men) ws not inlue. Eh ot represents n iniviul onor. P ¼.86. Error rs represent mens ± s.. (Fig. 5f), lthough fsting loo gluose onentrtions t this erly time point were unhnge, onsistent with equte et ell ompenstion (Fig. 5f). To provie itionl eviene tht the inflmmtion promote y T reg ell epletion in the ominl ipose tissue ws not merely response to ell eth, we use mie 38 in whih we rpily lte T reg ells y giving iphtheri toxin to femles rrying gene enoing DTR knoke into the lous on the X hromosome. This strtegy yiele 95% ltion in homozygous mie versus out 5% ltion in heterozygous mie (Supplementry Fig. 7). In the ltter se, rnom X hromosome intivtion protets out hlf of the T reg ells euse the DTR-rrying llele resies within the intivte hromosome. As expete, when most of the T reg ells were lte, proinflmmtory trnsripts (for exmple, RANTES n MCP-) were strongly inue in the ft tissue (Supplementry Fig. 7). In ontrst, when only hlf the T ells were kille, there ws no suh inution of proinflmmtory trnsripts, signifying tht sustntil iphtheri toxin inue ell eth ws not in n of itself the stimulus n tht hlf-omplement of T reg ells suffie to protet the ft from inflmmtion. A gin-of-funtion experiment involving trnsfer of ft-resient T reg ells into reipient fe high-ft iet oul potentilly further support for our proposl tht these ells re key meitors of metoli regultion. However, the lility n low reoverle numers of viserl ft resient T reg ells renere our mny ttempts t this pproh unsuessful; trnsfer of more limite numers of ft-erive T reg ells into lymphoefiient reipients lso prove prolemti euse the resultnt homeostti prolifertion ltere the phenotype of the introue popultion, notly its profile of ell-surfe homing reeptors (t not shown). Therefore, s n NATURE MEDICINE ADVANCE ONLINE PUBLICATION 7

8 29 Nture Ameri, In. All rights reserve. lterntive mens to hieve gin-of-funtion, we turne to in situ expnsion of T reg ells vi injetion of omplex onsisting of reominnt IL-2 n prtiulr IL-2 speifi monolonl ntioy (ma), previously shown to seletively eliit T reg ells 47,48. Dily injetions of the omplex for 6 into mie prefe on for 5 weeks i sustntilly inrese the frtion of T reg ells in the spleen n ominl ft s ompre with PBS-injete ontrols (verges from multiple experiments 37% ± 4% versus 2% ± 2% for spleen n 63% ± 2% versus 43% ± 7% for ominl ft; Fig. 5g). (The reution in the T reg ell frtion in PBS-injete -fe mie versus mie fe norml how is smller in Fig. 5 thn in Fig. 4 euse of the shorter time of.) To ssess insulin resistne in the omplex-injete mie, we mesure loo gluose onentrtions, whih were signifintly lower (Fig. 5h). As expete, IL- trnsript levels were signifintly higher in the -fe mie with more T reg ells (Supplementry Fig. 6). Wheres HOMA-IR ( stnr mesure of insulin resistne tht integrtes oth loo-gluose n loo-insulin onentrtions; Fig. 5i) n gluose tolerne (mesure vi n intrperitonel GTT; Fig. 5j) ll trene towr lower vlues in the T reg ell enrihe, -fe mie, these ifferenes fell short of sttistil signifine, proly owing to the greter experimentl vriility inherent in these ssys. In ft, given the very short experimentl winow tht we h to work in to voi expnsion of effetor T ells, we o not fin smll ifferenes t ll surprising. To enhne the power of the experimentl t, we injete numer of itionl -fe mie with the IL-2 se omplexes or PBS uner similr onitions, umulting totl of mie for eh group. The T reg ell frtion in the omplex-injete, -fe mie rnge from 4% to 83% (verge 68% ± 3%), wheres tht in the PBS-injete mie spnne 8% to 7% (verge 45% ± 3%). Both -fe groups were gluose intolernt s ompre with ontrol mie fe norml how; however, the omplex-injete group, with the highest levels of T reg ells, showe signifint improvement ompre with the PBSinjete group (Fig. 5j). We otine similr results in the A y / moel (Supplementry Fig. 8). Together, the loss-of-funtion n gin-of-funtion finings inite tht T reg ells gur ginst exessive inflmmtion of the ipose tissue n its ownstrem systemi onsequenes n strongly suggest tht T reg ells resiing in the ft re responsile. In vitro effets of T ell-erive ytokines on ipoytes A prole mehnism y whih T ells resiing in ipose tissue ffet neighoring ells is through solule meitors. Thus, we explore the influenes of the mjor ytokines ifferentilly proue y T reg n T onv ells in ft versus other tissues (IL- n IFN-g, respetively; Fig. 2). We pretrete fully ifferentite, lipi-len 3T3-L ipoytes for 48 h with IL- (or left them untrete) n susequently stimulte them for 24 h with TNF-, n estlishe metho for in vitro inution of insulin resistne. TNF- inue hnges in ipoyte expression of numer of trnsripts enoing inflmmtory meitors, for exmple IL-6, RANTES, SAA-3 n MMP-3; of note, IL- inhiite the TNF--inue expression of ll of these RNAs (Fig. 6,). TNF- hs lso een shown to ownmoulte insulin-epenent tyrosine phosphoryltion of insulin reeptor sustrte- n to inhiit gluose uptke meite y the gluose trnsporter Glut4 in 3T3-L ipoytes; these effets, too, were reverse y IL- (ref. 8), initing tht this ytokine reverts insulin resistne y mehnism iretly impinging on ipose tissue ells. In ontrst to the nti-inflmmtory effets of IL- me y viserl ft-resient T reg ells, mjor prout of the T onv ells t this site, IFN-g, ws proinflmmtory in the sme in vitro ssy system, s expression of SAA-3, RANTES n IL-6 trnsripts ws inue, n Glut4 RNA ws ownregulte (Fig. 6). Our mny ttempts to support these onlusions y performing oultures of ft-erive T reg ells n 3T3-L preipoytes hve so fr een unsuessful owing to rpi eth of the T reg ells uring the inution perio, proly s result of propoptoti ftors either in the meium or proue y the ipoytes, suh s TNF-. However, spleen T reg ells were less frgile in these oulture onitions, n we oul show tht they mpen levels of proinflmmtory trnsripts me y tivte ipoytes in ulture n, perhps most relevnt, inhiit their ownmoultion of Glut-4 trnsripts (Fig. 6 n t not shown). The greter frgility of ft-erive versus spleenerive T reg ells my e expline y the former ells higher expression of TNF reeptors (t not shown). FOXP3 trnsripts re reue in oese humn omentl ft Finlly, we sought to trnslte our finings to humn pthology. We h ess to set of pire frozen omentl n suutneous ft tissues from numer of iniviuls with n verge oy mss inex (BMI) of 44.85, thus (exept for one se) flling within the oese (3 39.9) n morily oese (44) rnge (humn sujet hrteristis given in Supplementry Tle 5). Given tht the smples were frozen, we oul not perform flow ytometri nlysis on or purifition of lymphoyte popultions, ut we oul etermine FOXP3 trnsript levels y PCR. FOXP3 RNA ws reily etetle in oth ft epots (Fig. 6e). Consistent with the oservtions on oese mie, we foun higher levels of FOXP3 trnsripts, presumly n initor of T reg ells, in the suutneous ipose tissue (Fig. 6e). We i not hve ess to nonoese ontrols for these stuies, owing to the rrity of ritri surgery on norml iniviuls. However, we i fin orreltion etween BMI n the rop in T reg ells in omentl versus suutneous ft (Fig. 6e). These t suggest tht our finings on mie my e trnsltle to humns, whih is enourging for future, more sophistite nlyses on purifie T ell susets from fresh ipose tissue. DISCUSSION Fining wht ppers to e unique popultion of regultory T lymphoytes enrihe in the ominl ipose tissue of norml, ut not oese, mie rises numer of questions. First, why n how o T reg ells umulte t this site? One ftor my e ntigen stimultion, s suggeste y the imprint of ntigeni seletion on the TCR repertoire of viserl ft T reg ells n y their unusully high stte of tivtion. Depening on the lotion of ny suh ntigens, they oul stimulte irulting T reg ells, provoking them to exit the lymph noes n inve the ft n/or restimulte T reg ells filtering through ipose tissue, therey promoting their retention. This notion is further supporte y the sequening n the gene-expression profiling t, whih rule out the possiility tht T onv ells re inue to onvert to T reg ells in the viserl ft. A seon ftor is lmost ertinly hemokines, given the unique pttern of hemokine hemokine reeptor gene expression y the T reg ells isolte from ipose tissue. Funtionl stuies will e neee to unrvel whih of these moleules re inee involve in ell ttrtion or ess to ft, their loliztion one therein or their reruitment of other leukoyte susets. Thir, there my e role for ipokines in nurturing T reg ell survivl in ipose tissue. A reent stuy highlighte the negtive effet of leptin on the prolifertive pity of T reg ells 37, whih fits well with their opposite proportions in norml n oese ft: mny T reg ells n little leptin in len ft tissue n few T reg ells n muh 8 ADVANCE ONLINE PUBLICATION NATURE MEDICINE

9 29 Nture Ameri, In. All rights reserve. leptin in oese ft. In ontrst, given the prllel high levels of T reg ells n iponetin in norml ft n low levels in oese ft, this ipokine is possile nite for positive ftor, espeilly s it inues IL- synthesis, t lest y mrophges 49,5. Next, wht funtion re T reg ells performing in norml ominl ipose tissue? Reent reports hve highlighte the interply etween ipoytes n popultion of nti-inflmmtory mrophges in this ft epot, suggesting role for resient mrophges in promoting tissue repir n ngiogenesis n in mintining insulin sensitivity 8,9. Our results rgue tht the tivities of oinient popultions of T onv n T reg ells nee to e e to the mix. It my e relevnt tht the T onv ells seem to e mking n ongoing T helper type response to some stimulus n tht their mjor prout, IFN-g, promotes synthesis of inflmmtory meitors y ipoytes. The T reg ells my e keeping this response in hek, s well s regulting the tivities of their mrophge n ipoyte neighors inee, their physil lotion in rown-like strutures t ipoyte juntions woul enourge intertion with oth ell types. IL- is one nite for hving role in T reg ell meite regultory tivities, given its ssoition with improve insulin sensitivity in numer of ontexts in oth roents n humns 5 53, lthough this ytokine is lso proue y the ft-resient nti-inflmmtory mrophges 8. Although it ws not fesile to more efinitively estlish the speifi funtion of T reg ells resiing in ipose tissue, given tht no ftresient T reg ell speifi regent exists n tht it hs not een possile to suessfully isolte n trnsfer ft-erive T reg ells in the requisite quntities, role for T reg ells, whtever their soure, in metoli homeostsis n its ysregultion in oesity is n unexpete fining. Lstly, wht provokes T reg ells to vte ominl ft in oesity, or, perhps more likely, to refrin from entering it? It my e seonry effet. Inresing iposity hs een ssoite with n influx of mrophges 6,7, in prtiulr the inflmmtory mrophge suset 8,9, into the ominl epot; s well s with inrese lol n systemi onentrtions of inflmmtory ytokines suh s TNF- n IL-6. In ition, reiprol inrese n erese in the ipokines leptin n iponetin, respetively, hs een reporte, fueling speultion of extensive rosstlk etween the two ell types in the oese onition. One possile senrio, then, is tht elements of this hnging environment re unfvorle for T reg ell entry, expnsion or survivl, leing to seonry eline of this regultory popultion; in ft, leptin 37 n IL-6 (refs. 54,55) re lrey known to hve suh properties. However, it is lso possile tht loss of T reg ells from the ominl ft is the primry effet. Wheres their exggerte numers in len ipose tissue my hve een suffiient to keep the hroni inflmmtion uner ontrol, lower T reg ell numers with inresing iposity oul open the gte to n invsion of inflmmtory mrophges n therey more roust synthesis of inflmmtory ytokines. Whether the influx of mrophges or the efflux (or eth) of T reg ells proves upstrem, oth proesses must e ownstrem of n inititing event, s yet unefine, though suggestions hve inlue lol hypoxi 56, inrese ipoyte eth 2 n ipoyte stress 57. METHODS Methos n ny ssoite referenes re ville in the online version of the pper t Aession oes. Mirorry t hve een eposite in the Gene Expression Omnius with ession oe GSE7852. Note: Supplementry informtion is ville on the Nture Meiine wesite. ACKNOWLEDGMENTS We thnk D. Littmn (New York University) for the DTR onstrut, L. Roser n K. Httori for ssistne with mie, S. Ruensky (Memoril Slon Kettering Cner Center) for proviing us with DTR mie, J. LVehio n G. Buruzl for flow ytometry n J. Hill, J. Perez n R. Melme for help with the mirorry nlysis. This work ws supporte y Young Chir funs to D.M. n C.B., y the US Ntionl Institutes of Helth (DK5729 n DK73547) n Aler Chir funs to S.S. n y Joslin s Ntionl Institutes of Dietes n Digestive n Kiney Diseses fune Dietes n Enorinology Reserh Center ore filities. Postotorl fellowship support for M.F. ws from the Germn Reserh Fountion (Emmy-Noether Fellowship, FE 8/-) n the Chrles A. King Trust Postotorl Fellowship, n for L.H. from the Ministry of Siene of Spin. J.W. n D.C. were supporte y preotorl fellowships from the US Ntionl Institutes of Helth (T32 DK726) n the Europen Shool of Moleulr Meiine, respetively. Pulishe online t Reprints n permissions informtion is ville online t reprintsnpermissions/.. Shoelson, S.E., Lee, J. & Golfine, A.B. Inflmmtion n insulin resistne. J. Clin. Invest. 6, (26). 2. Hotmisligil, G.S., Shrgill, N.S. & Spiegelmn, B.M. Aipose expression of tumor nerosis ftor-: iret role in oesity-linke insulin resistne. 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