© 2012 Nature America, Inc. All rights reserved. this this work. USA. Massachusetts, 2 1 have nevertheless emerged. The homeostatic distribution and logy is clear, notable parallels in the activity of NK cells and Although a central role for TCR-mediated activation in to specific neither NKRs are because and lineage cell NK the to than lineage cell T the to question by expressed i be also can (NKRs)) receptors’ cell (‘NK cells NK by (ref. NK1.1 marker cell of T cells helper subsets antigens differentiation cells, with somatic recombination T (MHC)-restricted complex nity, tumor rejection and inflammation immune the of arms system, critically adaptive affecting biological processes and in antimicrobial innate immu the both modulate cells CD1d by presented antigens lipid recognizes that (TCR) receptor antigen cell T semi-invariant a of subset A systems. immune adaptive relationships of the the into pheral invariant natural killer T cell ( text, we determined global gene-expression profiles for system thymicimmune mouse the in andnetworks regulatory and peri of comprehensivegene-expression definition erate a high-resolution, of rigorously standardized experimental and analysis pipelines, to gen immunologists and computational of biologists who consortium aim, a through is the use Project (ImmGen) Genome Immunological The after activation. Together our findings highlight a core effector program regulated distinctly in innate and adaptive lymphocytes. T cells. Notably, the program shared by NK cells and program transcriptional with NK cells, similar in magnitude to that shared with major complex histocompatibility (MHC)-restricted and programs subset-specific of gene expression by as part of the Genome Immunological Project. analyses comparative transcriptional High-resolution defined developmental To better characterize this population, we profiled gene expression in Invariant natural killer T cells ( Michael B Brenner Nadia R Cohen underlie the hybrid nature of Shared and distinct programs transcriptional nature immunology nature Received 19 June; accepted 6 November; published online 2 December 2012; NKT NKT cells Broad Broad Institute of MIT and Harvard, Cambridge, USA. Massachusetts, Department of Medicine, Division of Rheumatology, Immunology and Allergy, Brigham and Women’s Hospital, Harvard Medical School, Boston, USA. Massachusetts, The designation ‘NKT’ was coined to reflect expression of NK the expression to reflect coined was ‘NKT’ designation The 2 i 9 6 NKT cell transcriptional ‘landscape’, transcriptional cell and features NKT unique its , because A A list of members and affiliations appears at the end of the paper. should Correspondence be addressed to M.B.B. ( 3 8 , secrete cytokines characteristic of , characteristic the secrete cytokines T , , the of validity the ‘NKT’ designation has into been called i 4 NKT NKT cells are developmentally more closely related 1 Department Department of Pathology, University of Medical Massachusetts School, Worcester, USA. Massachusetts, i , NKT cells to other cell lineages of innate the and lineages to cell other cells NKT 5 , , Patrick J Brennan 1 i 7). Although many other receptors expressed expressed many receptors other 7). Although 5

NKT cells nor expressed on all all on expressed nor cells NKT , , and provide help to B cells & The Genome Immunological Project Consortium aDV 2 . By rapidly producing cytokines, these these cytokines, producing rapidly By . A NCE ONLINE PUBLIC ONLINE NCE i NKT NKT cells) are innate-like T lymphocytes that act as critical regulators of the immune response. i NKT NKT cell) subsets to gain insight 3 i α . Like major histocompatibility NKT cells undergo thymic thymic undergo cells NKT β T cells, cells, T 4 , recognize self and foreign 1 , 5 , , Tal Shay i H NKT cells express express cells NKT A 6 1, 1, T TION . i 1 NKT NKT cell bio . In this con this In . H i i NKT NKT cells. In addition, we found that 2 2 and T i NKT cells. cells. NKT

i NKT NKT cells also operated in constitutively NKT NKT cells 2 3

survival survival , Gerald F , WattsGerald Department Department of Pathology, Brigham and Women’s Hospital, Harvard Medical School, Boston, H 17 17 doi:10.1038/ni.249 ­ - - - ­

tions after activation. after tions popula lymphocyte adaptive other in induced in is and operates lymphocytes innate also that program effector core a represent cells patterns of by constitutively genes expressed both NK and cells Finally,T cells. we show that and transcriptional the MHC-restricted by shared those to breadth in similar are and appreciated and cells NK by shared cells and NK between similarities for basis transcriptional the assessed in expression gene of and developing mature of subsets comparison direct allows which pendium, CD4 of course the over operate cells presenting or cells NK and cells NK of responses the potentiate cells antigen-presenting the from derived cytokines tory inflamma which during cells antigen-presenting with interactions and cells NK Finally, antigens lipid self CD1d-presented in alterations tion-induced to inflamma are reactive which TCRs, their via stress cellular detect ligands stress-induced sense to dence suggests that requirement priming a without functions tor effec their h exert and 24–72 within of at infection sites accumulate cell chemokines, Both for inflammatory receptors express constitutively types kinetics. activation and trafficking their are similar Also of requirements In this report, we shed light on the transcriptional programs that that programs transcriptional the on light shed we report, this In i i NKT NKT cells during ontogeny and in peripheral subsets + NKT cells. Our data demonstrate that transcriptional programs programs transcriptional that demonstrate data Our cells. NKT i and CD4 NKT NKT cells 1 , , Manfred Brigl i 0 NKT cells, in turn, promote the maturation of antigen- of maturation the promote turn, in cells, NKT − 23–

i i NKT cell subsets. Using the ImmGen Project com Project Using ImmGen the subsets. cell NKT 6 NKT cells are similar to those of NK cells NK of those to similar are cells NKT 2 i 8 NKT NKT cells, like NK cells, can use activating NKRs . i i NKT cells engage in similar bidirectional bidirectional similar in engage cells NKT NKT cells are more extensive than has been been has than extensive more are cells NKT i i NKT NKT cells shared an extensive NKT cell development and in per in and development cell NKT 1 , g 3 15– , , Joonsoo Kang d i NKT cells to surface ligands, and and ligands, surface to cells NKT i T cells and in adaptive T cells NKT cells, NKT NK cells, cells and T we cells, [email protected] 1 5 9 These These authors contributed equally to . Furthermore, Furthermore, . 14 , e c r u o s e r 1 5 . In addition, evi addition, In . 4 , i NKT cells also also cells NKT

i NKT cells cells NKT i ). pheral pheral i 10– 20– NKT NKT

1 2 3 2  ------. .

© 2012 Nature America, Inc. All rights reserved. subsets, and the results were log were results the and subsets, two any between twofold over of expression in a change for and expression for prefiltered were genes 0.958); correlation, (mean analysis clustering Klrb1b ( (top), nomenclature (subset thymocytes differentiating ( gene. a single represents symbol Each considered. were subsets all in 0.5 than less of variation of coefficient lists, complete blue; light in differently regulated genes lists, complete in (left), thymocytes of transitions PBS-57, cytometry. 1 Figure adaptive in maturing in programs transcriptional NK1.1 and (ref. CD44 markers activation the dif of by expression in characterized ferences differentiation of stages sequential undergo maturation, During stage. (DP) positive MHC-restricted from diverge Like other T lymphocytes, to specific programs Developmental RESULTS e c r u o s e r  ( experiment each in mice more or three from f Activating Inhibitory e c a

) Surface expression of NKRs, determined by flow cytometry. ( cytometry. flow by determined NKRs, of expression ) Surface P value 10 10 10 10 10 10 10 –6 –5 –4 –3 –2 –1 4; 4; 0 , , Scl7 414

Rag1 PBS-57–CD1d tetramer Supplementary Fig. 1 Fig. Supplementary Klrb1c

F13a1 Cd8 Rag2 10 10 10 10 Rorc Upregulation of the expression of NKRs by by NKRs of expression the of Upregulation a1 Dntt 0 Aarp21 NKR-P1A 2 3 4 5 NKR-P1F NKR-P1D a 0.01 1 Ppr Aqp NKG2D NKG2C NKG2E Cd8 NKG2A Slc15ae NKG2I Ly49H NK1.1 i Ly49C Ly49E Ly49A Ly49F 11 Ly49J 0 Mark NKT cells. ( cells. NKT 11 Ly49I Expresssion, stage1/DP(fold) Klrg1 , , b Klri2 r Supplementary Tables 1–6 Supplementary Klri1 Themis 2B4 10 Wdr7 Cd24a Slc6a19 Klrb1f s 2 Bcl6 8 0.1 α Phxr 10 CD3

i PreT.ETP Dgkg NKT cells at stage 2 versus 2 versus stage at cells NKT -galactosylceramide analog. ( analog. -galactosylceramide 3 Arsi , , PreT.ETP-2A 2 α Il17r ε Klrc1 PreT.DN2A 10 β ETP toDP a Supplementary Tables 7 Supplementary c T cells, we profiled CD44 we T profiled cells, PreT.DN2B 4 ) Genes with a difference in expression (upregulated, red; downregulated, blue) in in blue) downregulated, red; (upregulated, expression in a difference with ) Genes i 1 1 PreT.DN2-3 NKT cells mature in the thymus, where they , , 10 i Zap7 Cd2 NKT cells but not in CD4 in not but cells NKT Klrc2 PreT.DN3A i 5 NKT cells relative to those that operate operate that to those relative cells NKT 2 8

PreT.DN3B 0 ). To characterize the developmental developmental the Tocharacterize ). α -transformed. Data are combined from at least three independent experiments for each population, with cells pooled pooled cells with population, each for experiments independent three least at from combined are Data -transformed. Irf1/4 Relb Cyld Egr2

PreT.DN3-4 Fyn β Nfkb1 Vdr , , 0 T.DN4 Tbx2 NK1.1 CD4 the at cells T Klrc3 10 10 10 10

T.ISP H2-K1 Il4 Plac8 1 0 Icos T.DP 2 3 4 5 Gmap . . ( Stage CD4 T.4 Samhd1 Chn2 Il2rb Art2b Naip3 Smpdl3 100 + 0 1,436 , , Il7 i

T.4SP69 Id2 Il18r d 8 int NKT cells NKT 10 Klrd1 4 r a ) Genes with a difference in expression in in expression in a difference with ) Genes L +

T.4SP24 Zbtb1 y6a 2 – 1 i 1 NKT cells su cells NKT

e + T.4SP24 a , i 10 CD44 g

int 6 CD8 NKT cells at stage 1 (middle) or in in or 1 (middle) stage at cells NKT T.8 , , ), or are representative of at least two independent experiments ( experiments independent two least at of representative are or ), Relative expression b 3 and + – Klri2 Low T.8SP69 ) Quantification of genes upregulated (Up) or downregulated (Down) during the developmental developmental the during (Down) downregulated or (Up) upregulated genes of ) Quantification 4 int i NKT cells at the end of thymic differentiation. ( differentiation. thymic of end the at cells NKT +

P value Stage

T.8SP24 10 10 10 10 10 Stage − 8 10

Supplementary Table 16 Supplementary + 4 + T.8SP24 NK1.1 –4 –3 –2 –1 CD8 ). For For ). and and 0 i

NKT int NKT.44 + 10 2 CD8 Septb1 Nrgn – 8 3 NKT.44 Tmod 5

int – Sox1 High Klrk1 NKT.44 .NK1.1 b Cd244 Klrk1 Klri2 Klre1 Klrc3 Klrc2 Klrb1f Klrb1c Klrb1a Klra8 Klri1 Klrg1 Klrc1 Klrb1b Klra10 Klra9 Klra6 Klra5 Klra3 Klra1 b − 1 T cells relative to their regulation in DP thymocytes (upregulated, pink; downregulated, downregulated, pink; (upregulated, thymocytes DP in regulation their to relative T cells + Hsn g sequently sequently + – (stage 1), (stage

.NK1.1 3

) Expression of a cluster of genes encoding NKRs ( NKRs encoding genes of a cluster of ) Expression – Expression, stage2/stage1(fold) d + double- .NK1.1 2 , only genes with expression above the detection limit in one or more subsets and a and subsets more or one in limit detection the above expression with genes , only – ; red) and other genes (black), derived from a Euclidean distance–based K-means K-means distance–based a Euclidean from derived (black), genes other and ; red) b + Genes (×102) 10 15 20 + + f 5 0 NKG2D cells (%) Ly49A cells (%) 1 DP to - 10 15 20 25 Stage 25 50 75 Stage

0 5 0 Stage Stage 1 1 1 tively by by tively selec modulated genes of subset a identified we point, branch DP ( function unknown of factors in involved be to known thymic between lated ( and respectively 2, 3, stage stage to 2 to stage 1 from stage from 1, stage to stage DP the from twofold least at of ( expression in change a had Project genes 697 and 155 ImmGen 1,850, that the of context the in cells CD44 1 to Down Up Stage Stage ); results were log were results ); 2 2 2 Il17re 3 3 Cxcr 2 to + i Il18rap 3 NK1.1

NKT cells at stage 3 versus 3 versus stage at cells NKT 3

+ Ccr + Klrb1 Klra3 i Ly49C/I cells (%) NKG2A cells (%) i NKT cells at stage 1 versus DP thymocytes; colors indicate indicate colors thymocytes; DP 1 versus stage at cells NKT 25 50 10 20 30 Stage 6 Stage CD4 early-stage by not but 1 stage at cells NKT 0 0 aDV Il1r c Cd44 Ror Stage Rny3 1 Stage Klra9 1 1 Klra10 c − d Fgl A Stage CD44 and 2) (stage Stage Rnu73b

2 147

NCE ONLINE PUBLIC ONLINE NCE 2 2 1

0 Expression, 2 3 3 + int -transformed, row-centered and locally color-scaled. color-scaled. locally and row-centered -transformed, CD4 CD8 /DP (fold) 10 10 a 10 10

) Identification of thymic thymic of ) Identification + + –2 –1

2B4 cells (%) Ly49E/F cells (%) P value 0 1 10 10 10 10 10 10 10 10 15 20 25 10 15 20 25 Stage Stage i 10 NKT cell populations encoded molecules molecules encoded populations cell NKT 0 5 0 5 –2 –1 –7 –6 –5 –4 –3 0 Stage Stage i NKT cell maturation cell NKT 1 1 476 Themi Ccr 534 0.01 Stage Stage Il17r i Ccr

2 0.1 NKT cells at stage 1 versus DP 1 versus stage at cells NKT Slamf e 2 9 Pdlim ) Expression of genes encoding NKRs in NKRs encoding genes of ) Expression b s Il23r 4 Supplementary Tables 1 Tables Supplementary 3 3 Vdr Marcks i Ror 6 Expression, stage1/DP(fold) NKT cells at stage 2 (right); 2 (right); stage at cells NKT CD24a Hells My 1 Expression, stage3/stage2(fold) f Klra3 Map4k5 c ; mean and s.e.m. of three mice). three of s.e.m. and ; mean b Ephb6 + g A Slamf1 0.1 NK1.1 Icos

TION Expression −4 −2 Fig. 1b Fig. Stat4 0 2 4 6 , , Klra5

DP + nature immunology nature (stage 3) thymic thymic 3) (stage 1 1 1 Other genes(76) NKRs (13) , i , , c NKT cells by flow flow by cells NKT Klra6 Stage modu Transcripts ). i. 1 Fig. 4 , as well as several several as well as , Fosl 1 Cd244 Stat Tbx21 Icosl Adh1 Il Vdr Ccr , , 4 2 0 4 Klra Klra9 2

Stage Cxcr a Ccl 3 Rbpms Klrb1c Fasl Itgae ). We found found We ). Plac8 Ccl 5 Smpdl3a 6 206 Klra – Gzma

2 Zbtb1 3 + , , 6 Klrk 100 adaptive adaptive 6 10 Klra10 Klra10 Fcerg1 ). At the the At ). 163 Il10r 6

Stage 1 Klri2 Klra9 Styk1 i Il12rb2 a NKT NKT

3 Klrc3 , - - © 2012 Nature America, Inc. All rights reserved. At the final maturation stage of of stage maturation final the At of development the the of lineage specification cell in functions important have to known Vdr as such (CD4 cells T ( experiments ( experiment each in mice more or three from pooled cells with population, each for experiments Table 16 ( ( gene. a single represents symbol Each included. are subsets all in 0.5 than less of variation of a coefficient and subsets more or one in limit detection the above expression with genes only CD4 in (blue) downregulated or (red) upregulated genes indicate CD4 splenic versus 2 Figure nature immunology nature c Memory CD8 Memory CD4 a ) Expression of genes encoding NKRs in peripheral MHC-restricted T cells and and T cells MHC-restricted peripheral in NKRs encoding genes of ) Expression

Naive CD8 Naive CD4 P value P value Thymic 10 10 10 10 10 10 10 10 10 10 10 10 10 (which encodes the receptor for vitamin D) encoded molecules molecules encoded D) vitamin for receptor the encodes (which 10 10 –8 –7 –6 –5 –4 –3 –2 –1 –5 –4 –3 –2 –1 0 0

); results were log were results ); Zbtb16 Cd4 Expression of NKRs in peripheral CD4 peripheral in NKRs of Expression i i NKT NKT NK + + + + Snord1 60 Cd4

Thymic 0.1 29– T T T T Snord6 0.1 b + Expression (NKT. Expression (NKT. ; mean and s.e.m. of three mice). three of s.e.m. and ; mean Rnu73b 0 CD8 3 c

i and PLZF) factor transcription the encodes (which NKT 1 1 , many encoded molecules not yet been linked to to linked been yet not molecules encoded many , Sgk Rny3 Il4 8 Ccr9 10 Snora34 − Snord82 Snord82/84

Ccr7 3 int i NKT cells (NKT.4 cells NKT Ccr 20 cells; cells; Snora69 i Sgk3 NKT cells ( cells NKT 9 Euclidean distance i

NKT 47.6 54.8 44.0 56.7 59.2 22.3

Chmp1b 30 iii 2 i 4 -transformed and gene-row-centered, and local color scaling was used. Data are combined from at least three independent independent three least at from combined are Data used. was scaling color local and gene-row-centered, and -transformed 4 – aDV 1 – .Sp/NKT.4 .Lv/ NKT.4 Fig. 1 Fig. 40 Klrb1f 1 A Klra3 50 NCE ONLINE PUBLIC ONLINE NCE Klrk1 Klrc1 i Rorc + NKT cells, many of the genes with with genes the of many cells, NKT d 59.2 63.1 72.1 72.0 76.3 .Sp fold Supplementary Tables 7 7 Tables Supplementary + Fcer1g

NK Ror ii .Lv fold) ). Although some of these genes, genes, these of some Although ). 60 i Cd7 Klrc3 Klrc2 − Klra10 b c .Sp; top) or in liver CD4 liver in or top) .Sp; Il23r Klra9 Naive CD4 CD4 liver and spleen on NKRs of expression ) Surface 70 Gzmb ) Il23r CD244 1 Gzma Klra10 Fcer1g 0 Gzmb 99 Klra9 Klra5 8 9 Klra5 76.3 56.7

0 Memory CD4 + T + and CD4 and

+ NKG2D+ cells (%) Ly49A+ cells (%) b 44.0

72.0 T 2.5 5.0 7.5 25 50 75 A 0 Spleen Spleen 0 iii TION ii

Naive CD8 −

i CD4 CD4 NKT cells. ( cells. NKT

54.8 Liver Liver Memory CD8 72.1

+ + –

+ i i − NKT NKT T cells relative to their expression in CD4 in expression their to relative cells i NKT cells (NKT.4 cells NKT and

i + + NKT NKT 63.1 47.6 NKG2A cells (%) Ly49C/I cells (%) 25 50 + 10 0 0 T 8 Spleen Spleen a ). ). ) Genes with a difference in expression in splenic CD4 splenic in expression in a difference with ) Genes

Liver Liver cells pooled from three or more mice in each experiment. each in mice more or three from pooled cells with population, each for experiments independent three least at from log were results included; were 120 above values expression mean with genes Only interest). of areas indicate (i–iii subsets intersecting between distance Euclidean average indicate areas outlined in numbers probes; variable i and T cells between relationship the to magnitude in similar is cells 3 Figure to secrete different types of cytokines In both mice and humans, CD4 peripheral of signatures Transcriptional of context broader the in in maturing occurred that changes gene-expression tinct dis and shared characterized comprehensively data these Together program. gene broader a of part be may NKRs of expression the of ( transition of over 3 NKRs the course the DP–to–stage as that of encoding genes kinetics expression same the followed genes of number large a that 2b Fig. maturation of Supplementary course the over NKRs surface of sion thymic of ( cytometry exceptions few a with development, cell T early in i developing in selectively occurred NKRs encoding genes of ulation that of NK ( splenic cells expression of transcripts NKRs encoding several similar in amount to ( receptor lectin family, which includes activating and inhibitory NKRs to that belong the killer molecules encoded upregulation the greatest NKT cells. Euclidean-distance matrix calculated with the 15% most most 15% the with calculated matrix Euclidean-distance cells. NKT NKT NKT cells but not T in maturing cells or MHC-restricted at any stage Fig. 1 Fig. i NKT cell subsets (subset nomenclature (top), (top), nomenclature (subset subsets cell NKT + +

+ 2B4 cells (%) Ly49E/F cells (%) 10 10 15 .Lv) versus liver CD4 liver versus .Lv) 0 Spleen 0 5 Spleen c

, bottom). By the final stage of ontogeny, ontogeny, of stage final the By bottom). , The global transcriptional relationship between NK and and NK between relationship transcriptional global The

Fig. 1 Fig. Liver Liver 2 -transformed and mean-centered. Data are combined combined are Data mean-centered. and -transformed + and CD4 and a NKR-P1D NKR-P1F g , c NKG2 NKG2 c NKG2E NKG2 ), which suggested that progressive upregulation upregulation progressive that suggested which ), NKG2I Nkp46 Ly49H Ly49C Ly49E Ly49A KLRI NK1. Ly49F ), or are representative of at least two independent independent two least at of representative are or ), Ly49J Ly49I Klrg1 2B i NKT cells confirmed the increase in expres in increase the confirmed cells NKT D C A 2 1 4 Supplementary Fig. 2a Supplementary ). K-means clustering analysis determined determined analysis clustering K-means ). − − + T.4Nve.Sp

α

cells (change in expression of over twofold); twofold); over of expression in (change cells i i NKT cells, determined by flow cytometry. cytometry. flow by determined cells, NKT NKT cells (NKT.4 cells NKT β T.4Mem.Sp T cell development. cell T +

CD4 T.4Mem44h62l.Sp and CD4 T.4Nve.LN + T T.4Mem.LN 5 , 32 T.4Mem44h62l.LN , 3 T.4Nve.PP 3 − . In addition,

i T.4Nve.MLN NKT cells have been reported T.8Nve.Sp − i .Lv; bottom); colors colors bottom); .Lv; CD8

NKT cells NKT T.8Mem.Sp Supplementary Supplementary +

T.8Nve.LN i + NKT cells (NKT.4 cells NKT ). ). In upreg addition,

e c r u o s e r T T.8Mem.LN

T.8Nve.PP Low i Relative expression i NKT cell subsets

NKT cells had had cells NKT T.8Nve.MLN Fig. 1 Fig. NKT.4 (Fig. 1 (Fig. i NKT

i NKT.4 + NKT cells cells NKT .Sp

NKT.4 i e + NKT NKT

). Flow Flow ). .Lv NKT. – Ncr1 Cd244 Klrk Klri2 Klre1 Klrc3 Klrc2 Klrb1f Klrb1c Klra8 Klrg Klrc1 Klrb1b Klra10 Klra9 Klra6 Klra5 Klra3 Klra1

High f .Sp + and and

.Sp) .Sp) 4 – 1 1 .L v  - - -

© 2012 Nature America, Inc. All rights reserved. used (subset nomenclature, nomenclature, (subset used in as (categorized genes of ( (bottom). category each in expression in a difference with genes of proportion and top), Results; in defined (as of plot) (above subsets ( T cells. and cells NK 4 Figure hypothesized that NKR expression by Because and cells NK by shared patterns Transcriptional i of CD4 expression ( number of genes were expressed differently in different similartissues to regardlesseach other than were CD4 counterparts showed that CD4 shown). In addition, comparison of liver than that in most MHC-restricted that in thymic of NKR-encoding mRNA in peripheral i NKR offrequency and withhigherexpression CD4in foundthatgenesencodingwereamongNKRsmodulated,most those from each other than are liver pulmonary and 3 Fig. CD4 in respondingsubsets in the lung, only 17 transcripts varied in expression cellsinthespleen, and 261genes were expressed differently inthecor Whereas 159 genes were expressed differentlyCD4 and in CD4 functional differences, we assessed the gene-expression profiles of CD4 characterizetranscriptionalthe distinct basisthatmay functionally underlie subset-specific be to suggested been have liver the from e c r u o s e r  experiment. each in mice more or three from pooled NKT cells subsets may serve organ-specific functions. NKTcells, as determined by flow cytometry (

C C B B A A a 2 1 2 1 2 1 SupplementaryFig. 3 19.2% NKT.4 and +

versusCD4 i

Characterization of shared and different gene expression in in expression gene different and shared of Characterization + − NKT.4 .Lu we features NK with cells, share NKT cells broad functional

i SupplementaryTables9 + mice.lungsofspleen,liverand thefromsorted cells NKT C

i .Lv NKT C NKT.4 1 i 2 NKT cells at stage 3, was maintained at higher amounts + A B .Sp i NKT cell subsets may be more functionally distinctfunctionally more be may subsets cell NKT 1 2 NKT.4 + Supplementary Fig.

– CD4amongcells

43.9% .Lu

a NKT.4 − A i ) Genes with a difference in expression in steady-state steady-state in expression in a difference with ) Genes hepatic B 2 NKT cells, NK cells and T cells, separated by category category by separated T cells, and cells NK cells, NKT – 1

36.9% NKT.4 .Lv a – ).In liver and spleen, this reflected greatera ) in relevant selected functional gene groups. In all heat maps, rows are mean-centered and normalized, and local scaling is scaling local and normalized, and mean-centered are rows maps, heat all In groups. gene functional selected relevant ) in Supplementary Table 16 Supplementary .Sp

NK NK.Sp − NK.Lv i NKT cell subsets. In all three tissues, we i − populations were more transcriptionally NKT subsets( NKT subsets thanCD4in T.4Nve.Sp α Relative expression β Low + T cell subsets (

T.4Mem.Sp – i NKT cells, across tissues. A small 14 − i T

i i NKT NKT cells might reflect a much 4

NKT cells, although lower than NKT cell subsets to their splenic T.8Nve.Sp i ), which supported the idea that ). That suggested that splenicthatsuggested That ). NKT cells than among CD4amongthan cells NKT High T.8Mem.Sp Fig. 2 Fig. Fig. or NK i i i NK NK NK T T T ). Data are combined from at least three independent experiments for each population, with cells cells with population, each for experiments independent three least at from combined are Data ). ≠ ≈ ≈ NK

2 + Fig. 2 T T versus CD4 b a b + , ).The expression subsets ( Supplementary i Tr NKT cells NKT anscription C C B A B A activity c factor 2 2 1 1 2 1 and data not

NKT.4

+ − NKT.4 Fig.2

.Lu 3 i i NKT 4 i NKT cells, cells, NKT + NKT

NKT.4 To . .Lv

NKT.4 + a + +

.Sp -

– NKT.4 .Lu whether the NK cell– NK the whether determine To shown). not (data variability greatest the with genes of 15% the just not and genes all analyzed we when maintained were CD8 and cells larger than that the distance separated Euclidean between distance probably included a substantial proportion of expression similar by cells, we sorted memory CD4 memory CD8 Although ii). area ( cells NK and cells T MHC-restricted all between distances in ( gene expression similarity in the greatest amongvariability the with analyzed subsets (presented as a matrix probes gene of 15% the using subsets, compared pairwise of patterns gene-expression the of similarity the of measure a tance, and T cells cells, NK state of steady- subsets between distances Euclidean we calculated T cells, cell– NK between is now appreciated. To relatedness the extent assess of transcriptional larger program transcriptional shared by NK cells and NKT cells than data data supported the idea of an substantial unexpectedly transcriptional cells was not limited and to the shared expression cells of NKRs. TogetherNK these between relationship transcriptional the Thus, the analysis remained unchanged essentially ( removing NKRs and related from molecules the data. The outcome of after matrix distance the of we recalculated NKRs, expression shared –

NKT.4 .Lv

– Fig. NK NK.Sp .Sp

NK.Lv Relative expression 3 Low T.4Nve.Sp ). Mature Mature ). i i NKT cells NKT and cells NK at cells and level, a to global compare the NKT cell relationship and the relationship of relationship the and relationship cell NKT aDV T.4Mem.Sp T

T.8Nve.Sp + + High T cells and to certain memory CD4 A T.8Mem.Sp ( cells T NCE ONLINE PUBLIC ONLINE NCE Rfx3 Klf7 Klf2 Zbtb16 Rora Rbpj Nr1d1 Npas2 Klf4 Hic1 Gata3 FosB Fo Etv5 Cebp Zeb2 Tshz3 Tcf4 Pbx Nfil3 Mef2c Lass6 Lass2 Klf12 Irf8 Hhex Esr1 Eome Elk3 Cbfa2t3 Thra Srebf2 Pcgf Notch1 Nfatc2 My Etv3 Ets2 Egr1 Bcl11b Zhx2 Trim28 Tcf7 Smad7 Rere Lef1 Id3 Foxp Foxo1 Foxk1 Bmy Bach2 Tbx21 Smad3 Rxra Nrid Klf6 Bhlhe40 Jund Junb Jun Jdp2 Aff3 Arntl s i c i 3 2 c 6 NKT cells had a somewhat closer relationship to to relationship closer somewhat a had cells NKT 1 NKT cells and NK cells showed a high degree of of degree high a showed cells NK and cells NKT s i NKT NKT cells (CD44 i NKT cell relationship was dependent on the the on dependent was relationship cell NKT d c Fig. Fig. Cytokine and TCR signaling component chemokine i signalin NKT cells. We determined Euclidean dis Euclidean We cells. NKT determined pathway i NKT NKT cells and NK cells was only slightly 3 + C C B B A A C C B B A A 2 1 2 1 2 1 T cells through the use of markers with Fig. Fig. , area iii). Those relative relationships relationships relative Those iii). area , 2 1 2 1 g 2 1 b s – NKT.4 d NKT.4 3 ) Expression ) Expression NKT.4 + + , area i), in to contrast the greater A .Lu NKT.4 .Lu TION

i +

NK NKT.4 + i .Lv i NKT.4 NK NKT cells NKT from cells naive CD4 .Lv + T NKT.4 + and CD62L .Sp T NKT.4 + .Sp NKT.4 – .Lu NKT.4 – nature immunology nature .Lu Supplementary Fig. Supplementary 5 – i

NKT.4 NKT cells. The average .Lv NKT.4 – .Lv NK.Sp – NK .Sp –

NK.Sp + NK .Sp NK.Lv T cells than to NK

Relative expression NK.Lv lo Lo T.4Nve.Sp Relative expression ) ) and thus they H w

Low T.4Nve.Sp i

T.4Mem.Sp to cells NKT T.4Mem.Sp T

T.8Nve.Sp T T.8Nve.Sp

ig T.8Mem.Sp High h T.8Mem.Sp Fig. i Ripk Pycar Il17rb Il12rb1 Gfi1 Cxcr6 Mapk3 Ki Jak1 Cx3cr1 Tnf Jak3 Il7r Il4ra Il27ra Il21r Egr1 Ptpn6 Il6st F2rl1 Ccr7 Klf6 Irak2 Il18ra Il12rb2 Ccrl Ccr5 Ccr2 Ccl5 NKT NKT t Ripk2 Gata3 Bcl2a1d Plcg Zap70 Thy1 Themis Skap1 Prkd2 Plcg Kcnn4 Itk Cd3 Ubash3 Ptpn Cd247 Ccr Ptpr 2 2 7 e j 2 1 + 6 T 3 ). ). - ,

© 2012 Nature America, Inc. All rights reserved. by NK cells, cells, NK by differently expressed be to known molecules chemokine-related or many transcription factors, TCR signaling components and cytokine- i (C that was higher expression and with genes cells; (B higher was that cells T and cells lower (A in NK and expression cells similar with genes subsets: three the of two in patterns common of on basis the categories six following the into genes modulated the classified ( groups three the in expression in differences had genes remaining the of (1,192) 20.2% that Wefound set. data Project ImmGen entire the over variability CD4 steady-state peripheral (ANOVA) of way variance of analysis to compare transcriptomes the by regulated concordantly genes specific the We to identify sought next distinct and Shared between that to nitude between relationship immunity (BP00157) NK cell–mediated Biological process category A Table 1 nature immunology nature categories ( Cell motility(BP00287) pathway (BP00107) mediated signaling Cytokine- andchemokine- (BP00255) mediated immunity Cytokine- orchemokine- in agivenbiologicalprocessrelativetorepresentationthatwouldoccurbychance. with DAVID software;enrichmentindicatestherepresentationofgenesincategoryA system). Enrichmentand in parentheses(leftcolumn)indicatemoduleidentifiers(PANTHER classification Functional biological process–enrichment analysis of genes in category A (BP00102) Signal transduction (BP00148:) Immunity anddefense NKT NKT cells than in both T cells and NK cells ( i NKT cells, NK cells and T cells. For this purpose, we used one- used we purpose, this For cells. T and cells NK cells, NKT + or CD8

2 Functional pathway–enrichment analysis Functional of pathway–enrichment genes in ) ) than that in in expression T genes with similar cells; 1 Fig. Fig. 4b i NKT cells and T cells partitioned as expected in these these in expected as partitioned cells T and cells NKT + T cells. We T low cells. with genes from analysis the excluded

– d P

). ). For example, genes encoding the values(fromamodifiedFisher’s exacttest)werecalculated i P

NKT cells and NK cells that was close in mag in close was that cells NK and cells NKT i Klrk1 Klrc2 Genes present Ccrl2 Ccr2 Ccrl2 Xcl1 Ccrl2 Dock5 Ccr5 Sema4a Rgs3 Dok2 Dock5 Ntng2 Irak2 Rhoc Plcb3 Ccrl2 Prkx Klrk1 Adora2a Gna15 F2rl2 Klrc1 Klrb1f Sh2d2a Il18rap Il12rb2 Klrk1 Cysltr2 F2rl2 NKT cell programs cell NKT < 0.05 (Bonferroni corrected)). We further further We corrected)). (Bonferroni 0.05 < i i

NKT cells and naive T cells. T naive and cells NKT NKT cells, NK cells and naive and memory memory and naive and cells NK cells, NKT aDV , , , , , , , , , , , , , , , , , , , Il12rb2 Ccl5 , Inpp5d Ccr2 , , , , Ccr2 Fasl Abr Dusp2 Il12rb2 Ccr5 Coro2a Coro2a , Klrb1c Klrc3 Abi2 Klrc2 Sema4a Ccl5 Klrc2 , Ifng , Smad3 , Xcl1 Diap1 Arhgap26 , Klrc3 , , Adora2a A Lgals3 Ifng Ccr5 Klrc1 Cysltr2 , NCE ONLINE PUBLIC ONLINE NCE , , , , Rxra , , , Inpp5d Anxa1 , , , , Chn2 Rhob Ifng Ccrl2 Itgb2 , , , , Klrb1c , , Ifng Klrc3 S100a6 , , i , Irak2 , Ccr5 Cd97 Xcl1 , NKT cells that was higher (A that NKT was cells higher Klrc1 Fgr Dok2 Zfp36l2 Ccr2 , 1 Ccr5 , Gem ) or lower (B lower or ) , , , Gzmb Anxa1 , , , , Gpr65 Tbx21 Ccr2 , , , , Dusp5 Klrb1f , , Fgl2 , , Xcl1 , Cd97 Ccl5 Abi2 , , , , , Rgs2 Fgr Ccl5 Ptprj , Klf6 Ifng , , , , , , , , , , , , , Fig. 4 , , , , , , Enrichment 13.70 (fold) 3.68 4.31 7.31 1.55 2.11 a 2 1 ) than that in NK NK in that than ) ). ). Genes encoding A ) ) or lower (C TION

transcription transcription 1 1.33 ×10 1.11 ×10 3.57 ×10 2.98 ×10 2.49 ×10 6.50 ×10 ; designations

P value i NKT NKT 1 2 ) ) or ) ) in 1 −5 −3 −3 −3 −4 −4

-

cells and T cells than in NK cells; cells; NK in than cells T and cells and cells NK in upregulated be to known Functional biological process–enrichmentanalysisofgenesin categoryC (BP00040) mRNA transcription cascade (BP00111:) Intracellular signaling (BP00281) Oncogenesis Apoptosis (BP00179) apoptosis (BP00263) Inhibition of Oncogene (BP00265) ANOVA the were ‘passed’ regulated uniquely in that genes the of fifth one about addition, In in patterns expression similar with genes of number the to similar was survival, which probably reflected the uniquely activated phenotype phenotype activated uniquely the reflected probably which survival, and proliferation including processes, biological distinct in involved C (category cells relative to their expression in NK cells and MHC-restricted T cells ( motility cell immunity, chemokine or cytokine responses, signal transduction, and cell–mediated NK including functions, encod effector with genes molecules for ing enrichment significant demonstrated Methods) database DAVID bioinformatics the from A category in genes of analysis process–enrichment segregated as anticipated into category B components CD3 signaling as such TCR encoding Genes cells). T in than cells A category into tioned Fig. 4 ( T-bet factor transcription the encoding genes The cells). T C into category in ( (IL-12 12 Fig. 4 factor PLZF ( as in (BP00116) Jnk cascade Biological process category C Table 2 C in the steady-state of 1 We found that the number of genes with similar expression patterns and C and i i NKT cells than in resting T cells or NK cells or NK T cells resting in than cells NKT NKT cells and T cells (523 genes (43.88%); categories B categories (43.88%); genes (523 T cells and cells NKT Table 1 c b i NKT and NK lineages (440 genes (36.91%); categories A ), ), and a component of the receptor high-affinity for interleukin ), ), as well as receptor for the IL-12RB2 IL-12 (

Functional pathway–enrichment analysis Functional of pathway–enrichment genes in 2 ). Jnk,kinase. ; ; 1

Il12rb1

Fig. 4 Fig. 1 ε Zbtb16 ) showed enrichment for genes encoding molecules molecules encoding genes for enrichment showed ) Table ( i Cd3e 1 NKT cells ( cells NKT (higher expression in expression (higher a ); ); and and 1 ; Fig. 4 Fig. ), ITK ( ITK ), Rbpj Dennd4c Nr1d1 Junb Fosb Dtx4 Dusp6 Jund Cxcl10 Rab4a Il4 Junb Lund Fos Gadd45b Bcl2a1a Cebpb Il4 Bcl2a1a Cebpb Il4 Jund Fos Junb Vav3 Genes present ). The group of transcripts upregulated in upregulated ). The group of transcripts Fig. 4 , , , Supplementary Table 15 Supplementary 1 , , Cap2 Bcl2a1d Bcl2a1d (higher expression in in expression (higher , , , , Vav3 Vav3 , , , , , , Cebpb Arntl Etv5 Dusp1 d Hic1 Gadd45b Emp1 Etv5 Cxcl10 , , , , , Fam129a b , i Table Socs2 Socs2 ), all known to be have higher expression expression higher have be to known all ), NKT NKT cells (229 genes (19.21%); categories Gata3 Rab27a Nfkbia Plk3 , ), the chemokine receptor CXCR6 ( Itk , , , , , Tnfsf14 Thoc4 , Emp1 Vav3 , , Lmo4 Lmo4 , klf4 ), PLC- ), Junb Zbtb16 Fos , , , , , , Lmo4 , , Jun Bcl2a1c Bcl2a1c Dusp1 , 2 Jun Bcl2a1b Bcl2a1b , , , junb , , , , ). Tbkbp1 Socs2 Npas2 Fig. 4c Fig. Etv5 , , Gfi1 Jun Jun , Jund Nfkbia , , Jund , , γ Nfkbia Rora i , , NKT cells than in NK cells or in than NK cells cells NKT , 1 ( 1 Jun , , , 1 , , , , , (higher expression in , , Plcg1 3 , , , , d 8 , (as described in Online Online in described (as ). Functional biological biological Functional ). Enrichment 29 i ) and Zap70 ( Zap70 and ) 10.35 14.49 (fold) 1.76 2.78 3.13 3.25 8.11 K cells NKT e c r u o s e r i ). Thus, in addition addition in Thus, ). NKT cells and NK NK and cells NKT , 30 , Il12rb2 35 1 , with software software with 3 6 , partitioned , partitioned 9.97 ×10 5.12 ×10 7.59 ×10 1.88 ×10 2.13 ×10 5.53 ×10 5.45 ×10 1 (presented 1 ; ; 3 P 1 and B and 7 Fig. Fig. 4 value and A parti , Zap70 Tbx21 Cxcr6 i i NKT NKT NKT NKT −3 −4 −3 −3 −4 −5 −5 d 2 2 ). ). ), ),  - - ) ) ; ;

© 2012 Nature America, Inc. All rights reserved. ferentiating CD4 ferentiating in dif and were not reactivated stage by DP thymocyte the regulated genes were these thymic precursors, then largely shut down or down in and NK cells upregulated genes and cells T in category A we analyzed by hierarchical clustering the expression patterns of genes tional program shared by mature To determine at what point during thymic development the transcrip Thymic induction of programs shared by NK cells and analysis. Euclidean-distance the with consistent cells, T by shared that to magnitude in similar and program with NK steady-state transcriptional cells that was extensive program, gene-expression distinct a having to e c r u o s e r  mature NK cells and and cells NK mature trast to genes in category A A category of set gene the between CD4 ( test In contrast, the distribution Kolmogorov-Smirnov calculated by comparison of naive the splenic by determined as genes, for expressed all distribution from different the baseline significantly with values higher toward shift increasing an showed progenitors, DP their in that with developing in gene each of expression the of comparison A category in genes of distribution the observation, 3 expressed more than 90% of the genes ( same those genes, upregulated cells so that thymic

a Genes expressed (%) PreT.ETP + 100 PreT.ETP-2A or CD8 40 60 80 PreT.DN2A

ETP toDP PreT.DN2B PreT.DN2-3 i NKT cells. Although approximately 75% of the shared shared the of 75% approximately Although cells. NKT

1 PreT.DN3A ETP toDP + over the course of the differentiation of MHC-restricted

T cells with DP cells showed no significant difference difference no significant showed DP with cells T cells PreT.DN3B

+ PreT.DN3-4 or CD8 or

i T.DN4 NKT cell maturation. All three distributions NKT were cell maturation. All three distributions T.ISP i NKT cells relative to their expression in T cells T cells in expression their to relative cells NKT T.DP T.4

CD4 +

+ T.4SP69 8 int 1 T cells. In contrast, differentiating differentiating contrast, In cells. T , , the genes downregulated in significantly

+ T.4SP24 T.4SP24 + Relative expression Low

CD4 T.4int8 int CD8 i NKT cells and NK cells was induced, T8SP69 –

i + + 1 NKT cells were expressed in early were NKT cells expressed +

and all expressed genes. In con In genes. expressed all and T.8SP24 + T.8SP24

CD8 int

i NKT.44 NKT Fig. 5 – High

+ NKT.44 – NK1.1

i NKT.44

NKT cells and other other and cells NKT +

a NK1.1 –

i + ). ). Consistent with that NKT NK1.1 – i NKT cells shared a shared cells NKT + i NKT cells NKT cells at stage 1 b 0.1 , calculated by calculated , Stage 2 Stage 3 Stage 1 i i NKT cells NKT cells cells NKT i. 5 Fig. Expression, i NKT NKT ( ( ( P P P =1.28 =9.14 =3.97 α 1 b ). ). β - - - -

× × × i NKT/DP 10 at steady state. at steady in differently and these data suggested that most of the gene program shared by NK cells in expression C (category subsets cell or NK T cell in not A (category cell– NK shared the for than prominent less was splenic in expressed were did than cells) T in sion and cells NK in (downregulated A category in of genes proportion smaller ( an even expressed T cells genes overexpressed for enrichment significant A category in genes ( resident IEL A of sion of in a category cluster genes large splenic CD44 in observed resting T cells. A subset of those genes was upregulated in CD44 splenic in expression low relatively of that to similar phenotype effector an have tions CD8 (IEL) lymphocyte intraepithelial Distinct V and V the whereas polarized, IL-17 δ TCR the of use their of basis the include subsets lymphocyte 10 10 Fig. 6 Fig. -chain variable region (V region variable -chain –19 –48 –15 The genes shared by NK cells and and cells NK by shared genes The ) ) ) i 1 NKT cells, as well as part of the program of genes upregulated upregulated genes of program the of part as well as cells, NKT (fold) 0 γ a 1.1 ). Distributions made by comparison of the expression of of expression the of comparison by made Distributions ). + aDV V 1 γ ), a portion of the genes upregulated in in upregulated genes the of portion a ), δ + δ i 6.3 V γ NKT cells, was also used by populations of of populations by used also was cells, NKT T cells than in the reference NK and A δ 100 NCE ONLINE PUBLIC ONLINE NCE γ T cell subsets ( subsets cell T − 2 subsets) secrete mainly T mainly secrete subsets) + and V 1 by splenic or IEL IEL or splenic by CD8 (category A experiment. each in mice more or three from pooled cells with population, each for experiments independent three least at from combined are Data test). (Kolmogorov-Smirnov P axis. vertical the along order rank in are genes expression); in change to (according thymocytes DP of that to 3 relative 2 and 1, A category in genes ( (below). maturation thymic of course the over expression for threshold the 4b– Fig. in as presented (top; correlation Pearson with i and T cells of maturation thymic the of course and cells NK peripheral in upregulated significantly genes of of maturation thymic and cells NK 5 Figure data extended our earlier observation that that observation earlier our extended data thymic ferentiating transcriptional program noted above. Innate above. noted program transcriptional with cytes innate features might also have the We next sought to determine if other lympho The maturation. thymic i part of the program transcriptional shared by end of development and indicated that a large i NKT cells, ordered by hierarchical clustering clustering hierarchical by ordered cells, NKT NKT cells and during NKT cells NK was acquired cells NKT cells acquired NKR expression at the the at expression NKR acquired cells NKT values, category A category values, i γ NKT cells, although most of those genes genes those of most although cells, NKT 2 γ δ − δ + i ). The subset bearing V bearing subset The ). cells. Notably, we found that the expres T cells (data not shown). Although it it Although shown). not (data cells T NKT cell programs in in programs cell NKT T cells ( T cells γ δ d

γ T 2 Transcriptional programs shared by shared programs Transcriptional ); and frequency of genes exceeding exceeding genes of frequency and ); Supplementary Fig. 7a Fig. Supplementary i − NKT cells relative to their expres their to relative cells NKT

γ subsets (including V (including subsets cells that can be categorized on on categorized be can that cells 2 -chain variable region (V region variable -chain A ) were not elicited as much in dif i i NKT cells (category A (category cells NKT during acquired are cells NKT TION Supplementary Fig. 6 Fig. Supplementary i NKT cells (category A (category cells NKT γ δ T cells and T cells showed showed cells T and cells T 1 − 1

in in

1 versus all expressed genes genes expressed all versus γ 1 i i ) also had relatively high high relatively had ) also NKT cells. ( cells. NKT NKT cells as in CD4 cells NKT was higher in all tissue- in all higher was nature immunology nature δ H i T cells, similar to that that to similar cells, T NKT cells at stages stages at cells NKT 1 or T or 1 α α i NKT cell program program cell NKT + b i i

) Distribution of ) Distribution NKT NKT populations i. 6 Fig. NKT cells NKT γ δ H gd i γ T cell popula cell T NKT cells but but cells NKT 2 cytokines 2 2 tends to be be to tends 2 a ) Expression ) Expression T cells T cells 1 γ ). Together ). 1.1 ) over the the ) over b γ ). IEL IEL ). δ ). These These ). +

T cells cells T 40 V γ 1 ) and and ) ) had had ) , δ

4 6.3 + 1 . or 3 γ 9 δ + 2 - - - - ­ .

© 2012 Nature America, Inc. All rights reserved. CD8 in maintained was program genetic this and expression, higher for enriched significantly expression) in change (of distribution a with cell– ( infection after h 48 category A expressing (OVA)-reactive ovalbumin an of expression transgenic have (which OT-I of mice spleens the from A categories in of genes expression the ined i and cells NK with characteristics functional shares that population i cell– NK shared the of genes of expression the We investigated next cell– NK The nature immunology nature cell– NK of the genes human homologous of the expression higher cantly ( virus stomatitis CD8 of genes of the NK cell– Fig. 8 CD8 A (category and in NK lated cells T by differently C of in genes category portion only In a limited contrast, NKT cells, including the expression of certain NKRs certain of expression the including cells, NKT NKT cell transcriptional program in CD8 in program transcriptional cell NKT a

cell populations ( populations cell T + + + i i NKT cell shared program (category A (category program shared cell NKT NKT cell shared program showed considerable upregulation, upregulation, considerable showed program shared cell NKT ). ). We obtained similar results when we examined the expression T cells from OT-I mice with infected OVA-expressing vesicular T cells, were partially repressed after infection ( memory T cells as late as day 100 after infection ( infection after 100 day as late as cells T memory NK

iNKT 1 Listeria monocytogenes Listeria became upregulated in effector CD8 2 γδ ), ), which by are definition in widely expressed naive OT-I T.V Spleen i i NKT cells) followed a similar pattern in effector CD8 in a pattern followed NKT effector cells) similar γδ NKT cell program in CD8 in program cell NKT γ 2 – T.V Low Relative expression γδ γ 2 + upeetr Fg 9 Fig. Supplementary T.V Supplementary Fig. 7b Fig. Supplementary γδ γ 2 – Fig. 7a Fig. i

NKT cells relative to their expression in T cells expression to their relative cells NKT T.V CD44

i γδ γ 2 + NKT cell shared transcriptional programs in NKT shared cell transcriptional T.V CD44 + aDV γδ IEL γ 5 – T.V + , High b γδ γ 5 + A ). By day 6, however, genes of the NK NK the of genes however, 6, day By ). T.V NCE ONLINE PUBLIC ONLINE NCE α γ 5 – β γδ CD44 T.V . Only a small fraction of genes in of genes fraction a small . Only TCR) after infection with OVA- with infection after TCR)

γ 5 + + CD44

+ . e lo on signifi found also We ). b 0. + T cells 1 1 ). The genes downregu genes The ). + 1 and A and effector T cells, a cell cell a cells, T effector ) in human peripheral peripheral human in ) + IEL Spleen T cells at 12, 24 and

A ( 2 P Supplementary Supplementary in CD8 in TION =3.90 ( P 4 =4.67 2 Expression, 1 . Weexam . (expressed (expressed 1

Fig. 7a Fig. × 10 + × –34 T cells T cells 10 ) –41

γδ , b )

T/ ). ). + - - - T

(fold 10 than is now appreciated. Our analyses showed that the transcrip the that patterns that tional showed analyses Our appreciated. now is than that NK and cells Wehypothesized relatives. distant developmentally are which cells, levels. at post-transcriptional controlled be thus may analyses The functional regulation of those and other genes not detected in our over variation of the course transcriptional Bcl-11b of ontogeny the affect tions both in upregulated were Egr2, and Ras-MAPK NF-kB, of subunits RelB and p50 the as such NF- the of members encoding genes cell were not expressed in an NKRs and pathways, NF- (MAPK) kinase protein factor activated transcription the of components T-bet, receptor, D vitamin the PLZF, encoding genes i of of maturation thymic the modulate specifically probably encod that but cells genes bi the of affect to number known previously large not a molecules ing identified thus have We point. by specifically expressed of genes programs transcriptional we have defined T cells, comparing developing NKT cells were consistent with published reports and included included and reports published with consistent were cells NKT ) Mature Many genes reported to be involved in the development of of development the in involved be to reported genes Many i NKT cells. NKT 4 (data not shown). Other genes encoding molecules known to to known molecules encoding genes Other shown). not (data 4 6 and the chromatin modifier Med1 (ref. i NKT NKT cells share several innate featuresfunctional with NK 100 i NKT cells at stage 1 shortly after the DP branch branch DP the after shortly 1 stage at cells NKT i NKT cells share a broader transcriptional NKT program share cells a broader transcriptional i NKT cells shared with NK cells made up nearly nearly up made cells NK with shared cells NKT i NKT cells with differentiating MHC-restricted i several days after their activation. their after days several effector in elicited was to be operating during the maturation of of maturation the during operating be to defined. incompletely remains attributes active in lymphocytes with similar functional involved relate programs to those scriptional of immunity cell T TCR, i functional a express they Although DISCUSSION Thus, a large proportion of the genetic genetic the of and cells NK by shared program proportion large a Thus, tions than in naive CD8 CD8 memory effector blood experiment. each in mice more or three from pooled cells with population, each for experiments independent three least at from in as (presented subset T cell the for values averaged the with IEL and splenic A category ( T cells). and cells ( categories ANOVA the define to used subsets the for values expression 4b– Fig. IEL A (category T cells and cells NK peripheral in upregulated significantly and cells NK by shared program the in genes express T cells 6 Figure NKT cells, such as the transcription factor factor transcription the as such cells, NKT NKT cells do not fit the paradigm of adaptive The transcriptional programs we found found we programs transcriptional The i i NKT cells are acquired and how tran the are acquired cells NKT NKT and non- and NKT γδ i i NKT cells relative to their expression in expression their to relative cells NKT NKT NKT cell specific–manner. For example, T cell subsets (top; presented as in as presented (top; subsets T cell d

Activated splenic splenic Activated ). Left three columns, averaged averaged columns, three Left ). 1 for averaged expression values for for values expression averaged for κ i ad Tae Ras–mitogen- GTPase and B NKT cells. ( cells. NKT κ γδ Fig. 5 Fig. B and Ras-MAPK pathway, Ras-MAPK and B 3 1 T cell subsets compared compared subsets T cell , b 1 ) in various splenic and and splenic various ) in 4 i ) Distribution of genes in genes of ) Distribution i NKT thymocyte popula thymocyte NKT . How the innate features features innate How the . NKT cell differentiation. NKT differentiation. cell b ). Data are combined combined are Data ). 47), showed little or no + a α e c r u o s e r T ) Expression of genes genes of ) Expression γδ β

T cells and IEL IEL and T cells cells T cells, but only only but cells, T i NKT cells, NKT NKT cells, NKT + o T logy of of logy 4

3 cell popula cell 4 ( i , 37 NKT cells cells NKT Fig. Fig. 7c , 44 , 4 i i

NKT NKT NKT NKT 5

. By By . ,

d γδ ). ).  - - - - -

© 2012 Nature America, Inc. All rights reserved. important for the regulation of CD8 of regulation the for important by program this of maturing program shared of the are part that molecules all CD38, and CCR5 B, Fas ligand, granzyme T-bet-deficient program. i lymphocyte effector shared the least at of part maintaining and eliciting in role important an has cells, T and cells NK both in upregulated significantly those among was transcript whose T-bet, and cells NK in both T-bet tor fac transcription the by driven is which expression, NKR of sition thymic i of end the at largely Acquired clues. several provided have data our Nevertheless, defined. fully be to remain program effector common. in have cells T MHC-restricted activated and consistent with the many abilities functional that core and effector program operational in lymphocytes of distinct lineages, cells NK by shared program the that but only days stimulation. These data after suggested antigen-specific The cell– NK responsiveness. innate-like rapid, for poised similarly subsets and cells NK shared by program the that found we Furthermore, cells. T restricted as large a of part the experiment. each in mice more or three from pooled cells with population, each for experiments independent A category in genes the of homologs human with cells, control naive the for that (1–3) donors various from subsets in as (presented cells control naive for expressing and cells NK peripheral 7 Figure e c r u o s e r  of IEL homeostasis forthe as well NKT cell maturation, the shared program mirrored the cells’ acqui cells’ the mirrored program shared the maturation, cell NKT K cls ae oe mN ad rti epeso o IFN- of expression protein and mRNA lower have cells NKT

a

The factors that regulate the expression of genes in this shared shared this in genes of expression the regulate that factors The T.8Nve T.8Eff.12 Listeria-OVA infectio T.8Eff.24

Low Programs shared by NK cells and and cells NK by shared Programs Relative expression i

NKT cell program was also induced in adaptive adaptive in induced also was program cell NKT T.8Eff. 48 i L. monocytogenes L. h NKT cells NKT T.8Eff.6 h T.8Eff.8 h T.8Eff.10d i NKT cells was also active in steady-state steady-state in active also was cells NKT T.8Eff.15d Hig i i T.8Mem.45 n NKT transcriptome as shared with MHC- those NKT transcriptome NKT cells. Furthermore, IL-15 and T-bet are are T-bet and IL-15 Furthermore, cells. NKT 4 d i h

5 i NKT cells relative to its expression in resting resting in expression its to relative cells NKT 4 T.8Mem.100 NKT cells (category A (category cells NKT , is also probably involved in the acquisition acquisition the in involved probably also is ,

4 d . IL-15, reported to act upstream of T-bet in of T-bet upstream to act reported . IL-15,

( d Listeria d Expression (CD8 4 0. b 3 i 1 (presented as in in as (presented NKT cells NKT γ -OVA; presented as in in as presented -OVA; δ Fig. 5 Fig. T cells T + effector T cell responses T cell effector i NKT cells are induced in activated CD8 activated in induced are cells NKT b ). ( ). 1 1 + 4 ) relative to their expression in antigen-specific CD8 antigen-specific in expression their to ) relative 3 i TListeria/CD8 9 7 NKT cells represents a represents cells NKT c . Thus, IL-15 and T-betand Thus,IL-15 . . Thus, it is likely that that likely is it Thus, . ) Expression of the human homologs of genes in category A category in genes of homologs human the of ) Expression i Fig. 4b– Fig. NKT NKT cells, NK cells 100 d 45 d 15 d 10 d 8 d 6 d 48 h 24 h 12 h ( ( P P 10 ( ( ( ( ( ( P P P P P = P = ( = = P + = = = = Fig. 4b– Fig. 1.93×10 4.85 Tnaivefold = 1.37 8.90 3.23×10 0.12) d 0.49) 0.10) 8.42 ). ( ). α γ β × δ 10 × × d T cells, T cells, T cells cells T 10 10 × ) Distribution of the gene set of category A category of set gene the of ) Distribution 10 –36 –42 d 100 4 –55 –41 –43 ). ( ). 8 –50 ) ) ) , , as ) ) ) ) b γ - - , ) Distribution of genes in category A category in genes of ) Distribution c vation and survival programs. For example, genes encoding several several encoding genes example, For programs. survival and vation acti cellular in involved molecules encoding those for enrichment genes in ‘preferentially’ upregulated GATA-3 were among the factors encoded by these genes. The group of specific to cells, NK in sion state. at steady cells these with acquisition of the differentiated terminally effector phenotype of and after the activation of T cells identity cell T of maintenance and establishment the for important are that pathway Wntsignaling Lef1 and cells NK in downregulated factors was other many encoding genes of expression the Also, cells. T and in NK both cells activation mediated by transforming growth factor- Smad3, a transcription factor important for the ‘tuning’ of lymphocyte CD4 in functions immunomodulatory with the transcription factor Bhlhe40, a regulator of the circadian rhythm in innate lymphocytes. For example, the expression of genes encoding and cells ated regulators transcriptional with similar expression patterns in NK shared program cell types. effector in several may have an important role in inducing the expression of genes in the 3 2 1 3 2 1 1 Nve. Approximately 20% of the genes with significantly different expres We have also identified genes encoding many previously unappreci (presented as in in as (presented Lef1 and + T cells. ( T cells. in NK cells and Ctr. Mem. Tcf7 i NKT cells that may help regulate the core effector program program that may core the effector cells NKT help regulate Donor i aDV Low NKT NKT cells. As expected, the transcription factors PLZF and Relative expression , which encode transcription factors downstream of the downstream factors transcription encode , which a A ) Expression of genes significantly upregulated in upregulated significantly genes of ) Expression NCE ONLINE PUBLIC ONLINE NCE i Eff. Mem. 2 1 NKT cells and T cells had transcriptional patterns patterns transcriptional had cells T and cells NKT Fig. 5 Fig. + T cells over the course of infection with OVA- with infection of course the over T cells i NKT cells relative to their expression in resting expression to relative their NKT cells Hig 3 b h i

). Data are combined from at least three three least at from combined are Data ). NKT NKT cells during development is consistent 54 1 1 1 in human peripheral blood CD8 blood peripheral human in , for human memory CD8 memory human for 5 5 0. for activated CD8 activated for d 3 5 1 ; Wnt signaling is typically repressed repressed typically is Wnt; signaling . . The relatively low expression of Expression (CD8 i A NKT cells (category C NKT cells (category i TION NKT cells. Among those were were those Among cells. NKT 1 nature immunology nature + + Tmemory/naivefold) Eff. Mem. Ctr. Mem. T cells T + T cells versus that that versus T cells β 10 , was upregulated + T cells versus versus T cells 51 ( ( P P , = 5 = 6.85 2 2.31 , as well as as well as , 1 + ) ) showed T cell T cell × × 100 10 10 –27 –17 Tcf7 ) ) 5 0 - - -

© 2012 Nature America, Inc. All rights reserved. 9. 8. 7. 6. 5. 4. 3. 2. 1. r R Published online at The authors declare no competing interests.financial to design andexperimental data collection. performance and data analysis; and The ImmGen Project Consortium contributed contributed to the manuscript design, all experimental and supervised with design andexperimental interpretation of the data; M.B.B. substantially experiments and analyzed the data; G.F.W. did experiments; M.B. and J.K. assisted of and did experiments and analyzed the data; T.S. conceived of and did analyzed the data; P.J.B. contributed substantially to the manuscript, conceived primarily N.R.C. wrote the manuscript, conceived of and did experiments and ImmGen Project consortium). to (R01AI063428 M.B.B., to T32AI007306 P.J.B, and to R24AI072073 the technical assistance. Supported by the US National Institutes of Health support; and S. Raychaudhuri, X. Hu and H. Li for advice, discussions and We thank the tetramer facility of the US National Institutes of Health for ongoing Note: Supplementary information is available in the codes. Accession the in v available are references associated any and Methods M programs. in transcriptional genes similar of expression modular through functions effector similar serve ultimately can ontogeny their in substantially differ that tions popula and how future research bring into focus lymphocyte clearer in programs transcriptional shared and distinct both defining By cells. T adaptive in activation after days several elicited also were that cells T innate-like other and cells NK with shared modules genetic extensive identified have data Our systems. immune adaptive and innate the of lymphocytes other to link both that grams pro gene-expression identified have we database, Project Genome Immunological Using the lineages. lymphocyte other with tionships phenotype. effector poised their with by factors transcription AP-1 of expression in expression high relatively had activation, Fos ( factors of AP-1the members of family transcription nature immunology nature C AUTH A e e eprints and permissions information is available online at at online available is information permissions and eprints ckn O p

ethods r Together our data offer a new view of of view new a offer data our Together r MP Godfrey, D.I., MacDonald, H.R., Kronenberg, M., Smyth, M.J. & Van Kaer, L. NKT L. Kaer,Van & M.J. Smyth, M., Kronenberg, H.R., MacDonald, Godfrey,D.I., NK1 Mouse A. Bendelac, Makino, Y., Kanno, R., Ito, T., Higashino, K. & Taniguchi, M. Predominant expression cells NKT invariant Harnessing M. Salio, & S.H. Masri, J.D., Silk, V., Cerundolo, family. cell NKT Coquet, J.M. the Raising A.G. Baxter, & S. Stankovic, D.I., Godfrey, cells. NKT of biology The L. Teyton, & P.B. Savage, A., Bendelac, cells, T lipids, CD1 by presentation Antigen Brenner,M.B. & S. Garg, N.R., Cohen, Heng, T.S.P. cells: what’s in a name? a in what’s cells: (1995). 1157–1161 V invariant of strategies. vaccination in USA population. cell NKT CD4–NK1.1- IL-17-producing an of Immunol. Immunol. immunity. microbial in cells NKT and cells. immune in s , , i n i Fosb o t s o O n E /

i n TING FINANCIA TING 105 w R R C

o d and and f e led

x t , 11287–11292 (2008). 11287–11292 , O

. 11 25 h h NTRIBUTI e t et al. m et al. gm , 197–206 (2010). 197–206 , (2007). 297–336 ,

Cebpb α p l 14 . a h p e t Diverse cytokine production by NKT cell subsets and identification The Immunological Genome Project: networks of gene expression Nat. Immunol. Nat. + GEO: microarray data, data, microarray GEO: t nts e TCR p r : / . ), which are normally induced in cells only after after only cells in induced normally are which ), / w O w L α Nat. Rev. Immunol. Rev. Nat. + w Nat. Rev. Immunol. Rev. Nat. i NS

INT hi i NK1.1 in chain T cells. T NKT cells and differentiate differentiate and cells NKT . n aDV a t u E

r i R 9 e NKT cells, our data open avenues for for avenues open data our cells, NKT A . , 1091–1094 (2008). 1091–1094 , E c Curr. Opin. Immunol. Opin. Curr. NCE ONLINE PUBLIC ONLINE NCE STS o m Adv. Immunol. Adv. / d o i + f i cl populations. cell T n

4 d 9 , 231–237 (2004). 231–237 , e , 28–38 (2009). 28–38 , G r o i / NKT cells and their rela their and cells NKT n 1 S l 0 i i i E NKT cells. Constitutive Constitutive cells. NKT n . NKT cells is consistent consistent is cells NKT

1 e 102 1 0

v 5 3 e

8 7 9 r h , 1–94 (2009). 1–94 , s / , 367–374 (1995). 367–374 , 0 t i n rc Nt. cd Sci. Acad. Natl. Proc. t o A p i 7 n . i 2 TION : NKT cells from from cells NKT .

Jun / o 4 / w f 9 n. Immunol. Int.

t w 0 h , , . . w e Junb

. p n a

nu Rev. Annu. a

p t

u e o r , , r . e Jund n . c l Nat. i o

n m 7 e - - - , , /

13. 12. 11. 10. 23. 22. 21. 20. 19. 18. 17. 16. 15. 14. 43. 42. 41. 40. 39. 38. 37. 36. 35. 34. 33. 32. 31. 30. 29. 28. 27. 26. 25. 24.

asd, J.L. Matsuda, Cooper,M.A. J.L. Matsuda, C. Grégoire, Reschner, A., Hubert, P., Delvenne, P., Boniver, J. & Jacobs, N. Innate lymphocyte Innate N. P.,Jacobs, P.,Hubert, & Delvenne, Reschner,A., Boniver,J. M. Salio, C. Paget, P.J. Brennan, T.Ota, by cells NKT of Regulation F. Takei, & T. Yamamura, S., Lohwasser, M., Maeda, T.Kawamura, Kuylenstierna, C. Lanier, L.L. Evolutionary struggles between NK cells and viruses. infection to respond cells T killer natural invariant How M.B. Brenner, & M. Brigl, Gattinoni, L. Gattinoni, past, the of witness cells: T on receptors NK-cell Inhibitory N. Anfossi, & Vivier,E. TCR into insights Biological A.C. Hayday, & E. Theodoridis, J., Shires, Fahrer,A.M. W.K. Born, & R.L. O’Brien, P.D. Thomas, M.J. Townsend, Johnston, B., Kim, C.H., Soler, D., Emoto, M. & Butcher, E.C. Differential chemokine M. Brigl, N.Y. Crowe, H. Watarai, distinct Functionally M.B. Brenner, & T. Yamamura, S., Miyake, J.E., Gumperz, Yu, S. & Cantorna, M.T. The vitamin D receptor is required for iNKT cell development. D. Kovalovsky, A.K. Savage, cell cell-dendritic killer Natural L. Zitvogel, & E. Vivier, M., Dalod, T., Walzer, M.S. Vincent, Fernandez, N.C. CD1d- of Mechanism Brenner,M.B. & J.E. Gumperz, S.C., Bry,Kent, M., L., Brigl, M.A. Degli-Esposti, & M.J. Smyth, W.M.,Yokoyama, A.A., Scalzo, D.M., Andrews, of natural killer cells. killer natural of tetramers. CD1d using antigen (2007). 169–182 (2007). activation. cell antigen-presenting 27 glycosphingolipids. charged and interferon I type requires cells dendritic signals. danger microbial by induced CD94/NKG2. 167 line. cell NKT of generation and cells NKT primary of analysis Ly49: Immunol. J. CD1d. by activation of co-stimulation and cytolysis NK-like of activation TCR-independent 8 (2010). by recognizing microbial or endogenous lipid antigens. (2002). 966–974 Nat. Med. Nat. future. the of actors (SAGE). TCR transcriptionaltheirprofile. Immunol. Semin. classification. Res. subfamily Acids Nucleic and family protein curated using function, biological by V and NK 171 TCR murine of patterns homing and responses 208 infection. microbial during activation cell T killer natural in dominate vivo in T producing Med. Exp. J. subsets of CD1d-restricted natural killer T cells revealed by CD1d tetramer staining. USA Sci. Acad. Natl. Proc. 9 functions. effector cell T killer natural invariant of development the lineage. cell NKT the responses. immune of (2005). S49–S59 1, Suppl initiation the in crosstalk (2002). 1163–1168 innatein anti-tumor immune responses 4 infection. microbial during activation cell T killer natural restricted Immunol. Nat. infection. viral during cells NK and cells dendritic between interactions Functional Immunol. Exp. Clin. response. immune the of regulation the in factor key a cross-talk: cell dendritic and , 259–268 (2008). 259–268 , , 1055–1064 (2008). 1055–1064 , (2003). 1230–1237 , , 597–609 (2007). 597–609 , , 4180–4186 (2001). 4180–4186 , , 2960–2969 (2003). 2960–2969 , (2011). 1163–1177 , α β . + Eur. J. Immunol. J. Eur. et al. et intraepithelial lymphocytes provided by serial analysis of gene expression gene of analysis serial by provided lymphocytes intraepithelial J. Exp. Med. Exp. J. Immunity et al. et et al. et α et al. et

17 14i NKT cells. NKT 14i h

etal. t al. et IFN-

t al. et 2- and T and 2- 182 et al. et et al. et t al. et 195 , 1290–1297 (2011). 1290–1297 , Blood t al. et et al. et t al. et

et al. et Innate and cytokine-driven signals, rather than microbial antigens, microbial than rather signals, cytokine-driven and Innate t al. et Modulation of human natural killer T cell ligands on TLR-mediated on ligands cell T killer natural human of Modulation t al. et t al. et Activation of invariant NKT cells by toll-like receptor 9-stimulated receptor toll-like by cells NKT invariant of Activation 4 t al. et et al. et al. et , 175–181 (2003). 175–181 , Attributes of gammadelta intraepithelial lymphocytes as suggested by , 250–258 (2009). 250–258 ,

et al. , 625–636 (2002). 625–636 , ifrnil niuo imnt mdae b NT el subsets cell NKT by mediated immunity antitumor Differential γ

22 eeomn ad ucin f nain ntrl ilr cells T killer natural invariant of function and Development

A human memory T cell subset with stem cell-like properties. cell-like stem with subset cell T memory human A -mediated negative feedback regulation of NKT-cell function by NKT-cellfunction of regulation feedback negative -mediated 15

h tasrpin atr LF iet te fetr rga of program effector the directs PLZF factor transcription The In vivo In The trafficking of natural killer cells. killer natural of trafficking The NKG2A inhibits invariant NKT cell activation in hepatic injury.hepatic in activation cell NKT invariant inhibits NKG2A

ATE: bosbe aaae f ee rdcs organized products gene of database browsable a PANTHER: 31 Tracking the response of natural killer T cells to a glycolipid a to cells T killer natural of response the Tracking

Dendritic cells directly trigger NK cell functions: cross-talk relevant D-eedn dnrtc el instruction. cell dendritic CD1-dependent 106 nain ntrl ilr cls eonz lpd ef antigen self lipid recognize cells T killer natural Invariant oesai o V lh 1i K cells. NKT 14i alpha V of Homeostasis

The BTB-zinc finger transcriptional regulator PLZF controls PLZF regulator transcriptional finger BTB-zinc The , 193–198 (2010). 193–198 , 152 Nat. Rev. Immunol. Rev. Nat. T-bet regulates the terminal maturation and homeostasis of homeostasis and maturation terminal the regulates T-bet h 202 , 419–434 (2001). 419–434 , Immunity NKG2D performs two functions in invariant NKT cells: direct Blood 17-cytokines. , 334–341 (2003). 334–341 ,

, 184–192 (2005). 184–192 , 41 , 219–226 (2008). 219–226 , evidence for a dependence on interleukin 15 for survival for 15 interleukin on dependence a for evidence , 1279–1288 (2005). 1279–1288 , Immunity

Proc. Natl. Acad. Sci. USA Sci.Acad. Natl. Proc. , 1913–1923 (2011). 1913–1923 ,

105 γ 100 δ cl sbes a ik ewe TR n function? and TCR between link a subsets: cell T J. Exp. Med. Exp. J.

, 5207–5212 (2008). 5207–5212 , 29 , 3633–3638 (2002). 3633–3638 , Proc. Natl. Acad. Sci. USA Sci. Acad. Natl. Proc. , 391–403 (2008). 391–403 ,

PLoS Biol. PLoS 20 Nat. Immunol. Nat. in vivoin , 477–494 (2004). 477–494 ,

4 , 190–198 (2004). 190–198 ,

. 192 Nat.Med.

α 10 β , 741–754 (2000). 741–754 , K cl subsets. cell NKT , e1001255 (2012). e1001255 ,

12 xet pn Bo. Ther. Biol. Opin. Expert Semin. Immunol.

98

, 1202–1211 (2011). 1202–1211 , 5 e c r u o s e r , 405–411, (1999). , 10261–10266(2001).,

muo. Rev. Immunol. 104 Nat. Rev. Immunol. a. Immunol. Nat. a. Immunol. Nat. , 20490–20495 , Nat. Immunol. Nat. Nat. Immunol. Nat. J. Exp. Med. Exp. J. . Immunol. J. J. Immunol. J.

22 Immunity , 79–86 γ δ

+ and 220

3 3  5 , , ,

© 2012 Nature America, Inc. All rights reserved. Medical Medical School, Boston, USA. Massachusetts, St. Louis, Missouri, USA. California, USA. Brigham and Women’s Hospital, Boston, USA. Massachusetts, Department, Stanford University, Stanford, California, USA. School, Boston, USA. Massachusetts, Boston, USA. Massachusetts, Department, Brown University, Providence, Rhode Island, USA. 7 Ayla Tata Nageswara Rao Miriam Merad Natalie A Bezman Tal Shay Shannon Turley Joonsoo Kang Jim Collins ImmGen Project PaulConsortium: Monach 49. 48. 47. 46. 45. 44. e c r u o s e r 1 Department Department of Medicine, Boston University, Boston, USA. Massachusetts, 0

ngk-hr, . Nsiua H, iai A & ohki Y Interleukin-15 Y. Yoshikai, & A. Mitani, H., Nishimura, K., Inagaki-Ohara, Antigen- Glimcher,L.H. & M. Herrath, von S.J., Szabo, A., Juedes, B.M., Sullivan, in Med1 subunit mediator of role Essential T. Borggrefe, & A. Izcue, X., Yue, P. Kastner, L.E. Gordy, Matsuda, J.L. gamma delta T cell receptor in mice. in receptor cell T delta gamma bearing lymphocytes intraepithelial intestinal of growth the promotes preferentially 100 T-bet.by regulated function cell T CD8 effector driven development. (2011). 17105–17110 T-cell killer natural invariant CD4 immature in cells. duringthedevelopment Valpha14iof NKTcells. E , 15818–15823 (2003). 15818–15823 , J. Immunol. J. rgun 15 , , Aviv Regev t al. et t al. et et al. 21 10 18 Icahn Icahn Medical Institute, Mount Sinai Hospital, New York, New York, USA. , , Michael Mingueneau , , Roi Gazit + T-bet concomitantly controls migration, survival, and effector functions L1 rglts oesai ad emnl auain f NKT of maturation terminal and homeostasis regulates IL-15 4

CD8 c1b erse a aue -el ee xrsin program expression gene T-cell mature a represses Bcl11b 187 18 , , Anne Fletcher 12 , , + , 6335–6345 (2011). 6335–6345 , 20 thymocytes. E , , Adam J Best 17 Joslin Joslin Diabetes Center, Boston, USA. Massachusetts, mmanuel , , Joseph C Sun 20 11 Immune Immune Diseases Institute, Children’s Hospital, Boston, USA. Massachusetts, , , Amy Wagers 15 11 , , Nadia Cohen , , Brian S Garrison 13 Eur. J. Immunol. J. Eur. Eur. J. Immunol. J. Eur. Division Division of Biological Sciences, University of California San Diego, La Jolla, California, USA. L 12 Gautier 13 , , Kutlu rc Nt. cd Si USA Sci. Acad. Natl. Proc. Blood , , Jamie Knell 17 21

20 , Charlie , C Charlie Kim 107

, , 40 , , Tracy Heng

Proc. Natl. Acad. Sci. USA Sci. Acad. Natl. Proc. D 27 16 , 2797–2805, (2006). 18 E , 2143–2154 (2010). 2143–2154 , iane iane Mathis , 2885–2891 (1997). 2885–2891 , , , Patrick Brennan , lpek 15 19 Broad Broad Institute, Cambridge, USA. Massachusetts, 17 , Claudia , JakubzickClaudia 10 11 Department Department of Microbiology & Immunology, University of California San Francisco, San Francisco, 7 Department Department of Biomedical Engineering, Howard Hughes Medical Institute, Boston University, , , Susan A Shinton 12 , , 13 D , , Angelique Bellemare-Pelletier , , Ananda Goldrath errick errick J Rossi 21 8

17 Fox Fox Chase Cancer Center, USA. Pennsylvania, Philadelphia, 21 108 , , Michio Painter , , & Christophe Benoist L ,

21 ewis ewis Division Division of Immunology, Department of Microbiology & Immunobiology, Harvard 55. 54. 53. 52. 51. 50. 16 , , Michael Brenner

Gattinoni, L. Willinger,T. B.N. Weber, and activation cell T Defective Taneja,R. & R.A. Flavell, R.Q., Li, B., Lu, H., Sun, K. Miyazaki, S. Honma, CD8 176 factor 7 (T cell factor-1) following antigen encounter transcription and 1 factor binding enhancer lymphoid factors transcription pathway differentiation. (2001). mice. Stra13-deficient in disorder autoimmune cells. T regulatory the in Nature L 11 18

8 L , 1439–1446 (2006). 1439–1446 , + 19 , , Kavitha Narayan , , Richard R Hardy memory stem cells. stem memory , , Gwendalyn J Randolph anier Department Department of Pathology & Immunology, Washington University,

419 aDV 13 et al. et , 841–844 (2002). 841–844 , , , Vladimir Jojic 21 et al. et et al. et al. et 17 t al. et A , , Jeffrey Nature Dec1 and Dec2 are regulators of the mammalian molecular clock. molecular mammalian the of regulators are Dec2 and Dec1 NCE ONLINE PUBLIC ONLINE NCE , , Jennifer Miller Human naive CD8 T cells down-regulate expression of the WNT the of expression down-regulate cells T CD8 naive Human Wnt signaling arrests effector T cell differentiation and generates The role of the basic helix-loop-helix transcription factor Dec1 factor transcription helix-loop-helix basic the of role The A critical role for TCF-1 in T-lineage specification and specification T-lineage in TCF-1 for role critical A

476 12 21 16 Dana-Farber Dana-Farber Cancer Institute and Harvard Medical J. Immunol. J. Nat. Med. Nat. 16 , 63–68 (2011). 63–68 , Division Division of Rheumatology, Immunology and Allergy, 12 E , , Taras Kreslavsky ricson , , D 4 8 , , Katelyn Sylvia , , Radu Jianu eepali Malhotra eepali 14

15

185 , , 21 , 808–813 (2009). 808–813 , D A 18 , 7330–7339 (2010). 7330–7339 , , , Scott TION aphne Koller 18 , , Brian Brown , 14 19 Computer Computer Science

, , Francis Kim a. Immunol. Nat. 9 in vitro Computer Computer Science nature immunology nature D 9 , , avis D 12 4 and , avid avid Koller 12 ,

21 , in vivo 14

,

18 2 ,

1040–1047 ,

. , J. Immunol.

20 ,

9 ,

© 2012 Nature America, Inc. All rights reserved. Where indicated, Pearson correlation with pairwise complete linkage was was linkage complete Institute). pairwise with (Broad correlation Pearson software indicated, Where GenePattern of module HeatmapViewer with produced the were maps Heat used. was normalization and mean) the of log were data For maps, heat overall. expression mean highest the with set probe sets the of Probe selection by used. consolidated were were symbol gene same sets) the with probe associated 22,268 with arrays (802 release Project visualization. and analysis Data Information). for Biotechnology for Center system (National homologs automated detecting HomoloGene the with identified were genes mouse Information Biotechnology for Center the of National repository database Omnibus) Expression (Gene GEO the from sets data from were Project ImmGen the from not sets website. data mouse Project and Human ImmGen the or documentation, pipeline Control Quality 16 Table Supplementary in available are details Additional center. processing Project ImmGen the at MoGene 1.0 ST zation (Affymetrix array) and data were were processing done hybridi microarray RNA below). control extraction, quality (disussed passed tion of the CD4 four replicates were obtained for each sample with a FACSAria, with the excep for was other negligible Contamination subset. for CD44 was estimated 0.9% that lack mice from CD1d-deficient directly into TRIzol (Invitrogen) at a purity of >99%. By staining of thym sorted double were and website) Project ImmGen the at available is strategy, gating including procedure, staining the about (information markers surface for cell were stained and lung cells cells mononuclear liver splenocytes, cytes, centrifugation. by gradient density isolated Ficoll filtered and washed. Livers were homogenized and liver mononuclear cells were 15 min at 37 °C in 7 U/ml of Liberase III enzyme (Roche) in DMEM, then were for digested were Lungs homogenized. mechanically were and PBS cold with non- of depleted then and cells, i blood red remove to (Lonza) buffer lysis bicarbonate were treated with ammonium disaggregated, chloride–potassium Spleens (Miltenyi). beads magnetic anti-fluorophore with and were separated non- of depletion for antibodies fluorophore-labeled with stained were then eBioscience), (2.4G2; anti-CD16-CD32 with blocked then and disaggregated were Thymocytes sample. per mice ten to five from liver and were cells lung isolated mononuclear splenocytes, thymocytes, cells, with parison about the thymic, NK cell, information Additional Project. ImmGen the of guidelines procedure ating oper standard to adherence strict in done was system immune of the of cells Cell isolation, microarray analysis and subset nomenclature. Health. of Institutes National US the of facility tetramer ( PBS-57 with loaded ers tetram CD1d Biosciences. BD from were (5F6) anti-Ly49c,1 and Ly49a (A1) anti-2B4 (ebio244F4), and anti-CD16,32 (2.4G2) were from eBioscience. Anti- (16a11), anti-NKG2A (CX5), anti-NKG2D (CM4), anti-Ly49e,f (145-2C11), anti-CD3 (102), anti-CD45 (GK1.5), anti-CD4 (IM7), anti- anti-CD44 (PK136), (N418), anti-CD8 anti-CD11c (A18), (M1/70), Ly6G/Gr1 anti-CD11b (TER119), anti-Ter119 Antibodies. Institute. Cancer Dana-Farber the of Use and Committee Care Animal the by approved were studies All conditions. under pathogen–free maintained specific were Mice collection. organ before week 1 Laboratories Mice. METHODS ONLINE doi:10.1038/ni.2490 NKT cells as described above. Lungs and livers were collected after perfusion above. Lungs after perfusion and NKT as livers cells were described collected 2 -transformed, and a relative color scale with row centering (subtraction (subtraction centering row with scale color a relative and -transformed, Male C57BL/6 mice 6 weeks of age were obtained from Jackson Jackson from obtained were age of weeks 6 mice C57BL/6 Male Antibody to CD19 (anti-CD19; MB19-1), anti-B220 (RA3-6B2), (RA3-6B2), anti-B220 MB19-1), to (anti-CD19; CD19 Antibody i NKT cells NKT is cells at available website. For Project the ImmGen −

i NKT subset NKT from subset the lungs, for only which a replicate single α (subset nomenclature key), Data Generation and and Generation Data key), nomenclature (subset γ -galactosylceramide analog) were provided by the the by provided were analog) -galactosylceramide δ T cell and CD8 α − 5/.) anti-TCR (53/6.7), NK1.1 Data from the March 2011 ImmGen ImmGen 2011 March the from Data i NKT cells, a false-positive rate of 2.7% a false-positive NKT cells, − (Stage 1) (Stage + T cell populations used for com i NKT cell populations. TwoNKT cell populations. to i NKT cells, the rarest thymic thymic rarest the NKT cells, i 43 NKT cell-enriched thymo NKT cell-enriched β , 5 (H58-597), anti-NK1.1 anti-NK1.1 (H58-597), 6 . Human homologs of of homologs Human . i NKT cell populations populations cell NKT All purification o i NKT NKT cytes cytes ± ε ------

expression values of of values expression of analysis. Euclidean-distance second, the not but first, the to contributed and were list gene variable most for 15% filtering after present Klrk1 Klrb1a ( ( analysis first the Cd244 org/cancer/so ( GenePattern of application ExpressCluster The analyzed. any subsets two between of twofold over expression in for a change value of GenePattern. of module Multiplot the by with produced were Volcano plots analysis. clustering for rows to applied 57. 56. tested. samples the of comparison selected gene set with with all genes meeting the Institute) criteria for expression (SAS (>120) in the software JMP with calculated were in maps heat the with ciated with processes Biological test. exact Tables 1 Relationships) Evolutionary Through Analysis (Protein function biological by organized products gene of database PANTHER the Health) of Institutes National US the of Diseases Integrated VersionDiscovery; 6.7; National Institute for andAllergy Infectious the from and Visualization software for DAVIDAnnotation, (Database database bioinformatics with done was analysis enrichment gene-set Functional in expression expression in NK cells; and genes with upregulated (C and cells T i in expression similar most the with genes cells; T in expression i and cells NK in expression similar most the with genes genes: of groups six gene each of cells, NK expression in average the of comparison By categories. gene 14 into separated then were genes 1,192 the analysis, this of results the of basis the other two, as indicated by the ‘multcompare’ of function MATLAB. On the from differed significantly populations three the of which determine to used 1,192 (of 5,900) genes the passed ANOVA test ( was applied to list correction and the of resulting the Bonferroni of ‘anova1’ comparison for (MathWorks) function software MATLAB with done was ANOVA genes. 5,900 Klrb1f Klra3 ( partners signaling and adaptors NKRs, inhibitory and vating ≥ log were genes selected The populations. multiple in vary that genes ‘weight’ to tions probes on the basis of a variation filters of ANOVAwhich with the GenePattern, geometric s.d. of of popula application PopulationDistances the with fied NKT cells that were upregulated (A upregulated were that cells NKT NKT cells that were upregulated (B upregulated were that cells NKT upeetr Fig. Supplementary 120, and centered on the mean before visualization. Genes encoding acti encoding Genes visualization. before mean the on centered and 120, ≤ For For ANOVA, log were data For For estimation of the significance of enrichment for the distributions asso for the distributions of enrichment of For the significance estimation For Euclidean-distance matrices, the 15% most variable genes were identi genes variable most 15% the matrices, For Euclidean-distance

Huang da, W., Sherman, B.T. & Lempicki, R.A. Systematic and integrative analysis integrative and Systematic R.A. W.,B.T.Lempicki, da, Sherman, & Huang H.D. Marshall, (2009). resources. bioinformatics DAVID using lists gene large of (2011). and memory Th1 CD4 0.5 across all ImmGen Project samples were Project 0.5 ImmGen across all removed and only genes with , , , , , , K , , Ncr1 , , Klra5 Fcer1g Klrc1 -means clustering analysis, genes were prefiltered for a mean expression Klrb1b ≥ 2 and -transformed, filtered for probes with a mean expression value of of value expression mean a with probes for filtered -transformed, 120 120 (cutoff above which genes have a 95% chance of expression) and , , Cd244

, , i 2 , , NKT cells and T cells, these categories were then sorted into into sorted then were categories these cells, T and cells NKT ftware/genepattern , , Klra6 . . i , , Klrc2 NKT cells relative to the expression in T cells or NK cells. cells. NK or cells T in expression the to relative cells NKT Tyrobp P Klrb1c values calculated with DAVIDwith Fisher’s a modified calculated represent values et al. et Fig. Fig. , , , , Fcer1g , , ≥ Klra8 Differential expression of Ly6C and T-bet distinguish effector T-betdistinguish and Ly6C of expression Differential Klrc3

120 in two or more arrays were considered, which left left which considered, were arrays more or two in 120 3 and and + 5 , , cell properties during viral infection. ) and were manually removed for the second analysis analysis second the for removed manually were and ) ). ). Klrb1f Klra1 , , , , , , Tyrobp Hcst Klra9 Klrd1 2 , , -transformed, low-variability genes with a s.d. s.d. a with genes low-variability -transformed, Figures 5 Figures Klrc1 ) were present in the initial gene list used in in used list gene initial the in present were ) , , / Klra3 i ) was used for clustering, with with clustering, for used was ) NKT cell, NK cell and T cell populations, populations, cell T and cell NK cell, NKT , , (but not , , Klra10 P Klre1 1 1 , , ) or downregulated (B downregulated or ) values less than 0.01 are presented. are 0.01 than less values ) or downregulated (A downregulated or ) Klrc2 , , Klra5 – , , 7 , , Klri1 , , Klra2 , Kolmogorov-Smirnov Kolmogorov-Smirnov , Klra17 Klrc3 , , P < 0.05). test A was secondary Klra6 , , , , http://www.broadins , , Klra17 Klri2 nature immunology nature 5 , , Klrd1 7 . Biological processes of of processes Biological . 1 Klrb1a ) ) or downregulated (C , , Klra8 , , Immunity a. Protoc. Nat. , , , , Klrk1 3 Klrg2 Klrg2 or Hcst Klre1 8 are presented in in presented are , , 2 2 , , Klrb1b ) relative to the the to relative ) ) relative to the the to relative ) Klra9 , , , , Klra1 P Klrg2

K Klri1 35 values, and values, = 10. =

, 633–646 , , , , 4 P 44–57 , , , Klra10 Klrb1c values values , , , , Klra2 ) ) were titute. Klri2 Ncr1 2 - - - - ) , , , , ,