CD1a-autoreactive T cells are a normal component of the human T cell repertoire

Transcription

1 -utorective T cells re norml component of the humn T cell repertoire Annemieke de Jong 1, Victor Peñ-Cruz 2, Tn-Yun Cheng 1, Rchel A Clrk 3, Ildiko Vn Rhijn 1,4 & D Brnch Moody 1 21 Nture Americ, Inc. All rights reserved. CD1 ctivtes T cells, ut the function nd size of the possile humn T cell repertoires tht recognize ech of the CD1 ntigenpresenting molecules remin unknown. Using n experimentl system tht ypsses mjor histocomptiility complex (MHC) restriction nd the requirement for defined ntigens, we show tht polyclonl T cells responded t higher rtes to cells expressing thn to those expressing CD1, CD1c or CD1d. Unlike the repertoire of invrint nturl killer T (NKT) cells, the utorective repertoire contined diverse T cell ntigen receptors (TCRs). Functionlly, mny -utorective T cells homed to skin, where they produced interleukin 22 () in response to on Lngerhns cells. The strong nd frequent responses mong geneticlly diverse donors define -utorective cells s norml prt of the humn T cell repertoire nd s trget of the T H 22 suset of helper T cells. Four memers of the humn CD1 system,, CD1, CD1c nd CD1d, ind nd present diverse lipids from mmmlin cells nd cteri. It is therefore thought tht the CD1 system llows T cells to survey CD1-expressing ntigen-presenting cells (APCs) for chnges in lipid content cused y infection, inflmmtion or mlignncy. This view is supported y severl types of experimentl studies. A prticulr supopultion of CD1-restricted T cells, known s invrint nturl killer T cells (NKT cells), ws discovered on the sis of their expression of conserved T cell ntigen receptors (TCRs) with limited vrile region β-chins (V β ) 1 or α-chins (V α ) 2,3. CD1d tetrmers specificlly ind the invrint TCRs of NKT cells, so the TCR serves s defining surfce mrker tht llows effector functions to e trcked nd mesured on single-cell or popultion sis 4 7. Also, germline deletion of CD1d or the joining segment of the invrint V α chin llows selective deletion of NKT cells 8,9, so the influence of NKT cells in mouse models of utoimmunity, infection, tumor growth nd other diseses hs een rodly ssessed 1. These pproches hve led to the conclusion tht invrint NKT cells re preprimed memory T cells tht secrete lrge mounts of interferon-γ () nd work directly or through modultion of NK cells or dendritic cells (DCs) to control infection nd promote tumor regression 1. However, for the reminder of CD1drestricted T cells tht do not express the invrint TCR or for T cells tht recognize the CD1 proteins in group 1 (, CD1 nd CD1c), there re no rodly useful cell surfce mrkers or genetic mens of deletion. Therefore, most insight into the immunologicl functions of diverse CD1-restricted T cells is limited to oservtions of long-term T cell clones. The evolution of gene fmilies encoding CD1 proteins in mmmls suggests tht humn, CD1 nd CD1c proteins nd their nonhumn orthologs hve importnt roles in the immune response. Although muroid rodents lck orthologs of, CD1 or CD1c, nerly ll mmmlin genomes hve one or more genes encoding, nd no mmmlin species is known to hve survived without genes encoding CD1 proteins The retention of lrge gene fmilies encoding CD1 proteins in most mmmls suggests tht ech type of CD1 protein hs nonredundnt nd physiologiclly importnt role 14. Tht evolutionry hypothesis is supported y moleculr nd cellulr evidence showing tht, CD1, CD1c nd CD1d differ in their trnscriptionl regultion 15, ptterns of tissue expression 16, sucellulr trfficking 17 nd ntigen groove size 18. Whether the differences in tissue distriution or cell iology of ech CD1 protein trnslte into functionl differences in the responding T cell popultions is unknown. Two decdes fter the identifiction of humn CD1-utorective T cells 19, nerly ll insight into their functions hs een inferred from severl T cell clones or lines rther thn direct mesurement of polyclonl T cells from lood nd tissues. Clonl nlysis hs een successful in ssigning certin unchnging spects of phenotype, such s TCR structure, expression of the CD4 nd CD8 coreceptors 2,21, ntigens recognized 22 nd moleculr mechnisms of ctivtion 18,23. However, long-term culture of humn T cells promotes the selective outgrowth of clones with in vitro growth dvntges tht re proly not representtive of in vivo repertoires. This longstnding nd sic gp in knowledge persists ecuse of limittions of even the est ville regents for 1 Division of Rheumtology, Immunology nd Allergy, Brighm nd Women s Hospitl, Hrvrd Medicl School, Boston, Msschusetts, USA. 2 Dn-Frer Cncer Institute, Boston, Msschusetts, USA. 3 Deprtment of Dermtology, Brighm nd Women s Hospitl, Hrvrd Medicl School, Boston, Msschusetts, USA. 4 Deprtment of Infectious Diseses nd Immunology, Fculty of Veterinry Medicine, Utrecht University, Utrecht, The Netherlnds. Correspondence should e ddressed to D.B.M. (moody@rics.wh.hrvrd.edu). Received 26 Mrch; ccepted 3 Septemer; pulished online 31 Octoer 21; doi:1.138/ni VOLUME 11 NUMBER 12 DECEMBER 21 nture immunology

2 21 Nture Americ, Inc. All rights reserved. trcking fresh polyclonl T cells, especilly CD1-utorective T cells. Autorectivity is key feture of CD1-restricted T cells tht proly represents their ility to recognize endogenous lipid ntigens 19, Lipid self ntigens hve een identified 22, ut the extent to which ny one ntigen cn e used to trck the lrger pool of utorective T cells is unknown. In ddition, trcking of utorective T cells with nturl ntigens loded onto CD1 tetrmers hs een limited, ecuse wek self gonists generlly do not relily confer dequte vidity to llow stining of TCRs 28. We sought to overcome those prolems y designing system in which the cells provide diverse pool of lipid ntigens for loding onto CD1. For the study of geneticlly diverse humns, second design considertion ws to minimize or remove the normlly strong llorective responses to mjor histocomptiility complex (MHC) clss I nd II proteins tht would confound CD1-rective responses. We trnsfected plsmids encoding humn CD1 molecules into humn myelogenous leukemi cells (K562 cells) tht hve low to sent expression of MHC 29,3, which resulted in APCs tht cn e universlly used with MHC-mismtched sujects. These cells presumly express wide rry of self lipid ntigens, which llows rod detection of CD1 utorectivity without prior knowledge of ntigen structures. This method detected stronger nd more frequent responses to thn to ny other humn CD1 ntigen-presenting molecule. Consistent with the hypothesis tht ech type of CD1 protein hs distinct immunologicl functions, we found tht -utorective T cells were fundmentlly different from CD1d-rective invrint NKT cells on the sis of their TCR ptterns nd effector functions. Furthermore, -utorective T cells expressed skin-homing mrkers nd could e isolted from the skin nd ctivted y epithelil -expressing Lngerhns cells (LCs). Our studies identify -utorective cells s suset of the HLA clss I HLA-DR relese (1 3 c.p.m.) DDM (ng/ml) humn T cell repertoire nd define s trget of cells of the T H 22 helper T cell suset, which suggests new model of LC T cell interctions in epithelil homeostsis. RESULTS -utorective T cells in peripherl lood CD1 utorectivity is defined s the specific recognition of CD1 in the sence of exogenous lipid ntigen. Studies of CD1d-rective NKT cells nd -, CD1- nd CD1c-restricted T cell clones suggest tht utorectivity involves the recognition of n endogenous lipid ntigen presented y CD1 protein 19,31,32. To study humn CD1-rective T cells, we developed system tht cn mesure the CD1 utorectivity of nonclonl T cell popultions ex vivo. Using K562 cells with low surfce density of MHC proteins, we trnsfected genes encoding, CD1, CD1c or CD1d; this resulted in high surfce expression of these proteins (Fig. 1). APCs trnsfected with n empty vector control (mock trnsfection) ctivted rndom donor humn T cells t frequency of pproximtely 1 in 1, (Supplementry Fig. 1), which confirmed the low MHC-llorective potentil of these APCs. We confirmed the T cell ctivting function of ech trnsfected CD1 molecule y the presenttion of known exogenous lipid ntigens to CD1-restricted control T cell lines (Fig. 1). To determine whether CD1-restricted T cells cn e detected in humn peripherl lood, we nlyzed lood from 14 lood donors. After one or two in vitro stimultion(s) with utologous DCs expressing, CD1, CD1c or CD1d, we ssessed the CD1 rectivity of polyclonl T cells y enzymelinked immunospot (ELISPOT) ssy of secretion, using CD1- trnsfected K562 cells s APCs (Fig. 1c). We considered donor T cells to e CD1 utorective when the numer of spots generted fter contct with CD1-trnsfected K562 cells ws significntly greter thn the ckground response to mock-trnsfected K562 cells, s ssessed y 8 K562-CD1 8 K562-CD1c K562-CD1d relese (1 3 c.p.m.) CD1 CD1c CD1d K562-CD1 K562-CD1c K562-CD1d CD1 CD1c CD1d GMM (ng/ml) c Spots (per cells) relese (1 3 c.p.m.) MPM (ng/ml) relese (1 3 c.p.m.) α-glcer (pg/ml) CD1 CD1c CD1d Figure 1 Popultion study of CD1-utorective cells in lood of humn donors. () Surfce expression of MHC nd CD1 on mock-trnsfected K562 cells () or K562 cells trnsfected with plsmids contining humn genes encoding vrious CD1 proteins (ove plots). Open histogrms, MHC or CD1 stining; filled histogrms, isotype-mtched control ntiody stining. Dt re representtive of three or more experiments. () Biossy of in superntnt of K562 cells incuted for 24 h with T cell lines recognizing nd dideoxymycoctin (DDM); CD1 nd glucose monomycolte (GMM); CD1c nd mnnosyl phosphomycoketide (MPM); or CD1d nd α-glctosylcermide. Dt re representtive of three or more experiments (men ± s.d.). (c) ELISPOT nlysis of relese y polyclonl cell cultures stimulted in vitro with utologous DCs nd nlyzed for CD1 rectivity, with K562-CD1 cells s APCs; results presented fter sutrction of ckground spots formed in response to mock-trnsfected K562 cells. Ech symol represents n individul donor (men of triplicte mesurements; n = 14 donors totl); smll horizontl lines indicte the men for the group. P <.1 (Dunnett s multiple-comprison test fter one-wy ANOVA). nture immunology VOLUME 11 NUMBER 12 DECEMBER

3 relese (1 3 c.p.m.) 3 2 Donor 2 Donor 5 Donor 15 Donor 19 No ntiody Anti- Control IgG 1 relese (1 3 c.p.m.) T cell clone BC2cl7 BC4cl45 BC5cl4 BC5cl71 BC5cl94 BC6cl35 BC6cl61 BC1cl159 BC23cl19 BC23cl37 BCTcl81 Bgpcl12 BC2cl7 BC4cl45 BC5cl4 BC5cl71 BC5cl94 BC6cl35 BC6cl61 BC1cl159 BC23cl19 BC23cl37 BCTcl81 Bgpcl12 c -rective clones Clones tested (per donor) Figure 2 Autorective T cells in the lood recognize. () Biossy of relese from polyclonl T cell cultures incuted for 24 h with mock-trnsfected K562 cells () or with cells lone (No Antiody) or cells pretreted with -locking ma (Anti-) or isotype-mtched control ntiody (Control IgG). Dt re representtive of three or more experiments (men nd s.d.). () Biossy of relese y T cell clones incuted for 24 h with mock-trnsfected K562 cells or cells. Dt re representtive of two or more experiments with ech clone. (c) -utorective T cell clones in pnel of 1,291 utorective T cell clones generted from 14 donors y ex vivo limiting dilution, ech ssessed in triplicte for relese s in or y ELISPOT (when the numer of cells ws limited). Clones were considered utorective if cytokine secretion incresed more thn threefold in the presence of unlocked. 21 Nture Americ, Inc. All rights reserved. Student s t-test (P <.5). By this criterion, three of fourteen donors responded to CD1 nd only one of fourteen donors responded to CD1c or CD1d. The low responses were not due to the inility of K562 to ctivte responses through CD1, CD1c or CD1d, ecuse K562 cells efficiently stimulted CD1-, CD1c- or CD1d-restricted T cell lines tht were utorective (Supplementry Fig. 2) or ntigen specific (Fig. 1) In ddition, we found tht K562 cells trnsfected with plsmid encoding CD1 (K562-CD1 cells) presented C8 GMM (dt not shown), n ntigen presented only fter endosoml recycling 33 ; this indicted tht K562 cells cn survey endosoml ntigens. Notly, 14 of 14 donors responded to cells, nd the men responses were significntly greter thn those to CD1, CD1c or CD1d (P <.1, Dunnett s Multiple Comprison Test fter one-wy nlysis of vrince (ANOVA)). Unlike NKT cells, -utorective T cells hve not een descried t the polyclonl level 4,5, so we undertook series of nlyses imed t confirming the moleculr trget of recognition nd more ccurtely mesuring the frequency of responding cells. In suset of five donors, we expnded popultions of responding T cells in the presence of DCs nd tested the restriction of the resulting shortterm T cell lines with cells in the presence or sence of -locking monoclonl ntiody (ma). In ll cses, the responses required on the APCs nd were locked to ckground expression with ma to (Fig. 2), which indicted tht the responses were dependent on nd not on other cell surfce determinnts. Next we nlyzed clonl precursor frequency ex vivo in seprte group of 14 donors using utologous DCs s stimultor cells. We generted pnel of 1,291 T cell clones tht detectly proliferted in response to utologous DCs nd determined wht percentge of utorective T cell clones secreted interleukin 2 () in response to the cells reltive to mock trnsfectnts (Fig. 2). We found tht 1 in every 5 utorective T cell clones, in smples from 12 of 14 donors, ws utorective (Fig. 2c). The first reported -utorective T cell clone expressed n αβ TCR nd lcked expression of the CD4 nd CD8 coreceptors 19, ut susequent exmples of -utorective T cell clones hve shown CD8 expression 34. Among the 12 clones tht we were le to unmiguously nlyze for coreceptor expression, we found 11 CD4 + single-positive clones, 1 CD8 + single-positive clone nd no CD4 CD8 clones. Invrint NKT cells tht recognize CD1d re defined y TCR α-chins tht contin V α 24 nd α-chin joining region 18, ut it is unknown whether T cells tht recognize re conserved or diverse, ecuse only two V α nd V β pirs hve een reported 27. Sequencing of the TCR α- nd β-chins showed tht none of the -rective clones expressed the invrint V α 24 (TRAV1) or V β 11 (TRBV25) found on humn NKT cells. Although severl utorective clones shred the sme vrile regions nd even joining regions in the TCRα or TCRβ chin, none of the clones expressed identicl complementrity-determining region 3 (CDR3) sequences (dt not shown). We conclude tht -utorective T cells re Spots (per T cells) K562 Anti- Control IgG Frequency Donor 3 Spots (per T cells) Donor 6 Spots (per T cells) Donor 7 Spots (per T cells) Donor 9 Spots (per T cells) Donor 11 1/1,951 1/254 1/584 1/1,17 1/1,818 1/313 1/6, 1/237 Spots (per T cells) Donor 12 Spots (per T cells) Donor 13 Spots (per T cells) Donor 15 Figure 3 Quntittive detection of -utorective memory T cells. ELISPOT ssy of -secreting cells mong CD45RO + T cells incuted with mock-trnsfected K562 cells (m) or with cells preincuted for 1 h with ma to or control IgG. Precursor frequency (ottom row): men numer of spots in response to cells plus control IgG minus men numer of spots in response to cells plus ma to /totl cells per well. P <.5 (two-tiled Student s t-test). Dt re presented s men nd s.d. of triplicte mesurements. 114 VOLUME 11 NUMBER 12 DECEMBER 21 nture immunology

4 21 Nture Americ, Inc. All rights reserved. common in the peripherl lood of humns nd do not hve the highly conserved TCR sequences seen in invrint NKT cells. Becuse -utorective T cells were undnt in these cultures, we hypothesized tht they might e preprimed T cell popultion tht ws detectle without expnsion y utologous DCs. By ELISPOT ssy, we detected responses to cells in purified CD45RO + memory T cells in eight of eight rndom donors (Fig. 3), nd ntiody-locking experiments showed tht ws necessry nd sufficient for the response in ll cses. On the sis of the frction of the response locked y ma to reltive to tht locked y control immunogloulin G (IgG), we estimted the frequency of -utorective T cells mong peripherl lood memory T cells to e etween.2% nd.4%. However, this could hve een n underestimte, given tht not ll -rective T cells my produce. In greement with our prior nlysis with DC-stimulted peripherl lood mononucler cells (PBMCs; Fig. 1c), this ex vivo nlysis of unexpnded cell popultions showed tht T cell responses to were of greter mgnitude thn those to other isoforms of CD1 (Supplementry Fig. 3). Collectively, these results showed tht -utorective T cells were undnt in the lood without immune stimultion nd were present in most or ll donors, so they cn e considered to constitute suset of the norml humn αβ T cell repertoire. Homing of -utorective T cells to the skin The expression of in humns is restricted to thymocytes, myeloid DCs nd LCs. In the periphery, is found predominntly in the skin, where epiderml LCs express t extremely high cell surfce density 35. Given their high cellulr density nd lrge surfce re 36, LCs form nerly contiguous network of high-density contining memrnes in the epidermis 37. The locliztion of in the skin led us to hypothesize tht -utorective T cells in the peripherl circultion might normlly home to nd loclize in the skin. We first determined whether -utorective T cells recovered from the lood expressed skin-homing mrkers. Becuse the surfce expression of homing mrkers is rpidly ltered during in vitro culture 38, we sorted peripherl lood T cells ex vivo immeditely fter isoltion on the sis of their expression of the memory mrker CD45RO nd cutneous lymphocyte ntigen (CLA), crohydrte epitope tht identifies T cells tht prticipte in immune responses in the skin 39. For this experiment we took dvntge of the serendipitous finding tht donor 8 hd -utorective T cell clone tht ws isolted from ech of three smples, which indicted tht these cells hd undergone clonl expnsion in vivo. We confirmed the clonlity y the expression of identicl TCRα nd TCRβ chins in ech of the isoltes nd lso ecuse this clone hd two rerrnged TCRα chins, one of which hd CDR3 out of frme (dt not shown). After vlidting clonotypic primers for the α- nd β-chin of this TCR, we were le to mesure the presence of this -specific clone in the nive T cell frction (CD45RO CLA ), memory T cell frction (CD45RO + CLA ) nd skin-homing memory T cell frction (CD45RO + CLA + ) of the peripherl lood. We found PCR products for the clonotypic primers lmost exclusively in the CD45RO + CLA + frction (Fig. 4), which indicted tht in this donor, -rective T cells were CLA + memory T cells nd further suggested tht rective T cells cn e prt of the skin-homing T cell suset. -dependent production Pulished studies hve descried -utorective T cell clones minly in the context of pthologicl conditions ssocited with Events PBMC (monocyte depletion) CD3 CD45RO CLA 2 3 utoimmunity 31,32 or llergy 4. However, the detection of polyclonl T cells tht recognized mong mny humn lood donors suggested tht they fulfill some physiologicl function without immune provoction or dysregultion. Lcking precedent for possile effector functions ssocited with -utorective T cells, we initilly screened for the possile existence of repertoire using (Fig. 1) nd (Fig. 2), ecuse these re somewht promiscuously produced y T cell susets nd so incresed the chnce of discovering the repertoire. We next screened roder pnel of cytokines tht might provide hints t specific effector functions. We tested for cndidte cytokines ssocited with the T helper type 1 (T H 1), T H 2 nd IL-17-producing (T H 17) susets of the MHC-restricted repertoire, s well s cytokines produced y CD1d-restricted NKT cells. The dominnt cytokine normlly produced y NKT cells ctivted with synthetic or foreign ntigens is. However, utorective NKT cell responses to endogenous ntigens cn show roder profile, including grnulocyte-mcrophge colony-stimulting fctor nd in the sence of (refs. 1,41). We stimulted -rective T cell lines with cells nd mesured cytokine mrna y rel-time PCR. To ensure tht ws oth necessry nd sufficient to induce mesured responses, we compred the mrna produced in response to cells treted with control IgG with the mrna produced in response to cells treted with the -locking ma. Two of eight donors tested showed dominnt upregultion of lone or oth nd, wheres five other donors showed n unexpected pttern chrcterized y sustntil upregultion of (Fig. 5). We lso detected secretion of protein in dose-dependent mnner in response to (Fig. 5). We considered tht might e n importnt effector function of the -utorective repertoire ecuse this cytokine is normlly restricted to limited suset of T cells nd ecuse oth nd hve een suspected of hving roles in skin immunity. is memer of the IL-1 cytokine fmily nd cts on nonhemtopoietic epithelil cells, such s kertinocytes of the skin 42, where it triggers the production of ntimicroil peptides nd expression of genes involved in cellulr differentition nd survivl 43,44. It is therefore thought to e involved in erly host defense ginst microil pthogens nd in the homeostsis of epitheli. In mice, is often detected in T H 17 cells 45, ut humn cells known s -only (T H 22) helper T cells cn produce in the sence of IL-17 (refs ). In our polyclonl nlysis, the lck of sustntil -dependent upregultion of IL-17 mrna in most donors suggested tht these two Gte: CD45RO CLA + Clonotypic α-chin Clonotypic β-chin TRAC Figure 4 -utorective T cells in the lood express skin-homing mrkers. RT-PCR nlysis (right) of RNA extrcted from CD45RO CLA (gte 1) T cells, CD45RO + CLA (gte 2) T cells or CD45RO + CLA + (gte 3) T cells (sorting, left nd middle), with primers confirmed to e specific for the CDR3 regions of the TCR α- nd β-chins of -utorective T cell clone. TRAC, TCRα constnt region primers (control for input cdna). Dt re representtive of three experiments (TCR sequences) or two experiments (sorted T cells). + + nture immunology VOLUME 11 NUMBER 12 DECEMBER

5 21 Nture Americ, Inc. All rights reserved. Figure 5 -dependent production. () Rel-time PCR nlysis of the upregultion of cytokine-encoding genes in polyclonl utorective T cell cultures incuted for 6 h together with cells pretreted with ma to or control IgG t K562 cell/t cell rtio of 1:1; results were normlized to β-ctin, nd results for incution with cells plus control IgG re presented reltive to those for incution with cells plus ma to. () Enzyme-linked immunosorent ssy of in superntnts of T cell lines showing upregultion tht were incuted for 24 h lone (No APC) or with incresing numers of mock-trnsfected K562 cells or cells. (c) Intrcellulr stining of IL-17 nd in T cell lines from the donors in nlyzed without stimultion (Unstim) or in response to stimultion with PMA nd ionomycin. Dt for protein re representtive of two or more experiments with two donors (men ± s.d. of triplicte mesurements). cytokines re not coordintely upregulted in humn -utorective T cells (Fig. 5). 4 Single-cell intrcellulr cytokine stining 3 showed tht even strong stimulus such s the phorol ester PMA nd ionomycin did 2 not induce sustntil production of IL-17 1 in two -producing -utorective T cell lines (Fig. 5c), wheres the sme stimulus induced the production of oth cytokines in the T cell frction of totl PBMCs (dt not shown). These dt support the ide tht IL-17 nd represent effector molecules produced y distinct T cell popultion in humns nd tht -utorective cells re in the -producing suset. -producing T cell popultions sometimes hve polyfunctionl cytokine response, producing not only IL-17 ut lso, nd, in some cses, Although it ws not s prominent s, ws detectle in -utorective popultions (Figs. 1 nd 5), which might hve een produced either y T H 1-like -restricted cells or from clones tht produce oth nd We found tht set of -utorective clones, selected on the sis of their production of nd confirmed for clonlity y V β PCR nlysis, expressed either lone or oth nd in vrious rtios in response to (Supplementry Fig. 4). This provided direct evidence of dul cytokine producing utorective T cells t the clonl level nd suggested tht our erlier mesurements of -dependent responses t the polyclonl level proly reflected oth T H 1 nd T H 22-T H 1 cells 49. Skin-homing T H 22 cells recognize The production of is only one feture of n emerging definition of the T H 22 helper T cell suset, which is lso chrcterized y expression of the ryl hydrocron receptor 5 nd expression of the chemokine receptors CCR6, CCR4 nd CCR1, which promote skin homing 47,48. Although ntigen-presenting molecules or other moleculr trgets recognized y T H 22 cells re not well chrcterized, represents good cndidte ecuse it is locted in skin, where T H 22 cells normlly home. We found tht -restricted T cell lines expressed the ryl hydrocron receptor (Supplementry -dependent cytokine mrna (fold) -dependent cytokine mrna (fold) (pg/ml) (pg/ml) Donor 2 IL-1 Donor 9 IL-1 Donor 19 APC (1 3 ) APC (1 3 ) Donor 5 IL-1 Donor 15 IL-1 No APC No APC c IL-17 Donor PMA-ionomycin.3.1 Fig. 5). To directly ddress whether -utorective T cells represent prt of the T H 22 suset nd their reltive undnce in this suset reltive to tht of T H 1 or T H 17 cells, we sorted memory CD4 + T cells into the following frctions: CCR6 + CXCR3 + CCR4 CCR1, CCR6 + CXCR3 CCR4 + CCR1 nd CCR6 + CXCR3 CCR4 + CCR1 +. The cytokine profiles mesured y RT-PCR mtched pulished results otined y enzyme-linked immunosorent ssy 47,48 nd confirmed tht these three sorted popultions were enriched for T H 1, T H 17 nd T H 22 cells, respectively (Fig. 6). After single in vitro popultion expnsion with DCs, we detected twofold or greter -dependent upregultion of cytokine mrna in five of six donors. The frction ssocited with T H 22 cells (CCR6 + CCR4 + CCR1 + ) showed the gretest nd most frequent responses (Fig. 6 nd dt not shown). In ddition, similr to our previous nlysis of -utorective T cell cultures (Fig. 5), nd were the most dominntly upregulted cytokines in CCR4 + CCR6 + CCR1 + CD4 + T cells. Thus, -utorective T cells re found in the humn T H 22 suset, which defines s trget for T H 22 cells. -utorective responses from skin The expression of CLA nd skin-homing chemokine receptors on the surfce of -utorective T cells indictes tht these cells should e present in the skin. To ssess this, we isolted T cells from humn skin iopsy specimens nd ssessed recognition 51. Similr to results otined with lood-derived T cells (Fig. 2c), initil screens isolted skin T cell clones whose ctivtion ws locked y ma to (dt not shown). We next ssessed -dependent production of in polyclonl T cells (Fig. 7). In two of three smples, 1.2 Unstim Donor 7 IL-1 IL-1 Unstim Donor PMA-ionomycin IL-1 Donor 23 IL VOLUME 11 NUMBER 12 DECEMBER 21 nture immunology

6 21 Nture Americ, Inc. All rights reserved. PBMC Memory CD4 + T cells CCR6 + CXCR3 + CXCR3 CCR4 CCR1 -dependent cytokine mrna (fold) Donor Donor CCR4 + CCR1 CCR1 + T H 1 T H 17 T H 22 -dependent cytokine mrna (fold) Cytokine/β-ctin ( 1 4 ) CCR6 + CXCR3 + CCR6 + CXCR3 CCR6 + CXCR3 CCR4 CCR1 CCR4 + CCR1 CCR4 + CCR1 + T H 1 1. T H 17 3 T H lymphocytes isolted from norml humn dermis, followed y popultion expnsion with DCs, showed sustntil production in response to cells, ut not to K562-CD1 cells or K562- CD1c cells, which demonstrted the dominnce of -medited responses in helthy humn skin nd their ility to produce. Expnding on the ide tht -utorective T cells might hve role in skin immunity, we mesured the ility of -utorective T cells to recognize nturl -expressing APCs. We isolted LCs from epiderml sheets of humn skin nd compred them with K562- trnsfectnts s well s monocyte-derived DCs, which mimic mny fetures of myeloid DCs. All three cell types ctivted T cell lines in -dependent mnner, with the highest potency of ctivtion using LCs (Fig. 7). Similr to cells, LCs induced Cytokine/β-ctin ( 1 4 ) CCR6 + CXCR3 + CCR4 CCR1 T H 1 CCR6 + CXCR3 CCR4 + CCR1 + T H 22 Cytokine/β-ctin ( 1 4 ) Figure 6 The T H 22 suset contins -utorective T cells. () Reltime PCR nlysis of the cytokine profiles of memory CD4 + T cells sorted into CCR6 + CXCR3 + CCR4 CCR1, CCR6 + CXCR3 CCR4 + CCR1 nd CCR6 + CXCR3 CCR4 + CCR1 + frctions (fr left) nd stimulted for 6 h with nti-cd3. Dt re representtive of experiments with three donors. () Rel-time PCR nlysis of the upregultion of cytokine-encoding genes in sorted T H 1 (top) or T H 22 (ottom) T cell frctions tht underwent single in vitro expnsion with DCs nd were cultured for 6 h with K562- cells preincuted with ma to or control IgG, t K562 cell/t cell rtio of 1:1; results were normlized to β-ctin, nd results for cells plus control IgG re presented reltive to those for incution with cells plus ma to. considerle upregultion of in the sence of sustntil upregultion of either or IL-17 (Fig. 7c), which indicted tht the pttern of -dependent induction of cytokine-encoding genes ws similr when either K562 cells or freshly isolted LCs were used s stimultor cells. DISCUSSION More so thn the T cells of other orgns, T cells tht reside in the skin hve repertoires tht differ fundmentlly etween mice nd humns. For exmple, mouse skin contins lrge numers of γδ T cells with V γ 5 + V δ 1 + invrint TCRs, known s dendritic epiderml T cells, whose development requires Skint1, immunogloulin-like derml protein 52. Humn skin lcks Skint1 nd dendritic epiderml T cells nd insted contins predominntly T cells tht express rerrnged αβ TCRs 53,54. During the evolution of muroid rodents, rek in chromosome 1 resulted in loss of genes encoding CD1 proteins of group 1, so humn skin, ut not mouse skin, contins LCs tht express t very high density 13. These generl oservtions highlight two key resons for developing experimentl systems for studying skin T cell immunity in humns. First, the nturl functions of cnnot e studied in unmodified mice. Second, nd other receptors of the immune response, which nturlly differ etween these two species, represent cndidte molecules for controlling the dichotomously different orgniztion of skin T cell popultions seen in these species. Although overexpression of Notch results in production of in the sence of IL-17 (ref. 55), mice typiclly secrete in tndem with IL-17, nd such doule cytokine production is usully understood s hllmrk of the T H 17 helper T cell suset 56. Humn αβ T cells tht produce in the sence of IL-17 hve een frequently (ng/ml) Donor Mock CD1 CD1c Derml T cells Mock CD1 CD1c Mock CD1 CD1c relese (1 3 c.p.m.) LCs DCs LCs + nti- DCs + nti- + nti Donor APC 6 4 LC DC 12,722 1, c Cytokine mrna (fold) IL-17F Figure 7 producing -utorective T cells in humn skin. () Enzyme-linked immunosorent ssy of relese y lymphocytes otined from norml humn dermis smples, followed y popultion expnsion in vitro with DCs nd incution for 24 h with CD1-trnsfected K562 cells Dt re representtive of one experiment with three donors (men nd s.d.). () Biossy of in superntnts of freshly isolted LCs (left), monocytederived DCs nd cells incuted for 24 h with -utorective T cell line. Right, flow cytometry of on APCs; numers in plots indicte men fluorescence intensity (open histogrms, stining; filled histogrms, isotype-mtched control ntiody stining). Dt re representtive of three experiments (men nd s.d.). (c) Rel-time PCR nlysis of the upregultion of cytokine-encoding genes in -utorective T cell line incuted for 18 h together with freshly isolted LCs pretreted with control IgG or ma to ; results were normlized to β-ctin, nd results for incution with LCs plus control IgG re presented reltive to those for incution with LCs plus ma to. Dt re presented s men ± s.d. of triplicte mesurements. nture immunology VOLUME 11 NUMBER 12 DECEMBER

7 21 Nture Americ, Inc. All rights reserved. oserved nd re now considered the T H 22 helper T cell suset. Although the ntigen-presenting molecules or ny moleculr trgets recognized y T H 22 cells were unknown t the outset of this study, represented good cndidte ecuse it is sent in mice nd hs high expression in tissues enriched for T H 22 cells. Here we hve shown tht -utorective T cells hve ll of the known properties of T H 22 cells, including the ryl hydrocron receptor nd skinhoming molecules (CCR4, CCR1 nd CLA), therey identifying s moleculr trget of T H 22 cells. Determining the reltive contriution of nd MHC ntigen-presenting molecules in T H 22 cell ctivtion will e importnt for understnding nd intervening in skin immunopthology cused y in psorisis nd other skin diseses 46,49. More generlly, our dt hve shown tht popultions of -utorective T cells were undntly present in the lood nd skin of most humn donors tested, so we conclude tht they re norml prt of the humn αβ T cell repertoire. The experimentl system we hve generted is unised in the sense tht it is le to detect responses to ny CD1 protein, ut T cell ctivtion y dominted in mgnitude of response nd the percentge of people whose lood- nd skin-derived T cells responded. An ovious possiility tht would explin these findings is tht utorective T cells my e s common s or more common thn T cells tht recognize other CD1 isoforms in unchllenged humn hosts. The estimted precursor frequency for -utorective T cells mong memory T cells in the lood mesured here with expressing cells (.2% to.4%) is similr to tht found for humn NKT cells mesured with CD1d α-glctosylcermide tetrmers (undetectle to 1.%) 5,7. Tetrmer-sed detection of NKT cells is direct, ut this pproch necessrily requires priori knowledge of ntigens, mesures responses to only one ntigen nd typiclly uses the synthetic supergonist α-glctosyl cermide. The detection system reported here differs in tht it presumly mesures responses to diverse nd nturl cellulr self ntigens, specultion supported y dt showing tht lipid extrcts of K562 cells ctivte -utorective T cells (dt not shown). Given the incomplete understnding t present of the diversity of self ntigens for CD1 proteins, the fetures of this detection system re not only desirle ut lso necessry for glol mesurement of CD1 utorectivity ex vivo in humns. However, ecuse the detection method descried ove requires n ctivtion response, the numer of cells detected depends on the ctivtion threshold. Therefore, the second possiility for the detection of more -utorective cells is tht they hve lower threshold of ctivtion thn do T cells tht recognize other CD1 proteins. Becuse is generlly sent from or low in undnce in peripherl lood nd tissues other thn skin, contct of -utorective T cells with -expressing APCs is not expected to e common event in the lood or most other tissues. This sitution contrsts with tht for CD1d, which is expressed on the peripherl lood B cells, monocytes nd other cells in lood nd tissues 16. These considertions suggest model in which -rective T cells might hve low intrinsic resistnce to ctivtion. Their function my e controlled y their direct ccess to -expressing APCs in specilized tissue environments such s the skin. Three relted oservtions suggest tht recognition of occurs s mechnism of skin homeostsis: restriction of expression to the skin; homing receptors (CLA, CCR6, CCR4 nd CCR1) tht drive T cells to the skin; nd linkge of utorectivity to the T H 22 phenotype. Rther thn stimulting other hemtopoieticlly derived immune cells, cts on heterodimers of the receptors IL-1R2 nd R1 expressed on epithelil cells, including kertinocytes 42. Therefore, secretion of resulting from chronic interctions with -utorective TCRs on LCs might promote remodeling of the epithelil mtrix in seline stte without necessrily eliciting roder immune ctivtion or immunopthology 44. However, strong ctivtion of utorectivity is expected to cuse overproduction of, which promotes the cnthosis oserved in psorisis 57. Also, other cytokines relesed y -utorective cells (, nd ) cn promote immunopthology. These considertions drive ongoing efforts to understnd the regultion of such seline utorectivity. Our preliminry dt suggest tht -utorective T cells re present in dermis (s reported here, nd dt not shown), yet LCs loclize minly to the epidermis. Therefore, one model is tht these ner neighors cross the dermlepiderml junction to contct nd ctivte ech other. Also, skin is rich source of tissue-specific oils nd wxes tht might function s -presented lipid utontigens. Methods Methods nd ny ssocited references re ville in the online version of the pper t Note: Supplementry informtion is ville on the Nture Immunology wesite. Acknowledgments We thnk D.C. Brrl, M. Brenner nd M. Relloso (Hrvrd Medicl School) nd M. Sugit (Kyoto University) for CD1 plsmid constructs; J. Gumperz (University of Wisconsin) nd M. Brigl (Hrvrd Medicl School) for humn NKT cell lines; G. Losyev for cell sorting; nd K. Mglhães, S. Hung, I.C. Ho nd R. Grenningloh for technicl dvice. Supported y the Ntionl Institute of Arthritis, Musculoskeletl nd Skin Diseses (48632 to D.B.M. nd AR5672 to R.A.C.), the Ntionl Institute of Allergy nd Infectious Diseses (AI71155 to D.B.M., nd AI54456 nd AI56299), the Dmon Runyon Cncer Reserch Foundtion (R.A.C.), the Burroughs Wellcome Fund (D.B.M.) nd the Ntionl Psorisis Foundtion (A.d.J.). AUTHOR CONTRIBUTIONS A.d.J. designed nd did the experiments; A.d.J. nd D.B.M. prepred the mnuscript; D.B.M. supervised the experiments; V.P.-C. isolted LCs from humn epidermis nd lymphocytes from the dermis; T.-Y.C. did T cell culture nd immunolot nlysis; I.V.R. ssisted in experiments; nd R.A.C. provided T cells isolted from humn skin iopsies. COMPETING FINANCIAL INTERESTS The uthors declre no competing finncil interests. Pulished online t Reprints nd permissions informtion is ville online t reprintsndpermissions/. 1. Fowlkes, B.J. et l. A novel popultion of T-cell receptor α et-ering thymocytes which predominntly expresses single V β gene fmily. Nture 329, (1987). 2. Lntz, O. & Bendelc, A. An invrint T cell receptor lph chin is used y unique suset of mjor histocomptiility complex clss I-specific CD4 + nd CD4 8 T cells in mice nd humns. J. Exp. Med. 18, (1994). 3. Porcelli, S., Yockey, C.E., Brenner, M.B. & Blk, S.P. Anlysis of T cell ntigen receptor (TCR) expression y humn peripherl lood CD4 8 α/β T cells demonstrtes preferentil use of severl Vβ genes nd n invrint TCR α chin. J. Exp. Med. 178, 1 16 (1993). 4. Benlgh, K., Weiss, A., Bevis, A., Teyton, L. & Bendelc, A. In vivo identifiction of glycolipid ntigen-specific T cells using fluorescent CD1d tetrmers. J. Exp. Med. 191, (2). 5. Gumperz, J.E., Miyke, S., Ymmur, T. & Brenner, M.B. Functionlly distinct susets of CD1d-restricted nturl killer T cells reveled y CD1d tetrmer stining. J. Exp. Med. 195, (22). 6. Mtsud, J.L. et l. Trcking the response of nturl killer T cells to glycolipid ntigen using CD1d tetrmers. J. Exp. Med. 192, (2). 7. Krdimitris, A. et l. Humn CD1d-glycolipid tetrmers generted y in vitro oxidtive refolding chromtogrphy. Proc. Ntl. Acd. Sci. USA 98, (21). 8. Smiley, S.T., Kpln, M.H. & Grusy, M.J. Immunogloulin E production in the sence of interleukin-4-secreting CD1-dependent cells. Science 275, (1997). 9. Cui, J. et l. Requirement for V α 14 NKT cells in IL-12-medited rejection of tumors. Science 278, (1997). 118 VOLUME 11 NUMBER 12 DECEMBER 21 nture immunology

8 21 Nture Americ, Inc. All rights reserved. 1. Godfrey, D.I. & Kronenerg, M. Going oth wys: immune regultion vi CD1ddependent NKT cells. J. Clin. Invest. 114, (24). 11. Vn Rhijn, I. et l. The ovine CD1 fmily contins group 1 CD1 proteins, ut no functionl CD1d. J. Immunol. 176, (26). 12. Looringh vn Beeck, F.A. et l. Two cnine proteins re differentilly expressed in skin. Immunogenetics 6, (28). 13. Dscher, C.C. Evolutionry iology of CD1. Curr. Top. Microiol. Immunol. 314, 3 26 (27). 14. Ksmr, A., Vn Rhijn, I. & Moody, D.B. The evolved functions of CD1 during infection. Curr. Opin. Immunol. 21, (29). 15. Rour-Mir, C. et l. Mycocterium tuerculosis regultes CD1 ntigen presenttion pthwys through TLR-2. J. Immunol. 175, (25). 16. Dougn, S.K., Kser, A. & Blumerg, R.S. CD1 expression on ntigen-presenting cells. Curr. Top. Microiol. Immunol. 314, (27). 17. Sugit, M., Cernds, M. & Brenner, M.B. New insights into pthwys for CD1- medited ntigen presenttion. Curr. Opin. Immunol. 16, 9 95 (24). 18. Moody, D.B., Zjonc, D.M. & Wilson, I.A. Antomy of CD1-lipid ntigen complexes. Nt. Rev. Immunol. 5, (25). 19. Porcelli, S. et l. Recognition of cluster of differentition 1 ntigens y humn CD4 CD8 cytolytic T lymphocytes. Nture 341, (1989). 2. Rost, J.P. et l. CD1-restricted microil lipid ntigen-specific recognition found in the CD8 + αβ T cell pool. J. Immunol. 162, (1999). 21. Sieling, P.A. et l. Evidence for humn CD4 + T cells in the CD1-restricted repertoire: derivtion of mycocteri-rective T cells from leprosy lesions. J. Immunol. 164, (2). 22. Moody, D.B. The surprising diversity of lipid ntigens for CD1-restricted T cells. Adv. Immunol. 89, (26). 23. Borg, N.A. et l. CD1d-lipid-ntigen recognition y the semi-invrint NKT T-cell receptor. Nture 448, (27). 24. Shmshiev, A. et l. The αβ T cell response to self-glycolipids shows novel mechnism of CD1 loding nd requirement for complex oligoscchrides. Immunity 13, (2). 25. Shmshiev, A. et l. Presenttion of the sme glycolipid y different CD1 molecules. J. Exp. Med. 195, (22). 26. Sieling, P.A. et l. Humn doule-negtive T cells in systemic lupus erythemtosus provide help for IgG nd re restricted y CD1c. J. Immunol. 165, (2). 27. Vincent, M.S., Xiong, X., Grnt, E.P., Peng, W. & Brenner, M.B. -, -, nd c-restricted TCRs recognize oth self nd foreign ntigens. J. Immunol. 175, (25). 28. Zhou, D. et l. Lysosoml glycosphingolipid recognition y NKT cells. Science 36, (24). 29. Klein, E. et l. Properties of the K562 cell line, derived from ptient with chronic myeloid leukemi. Int. J. Cncer 18, (1976). 3. Britten, C.M. et l. The use of HLA-A21-trnsfected K562 s stndrd ntigenpresenting cells for CD8 + T lymphocytes in ELISPOT ssys. J. Immunol. Methods 259, (22). 31. Shmshiev, A. et l. Self glycolipids s T-cell utontigens. Eur. J. Immunol. 29, (1999). 32. Rour-Mir, C. et l. nd CD1c ctivte intrthyroidl T cells during Grves disese nd Hshimoto s thyroiditis. J. Immunol. 174, (25). 33. Moody, D.B. et l. Lipid length controls ntigen entry into endosoml nd nonendosoml pthwys for CD1 presenttion. Nt. Immunol. 3, (22). 34. Vincent, M.S. et l. CD1-dependent dendritic cell instruction. Nt. Immunol. 3, (22). 35. Meunier, L. et l. Quntifiction of, HLA-DR, nd HLA clss I expression on vile humn Lngerhns cells nd kertinocytes. Cytometry 26, (1996). 36. Yu, R.C., Arms, D.C., Alic, M. & Chu, A.C. Morphologicl nd quntittive nlyses of norml epiderml Lngerhns cells using confocl scnning lser microscopy. Br. J. Dermtol. 131, (1994). 37. Chu, A. et l. Immunoelectron microscopic identifiction of Lngerhns cells using new ntigenic mrker. J. Invest. Dermtol. 78, (1982). 38. Armerding, D. & Kupper, T.S. Functionl cutneous lymphocyte ntigen cn e induced in essentilly ll peripherl lood T lymphocytes. Int. Arch. Allergy Immunol. 119, (1999). 39. Fuhlrigge, R.C., Kieffer, J.D., Armerding, D. & Kupper, T.S. Cutneous lymphocyte ntigen is specilized form of PSGL-1 expressed on skin-homing T cells. Nture 389, (1997). 4. Age, E. et l. Humn CD1-restricted T cell recognition of lipids from pollens. J. Exp. Med. 22, (25). 41. Wng, X. et l. Nturl killer T-cell utorectivity leds to specilized ctivtion stte. Blood 112, (28). 42. Wolk, K. et l. increses the innte immunity of tissues. Immunity 21, (24). 43. Bonifce, K. et l. inhiits epiderml differentition nd induces proinflmmtory gene expression nd migrtion of humn kertinocytes. J. Immunol. 174, (25). 44. Wolk, K. & St, R. Interleukin-22: novel T- nd NK-cell derived cytokine tht regultes the iology of tissue cells. Cytokine Growth Fctor Rev. 17, (26). 45. Ling, S.C. et l. Interleukin (IL)-22 nd IL-17 re coexpressed y Th17 cells nd coopertively enhnce expression of ntimicroil peptides. J. Exp. Med. 23, (26). 46. Nogrles, K.E. et l. -producing T22 T cells ccount for upregulted in topic dermtitis despite reduced IL-17-producing TH17 T cells. J. Allergy Clin. Immunol. 123, (29). 47. Duhen, T., Geiger, R., Jrrossy, D., Lnzvecchi, A. & Sllusto, F. Production of interleukin 22 ut not interleukin 17 y suset of humn skin-homing memory T cells. Nt. Immunol. 1, (29). 48. Trifri, S., Kpln, C.D., Trn, E.H., Crellin, N.K. & Spits, H. Identifiction of humn helper T cell popultion tht hs undnt production of interleukin 22 nd is distinct from T H -17, T H 1 nd T H 2 cells. Nt. Immunol. 1, (29). 49. Eyerich, S. et l. Th22 cells represent distinct humn T cell suset involved in epiderml immunity nd remodeling. J. Clin. Invest. 119, (29). 5. Veldhoen, M. et l. The ryl hydrocron receptor links T H 17-cell-medited utoimmunity to environmentl toxins. Nture 453, (28). 51. Clrk, R.A. et l. A novel method for the isoltion of skin resident T cells from norml nd disesed humn skin. J. Clin. Invest. 126, (26). 52. Strid, J., Tigelr, R.E. & Hydy, A.C. Skin immune surveillnce y T cells new order? Semin. Immunol. 21, (29). 53. Foster, C.A. et l. Humn epiderml T cells predominntly elong to the linege expressing α/β T cell receptor. J. Exp. Med. 171, (199). 54. Clrk, R.A. et l. The vst mjority of CLA + T cells re resident in norml skin. J. Immunol. 176, (26). 55. Alm, M.S. et l. Notch signling drives secretion in CD4 + T cells y stimulting the ryl hydrocron receptor. Proc. Ntl. Acd. Sci. USA 17, (21). 56. Korn, T., Bettelli, E., Oukk, M. & Kuchroo, V.K. IL-17 nd Th17 Cells. Annu. Rev. Immunol. 27, (29). 57. Bonifce, K. et l. A role for T cell-derived interleukin 22 in psoritic skin inflmmtion. Clin. Exp. Immunol. 15, (27). nture immunology VOLUME 11 NUMBER 12 DECEMBER

9 21 Nture Americ, Inc. All rights reserved. ONLINE METHODS Antiodies. Detils of ntiodies used in flow cytometry nd neutrliztion ssys re in Supplementry Tle 1. CD1-expressing APCs. PBMCs were otined from lood donted t Msschusetts Generl Hospitl, s pproved y the Prtners Helthcre Institutionl Review Bord, nd were isolted from uffy cots y density centrifugtion over Ficoll Hypque. Monocyte-derived DCs expressing CD1 were isolted y dherence to plstic nd tretment for 72 h with grnulocytemcrophge colony-stimulting fctor (3 IU/ml) nd (2 IU/ml), followed y γ-irrdition (5, rds). K562 cells were trnsfected with pcdna3.1 or pcdna3 vector contining no insert or cdna encoding, CD1, CD1c or CD1d. The, CD1c nd CD1d constructs contined cdna encoding the hevy chin only, wheres the CD1 construct contined cdna encoding the CD1 hevy chin linked to humn β 2 -microgloulin. K562 clones were selected for high CD1 expression nd low expression of HLA clss I. Surfce expression of HLA-DR ws undetectle on ll clones. Polyclonl T cell cultures. Autologous monocyte-derived DCs expressing CD1 were cultured with nondherent cells t rtio of 1:5 in 96-well pltes (1 1 5 cells per well) in RPMI medium supplemented with 1% (vol/vol) FBS (Hyclone) nd 2% (vol/vol) humn AB serum (Gemini), essentil nd nonessentil mino cids (Gico), penicillin-streptomycin (Gico) nd β-mercptoethnol (complete medi). On dys 2 nd 7, (.2 nm; Chiron) nd IL-15 (5 ng/ml; Peprotech) were dded to the cultures, which were split when the wells ecme full. After 1 14 d, cells were tested for rectivity to CD1-trnsfected K562 cells. CD1-utorective T cells were mintined in culture y restimultion with utologous DCs. -utorective T cell clones. For ex vivo limiting dilution, 96-well pltes were seeded with 1 utologous DCs per well nd 1 nondherent cells per well. After 24 h, utologous irrdited PBMCs were dded (1 1 5 cells per well) in complete medium contining (.5 nm) nd IL-15 (2 ng/ml). Pltes were screened for clones fter 14 d nd ll clones were tested for rectivity to. The numer of replictes per clone depended on the ville mount of cells; rectivity ws mesured y secretion or ELISPOT, with the ltter generlly used when cells were limiting. The clonlity of cell cultures ws determined y PCR for TCR α- nd β-chins with primer pnel covering the TCR V regions (Interntionl Immunogenetics primer dtse) comined with constnt-region primers. For ex vivo limiting dilution of -utorective T cell clones from the skin, skin T cell clones were generted y the method descried ove, except tht llogeneic DCs were used s APCs. DCs were pretreted with ntiody to HLA clss I nd ntiody to HLA-DR (2 μg/ml) efore incution with T cells. -utorective T cell clones were derived from polyclonl cultures y limiting dilution. T cells were seeded t density of one cell per well in 96-well pltes in 1 μl cell suspension contining irrdited trnsformed B cells (1 1 5 cells per well), PBMCs (1 1 6 cells per well) nd cells (5 1 5 cells per well), (.5 nm) nd IL-15 (2 ng/ml) nd phytohemgglutinin (.5 μg/ml; Sigm-Aldrich). After 14 d, pltes were screened nd ll clones were tested for rectivity to. Cell purifiction nd sorting. Memory CD4 + T cell were purified from fresh PBMCs with CD4 + T cell Isoltion kit (Miltenyi Biotech), nd helper T cell susets were sorted fter stining with nti-cxcr3, nti-ccr6, nti- CCR4 nd nti-ccr1. Cells were sorted on n 11-color FACSAri (Becton Dickinson). Totl memory nd CLA + T cells were sorted fter stining with nti-cd3, nti-cd45ro nd nti-cla. Norml humn skin smples were otined s discrded mteril fter cosmetic surgery under rules of the Institutionl Review Bord of Prtners Humn Reserch Committee. LCs nd derml lymphocytes were isolted s descried 58. Totl skin T cells were isolted from skin iopsies y pulished method 51. Cellulr ssys. T cell cultures were nlyzed for utorectivity to CD1 y coculture with APCs (mock-trnsfected K562 cells, CD1-trnsfected K562 cells, DC or LCs) t n APC/T cell rtio of etween 1:1 nd 1:5 (1:1 for rel-time PCR). For confirmtion of the CD1 dependence of the response, CD1- trnsfected K562 cells were preincuted for 1 h t 37 C with locking ntiody to the corresponding CD1 molecule or control IgG (1 μg/ml). ws ssessed y iossy. Superntnts from T cell ssys were dded to wells contining dependent HT-2 mouse tumor cells in 1 μl T cell medi, then were cultured for 24 h efore the ddition of 1 μci [ 3 H]thymidine for n dditionl 6 h of culture, followed y collection nd counting of β-emissions. For ELISPOT ssy, cocultures of APCs nd T cell were incuted for 16 h in Multiscreen-IP filter plte (96 wells; Millipore) coted with nti- ccording to the mnufcturer s instructions (Mtech). For intrcellulr cytokine stining, T cells were stimulted with PMA (phorol 12-myristte 13-cette; 1 ng/ml) nd ionomycin (25 ng/ml). After 2 h, refeldin A (1:1, dilution; Golgiplug; BD Biosciences) ws dded, followed y incution for 6 h. Then, cells were fixed with 4% (vol/vol) prformldehyde, mde permele with Cytoperm/Cytowsh uffer (BD Biosciences) nd stined for intrcellulr IL-17 nd. protein in cell culture superntnts ws mesured y enzymelinked immunosorent ssy ccording to the mnufcturer s instructions (Peprotech). PCR. RNA ws isolted from cells with n RNesy kit (Qigen), nd cdna ws synthesized with Quntitect reverse trnscription kit (Qigen), including genomic DNA removl step. CDR3 clonotypic primer sequences were s follows: 5 -TCAACGTTGCTGAAGGGAATCCTC-3 (V α forwrd), 5 -CCTGCACTCTCCTGGGGGA-3 (CDR3α reverse), 5 -GCAGGGTC CAGGTCAGGACCCCCA-3 (V β forwrd primer) nd 5 -CTCCCGCTA TCCATCCCAGGC-3 (CDR3β). The specificity of these primers ws confirmed y cdna from the specific T cell clone nd showed miniml or no nneling with cdna from PBMCs from rndom donors. Cytokine nlysis y rel-time PCR. cells were preincuted with ma to or control IgG nd then were cultured for 6 h with utorective T cell cultures t K562 cell/t cell rtio of 1:1. RNA ws extrcted nd, fter reverse trnscription, rel-time PCR ws used to determine the -dependent upregultion of cytokine-encoding genes. The sequences of primer pirs for cytokine mrna were derived from the PrimerBnk dtse nd their specificity (melting-curve nlysis) nd priming efficiency ws confirmed. The Strtgene Mx3P system nd PerfeCT SYBR Green SuperMix (Qunt Biosciences) were used for rel-time PCR. Bckground trnscripts for were mesured nd were less thn 1% of those in unstimulted T cells for ll cytokines tested. Sttisticl nlysis. An unpired two-sided Student s t-test ws used to determine if response to CD1-trnsfected APCs ws significntly different from tht of the mock-trnsfected control. Responses to APCs trnsfected with, CD1, CD1c or CD1d were compred y one-wy ANOVA nd Dunnett s multiple-comprison test. 58. Pen-Cruz, V. et l. Extrction of humn Lngerhns cells: method for isoltion of epidermis-resident dendritic cells. J. Immunol. Methods 255, (21). nture immunology doi:1.138/ni.1956