© 2012 Nature America, Inc. All rights reserved. this this work. New of Experimental Medicine, University of California, San Francisco, San Francisco, California, USA. 1 a represent simply might cells NK that proposal the to led have interferon- and A granzyme perforin, CD90, CD7, CD2, as (such molecules effector and antigens cell-surface many of cells T and cells NK by expression shared the as well as cytolysis, cell–like’ bearing cells T as such populations, cell T certain ability of The debate. of topic a been has cells myeloid and lymphoid antibodies. and chemokines cytokines, of tion and the rapid secre such as cytolysis responses mount effector quick antigens self and microbes from structures specific recognize to genes rearranged somatically features innate and adaptive both have cells, B marginal-zone and cells B-1 include ( cells which NKT invariant lymphocytes, innate-like and cells NK response requires cell division and adaptive the differentiation of effector cells. the whereas agents, infectious to rapidly responds system immune innate The branches. adaptive and innate into divided cally classi is vertebrates of system immune The biology. cell investi NK for gating resource a generate to infection viral a to response the during and state steady the in this analyzed have and cells) (NK cells tion, we have identified the gene-expression programs of natural killer system immune mouse the in expression gene of database comprehensive a at establishing aimed laboratories of consortium a is Project (ImmGen) Genome Immunological The expression of NK cells in various states. and previously unknown molecular aspects of NK cell identity and function by delineating the genome-wide repertoire of gene T cells, which allows NK cells to respond more rapidly to viral infection. Collectively, our data provide a global context for known many encoding molecules with unknown functions. Resting NK cells demonstrate a ‘preprimed’ state compared with naive with cytotoxic CD8 encoding molecules with similar signaling functions. Whereas resting NK cells are known to share expression of a few genes relationship between NK cells and T cells than between any other leukocytes, by distinguished their shared expression of genes Using whole-genome microarray data sets of the Genome Immunological Project, we demonstrate a closer transcriptional Project Consortium Yosuke Kamimura Natalie A Bezman natural killer cells Molecular definition of the identity and activation of nature immunology nature Received 9 April; accepted 12 July; published online 19 August 2012; Department Department of Microbiology and Immunology and the Cancer Research Institute, University of California, San Francisco, San Francisco, California, USA. ic te is dsrpin f K cells NK of description first the Since

York, New York, USA. 2 6 , 3 Full Full list of members and affiliations appears at the end of the paper. should Correspondence be addressed to L.L.L. ([email protected]). . These innate B cells and T cells use receptors encoded by encoded receptors use T cells and B cells innate . These α β T cell antigen receptors (TCRs), to mediate ‘NK ‘NK mediate to (TCRs), receptors antigen cell T + T cells, our analysis demonstrates that transcriptome-wide the extend commonalities to hundreds of genes, i NKT cells), intestinal epithelial lymphocytes, lymphocytes, epithelial intestinal cells), NKT 1 4 1 Division Division of Biological Sciences, University of California San Diego, La Jolla, California, USA. ,

5 , , J Adam Best 6 , Charles , C Charles Kim aDV 2 . Functionally, innate-like lymphocytes lymphocytes innate-like Functionally, . A NCE ONLINE PUBLIC ONLINE NCE γ δ T cells and some activated activated some and cells T 4 1 , , Ananda W Goldrath . As part of this collabora this of part As . 4 , 5 2 , their relationship to to relationship their , , 5 , , Joseph C Sun A TION γ γ

(IFN- δ T cells, cells, T doi:10.1038/ni.239 γ 3 )), )), , 5 - - - - , , Gundula Min-Oo

4 , Lewis L , Lanier Lewis that documents the transcriptome of NK cells in various states. new insights into the nature of NK cells and a publicly availablemolecular resourcedefinitions of NK cell identity and function and provide both compared types) simultaneously. The findings presented here provide cell andconditions as (such sets data of number large the of because analyses previous from different study multidi this makes was which mensional, technique profiling transcriptional Our consortium. ImmGen the by profiled populationsmyeloid and lymphocyte other all torelative and statesactivation includingcontexts, several in cells study,wehave systematicallytranscriptome the defined ofmouse NK changes that occur during the activation of NK cells sets of genes specifically expressed in NK cells, as well as transcriptional identified cells NK mouseapproachhumanandanalyze this to using new insights into the biology of specific cellular subsets cells myeloid with than rather lymphocytes with shared are progenitors from derived cells NK most that demonstrated have populations progenitor However, subsequent studies defining the properties of hematopoietic shared expression of markers, cell-surface such as CD11b and CD11c. NK cells and between myeloid cells has on proposed been the ofbasis of cells lymphoid lineage as distinct a NK cells third, distinguish unambiguously for TCR required rearrangement binases recom or the a thymus lacking mice in cells of NK development the and cells NK mature in genes TCR of rearrangement productive of lack the However, cells. T of stage differentiation or developmental Global transcriptional analysis is a powerful approach that has yielded 5 3 Immunology Immunology Program, Memorial Cancer Sloan-Kettering Center, 7 . 1 & The Genome Immunological 1 , Deborah , W Deborah Hendricks 5 These These authors contributed equally to e c r u o s e r in vitro 1 , 6 8 . A relationship . A relationship

, 9 . Early studies 10– 2 Division Division

1 2

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 - - © 2012 Nature America, Inc. All rights reserved. naling naling constitutes a large component of the overall between similarity and cells T cells andNK provides of confirmation that this biology functional similarity in shared sig the reflects evaluation unbiased by molecules signaling for these enrichment prominent of such finding ( ( kinases Tec such as Zap70, Lck, cells, of NK cells–T activation the to regulate vation motif pathway,signaling including known molecules signaling encoding below) the components of parentheses the immunoreceptor (in tyrosine-based acti genes for enrichment considerable showed and cells, NK resting of subsets distinguished signature that 24-gene a identified comparisons, pairwise all of section inter the of by identification followed DCs), plasmacytoid and DCs lation with B cell populations and myeloid populations (macrophages, resolution. of level at this populations T cell adaptive the from segregate not did discernibly T lymphocytes ‘NK cell–T cell complex’ here). Notably, NK cells and other innate-like cells, NK of subsets containing cluster a and subsets cell B containing cluster a of ration sepa distinct a was there cells, lymphoid Among subsets. cell phoid lym and macrophages between clustered populations cell dendritic CD11c conventional macrophages, to close grouped cells dendritic plasmacytoid Whereas plot. PCA the in macrophages from distant cells, ( clusters discrete five into populations the of segregation showed populations, leukocyte splenic all across expression variable most the with of genes 15% the by as defined components, principle relationships informative most three the with population of Delineation set. data a across variance explain best that components) (principal patterns gene-expression identifies that a method (PCA), analysis principal-component using investi gated the we relatedness of naive NK identity, cells to populations other leukocyte cell NK of definition molecular a establish To Transcription-based organization of the main leukocyte subsets RESULTS type. cell per replicates independent Wnt– the in molecules blue, pathway; signaling motif activation tyrosine-based immunoreceptor the in molecules Green, populations. analyzed all of value expression median the ( significance of order decreasing in map a heat as presented macrophages, and cells, dendritic B cells, in and complex cell cell–T NK the in CD4 CD8 i T cells; T cells; NK 8 8 DC DCs; 4 DC macrophage(s); 8 8 pDC B cells; center germinal B GC, B cells; zone marginal B MZ, cells; B-1a B1a, B cells; follicular B Fo, 1–3; types transitional of B cells T3, B T1–B parentheses). (in variation intersample to contribution their and (PC1–PC3) components principal three top the showing populations, leukocyte splenic of ( level. transcriptome the at similarity 1 Figure e c r u o s e r  cells. T and cells NK NKT cells; T 4 cells; NKT δ Rnf125

− + score; left margin); expression is relative to relative is expression margin); left score; 4 , CD8 Comprehensive pairwise comparison of comparison each NK cell popu cell–T Comprehensive pairwise − + + α 11b or CD8 or T 4 T cells; β i NKT cells, cells, NKT T cells from other populations leukocyte ( i −

NKT 4 NKT ), ), and adaptors ( − or CD8 or NK cells and T cells show close close show T cells and cells NK Itk , , CD8 − γδ 4 − and and + , , TCR 11b T cells. ( T cells. + + − Nve or T 8 or Nve or CD4 + + plasmacytoid DCs; MF, DCs; plasmacytoid + Mem or T 8 or Mem Txk γδ , , CD8 i NKT 4 NKT γ T cells; Treg, regulatory Treg, regulatory T cells; − + δ CD11b or DC 8 DC or i T cells and and cells T ), PKC- ), NKT cells, cells, NKT b Fig. 1 Fig. − ) Gene expression expression ) Gene CD4 Cd247 − + , , CD4 β Nve, naive CD4 naive Nve, − -catenin pathway; protein designations in parenthesis. Data presented are based on the analysis of a minimum of three three of a minimum of analysis the on based are presented Data parenthesis. in designations protein pathway; -catenin − a DCs; NK, NK, DCs; + + CD11b θ ). Lymphoid cells, including B cells, NK B cells, including Lymphoid cells, ). , , CD4 Mem, memory memory Mem, ( + , , or CD4 or Prkcq Lat γ δ α + − a T cells and and cells T + or CD8 or or pDC pDC or , , β ) PCA ) PCA DCs; DC DCs; Skap1 T cell subsets, formed groups groups formed subsets, cell T

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T B1a 8 B GC 4 B MZ T + + –0.1 Mem T Nve 8 pDC + NK cells had higher expression of genes encoding effector molecules, NK cells had expression of molecules, higher effector genes encoding T cells respond rapidly to stimulation, it was not unexpected that resting stained positive for these markers ( were reflected in the frequency of NK cells, lular molecules showed that in most cases, the transcript measurementsintracel andantigens cell-surface of representative set a for Staining unknown. is lineages these in products gene the of function the but with a distinctive expression pattern in NK cells and innatewe found an lymphocytes,additional set of genes ( phocytes, which provides confirmation of these data expressionshownhavehavetoexpression been higher innatein lym (NTAL), genes encoding adaptors, such as and Fasl ing transmembrane proteins and other surface receptors,(CRACC), such as Fcgr3 as such parentheses), in encoded products (NKRs; receptors cell NK molecules and ( receptors signaling and surface encoding genes for in cell expression their innate to relative other subset one least at and cells NK resting by upregulation significant most the with genes 112 of group The comparisons. four the across conservation assessed and cells T adaptive in expression their torelative populationinnate each in expressiondifferentcantly To investigate these shared programs, we identified genes with signifi distinguishwouldthatinnateexist populations adaptivefromcells. T would commonalities transcriptional complex, cell cell–T NK the of populations innate the of diversity the despite that Wehypothesized ( clustering hierarchical by organization similar a observed (CD4 the onlytogether, groupwith to failed populations ( cells of T adaptive grouping of populations showed the populations complex cell cell–T NK across nine expression the variable most the with genes of 15% the of PCA complex cell cell–T NK the of organization Molecular 4 Nve + Fig. 2 Fig. Me Given the observation that unprimed NK cells, cells, NK unprimed that observation the Given m , , pDC NK pDC Itgax i NKT (CD16), Kit + n CD4 and Treg c 8 8 i DC NKT 4 + – DC ). These included genes encoding activating and inhibitory inhibitory and activating encoding genes included These ). – and T Sh2d1b1 γδ (CD11c); genes encoding kinases, such as such kinases, encodinggenes (CD11c); –0.1 4 8 + 4 – aDV 4 + Klra5 – 11b MF 0 DC DC + Cd7 (30.18% Ncr1 8 A MF 8 – 0.1 4 + PC1 (Ly49E), NCE ONLINE PUBLIC ONLINE NCE – 11b (EAT-2) and cDCs − ; genes encoding integrins, such as as such integrins, encoding genes ; –

(NKp46), 0.2 i ) K cls soig coe eainhp We relationship. close a showing cells) NKT b 1.0 1.5 0.5 2.0 2.5 δ Klra9 –3 Expression (log 2 Klrc2 B 0 fold) Clnk (Ly49I), Fcer1g 3 Fig. 2d (NKG2C), . Many of these genes with different Fgl2 A Fig. 2 Fig. DC α TION (FcR β Klrc1 , T cell subsets was enriched enriched was subsets cell T Sulf2 , e MF i

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© 2012 Nature America, Inc. All rights reserved. the requirement for these factors in the development and function function and development the in factors these for requirement the in than ages and including factors, transcription encoding genes tion, ( granules lytic of release or generation the affect that changes in orvesicle rearrangements trafficking cytoskeletal may and/or as (such of the cytoskeleton vesicle transport (such as Serpinb9 and IFN- including ( type cell per replicates independent three of a minimum of analysis the on ( bars (error experiments two of representative are Data here. presented not is and population any in background populations. analyzed all of value expression median the to relative presented (above), populations leukocyte splenic i TCR among T-bet ( Syk or ( factor. transcription blue, molecule; surface purple, molecule; effector green, encoded: molecule of function indicates cells, NK resting to unique (genes 0.5 of ( T cells ( (NK), ( analysis. the in included populations these of any in expressed genes all with a population, of arrays averaged of distances Euclidean on based populations, complex cell cell–T NK the of clustering in as αβ adaptive and T cells innate-like cells, ( complex. cell cell–T NK the of branches adaptive and 2 Figure nature immunology nature cells these of NKT cell and and cell NKT i c b (14.61%) a NKT 4 NKT ) Overlapping of genes expressed in NK cells cells NK in expressed genes of ) Overlapping T cell populations (presented (presented populations T cell PC3 (19.87%) Id2 Ctsd Fig. 1 Fig. PC2 γδ –0.2 –0.2 + αβ i T cell ( T cell

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© 2012 Nature America, Inc. All rights reserved. toxic lymphocytes from granzyme B–mediated cell death cell B–mediated granzyme from lymphocytes toxic granzyme B in an irreversible manner cells. inhibitor The ( Serpinb9b serine-protease may we of ability which for be important speculate NK the migratory tease ADAM14 ( pro The to our knowledge. of lymphocytes, subset by this expressed Adamts14 in NK cells, higher uniquely was expression whose genes the Among Itga2 ( receptor 1-phosphate sphingosine a encoding those include identified been have that cells ROR transcription factor the express that cells lymphoid innate of population cells, NK in expression subsets cell T rare exceptions: two with selective have to shown been ( has population NKp46 leukocyte mice, other any C57BL/6 in in detectable cells not NK was it of 50% only in expressed was Ly49H were which of in expression NK cells than higher in all other encoded NKRs, leukocytes the most specific significantly with genes 25 the of half Nearly expression higher in NK cells than in populations. all other leukocyte therefore defined a resting NK cell signature by identifying genes with We types. cell these among innate-like distinctions other the blurs and populations cell T cells NK by expressed factors transcription and molecules signaling receptors, surface of repertoire shared The A signature transcriptional that defines resting NK cell identity populations. lymphocyte innate-like of components molecular unknown previously identified and cells T innate-like and cells NK of functions known confirmed analyses These lymphocytes. innate of program activation or developmental a of regulator additional an signaling receptor–mediated antigen limit to mechanism are in feedback proteins a involved negative that these cell, NK in sion only that found we but tebrates, ver in higher are genes such four there regulators; of transcriptional in SproutyDCs. homolog 2 ( cell cell–T NK innate ( to ­subsets specific was Tmem176b) protein brane Tmem176b of independent experiments ( cell type ( minimum of three independent replicates per Data presented are based on the analysis of a in decreasing degree of influence (left to right). predicted to do so (significantly linked), presented genes along the horizontal axis) most ‘strongly’ by the transcriptional regulators (encoded by the of the 93 NK cell signature genes in indicates TCR and TCR percent Ly49H (left; number above bracketed line indicates ( (full list of genes, (as in relative to all other cell populations, presented significant difference in expression in NK cells cells. ( Figure 3 e c r u o s e r  molecules. cytolytic own their by killed being from cells NK protect b

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© 2012 Nature America, Inc. All rights reserved. for Thesefindings demonstrate that the preprimed state before described sion of these effector molecules as part of their persistently ‘alerted’in terms of transcriptional prepriming,state. NK cells have maximal expres naive NK cells than in effector CD8 mol CD8 effector of differentiation the regulating in factor transcription this of role the given notable, was program. The higher expression of CD8 Prdm1 as (such factors transcription encoding CD8 effector and cells NK of distribution tissue ate appropri the may influence molecules of these action concerted the CD8 effector in induction requiring genes ( CD11b ( pro CCR2 teins chemotactic the encoding genes of expression basal high had chemokine cells of NK naive that We observed expression molecules. adhesion and surface receptors the in increase an to attributed largely is this and infection, of sites to migrate to ability the acquire tions and and ing several activating NKRs (such as NK CD8 and cells effector Ifng shared expression of genes encoding other such effector molecules, as CD8 effector ( type ( cell per experiments replicates independent independent three of minimum a of analysis the on and based line are regression presented the of Data slope the interval. indicate plot confidence in 95% the numbers (solid); line regression linear the of intervals confidence 95% indicate lines dashed or (left) VSV with infection after 6 day ( TCR. ovalbumin-specific an of expression transgenic with T cells OT-I, NKG2E; and NKG2C NKG2A, NKG2A/C/E, B; granzyme GzmB, CD8 effector and cells NK naive in in identified genes the by encoded molecules ( presented are expression different significantly most the with genes Only (gray). other or (blue) factor transcription (brown), molecule regulatory (red), integrin (purple), receptor (green), molecule effector secreted encoded: molecule of function indicates CD8 naive in that to relative presented (LM), CD8 ( 4 Figure nature immunology nature to many additional genes encoding putative effector molecules. a c δ a score > 2.0; complete list, list, complete > 2.0; score ) Gene expression in resting (Naive) NK cells and in effector effector in and cells NK (Naive) resting in expression ) Gene ) Gene expression in naive NK cells and in CD8 in and cells NK naive in expression ) Gene

A comparison of the expression of genes encoding effector effector encoding genes of expression the of comparison A Expression (log fold)

Ifng 2 and e + Slamf7 Klrg1 0 2 4 6 8 + T cells at day 6 after infection with VSV or or VSV with infection 6 after day at T cells cules showed that on average, their expression was higher in in higher was expression their average, on that showed cules 3 T cells ( cells T 3 (Blimp-1)) were also expressed in naive NK cells and effector effector and cells NK naive in expressed also were (Blimp-1)) . For effector cells to respond to foreign invaders, they must must they invaders, foreign to respond to cells effector For . and genes encoding granzymes also applies transcriptome wide

Ccl5 Naive NK cells are primed for effector responses. responses. effector for primed are cells NK Naive Itgam (KLRG1)); (KLRG1)); (day 6) (CRACC)) and inhibitory NKRs and (such as (CRACC)) inhibitory VSV (RANTES; (RANTES; + Ccr2 T cells. Naive NK cells and effector CD8 effector and cells NK Naive cells. T Fig. 4a Fig. ), CD11c ( CD11c ), CD8 ) and CCR5 ( CCR5 and ) +

T , Fig. 4a Fig. b (day 6) ), which suggested a common differentiation differentiation common a suggested which ),

LM Fig. 4 Fig. b Itgax aDV + ). T of shared expression cells genes encod Supplementary Table 2 Supplementary , b A ) and CD29 ( CD29 and ) a ), consistent with published observa published with consistent ), NCE ONLINE PUBLIC ONLINE NCE Ccr5 + and T cells but not naive CD8 naive not but T cells + T cells ( Prdm1 + T cells T L. monocytogenes L. Klrc2 ) and the cell-adhesion proteins proteins cell-adhesion the and ) Naive Supplementary Table 2 Supplementary N K Tbx21 + (Blimp-1) in naive NK cells (NKG2C), T cells. Color (key) (key) Color T cells. Fig. 4 a + 34 as being expressed expressed being as T cells ( T cells Itgb1 , L. monocytogenes L. 3 + 5 T cells at at T cells (T-bet), (T-bet), . c ). This suggested that ). ( ). Gzmb Gzma Itgb1 Itgam Itgax Slamf7 Ccr2 Klrg1 Klrk1 Ccr5 Klrc2 Klrc1 Ccl5 ), rather than these these than rather ), b A Fig. 4a Fig. ) Staining of ) Staining + TION Klrk1 (right), presented as normalized log values for genes with significantly different expression; expression; different significantly with genes for values log normalized as presented (right), Klrc1

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The expression of genes encoding the IL-12 receptor ( receptor IL-12 the encoding genes of expression The IFN- promotes factors STATtranscription through cytometry flow by molecules these ( of set a of expression higher GzmB as (such as inflam of (such indicators encoding mation those included infection after 1.5 ( populations cell the among NK distance various Euclidean the on based at points cells times NK other of all profile transcriptional the from distinct clearly was cells NK effector or naive of that from different state a unique reflects that NK memory cell the proposal ( supported which cells NK memory in upregulated specifically were genes terns than was any other population pair ( infection were more similar to each other in their gene-expression pat at after day 27 cells NK memory and infection at day 7 after cells NK although On late the whole, effector was genes sustained. of expression certain infection, after 7 day by diminished was response this of (upregulation of 875 genes; Ly49H activated in of many genes expression in difference as by shown the infection, during early occurred changes The largest (day cells and 7 (day cells 27 memory after infection) after infection). effector late infection), after 1.5 (day cells effector early as infection MCMV after and (MCMV), cytomegalovirus mouse with infection Ly49H by profiling expression of gene portrait kinetic a generated we activation, pathogen-specific during (the resting state) of an NK cell’sstate single existence. a To represents explore above NK cell changes defined baseline transcriptional The infection during cells NK of profile Transcriptional Intensity in

Fig. Fig. 5 + CD8 T cells, VSV day 6 (log2) The transcriptional profile of NK cells at day 1.5 after infection infection after 1.5 day at cells NK of profile transcriptional The 0 4 8 CD11c GzmB 0 ; ; c 0.66 ). ). Published studies have demonstrated that signaling by IL-12 i. 5 Fig. Intensity innaiveNK(lo Il2ra ± 0.08 a (CD25)) and effector function (such as as (such function effector and (CD25)) and and 4 Cd69 NKG2A/C/E upeetr Tbe 3 Table Supplementary CD11b , , g Ifih1 Fig. 5 2 ) , , Fig. Fig. 8 a Ifitm1 and Intensity in 5 CD8+ T cells, LM day 6 (log ) b 2 0 4 8 Supplementary Table 3 + NKG2D ). Genes upregulated at day day at upregulated Genes ). NK cells as naive cells before before cells naive as cells NK T-bet and and 0 Fig. 5 0.76 Intensity innaiveNK(log ± ). We also confirmed confirmed also We ). Ifitm3 b 0.09 ). ). Additionally, many e c r u o s e r a Naive NK Naive CD CD OT- Naive , ), proliferation proliferation ), 3 γ c 4 6 production ) or two two or ) 8 8 . + + Il12rb1 T LM (day 6) (day LM T 6) (day VSV T KLRG1 + NK cells cells NK I Ifng Fig. 5 Fig. ). Most 2 ) ) and and ) and and a 3 8 7 ), ),  - - .

© 2012 Nature America, Inc. All rights reserved. genes encoding transcription factors shown to the prolifera factors regulate transcription encoding genes ( 7 day at higher was (MKI67) proliferation cellular with associated protein a and proteins) cycle) (cell-division (CDC cycle cell the of regulators MCMV with infection during 1,000-fold to 100- of expansion population of capable are cells NK for Ly49H MCMV. with infection after early cells NK of function and effector activation the on influence their exerted molecules regulatory pathology limit and response infection ( MCMV after early IL-10 expressed and transcribed cells proteins of SOCS activity of the because probably not but (data shown), abrogated was at rapidly day infection 1.5 after early this ( point at time SOCS3 and SOCS1 signaling cytokine of suppressors the encoding genes of the expression higher the in reflected was this in the environment is not inflammation that gen uncontrolled cells when activated, these cells must also be regulated immediately so effector potent are such cells NK Because determined. be to remains of this transcription factor influences the effector function of NK cells ( after upregulated infection ( cells NK resting ( in NK naive cells STAT1, STAT3than phorylated STAT4 and infection after 1.5 at day production of such cytokines effector as IFN- sensitized to signaling via IL-12 and STAT proteins to mediate optimal ( infection ( STAT2 and STAT1 e c r u o s e r  including lymphocytes, T of survival and tion Fig. Fig. 5

d c a Day 7 after MCMV infection marks the peak of clonal expansion expansion clonal of peak the marks infection MCMV after 7 Day Cells (relative) Cells (relative) Cells (relative) –2 Naive 100 100 100 50 50 50 0 0 0 a 0 0 0 Early effector Late effector ), ), which would serve to regulate the magnitude of the immune Day 1.5 2 p-STAT1 Fig. Expression (log + Fig. 5 Fig. CD69 KLRG1 NK cells Fig. 5 Fig. Day

5 M 7 10 10 a 10 a and and 5 5 5 ), which suggested that activated NK cells become become cells NK activated that suggested which ), Fig. 2 Fig. em Fig. 5 Fig. a 2 fold) ). Indeed, IFN- Indeed, ). 100 100 100 4 50 50 50 Stat1 0 0 0 0 . Consistent with the observation that Ly49H that observation the with . Consistent Supplementary Table 3 Table Supplementary Induced atday27 Memor Induced atdays1.5,7and27 Infection signatur Induced atdays1.5and Sustained effector Induced atday Late effector Induced atday1. Early effector Response pattern 0 0 0 e c ), both transcript and protein were further further were protein and transcript both ), ). Although ). Although p-STAT3 and and CD25 CD11b y Fig. Fig. 5a 3 4 9 10 10 Stat2 0 10 7 . Thus, both proinflammatory and and proinflammatory both Thus, . , the expression of genes encoding encoding genes of expression the , e 5 5 5 5 , γ c 100 100 100 production by NK cells peaked peaked cells NK by production 50 50 50 ); whether the higher expression the ); higher whether 0 ) increased early after MCMV MCMV after early increased ) 0 0 Tbx21 7 0 0 0 p-STAT4 CD90 IFN- (T-bet) was expressed in expressed was (T-bet) γ γ Genes Ly6c1, Ptpn Casp1, Dgkz,Fasl,Klra6,Klra10, Gp49a, Hopx,Nlrc5 Runx Foxm1, Jak3,Mki67,Nlrc5,Prkcz, St3gal3, St6galnac6 Cdc20, Foxk1,Klf11,Klf13,Klrg1, Socs3, Stat1,Stat2,Tbx21 Myd88, Oas1,Oas3,Oasl2,Socs1, IIl10, Il12rb1,Il2ra,Irf1,Irf7,Irgm1, Gbp4, Gzmb,Ifih1,Ifitm1,Ifitm3,Ifng, Bcl2, Ccl3,Ccl4,Ccr5,Cd69,Cxcl10, . . We found more phos 10 10 10 ). The expression of of expression The ). Foxm1 3 5 5 5 3 8 100 100 100 . In addition, NK . In addition, 50 50 50 0 0 0 4 0 0 0 (ref. (ref. GzmB Bcl-2 T-bet 41) and and 41) e rated; rated; 10 10 10 5 5 5

+ - -

e b to that of CD8 of that to relative infection MCMV after 7 and 1.5 on days cells NK in changes for comparison, we calculated correlations between the gene-expression T cells in response to infection. To identify the appropriate time points in gene expression common to the differentiation of NK cell and CD8 tional program central to effector differentiation, we examined changes cell–derived signals to become fully functional dendritic and organs lymphoid secondary to migration on rely cells NK preprimed, are functions effector some Although of cells. infected cytolysis the and cytokines antiviral of secretion the expansion, of cells from effector lymphoid to tissues nonlymphoid clonal tissues, migration the by characterized is infection to response effector The CD8 and cell NK Effector and mechanisms. effector effector some of prepriming the into despite cells, memory cells NK naive of differentiation the in involved are processes cellular many that suggested further and MCMV with infection during cells NK of dynamics transcriptional the explored ( staining surface cell by confirmed ( cells NK resting in than cells NK memory in ( infection after times earlier at than 7 day at lower was Bcl-2 protein at approximately day 7 expression after of infection, the antiapoptotic response cell NK the of phase contraction the of beginning the with pool that persists for months after the initial infection cells undergo cell death and leave behind a long-lived memory NK cell effector most which in occurs MCMV, with phase tion a contraction Ly49H all resented changes for markers rep these that the transcriptional demonstrated ( cells NK resting in than infection 7 after day

Fig. 5 Fig. Cells (relative) 100 50 After After the of peak the response of Ly49Heffector 0 0 d Memory ). At day 27 after infection, expression of of expression infection, after 27 day At ). Ly6c Memory Naiv MCMV day MCMV day1. aDV e 10 + A 5 T cells at each time point after infection with either either with infection after point time each at cells T NCE ONLINE PUBLIC ONLINE NCE 7 5 + NK cells rather than representing a subset. a representing than rather cells NK three independent experiments. independent three of representative are Data mice. uninfected Ly49H indicate curves shaded Ly49H of cytometry ( MCMV with infection ( MCMV with infection 7 after day at Bcl-2 of expression intracellular and CD90, and CD11b ( MCMV with STAT4, infection and after T-bet 1.5 and day at STAT1, (p-) STAT3 phosphorylated B (GzmB), IFN- of expression intracellular and ( MCMV. with infection 7 after and 1.5 days at Ly49H of transcriptome ( type. cell per replicates independent three of minimum a of analysis the on based are presented Data memory. Mem, clustering. hierarchical by Ly49H naive in expression their to relative infection after point time any at induced significantly right) at listed (subsets ( responses. memory and effector by followed response, activation early an by dominated is infection MCMV to 5 Figure cit n poen i Ly49H in protein, and script and (CD11b) of expression higher ( cells Ly49H naive in than 7 day at cells NK Klf13 d c – ); and expression of Ly6c at day 27 after after 27 Ly6c of day at expression and ); e ) Surface expression of CD69 and CD25 CD25 and CD69 of expression ) Surface + T cell differentiation T cell (ref. Fig.

The response of Ly49H of response The b

) Hierarchical clustering of the the of clustering ) Hierarchical 42), was also higher in Ly49H in higher also was 42), 5 c Fig. 5 Fig. A a ); surface expression of KLRG1, KLRG1, of expression surface ); TION ). Additionally, we observed observed we Additionally, ). Thy1 e +

a ). In summary, these data data these summary, In ). NK cells (black lines); gray gray lines); (black cells NK ) Heat map of all genes genes all of map ) Heat C9) a bt tran both as (CD90), e nature immunology nature 2 ), assessed by flow flow by assessed ), Klrg1 + 4 Fig. 5a Fig. NK cells before and and before cells NK . To define a transcrip Fig. 5 Fig. + NK cells, grouped grouped cells, NK + NK cells to infec Ly6c1 (KLRG1), (KLRG1), + NK cells from from cells NK 40 , + a d , + NK cells cells NK 4 ), a result we we result a ), ). These data data These ). NK cells at at cells NK 3 γ was higher higher was . . Consistent , granzyme , granzyme Itgam + NK NK + + - - - -

© 2012 Nature America, Inc. All rights reserved. 7 d, followed by a contraction phase and the generation of long-lived long-lived of generation the and phase contraction bya followed d, 7 massive population expansion over underwent NK cells ofcourse the Ly49H-deficient hosts ( adoptive-transfer published system a Using differentiation. memory-cell accompany that changes transcriptional the identified we functions, these establishes that program underlying the understand Tobetter effector functions much more rapidly after reexposure to antigen to seed a pool of long-lived memory cells that can subsequently acquire and CD8 cells NK both of responses immune the of phase second the During memory underlying program A conserved CD8 induction was generally smaller than the responses specific of effector transcriptome-wide their of magnitude the pathogens, to responses rapid for primed naturally are cells NK because that Weconcluded CD8 naive in than higher (67-fold cells NK naive in expression high with degree, greater a to but trend, this followed ( CD8 naive in expression their cells; NK in genes these of expression line base to was due higher mainly of induction magnitude That smaller no gene reached an induction greater than twofold in NK cells ( genes seven had an in induction CD8 two-tailed (Student’s in gene induction NK was cells 85% of that in CD8 was tion lower in in than NK CD8 cells induc gene of magnitude the that showed basis gene-by-gene a on tissues. host to damage collateral limiting of provide a means of regulating cells undergoing rapid cell division and CD8 specific KLRG1 on the surface of both Ly49H (such as E2f2 as (such factors transcription encoding genes included These ( counterparts naive their in tor NK cells and CD8 at day 7. cells NK with for at and comparison 15) 10 8, days the with infection after and 15, and points 8 6, (VSV at days correlation strongest time three the used therefore We response at day 6 after with infection either VSV or CD8 the resembled closely most response this Furthermore, similar more CD8 effector to transcriptionally whole, the on were, infection after VSV or type. cell per replicates independent three of a minimum of analysis the on based are presented of induction ‘preferential’ indicates red intervals; CD8 in and cells NK in ( genes. selected additional indicates black twofold; than greater of regulation in a difference with genes indicates Red cells. CD8 and cells NK in ( correlation. strongest or VSV with infection CD8 of response the to relative MCMV with infection 7 after ( 6 Figure nature immunology nature memory NK cells isolated at day 27 after infection ( a Fig. Fig. 6 ) Pearson correlation of the response of NK cells at day 1.5 and day day and 1.5 day at cells NK of response the of correlation ) Pearson Comparison of the effector responses of of responses NK and the cells Comparison effector CD8 We identified 32 genes with significantly altered expression in effec , , + + Hmgb2 T cells was (on average) 58% their expression in T naive was expression (on NK cells 58% their average) cells T cells, as observed before for for before observed as cells, T c 4

; slope = 0.58; 95% confidence interval, 0.36–0.79). 0.36–0.79). ; interval, = slope 95% 0.58; confidence L. L. monocytogenes 0 Itga1 , we transferred purified NK cells from wild-type mice into mice wild-type from cells NK purified transferred we , Common effector mechanisms of NK cells and CD8 and cells NK of mechanisms effector Common + T cells, most effector cells die, but those that survive go on that survive but die, those cells effector most T cells, + , , and T cells undergoing clonal expansion ( Zmiz1 + T cells than to NK cells on day 1.5 after infection. infection. after 1.5 day on cells NK to than cells T Ccl9 + effector cells, relative to their expression in naive naive in expression their to relative cells, effector L. monocytogenes L. and and b + + t ) Induction of genes encoding effector molecules molecules effector encoding genes of ) Induction T cells; dashed lines indicate 95% confidence confidence 95% indicate lines dashed T cells; ) and an effector molecule ( T cells relative to the expression of genes those -test assuming unequal variances)). Whereas Whereas variances)). unequal assuming -test Fig. Fig. 7

( HopX aDV Fig. Fig. 6 a Fig. 6 Fig. A ). ). After with infection MCMV, Ly49H ), migration and adhesion molecules molecules adhesion and migration ), NCE ONLINE PUBLIC ONLINE NCE a ); ); this showed that NK cells on day 7 b . Red indicates responses with the the with responses indicates . Red + Ifng + and and NK cells ( T cells of greater than twofold, + T ( cells and and Supplementary Table 4 Table Supplementary Gzmb Klrg1 Fig. Fig. 5 c Fig. 6 Fig. Gzmk ) Gene expression expression ) Gene Fig. 7 + + L. L. monocytogenes in NK cells. Data Data cells. NK in L. monocytogenes L. T ( cells Fig. Fig. 4 A T cells; cells; T 4 4 TION d . b ). We detected ) ) and antigen- b + ); the median ); the median T cells to T cells + ). In contrast T cells. T cells. b

); ); this may Klrg1 P Fig. 6 Fig. Fig. 6 + = 0.039 + Runx3 T cells T cell cell T 40 also also , b c 4 5 ). ). ). ). ). + - - - . . ,

expressed CD27 ( expressed frequency of memory NK cells expressed CD11b and a lower frequency Ly49H naive to program conserved in the memory differentiation of NK cells and and CD8 cells NK of transcriptional differentiation memory the in common ( a conserved program 1.28–1.72) demonstrated results interval, these confidence Together 95% 1.50; = (slope CD8 memory in greater generally was induction of magnitude the populations, gene to effector in similar induction that showed intensity expression of Analysis infec tion. after cells memory NK of persistence the promote also may mouse activated cells of 1 effector-memory type T helper survival the for critical is and cells T memory Hopx factor, that transcription and a cells identified NK ( we memory addition, of In makers cells. T cell-surface ( identified cells newly NK memory both CD8 downregulation in ­memory and CD55 CD49a and and CD62L Ly6C of of upregulation the of firmed CD8 model of virus differentiation choriomeningitis memory lymphocytic the in cells including T genes, identified Itga1 those of subset A signaling in Itga1 involved as molecules as (such encoding potential ( genes cells memory included into cells naive and of differentiation the during regulated coordinately were that genes 47 of set common a Ly49H naive of VSV with or infection after (generated CD8 memory and MCMV) with infection 27 after (day cells Hopx b a

We of profiles compared Ly49H the memory gene-expression + Gzmb

Induction in CD8 T cells (log2 fold) Time (d Hopx 0 2 4 + Correlation , , (CD49a)) and apoptosis (such as as (such apoptosis and (CD49a)) upeetr Tbe 5 Table Supplementary T cells in response to infection. to response in cells T 0 ), that was upregulated in memory cells. Published reports reports Published cells. memory in upregulated was that ), –0. Ly6c1 Hopx Dock5 0. 0. 0. 0. Stard10 Induction inNKcells(lo 2 Klrg 0 2 4 6 8 ) , , is upregulated in induced regulatory T cells and effector effector and cells T regulatory induced in upregulated is Fasl Gna15 1 Smpdl3b Runx3 and and + and and E2f 5 + T cells ( cells T VSV +

Ccl 6 NK cells or endogenous Ly49H endogenous or cells NK 2 2 NK and CD8 and NK MCMV (day1.5) Gzmk Itga1 8 Fig. Fig. 7 9 15 Gzmb 45 106 S100a6 Sytl2 0.5 c 1 ), ), consistent with published observations Fig. 7 Fig. ae lo peuae i mmr CD8 memory in upregulated also are , 2 LM g ), migration (such as as (such migration ), 6 2 8 4 fold

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© 2012 Nature America, Inc. All rights reserved. also have properties associated with adaptive CD8 adaptive with associated properties have also exploration. targeted further for mechanisms a signaling of ­demonstrates wealth unknown previously innate-like T cells is consistent with their role as primary sentinels and and by NK cells expressed molecules and signaling receptors surface of cell- abundance The similarity. a developmental than rather larity which simi a functional reflect cells, profiles gene-expression T their that suggests innate-like and cells NK naive between relatedness We transcriptional of a NK cells. close states tion have demonstrated activa the regulate that pathways of discovery the accelerate should and of functions NK cell for interpretation context the genome-wide a provide data Our cells. T particularly system, immune the of types cell other with shared many with to being NK cells, specific uniquely were transcripts few that found we addition, In cytometry. flow by far populations beyond the few leukocyte specific markers commonly used other to identify these cells and cells NK between differences lished analyses cells in a more robust and in-depth than perspective provided by pub the from available of the identity NK that define to transcripts Project explore ImmGen populations cell of breadth the used We tions. func cell NK with involved genes specific the describing resource a and insights new generate to states various in cells NK perspective, transcriptome-wide the from to work was define, of this goal A chief DISCUSSION ( type cell per replicates independent ( bars (error experiments independent two least at of representative (Memory; MCMV with infection after 28 day or (Naive) Ly49H in CD55 and CD62L ( counterparts. naive their in expression to relative presented in mice recipient the in cells NK and cells T CD8 memory in genes induced commonly of MCMV. ( with infection after 21 day CD45.1 Ly49H transferred among cells CD11b-expressing and MCMV. ( with infection in mice recipient the of population CD45.1 Ly49H transferred MCMV. ( with mice recipient (CD45.2 (Ly49H Ly49H-deficient into (CD45.1 C57BL/6 10 × (1 cells NK wild-type protocol: CD8 and cells 7 Figure e c r u o s e r  systematic a provided has work Our memory. and cytotoxicity ing Purify splenic

Although NK cells are distinct in their innate properties, they they properties, innate their in distinct are cells NK Although a NK cells Adoptive transfer + CD45.1 + + Wild-type B6

+ NK cells in the recipient mice in in mice recipient the in cells NK NK1.1 total the in cells NK (CD45.1 Common memory responses of NK NK of responses memory Common (CD45.2 ) mice, followed by infection of of infection by followed mice, ) e Ly49H ) or OT-I or CD8 ) MCMV + e T cells. ( cells. T + – 10– , + + ) f ) and endogenous Ly49H endogenous and ) Expression of Ly6c, CD49a, CD49a, Ly6c, of Expression ) + + 1 CD45.1 ) mice were transferred transferred were mice ) 2 . . Our results have shown extensive transcriptional 27 c ) Frequency of CD27- CD27- of Frequency ) + a d T cells before or 60 d (Ly6c, CD49a and CD55) or 100 d (CD62L) after infection with VSV ( VSV with infection after (CD62L) d 100 or CD55) and CD49a (Ly6c, d 60 or before cells T ) Experimental Experimental ) microarray analysis cells (CD45.1 + + Sort Ly49H b NK cells before before cells NK and Ly49H and − ) Frequency of of Frequency ) ) C57BL/6 C57BL/6 ) a d 5 after after ) Expression Expression ) + + ) from from ) d NK + TCR ) for ). −

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T cells, includ cells, T Events (% of max) Events (% of max) Time afterinfection(d) 100 100 50 50 + – 0 0 CD45.1 CD45.1 8 0 0 Naive CD8 Naive NK + + b , Ly6c Ly6c c ), s.e.m.). Data presented are based on the analysis of a minimum of three three of minimum a of analysis the on based are presented Data s.e.m.). ), 16 - - - - - + T 1 1 the expression pattern of the memory repertoire, rather than the the than rather repertoire, memory the of pattern expression the that We antigen. speculate to exposure quick secondary after allow reexpression to possibly differentiation, of stages prior from tained of by and cells genes expressed main long-lived uniquely those these composite a represents cells NK memory of transcriptome the suggests that This study. further of worthy are and memory cell NK in role potential a having as appreciated previously been not have that as (such cells memory in and genes both to included unique state the memory (such as potential. logical immuno confer to potential greatest the with cells NK memory for ferentiation is that genes specific could be as useful surrogate markers dif memory to corresponds that a signature of gene-expression implication defined one infancy, the its in is understanding memory cell Although NK of cell. function memory to cell effector late cell to effector early to cell naive from differentiation of stage each at transcriptomes cell NK distinct the by shown as state, activation in However, in changes of context the must dynamic this be interpreted CD8 and cell NK CD8 and memory memory NK cell differentiation signature that and were shared by effector effector the of elements Wefound functions. these with ciated and has identified hundreds of genes not activities previously common known to be these asso with associated genes the of ­identification 0 0 5 5 As for for As 100 100 50 50 Ly6c1 0 0 24 0 0 Memory NK Memory CD8 c α ) and initially expressed in effector cells and maintained maintained and cells effector in expressed initially and ) aDV Positive NK cells (%) β 100 50 CD49a CD49a T cells, the memory NK cell differentiation signature signature differentiation cell NK memory the cells, T 0 A Ly49H Ly49H NCE ONLINE PUBLIC ONLINE NCE CD27 + + +

T cell lineages of some activation mechanisms. mechanisms. activation some of lineages cell T T cells, which suggests conservation between the between conservation suggests which T cells, T – + 1

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–3 2 0 3 6 1 1 Cd55 0

0 –3 ), which represent genes genes represent which ), 5 5 nature immunology nature 100 100 Expression inmemory 50 50 b 0 0 NK cells(log , Sell c Ptpn4 0 0 Sytl2 , Hopx e Gzm Fasl , 0 f ) are are ) b Pmaip1 Ly6c1 S100a6 CD55 CD55 Casp1 2 Casp1 fold) Itga1 3 1 1 , , 0 0 Fasl 5 5 6 - - - - ,

© 2012 Nature America, Inc. All rights reserved. 15. 14. 13. 12. 11. 10. 9. 8. 7. 6. 5. 4. 3. 2. 1. reprints/index.html. R Published online at The authors declare no competing interests.financial and L.L.L. studies designed and wrote the paper. J.A.B. and A.W.G. anddesigned did the T cell studies; and N.A.B., C.C.K., J.C.S. experiments and analyzed data; G.M.-O., D.W.H. and Y.K. did experiments; C.C.K. analyzed data; N.A.B. and J.C.S. sorted cell subsets, did follow-up Research (G.M.-O.) and the Searle Scholars Program (J.C.S.). American (L.L.L. andCancer Society N.A.B.), the Canadian Institutes of Health to T32AI060537 D.W.H.; to T32AI060536 J.A.B.; and AI072117 to A.W.G.), the of the US National Institutes of Health (R24 AI072073 and R01 AI068129; Project. Supported by the National Institute of and Allergy Infectious Diseases andand ExpressioneBioscience, Affymetrix Analysis for support of the ImmGen A. J.Beaulieu, Karo and S. Madera for data from MCMV infection experiments; and processing; J. Jarjoura and J. Arakawa-Hoyt for assistance in cell sorting; the ImmGen core team (M. Painter, J. Ericson and S. Davis) for data generation the members of the ImmGen Consortium and M. Dozmorov for discussions; We thank A. Weiss (University of San California, Francisco) for antibody to Syk; Note: Supplementary information is available in the codes. Accession the pape the of in version available are references associated any and Methods M biology. cell NK of investigation further the for resource unique a providing while findings published extend and support simultaneously data Our of T lymphocytes. related context closely the in function cell NK of stages various on perspective state. In sum, our study has a provided comprehensive transcriptome NK cell the memory to is define needed themselves, genes ­individual nature immunology nature COMP AUTHOR CONTRIBUTIONS A eprints and permissions information is available online at http://www.nature.com/ at online available is information permissions and eprints cknowl

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© 2012 Nature America, Inc. All rights reserved. and and Program in Cellular and Molecular Medicine, Children’s Hospital, Boston, USA. Massachusetts, USA. University, Providence, Rhode Island, USA. USA. Massachusetts, California, USA. USA. 7 Michio Painter Natalie A Bezman Francis Kim Jim J Collins Angelique Bellemare-Pelletier Nadia Cohen Ananda Goldrath E ImmGen Consortium: e c r u o s e r 1 Harvard Harvard Medical School, Boston, USA. Massachusetts, & Immunology, University of California, San Francisco, San Francisco, California, USA. Chase Cancer Center, USA. Pennsylvania, Philadelphia, Icahn Icahn Medical Institute, Mount Sinai Hospital, New York, New York, USA. 0 mmanuel L Gautier

16 9 Division Division of Biological Sciences, University of California San Diego, La Jolla, California, USA. University University of Medical Massachusetts School, Worcester, USA. Massachusetts, 11 18 15 Division Division of Rheumatology, Immunology and Allergy, Brigham and Women’s Hospital, Boston, USA. Massachusetts, 11 , , Tata Nageswara Rao , , Kavitha Narayan 13 22 , , Patrick Brennan Dana-Farber Dana-Farber Cancer Institute and Harvard Medical School, Boston, USA. Massachusetts, , , Jeffrey 9 21 , , Jennifer Miller , , Joseph C Sun 7 , 8 , Claudia , JakubzickClaudia E ricson 15 13 Department Department of Biomedical Engineering, Howard Hughes Medical Institute, Boston University, Boston, Massachusetts, , Deepali Malhotra , Deepali 16 11 22 , , Katelyn Sylvia 21 , , Michael Brenner 7 , , Scott Davis 18 , , Brian Brown , Charlie , C Charlie Kim , , Amy Wagers 20 Department Department of Medicine, Boston University, Boston, USA. Massachusetts, 7 , , Gwendalyn J Randolph 22 , , Diane Mathis 7 16 , , Miriam Merad 13 8 18 , , Joonsoo Kang Department Department of Pathology & Immunology, Washington University, St. Louis, Missouri, 21 11 , , Shannon Turley , , Susan A Shinton , Lewis L , Lanier Lewis , , Tal Shay 17 22 Department Department of Stem Cell and Regenerative Biology, Harvard University, Department Department of Microbiology & Immunobiology, Division of Immunology, 22 12 & Christophe Benoist , , Aviv Regev 7 aDV 16 , , Vladimir Jojic 10 18 7 , , Roi Gazit , Computer Computer Science Department, Stanford University, Stanford, Joslin Joslin Diabetes Center, Boston, USA. Massachusetts, 8 21 A 13 , , Adam J Best 19 NCE ONLINE PUBLIC ONLINE NCE , , Tracy Heng , , Radu Jianu , , Richard R Hardy 14 12 Department Department of Computer Science, Brown 17 , , Anne Fletcher , Derrick , J Derrick Rossi 10 9 14 22 , , Daphne Koller , , Jamie Knell , , David Laidlaw , , Taras Kreslavsky A 22 TION 21 19 12 Department Department of Microbiology Broad Broad Institute, Cambridge,

, , Paul Monach nature immunology nature 13 , , Kutlu 17 9 , ,

10 14 ,

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lpek , 19 20 Fox Fox , 13 , © 2012 Nature America, Inc. All rights reserved. prepared for hybridization to the Affymetrix MoGene 1.0 ST array with with array ST accordance in Assay 1.0 TargetLabeling Sense MoGene Transcript Whole GeneChip the Affymetrix the to hybridization for prepared analysis. and hybridization Microarray mice. 16 from Ly49H memory for except sorted, were samples triplicate independent population, each For (BD). FACSAria a with (Invitrogen) Trizol into directly double-sorted were 10 × ~1 and markers, cell-surface for stained and mice three from pooled were samples All infection. MCMV after 27 day on mice Ly49H-deficient of from NK were spleens reconstituted cells isolated from C57BL/6 mice on days 1.5 and 7 after MCMV infection. Memory Ly49H Ly49H Effector mice. C57BL/6 uninfected of spleens Ly49H or NK Naive Project. ImmGen the of sorting. Cell Francisco. San Francisco, San California, of the Institutional Animal Care and Use of Committee guidelines the University were purified from mice 2 d after transfer. Experiments were done according to OT-I cells, 5 × 10 10 × 5 5 × 10 × 5 with infected were mice transfer, after d At 1 recipients. C57BL/6 into ferred viral In infection. the case before of CD8 d 1 mice C57BL/6 Ly49H-deficient into transferred adoptively described as NK cells of memory for generation the was used system An adoptive-transfer 10 × (5 strain Smith MCMV of injection peritoneal under specific pathogen–free conditions. C57BL/6 mice were infected by intra B6.SJL- and C57BL/6 Pepc male week-old eight to six- Project, ImmGen infection. and Mice MET ONLINE doi:10.1038/ni.2395 b /BoyJ congenic CD45.1 congenic /BoyJ 3 3 plaque-forming units of ovalbumin-expressing VSV. naive To obtain ovalbumin-expressing of units plaque-forming colony-forming units of ovalbumin-expressing ovalbumin-expressing of units colony-forming 4 Cells were prepared according to standard operating protocols protocols operating standard to according prepared were Cells 0 . Purified NK cells from B6.SJL- from cells NK Purified . + 6 H NK cells, which independent duplicate samples were sorted were sorted samples duplicate independent which NK cells, CD45.1 ODS In accordance with standard operating protocols of the the of protocols operating standard with accordance In + OT-I cells were transferred into C57BL/6 mice and + T cells, 5 × 10 + mice (Jackson Laboratory) were maintained maintained were Laboratory) (Jackson mice Isolated RNA was amplified and and amplified was RNA Isolated 3 CD45.1 + 4 to 3 × 10 × 3 to NK cells were isolated from from isolated were cells NK Ptprc + 4 a + OT-I T cells were trans plaque-forming units). units). plaque-forming

NK cells were isolated isolated were cells NK Pepc L. monocytogenes L. 4 cells (>99% pure) pure) (>99% cells b /BoyJ mice were were mice /BoyJ Ptprc or or + - - a

STAT4 (38/p-Stat4; all from BD); Live/Dead Fixable Near-IR dye (L10119; (L10119; dye Near-IR Fixable Live/Dead BD); from all (38/p-Stat4; STAT4 STAT3 to body phosphorylated (4/P-STAT3) and to antibody phosphorylated (GL3), anti-granzyme B (GB11), antibody to phosphorylated STAT1 (4a), anti anti-CD49a (Ha31/8), anti-CD62L BioLegend); (MEL-14), anti-Ly6C (AL-21), from anti-TCR all (4B10; anti-T-bet and (30-H12) anti-CD90 (H1.2F3), anti-CD69 (RIKO-3), anti-CD55 (N418), anti-CD11c eBioscience); from all anti- (104), IFN- anti-CD45.2 (A20), anti-CD45.1 (2F1), anti-KLRG1 (H1.2F3), DX5), anti-NKG2A-NKG2C-NKG2E (20d5), anti-CD11b (M1/70), anti-CD69 or (MI-6 anti-NKG2D anti-Ly49I (YLI-90), (3D10), Ly49F anti-y49H (CM4), anti-Gr-1 (RB6-8C5), anti-B220 (RA3-6B2), anti-Ter119 (Ter119),(PC61.5), anti-Ly49E-anti-CD25 (MB19-1), anti-CD19 (53-7.3), anti-CD5 eBio500A2), 597), anti-CD4 (RM4-5 or anti-CD8 anti-CD3 GK1.5), or (53-6.7), (145-2C11 anti-TCR anti-NKp46 (29A1.4), PK136), to body (anti-NK1.1; NK1.1 anti used: were proteins intracellular and markers surface cell to antibodies (10 cytometry. Flow population. details one Additional least at of replicates all in expressed were they if only sons and above probes were in expression background, indicate compari included to taken was 120 of threshold post-normalization ­consortium-standardized ( base data GEO the in available are samples ImmGen all from data Raw Pattern). (Gene module Creator’ File ‘Expression the in algorithm average multichip robust the with normalized were data Raw instructions. manufacturer’s with 49. (Tree Star). software FloJo with (BD) II LSR an on lyzed ana were cells All (BD). instructions manufacturer’s to according done was staining Intracellular Weiss). A. by provided (5F5; anti-Syk and Invitrogen);

ahta D. Malhotra, lymph nodes defines immunological hallmarks. (2012). immunological defines nodes lymph µ γ g/ml; g/ml; 2.4G2; UCSF Antibody Core) before surface staining. The following (XMG1.2), anti-Bcl-2 (10C4), anti-CD45.1 (A20) and anti-CD45.2 (104; GSE1590 7 t al. et ); processed data are available on the ImmGen website. The The website. ImmGen the on available are data processed ); Fc receptors were blocked with mAb to CD16 and CD32 CD32 and CD16 to mAb with blocked were receptors Fc 4 9 are provided as as provided are Transcriptional profiling of stroma from inflamed andresting inflamed from stroma of profiling Transcriptional Supplementary Notes 1 and 2 and 1 Notes Supplementary a. Immunol. Nat. nature immunology nature

13 499–510 , . β (H57- γ δ - - - - -