© 2012 Nature America, Inc. All rights reserved. that that program effector function. Received Received 10 November 2011; accepted 17 January 2012; published online 1 April 2012; be addressed to J.K. ([email protected]). Massachusetts, USA. Pennsylvania, USA. USA. Massachusetts, Worcester, School, Medical 1 memory to similar factors, infection, After activation. with cellular markers associated playing cell-surface dis cells, memory-like ‘pre-made’ as thymus the from exported are lymphocytes innate other to Similar paramount. is tion are first encountered pathogens where epithelia mucosal the in mainly located are cells T system, immune the of sentinels As unclear. is specialization tor cytokines effector of cluster discrete a secrete to programmed subsets distinct of composed also innate the that are indications There lineages. cell distinct considered often are that cells effector cytotoxic and cells T regulatory T cells, helper follicular T cells, helper (IL-17)-producing 17 into 2 1 T interleukin T type type divided cells, helper helper α either of expression the of basis the on lineages two into separated are vertebrates in cells T cells. T adaptive conventional in operate that those from distinct subsets effector-cell innate of tion produc the control that networks gene identify to and lineage T cell ( receptor antigen T cell the expressing cells intrathymic of subsets of profiles expression mouse system the immune the of in networks regulatory gene define hensively compre to assembled biologists computational and immunologists of consortium a is Project (ImmGen) Genome Immunological The subtypes of g is not the main determinant of the of differentiation function correlates with genomically encoded T cell antigen receptor (TCR) chains, which suggests that clonal TCR selection cytokines, analogous to adaptive helper T cell subsets. However, unlike the function of adaptive T cells, homogenous population, they can be functionally classified into effector subsets on the basis of the production of signature Innate The Genome Immunological Project Consortium Hardy Kornfeld Kavitha Narayan molecularly distinct Intrathymic programming of effector fates in three nature immunology nature Department of Pathology, University of Massachusetts Medical School, Worcester, Massachusetts, USA. USA. Massachusetts, Worcester, School, Medical Massachusetts of University Pathology, of Department d β thymocyte subsets segregated on the basis of use of the TCR TCRs on their cell surface. The adaptive adaptive The surface. cell their on TCRs g d γ T cells function in the early phase of immune responses. Although innate δ T cells rapidly produce effector cytokines and growth growth and cytokines effector produce rapidly cells T g 1 d I ta cnet w dtrie te lbl gene- global the determined we context, that In . effector cells in the thymus. The immature 4 Brigham and Women’s Hospital and Department of Medicine, Division of Rheumatology, Immunology and Allergy, Harvard Medical School, Boston, 5 These authors contributed equally to this work. 2 4 1 , , Na Xiong , , and thus the rapidity of their response to infec , 5 , Katelyn E Sylvia

γ δ aDV TCR) to ascertain the heterogeneity of the heterogeneity the to ascertain TCR) α 2 β , 3 A T cells T , but the mechanism of innate effec innate of mechanism the but , NCE ONLINE PUBLIC ONLINE NCE 3 , , Nadia R Cohen 7– 9 . 3 1 α Department of Veterinary and Biomedical Sciences, The Pennsylvania State University, University Park, Park, University University, State Pennsylvania The Sciences, Biomedical and Veterinary of Department , 5 β , Nidhi Malhotra T cell lineage is sub is lineage cell T γ A δ TION T cell lineage is lineage T cell gd 4 5 g , , Michael B Brenner , 6 γ

, , d δ 6 γ effector cells. A analysis transcriptome high-resolution of all emergent Complete list of authors and affiliations appears at the end of this paper. Correspondence should TCRs or or TCRs δ T cell subtypes T cells cells T 6 g d subsets were by distinguished modules unique transcription-factor γ γ δ δ 1 ------, Catherine C Yin

g -chain -chain or mice segregated on the basis of the use of genes encoding V of profiles emergent gene-expression V of use by defined subsets distinct total of analysis by far, So understood. well not is thymus the in fates effector-cell distinct toward selected in Howtissues. pathogens peripheral and when encountering after subsets effector into differentiate in the thymus programmed of function the Second, subset. for each diverse vastly is which repertoire, TCR by not produced, cytokines on of of the basis effector subsets the repertoire into functional sified conventional In comparison, respectively. subset, with the V interferon- IL-17, of production the γ γ of functions effector of segregation the First, critical. particularly is of features unique two underpin that circuits true (ref. SOX13 factor transcription the and TCR the than other them, distinguish that molecules known of city of function and fied contemporaneously, studies delving into the distinct development δ -chain variable region (V region variable -chain T cells from adult mice is based on the use of genes encoding the the encoding genes of use the on based is mice adult from cells T Although the genes that encode the γ doi:10.1038/ni.224 δ T cell lineage–specific markers. Identification of the genetic genetic the of Identification markers. lineage–specific cell T 4 d γ , Leslie , J Leslie Berg -chain -chain indicated the existence of three separate 2 + (V2), V γ δ 1 , Gregory Martens T cells have been greatly hampered by the scar the by hampered greatly been have cells T g 2 γ Department of Medicine, University of Massachusetts Massachusetts of University Medicine, of Department γ d δ δ γ T cells have often been studied as one TCR 7 1.1 T cell development has been studied mainly mainly studied been has development cell T + V + γ 12– 1 ) and/or and/or ) cells as one uniform population, not as as not population, uniform one as cells δ , , Joonsoo Kang 6.3 1 4 − . In contrast, conventional . In conventional contrast, (V1) or V γ δ γ subsets are subsets and/or programmed or V or δ γ γ γ δ δ (IFN- -chain variable region (V region variable -chain TCR and TCR γ g 10), that can be classified as classified be can that 10), 2 δ δ d , Therese Vallerskog T cell subsets seems to be be to seems subsets T cell chains. We compared the We the chains. compared γ effector T cell 1.1 + γ thymocytes from adult thymocytes ) or IL-4 is associated associated is IL-4 or ) γ + δ 1 V e c r u o s e r & T cell development development cell T α δ α β 6.3 β TCR were identi

T cells are T clas cells + (V6) γ and/or V α β γ δ T cells T cell δ ) 1 2 1  δ - - - , ;

© 2012 Nature America, Inc. All rights reserved. trary trary threshold of a twofold change in expression, the number of genes other all of that with compared the V2 subset (immV2) of cells from adult mice, the largest population in (V V4 cells), T epidermal subsets of (CD24 immature three following the of profiles gene-expression established (CD24 of mature paucity The by ImmGen. profiled lineage T cell of the sets with that subsets of thymocyte other thymic sub lymphocyte) thelial (V V1 Methods), total among frequency and repertoire (V V2 the of We expression compared antigen). on the basis of expression of the cell surface marker CD24 (heat-stable the basis of expression of TCR of of four subsets signatures expression encoded germline- unique of expression the by distinguishable subtypes cell populations in the thymus are composed of molecularly heterogeneous V A correlation between effector function and use of the TCR V emerging among subtypes Distinct RESULTS expression. TCR with concurrent or before thymus the that indicates which tion, func effector subset-specific direct that programs gene unique with immature emergent the of they were from as other each from distinct as were subsets main the that found and T.DN4.Th; DN4, T.4SP24-.Th; ETP, MatCD4, preT.DN3A.Th; preT.ETP.Th; vg4.e17.Th; DN3A, preT.DN2.Th; immTgd. DN2, ImmV4.e17, immTgd.vg3.e17.Th; T.ISP.Th; CD4 T.8SP24-.Th; positive), MatCD8, DP, T.8SP24int.Th; T.DP.Th CD69 DP Semi-matCD8, (CD69-); matTgd.vg1+vd6+.Th; MatV6, duplicate); in (sorted matTgd.vg1+vd6-.Th MatV1, duplicate); in (sorted matTgd.vg2+.Th MatV2, duplicate); in (sorted immTgd.vg5+.Th ImmV5, immTgd.vg1+vd6+.Th; ImmV6, immTgd.vg1+vd6-.Th; ImmV1, immTgd.vg2+.Th; ImmV2, follows: as populations ImmGen with correlate axes along CD8 of comparison the for obtained were results Similar spleen. the from isolated CD4 genes. these of number total indicate plots above parentheses in numbers subset); one in > 120 (MEV) ( twofold than more by changed expression with genes indicates red sets); probe all of (mean gene one represents dot Each mice. C57BL/6 from thymocytes mature and mice fetal or adult from thymocytes immature of 1 Figure e c r u o s e r  that the immV2 distinguished subset from other immature

δ Total γδ ImmV6 Pairwise comparisons showed that the earliest identifiable immature 10 10 10 10 10 10 10 10 chain γ 1 2 3 4 1 2 3 4 δ 10 10 T cell lineage, was distinct in its global gene-expression profile profile gene-expression in its global was distinct T lineage, cell lo 1 1

) V5 thymocytes prohibited their analysis. In addition, we we addition, In analysis. their prohibited thymocytes V5 ) Distinct global gene-expression profiles of the the of profiles gene-expression global Distinct 1 γ 10 10 1 δ i ImmV2 (1568 2 2 raised the possibility that the earliest identifiable identifiable earliest that the possibility the raised NKT, NKT.44+NK1.1+.Th (unless otherwise specified). Data are from two to three independent experiments with 4–30 mice each. mice 4–30 with experiments independent three to two from are Data specified). otherwise (unless NKT, NKT.44+NK1.1+.Th (905) TCRs. TCRs. To address this possibility, we determined the gene- DP 10 10 3 3 ) α 10 10 β γ 4 4 TCR 1.1 hi + ) ) Total γδ ImmV1 ), V6 (V V6 ), MatV2 γ 10 10 10 10 10 10 10 10 + 10 10 10 10 δ thymocyte subsets. Most notably, the profiles 3 4 1 2 3 4 1 2 1 2 3 4 T cells from fetal mice: V3 (V mice: from fetal T cells 10 10 γ 10 γ 4 1 1 δ γ 1 + function is molecularly programmed in programmed is molecularly function δ ) and V2 (V V2 and ) 10 10 10 T cell subsets were already embedded embedded already were subsets cell T γ ImmV2 DP69 2 2 ImmV2 and/or TCR (866) (719) (1190) γ 2 1.1 γ 10 10 δ 10 3 + 3 T cell subsets ( subsets cell T 3 + V 10 10 10 4 δ 4 4 6.3 gd γ γ δ γ δ 2 + TCR TCR T cells from adult mice on mice from T adult cells Total γδ ImmV5 10 10 10 10 10 10 10 10 + ) and V5 (V V5 and ) MatV1 δ 10 10 10 10 + ). 1 2 3 4 1 2 3 4 and FoxP3 1 2 3 4 10 10 10 + + 1 1 thymocytes 1 thymocytes, Online Online thymocytes, γ

10 10 ImmV2 2 maturational maturational stages 10 Fig. Fig. (1509) + 2 2 − (843) IS ImmV1 2 (811) γδ , T.4FP3-.Sp CD4 , T.4FP3-.Sp ; partner partner ; P 10 10 10 cell subsets defined by TCR repertoire. Expression of consolidated probe sets by populations populations by sets probe consolidated of Expression repertoire. TCR by defined subsets cell 3 3 1 3 γ γ ). At an arbi At an ). 3 10 5 10 10 + + 4 4 ; dendritic ; dendritic 4 ; intraepi ; γ δ γ γ δ subsets δ and/or

-chain -chain MatCD8 MatCD8 P 10 10 10 10 10 10 10 10

T cell MatV6 10 10 10 10 < 0.05 (Student’s (Student’s < 0.05 1 2 3 4 1 2 3 4 1 2 3 4 10 10 10 1 1 1 - - - - +

10 10 FoxP3 10 ImmV ImmV DP6 2 2 (825) (920) 2 (520) at the immature developmental stage: V3, V4-V2 and V1-V6-V5. V4-V2 V3, stage: at immature developmental the three distinct subtypes on the basis of unique gene-expression profiles ments consistent with their known distinct developmental origin and require T cells, were distinct from other epidermal of dendritic precursors the mice, from fetal cells (immV3) V3 immature contrast, In phenotype. effector IL-17-producing mon com their with correlated which profiles, gene-expression global lar from fetal mice programmed for IL-17 production cells of subset (the mice fetal from cells (immV4) V4 immature and of phenotype in the heterogeneity siderable different expression ( ( whose genes had different expression factors in of subsets transcription selected of amount the confirmed We ( counterparts peripheral differentiated fully their of features molecular the with marked already were they V5 (immV5) cells, respectively, suggested that despite their similarity, intraepithelial lymphocytes (encoded by expression of the natural T killer cell (NKT cell) lineage marker PLZF higher the However, genes. 54 most at of expression the in differed among of immature the similarity subsets V1, V5 and V6 which cells, to contrast in was cells immV2 of signature gene-expression distinct ( genes ~400 of expression the in differed spleen the in Foxp3 lineage, numbered and ~1,600 ~900, respectively. thymocytes In the DP CD69 DP CD4 immature in expression different with genes context, proper the in CD8 and subset immV2 the between difference the to equivalent roughly was 10 10 Fig. 10 9 6 3 3 2 + + 3 + , T.4FP3+25+.Sp; ImmV2.e17, immTgd.vg2.e17.Th; ImmV3.e17, ImmV3.e17, immTgd.vg2.e17.Th; ImmV2.e17, , T.4FP3+25+.Sp; 10 10 CD24 + 10

2 4 4 CD8 17 4 a , ) and obtained confirmatory data for additional genes with with genes additional for data confirmatory obtained and ) 1 + 8 + + t int + . Thus, the thymocytes of the of the thymocytes -test); coefficient of variation < 0.5; mean expression value value expression mean < 0.5; variation of coefficient -test); CD4 Foxp3 ImmV6 + aDV 10 10 10 10 MatV6 10 10 10 10 cells that had received TCR signals versus total total versus signals TCR received had that cells 10 10 10 10 thymocytes and immV2 cells (615 genes). Designations Designations genes). (615 cells immV2 and thymocytes double-positive (DP) CD69 (DP) double-positive 1 2 3 4 1 2 3 4 Zbtb16 1 2 3 4 10 10 10 + + 1 A 1 , T.DP69+.Th; Total , T.DP69+.Th; 1 regulatory T cells versus conventional CD4 conventional versus cells T regulatory CD NCE ONLINE PUBLIC ONLINE NCE 10 10 10 ImmV1 4 2 2 (392) MatV2 2 + (894) (40) ) and granzyme A, which has shown enrichment in Foxp3 10 10 γ Supplementary Supplementary Fig. 1 10 δ 3 3 3 T cell lineage can be divided into the following – 10 10 10 4 4 4 1 5 + , in immature V6 (immV6) and immature Foxp3 α ImmV3.e17 ImmV5 γ

MatV1 10 10 10 10 β 10 10 10 10 10 10 10 10 δ lineage ( lineage 1 2 3 4 1 2 3 4 T cell subsets from fetal mice ( 1 2 3 4 10 10 γδ 10 − 1 1 and CD4 and 1 , Tgd.Th; ISP (immature single- (immature ISP , Tgd.Th; A ImmV2.e17 10 10 TION 10 ImmV 2 (307) 2 MatV2 2 (427) (13) Fig. 2 Fig. − 10 10 10 Fig. Fig. cells of the the of cells 3 1 3

3 ), ), which captured the con γ γ + 10 δ 10 nature immunology nature 10 Foxp3 δ 4 T cells from adult mice a 1 4 subtypes. The immV2 The immV2 subtypes. 4 and data not shown). shown). not data and ). Putting this finding finding this Putting ).

11 ImmV4.e17 ImmV5 + MatV6 10 10 10 10 10 10 10 10 10 10 10 10 , samples were were samples 1 1 2 3 4 6 1 2 3 4 1 2 3 4 10 ) had very simi 10 α 10 1 1 β 1 lineage and and lineage Fig. Fig. ImmV2.e17 10 10 ImmV 10 2 MatV1 2 2 (301) (54) (16) α + γ 10 10 10 6 1 β T cells cells T δ Fig. 3 3 3 ). The The ). T cell TCR 10 10 10

4

4 1 4 ), + - - - -

© 2012 Nature America, Inc. All rights reserved. files of nonsegregated total total nonsegregated of files pro gene-expression the compare to PCA used Wefirst complete. of subsets on Wefocused to data among samples. the of main the components variability gene-expression demonstrate of dimensionality the decreases that tion transforma mathematical a (PCA), analysis ­principal-component ( each mice 4–30 with ( experiment per mice three least at with experiments independent three least at of representative ( cells. the by (NK) NK1.1 and (44) CD44 of Expression axes). along parentheses (in component each by represented variability total the of proportion the with along here, presented are (PC1–PC3) components principal three first The genes). 1,594 population; one least at in > 120 MEV population; indicate labels and bars of (colors cells of populations various in genes ( marker. the expressing cells percent indicate lines bracketed the among expression in difference relative the present best to expression high with cells on drawn were gates cases, some In maturation. after downregulated is that subsets immature among expression similar with factor transcription ROR for negative cells ROR for used; was control a flow-cytometry-minus-one GATA-3, For antibody. control isotype-matched with staining histograms, shaded Gray cells. V total on gated are columns V6 and V1 the ‘straddling’ Plots subset. each in cells V and V of expression of basis the on TCR total on V6 and V1 (CD24 immature ( of divergence and factors 2 Figure nature immunology nature a

b a PC2 (23.26% PC2 in factors transcription of ) Expression

PC3 (11.60%) by visualized be can subsets diverse among relatedness Lineage −0. −0. −0. −0. 0. 0. 0. 0. −0. DN2 δ 0 1 2 3 4 4 3 2 1

6.3, and gating on CD24 on gating and 6.3,

c 2 ) in as cells of populations various in genes variable most 15% the of ) PCA γ DN3A

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PC1 (32.89%) 0. ImmV5 DN3 ImmV6 ImmV1 γ b 1 t expression). SMO, SMO, t expression). , + 0 ET c A −0.1 ). γδ

P ImmV5 T cell subsets. subsets. T cell aDV γ −0.2 DN2 i NKT cell subsets is included in parentheses. Semi-matCD4, semi-mature CD4 semi-mature Semi-matCD4, parentheses. in included is subsets cell NKT δ γ γ cells from adult mice, as this data set is is set data this as mice, adult from cells 2, V 2, hi δ −0.3 A or CD24 or TCR NCE ONLINE PUBLIC ONLINE NCE γ 1.1, 1.1, c lo + γ thymocytes with those of three three of those with thymocytes DN2 ) V2, 1.1

lo

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hi Lo

ETP Expression

ImmV6 a Events (% of max) A 100 100 100 100 100 100 20 40 60 80 20 40 60 80 20 40 60 80 20 40 60 80 20 40 60 80 20 40 60 80 TION ImmV1 Hi 0 0 0 0 0 0 EOME SMO PLZ LEF-1 GATA-3 ROR 1 0 1 0 1 0 1 0 1 0 1 0 ImmV5 10 10 10 10 10 10 2 2 2 2 2 2 10 10 10 10 10 10 F V2

γ 3 3 3 3 3 3 t 10 10 10 10 10 10 27 32 88 2.6 S 1.8 ImmV2 78 4 4 4 4 4 4 Immature (CD24 0 0 0 0 0 0 Zcchc1 Etv5 Eome Nr4a Fhl2 Id Egr Id Phf Ma Sox13 Rcor Rbl Etv6 Notch Runx Phf21 Zbtb8o Churc1 Yaf2 Zbtb1 Rorc Tox Foxj2 Klf3 Hdac Gata3 Tcf12 Tcf Bcl11b E2f4 Zfp65 Bcl9 Foxp1 Crtc Ci Kdm6 Stat Arid1 5 5 5 5 5 5 2 3 c 7 f 1 2 2 2 4 3 l 3 2 3 a s 6 b 7 2 a V1 3 s 55 2 44 17 82 13 - - 10 hi experiments with 4–30 mice each. mice 4–30 with experiments independent three to two from are Data 3A. stage double-negative (CD4 double-negative DN2, together. grouped In text. in discussed factors transcription indicates font Red subset. and gene by clustered hierarchically and row gene by centered transformed, log were data cells; immV6 or immV1 and immV2 among regulation in differences ( factors transcription ( processes metabolic in involved molecules encoding genes of and genes; (1,006 cells immV6 or immV1 and immV2 ( subsets. distinguishes processes metabolic in involved molecules 3 Figure distinct other all was from subset immV2 the that clear was it however, analysis, ( PCA for 2a the used Fig. with regulation genes in of difference 15% greatest the for and coefficients distance correlation Euclidean Pearson’s by relatedness subset measured we when of ters total other from separately clustered maturation of Innate-like rity. CD8 single-positive (immature subsets precursor from displaced increasingly together, ( popu the lations across variance and expression of basis the on the samples, among expression in difference greatest the with genes of 15% α ) β V6 67 7.6 45 thymocyte subsets ranging in maturity and lineages using the the using lineages and maturity in ranging subsets thymocyte 83 30 Supplementary Tables 1 Supplementary b (MEV > 120 in at least one population; 1,597 genes). Data are are Data genes). 1,597 population; one least at in > 120 (MEV γ δ α ,

T cells were positioned away from the four distinct clus distinct four the from away positioned were cells T b β Expression of transcription factors and genes encoding encoding genes and factors transcription of Expression i. 2 Fig. a ). When we used the segregated segregated the used we When ). ) PCA (as in in (as ) PCA thymocyte subsets ( subsets thymocyte V2 1.8 56 23 11 3.3 5.7 Mature (CD24 γ b δ α ). Mature Mature ). thymocyte subsets, positioned uniquely in relation relation in uniquely positioned subsets, thymocyte β a V1 b ) or are from two to three independent experiments experiments independent three to two from are ) or invariant NKT cells ( cells NKT invariant c 23 12 20 20 , interspersed immV1 and immV6 replicates are are replicates immV6 and immV1 , interspersed + 27 ) in thymocyte subsets, showing genes with with genes showing subsets, thymocyte ) in Fig. 2 Fig. and DP) as a function of their relative matu relative their of function a as DP) and lo 5.9 ) b and V6 + α 29 42 ) of genes with different regulation among among regulation different with genes ) of 4 56 cells; Semi-matCD8, semi-mature CD8 semi-mature Semi-matCD8, cells; 68 β 2 thymocyte subsets were positioned positioned were subsets thymocyte Fig. 2 Fig. ). ( ).

b c PC2 (21.28%) PC2 PC3 (13.27%) PC3 (12.34%) b

−0.2 −0.1 −0.3 −0.2 −0.1

, 0. 0. 0. 0. 0. (24.89%) PC2 0. 0. 0. 0. c 0. b ) Heat maps of the expression expression the of maps ) Heat 0. 1 2 3 4 5 0 0 1 2 3 4 0. 4 ). We obtained similar results results similar Weobtained ). 3 2 −0.2 0. 0 i MatCD4 NKT i NKT 2 0. i − NKT cells) at three stages stages three at cells) NKT 1 CD8 γ (4 matCD4 Semi- i δ 0 (4 NKT −0.1 −0.2 i MatCD8 4 MatCD8 ImmV T cell subsets for this this for subsets cell T NKT 4 + −0.2 Supplementary Fig. 3a Fig. Supplementary + NK − MatCD4 NK matCD8 Semi- ) stage 2; DN3A, DN3A, 2; ) stage α (4 −0.1 e c r u o s e r Total + (4 4

PC1 (33.80%) 2 + β ) ) + DP 4 NK cell subsets, and and subsets, cell – 0 −0.2 NK ImmV Supplementary Supplementary γδ γδ – 0. ) −0.1 – thymocyte thymocyte Semi-matCD4 ImmV ImmV6 ) IS 1 Semi-matCD8 PC1 (35.91%) 1 0. P 0 2 b IS 5 0. 0. ) ) or DP P 1 3 0. 0. 2 4 0.

3 + 0.

4 

- - - © 2012 Nature America, Inc. All rights reserved. product, SOX13, interacts with TCF1 (encoded by by (encoded TCF1 with interacts SOX13, product, system, ( factors and transcription SOX-TCF1-TOX of the family to box belonging high-mobility-group those of prominent factors, most transcription the most of expression higher much had subset immV2 the that indicated cells immV6 and immV5 immV1, versus cells immV2 in expression different with factors transcription of sis emerging in evident already was function effector off subset-specific in factors transcription turning dominant and the of programming genes Distinct genes. ­lineage-mismatched lineage-associated on turning by process maturational with unique transitions. distinctly develop cells immV2 marker of apoptotic cells (data not shown). These results suggested that G0 state of the cell cycle ( of expressing a Ki67, cells marker frequencies of non-had the similar and similarly BrdU analog thymidine the incorporated subsets ture imma the as to death, or susceptibility properties cell-cycle different of signatures gene distinct The ( populations age, which provided the main element of similarity between these two cells to corresponded the pattern in immature DP cells of the sets. The expression of genes encoding metabolic molecules in immV2 immV2 cells ( in production and energy lower metabolism suggested which genes), (~270 translation protein and phosphorylation oxidative biogenesis, RNA DNAsynthesis, in involved proteins encoded cells V6 or V1 in About one third of the genes with lower expression than cells immV2 subtypes immature in circuits regulatory Distinct type. cell unique a represented sity, nevertheless diver junctional (VDJ) (VJ)–variable-diversity-joining able-joining TCR their from generated diversity clonal TCR the encoding that conversely, immature other from different were domains TCR by germline-encoded distinguished cells that showed immV2 results cells were most similar immV2 to DP CD69 that indicated PCA segregated, were populations DP T precursor subsets ( from subsets distantly precursor T segregated and variance the of >50% and shared which immV2 between similarity close of the idea supported scores correlation and distances Euclidian PCA, by 3a Fig. immature other in that from different subsets immV2 in expression with genes for Second, other to each. mice 4–30 with experiments independent three to two from are Data together. grouped are replicates in as (presented cells ( processes metabolic in involved molecules encoding genes of expression ( clustering. hierarchical on based populations γδ the of genes 495 ( cluster. mature oval, red cluster; ( of maturation the after regulation different ( maturation. after 4 Figure e c r u o s e r  b 5 Fig. Supplementary

) or transcription factors ( factors transcription ) or T cell subsets. Dendrogram (above) shows the relatedness of the the of relatedness the shows (above) Dendrogram subsets. T cell Lineage-specific transcription factors propel the lineage-commitment Supplementary Tables 2 Supplementary and and

Sox13 Convergence of the gene-expression profiles of of profiles gene-expression the of Convergence γ δ and and Supplementary Tables 1 1 Tables Supplementary Fig. 3 Fig. Supplementary TablesSupplementary 1 γ expression is restricted to developing developing to restricted is expression -chain constant region 1 (C 1 region constant -chain γδ α γ a δ ) PCA (as in in (as ) PCA β maturation gene signature in precursor, precursor, in signature gene maturation T cells expressing the V the expressing cells T subsets ( subsets b Fig. 3b, Fig. and and , , γ Fig. 3 Fig. δ Supplementary Table4 Supplementary T cell subsets. Hierarchical clustering analy clustering Hierarchical subsets. cell T γ δ Supplementary Fig. 4a Supplementary Supplementary Table 7 Supplementary c cell subsets (1,006 genes; genes; (1,006 subsets cell ) in subsets of immature and mature mature and immature of subsets ) in c a ). In In ). Fig. 2 Fig. and and Fig. 2 Fig. , + 5 5 b cells ( γ ) Heat map of the expression of the the of expression the of map ) Heat δ and c subsets were not a consequence of consequence a not were subsets , interspersed immV1 and immV6 immV6 and immV1 , interspersed Supplementary Fig. 3b Fig. Supplementary b c ) of the 495 genes with with genes 495 the ) of and and 6 Supplementary Fig. Supplementary 3d ). Among cells of the immune immune the of cells Among ). , 3 and γδ Supplementary Fig. 2c Fig. Supplementary and T cells from adult mice mice adult from T cells c γ 2 , 1), despite the extensive extensive the despite 1), γ d 4 2 chain from the locus locus the from chain 2 ), the results obtained obtained results the ), ) Heat maps of the the of maps ) Heat and data not shown). shown). not data and ). Blue oval, immature immature oval, Blue ). ) ) than in other α δ , b partners and vari and partners β ), ), and annexin V, a DP thymocytes, thymocytes, DP γδ Supplementary Supplementary Tcf7 γ subsets subsets δ γ αβ T cells and, and, cells T δ ) and LEF1 LEF1 and ) T cells. Its cells. T , and , and , c ). When When ). α γδ ). ). These i. 3 Fig. γ β δ

line sub , d ). ). c ------

of IL-17, of IFN- of IL-17, of by ROR factors The transcription were the predominant producers of IL-17 ( all with mice of the during pattern effector this fied CD27 V1 whereas IL-17, Table 6 Supplementary ( cells immV2 in than cells immV6 and immV5 immV1, development cell NK of regulator negative a encodes cells immV2 in than ( regulator HEB (encoded by by E47 (encoded factors transcription E helix-loop-helix expression of pan– the part, in by (encoded T cell subset–specific functions ( functions T subset–specific cell three these peripheral of their with correlated which genes, factor expression transcription selective by distinguished were subsets cells T a c b In the periphery, V2 CCR6 Eomes γ MatCD4 PC3 (10.31%) −0. −0. δ

0.1 0.2 0.3 subsets in the lung synthesized IFN- synthesized lung the in subsets MatCD8 (14.21% PC2 0.3 ImmV6 ImmV1 0 2 1 22– 0. + iNKT 2 DN2 0.1 2 ) and PLZF (encoded by by (encoded PLZF and )

5

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DN2 A −0. γ Tcf12 δ DP and of both IL-4 and IFN- and IL-4 both of and

lineage gene expression signature expression gene lineage ETP 2 −0.1 19 CD4 Id3 Mat DN3A PC1 (44.84%) CD8 Mat , 0 ), ), along with the gene encoding their upstream 2 MatV Mature 0

). ImmV2 , , which encode the negative regulators of the . Furthermore, expression of of expression Furthermore, . 0.1 + MatV6 MatV2 MatV1 i NKT T cells secrete IFN- secrete cells T

6 DP 0.2 MatV

+ DN4 0.3 CD27 γ

t (encoded by t (encoded ImmV6 M 1 Figs. 2a Figs. ImmV1 atV Zbtb16 A − ImmV5 2

γ TION γ δ δ d T cell response to infection infection to response cell T T are cells to biased produce and Supplementary Supplementary Fig. 4c 7

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γ ImmV1 c γ 8 Expression ). As further support support ). As further , respectively, in in respectively, , , 1 4 ), Eomes (encoded ), Eomes (encoded ImmV2 . We further veri We . further 2 1 1 MatV6 Bcl11b 0 , was lower in in lower was , . Conversely, Conversely, . Hi Tcfe2a Fig. 3 Fig. MatV1

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© 2012 Nature America, Inc. All rights reserved. Runx3 by of ferentiation dif the for required factors other encoding genes of expression The immV2 cells than in other subsets ( (ref. SCART2 and SCART1 receptors scavenger T cells—the signaing molecule Blk IL-17-producing of expres markers three the cells, encoding genes the of sion immV2 of programming thymic of proposal the for ( each mice three least at with six of representative experiment ( each mice 4–30 with experiments independent three to two from are Data cytokines. of staining intracellular and surface by followed ionomycin, and PMA ester phorbol the 4 h with for stimulated ROR of expression V mature in only discerned be could IL-17A and Blk of expression The overlays. in expression marker CD24 on gated and TCR total on gated (mat) mature and (imm) immature in IL-17A and in as (presented receptors factor ( (red). subsets differentiated fully in maintained and maturation during precipitously as downregulated or extinguished and subsets immature in expressed receptors, chemokine encoding ( programs. function effector of 5 Figure nature immunology nature by divergent transcription-factor networks that are predicted to pro that are predicted networks by transcription-factor divergent TCRs, of various expression with ( in in the execution of effector their peripheral subset–specific involved directly of proteins expression the modulated subsets T cell with V1 cells ( associated expression of amount the to factors transcription specific ( distinct 8 Table remained cells V6 mature (matV6) whereas converged, cells (matV1) V1 mature and cells molecules encoding genes involved of in metabolism pattern or RNA-DNA expression biogenesis the in First, mature V2 ration. (matV2) the in maturity to transition the of demarcation a as CD24 of loss the established results These shown). during modulated uniquely were mature CD4 after the maturation of 495 ( of less T lineage cell cluster, from regard of a the cluster separate distinct mature subsets, T cell maturation as well, as and precursors immature formed subsets the that showed these of scores correlation and distances Euclidian clustering, hierarchical all of maturation T after cells ( regulated coordinately were that genes among differences specific immature gene subset–specific networks the ( of effect and duration the assess to V6) and V1 (V2, (CD24 mature of profiles expression gene- the We determined studied. systematically been not have tion thymic with associated processes biological The markers functional and homing induces Maturation functions. effector distinct gram Figs. Figs. 2 Four overriding themes characterized characterized themes overriding Four Maf γ δ γ -maturation genes identified, ~78% were coordinately regulated ) ) δ 3 Supplementary Fig. Supplementary 5a

). Second, after maturation, V2 cells downregulated subset- downregulated cells V2 maturation, after Second, ). 2 and 1 maturation–dependent genes in other thymocyte subsets subsets thymocyte other in genes maturation–dependent Generation of mature mature of Generation 8 , was also higher in immV2 cells than in other other in than cells immV2 in higher also was , , GATA-3, by (encoded 3 + cells, mature CD8 c b Fig. Fig. 4 ). ). These results demonstrated that before or concurrent ) Heat map of the expression of cytokine and growth growth and cytokine of expression the of map ) Heat α β γ γ δ t t ( hi δ effector helper T cells, including c-Maf (encoded (encoded c-Maf including cells, T helper effector + cells, separated into V into separated cells, (light gray lines) or CD24 or lines) gray (light maturation signature was conserved during during conserved was signature maturation Fig. Fig. d Fig. 4a Fig. and α

2 β γ ). For analysis of IL-17A expression, cells were were cells expression, IL-17A of analysis For ). 2 T cells subsets (comparison of DP cells with aDV Supplementary Table Supplementary 9 + thymocytes, whereas immV2 cells had low low had cells immV2 whereas thymocytes, γ , b a γδ δ ) Heat map of the expression of genes genes of expression the of map ) Heat and A T cells, there was a shared set of 495 495 of set shared a was there cells, T Fig. 3b, Fig. cell subsets poised for the elaboration elaboration the for poised subsets cell , Fig. Fig. γδ + NCE ONLINE PUBLIC ONLINE NCE b cells and and Gata3 T cells mature (blue) or induced induced or (blue) mature T cells γ γ 2 Supplementary Supplementary Tables 2 δ δ Supplementary Fig. 5c Fig. Supplementary 6 γ 1 ( T cell maturation only (data not (data only maturation T cell thymocyte subsets are regulated are regulated subsets thymocyte δ ). ). Despite the substantial subset- Supplementary Supplementary Fig. 4f T cell lineage. cell T Supplementary TableSupplementary 7 c i. 4 Fig. ). ( ). ) lo 29 γ ) thymic thymic ) 2 c , i + 3 ) Expression of Blk, ROR Blk, of ) Expression NKT NKT cells), whereas ~22% γ lo and V and 0 δ and Runx3 (encoded by (encoded Runx3 and (black lines) to show show to lines) (black c subset–specific matu subset–specific and and γδ thymocyte subsets, subsets, thymocyte γ 27)—was higher in in higher 27)—was 2 ). ). Third, mature a γ γ − , A δ populations, populations, b δ Supplementary Supplementary TION ) or are from one one from are ) or T cell matura cell T T cell subsets subsets cell T

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genes encoding cytokine receptors dedicated to IL-17 production production IL-17 responsiveness: and to dedicated receptors cytokine encoding genes of super-induction abrupt by phase effector-poised the entered cells V2 maturation, thymic After functions. effector programmed their subsets with the ability to sense environmental cues that fully engaged to specific also were expression duction in T cells and suggests that the molecular program that specifies IL-17 promaturation further emphasizes the separation of V2 cells from other The finding of dynamic subset-specific alterations in tissue tropism lymphoid aftercells, some with the ability to secrete IL-17 or IL-22 (ref. ROR hi tsus Ntby mt2 el ae ITGB7 are cells matV2 Notably, tissues. phoid Eomes by part in controlled is lated with IL-17-producing lymphocytes Fig. 1 ated with maturation of the V6 and V1 subsets ( stage, whereas induction of the chemokine receptor CXCR3 was associ and uniquely upregulated in the V2 subset after transition to the mature Fig. 1 CCR10 by the promoter of the gene encoding CCR10; the greatest frequency of by analysis of reporter mice that express green fluorescentWe protein confirmed driventhe expression pattern of the chemokine receptor CCR10differentiated( subsets fully in and expression induced precipitously during maturation subsets that and was extinguished or downregulated as maintained immature in expression expression: chemokine-receptor of patterns loss of chemokine and integrin receptors. We noted the following two mice with unique homing properties, as shown by the below. detail of features two last PLZF of profile gene-expression the acquiring the with state, mature the to transition after diverged cells V6 including c a Maturation stage–dependent alterations in cytokine-receptor cytokine-receptor in alterations stage–dependent Maturation Transition to the mature stage endows subsets of Events (% of max) DN3A 100 100 100 20 40 60 80 20 40 60 80 20 40 60 80 γ 0 0 0 and ). ). Expression of the chemokine receptor CCR6 was considerably t

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© 2012 Nature America, Inc. All rights reserved. induction of of induction including IL-13), and (IL-5 2 T type that cytokines produce helper lymphocytes innate ( maturation Table 11 Supplementary after cells, V2 and V1 in lower but cells, γ maturation was the fine-tuning response. The main gene upregulated by V1 cells after for receptors additional multiple with IL-23, IL-17 paracrine by on turned autocrine to respond to programmed are cells matV2 Thus, in ( immature cells expression the of respectively, 25%, and 10% was cells matV2 in IL-12R) for tor and for receptor the IL-25) of expression the whereas IL-2 IL-27, and encoding those of expression higher having cells matV2 with maturation, after expression divergent had production IL-17 of tors ( IL-17 of ROR of expression enhanced by accompanied was super-induction This ( cells matV2 in tion (ref. CD24 in expression ( from or pooled ( each mice 4–30 with experiments Student’s (two-tailed ( mean the represent bars horizontal small mouse; individual an represents (right) symbol Each each. in cells Ctsl and mice among cells V6 of frequency and Cd74 V for staining of (left) CD4 mature versus cells ( in expression in expression higher had genes 292 of total A subset. one least at in >120 CD8 CD4 mature (vs) versus cells both in (FC)) change’ (‘fold more or twofold by altered expression ( 6 Figure e c r u o s e r  of acquisition the like that indicated cells V6 IL-4-producing c a δ c a

) PCA (as in in (as PCA ) , T cell subsets. Expression of of Expression subsets. cell T i b ) PC3 (9.90%) P value NKT cells than mature CD4 mature than cells NKT −0.4 −0.2 −0.3 −/− 10 10 10 10 10 10 10 10 10 ) Identification of of Identification ) −0.2 0.2 0.4 + −0.1 35) 35) and 0 cells ( cells γ −/− PC1 (52.23% –9 –8 –7 –6 –5 –4 –3 –2 –1 0.1 0.2 mice). Numbers adjacent to outlined areas (left) indicate percent percent indicate (left) areas outlined to adjacent Numbers mice). t and the signaling molecule Blk dedicated to the production production the to dedicated Blk molecule signaling the and t 0 0.3 MatV6 i 0.4 NKT

Igfbp4 10 γδ Sel Common features of of features Common n S1pr1 −0.4 –3 Fig. 5 Fig. thymocytes ( thymocytes l Themis = 9 9 = MatV1 Ccr7 b i NKT vsMatCD4(FC) Downregulated in Upregulated in −0.2 ), plus a coefficient of variation of <0.5 and an MEV of of MEV an and <0.5 of variation of coefficient a plus ), 10 Tnfrsf25 i Il17rb, Fig. 2 Fig. NKT cells than in mature CD4 mature in than cells NKT PC2 (20.58% –2 d MatCD8 Cd74 MatCD4 0 c , MatV2 0.2 Il18r1 e ). Receptors for four counter-regulators or modula or counter-regulators four for Receptors ). 10 , right) two independent experiments. independent two , right) α 0.4 Cd4 b –1 β t which encodes part of the receptor for IL-25, in in IL-25, for receptor the of part encodes which −/− γ 0.6 lo ) of the 540 genes with different regulation in in regulation different with genes 540 the of ) -test). Data are from two to three independent independent to three two from are Data -test). NKT cells and TCR Zbtb16 i 1.1 and V and 1.1 4

(ref. ) NKT cell signature genes on the basis of of basis the on genes signature cell NKT Fig. 5 Fig. cells than in CD24 in than cells , , and this pattern was shared among the mature 0.8 mice; in in mice; d 10 i + NK Tbx21 ) or wild-type mice and and mice wild-type ) or or CD8 or 0 i NKT T ). IL-25 drives the population expansion of expansion population the drives IL-25 ). Klra10 36) 36) were the genes with the induc greatest d e + Gzm Klra 10 b Il12rb2 cells ( cells V 1.1 αβ V 1.1 γ + and and γ Klrb1c 1 9 10 10 10 10 10 10 10 10 b Klra or CD8 or δ Fig. Fig. 5 0 0 6.3 on C57BL/6 wild-type (WT) and and (WT) wild-type C57BL/6 on 6.3 2 3 4 5 2 3 4 5 +

γδ e i 5 V V cells. ( cells. 0 0 NKT cells and and cells NKT 25 26 , , a 10 δ δ 10 Il17rb T cells (right; in in (right; cells T 6.3 6.3 – n ± a Supplementary Tables 7 7 Tables Supplementary 2 2 10 WT WT 10 c = 5 wild-type mice and and mice wild-type 5 = (which encodes part of part recep the encodes (which ) and and ) s.d.). * s.d.). ) or are representative of ( ) representative or are 3 b 8.5 3 10 10 6.3 + b and cells (red); 248 genes had lower lower had genes 248 (red); cells 4 4 10 10 Il17rb

d P value was selectively higher in V6 V6 in higher selectively was 10 10 10 10 10 10 10 10 10 5 5 , αβ e − 23 34 hi –9 –8 –7 –6 –5 –4 –3 –2 –1 ) Flow cytometry analysis analysis cytometry Flow ) + Cd74 type 2 type cells Supplementary Table 11 Supplementary Ctsl P or CD8 or cells), cells), NKT cells versus mature mature versus cells NKT = 0.04 and ** and 0.04 = Sell 10 (which encodes part of of part encodes (which 14 –/ S1pr1 2.9 –3 –/ Ctsl γδ Themis – – Igfbp4 i Downregulated in Upregulated in NKT vsMatCD8(FC Ccr7 matV6 cells. cells. matV6 10 −/− d + Il17re, Il1r1, Il18r1 Il1r1, Il17re, –2 , , cells (blue). (blue). cells V6 (% of total γδ)

n V6 (% of total γδ) 10 20 30 γδ 0 0 2 4 6 8 = 6 wild-type wild-type 6 = 3 10 4 thymocytes ( thymocytes . The selective . The selective –1 Cd4 WT Tbx2 WT Fig. 5 Fig. 4 P n i NK 10 = 5 = = 0.005 0.005 = 1 d Zbtb1

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cells that make up the residual ~5% of total total of ~5% residual the up make that cells (V thymus adult the in repertoire TCR of basis the on remaining two only the examine directly not could cells from fetal mice V3 mice; fetal from cells V4 and mice fetal and adult from cells V2 IL-17-producing profiles: gene-expression cell distinct three types in the following the identified have We cells. T innate emergent of programs gene-expression in divergence early the that onstrated TCR by distinguished subsets mocyte of profiling gene-expression global Systematic DISCUSSION fate. effector-cell specifies mainly type TCR the of independent features TCR-signaling in similarity a or works net transcription-factor similar by programming molecular either of generation the regulated esses these results indicated that common gene networks and cellular proc cells V6 fewer of production the to led all but V6 not cells, affected specifically also The gene same deficiencies We predicted that matV6 cells would overlap overlap would cells matV6 that Wepredicted the of Acquisition systems other in differentiation cell control that networks gene-regulatory established the with well fits overall pattern of expression program a dynamic transcription-factor that established the factors effector gene circuits as transcription of activities the of dampening overall an ( molecules effector induce rapidly will sites, tissue at engaged once γ thymus the equipped that stage mature the at phase primed a and stage; immature the at phase establishment an steps: two ing dictated ( IL-17A produced that cells with abundant pattern was matV6 of with expression cells high PLZF that correlated this of illustration best The stage. mature the at specifically initiated matured, the expression of genes encoding effector cytokines was also the expression of overall as modulation the cytokine-receptor subsets of expression by matV2 cells, the responsiveness quintessential cytokine chain) has a negative effect on the development of of development the on effect negative a has chain) L) and cathepsin encodes (which kinase Itk), PLZF), and cells ( of acquisition the ( 2 and were nearly identical in their placement along and principal components matV6 1 as signature, expression this shared of 540 the scores tion and conventional mature other versus thymic in expression different with genes identifying by of the signature gene the derived larity, we first simi this of extent the To and determine properties. PLZF of functional similar expression shared their given level, gene-expression Supplementary Fig. 6a Fig. Supplementary α δ thymocytes with tissue-migratory cues as well as sensors that, that, sensors as well as cues tissue-migratory with thymocytes γ In sum, our results indicated that the unfolding gene programs that β δ versus versus Supplementary TableSupplementary 12 effector subsets is intrathymically programmed. Coincident with Sh2d1a Fig. 6 Fig. γ α δ γ aDV subset–specific effector functions occurred by the occurred follow functions effector subset–specific β γ Id3 δ γ δ

δ cell subsets underpins the effector diversification of of diversification effector the underpins subsets cell i ). Genetic studies also supported the relatedness of V6 V6 of relatedness the supported also studies Genetic ). c NKT cells: cells: NKT T cell lineage on the basis of TCR repertoire and shared (which encodes the inhibitor transcriptional Id3), and and (which encodes SAP), (which encodes A Il4 NCE ONLINE PUBLIC ONLINE NCE transcription ( transcription i NKT gene signature was independent of NKT gene was TCR signature independent type 17 Supplementary Fig. 6b Fig. Supplementary i NKT cell signature by matV6 cells matV6 by signature cell NKT , i 1 NKT signature genes showed that matV6 cells cells matV6 that showed genes signature NKT 8 ; ; and V1, V5 and V6 cells from adult mice. We ). The phase transition was accompanied by accompanied was transition phase The ). in vivo in Fig. 5 Fig. ). ). PCA, Euclidian distances and correla , the loss of of loss the , Supplementary Fig. 5e Supplementary Cd74 γ c α δ ). 3 A V6 and and V6 7 β TION . Itk thymocyte subsets ( subsets thymocyte γ γ (which encodes the invariant invariant the encodes (which and TCR and δ (which encodes the (which encodes tyrosine thymocytes first arose. This

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© 2012 Nature America, Inc. All rights reserved. subset diversification of innate diversification subset effector of programming thymic the of mechanism the identify will nal cues. Elucidation of the upstream regulators of these gene networks gests that γ of subsets of innate molecular the of heterogeneity underpin may precursors the of activities gene tive γ TCR functional of onset the in difference the that suggested which 1 ligand (N.M. Delta-like expressing mal cells CCR6 of tion c-Kit earliest the independently and distinctly lated C functional three the that indicating studies several by supported is maturation precursor of stages different at originate resultant T cell properties of the definition for the critical is type, TCR than rather maturation, well that documented the timing of TCR expression during precursor cells T epidermal dendritic generates that mice fetal from progenitors T cell early in exclusively rearrangement gene programs. The most clear-cut example of this is the V programmed fates by inheritance and fixation of distinct precursor gene-expression V ferent diversification. subset effector of discriminator potential one is signaling TCR unique that possibility the of cells DP and cells V2 in metabolism cellular in involved molecules encoding cluster gene a of expression manner dimerization–dependent TCR precedent that the V manner. Similarly, is there in an a occurs ligand-independent α in presented ligands selecting of independently ization, of intrinsic differences in TCR conformation and/or membrane local either by unique interactions with ligand(s) selecting V TCR encoded genomically of diversification the of regulator tial poten another are Wnt ligands canonical TCF1-LEF1, on signaling (N.M. from cells adult mice, as IL-17-producing of differentiation the in involved circuit gene tral cen one are targets gene their and TCF1-LEF1, its partners SOX13, interacting factor transcription high-mobility-group The cells. tor effec programmed of generation the for mechanism possible one is subtypes effector define that factors transcription the the of control expression that factors growth and cytokines to cells precursor of responsiveness distinct The factors. transcription encoded subsets immature emergent the distinguished that modules in cells T of differentiation effector the alter not appreciably do thymocytes sibilities can now be narrowed down. Data indicate that that indicate Data down. narrowed be now can sibilities immature distinct shown). not (data expression gene in thymocytes immV6 V immature that suggested functional observed the V ~85% of ing V immature of analysis However, available. not are cells these sort to antibodies nature immunology nature δ -chain expression with the attendant inheritance of distinct collec distinct of inheritance attendant the with expression -chain β thymocyte differentiation and fixed during thymic maturation sug The identification of gene networks that specify the effector functions dif generation of the timing distinct the that possible Finally, it is The biological processes that govern the generation of these three three these of generation the govern that processes biological The T cells, the pre-TCR-mediated transition to the immature DP stage et al. et γ TCRs during precursor maturation leads to alternative cell cell alternative to leads maturation precursor during TCRs trans γ δ T cells probably do not respond homogeneously to exter , data not shown). Given the modulatory effect of Wnt of effect modulatory the Given shown). not data , γ 1.1 + V2 cells in cultures of OP9 mouse bone marrow stro marrow bone mouse OP9 of cultures in cells V2 γ (K.N. (K.N. 1.2 − + γ V T cell progenitors were skewed toward the genera toward the were skewed T progenitors cell δ + γ γ γ subsets. γ and 15% V 15% and 2 δ 2V δ − T cells that are established at the earliest stage of cell subsets are unknown, but the list of pos of list the but unknown, are subsets cell et al. et V δ

γ 5 TCR can also signal in a ligand-independent, Sox13 Tcrg 5 43 aDV − , data not shown). The most notable gene gene notable most The shown). not data , ,

γ 4 γ 1.2 4 δ gene-rearrangement frequencies gene-rearrangement . . The proposal that γ thymocytes from adult mice (consist mice adult from thymocytes A −/− chains may transmit distinct signals, signals, distinct transmit may chains γ + γ NCE ONLINE PUBLIC ONLINE NCE δ 4 cells resemble immV1, immV5 and and immV5 immV1, resemble cells mice lack V2 IL-17-producing cells mice lack V2 IL-17-producing T Moreover,cells. developmentally + cells, as estimated on the basis of of basis the on estimated as cells, 45 , 4 6 γ . Most directly, we found that that found we directly, Most . δ effector cells. Alternatively, Alternatively, cells. effector 1 3 . The finding of similar similar of finding The . α β et et al. lineage supported the the supported lineage γ 1 δ 8 T cell subsets may , , data not shown), . It has also been been also has It . A 1 TION 3 γ or on the basis loci are regu are loci γ δ trans thymocyte thymocyte

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3. Project. Core resources supported by Research the Diabetes Endocrinology and ExpressioneBioscience, Affymetrix Analysis for support of the ImmGen J. Ericson and S. Davis) for help with data generation and processing; and of the ImmGen Consortium for discussions; the ImmGen core team (M. Painter, PCA program; A. Hayday (King’s College, London) for anti-V Medical forSchool) the ConsolidateProbeSets module and PopulationDistances (University of Massachusetts Medical forSchool) (University of Massachusetts Medical forSchool) reporter mice; V. Lefebvre (Cleveland Clinic) for H. Birchmeier (Max-Delbrück-Center for Molecular Medicine forBerlin) We thank M. Mohrs (Trudeau Institute) for IL-4–GFP reporter mice; Note: Supplementary information is available on the code. Accession online the at http://www.nature.com/natureimmunology/. paper of the in version available are references associated any and Methods M subsets. effector of lymphoid innate origin unified T cells IL-17-producing in operate that use those to similar networks to genetic predicted are gut the in subsets lymphocyte innate other by hardwired programs for the production of cytokines of the IL-17 family 12. 11. 10. 9. 8. 7. 6. 5. 4. 2. 1. reprints/index.htm R Published online at http://www.nature.com/natureimmunology/ The authors declare no competing interests.financial studies and wrote the paper. of some results; K.N. analyzed gene-expression data; and J.K. and K.N. designed cell subsets; L.J.B. provided reagents and shared data used in the interpretation mutant strain; andN.R.C. M.B.B. generated the gene-expression profiles of NKT the studies ofsupervised and analyzed data; G.M., T.V. and K.N. did the studies of K.E.S. sorted cell subsets; K.N., N.M., K.E.S. and C.C.Y. did follow-up experiments ImmGen group, and CA100382 to J.K.). Infectious of Diseases the US National Institutes of Health (R24 AI072073 to the Center (DK32520) were used. Supported by the National Institute of and Allergy A C AUTH eprints ckn O

ethods

MPETING FINANCIAL INTERESTS FINANCIAL MPETING Stark, M.A. Stark, Azuara, V., Levraud, J.P., Lembezat, M.P. & Pereira, P. A novel subset of adult of subset novel P.A Pereira, M.P.& J.P.,Lembezat, V.,Levraud, Azuara, W.K. Born, & R.L. O’Brien, Melichar,H.J. C.E. Sutton, Interleukin-17- M. Veldhoen, & B. Stockinger, D.J., Cua, K., Hirota, B., Martin, by dominated is production IL-17 J.L. Flynn, & A.M. Green, E., Lockhart, cell T CD8 the in memory Alternative K.A. Hogquist, & S.C. Jameson, Y.J., Lee, Berg, L.J. Signalling through TEC kinases regulates conventional versus innate CD8 by tissues the in Immunoregulation Tigelaar,R. & A. Hayday, D.A. Ferrick, gene of networks Project: Genome Immunological M.W.The Painter, T.S.& Heng, lineage. T-celldevelopment. Immunol. IL-17. and IL-23 (1995). 255–257 by pathogens Th2-stimulating and Th1- to response cells. immune in expression hmcts ht erts dsic pten f yoie ad xrse a very a expresses and cytokines of repertoire. receptor pattern cell T restricted distinct a secretes that thymocytes Immunol. Semin. SOX13. factor transcription autoimmunity. (2009). and responses Th17 amplifying cells, T signals. environmental producing 177 during cells T CD4 than rather ow O , 4662–4669 (2006). 4662–4669 , 34

R R C and , 4 le 7 TrendsImmunol. , which indicates a possible route for the identification of route a a for possible indicates , identification the which O

3 permissions d γ NTRIBUTI , 233–242 (2003). 233–242 , et al. et δ et al. et gments cls eetvl epn i rsos t ptoe pout and products pathogen to response in expand selectively cells T t al. et et al. et l . GEO: microarray data, GSE15907. data, microarray GEO: Phagocytosis of apoptotic neutrophils regulates granulopoiesis via granulopoiesis regulates neutrophils apoptotic of Phagocytosis

Interleukin-1 and IL-23 induce innate IL-17 production from production IL-17 innate induce IL-23 and Interleukin-1 Immunity 22 Regulation of Regulation ifrnil rdcin f interferon- of production Differential Nat. Rev. Immunol. Rev. Nat. , 193–198 (2010). 193–198 , M. M. tuberculosis

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© 2012 Nature America, Inc. All rights reserved. of Immunology, Department of Microbiology & Immunobiology, Harvard Medical School, Boston, Massachusetts, USA. Massachusetts, Boston, School, Medical Harvard & Immunobiology, of Microbiology of Immunology, Department USA. Massachusetts, University, Boston, Boston Medicine, 18 16 USA. Island, Rhode Providence, USA. Massachusetts, USA. Massachusetts, USA. California, 7 Goldrath Ananda Jakubzick Claudia Tal Shay D Joonsoo Kang Angelique Bellemare-Pelletier Lanier L Lewis Yan Zhou The complete list of authors is as follows: 26. 25. 24. 23. 22. 21. 20. 19. 18. 17. 16. 15. 14. 13. e c r u o s e r  29. 28. 27. 30. Fox Chase Cancer Center, Philadelphia, Pennsylvania, USA. USA. Pennsylvania, Center, Philadelphia, Cancer Chase Fox

Division of Rheumatology, Immunology and Allergy, Brigham and Women’s Hospital, Boston, Massachusetts, USA. Joslin Diabetes Center, Boston, Massachusetts, USA. USA. Massachusetts, Center, Boston, Diabetes Joslin avid Laidlaw avid

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© 2012 Nature America, Inc. All rights reserved. sorted from fetal mice by gating on TCR the ImmGen Project). As antibodies to V cates were sorted unless noted otherwise (complete sorting details available from 3 × 10 and an autoMACS, then were stained and sorted with a FACSAria (~2 × 10 for enriched were Sample preparation for microarray analysis. School. Medical Massachusetts of University the of Committee Use and Care Animal Institutional the by approved were Experiments facility. barrier rodent pathogen–free specific a in housed were and Rock, K. by provided mice. reporter of generation the for used was Consotrium) Trap Gene (International clone cell stem embryonic gene-trap reporter LacZ A described. been have row and column, complete linkage). Data were visualized with a global color color global a with visualized were Data linkage). complete column, and row Pearson’s of GenePattern, module for correlation ing (HierarchicalClustering cluster hierarchical by generated were Pearson’sand coefficients correlation Heat distance maps of for Euclidian of and a set subsets. genes given matrixes correlation and distance generate to R in ‘cor’commands ‘dist’and the with and Pearson’s distance Euclidian tion. were calculated coefficients correlation an for with MEV visualiza genes of before >120 and filtered and gene subset, by normalized transformed, log were Data populations. multiple in genes vary that weight to populations of deviation standard geometric the with ance vari of analysis of variation a of basis the on filtering by subsets among sion for the identification of the 15% of genes with the greatest difference in expres edu/LabMemb PCA program ( done with the PopulationDistances was analysis component Principal GenePattern. of module HeatMapViewer the with visualization before rows on centered were Data linkage. complete distance measurement. Data were log transformed and clustered with pairwise for correlation Pearson the with (column) subset and (row) gene of basis the on data of GenePattern) of module (HierarchicalClustering clustering chical by Heat hierar maps were generated in comparison. the in at one subset least P a had of had a 0.5, among than less of replicates Student’svariation twofold, coefficient than more by expression in differed they if differently lated regu be to considered were genes otherwise, indicated Unless replicates. all of average the of use the through GenePattern of module Multiplot the with identified were regulation in differences with Genes gene. each for value set was used for the consolidation of multiple probe sets into a single mean probe- School), Medical (Harvard Davis S. by written module GenePattern custom a ConsolidatedProbeSets, GenePattern). of module (ExpressionFileCreator array average method multi- with quantile normalization and robust background correction the by normalized were data Raw platform. analysis genomic 1 Notes org/ procedures; operating standard ImmGen (by center with the MoGene Affymetrix 1.0 ST array was done at the ImmGen processing visualization. and analysis Data frequencies); and V3 and V4 thymocytes from fetal mice coexpressing V V5 (~5% of total quencies, all V paired wth diverse V pairedV with TCR atedsorted with (estimated purity, ~95%). The approximate frequencies of TCR mice IL-4–GFP reporter CCR10-GFP arrival. of week 1 within analysis microarray Mice. METHODS ONLINE doi:10.1038/ni.2247 value of less than 0.05, and had a mean expression value of more than 120 120 than more of value expression mean a had and 0.05, than less of value Protocols/ImmGe Male C57BL/6J mice 5 weeks of age (Jackson Labs) were used for for used were Labs) (Jackson age of weeks 5 mice C57BL/6J Male 4 cells; >99% pure) directly into TRIzol (Invitorgen). Independent tripli and and 4 9 Ai2Lc rpre mice reporter Axin2-LacZ , δ δ 4 (50%), V (50%), 4 2 ersPges/SD.htm 6.3 ). Data were analyzed with modules of the GenePattern GenePattern the of modules with analyzed were Data ). γ δ − γ cells; V ); V6 (~8% of total δ δ chains including V T cells by depletion of CD8 of depletion by cells T γ subsets were as follows: V2 (~45% oftotalwerefollows: (~45% subsetsas V2 n Cell prep and sorting SOP.pdf; SOP.pdf; sorting and prep Cell δ Cenpk 5 (40%), all V all (40%), 5 γ 5 paired with V Cd74 l ). ). Where indicated, the PCA program was used ecdn te etoee rti SOLT) protein centromere the (encoding −/− RNA processing and microarray analysis analysis microarray and processing RNA mice were provided by E. Huseby. Mice Mice Huseby. E. by provided were mice γ δ δ δ cells; V δ 4 (25%), V γ + 6.3 4 are not available, immV4 cells were cells that were V 5 δ 0 5 (40%) and various others at lower n Sx-aZ eotr mice reporter Sox5-LacZ and Pooled thymocytes from 4–30 mice − ); V1 (30% oftotal(30% V1 ); γ 1.1 paired with V + δ cells with magnetic beads beads magnetic with cells 5 (15%), others at lower fre http://cbdm.hms ht γ tp://www.immgen. 1.1 Ctsl Supplementary Supplementary − δ V -chains associ γ −/− γ δ δ γ 6.3 (100%)); 2 cells; V cells; δ mice were were mice − cells; V cells; V .harvard. γ 3 δ − 1. t V -test -test γ 4 γ 1.1 to 4 5 γ 5 8 2 − 1 ------,

anti-GATA-3 (L50-823; all from eBioscience); anti-Blk (3262) anti-GATA-3 and (3262) anti-LEF-1 anti-Blk all from eBioscience); (L50-823; anti-ROR (Dan11mag), anti-EOMES (TXRX10), anti-TOX antibodies: with the done following with the Foxp3 Kit was Staining (eBioscience) staining Intranuclear Biosciences). (BD kit Cytofix/Cytoperm the with IFN- and eBioscience) (17B7; Anti-V (Invitrogen). Bio-XCell and biotinylated with the FluoReporter Mini-Biotin-XX Labeling Kit (IFN- anti-CD119 and BioLegend); (D7715A7; anti-IL-6R eBioscience); streptavidin-phycoerythrin-indotricarbocyanine from (all and (37.51) anti-CD28 (HM48-1), (DTA-1), anti-CD48 anti-GITR (20D5), anti-NKG2ACE 244F4), (2B4, anti-CD244.2 2B11), (CXCR4, anti-CD184 174), (CXCR3, anti-CD183 4B12), (CCR7, anti-CD197 anti- CW-1.2), (CCR9, (J43), anti-CD199 anti-PD1 (TY/11.8), CD73 (MEL-15), anti-CD62L (CX5), anti-NKG2D (GL3), (IL-7R antigen; anti-c-Kit anti-TCR (heat-stable M1/69), anti-CD24 (2B8), anti-CD127 A7R34), (5H4), anti-CD122 (IM7), anti-CD44 (53-6.7), anti- CD8 (RM4-5), anti-CD4 (145-2C11), anti-CD3 Biosciences); BD from (all streptavidin-allophycocyanin and (KMC8) anti-CD9 (53-2.1), anti-Thy-1.2 V anti-V (LG.3A10), anti-CD27 (PC61), anti-CD25 used: cytometry. Flow classifications. functional some for used were Genomes) and Genes of (Kyoto Encyclopedia pathway KEGG and Project) Ontology (Gene pathway AmiGO The for analysis. pathway were used inspection and manual software Ingenuity GenePattern. of module HeatMapViewer the in scheme 51. 50. 49. 48. Student’s analysis. Statistical ionomycin. and PMA with culture diately, after or imme stained and digestion) (enzymatic lungs and spleen the from isolated were cells and killed were mice infection, after Two weeks route. aerosol the C57BL/6 8-week-old to 10 × 2 6- with Labs) male (Jackson mice ten to four of infection the for used were Infection with (BD Plug protocols. manufacturers’ the to Golgi according h, 1 after and added Stop Biosciences) Golgi with ug/ml), (1 Ionomycin and ng/ml) 13-acetate; 10 12-myristate (phorbol PMA with °C 37 at h 4 for well) per cells Ex vivo histograms. in visualization for FlowJo in samples independent multiple In (Treestar). FlowJo with analyzed and LSRII BD a on acquired were Data protocols. standard to di- fluorescein for Staining Cruz). Santa and from both (N-19; Biosciences); anti-SMO and BD (D-9) (B56; anti-PLZF anti-Ki67 Signaling); Cell from both (C12A5; in Fisher’s exact test. Fisher’sin exact the program’swith was determined built- significance and statistical Analysis unpaired two-tailed was Data were used. for with F-tests tested and with were normality then Software) analyzed (GraphPad Prism data, cytometry flow of analysis statistical γ 3 (536), anti-V (536), 3

Smits, P.Smits, 2 type of Analysis R.M. B. Lustig, Locksley, & K. Mohrs, K., Shinkai, M., Mohrs, the for important is CCR10 N. Xiong, & C.M. Saylor, A., Sun, M., Xia, Y., Jin, formation. (2002). tumors. liver and colorectal in conductin/axin2 reporter. IL-4 bicistronic a with (2001). vivo in immunity Immunol J. skin-specific of development γ R stimulation. α ; GR20; RDI–Fitzgerald Industries). Anti-V t -test -test et al. et et al. et Dev. Cell Dev.

β 185 M. tuberculosis - P The transcription factors L-Sox5 and Sox6 are essential for cartilage for essential are Sox6 and L-Sox5 factors transcription The d values were generated with Multiplot (Genepattern). For (Genepattern). Multiplot with generated were values The following antibodies to cell surface molecules were were molecules surface cell to antibodies following The δ Negative feedback loop of Wnt signaling through upregulation of upregulation through signaling Wnt of loop feedback Negative , 5723–5731 (2010). 5723–5731 , -galactopyranoside (F1179; Invitrogen) -galactopyranoside was done according 6.3 (8F4H7B7), anti-Ly49C/I (5E6), anti-CCR6 (140706), (140706), anti-CCR6 (5E6), anti-Ly49C/I (8F4H7B7), 6.3 Cells were stimulated t For the identification of genes with different expression, expression, different with of genes identification For the δ -tests. -tests. Pathway analysis was done with Ingenuity Pathway

Supplementary Figure 1 Figure Supplementary 1 (17D1) 1 was (17D1) provided by A. Hayday. IL-17 Intracellular 1 , 277–290 (2001). 277–290 , and analysis. γ δ γ T cells by regulating their migration and location. and migration their regulating by cells T (XMG1.2; BD Biosciences) were detected detected were Biosciences) BD (XMG1.2; 3 colony-forming units of of units colony-forming ex vivo Wild-type strains of , data were concatenated from from concatenated were , data o. el Bio. Cell. Mol. by culture of total cells (2 × 10 nature immunology nature γ 1.1 (2.11) was purified by Immunity γ 2 (UC3-10A6), anti- 2 (UC3-10A6), M. tuberculosis M. γ t (AFKJS-9) and and (AFKJS-9) t

22

M. tuberculosis 15 1184–1193 , , 303–311 303–311 , by α δ 6 - ;