© 2012 Nature America, Inc. All rights reserved. paper. should Correspondence be addressed to M.M. ([email protected]). USA. California, USA. 6 New York, USA. York, New York, USA. 1 lymph nodes tissue-draining the to migrate to ability superior their with surface cell the on II class MHC and I class (MHC) plex in com and major histocompatibility of form of the peptide complexes foreign and self present and process to ability their on antigens self tissues the breach that antigens foreign to responses immune adaptive -specific initiate to is of DCs function main repair, the tissue promote and microbes genic macrophages, whose main role is to scavenge damaged cells or patho responses immune tissue of induction the antigens, sense environmental extracellular injuries and contribute to in most and tissues ability localization their to their including sample macrophages, tissue with properties share that cells hematopoietic lineage cell hematopoietic the to addition recent most the are DCs ago, years (DCs). 50 only cells Discovered dendritic of development lineage the of function and the control that networks regulatory and profile tional transcrip the to define we sought pipe database, extensive With this line. data-generation controlled rigorously same the with lineage networks across and the entire regulatory- hematopoietic of gene-expression database an to exhaustive create have united who from many institutions biologists and computational immunologists of consortium a is Project (ImmGen) Genome Immunological The steady-state migration of tissue DCs to the drainingsignature lymph nodesthat thatdistinguished may control tissue tolerance DCs from to macrophages.self tissueof myeloid Weantigens. alsoprogenitor identified cells a totranscriptional the DC lineage program and precursors,expressedpredicted specificallyregulatorsacross the ofduring entireDC functionaltheimmune system.diversity Here in wecomparative tissues.characterized We analysis identifiedcandidate of CD8atranscriptional molecular activators involvedregulation in ofthe thecommitment lineage commitment and functional Althoughspecialization much ofprogress DCs has been made in the understanding of the ontogeny and function of dendritic cells (DCs), Gwendalyn J theRandolph transcriptional Jeremy Price Gaurav Pandey Jennifer C Miller dendritic cell lineage Deciphering the network transcriptional of the nature immunology nature Received 12 March; accepted 8 June; published online 15 July 2012; Department Department of Pathology & Immunology, Washington University in St. Louis, St. Louis, Missouri, USA. Immunology Institute, Mount Sinai School of Medicine, New York, New York, USA. 10 6 Present Present address: Department of Computer Science, UNC, Chapel Hill, North Carolina, USA. and localize together with T lymphocytes and B lymphocytes 3 . The unique role of DCs in adaptive immunity relies relies immunity adaptive in DCs of role unique The . 4 8 Broad Broad Institute, Cambridge, USA. Massachusetts, Cancer Cancer Immunology and AIDS, Dana Farber Cancer Institute, Boston, USA. Massachusetts, 1 , 2 3 , , Melanie Greter 3 Department Department of Genetics and Genomic Sciences, Institute for Genomics and Multiscale Biology, Mount Sinai School of Medicine, New York, , Marylene Leboeuf , Marylene 1 , 2

, , Brian D Brown aDV 1 + A , 1 , CD103 5 . DCs represent a small population of population small a represent DCs . NCE ONLINE PUBLIC ONLINE NCE , 2 6 , as well as to maintain tolerance to to tolerance maintain to as well as , , , Shannon J Turley 1 + , , CD11b 2 , 7 1 , , Milena Bogunovic . However, in contrast to to contrast in However, . 1 1 , , 3 2 , , Tal Shay , , Kutlu G Elpek + and plasmacytoid DC subsets, as well as macrophage DC precursors and common DC A TION 8 4 5 , , Miriam Merad ,

Department Department of Regenerative Biology, Mount Sinai School of Medicine, New York, New York, USA. 5 4 , together together , doi:10.1038/ni.237 , , Emmanuel L Gautier 8 7 , , Julie Helft 1 - - - - .

, 2 , , Angelique Bellemare-Pelletier 2 Department Department of Oncological Sciences, Mount Sinai School of Medicine, New in the bone marrow are starting to be characterized. A myeloid myeloid A characterized. be to starting are marrow bone the in intestine the in cells T regulatory of induction the facilitate CD8 stimulating at potent most the are and CD103 cDCs DCs also include two cDC subsets: the CD103 antigens to viral immunity and initiate interferon- antiviral the of amounts large uniquely produce to able are pDCs The (pDCs). DCs plasmacytoid the are DCs of CD4 at potent most the stimulating CD8 to antigens cell-associated of are functionally specialized; CD8 CD8 tissue–resident lymphoid as subcategorized further are and subset lymphocytes tor antigens, respond to environmental stimuli and engage effec distinct to debated DCs still is responses and immune tissue macrophages of contribution exact the consequently, and, and functions macrophages phenotypes overlapping of partially their and because DCs still between distinction controversies overall the However, about exist an of response. induction immune the control to antigen-specific poised uniquely DCs makes This The successive steps that lead to commitment to the DC lineage lineage DC the to commitment to lead that steps successive The process to abilities different with subsets distinct of consist DCs + in vivo 0 1 1 cDCs and CD8 3 , + 2 . Similar . to Similar CD8 lymphoid-tissue cDCs are efficient cross-presenters of cell-associated antigens & the Genome Immunological Consortium 1 , 2 remains poorly understood. We made a comprehensive , , Daigo Hashimoto 11 Full Full list of members and affiliations appears at the end of the 7 1 Computer Computer Science Department, Stanford University, Stanford, 1 , 5 0 . Classical DCs (cDCs) form the predominant DC DC predominant the form (cDCs) DCs Classical . , 6 , , Vladimir Jojic 9 – Albert Albert Einstein College of Medicine, Bronx, New York, cDCs 1 1 . Lymphoid tissue–resident cDC subsets + cDCs excel in the cross-presentation + + T cells, whereas CD8 whereas cells, T 1 T cells. The second main subset main subset The second T cells. , 8 2 , , Paul S Frenette , , Andrew Chow 7 + , 8 10 cDCs, nonlymphoid-tissue nonlymphoid-tissue cDCs, , 9 . , Ariella , Cohain Ariella + 1 + 2 T cells T cDCs and the CD11b . Nonlymphoid-tissue . Nonlymphoid-tissue e c r u o s e r 1 0 but may also also may but – 1 9 cDCs are are cDCs , 1 , 2 4 , . 9 , 3 11 , α  + -

© 2012 Nature America, Inc. All rights reserved. For of analysis site whether or batch may effects have the confounded 2, expression. (population CD11c of basis the on mainly DCs purified Boston) in spleen sorted to identical be to found was and B220 or F4/80 of expression of lack and CD11c and II class MHC of sion in 1, New sorted York)(population on of was the sorted basis expres New York, and Boston, Massachusetts. One population of spleen DCs The DC purified subsets were isolated from laboratories in New York, before described as control quality and generation data with line, Affymetrix ST1.0 microarrays according to the ImmGen Project pipe described as arrays micro to hybridized and lysate TRIzol the from prepared was RNA Massachusetts. Boston, in Project ImmGen the of team core the to them sent and 2 min after samples the froze TRIzol, in directly cells) ( purity of over 99% on a the basis of to the appropriate cell surface markers populations cell Wedouble-sorted circulating blood. the isolated from and monocytes marrow bone the from CDPs (GMPs), and MDPs precursors granulocyte-macrophage purified We nodes. migratory CD103 tissue- We isolated epidermis. the from (LCs) cells Langerhans mal epider isolated and ; and intestine , small , the from CD103 purified nodes; lymph and mus CD8 Weisolated to according the protocol standard operating of the Project. ImmGen immunological specialized function with subsets DC discrete represent to thought of on of sues on markers surface basis the expression cell the tis nonlymphoid and tissues lymphoid secondary tissues, lymphoid primary from isolated populations DC distinct 26 Wecharacterized lineage DC the of characterization Transcriptional RESULTS origin. lineage or tissue their of regardless nodes, lymph draining the to migrated have that cDCs nonlymphoid-tissue all by expressed of signature the a tify identifies as well as study subsets iden diversity. also of DC results Our tissue regulators predicted the DC Our tissue various tissues. between relationship in lineage macrophages from distinguishes cDCs that signature our DC-specific a of results characterize as The help study state. well as migratory a in DCs, DCs nonlymphoid-tissue nonlymphoid-tissue and lymphoid-tissue cells, system. and immune the across vertically horizontally both analysis comparative comprehensive, for able avail data and limited the of because DCs part in unanswered remain tions ques lymphoid-tissue These understood. between poorly remain DCs, relationship nonlymphoid-tissue the DCs as of well specialization as functional and and lineage DC diversification the the the to DCs, of commitment of function regulation and transcriptional ontogeny the understanding in made cDCs tissue–resident phoid CD8 tissue–resident lymphoid into pDCs dif to into potential ferentiate the lost have that cells progenitor cDC-restricted into and pDCs cDCs to differentiate exclusively to rise give and potential macrophages the or monocytes lost have that cells precursor (CDP) precursor DC common the monocytes to to and rise give to shown been has and identified been has (MDP) precursor’ DC and ‘macrophage the called cell ­precursor e c r u o s e r  CD8 signals, Table

Here we delineate the transcriptional network of DC progenitor progenitor DC of network transcriptional the delineate we Here 17 1 , 1 ). We did the final sorting by flow cytometry (10,000–30,000 ). (10,000–30,000 We by cytometry flow sorting did the final 8 in vivo in . CDPs also produce pre-cDCs, which are circulating circulating are which pre-cDCs, produce also CDPs . + and CD8 and 1 + + 3 and CD8 and ( DCs and CD11b 2 Table 1 1 . Expression profiling data were generated on on generated were data profiling Expression . 6 and home to tissues to differentiate locally locally differentiate to tissues to home and – spleen cDCs were sorted independently at independently sorted were cDCs spleen 1 ). We sorted each subset to high purity purity high to subset each sorted We ). – 1 cDCs and pDCs from the spleen, thy spleen, the from pDCs and cDCs 9 . Although much progress has been been has progress much Although . + DCs from tissue-draining lymph + or CD8 or + cDCs and CD11b and cDCs 1 6 . CDPs are clonogenic clonogenic are CDPs . – cDCs 1 6 and nonlym and n vivo in + cDCs cDCs 2 1 1 5 ------, .

known to have a role in cell fate and the initiation of transcrip of initiation the and fate cell in role a have to known This macrophages. included and group DCs of development global the influence more or GMPs; in than MDPs in 1.5-fold higher (expression MDPs into GMPs of the during differentiation specifically upregulated was group first The differ DC entiation. during stages different at induced were that encoding genes regulators of groups identified thereby and step dif ferentiation each with correlated that tran and for activators search to scriptional tree lineage myeloid-DC the along expression potential pDC of loss the poten monocyte of loss tial the with CDP, to MDP from transition fates precluded been granulocyte and have lymphoid , erythroid, point MDP,which at the of stage the at determined is lineage phagocyte mononuclear to the lineage myeloid of the of cells commitment The lineage DC the to commitment of control Transcriptional ( CD8 between for differences the influences, excellent correlation in each subset, with little evidence of lab-specific (New York locations showed data The two different the and Boston). expression; expression; lower (67% downregulated was group third A Zbtb46). factor tion of expression low as such development, pDC ulate reg to known molecules encoding genes included group CDP-versus-monocyte This fate. control probably that molecules ( encoded monocytes dif circulating the into MDPs during of not but ferentiation GMPs) or MDPs in than CDPs in higher the more or 1.5-fold during (expression CDP to GMP or MDP from upregulated transition was group second The respectively. tion, tified. The factors E2-2 (encoded by by (encoded E2-2 factors transcription The tified. iden be to starting also are diversification DC regulate that Factors by Flt3 and signaling DC mediates differentiation Flt3L-dependent by (encoded PU.1 factor transcription the by controlled partly is sion Flt3L its and Flt3 cytokine the Ikaros zinc-finger the on dependent is cDCs and pDCs of differentiation the example, For development. DC control to shown liver fetal in hematopoiesis definitive during function as well CD103 as such genes expected CD8 in expression CD8 their in more or 1.5-fold factors upregulated transcription were that encoding genes identified also We factors). of of upregulation downregulation the with associated TGF- of the tor with associated was of pDCs downregulation into CDPs of differentiation The lymphoid-tissue pDCs, into CDPs CD8 of differentiation the during regulators encoding genes of candidate expression the CDPs, we examined of fate pDC-versus-cDC the to contribute might that lators Trim13 factor- included Supplementary Fig. 1 Fig. Supplementary Several transcription factors identified in our analysis have been been have analysis our in identified factors transcription Several Sfpi1 1 6 + , whereas cDC commitment occurs at the pre-cDC stage, with with stage, pre-cDC the at occurs commitment cDC , whereas cDCs or cDCs CD8 lymphoid-tissue β + (which encodes an E3 ). To search for regu for To search ligase). ubiquitin E3 an encodes (which Pbx1 ) (TGF- cell–specific IRF8 (refs. (refs. IRF8 factor transcription cell–specific 2 Tgif1 7 aDV ), and the transcription factor STAT3, which is activated activated is STAT3,which factor transcription the and ), Fig. 1 Fig. β , which encodes a Hox transcription factor shown to to shown factor transcription Hox a encodes which , ), whereas the differentiation of CDPs into cDCs was was cDCs into CDPs of differentiation the whereas ), A (a gene induced by transforming growth growth transforming by induced gene homeobox (a β Batf3 Sox4 NCE ONLINE PUBLIC ONLINE NCE )), )), a , iii) during the transition from GMP to MDP and , the transition during iii) Id2 Zbtb46 Tcfec and and , , 15 Bcl6 , , ). Zbtb46 , 16 (which encodes a transcription factor) and factor) a transcription encodes (which – Taf4b Irf8 17 , cDCs (and vice versa). These included included These versa). vice (and cDCs and 2 (which encodes zinc-finger transcrip zinc-finger encodes (which 2 , 1 . DC commitment occurs during the the during occurs commitment DC . 8 wih noe te CD8 the encodes which , . We probed the pattern of regulator regulator of pattern the We probed . and and Ciita , which transcription factors factors transcription encode , which Fig. 1 Fig. Irf8 Cited2 A (which all encode transcription transcription encode all (which – TION cDCs ( cDCs , , a Bcl11a , i), which would potentially potentially would which i), , Irf8

(which encodes a regula encodes (which – nature immunology nature Supplementary Fig. 2 Supplementary and CD8 , , and and Tcf4 2 6 + (and Flt3 expres Flt3 (and cDCs relative to to relative cDCs Runx2 and and Tcf4 Fig. 1 Fig. + cDC subsets subsets cDC ), Spi-B and and Spi-B ), 2 Runx2 4 + 19–23), as as 19–23), , as well as as well as , . cell– and and cell– a , ii) and and ii) , and and 2 2 5 8 ). ). ------, .

© 2012 Nature America, Inc. All rights reserved. CD103 CD11b CD103 Nonlymphoid-tissue DCs pDC LC CD11b CD103 Migratory DCs CD8 CD8 Resident DCs Lymphoid-tissue DCs Population Table 1 GMPs Precursors LCs MA, DCsisolatedfromlaboratoriesinNewYork, NewYork, andBoston,Massachusetts,respectively. Dataarerepresentativeofatleastthreeexperiments. dures oftheImmGenProject.ThespleenpDCsincludeaCD8 Expression ofcell-surfacemarkersbyDCspurifiedflowcytometryfromthebonemarrow (precursorcells)ortissues(differentiatedaccordingtostandardoperationproce Monocyte CDPs MDPs control into differentiation the or monocyte DC lineage as well as DC that cues molecular the to identify and of helped DCs generation the in checkpoints key accompanied that regulators potential unknown a provided results these map of and known regulators previously also phagocytes tissue other from cDCs distinguishing for marker useful a as serve can Zbtb46 that and cDCs tissue and cells that have shown studies published two lineage, of cDC ment CDPs to the that finding our CD103 intestinal of ferentiation (ref. IRF4 of CD8 tiation development of CD8 IRF8 BATF3, factors transcription The pDCs of not but cDCs of development the controls IRF8 have been shown to control pDC differentiation nature immunology nature diversification − + Zbtb46 + + + + + CD11b

Expression Expression of cell surface markers by DCs and DC precursor populations 8) and Notch2 (ref. (ref. Notch2 and 8) expression is restricted to cDC-committed precursor precursor cDC-committed to restricted is expression + SI Kidney SI Liver Lung SI Liver Lung Spleen SDLN MLN Spleen SDLN MLN SDLN MLN SDLN Spleen Spleen (MA) Spleen (NY) MLN SDLN Thymus Spleen (MA) Spleen (NY) MLN SDLN BM Blood BM BM – in vivo in cDCs is controlled by the transcription factors IRF2 factors by transcription the is controlled cDCs Location Zbtb46 + cDCs and CD103 . expression was associated with the commit the with associated was expression aDV + + + + + + + + + + + + + + + + + + + + + + + + + + + + A

20), a factor that also controls the dif the controls also that factor a 20), CD45 NCE ONLINE PUBLIC ONLINE NCE + hi hi hi hi hi + + + + + + + + + + + + − CD11b MHCII int int int int int + , + + + + + + + + + + + + + + + + + + + + + + + Id2 and mTOR control the the control mTOR and Id2 cDCs, whereas cDCs, the differen

+ CDllc cDCs − − − − − + + + − − − − − − − − − − + + + + + − − CD8 + 2 subsetandaCD8 − − − − − − − − − − − + + + + − − − − − − 0 . Consistent with with Consistent .

A CD4 2 3 TION 9 0 , whereas Bcl-6 + + + + − − − + + + − − + + + + − − − − − + + − − in the spleen. spleen. the in 31

, CDllb 3 2

. Together . − − − + + + + − − − − + + + − CD103 − lo − − − + − subset.CSF1R,colony-stimulatingfactor1receptor;Lang,;BM,bonemarrow;NYand F4/80 - - - , + + − − − − − − − − + + − − − + −

Gr-1 transcripts absent from four prototypical macrophage populations populations macrophage prototypical four from absent transcripts CD103 nonlymphoid-tissue and cDCs CD8 lymphoid-tissue whether investigated and population CD11b nonlymphoid-tissue As macrophages. from absent but subsets cDC all in present genes and cDCs formed distinct populations at the transcriptome level. organ ( same the from isolated leukocytes steady-statevarious byexpression componentanalysis (PCA) of the15% of genes with themost variable ers clustered as one population or as separate populations by whether cDCs and macrophages sorted on the basis of published mark cific phenotypic markers for defining tissue cDCs. We first ascertained cDCs versus macrophages in tissue immunity has been the lack of spe One of the main challenges in understanding the exact contribution of signature gene core a cDC of Identification + + − − + + − − − − We then sought to determine whether cDCs expressed a set of of set a expressed cDCs whether determine to sought then We B220 Marker Supplementary Fig. 1d − − − Sca-1 8 , hi hi 9 lo , we excluded those cells from our comparative analysis analysis from our comparative cells , those we excluded c-Kit + + + CSF1R + + Flt3 − − − − − − − − − − − − − − − + ). These results showed that macrophages cDCs probably form a heterogeneous heterogeneous a form probably cDCs CD3 − − − − − − − − − − − − CD19 − − − − − − − − − − − +

cDCs shared specific cDC cDC specific shared cDCs Ter119 − − − − − − − − − − − − − − − − NK1.1 e c r u o s e r CD16-CD32 + NKp46.1 CD34 Ds n CD8 and cDCs CD43 Lang Lang Lang Lang Other − − − − − − − − + + − + + & − −

hi principal- Replicate Number 7 3 2 4 2 5 3 3 2 3 3 3 3 3 3 3 5 6 2 3 3 5 3 3 3 3 3 2 4 3 3 3 -  – - -

© 2012 Nature America, Inc. All rights reserved. CD26 were expressed as on spleen CD8 spleen on proteins as expressed were CD26 confirmed that Flt3, c-Kit, the we cytometry, flow By unclear. remains function DC in role whose ( as well as molecules, II class MHC encoding genes many ing ( Table 1 CD8 in in CD8 and T cell antigen receptors by mediated signaling attenuates that superfamily immunoglobulin ferentiation dif mast-cell as well as hematopoiesis in role its for known ligand), as biology, such role in cDC no identified with products encoded genes subsets cell hematopoietic ( other all in expression cDCs their to in relative upregulation substantial showed CD13) aminopeptidase cell hematopoietic other populations, in present also were macrophages from absent were and cDCs in enrichment showed that genes many of the DCs of homeostasis and differentiation the control ( draining the to lymph cDCs nodes of migration the control to shown been has ( gene the Table expressed; being of chance 95% a has gene the which at value (the value ‘QC95’ the below values expression by determined ( less or 0.05 of (FDR) rate false-discovery a and macrophages, and cDC between more or fold two of expression in change a (with cDCs in expressed (Online genes 24 Wefound Project Methods). ImmGen the by profiled in otherwise noted (unless replicates more or three with experiments three least at of representative are Data correlation. Pearson the with linkage centroid pairwise by clustered were genes and populations and centered, and samples) across zero of expression mean the (to normalized log-transformed, were results CD8 versus CD8 and pDCs, versus (cDCs checkpoint that of neighbor developmental nearest the in expression their to relative checkpoint cellular each at 1.5-fold least at by upregulated in results ( here. presented are both for transcripts two were There families. (Mono) MDP, monocyte and CDP GMP, the across expression gene of patterns common by plots in grouped and samples all across expression mean to normalized (iii), CDPs in expression their to relative MDPs in higher or 1.5-fold or, (ii) conversely, GMPs and/or MDPs in expression their to relative CDPs in more or 1.5-fold upregulated or (i) GMPs in expression their to relative MDPs in more or 1.5-fold upregulated regulators transcriptional ( lineage. DC the along factors transcription 1 Figure e c r u o s e r  CD8 a Supplementary Fig. 3 Supplementary Fig. Fig. 2 t Fig.

-test)) in cDCs relative to their expression in macrophages, includ in macrophages, expression to their relative in cDCs -test)) ) Kinetics of the expression of genes encoding encoding genes of expression the of ) Kinetics Kit –

1a cDCs, as well as nonlymphoid-tissue CD103 nonlymphoid-tissue as well as cDCs, , which encodes c-Kit, the receptor for stem cell factor (Kit (Kit factor cell stem for receptor the c-Kit, encodes which , 2 a + ). Several ). genes Several were upregulated twofold or more (FDR –

). This group formed the ‘core cDC signature’ and included included and signature’ cDC ‘core the formed group This ). and ) cDC and cDC CD103 cDCs ( cDCs Expression of genes encoding encoding genes of Expression ; and and ; a Ccr7 , along with those of transcripts transcripts of those with , along + cDCs; cDCs; 3 Table 5 3 , and and , 3 Zbtb46 Flt3 ; ; ) that encodes the chemokine receptor CCR7, which which CCR7, receptor chemokine the encodes that ) Zbtb46, Fig. Fig. Supplementary Fig. 2 Fig. Supplementary , which encodes the known to to known receptor cytokine the encodes which , 3 ), which encodes the dipeptidyl peptidase CD26, CD26, peptidase the dipeptidyl encodes ), which Btla , , 2 , , l3 Pvrl1 Flt3, and which was first upregulated at upregulated was the CDP first which stage b Supplementary Fig. Supplementary , which encodes CD272, a member of the the of member a CD272, encodes which , ) Heat map of the the of map ) Heat + cDC populations relative to relative its populations expression cDC t -test)) and absent from macrophages, as as macrophages, from absent and -test)) Runx2 Supplementary Table 1 Supplementary

inhibitory 3 and and and and 6 Table . . Btla – ); ); cDCs cDCs

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Anpep was specifically upregulated was specifically receptor CD272 (BTLA) and ).

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3 (which encodes the the encodes (which and and + cDCs and spleen spleen and cDCs + ). ). Notably, many cDCs, and were were and cDCs, Supplementary Supplementary iii ii a i 22 , 3 Tcfec Chd7 Tgif1 Trim13 Kdm5b Tcf4 Hmgn3 Zeb1 Irf8 Bach2 Hdac9 Runx2 Zbtb46 Tcf19 Spib Runx2 Klf8 Hes6 Bcl11a Nsbp1 Erg Taf4b Sox4 Mtf2 Npm 4 Fig. 2 Fig. . Although Although . 1 a ≤ Dpp4 and and 0.05 0.05 GMP Expression (normalized) Expression (normalized) Expression (normalized) Expression (normalized) - -

MDP and different subsets exist in lymphoid and nonlymphoid tissues. tissues. nonlymphoid To and understand lymphoid the relationships in among these exist various DCs subsets different and distinctcell-surfaceofmarkers,basis the classified areonsubsets DC clusters DC tissue distinct of signatures gene Unique tissues. in macrophages identified a core DC signature that helped distinguish tissue DCs from ( microglia and macrophages toneal absent from red-pulp macrophages, lung alveolar macrophages, peri

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+1 populations, Mycl Hmgn3 Irf7 Stat Irf8 Myb Bcl11a Sp3 Notch3 Cbx4 Hdac5 Notch1 Ikzf Foxp1 Hhex Eya1 Hoxa5 Runx2 Mef2c Ets1 Hivep3 Ikzf Spi Sfp691 Pml Tcf12 Tcf Trim2 Tsc22d Mxd4 Xbp1 Sp110 Zeb2 Hmgb1 Cited2 Hmgb2 Uhrf Tcf19 Ctbp2 Zfp367 Bhlhe40 Chd3 Bcl6 Zbtb46 Batf3 Id Egr2 Egr3 Etv3 Nr4a3 Egr1 Trerf Tcf Ciit Cbfa2t Nfatc2 Klf3 Nfat Nr4a1 Relb Chd3 Trps1 Trps1 Klf3 Nab2 E2f Hlx Kcnip3 Tgif1 Pbx3 Sfpi Nfix Ehf Prdm1 Tbx21 Notch4 Pbx1 Mllt Ncoa7 Rbpj Zfp318 Hivep3 Elk3 Stat Pou2f Zfp36l Creb3l Junb Aff1 Arid3b Aff3 Arid5a Arid3a Aof Atad2 Asf1b Ahr Atf3 Aebp2 Arid5b 2 4 7 a 8 2 b 1 2 3 3 1 2 4 5 1 1 1 5 2 2 2 3 1 -

© 2012 Nature America, Inc. All rights reserved. Fig. 4 Fig. including pDCs, other whereas clusters the twocDCs, cDC expressed 125 genes from absent from absent genes 93 expressed cluster pDC The signatures. ( by lymphoid-tissue CD8 nonlymphoid-tissueandCD103 CD8 lymphoid-tissue by formed was cluster one clusters: DC distinct three identified components principal main The intestine). CD103 nonlymphoid-tissue CD8 spleen thymusand CD8 spleen (, cDCs skin- spleen, the (from ­draining lymph nodes and mesenteric pDCs lymph nodes), lymphoid lymphoid-tissue of populations expression in variable most the with genes of 15% the of PCA did we in otherwise noted (unless replicates more or three with experiments independent three of representative are Data . control isotype-matched curves), shaded (grey Isotype populations. macrophage tissue and cDCs CD8 spleen gated in BTLA and c-Kit CD26, Flt3, of expression the assessing , and cavity peritoneal liver, ( Massachusetts. Boston, from sample York; (2), York, New New from sample (1), MF, intestine; macrophage; small SI, node; lymph mesenteric MLN, node; lymph skin-draining SDLN, signature. cDC core the form in as presented CD103 nonlymphoid-tissue and cDCs lymphoid-tissue in expression 2 Figure nature immunology nature CD8 by shared genes 28 showed subsets Fig. 3 Fig. a CD8 and

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Identification of genes significantly upregulated in cDCs relative to their expression in macrophages. ( macrophages. in expression their to relative cDCs in upregulated significantly genes of Identification + CD8 spleen DC (1) Supplementary Table Supplementary 1 CD8 + – spleen DC (2) CD4 + CD8 SDLN DC Fig. 1 Fig. + CD103 MLN DC Zbtb46, Pvrl1 Zbtb46, + CD103 cDCs; and spleen CD8 spleen and cDCs; +

b lung DC CD103

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Anpep Peritoneal cavity MF + Lung MF spleen DC +1 Microglia + Tbc1d8 Spint2 Runx3 Pstpip1 Napsa Jak2 Haao H2-Q6 H2-Eb1 H2-DMb2 H2-Ab1 H2-Aa Fgl2 Dpp4 Cnn2 Ciita Cbfa2t3 Bri3bp Zbtb46 T Slamf7 Sept6 Rab30 Pvrl1 P2ry10 Km Klri1 Kit Hmgn3 H2-Eb2 Gpr82 Gpr68 Gpr132 Gpr114 Flt3 Ccr7 Btla Bcl11a Anpep Ass1 Ass1 Ap1s3 Amica Adam19 cDCs and CD103 and cDCs ( – raf1 CD4 + Fig. 3 Fig. o cDCs; lymph node and lymph node cDCs; – CD11b b , , A TION Supplementary Supplementary + cDCs) and cDCs)

Brain cavity Peritoneal Lung Spleen Liver propria Lamina Kidney Lung Spleen b b ) Flow cytometry of single-cell suspensions of the spleen, lung, kidney, lamina propria, propria, kidney, lamina lung, spleen, the of suspensions single-cell of cytometry ) Flow + + - cDCs cDCs + cDCs cDCs tissue cDCs than in four prototypical macrophage populations (values, (values, populations macrophage prototypical four in than cDCs Table

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1 (% of ) 100 20 40 60 80 ). 0 and and ( cDCs other from absent and together with several genes specific to monocytes and monocytes to specific genes several with together Lymphoid ( lineage cell CD8 in ofCD8 Notably, results for lung CD103 response to the TLR3 ligand poly(I:C) also the only lymphoid-tissue DC subset that produces interferon- ands CD103 CD8 TLR3, expressionofunique their agreementwith In (TLR3)) and CD103 Flt3 0 10 2 37– upeetr Tbe 1 Table Supplementary 10 3 + + + 10 4 + CD11b cDCs included included cDCs 0 T effector cells in mice andhumans mice in cells effector T Ds n CD103 and cDCs 4 Xcr1 . In. addition, published data have shown that CD8 10 - tissue CD8 5 CD26 Xcr1 Supplementary Fig. 4c 4c Fig. Supplementary 0 (encoding CCXCR1, which controlsdifferentiation the which CCXCR1, (encoding 10 – 2 cDCs share a superior ability to respond to TLR3 lig 10 (which encodes the chemokine receptor CCXCR1). 3 10 4 10 – + cDCs 5 CD11b c-Kit 0 Tlr3 10 2 + 10

populations coexpressed core cDC genes Ds cos h etr hemat entire the across cDCs + 3 . ee seii t CD8 to specific Genes ). wih noe Tl-ie eetr 3 receptor Toll-like encodes (which – Fig. 3 Fig. cDCs or CD8 or cDCs 10 a DCs (J.H. and M.M., data not shown). ) Heat map of transcripts with different different with transcripts of map ) Heat 4 10 5 b BTLA 4 0 , , 1 and 10 Supplementary Figs. Supplementary , and we have obtained similar 2 10 upeetr Table Supplementary 3 10 – cDCs, tissue CD103 tissue cDCs, 42 4 10 , 4 5 3 ) was expressedonlywas ) e c r u o s e r Isotype MF Red-pulp MF Isotype CD103 CD103 Isotype CD103 CD103 Isotype CD103 CD103 Isotype CD103 CD103 Isotype CD Isotype CD CD CD Isotype microgli Microglia Isotype MF Peritoneal MF Isotype MF Alveolar MF Tables 2 8 8 + – DC DC

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© 2012 Nature America, Inc. All rights reserved. tinal CD103 tinal ­significant upregulation in CD8 ( module this of regulators predicted Ds eaie o hi epeso i CD8 in expression their to relative cDCs ( ( ment for of genes CD8 the CD8 in tion and ( cDCs to CDPs of commitment the during upregulated also of this included BATF3module and RelB ( ( (hypergeometric test)), genes such as cDC core for enrichment significant and Module F156 ( showed significant pDCs to upregulation CDP in of cDCs ( mitment Tsc22d1 by (encoded Egr5 STAT2, and IRF8, Runx2 included module this of ( pDCs in upregulation from the ImmGen Project ( samples of rest the the in expression to their relative subsets DC cific modules.htm ( compendium Project ImmGen entire the for identified regulators predicted their and coexpression substantial with genes of modules fine 334 the among searched we axon guidance Table1 ( monocytes or phages were expressed only by CD8 genes, including genes macrophage-related of expression different published results showing that CD8 ( are representativeofatleastthreeexperiments(average ­according totheQC95value;resultsarepresentedas‘relativeexpressionunits’.Data populations: red-pulp,alveolarandperitoneal-cavitymacrophagesmicroglia), cDC subsets;FDR Genes ofthetranscriptomeexpressedincDCs(excludingnonlymphoid-tissueCD11b Zbtb46 Traf1 Slamf7 Sept6 Rab30 Pvrl1 P2ry10 Kmo Klri1 Kit Hmgn3 H2-Eb2 Gpr82 Gpr68 Gpr132 Gpr114 Flt3 Ccr7 Btla Bcl11a Ass1 Ap1s3 Amica1 Adam19 Gene Table 2 e c r u o s e r  SupplementaryFig. 4 Fig. 3 Fig. P

< 1 × 10 × 1 < To delineate the gene ‘architecture program’ of these subsets of DCs, Supplementary Fig. 2 e ). Dscam has enormoushasdiversity Dscam ). molecular andinvolved is in

). IRF8 and Pbx-1, encoded by genes upregulated in CD8 in upregulated genes by encoded Pbx-1, and IRF8 ). ; ; Core Core cDC transcripts Fig. 3 Fig. –13 + + cDCs and CD103 cDCs l CD11b ) to identify those with significant upregulation in spe upregulation significant with those ) to identify 4 ≤ (hypergeometric test)), such as as such test)), (hypergeometric c 4 0.05(t-test))andnotexpressedinmacrophages(fourmacrophage Dscam and pathogen recognition ), whose genes were also expressed during the com the during expressed also were genes whose ), 1,007.6 3,408.4 1,481.7 1,044.4 1,727.4 1,080.3 1,160.4 2,368.3 519.2 527.4 782.2 406.6 1,012 940.1 642.4 702.6 1,119 cDC expression 289.4 400.8 289.2 475.6 153.6 + 67.7 cDCs, a nonlymphoid-tissue cDC subset that that subset cDC nonlymphoid-tissue a cDCs, (which encodes the adhesion molecule Dscam), 905 and upeetr Fg 4 Fig. Supplementary P = 4.77 × 10 × 4.77 = )). Module 152 showed significant upregula + ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± Fig. 3c DC and DC CD103 – 234.2 SupplementaryTable 1 152.7 204.7 371.1 18.2 132.9 251.5 262.9 111.2 216.9 259.2 54.2 52.8 54.3 25.3 61.4 224.5 145.6 202.3 212.6 375.7 127.9 370.3 413.3 cDCs andcDCs were not expressed in macro i. 1 Fig. Zbtb46 + – http cDCs ( cDCs cDCs ( – e – b cDCs consist of two subsets with ). ). Module 150 showed significant ://www.immgen.org/Mo and and and Fig. 3 Fig. –11 P P = 1.08 × 10 = 1.34 × 10 = 1.34 ), and predicted regulators regulators predicted and ), Fig. Fig. 3 4 Pvrl1 upeetr Fg 2 Fig. Supplementary 5 + . – ± DC transcript signature transcript DC e Macrophage expression Ds ( cDCs s.e.m.). ). Module 154 showed showed 154 Module ). and and . . Predicted regulators d Tlr3, Xcr1 Tlr3, ), whose ), genes whose were 93.8 99.2 2 51.6 31.7 28.7 74.4 19.6 20.7 17.2 67.7 23.1 43.4 11.7 52.5 84.1 57.1 81.3 49.4 95.3 43.7 45.5 94.3 66.3 69.8 0 P ), consistent ), with . However, some some However, . –15 41 × 10 × 4.18 = Supplementary Supplementary –25 P i. 1 Fig. = 7.01 × 10 ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ) ) and in intes ) ) and enrich 10.3 12.4 2.8 5.2 3.6 6.1 2.2 1.1 0.5 6.1 2.5 5.5 0.8 4.8 5.2 3.9 7.2 3.9 9.3 7.1 3.1 6.1 11 2 and and b dsRegs/ ), were were ), Fig. 1 Fig. Fzd1 –35 –10 )). )). +

b + - - - - - ­ )

and and kidney, and did PCA of the along with rest of those sub the cDC as well as intestine, CD11b from the small CD103 we purified above, identified signature cDC core of the of genes sion cDCs to than macrophages to closely more migrates poorly to the draining lymph nodes 1 factor for receptor its for development ligand colony-stimulating monocytes lating cDCs to efficiently the draining lymph nodes CDPs and pre-DCs CD103 functionally. and CD103 of CD11b. expression the integrins different with subsets and ontogenically both The intestine small is cDC populated by distinct three phenotypically subset cDC characterized CD11b Nonlymphoid-tissue CD11b specialization functional of DC regulators as potential well as genes unknown previously identified signature, gene core the CD8 has been shown to share development properties with lymphoid experiments (average macrophages (populations asin tissue CD11b Genes ofthetranscriptomesignificantly upregulated incDCs(excluding Tbc1d8 Spint2 Runx3 Pstpip1 Napsa Jak2 Haao H2-Q6 H2-Eb1 H2-DMb2 H2-Ab1 H2-Aa Fgl2 Dpp4 Cnn2 Ciita Cbfa2t3 Bri3bp Anpep Gene Table 3 signaling molecule (FzD1) that controls activation of ( identified expressed all transcripts specific to the CD8 6 Fig. scripts of CD8 clustered near lymphoid CD8 CD8 CD103 the with clustered population. heterogenous a nonlymphoid-tissue CD11b ( genes core cDC of number variable a PCA plot between the cDCs and macrophages ( CD11b The peritoneum. and brain lung, spleen, the from isolated macrophages with and sets To determine whether those subsets had differences in their expres In the PCA plot, CD103 plot, PCA the In – 4 cDCs ). ). Accordingly, CD103 9 + + + + . In contrast, the CD103 the contrast, In .

CD11b CD11b CD11b DC heterogeneity delineates the cDC core gene signature Genes Genes upregulated in cDCs + aDV cDCsubsets;FDR Fig. Fig. 2 0 ( + A Supplementary Fig. 5 Supplementary cDCs and CD103 + – – NCE ONLINE PUBLIC ONLINE NCE 3 , , CD103 cDCs and CD103 and cDCs cDCs from the lamina propria of the propria from of intestine the lamina cDCs the small ± 46 ), including including ), s.e.m.). 46 , 4 10,299.7 13,267.3 , 7 4 1,276.4 2,567.1 1,481.4 2,491.8 1,884.8 1,266.3 1,682.8 , develops independently of Flt3L, requires the the requires Flt3L, of independently develops , 1,616.4 7,165.4 7 2009.8 DC expression , require Flt3L for their development 475.1 653.2 814.3 773.6 672.8 2,300 1,692 + Table + CD11b cDC subsets were distributed across the the across distributed were subsets cDC ≤ + + 0.05(t-test))relativetotheirexpression in + Ds n CD103 and cDCs + CD11b cDCs, as defined at present, represented – ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± CD11b 2 cDCs and did not express unique tran 209.5 42.7 59.4 86.3 326 360 166 149.9 305.6 271.2 100.8 197.7 490.6 555.7 237 608 151.1 234.7 190.1 Fzd1 ). Dataarerepresentative ofatleastthree + – Ds ean h least-well- the remain cDCs + CD11b + cDCs ( and CD103 + , which encodes a Wnt-receptor Wnt-receptor a encodes which , – A CD11b cDCs from the small intestine intestine small the from cDCs ). These modules, together with together ). modules, These – TION cDCs from the small intestine intestine small the from cDCs 4 Fig. 4 Fig. 8 and are thought to represent + Fig. Fig. 4 subset derives from circu from derives subset +

+ cDCs from the lung, liver cDCs 4 + 4 cDCs and CD103 9 b nature immunology nature cDCs are derived from from derived are cDCs 8 . and is thought to relate Macrophage expression ), which indicated that that indicated which ), – Fig. 4 a CD11b 1,093.6 1,632.6 and 704.4 139.4 219.3 100.9 104.4 190.9 331.9 152.3 400.5 230.7 121.4 208.5 + 90.2 72.3 87.1 cDCs, whereas whereas cDCs, 288 177 β a Supplementary Supplementary ) ) and expressed -catenin and its + in in vivo ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± cDC subsets subsets cDC

755.3 48.4 44.1 458.9 26.3 29.6 20.7 8.4 1173.7 75.8 11.3 16.4 49.7 51.5 24.9 67.6 54.7 46.8 52.5 nonlymphoid 4 7 , migrate . +

- cDCs tissue 4 7 - - - - ,

© 2012 Nature America, Inc. All rights reserved. 40% of the cDC genes and lacked the cDC proteins c-Kit, Flt3, BTLA Flt3, c-Kit, proteins cDC the and 40% of genes lacked cDC the ( plot PCA the in cDCs from away CD103 In contrast, lineage. the to belonged subsets two these that suggested which ( surface cell the on CD26 and BTLA Flt3, c-Kit, proteins cDC-specific the expressed also and genes cDC core of the 100% expressed intestine small the from DCs the PCA we results, found that CD103 ( plot PCA in the DCs other away and from CD103 of function of the contribution examine should FzD1 to the immunomodulatory intestine the in tolerance cell T promote to DCs of β ( intestine from the small cDCs tine and was absent from CD103 in CD103 specifically expressed translocation to the nucleus ( in otherwise noted (unless replicates more or three with or T8 cell; pre-B T, preB, CD8 CD8 monocyte; MO, progenitor; MEP, megakaryocyte-erythrocyte CMP, progenitor; cell; stem lympoid CLP, common hematopoietic progenitor; HSC, myeloid common repressors). predicted blue, activators; predicted (red, box in outlined algorithm Ontogenet the with predicted ( F152 ( CD8 and pDCs, versus cDCs (FDR population comparison CD8 3 Figure (5.13% nature immunology nature ( and on CD26 surface the cell F150 c GN PC3 c a -catenin and its translocation to the nucleus can control the ability ability the control can nucleus the to translocation its and -catenin – In contrast, the CD103 e F630111l10rik, Gm10790,Gsn,Havcr1,Ifi205,Kdr,Klra17,Met,Mycl1,Myh10, P2ry14, Pde1b,Pdia5,Plxdc2,Ppt1,Runx2,Siglech,Sspn,Tmprss8,Zfp691 ) ImmGen Project fine modules, consisting of genes with high coexpression with projection of module F150 ( F150 module of projection with coexpression high with genes of consisting modules, fine Project ImmGen ) + Amica1, Cadm1,Cd209d,Cd209e,Cd300c,Clec1a,Clec9a,Cox6a2,Ece1, MF ) cDCs, CD8 cDCs, –0.100 0.100 0.200 e Smad3, Foxf1a,Esr1 MO LC –0.100 ) across data from the ImmGen Project (mean of each module). Below, genes expressed in each module, with genes encoding regulators regulators encoding genes with module, each in expressed genes Below, module). each of (mean Project ImmGen the from data across ) Mafb, Rbpj,Insm1,

0 Unique gene signatures characterize distinct tissue DC clusters. ( clusters. DC tissue distinct characterize signatures gene Unique CD11 CD8 CD8 CD8 CD8 CD8 CD8 CD8 CD8 CD8 DC CD8 MEP + (8.97%) b CD8 T cell; T4 or CD4 T, CD4 or CD4 T4 cell; T + 0 PC2 + + + + + + + + – CD11 CD10 spleenpDC SDLN MLN spleenDC(2) spleenDC(1) thymusDC SDLNpDC MLNpDC spleenpD + cDC DC – 0.100 cDC – GMP MDP CMP b 3 cDCs and CD103 and cDCs + + CD103 – CD10 CD11b DC 0.200 – C CD11b 3 0.300 + + CD8 cD

DC

– C 0.146 CD CD8 – DC + aDV + CD4 CD103 CD103 CD103 CD4 CD4 CD4 CD4 CD11b P HSC cDCs and CD103 and cDCs cDCs clustered near macrophages and and macrophages near clustered cDCs ≤ + + 0.140 Pbx1, Mycl1,Tsc22d1 Fig. 4b Fig. Batf3, Ciita,Stat2,Irf8,Runx 0.05 ( 0.05 cDCs in the small intestine. small the in cDCs Supplementary Figs. Supplementary 4 + – + + + pDC CD11b spleenDC(1) MLNDC SDLNDC spleenDC(2) A Fig. 4b Fig. + + + SIDC lungDC liverDC Supplementary Fig. 6 Supplementary NCE ONLINE PUBLIC ONLINE NCE 0.133 + + preB + CD11b CD11b + B + cDCs. ( cDCs. spleenDC cDCs clustered cDCs near macr t Fig. 4 Fig. + – 0.126 -test)) and not expressed in the exclusion population (according to the QC95 value), for pDCs versus cDCs, cDCs, versus pDCs for value), QC95 the to (according population exclusion the in expressed not and -test)) NK T cell; GN, granulocyte; B, B cell; NK, natural killer cell. Data are representative of at least three experiments experiments three least at of representative are Data cell. killer natural NK, cell; B B, granulocyte; GN, cell; T (79.96% d CLP – Naive T8 PC1 + and and CD8 d pDC CD11b 0.119 and – + a T cDCs from the small intes from small the cDCs b ) cDCs and CD103 ), and they expressed only only expressed they and ), ) Heat map of genes significantly upregulated by at least twofold relative to their expression in the the in expression their to relative twofold least at by upregulated significantly genes of map Heat ) CD4 Naive T4 2 0.112 + Supplementary Fig. 6 Fig. Supplementary Fig. 4 Fig.

cDCs versus pDCs or CD8 or pDCs versus cDCs Supplementary Fig. 6 Fig. Supplementary T +1 –1 0 – and CD103 b a ). Consistent with with ). Consistent 5

A p 0 Table Table

and DC TION . Future studies studies Future . ). Activation of). Activation 6 ). ). 1

). ). + –

Fzd1 CD8 CD11b CD11b o + phages phages cDC was

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– + cDCs (full gene list, list, gene (full cDCs a cDC ) PCA of the 15% of genes with the most variable expression in pDCs, pDCs, in expression variable most the with genes of 15% the of PCA ) in response to tissue injury or tissue immunization are starting to be be to are starting immunization or tissue injury tissue to response in nodes lymph draining the in cDCs nonlymphoid-tissue of function cDCs’ ­migratory ‘tissue- called also tissue, drained the from migrated have that cDCs CD103 nonlymphoid-tissue as well as DCs’, CD8 pDCs, Tissue-draining lymph nodes contain blood-derived DCs that include DCs migratory of signature transcriptional Unique tissues. nonlymphoid in macrophages from cDCs CD11b signature of distinguishing means gene a new provide may here cDC the identified of use the that and DCs tissue sufficient not identify is to CD11c, and II class MHC of DCs, of expression on based definition phenotypic present the that established results (E.L.G. macrophages with the that indicated CD103 transcripts macrophage-associated of analysis focused Accordingly, lineage. macrophage the to belonged intestine CD103 the that suggested which 0 CD8 – cDC +1 ) – CD11b Tlr3 Cd36 Hepacam2 Ifi205 Xcr1 Clec4g Card6 Il17rb Duxb Klra17 Cdh1 Duxb Prkc Grm8 Blnk Cmah Klk1 Mctp2 Zfp521 Pacsin1 Bcl2a1a Dock5 Ptpn22 Cd83 Tlr13 Bcl2a1c Fgl2 Pak1 Cd Anpe Slamf8 Id Cxcl16 Ddef2 Cd86 E2f8 Batf3 Myc Tlr11 Nlrp1c 2 9

+ a l l p

cDCs and CD8

+ cDC population from the small intestine clustered clustered intestine small the from population cDC 6 Supplementary Table 1 Supplementary . The mechanisms that control the migration and and migration the control that mechanisms The . e d 6030429G01rik, A530099J19rik,Adam23,Apol7c,Avpi1,C130092O11rik,Cadm3,Dnase1l3, Eg240055,Fam149a,Fndc7,Fzd1,Gcet2,Gpr120,Gpr33,Gramd2,Hepacam2,Htr7,Ildr1, Rogdi, Slc46a3,Tbc1d4,Tbc1d9,Tlr11,Tmeff1,Tspan33,Ttc39a,Zbtb46,Zc3h12c,Zfp366 F152 F156 Ehf, Fndc4,Gm11545,Gpr124,Gpr149,Gpr157,Gpr4,Hap1,Il12b,Mpzl2,Ptchd1,Pvrl1, Itga8,Naalad2,Notch4,Plce1,Ppef2,Praf2,Sept3,Snx22,Tlr3,Tmem27,Xcr1,Zfp57 1700009j07Rik, C530028O21Rik,CD207,Cldn1,Clec4b2,Cxx1b,Cxx1c,Dbn1,Dkk3 GN GN

MF MF Elk3 MO MO LC LC Rbpj, Ebf1,Nr4a2 – et al. et CD11b CD11b cDCs, also cDCs, ‘lymph called node DC DC ME ME + P P + CD11b CD103 CD11b CD103 c , data not shown). Together these these Together shown). not data , DC DC ), module F156 ( F156 module ), Rarb, Hic1,Pax5 Zeb2, Ciita,Klf8, GM CM GM MD MD CM – + + CD11b + + CD103 P CD103 P P P P P DC DC ; presented as in in as presented ; + + CD CD DC DC 8 8 – – , + CD CD CD CD cDCs from the small small the from cDCs DC DC P P HS HS 8 8 + + + Ciita, Pbx1,Relb,Mycl Cbfa2t3, Klf8,Batf3, Ds n CD11b and cDCs C C e c r u o s e r pD pD d C C ) and module module and ) preB preB B B Ncoa7, Batf3 Pbx1, Irf8,Mycl Fig. 1 Fig. NK NK CL CL Naive T8 Naive T8 CD8 P CD8 P – 1 b resident resident T T ). ). 1 Naive T4 CD4 Naive T4 CD4

+1 T –1 T , +1 –1

0 0  + +

© 2012 Nature America, Inc. All rights reserved. had had moderate expression of gene the specific CD11b to tissue in contrast we that found In addition, that in migrated the steady state may have represented the cDCs. true ( CD103 with together clustered ( program ( tissue drained populated that the populations cDC parent the from away segregated origin, tissue or cellular their of regardless plot PCA the in together segregated cDCs migratory that found we Notably, cDCs. resident node– lymph of that as well as nodes, lymph the to migration after and population) DC (parent nodes lymph draining the to migration their before cDCs of tissue program Wetranscriptional the analyzed state in of cDCs the uninflamed function migratory immunological the control that regulators gene the or nodes lymph draining the to migrate and tissues peripheral leave to cDCs of ability the controls that program gene the about known is less far however, elucidated; CD11b and on BTLA c-Kit CD103 CD26, and Flt3, left); dye (far DAPI the with DNA-intercalating staining on and CD11b CD45 CD103 by cytometry: flow analyzed intestine, small ( in macrophages. to expression their in relative cDCs upregulated in (identified above those match and cDCs CD8 4 Figure e c r u o s e r  always had cDCs of expression high Fig.

a

CD103 d

10 10 10 5 10 0.400 + 0 a 3 4 5 2 cells. Data are representative of three independent experiments with three or more replicates (unless noted otherwise in otherwise noted (unless or replicates three more with experiments of independent three are Data representative cells.

), which suggested that among tissue CD11b tissue among that suggested which ), CD11b Heterogeneity of nonlymphoid-tissue CD11b of nonlymphoid-tissue Heterogeneity 0 MF 10

Fig. 5b Fig.

Isotype CD103 CD103 CD103 PC 2 (5.93%) 2 PC 2 Fig. Fig. 0.200 – 10 cDCs, nonlymphoid-tissue CD103 nonlymphoid-tissue cDCs, – + + 3

CD11b CD11b CD11b 2 10 c ) ) in CD11b – CD103 4 ). ). ( LC e 0 ). Migratory CD11b Migratory ). + + 10 – i. 5 Fig. DC DC b DC 5 – ) Frequency of core transcripts and transcripts significantly upregulated in cDCs relative to their expression in macrophages in macrophages to expression their in relative cDCs upregulated significantly and transcripts of transcripts core ) Frequency CD11b –0.200 + a Events (% of max) + SI DC 100 ) and shared a similar transcriptional transcriptional similar a shared and ) cDC subsets and red-pulp macrophages, among all transcript present (in each subset). ( (in subset). each present all transcript among macrophages, and red-pulp cDC subsets 20 40 60 80 0 0.168 CD8 0 Flt3 CD11b + 10 migratory cDCs in the PCA plot plot PCA the in cDCs migratory + 2 CD103 0.180 CD8 10 Flt3 + CD11b

liver DC 3 CD103 – 10 CD11b + cDC 0.191 cDC . Specifically, epidermal LCs, . epidermal Specifically,

4 PC 1 (77.61%) + + 10

lung DC + cDCs and migratory LCs LCs migratory and cDCs CD11b 5 + 0.203 kidney DC CD2 Flt3 0 + cDCs, nonlymphoid-tissue CD11b nonlymphoid-tissue cDCs, 10 +

6 2 SI DC 0.215 , migratory , CD11b migratory + 10 cDCs. ( cDCs. 3 10 –0.200 0 0.20 0.40 0.22 4 + + 10 0 0 (3.10%) cDCs, which which cDCs, 7 cDCs, those those cDCs, 5 PC a c-Kit ) PCA of the 15% of genes with the most variable expression in lymphoid-tissue CD8 in lymphoid-tissue ) expression PCA of the 15% the variable of with most genes 3 0 d 10

) Expression of the products of core cDC genes in single-cell suspensions of the suspensions of in cDC core genes single-cell of the products ) Expression CD4 2 – 10 CD11b CD103 3 10 b + + 6

CD11c + 4

+ spleen DC cDC transcript (%) . CD11bCD11b 100 10

20 40 60 80 0 5 + +

BTLA CD11b that were upregulated in steady-state migratory cDCs included included cDCs migratory steady-state in upregulated were that cDCs ( poly(I:C)-activated did than molecules immunomodulatory ( tory inflamma encoding genes upregulate not did cDCs migratory state been has that CD40 LCs migratory molecule on steady-state reported costimulatory the encodes which upregulated cDCs tissue-migratory and steady-state activated cDCs with poly(I:C)-activated migratory cDCs ( steady-state compared further we injury, to response in responses ( immune of dampening the to dedicated molecules encoding genes of function cDCs. or migratory steady-state homeostasis the in Flt3 for role critical a suggested ( nodes lymph the reached they once expression of Flt3L and Flt3 of independently develop which kidney SI DC i. 6 Fig. 0 +

10 CD11b We also found that tissue-migratory cDCs upregulated some some upregulated cDCs tissue-migratory that found also We

cDCs from the lamina propria that expressed MHC class II MHC class and lacked expressed that propria the from lamina cDCs + CD103 2 lung DC

10 +

+ – liver DC 3 Red-pulp spleen MF cDCs, epidermal LCs and macrophages (population means; colors colors means; (population LCs and macrophages epidermal cDCs, CD11b 10 a ). The genes encoding immunomodulatory molecules molecules immunomodulatory encoding genes The ). 4 + 10 aDV SI DCLC Fig. Fig. 5 Flt3 A 6 Fig. Fig. 6 NCE ONLINE PUBLIC ONLINE NCE ). ). As such ‘dampening genes’ can also be upregulated in tissues, showed considerable upregulation of + CD8 c CD11b + CD8 thymus DC b +

) ) and had expression higher of genes encoding CD8 spleen DC (1) + CD8 spleen DC (2) –

cells, CD103 cells, + CD8 SDLN DC CD103+ MLN DC CD103 + CD103 lung DC + CD4 liver DC + + CD11b CD4 spleen DC (1) A + Fig. 6

c –

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cells and CD103 cells + kidney DC 1 CD11b + + a 9 lung DC SI DC and have very low low very have and ); ); however, steady- CD103 + 0 1 LC liver DC

). – Red-pulpCD11b MF

Peritoneal cavity MF + SI DC

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Microglia Tbc1d Spint2 Runx3 Pstpip Napsa Haao H2-Q6 H2-Eb1 H2-DMb2 H2-Ab1 H2-Aa Fgl Dpp4 Cnn2 Ciita Cbfa2t3 Bri3b Zbtb4 Traf1 Slamf7 Sept Rab30 Pvrl P2ry10 Km Klri Hmgn H2-Eb2 Gpr8 Gpr6 Gpr132 Gpr114 Ccr Bcl11a Jak2 Anpe Ass1 Ass1 Ap1s3 Amica Adam19 Ki Flt3 Btl ), which which ), t a 2 1 o 7 1 – 6 Cd40 2 8 p p + 6 3

1 8

Flt3 - ,

© 2012 Nature America, Inc. All rights reserved. not expressed in nonlymphoid-tissue cDCs, according to the QC95 value). LuLN, lung-draining lymph node. Data are representative of three experiments of experiments three are Data representative node. lymph lung-draining LuLN, to the value). QC95 according cDCs, in nonlymphoid-tissue not expressed ( as in ( CD103 in 50% or migratory (blue) more ( means). (population nodes lymph and lung-draining CD103 and migratory nodes, lymph the from skin-draining LCs isolated (Mig) migratory LCs, epidermal CD8 tissue-resident by lymphoid expression variable 5 Figure TGF- the as such cytokines, immunosuppressive of activation or duction by (encoded PD- as such directly, either function cell T suppress to known molecules encoding those nature immunology nature in otherwise noted (unless or replicates three more with Supplementary Table Supplementary 2 t -test). ( -test). 0.230 b (75.16% a β ). ). ( -activating -activating integrin PC1 0.217

c Tissue-migratory cDCs upregulate a unique gene signature regardless of tissue or cellular origin. ( origin. or of cellular tissue regardless signature a gene unique Tissue-migratory upregulate cDCs d ) in migratory CD103 in migratory ) expression Gene ) Gene expression in migratory LCs, relative to that epidermal LCs (colors as in LCs (colors to epidermal that relative LCs, in migratory ) expression Gene 0.203 ) 0.189 0.176 d b Migratory DC c + Mig CD11b+ cDC Mig CD11b cDC CD103

(fold) (fold) 0.162 CD8 0.01 0.01 + ) upregulated at least fivefold in migratory cDCs relative to the mean expression in nonlymphoid-tissue cDCs (red font, transcripts transcripts font, (red cDCs in nonlymphoid-tissue to the expression mean relative cDCs in migratory at fivefold least ) upregulated CD11b 10 10 + 1 1 CD8 P value 0.01 aDV 0.05 0.400 β – – 0.01

8 cDC (encoded by A Mig CD103 Mig CD103 0.01 NCE ONLINE PUBLIC ONLINE NCE 0.200 Mig LC(fold) 0.5 0.5 + 0.5 cDCs and cDCs CD11b Cd274 (7.82% + + CD11b 1 1 cDC(fold) cDC(fold) PC2 1 0 1 2 1 2 Itgb8 LC 2 ) + + ) cDC, cDCs and cDCs CD11b 5 2 –0.200 10 ) , or through the pro the through or , + 0 5 0 b cDCs and cDCs CD8 3 . . Other ) Genes with significant upregulation of twofold or more (red) or significant downregulation by downregulation or or (red) significant more of twofold upregulation ) significant with Genes 0.400 0.200 0 –0.200 Table A TION + cDCs, relative to that in nonlymphoid tissue–resident CD103 tissue–resident to in that nonlymphoid relative cDCs, (4.47%

1 upregulated PC3 ).

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CD8 -

+ CD8 Thymus DC

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b + ). ). ( CD4 Spleen DC (1) 5 + 4 e

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a lung DCspleen DC

) ) PCA of the 15% the of with most genes CD103 + liver DC CD103 + CD11b –1 CD11b +

CD11b – + CD11b + SI DC cDCs isolated from skin-draining skin-draining from isolated cDCs liver DC Expression (fold) + CD103 + SI DC lung DC + LC – and CD11b + b CD11b

0 Mig CD11b cDCs and cDCs CD8 –

d +

; right margin, genes genes ; margin, right Mig CD103 SI DC Mig CD103 + SDLN DC + Mig CD11b e c r u o s e r +1 SDLN DC

+ +

cDCs. cDCs. Mig LC LuLN DC Spsb1 Extl1 Adora2a Rnf115 E130311K13Ri Arhgap28 Anxa3 Insm1 Ankrd33b N28178 Stap2 Il1 Socs2 Cacnb3 Il15ra Eno2 Adcy6 BC062109 Itgb8 Nudt17 Gal3st2 Ktelc1 Zfand6 Cbl Hivep1 Gm10851 Pias3 Serinc5 Pfkfb3 Fam164a Bmp2 Cdkn2b Samsn1 Pcgf5 Fscn1 Cl Fah Slco5a1 Gpr52 Net1 Tmem123 BC023744 Gcap14 Ogfrl1 Dap Gyg Gm10432 Rabgap1l Slc22a23 + LuLN DC u – 5 + cDCs (colors (colors cDCs b cDCs, cDCs, k P

≤ 0.05 k 

© 2012 Nature America, Inc. All rights reserved. CDP to either pDC or CD8 or pDC either to CDP checkpoints: common myeloid progenitor to MDP; MDP to CDP; and with three key DC-differentiation network associated transcriptional the analyzed we lineage, DC the to cells myeloid of commitment the lymph to nodes. draining the DCs tissue of steady-state migration tional immunomodulatory program during the expressed specifically subsets and predicted regulators of DC diversity, as well as a transcrip the relationship between lymphoid-tissue and nonlymphoid-tissue DC guishes from cDCs macrophages in They tissues. have also elucidated distin that signature DC-specific a as well as DCs, of diversification transcriptional network that accompanies the lineage entire commitment the and the identify to helped have study our across of results The system. immune DCs tissue and precursors DC of transcriptome the of analysis comparative comprehensive a provided have we Here DISCUSSION experimentally. confirmed be must DCs tory migra of signature immunomodulatory the of relevance functional The antigens. tissue self to response immune adaptive of induction the may that prevent program immunomodulatory a transcriptional tissues in the steady state upregulate genes that encode nonlymphoid components ofleave that cDCs that speculate we data, these of basis ( cDCs tissue-migratory steady-state in protein CD40 and PIAS3 PD-L1, CD200, Fas, of expression confirmed we Fas (CD95) receptor cell-death including DCs, of survival the diminishing in important molecules genes encoding upregulated cDCs migratory receptor, which is also expressed on DCs to diminish transcription the proinflammatory activation of DCs after the binding to its of expression NF- factor and STAT3 of in otherwise noted (unless replicates more or three with independent three of experiments representative are Data ×63. magnification, Original monoclonal or PIAS3. to alone antibody antibody monoclonal secondary with stained cDCs migratory and cDCs resident ( antibody. control isotype-matched of with above staining curves, populations the shaded Gray DC). LN Resident left; at oval left (top II class MHC of expression CD45 and DC) Mig left; at oval right top (MHCII; II class CD45 gated in PD-L1 and CD40 Fas, CD200, CD103 CD8 (LN-resident) tissue–resident ( molecules inflammatory and ( state. steady the in 6 Figure e c r u o s e r 1 genes encoding a group of transcriptional activators, including a 0 104 120 To gain knowledge of the transcriptional network that controls controls that network transcriptional the of knowledge gain To

Expression (fold) 24 40 56 72 88

Itgb8 1 5 7 8

+ Socs2 cDCs; results are presented relative to the minimum value of all samples. ( samples. all of value minimum the to relative presented are results cDCs; 57

Tissue-migratory cDCs express genes encoding –dampening molecules molecules response–dampening immune encoding genes express cDCs Tissue-migratory , 5

κ Fas 8 . By flow cytometry and analysis, analysis, immunofluorescence and cytometry flow By . B Spred1 5 5

; ; and CD200 Pias3 Cd200 a , b Etv3 ) Expression of genes encoding immunomodulatory molecules and CD40 ( CD40 and molecules immunomodulatory encoding genes of Expression ) PD-L1 Tnfaip3 , b +

an immunoregulatory molecule known known molecule an immunoregulatory Sbno2 or CD8 or ) in purified lung-migratory (Lung mig) CD103 mig) (Lung lung-migratory purified in )

+ Cd40 cDCs, lung-resident CD103 lung-resident cDCs, – b cDC. This analysis identified identified analysis This cDC. 110 120 Expression (fold) 100 10 15 20 25 40 50

5 Cxcl10 1 5 6 . . Furthermore, steady-state +

CD11c Ccl3

Cxcl9 Fig. 6c Fig. int Il6 Poly(I:C)-activated lung-residentCD103 Lung-resident CD103 LN-resident CD8 Lung migCD103 migratory cDCs with high expression of MHC MHC of expression high with cDCs migratory +

CD11c Il1b

, Il1a d ). On the the On ). d + ) Microscopy of sorted lymph node– lymph sorted of Microscopy )

cDCs and poly(I:C)-treated lung lung poly(I:C)-treated and cDCs Tnf hi Runx2 resident cDCs with intermediate intermediate with cDCs resident + Ccrl2 + DC DC + Table Table

Ccl5 DC - - - ,

Ccl2

1 Ifnb1 tissue macrophages. The cDC-specific genes included included genes cDC-specific The macrophages. tissue CD8 cDCs and CD103 the tissue environment in which they resided. nonlymphoid-tissueThe gene CD103signature of CD8 populations DC define to used now markers phenotypical the with detected not have been would that population CD11b tissue nonlymphoid- of heterogeneity the delineate helped signature gene of and DCs homeostasis function ies are needed to identify the role, if any, of c-Kit in the differentiation, to have an intrinsic role in cDC development nature as was c-Kit unexpected, and its ligand have never been shown Kit CD8 by lymphoid-tissue shared signature gene cDC core function macrophage-specific or cDC- assess to use ‘promiscuous’ markers such as MHC class II, CD11c and has which two these led cell types, researchers between to F4/80distinguish to available of of markers paucity the a consequence is partly confusion This immunity. tissue to macrophages versus cDCs of contribution diversification lineage DC drive may products whose candidates gene several identified and subsets CD8 pDC, into CDPs of differentiation the We networks the that transcriptional also characterized accompanied CDPs. for ‘default’ pathway the represents pDCs into differentiation that suggested which lineage, pDC the control to shown molecules and monocytes from away precursors DC-restricted to cells myeloid driving of in commitment role the key potential their suggested which monocytes, to commitment their during not but CDPs to MDPs of commitment Bcl11a ). + cDCs, lymph node node lymph cDCs,

We also established that among cDCs, lymphoid-tissue CD8 distinct the is literature DC the in controversies main the of One Il12b and + DC + Cxcr3 cDCs and nonlymphoid-tissue CD103 and nonlymphoid-tissue cDCs c and and c

Ccr7 ) Expression of of Expression ) aDV CD11c Klf8 10 10 10 10 . The identification of . The identification 0 2 3 4 5 + Isotype residentLNDC Isotype migDC Resident LNDC Mig DC A MHCII cDCs and identified a contaminating macrophage macrophage contaminating a identified and cDCs , whose expression increased specifically during the the during specifically increased expression whose , 0 NCE ONLINE PUBLIC ONLINE NCE + 10 cDCs was absent from the rest of the DCs, including 2 a 10 ) 3 in vivo in 10 Control 4 PIAS3 d 10 + cDCs shared a gene signature regardless of 5 . Notably, most of these genes encoded encoded genes these of Notably,. most Events (% of max) Events (% of max) 100 100 20 40 60 80 20 40 60 80 in vivo in 0 0 in in vivo CD200 Kit CD40 Resident cDC 0 0 A 10 10 TION as of part the cDC 2 2 . 10 10 . . Notably, the use of the cDC 3 3

10 10 4 4 nature immunology nature in vivo + in vivo in + cDCs and from absent cDCs 9 10 10 cDC and CD8 and cDC . We have identified a We . identified have 5 5

Events (% of max) Events (% of max) 100 100 . . Additional stud 20 40 60 80 20 40 60 80 . 0 0 Migratory cDC Fas PD-L1 0 0 10 10 Zbtb46 - specific sig specific 2 2 – + cDCs and cDCs 10 10 cDCs and 3 3 10 10 – , , cDC cDC 4 4 Flt3 10 10 5 5 + - - ,

© 2012 Nature America, Inc. All rights reserved. R Published online at The authors declare no competing interests.financial G.J.R. and S.J.T. provided intellectual input for design. experimental andDC purified macrophage populations; P.S.F. provided intellectual input; and populations;DC purified M.G. macrophagepurified populations; M.B. and A.B.-P. did computation analyses; M.L. did experiments; K.G.E., J.H., D.H., A.C. and J.P. computation analyses and, together with T.S., Ontogenet;designed A.C. and G.P. E.L.G. macrophagepurified populations and provided intellectual input; V.J. did helped write T.S.the paper; did computation analyses and all identified modules; wrote B.D.B.the paper; provided intellectual input, did computational analyses and J.C.M. did experiments and wrote M.M. the experiments designed paper; and HL097819 and HL097700 to P.S.F.; and U54CA149145 to V.J.).DP2DK083052-01 to B.D.B.; DK074500 and AI045757 to S.J.T.; HL69438, DK056638,Project (led by C. Benoist); AI080884 and HL086899 to M.M.; JDRF172010770 and Diseases of the US National Institutes of Health (R24 AI072073 to the ImmGen of the ImmGen Project. Supported by the National Institute of Allergy and Infectiousthe design and writing of the manuscript; and eBioscience and Affymetrix for support M. Painter, J. Ericson and S. Davis, for contributions; C. Benoist forWe contributions thank colleagues to of the ImmGen Project, especially the technical team, including Note: Supplementary information is available in the codes. Accession the pape the of in version available are references associated any and Methods M gram that controls DC function in the steady state and injured state. DC gene-expression networks and help delineate the transcriptionalsets from the proImmGen Project will now permit further investigationcomplexityphagocytetheofsystem. Moreover, into availabilitythe dataof myeloid precursor populations, to further elucidate the developmental gene regulation developmentinducibletheasgeneticsuchtools, innewshould aid of findingslineage. Our transcriptional DC thethe zationnetwork ofof antigens self to tolerance of control the in participate may response cell T regulatory the of maintenance or induction the in of cDCs role migratory steady-state potential the with consistent were results These cell T activation. of dampening the to lead to known and survival DC activation diminished DC of dampening DCs, by cytokines nosupressive immu of production the to linked been have upregulation greatest steady the in nodes a of Some state gene shared upregulated signature. the the with genes lymph draining the to migrated that LCs mal CD103 lymphoid-tissue may drive the functional specialization of DCs grams that could be used to predict their expressionand CD103pattern and therefore F150, F156 and F152 were upregulated in pDCs, cDCs and CD8 ferent expression in each DC subset. Specifically, we found that modulesmodules of genes with substantial coexpression that had specificdata setand ofdif the ImmGen Project (V.J. algorithmfunction.Ontogenet,Withtheandstasis thedeveloped for identified the gene regulators that may drive theirestablished differentiation, CD8 homeo pDCs, CD8 nature immunology nature reprints/index.html. C AUTH A eprints and permissions information is available online at http://www.nature.com/ at online available is information permissions and eprints ck O ethods Theresults ofour study have provided comprehensivea characteri non origin, cellular or tissue of regardless Notably,that found we MPETI now O R R C N ledgme ON + G G FI – cDCs, respectively, and identified candidate regulatory pro cDCs and CD103 TRIBUTI N in vivo + http://www.nature.com/doifinder/10.1038/ni.237 A cDCs and CD103 GEO: microarray data, data, microarray GEO: N n r CIAL CIAL I ts . and lineage tracing of genetically marked, defined ON

+ N S cDCs and CD11b and cDCs aDV 3 TERESTS and identified candidate molecules that that molecules candidate identified and – A cDCs in the same tissues. These results NCE ONLINE PUBLIC ONLINE NCE + cDCs as a distinct lineage subset and et al. , data not shown), we identified GSE1590 online version of the pape + in vivo cDCs as well as epider as well as cDCs 7 . A . TION 0 .

in vivo in

+ online online r cDCs . . ------­ 16. 37. 36. 35. 34. 33. 32. 31. 30. 29. 28. 27. 26. 25. 24. 23. 22. 21. 20. 19. 18. 17. 15. 14. 13. 12. 11. 10. 9. 8. 7. 6. 5. 4. 3. 2. 1.

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© 2012 Nature America, Inc. All rights reserved. upregulated genes encoding molecules important to the DC lineage alone. alone. lineage DC the to important molecules encoding genes upregulated the for monocyte nearest-neighbor the of that than greater 1.5-fold least at expression with genes for filtered further were results Those was upregulated 1.5-fold or more in MDPs relative to their expression in CDPs. Conversely, genes encoding regulators were filtered for those whose expression be increasing in the MDP population as well and thus may still be informative. ‘MDP The and/or GMPs. GMP’ classification allows and/or the inclusion ofMDPs genes whose by expression may more or 1.5-fold upregulated was sion expres whose for those by CDPs filtered were expressed regulators encoding Genes CDPs. and with GMPs in expression genes their than greater those 1.5-fold were expression selected MDPs by expressed regulators The licates. regulators com diversification. and lineage mitment DC with associated factors transcription of Identification Documentation_ QC RNA Ext ( of Project the ImmGen procedures Affymetrix MoGene 1.0 ST array was prepared according to standard operating suite GenePattern the algorithm average multi-array robust the by normalized and preprocessed were populations all for data Raw procedures. manufacturer’s the to according array ST 1.0 Gene as reagent Trizol with mice described C57BL/6J from sorted populations cell from analysis. data and normalization analysis, Microarray software. AxioVision Zeiss with analyzed were and camera color MRc AxioCam and a Zeiss microscope 2IE Axioplan a of Zeiss with ×63 nification mag a at acquired were Images Sciences). Microscopy (Electron Trisbuffer in with Fluoro-gel were mounted with DAPI (4,6-diamidino-2-phenylindole) mouse for Slides 1 h to slides at to temperature. added room was Invitrogen) (A-11005; antibody goat secondary 594–conjugated Fluor Alexa 1:2,000. of room temperatureat with goat incubation mAb of to h mouse PIAS31 by (P0117; Sigma) at followed a dilution °C, 4 at h 48 for stained were samples then Triton PBS, in 0.1% in BSA and 0.1% serum goat for 1 10% h with incubation by blocked was binding nonspecific PBS, in paraformaldehyde 4% with for h 1 fixed were Slides overnight. dried and Cytospin) (with slides glass onto spun sorted, were CD11c and II class MHC of expression to according lated cells. sorted of immunofluorescence and Cytospin IgG2a, to mAb (H1940112), CD26 to mAb Jo2), (Fas; CD95 to mAb eBioscience; from IgG, IgG2a, rat to mAb mAb MIH5), mAb (OX90), to to (CD274; CD200 to PD-L1 (HM40-3), CD40 (c-Kit; 2B8), mAb to CD135 mAb(Flt3; A2F10), to 6F7), BTLA mAb(CD272; mAb (M1/70), mAb to I-AI-E (M5/114.15.2), to CD103 (2E7), mAb to CD117 mAb to mAb CD11b (N418), to mAb CD11c to (30-F11), CD45 (L3T4), CD4 ware (Tree Star). Monoclonal antibody (mAb) to mouse CD8 mAb(53-6.7), to eter analysis of stained cell followed suspensions, by analysis with FlowJo soft website ( Project ImmGen on the are available populations of specific sorting for the used combinations Cytometry Flow Sinai Mount the with a Facility Shared FACSAriaResource at II (BD) or The (BD). Influx marker sorted were Cells below). (identified Im ( Project of ImmGen protocol the sorting cytometry. flow and sorting Cell Medicine. of School Mount Sinai the of Use and Committee Care Animal of School involving facility.Medicine mice were approvedExperiments by the Sinai Mount the at facilities pathogen–free specific in housed were mice All enhanced to LCs of protein to langerin-positive aid linked in fluorescent green the purification langerin of expression transgenic with mice C57BL/6J from were isolated which LCs, LCs and except Laboratory, migratory Jackson Mice. METHODS ONLINE doi:10.1038/ni.2370 Gn el rp n s and prep Cell mGen κ , mAb to rat IgG2a, rat to mAb , All cells were obtained from 6-week-old male C57BL/6J mice from mice The C57BL/6J male from were 6-week-old cells All obtained raction with raction Trizol. 6 6 0 3 . RNA was amplified and hybridized on the Affymetrix Mouse Mouse Affymetrix the on hybridized and amplified was RNA . with a coefficient of variation of less than 0.5 in population rep population in 0.5 than less of variation of coefficient a with κ and Armenian hamster IgG2, hamster Armenian and 6 κ 1 implemented in the ‘Expression File Creator’ module in in module Creator’ File ‘Expression the in implemented , mAb to IgG2b, to mAb , ALL-DataGeneration_0 6 2 . RNA processing and microarray analysis with the the with analysis microarray and processing RNA . λ Table rig SOP.pd orting , and mAb to Armenian Hamster IgM, Hamster Armenian to mAb and , pd f The data set was filtered for genes encoding encoding genes for filtered was set data The ; ; 1 http://www.immge ). ). An LSR II (BD) was for used multiparam All cells were purified by the standardized standardized the by purified were cells All http: κ , mAb to rat IgG2a, rat to mAb , //www.immgen.org/Pro f http://www.imm ) with the appropriate antibodies appropriate the with ) λ 612.pd were all from BD. from all were n.org/Protocols/ImmG f Viable sorted cells iso cells sorted Viable ). RNA was prepared prepared was RNA gen.org/Protocols/ κ

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of the inclusion group over any single population mean value of the exclusionthe of inclusion valuepopulationgroup anymeanoverthesingle of probes for which the change in expression of any single population mean value Benjamini and Hochberg FDR of <0.05. The data set was then filtered for those The data.expression ized assessed by an unpaired two-way analysis of variance Student’s Generation of the cell-specific gene signatures. populations. cDC and pDC in expression their to relative genes expression whose was upregulated 1.5-fold or more in the precursor cell identify to CDP precursor their of those with compared then were cDCs and pDCs for results The comparison. each in 1.5-fold least at upregulated for genes filtered were results and DCs node lymph mesenteric and nodes lymph and CD8 CD8 with compared were pDCs Thus, organ. each in 1.5-fold least at by upregulated of selection the for analyzed further were and <0.05 of FDR Hochberg and Benjamini a with corrected data expression Student’s variance of analysis two-way a by neighbor tal developmen nearest their of those with compared were subsets DC entiated populations these in 0.5 than less of variation GMP, the across populations. MDP, monocyte and CDP L ( ExpressCluster module into GenePattern the of identification the for along the Gene lists DC lineage. were expression decreased genes whose input selected were CDPs in in more that or to relative 1.5-fold monocytes upregulated was expression whose genes Conversely, by subtraction of the mean and division by its standard deviation across all all across deviation standard its by division and mean the of subtraction by standardized was gene each of expression The here. F1–F334’ modules ‘fine called modules, fine 334 in resulted which clusters, finer into clustering cal ‘coarse called are Those modules C1–C81’ here. Each coarse cluster was clustered further by (C81). hierarchi cluster separate a into grouped were unclustered genes remaining The clusters. stable 80 in resulted which clustering, clustering. for the used were set data entire the deviation across 0.5 than higher standard a with sets probe 7,996 the only those, Of retained. was expression mean highest the with set probe only the more one than set, probe Data were log release). 2011 (March pipeline Project ImmGen the of part as normalized were data Generation of gene modules and prediction of module regulators. Multiplot module than 0.05 for of the signatures. The identification downregulated GenePattern the satisfied that DCs resident in that to or relative twofold DCs more upregulated expression in with genes migratory t the satisfied that more or twofold of expression in increase an with genes of versus resident skin-draining lymph node migratory populations, for again with compared the selection were genes remaining the for results environment, node lymph the by created be could that signature potential any of removal Student’sthe satisfied that more or twofold of expression in change a with genes and equivalents, tissue-resident their of those with compared directly were cDCs CD103 lung LCs, migratory of transcriptome the in 0.5 than less of population replicates. variation For of the creation of the coefficient gene signature a of migratory with cDCs, expression with genes for Generation of the gene signature of DCs. migratory analyses are provided in for multiple hypotheses testing with the Bonferroni adjustment. Data from these each population were analyzed by a for Data analysis.log-transformedbeforewere computing. statistical Data for with the analysis of variance (ANOVA) command in the software turesof the wereR projectalso analyzed for variance across all steady-state leukocyte would populationshave a predicted 95% certainty of expressing the gene). These gene signa expression value less than the QC value of 95 (the value at which each data populationset was further filtered for genes for which the exclusion populations had an group was twofold or greater to create signatures of upregulated transcripts. The -test criterion of a criterion -test abMembersPges/SD.htm The data set was filtered for genes encoding regulators with a coefficient of a with coefficient regulators for encoding genes was The filtered data set ue Prmgei Clustering Paramagnetic Super + t cDCs were cDCs compared with CD8 -test criterion of a a of criterion -test 6 2 P was used for these analyses. these for used was value of less than 0.05. This same method was applied to was applied method same This of value 0.05. than less 2 -transformed. For on genes represented -transformed. the array with Supplementary Table 1 t l -test was controlled for multiple hypothesis with a with hypothesismultiple for controlled was -test ) for the identification of patterns of expression expression of patterns of identification the for ) P value of less than 0.05 were selected. For the the For selected. were 0.05 than less of value post-hoc 6 4 with default parameters was used for for used was parameters default with – t cDCs in the spleen, skin-draining in skin-draining cDCs the spleen, -test criterion of a a of criterion -test pairwise Student’s http://cb 6 Differences in expression were . 3 . Results obtained for differ for obtained Results . nature immunology nature + cDCs and lung CD11b lung and cDCs + The data set was filtered cDCs and CD8 and cDCs dm.hms.harvard.edu/ t -test of normalized normalized of -test t -test on normal t -test, corrected P value of less less of value Expression – cDCs, cDCs, + - - - - -

© 2012 Nature America, Inc. All rights reserved. were were centered on rows before The visualization. Immgen PopulationDistances Data as type. a linkage clustering complete-linkage as a and pairwise measure correlation Pearson with clustered and mean the around centered scaled, log GenePattern genomic analysis platform with the HeatMap visualized Viewer or tool of Clustering the the Hierarchical Data analysis and visualization tools. P of FDR Hochberg Benjamini A signatures. gene four the for Project ImmGen the of modules fine of enrichment the of estimation for used was groups two for test hypergeometric A modules. fine Hochberg Benjamini A of FDR Project. ImmGen the from samples the of rest for CD103 and cDCs CD8 cDCs, pDCs, were groups cell The groups. cell specific in Project for the of identification induced modules with expression significantly ImmGen the of module fine each of expression mean the to applied was test Kolmogorov-Smirnov two-sample one-sided A tree. lineage the of structure the and expression regulator of basis the on module fine and coarse each for Project (V.J. tree. the on projected was each module of expression mean The averaged. were replicates of Results sets. data nature immunology nature values of all four groups across all fine modules. fine all across groups four all of values The Ontogenet algorithm was developed for the data set of the ImmGen ImmGen the of set data the for developed was algorithm Ontogenet The ≤ 0.05 was applied to the table of of table the to applied was 0.05 et et al. , , data not shown). This finds a algorithm program regulatory + cDCs. The background for each was the result obtained obtained result the was each for background The cDCs. Signature transcripts were clustered and 6 2 . For hierarchical clustering, data were P values of all four groups across all all across groups four all of values ≤ 0.05 was applied to the table of of table the to applied was 0.05 – DCs, CD8 DCs, +

64. 63. 62. 61. 60. 59. Software. Prism with created were genes individual GenePattern of module Multiplot the with visualized versus expression in change of or expression in change sample versus of expression in set change of each Comparisons in 0.5 visualization. before than replicates less of variation of coefficient a with genes for filtered and subset and gene for normalized log-transformed, were Data standard of to geometric populations ‘weight’ deviation in genes that multiple varied populations. the with analysis of variation a of basis the on filtering by subsets among expression in difference greatest the with genes the the of 15% identify to used was program PCA the indicated, Where PCA. for used PCA program (

lt, . Wsmn S & oay E Spraaantc lseig f data. of clustering Superparamagnetic E. Domany, & S. Wiseman, M., Blatt, N. Novershtern, M. Reich, R.A. Irizarry, Yamagata, T., Mathis, D. & Benoist, C. Self-reactivity in thymic double-positive cells A. Kissenpfennig, Phys. Rev. Lett. Rev. Phys. hematopoiesis. human in states Res. Acids Nat. Immunol. Nat. CD8 a to cells commits cells. dendritic cells colonize lymph node areas distinct from slower migrating Langerhans Immunity

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38 , 500–501 (2006). 500–501 , 6 2 . Plots of results from from results of Plots . P doi:10.1038/ni.2370 value ( value in vivo in t -test) were were -test) : dermal : Nucleic l ) ) was