The Transcriptional Landscape of Ab T Cell Differentiation

© 2013 Nature America, Inc. All rights reserved. the the paper. of Microbial and Molecular TexasPathogenesis, A&M Health Science Center, College Station, Texas, USA. University, Clayton, Victoria, Australia. Division, The Walter and Eliza Hall Institute of Medical Research, Parkville, Victoria, Australia. Boston, USA. Massachusetts, 1 thymus the from emigration subsequent and progenitors DN γ (ref. CD28 and (ref. CD44 CD25, markers the of expression with cells T becoming to commitment the track that subsets into subcategorized CD4 thus are (and CD8 and CD4 coreceptors the of expression lack thymocytes immature most The molecules. cell-surface of certain expression the not do that and to These steps overreact are by self (negative selection). distinguished selection) (positive specificities TCR useful tially poten bearing thymocytes of those only maturation allows that tion T encoding cell antigen receptors and selec (TCRs) subsequent their genes of rearrangement random the to lineage, cell T the to cursors types cell mammalian among cascade differentiation any of parsed finely decades several thymus. This differentiation process has been studied intensively over similarities. unanticipated and breakpoints sharp changes, gradual of all ImmGen data. Through this analysis, we identified unexpectedly pathway of T in the backdrop the cells thymus against differentiation entire the through transcriptome the tracked we Here mouse. the of system immune the in regulation its and expression gene of section collaboration of laboratories that perform a collectively thorough dis international an is (ImmGen) Project Genome Immunological The candidate mediators of key transitions help define the ‘known unknowns’ of thymocyte differentiation. T cells and their activation, demonstrated by mass The cytometry phosphoproteomics. transcripts newly identified as encoding unexpectedly few genes accompanied commitment to the CD4 distinct signatures that marked cells destined for positive selection versus apoptotic deletion. Differences in the expression of driven by major complex histocompatibility (MHC) molecules promoted a Wereactivation. large-scale transcriptional identified is rare among cells of the immune system and correlated tightly with expression of the factor transcription c-Myc. Selection gradual changes. In contrast, transit through the CD4 precursors gives rise to mature T cell Wetranscriptomes. found that early T cell commitment was driven by unexpectedly the breadth of expression data sets from the Genome Immunological Project to analyze how the of differentiation thymic The of differentiation Bendall Sean Michael Mingueneau The transcriptional landscape of nature immunology nature Received 30 November 2012; accepted 19 March 2013; published online 5 May 2013; corrected online 13 May 2013 (details online); Christophe Christophe Benoist Division Division of Immunology, Department of Microbiology and Immunobiology, Harvard Medical School, Boston, USA. Massachusetts, δ TCR promotes proliferation accompanied by early maturation of of maturation early by accompanied proliferation promotes TCR Hematopoietic precursors differentiate into T cells in the vertebrate 2– 9 . The sequence runs from the commitment of lymphoid pre of lymphoid commitment the from runs . sequence The 9 These These authors contributed equally to this work. should Correspondence be addressed to C.B. ( − CD8 1 6 1 , which has resulted in what is arguably the most most the arguably is what in resulted has which , , , Matthew H Spitzer 2 ). Functional rearrangement and expression of a of expression and rearrangement Functional ). − double-negative (DN) cells) and can be further further be can and cells) (DN) double-negative a

1 b & the Genome Immunological Consortium 3 advance online publication online advance Department Department of Medical Biology, University of Melbourne, Parkville, Australia. 1 T T cells from thymic precursors is a complex process essential for adaptive immunity. Here we exploited , 9 , , Taras Kreslavsky 6 Department Department of Microbiology and Immunology, Stanford University School of Medicine, Palo Alto, California, USA. 6 , Garry P , NolanGarry 2 , 9 , , Daniel Gray 1 +

0 CD8 ), c-Kit ), 1 3 6 . + , , Koichi Kobayashi stage involved a global shutdown of housekeeping genes that 1 + 1 - - - - or CD8

3 , mocyte transcriptomes mocyte thy have many profiled studies Although system. in an unperturbed those and other pathways integrate to direct thymocyte differentiation by genetic approaches. What is lacking is a of general perspective how through certain stages of thymocyte differentiation have been defined ration to become functional CD4 are exported from the thymus and undergo a phase Foxp3of post-thymic matu tion) or are diverted into alternative lineages such as CD8 apoptosiseliminatedthymocytesareby(clonaldelehigh, too is tion extensive proliferation. If the affinity of the TCR-peptide-MHC interac tive selection process differs from ­single-positive (CD4SP) thymocytes (lineage commitment) differentiationandthegeneration ofCD4 nl mtr a CD4 as mature ingly class I or MHC class II molecules are positively selected andThosethymocytesDP thatexpress correspond TCRthata can interact withMHC peptide–MHC(ref.majority)thed(thought within 3–4 bedie to (MHC) interact with complexes of peptide and major histocompatibilityscreenthymiccorticalepithelial potential to TCRforthe cellstheirof complex thymocytes subsetDNinitiatesthe severalrounds proliferationof essential for 4 , 9 Several of the molecular mediators required for progression progression for required mediators molecular the of Several Incontrast, successful rearrangement of the gene encoding TCR , , Tracy Heng + + lineage, a similarity that carried through to peripheral regulatory T cells 15– 8 5 ab Department Department of Anatomy and Biology,Developmental Monash 1 8 1 . Those DP thymocytes bearing TCRs that cannot bind self 4 , in which TCR 2 , T cell differentiation [email protected] 7 8 , , Harald von Boehmer A A full list of members and affiliations appears at the end of 5 4 , Molecular Molecular Genetics of Cancer Division and Immunology 9 − , , Richard Cruse CD8 24– 21 3 , 2 α 3 + 2 , they focused on , only focused limited they igepstv (DS) r CD4 or (CD8SP) single-positive rearrangement takes place. DP thymocytes . Thymocytes that survive these processes + β or CD8 -selection, as it occurs in absence of ) ) or D.M. ( 2 Dana-Farber Dana-Farber Cancer Institute, + + 1 T cells CD8 , , Jeffrey Ericson doi:10.1038/ni.259 2 [email protected] + , , Diane Mathis double-positive (DP) 2 e c r u o s e r 3 . α 2 α 0

7 transitions transitions . This posi + Department Department

T cells or 0 + 1 CD8 , β 1 1 α 9 in , ). ). β  − ------

© 2013 Nature America, Inc. All rights reserved. minor changes, and CD4 and changes, minor or CD4SP involved comparatively cell CD8SP to terminal thymocyte CD69 from transition the contrast, major In change. another yet transcriptional initiated thymocytes DP in signaling TCR that were also different very from CD69 DP small populations, cells were Small different. DP very thymocytes ( changes DN2a-to-DN2b cells) (OP9-DL1 1 Delta-like ligand Notch the expressing cells mouse stromal marrow OP9 bone with cultured when develop that cells for shown has been what to Contrary compartment. DP the around shift) such est strong (the and DN3a; through pre transition the marrow at ETPs; and bone cursors between shifts’: ‘tectonic main three identified ( transition each at repressed or induced ( populations between tance dis Euclidean cumulative the plotting by better differentiation out both incorporates (which excluded we For analyses, stages. subsequent DP and (ISP) SP intermediate the through component stage 3 (DN3), whereas of cells maturation marrow bone of analysis cytometry in mass observed multivariate component ‘differentiation’ major the of reminiscent with nant ­component Principal- cells. SP mature of group distinct knit into tightly the stages as ‘pods’, such grouped also but sequence differentiation the ( space three-dimensional in tions along differentiation. relationships do evolve linearly tional transcrip that and decades three past the over determined tree etic hidden, unexpected findings in the ( accuracy notable with hierarchical clustering reproduced Unsupervised the known differentiationpopulations. sequence between relationships the determine to Wefor shown). tools analysis multivariate mathematical several used ( patterns expression as such ferentiation, dif thymocyte in events key mark that genes well-characterized 50 male mice. To data, validate these we of the activity first assessed over in duplicate or triplicate from cells obtained from 6-week-old C57Bl/6 were generated sets data All cells. naive T progenitors peripheral and Supplementary Table 1 sorting, cell for of used markers list and abbreviations (complete names, thymus population the from export for ready thymocyte SP mature most the to (ETP) progenitor thymic early from maturation, We generated gene-expression profiles from stage every of thymocyte differentiation T cell of perspective Overall RESULTS maturation. and transitions hinted at selection during important thymocyte functions certain at regulation coordinated whose genes candidate identified of and T aspects cell differentiation showed unexpected This analysis of data sets. breadth the ImmGen using standpoint, a transcriptional the in states ferentiation of dif Here the entire spectrum we checkpoints. revisited or selected e c r u o s e r  ( perspective this from

We visualized the magnitude of transcriptional changes through changes transcriptional of magnitude the We visualized popula cell map to analysis principal-component used Wethen

maturation in lineages of the immune system (data not shown), not shown), (data system of immune the in lineages maturation component (PC1; 73% of variance) that tracked ‘generically’ ‘generically’ tracked that variance) of 73% (PC1; component Fig. 1d Fig.

analysis of analysis the a entire data ImmGen domi set identified γ δ

, 3 transition was associated with relatively moderate moderate relatively with associated was transition e T cells proceeded directly from precursors at DN DN at precursors from directly proceeded cells T 1 ). Although DP blasts resembled earlier earlier DP resembled blasts ). Although , , in vivo in Rag1 i. 1 Fig. Fig. 1d Fig. ). We also included profiles from bone marrow α Fig. 1 Fig. α β , , + α T cells first ‘regressed’ along this principal β commitment to the T cell lineage at the the at lineage cell T the to commitment Tcra or CD8 or β T cell branch of the lymphoid tree from from tree lymphoid the of branch cell T a Fig. 1 Fig. TCR and and TCR , , b , upeetr Fg 1 Fig. Supplementary e ), which indicated that there were no no were there that indicated which ), and and ). Finally, egress from the thymus to to thymus the from egress Finally, ). α + d γ + DP which indicated thymocytes, β ) and the number of transcripts transcripts of number the and ) Tcrg i. 1 Fig. δ cells looked extremely similar similar extremely looked cells T cell branch of the hematopoi T cells T γ δ , and observed the expected expected the observed and , Fig. 1 Fig. TCR thymocytes). TCR c ), which again matched matched again which ), 3 5 e and the DN4 subset subset DN4 the and ). These two metrics metrics two These ). and data not not data and β -selected -selected 3 4 . The The . + DP DP ------mocyte differentiation, we applied k-means clustering to the set of of set the to clustering k-means applied we differentiation, mocyte intermediates. differentiation identify to used markers cell-surface few the in not changes are with they ­synchronized that prosaically, more or, degrees by proceed but events instead ‘radical’ not are transitions these that indicate may and mon feature below) throughout early T (discussed cell differentiation 1 Fig. (ETP to DN2a) and after (DN2b to DN3a) that time ( before also but occurs, actually commitment when stage, DN2b the commitment factor of expression the example, For gradual. were regulators, key many encoding those including genes, manner discrete rather a in happen transition) DN2a-to-DN2b the at lineage cell T the to commitment irreversible and stage ETP the at of potential cell B loss is, (that differentiation cell T early of landmarks major The differentiation T cell Early transitions. key of basis transcriptional the into insights new mental progression of thymocyte differentiation and provided several develop accepted the recapitulated transcriptomes the among ships had a on the Thus, effect small periphery very relation transcription. ters (named by a characteristic gene or group of genes; of genes; or group clus gene by a characteristic (named resulting ters The expression. variable most the with genes 2,088 differentiation ( differentiation thymocyte during family DGK in entire the shifts were notable there DGK- for suggested been has as stage, this at signaling TCR regulate also may acid, phosphatidic to DGK kinase (DAG) erol onward selection after and positive downregulated from of DN2 to isoform protein is this be expressed reported specific thymocyte- A signaling. TCR of regulator negative putative a thus calcineurin including enzymes, calmodulin-dependent of ( clusters example, For these in also were functions signaling probable Fig. Supplementary 2 Cd3e ( signaling TCR to related molecules encoding genes of subset ing involved molecules in recombination ( DN3a stage the of T cell ( differentiation around plateau a reached and thymocytes early in upregulated ‘ The machinery. ing of rangement Bcl11a Transcriptional regulators in these clusters included those encoded by of genes typical of non-T cell lineages (B cells, NK cells or myeloidtor cells,cells). such as DN3 cells, including those encoding markers of hematopoieticto cells stem progenihematopoietic from downregulated gradually were that ‘PU.1’ clusters ( programs of progenitor and non-T cell lineages differentiation. cyte in thymo participants molecular additional for resource discovering rich a provided which knowledge, our to cells T in studied been yet and ‘ the for probes the of half about example, (for differentiation thymocyte in functions assigned previously with Supplementary Table 2 To group those transcripts with similar behavior during early thy early during behavior similar with To transcripts those group omtet o h T el ieg i ascae wt te rear the with associated is lineage cell T the to Commitment An important feature of early T cell differentiation is the shutdown of Cd8 , , ). The gradual nature of gene expression changes was a com a was changes expression gene of nature gradual The ). Cd3g , Hhex ’ clusters) but also many genes whose functions have not not have functions whose genes many also but clusters) ’ advance online publication online advance , , Lck , Arpp21 Jun Tcrb Fig. 2a Flt3 Fig. 2 Fig. , , 9 , Itk . In contrast, the transcriptional changes in most most in changes transcriptional the contrast, In . Meis1 , , , 32 Tcrg , , Cd34 Gata-3 encodes RCS, which is a competitive inhibitor inhibitor competitive a is which RCS, encodes , Grap2 36 , d ). Several ). notable Several genes encoding proteins with b ). , and ) ) included not only genes encoding molecules 3 , and and 7 ε Mef2c , , , not only from increased the DN2a stage to (encoded by by (encoded Ly6a (GADS), (GADS), ’ and ‘ and ’ Supplementary Table 2 Tcrd (Sca-1) and and Bcl11b α loci and induction of TCR signal TCR of induction and loci Tcf-1 and DGK- and Sfpi1 Fig. Fig. 2a Rasgrp1 , which encodes a T lineage– T a encodes which , and and Dgke (PU.1) ( Rag1

Kit Tcf-1 , b Lef-1 nature immunology nature ) ) included genes encod ), which converts DAG converts which ), 3 , as well as a small group , , 8 ζ and . The ‘ Zap70 41 and and Fig. 2 , ’ clusters that were were that clusters ’ 4 4 2 0 Lig4 . More generally, generally, More . ) contained genes . . The diacylglyc Supplementary Supplementary c-Kit Lef-1 , , c Cbl Fig. 2a Fig. ) ) and a major ). ’, ‘ and and ’ and ‘ and ’ Cd34 3 Fig. 2 Fig. 9 , and is is and , , Cd3d b Nck2 ’ and and Cd4 b ). ). ------; ,

© 2013 Nature America, Inc. All rights reserved. the idea that DN3a cells receive the strongest Notch signaling Notch strongest the receive cells DN3a that idea the and ‘ pre-TCR as such targets, Notch canonical Many sion. tributed across multiple clusters with very different patterns of expres stage DP the before differentiation at of steps all T cell and to is mitment T the indispensable lineage cell signature. proliferation the of part not probes symbols, open probes; total symbols, Filled differentiation. T cell during axis) horizontal (arrows, transitions various at more or twofold by (blue) ( analysis. the before row-standardized and log were values Expression subsets. the of one least at for >120 values expression with and subsets these among probes with the greatest difference in expression of 15% the with calculated margins), right distances between various populations ( component analysis ( mean. ( Table 1 abbreviations, axis; (horizontal populations thymocyte and marrow bone ( differentiation. T cell of course the during changes 1 Figure nature immunology nature then their expression reached a reached expression then their plateau by DN3a and was maintained ‘ the in genes factor–encoding tion ( were upregulated and reached a plateau as early as in ETP thymocytes as such targets, few A e a Fig. Fig. 2 c (5.89% a ) Probes upregulated (red) or downregulated downregulated or (red) upregulated ) Probes of ) Expression PC3 Signaling via the Notch family of receptors is necessary for com for necessary is receptors of family Notch the via Signaling LT-HSC Relative expression 0 1 Il7r −0.15 ST-HSC –0.2 –0.1 ) a ), ), presented as the maximum-normalized 0.2 b Tcf-1

). ). Two were interest the transcrip Notch of targets particular – 0 1 −0.1 ’ ’ clusters that around peaked DN3a ( ‘Birds-eye’ view of transcriptome transcriptome of view ‘Birds-eye’ α (12.74% d MPP −0.05 ) ) Hierarchical clustering ( ) PC2 43 and and CLP , 4 4 ) 0 , , ETP-DN2a 0.05 ETP Tcra Dtx1 Lef-1 0.1 8SP24 8SP24 8SP69 4SP24 4SP24 4SP69 4p8int DP69 c , , ) ) and heat map of Euclidian 0.15 Tgd

Rag1 DN2a , , Il7r ’ cluster). These genes were upregulated early, upregulated were genes These cluster). ’ + Nrarp 0.2 – in + – in +

DN2b 2 t t -transformed -transformed 4 and and advance online publication online advance

5 DN3a DN4 and Tcf7 Supplementary Supplementary and and DN3b-DN4DN3b DN3-4 DN3b DN3a Kit (refs. (refs. Notch1 0.13 DPsm in various various in b ), ), principal- Hes1 8 , d 9 DN2b

0.125 ISP ; ; top and . Direct Notch targets were dis were targets Notch Direct . ISP

48

DPbl itself (in corresponding clusters), clusters), corresponding (in

, DPsm 0.12 4 9

) ) and

DPbl DP69

4 Fig. Fig. 2

0.115 DN2a ETP-DN2a ETP 6

+ were in the ‘preTCR Ptcra 4 +

4SP698 int (72.61% Bcl11b 0.11 a

PC1 4SP24

), ), consistent with + (which encodes encodes (which Number of probes with fold 2 e CLP change > 2 (×10 ) 0.105 4SP24 int ) 32 8SP69 –

, 0.1 36 MPP ST-HHC LT-HSC 10 15 15 10 0 5 5 8SP24 , + 3 7 MPP (both (both 0.095 8SP24int Kit Rag1 Tcra 46

, – 4 α 0.09 7 - - - - . ’ LT-HSC ETP d b ST-HSC locus locus combine with the pre-TCRgermline-encoded TCR by induced program T cell differentiation. thymocyte early in patterns of expression diversity observed the in results which targets, Notch of expression regulate cytes. It is therefore very likely that additional factors other than Notch thymo DP in expression in decrease abrupt an and early expression Myc entiation but is for dispensable maintenance of T cell identity. Finally, consistent with the finding that Notch is required early in T cell differ differentiation, cell T of regulators key these of maintenance the not β after signaling Notch of downmodulation after even thereafter, high -selection. Thus, Notch signaling is required for the induction but induction the for required is signaling Notch Thus, -selection. DN2a

marrow MPP LT-HSC Bone 5 β 0 ST-HSC CLP (from the ‘ the (from proteins formed after productive rearrangements of the the of rearrangements productive after formed proteins MPP

DN2b ETP-DN2a CLP ETP ETP ETP-DN2a DN2a DN2a

DN3a DN2b DN2b DN3a

c-Myc DN3a DN3b

DN3 DN3b-DN4 DN3b-DN4DN3b DN4 I b ’ cluster) had yet another pattern of steady steady of pattern another yet had cluster) ’

ISP Thymus

DPbl SP ISP

b DPsm DPbl

-selection DP69 DPsm

DPbl 4 + + 8 int 4SP69 DP69 4SP24 + + DPsm 4 + 4SP24 int 4SP698 int Down (notinproliferationsignature) Up (notinproliferationsignature) Down Up 8SP69 – 4SP24 + 8SP24 + DP69 int 8SP24 int 4SP24 LN e c r u o s e r + T4Nve – 8SP24 –

α TT8Nve 4SP6 MPP chain (preTCR) ISP DN3b-DN4 DN3b DN3a DN2b DN2a ETP-DN2a ETP CLP ST-HSC LT-HSC T8Nve T4Nve 8SP24 8SP69 4SP24 4SP24 4SP69 4 DP69 DPsm DPbl DN4 8SP24 8SP69 int + 8 9 in + 8SP24 + t + 4SP2 – + – in + in t t – T4Nve 4 – LN Thymus marrow Bone T8Nve T4Nv Tcrb e  - -

© 2013 Nature America, Inc. All rights reserved. (right margin) whose expression changed by threefold or more during early T cell differentiation. T cell early during more or threefold by changed expression whose margin) (right ( margin). (left clusters various from margin) (right regulators transcriptional ( clusters. right) (bottom CD4-CD8 and right) (top TCF-1–LEF-1 (left), PU.1 the in margin) (right each, in genes of list complete cluster; the in genes of group or gene a characteristic ( differentiation. T cell 2 Figure e c r u o s e r 

PU.1 cluster

a b

Dynamics of gene expression during early T cell differentiation. K-means clusters of genes with different expression profiles during early early during profiles expression different with genes of clusters K-means differentiation. T cell early during expression gene of Dynamics LT-HSC ST-HSC

MPP ETP-DN2a ETP No info(non–proteincoding) No info(proteincoding) Known functio MPP DN2a ETP DN2b ETP-DN2a DN3a a DN2a ) Expression of genes (log genes of ) Expression DN3-DN4DN3b DN2b DN3a ISP n DN3b DPbl DN3b-DN4 DPsm ISP LT-HSC DPbl ST-HSC DPsm Tes Dusp6 Vim Gpr17 B3gnt5 Ctsg Rgs Sfpi1 Clec4e Cd24 Naps 9030619P08Rik Cd44 Gpr97 Neurl3 Gm10759 Lyl1 Fcer1g Il18rap H2-O Mef2 Bcl11a Mlkl Btk Hhex Cd72 Ncf1 Tyrobp Lpcat2 Cd18 Cd33 Bex6 Il1r1 Ccl3 Hbb-b1 Hbb-b1 Myc Ptpr Nfam1 Trf Lat 5430427O19Ri Zbtb10 Rhob Slc16a7 Pik3ap Ge Golm1 Chn2 6330407A03Rik Ier2 Myo1f MPP m 2 ETP-DN2a 1 e n ETP c a b 4 0 1 1 DN2a TCF-1–LEF-1 DN2b DN3a k CD4-CD8 DN3-DN4DN3b cluster cluster 2 ISP -transformed mean-centered; black lines) and cluster centroids (gray line) for each cluster (named for for (named cluster each for line) (gray centroids cluster and lines) black mean-centered; -transformed DPbl DPsm MPP ETP LT-HSC ST-HSC ETP-DN2a DN2a MPP DN2b ETP-DN2a ETP DN3a DN3b DN2a DN3b-DN4 DN2b ISP DN3a DPbl DN3-DN4DN3b DPsm Tcrb- Sh2d1a Rag2 Nsg2 Nebl Slc16a5 Rag2 Ccr9 Txk Slc7a11 F13a1 Fas Tdrd5 Ets2 Glcci Cd Cd Glcci Rorc Ikzf Camk4 Cd8b1 Cd8a Spats2 Gm668 Gzma Fam169b Tcf7 Ly6 Lc Grap2 Themi Rasgrp1 Thy1 Skap1 Bcl11b Gbp4 Pp11r It Slamf6 Prkc Ets1 Dgke Edem1 Cd28 Rag1 Lef1 Cd3d Cd3e Cd3g A930002I21Rik Arpp21 Aqp11 A930013B10Rik ISP k

k DPbl 2 4 3 d DPsm b 1 1 J s d LT-HSC 3 ) Heat map of the expression of genes encoding diacylglycerol kinases kinases diacylglycerol encoding genes of expression the of map ) Heat ST-HSC

TCF-1–LEF-1 MPP PU.1 clusters ETP-DN2a c-Kit, CD34

Gata-3 and ETP TC CD4-CD8 Supplementary Table 2 Supplementary clusters clusters

R DN2a d c

α DN2b and an DN3a advance online publication online advance

d DN3-DN4DN3b

MPP MPP ISP DPbl c ETP DPsm ) Heat maps of the expression of genes encoding encoding genes of expression the of maps ) Heat ETP LT-HSC ST-HSC ETP-DN2a ETP-DN2a

MPP DN2a DN2a ETP-DN2a ETP

DN2b DN2b

). ( ). DN2a DN2b

b DN3a DN3a DN3a ) Heat maps of the expression of genes genes of expression the of maps ) Heat DN3-DN4DN3b

DN3b DN3b ISP DPbl DPsm

DN3b-DN4 DN3b-DN4 LT-HSC ST-HSC

ISP ISP MPP nature immunology nature ETP-DN2a ETP

DPbl DPbl DN2a DN2b DN3a DPsm DPsm DN3-DN4DN3b Dgkg Dgkh Dgkz Dgkd Dgke Dgka Nfatc3 Mef2 Suhw Chd1 Meir Klf7 Ssbp Pou6f1 Bcl6 Rorc Ikzf Camk Ets2 Meis Tsc22d Myc Er Sfpi Bcl11a Lyl1 Mef2 Hhex Arnt Ju Lef1 Tcf7 Ets1 Bcl11 Id Tcf1 Hivep Gata Klf3 Hdac E2f4 Satb E2f2 ISP g 3 n DPbl 3 1 l n 1 2 1 1 3 a

c DPsm 2 7 4 4 2 b 1

© 2013 Nature America, Inc. All rights reserved. unchallenged OT-I unchallenged TCR) mice express (which an ovalbumin-specific DP–to–CD69 to-DN3b transition) or the overlap between genes induced by signaling from the pre-TCR (DN3a- Zfp280d ( differentiation thymocyte of context the in recognized yet not regulators several encoding genes Cd8 β the of components known encoding Genes shown). not (data lism genes; upregulated of (>48% proliferation to thy the in ( shifts transcriptome prominent mocyte most the of one with associated was cells DN3a unselected from post- distinguishes which of expression, CD25 marks of CD8) and expression) and DP (with upregulation of the expression of both CD4 of of ISP upregulation (with CD8 CD25 expression), downregulation (with DN4 stages to the progression phenotypic and of proliferation to allow in DN3 thymocytes T early differentiation. cell during in and c-Myc T IL-7R physiology cell Notch1, pre-TCR, ( experiments. of independent three are Data representative staining. control Ctrl, (right). of (maximum-normalized) ( mean. as maximum-normalized presented of 2. a mark ( change’ lines Blue ‘fold for clarity. removed been have signature to the proliferation belong OT-Inaive versus (OT-Icells that Genes axis). vertical T8Nve); (OT-IListeria-OVA with infection after T8Eff.24hr.LisOva) OT-I T (bottom; cells or in peripheral T 24 cells h axis) vertical (DPsm); DP small thymocytes versus CD69 (top; thymocytes by induced and those axis) horizontal DN3a; versus (DN3b thymocytes in DN signaling by pre-TCR induced expression ( right). (bottom or left) >2 of (bottom <0.5 a change’ ‘fold with (black) genes (blue)/total genes associated proliferation- indicate in corners Numbers plot. as a volcano presented DN3a, stage thymocyte (vs) versus DN3b stage in (red) thymocyte (blue) and components of Notch signaling pathway those to corresponding the signature proliferation P ( Figure 3 nature immunology nature ovalbumin-expressing with challenged OT-I from mice those versus a a value ( -selection program were also included in the ‘ the in included also were program -selection ) ) Change in expression (horizontal axis) versus P (–log ) Do all TCR-mediated signals proceed similarly? We analyzed the the We analyzed similarly? proceed signals TCR-mediated all Do 10 6 5 4 3 2 1 0 0.01 ’ clusters (for example, example, (for clusters ’ 33 Notch3 /4

Lfng t Ma 20 (which encodes Suhw4); Suhw4); encodes (which Transcriptional footprint of -test; -test; vertical axis) for all probes (black), Dt β Pt Dt ml 5 x1 cr -selection and occurs before the complete downregulation before and the complete downregulation occurs -selection x3 Dt 1 2 Il2 a 0. . Upregulation of CD28 expression is one of the earliest earliest the of one is expression CD28 of Upregulation . x3 l ra Hes 1 1 + DP transition) or in peripheral T cells (CD8 b 1 ) Comparison of changes in gene of changes ) Comparison Notch1 DN3b vsDN3a Fold change 0. 2 5 αβ + DP thymocytes (DP69 DP thymocytes TCR signaling in DP TCR signaling Notch signalin Notch Proliferation signatur Proliferation

Myc Fig. 1c Fig. advance online publication online advance α mRNA and mRNA encoding negative regulators of c-Myc, as well as c-Myc protein (key), in various thymocyte populations populations thymocyte in various (key), as of as protein well c-Myc, c-Myc regulators negative and encoding mRNA Cd4 β 1 β TCR in immature DP thymocytes (small 2 10 -selection, a process that drives a that burst a drives process -selection, Chd1 . The transition from DN3a to DN3b DN3b to DN3a from transition The . – , , e Cd8a β g 337 ). Much of this change was related related was change this of Much ). -selection. -selection. c Fig. 2b Fig. /7 ) Expression of ) Expression , , 100 02 Klf7 e , ,

d Cd8b1 ) Expression of c-Myc in various cell subsets (key), detected by flow cytometry (left), and expression and expression (left), by cytometry flow detected (key), subsets cell of in c-Myc various ) Expression , ,

, Fold change c Mef2a + b

Fold change

). )

OT-I T8Eff.24hr.LisOva +

DP69 vs DPsm

and and vs OT-I T8Nve β 0. 0. 0. 0. Fig. 3 Fig. TCR 10 10 -selected DN3b cells cells DN3b -selected , , 1 5 1 5 1 2 1 2 0. Notch1 e DN3a Meir1 Serpinb6b 1 Rorc IL-7R Serpinb9 α

Nlrc Tnfrsf9 a H2-K Tnfsf14 ’ and ‘ ’ and ) or metabo or ) ), along with with along ), 5 + , , and various Notch target genes (key) in various thymocyte subsets (horizontal axis), axis), (horizontal subsets thymocyte in (key) various genes target Notch and various c-Myc

1 T cells from Pou6f1 Minus proliferationsignature Notch1

Ccl4 0. Cd4

5 DN3b vsDN3a Fold change Pre-TCR and and and 1 Irf8 Il7r - - Serpine2

α 2 Nr4a1 Cd5 was lower during during lower was and DP thymocytes, respectively, but the magnitude of their induction were induced downstream of both the pre-TCR and the ( strength signal TCR of markers several ing the from distinct Ikzf3 included (those pre-TCR by the induced selectively genes few very with considerably, overlapped activation transcriptional programs induced by ( analysis this from those excluded therefore and stage DN3 the the at pre-TCR the both by induced strongly were genes signature Proliferation differentiation. cell T of stages various at receptors these of downstream engaged was program scriptional monocytogenes Listeria Notch regulators; and and regulators; Notch Dtx1 ligands; Delta-like to receptors Notch of sensitivity the enhances that ferase after Notch1 the downregulated of were components pathway of array Notch wide a encoding genes Accordingly, IL-7R Cd8a Pre-TCR signals are known to antagonize Notch signaling Notch antagonize to known are signals Pre-TCR Cd8a Cd5 Gzma Cd69 α c-Myc , , ). In contrast, TCR signaling at the DP stage seemed to be very very be to seemed stage DP the at signaling TCR contrast, In ). Cd8b1 β Notch Zap70 Camk4 Cd8b1 Dtx3 Camk4 Pr Rorc TCR in peripheral T cells but not in DP thymocytes, and we we and thymocytes, DP in not but cells T peripheral in TCR and and 1 olif Maml2 0 Ikzf Etv e Ikzf 1 Etv er ) Proposed model of the interplay among the functions of the the functions among of model the interplay ) Proposed Pre-TC 5 3 atio 3 5 and and Notch3 n R , which encodes a Notch transcriptional coactivator; coactivator; transcriptional Notch a encodes which , α d c Dtx3l -TCR β β -selection transcriptional program. Genes encod Genes program. transcriptional -selection themselves; themselves; -selection ( -selection

c-Myc Relative expression β E ET TP 0 1 P

, which encode members of Deltex family of of family Deltex of members encode which , - DN3b DN3a DPsm Ctrl Hes1 DN2a IL-7 t dtrie hte a omn tran common a whether determine to )

R DN2a , , c-Myc Il2ra Notch DN2b Pr Fig. 3 Fig. olif Lfng

1 DN3a er , , Pre-TC atio Ptcra

, which encodes a glycosyltrans encodes , which DN3b b n β D DN N ). -selection -selection and peripheral T cell 0 Relative expression 1 3b-DN4 R 2 α (pre-TCR

-TCR DN3a Rorc

β DN3b-DN4DN IS

Fig. 3 Fig. P c-Myc (protein) My Cd69 3 β , , b DPbl Zap70 slcin ( -selection IL-7 c e c r u o s e r (RNA) DPsm R α b , ,

c-Myc I Cd5 ). Unexpectedly, Unexpectedly, ). SP ), which encode encode which ), Notch , , α Cd8 DPbl Il7 Ptcr Notch1 My β 1 and and r Pre-TC TCR in DN

DPsm c a , , i. 3 Fig. Etv5 DP Trpc4ap Sin3b Mxi1 Nr4a1 R 69 α DPsm + -TCR 46 and 4S , a 4 P β 7 ): ):  - - - ) .

© 2013 Nature America, Inc. All rights reserved. size characteristic of the resting small DP stage ( stage DP 3a Fig. small resting the of characteristic size of of shutdown the we prevented ( stage DP small quiescent the to sition tran with associated ultimately changes in sets transcriptional the that motion timer Thus, molecular a as pathways. serve may shutdown mitogenic and delayed this those signaling regulated Notch negatively down shut therefore expression pre-TCR However, and is augmented by signals from the receptor for interleukin 7 (IL-7R). by activity. and expression c-Myc that mechanisms hinted regulate at observations post-transcriptional dimers c-Myc–MAX of functions tion Mxi1 which encodes a molecule that targets c-Myc for ubiquitination c-Myc: of regulators functional and several post-transcriptional of encoding genes of expression the of upregulation the with mRNA in ( decrease substantial a without stage ISP the at protein of abundance the in decrease this was almost completely undetectable by the DP stage. We first observed with its mitogenic role at role the mitogenic its with after threefold increased actually regulation of c-Myc protein ( ( kinetics delayed with but and Notch targets Data are representative of two independent experiments ( intensity of eU incorporation by various thymocyte subsets (horizontal axis).cytometry, to assess transcriptional activity (left), and mean fluorescenceright corner) incubated for 2 h in the presence of eU, followed by subsetsflow (horizontal axis). ( intensity (MFI) of pyronin Y (bottom) in 2N cells of various thymocyte subsets (key), quantified by staining with pyronin Y, and mean fluorescence(vertical) for populations in in the proliferation signature (horizontal axis) or translation and ribosomecalculated categories by averaging of row maximum-normalized expression values for eachin smallprobe DP cells (DPsm). ( progenitor, B cell, myeloid and T cell subsets. Probes are sorted (GO:0005739;by increasing middle) value and cell cycle (GO:0007049; bottom) in bone marrow of ribosome and translation (GO:0005840 and GO:0006412; top), mitochondrion normalized expression values for probes belonging to the gene-ontology categoriesFigure 4 e c r u o s e r  Mitochondrion a and ribosome

In addition to In c-Myc activity addition Myc β Translation Cell cycle -selection requires via signaling -selection both the pre-TCR and Notch Myc and at the DP stage, DP thymocytes did not acquire the small cell cell small the acquire not did DP thymocytes DP at stage, the

). The positive correlation between between correlation positive The ). Transcriptional shutdown in small DP thymocytes. ( 14 Sin3b , progenitor 50 Stem , 5 4 2 4 Fr Dand , were also downregulated at the transcriptional level, level, transcriptional at the downregulated also , were , . Genes encoding other . Notch encoding Genes such targets, as , , which encode molecules that antagonize transactiva B cells B macrophages E Red pulp d b ) Incorporation of eU by thymocytes (subset, top a ) Scatter plot of proliferation and translation indexes, . ( Myeloid Fig. 3 Fig. c ) Total RNA abundance (top) in various thymocyte Fig. 3 Microglia β Neutrophils -selection checkpoint -selection Myc 5 Fig. 3 Fig. 3 c , the burst of proliferation triggered triggered , of the burst proliferation β ). We also observed delayed down delayed We). observed also d -selection ( -selection ). The abundance of c-Myc protein expression by ectopic expression expression by ectopic expression NK d 55 ), a divergence that coincided coincided that a ), divergence Fig. 3 Fig. DPsm , 5 6 ( Myc Fig. 3 Fig. Fig. Fig. 3 e ). Accordingly, when when Accordingly, ). T OT-I d6-15 expression and the the and expression SV-Ova c d , d d ). Together these these Together ). Supplementary Supplementary 14 ; error bars ( a ), ), in accordance ) Heat maps of , 50 , 52 , 5 b Trpc4ap 3

Il7r , then it , then Translation index 0.2 0.3 0.4 0.5 0.6 5 4 d

; ; and ), s.d.). 0.2 45 57

, , 5 5

8 0 - - - ,

T cells T at cells of phase neutrophils, the the contraction immune response, included cells DP small of that to similar extent an to transcriptome ( nature sig metabolism’ ‘low the had few very but phenotype, proliferation’ ‘low had many data the the set, expected entire ImmGen populations metabolism and proliferation-related gene-ontology categories across cycle. When we simultaneously analyzed the cell expressionthe offrom genesexit of in result the a merely not was cells DP small in ecules ( levels protein and transcriptional the at subunit) ribosomal 40S the of part (a S6 protein ribosomal the of data not shown). This observation was exemplified by downregulation processing and many metabolic processes ( RNA translation, as such activities ‘housekeeping’ various and tion prolifera controlling genes affected shutdown transcriptional this a ‘fold change’ with of >2; probes (225 upregulated were than <0.5) change’ of ‘fold a with probes (1,448 downregulated An unusual feature transcriptome. of this shift was that it many seemed more genes thymocyte were the to change greatest the with ated associ was stage DP small the to stage blast DP the from Transition cells DP Small thymocytes to smaller lead much stage DN3 the at MAD1 inhibitor c-Myc the of expression of phenotype. deletion DP both small results, these with quiescent Consistent the toward transition the drives of regulation ( cells DP of size

The broad shutdown of genes encoding metabolism-related mol metabolism-related encoding genes of shutdown broad The 0.3

Proliferation index d Neutrophils eU Fig. 4 Fig. DPsm advance online publication online advance 0.4 ISP DN DPbl a Myc ). Cell types that shut down their metabolism-related metabolism-related their down shut that types Cell ). DN3b Supplementary Fig. 3b Fig. Supplementary may be one of the key molecular events that that events molecular key the of one be may 0.5 DPbl 4SP DPsm 8SP 53 , 5 9 c

. Pyronin Y (MFI) Events (% max) 100 1.0 2.0 eU incorporation (MFI) 0.5 1.5 2.5 20 40 60 80 1,500 3,000 0 0 250 500 750

DN Pyronin DN Supplementary Fig. 5 Fig. Supplementary ISP

ISP Y Supplementary Supplementary Fig. 4 DPbl ) indicated that down that indicated ) nature immunology nature DPbl DPsm DPsm Myc

8SP

Fig. 1 Fig. 4SP69 and transgenic transgenic and eU UT 4SP69 + 4SP ( n e – =3)

). Much of ). 8SP69

+ ). 8SP69

8SP 4SP DPsm DPbl ISP DN

and – - - - - - © 2013 Nature America, Inc. All rights reserved. was downregulatedwas substantially ( ofscription upregulated substantially in CD69 ( genes signature upregulated the among were families) of NFAT, the members (including NF- regulators transcriptional TCR canonical encoding genes and immediate-early-response genes of number large a Accordingly, works. net gene of TCR-controlled to ‘generic’ branches belonged thus and of T in in peripheral cells TCR (data particular not signaling, shown), as were were examples they other genes) shared with not DP specific, signature upregulated the of (75% genes TCR-related those of Most ( thymocytes DP TCR-stimulated characterizing genes ture of signa the partitioning as by shown distinct the in TCR activation, CD69 in induced ( expression differ ent with genes of number large a showed thymocytes DP small of parison of profiles the gene-expression CD69 throughout thymocyte differentiation. Accordingly, side-by-side com transcriptome the in change greatest second the with associated was CD69 to thymocyte DP small from transition The (ref. CD69 marker of activation the expression MHC upregulate DP thymocytes that receive a TCR signal after engagement of peptide- differentiation blockade enforced at the DP stage by encodingE the proteinsinhibitory molecules Id2 and Id3, proposed to relieve the possiblefunctions in thymic positive selection. For example, the genes part of this signature ( rearranged newly their with interactions self peptide–MHC for scanning by selection positive for ‘audition’ cells DP Small CD69 activities were disproportionally affected during this shutdown. genes encoding molecules associated with metabolic and housekeepingthat and stage DP small the at suppressed globally was transcription RNA that indicated data Togetherthese cells. DP small in observed stage and was thus a major contributor to the transcriptional shutdown An abrupt decrease in (5-ethynyluridine; eU analog uracil incorporationbythe of cytes relative to that in DN4 cells report that detected 10% the amount of total RNA in small DP thymo subsets (DN, DP blast, CD4SP and CD8SP), in accord with a published etry ( RNAtotalquantified by analyzing pyronin stainingcytom by Y flow widespreadvariationbutmiss can cell a microarray data, which assesses only relative amounts of transcripts in of normalization after surmised than greater even be mayshutdown small DP stage ( allel upregulation of genes encoding negative regulators of c-Myc at the Therefore, the considerable downregulation of system. the immune adaptive an of generating in waste cost inevitable energy the diminishes that adaptation an be may state’ express a ‘useful’ antigen receptor, acquisition of this ‘low metabolism to failure their of because die to destined are subsets these in cells of numbers huge that Given checkpoint. receptor–controlled antigen ( extent a lesser to although categories, proliferation-related and metabolism- the in genes of shutdown similar a underwent also D) (fraction cells Pre-B ished transcription and translation and activities few mitochondria have DP small a thymocytes, of days lifespan and several have dimin ( macrophages pulp red and microglia nature immunology nature Analysis of genes encodinggenestranscriptionalAnalysisof regulators werethatnot Studies have described c-Myc as a global amplifier of transcription Fig. 4 + DP thymocyte: crossroad of multiple fates multiple of crossroad thymocyte: DP c ). Small DP cells had less RNA than did their ‘neighboring’ Tcf12 Fig. 4 Fig. Fig. 3 Fig. 5 Fig. + (which encodes the transcriptionalregulatorHEB)the encodes (which DP thymocytes affected transcripts induced early early induced transcripts affected DP thymocytes Fig. 5 d a de novo ), and like small DP cells, are of on DP cusp an the cells, small ), and like a ) suggested that the extent of the transcriptional ). As expected, a major part of the program program the of part major a expected, As ). advance online publication online advance b ) identified genes encoding molecules with RNA synthesis occurred at the small DP 6 3 + . We also assessed actual transcription DP thymocytes. Symmetrically, tran Fig.5 κ b 6 B, EGR, Fos, Rel and NUR77 and Fos, Rel NUR77 B, EGR, Fig. 4a Fig. and 2 . To . thathypothesis,testwe Myc SupplementaryFig.1 , + b DP thymocytes and DP thymocytes ). Neutrophils, like like Neutrophils, ). expression and par + DP thymocyte thymocyte DP Fig. 5 Fig.

α 66– β Fig. 4 Fig. Fig. 5 Fig. 6 TCR a 8 ). , were 6 d 5 a 6 6 6 4 0 ). ). ). ). 1 ). ). ). ------. . .

lt oprn te CD69 the comparing plot and NUR77), ( TCR-transgenicmodels innegative selection with associatedreproducibly been have that genes those highlighted ligands peptide-MHC selecting negatively with TCR the of engagement after die to doomed is TCR CD69 of fraction any) (if which interactions posi TCR-peptide-MHC in selecting engaged tively recently thymocytes DP identifies CD69 of sion CD69 keeping functions downregulated at the small DP stage. DP stage was characterized by the reactivation of most cellular house the upregulation was only at partial the CD69 ( teins and key regulators of translation, such as the ribosomal S6 kinase ple was the upregulation of genes encoding 20 ribosome structural pro genes; upregulated genes); and cytoskeleton-based motility (6% of upregulated thesis (10% of upregulated genes); nucleocytoplasmic transport (2% of proteinsyn restoration active of the involved in molecules encoding was genes that included These signature.TCR-activation early the of transcriptome part not upregulated the of 30% almost composed together which activities, housekeeping cellular other of reactivation genes genes ( of expression increased simultaneously and complex, dehydrogenase pyruvate the of inactivation and phosphorylation the catalyze which isozymes, kinase dehydrogenase pyruvate active most the of two ing simple shift in the balance of pro- and antiapoptotic factors, the CD69 ( cells SP mature into thymocytes rates, which onlyincreased gradually transcriptional as well as content,RNA cellular in reflected also was was completed only by the mature SP stages. Such stepwise reactivation isozymes) hexokinase regulates negatively product reaction downregulation of and thymocytes) in mutase 3-phosphoglycerate abundant most the colytic pathway ( cells starving Pdk1 ( reaction site CD69 complex. dehydrogenase pyruvate the of regulators key of regulation DP thymocyte to CD69 hub from in transition small metabolic the as emerged a acid cycle critical tricarboxylic The pathways. oxidative and biosynthetic major the induced genes, with genes involvedencoding molecules in several 3 Table ( represented highly were activation signature other several categories identified functional that tome of CD69 differentiation and warrant future exploration ( thymic in function reported no regulatorstranscriptionalhave other other factors modulate MHC class I expression at this step. Many of the and wild-type in deficientmice,increasethe after positive selectionlargelywas parallel expression of MHC class I molecules was considerablyNlrc5 impaired in Another hallmark of positive selection was the fourfold upregulation of Rps6ka1 s hr eiec o ngtv slcin n h tasrpoe of transcriptome the in selection negative of evidence there Is the with associated also was above noted switch metabolic The Gene Gene ontology and manual curation of the fraction of the transcrip and , which encodes an MHC class I transactivator Gadd45b + + Supplementary Fig. 6 Pdp1 DP Although thymocytes? it is generally accepted that expres DP genes downregulated ( thymocytes ). In particular, the ‘metabolic’ category represented 11% of 11% represented category ‘metabolic’ the particular, In ). ; ; Bcl2l11 Pdk2 Fig. 5 Fig. and 7 0 Fig. 5 Fig. + wih noe GADD45B) encodes (which ), ), CD69 with that of with DP that thymocytes was not part of the ‘generic’ TCR- e Pdp2 Fig. 5 (which encodes Bim), Bim), encodes (which ). Notably, for most of the genes in these categories, these Notably, in ). genes the of most for Nlrc5 Pgm2l1 d ). In addition to that coordinated regulation of regulation coordinated that to addition In ). ) ) encoding phosphatases that catalyze the oppo d + -deficient mice ( mice -deficient + ), ), with upregulation of DP reactivated the thymocytes upstream gly DP thymocyte, with concerted transcriptional Supplementary Fig. 6 Fig. Supplementary (which encodes glu-1,6-biP synthase, whose + Pdp1 P tg ad ml D sae ( stage DP small and stage DP and and 72– Supplementary Table 3 + Fig. DP thymocytes triggered via the the via triggered thymocytes DP 7 7

T ades hs usin we question, this address To . during the differentiation of DP Pdp2

4c Fig. 5 Fig. Pdcd1 , d (which is also observed in observed is also (which ). Therefore, rather than a than rather Therefore, ). c + Pdk1 (which encodes PD-1) PD-1) encodes (which 29 Fig. 5 ), which indicated thatindicated which ), Pgam1 Nr4a1 DP stage ( , and and 30 6 e c r u o s e r 9 3 ( , , 7 and 6 Fig. 5 8 b 5 o a volcano a on ) (which encodes encodes (which , it is not clear clear not is it , Supplementary Supplementary ). (which encodes Pdk2 ). One exam b Fig. 5 ). Although 7 i. 6 Fig. 1 ) ) encod . e Nlrc5 ) ) and a ).  + ------

© 2013 Nature America, Inc. All rights reserved. selected cell population that ultimately gives rise to both CD4 both to rise gives ultimately that population cell selected positively earliest the to correspond which thymocytes, CD4SP) ate tran the compared CD69 of next scriptomes we level, transcriptional the at selection ‘footprint’ on the transcriptome of CD69 which suggested that negative mayselection indeed have a substantial CD69 in substantially induced were genes These thymocyte. Genes encoding structural ribosomal proteins: normalized mean) of 20 genes encoding ribosomes and proteins regulated translation-related at the transition from small DP thymocyte to CD69 cycle pathways (right). Glu, glucose; P, phosphate; G, glycerate; Pyr, pyruvate; PDH, pyruvate HK, dehydrogenase; hexokinase. ( at the transition from small DP thymocyte to CD69 and presented relative to expression in small DP thymocytes. ( (horizontal axis) from (among 1,680 known or putative regulators, transcriptional after TCR stimulation among DP signature genes (shown in activation signatures in the induced (right) or repressed (left) quadrants. ( growth platelet-derived factor with splenic mimotope-pulsed APCs; genes orange (derived indicates immediate-early-response from analysis transcriptome of a cell line stimulated with with a ‘fold change’ threshold of 2, at 3 h or 7 h relative to their expression untreated cells after from a published analysis transcriptome of BDC2.5 DP thymocytes (DPsm), presented as a volcano plot. Colors indicate signature genes of DP TCR-stimulated thymocytes (derived (DP69 axis) in CD69 axis) and in gene expression (horizontal positive selection. ( housekeeping activities after Figure 5 e c r u o s e r  CD8

c a To determine whether we could distinguish positive and negative negative and positive distinguish could we whether Todetermine MHCI expression (fold) + 100 0.1 10 + lineages P (–log )

1 10 ) ) versus small DP thymocytes 6 5 4 3 2 1 0

Reactivation Reactivation of P 310 Rag2 values ( Rag1 DPsm 33 0.01 Rorc Dntt Arpp21 Aqp11 + Spo11 DP thymocytes

7 DP69 Down Nlrc5 Nlrc5 Nlrc5 Nlrc5 ( Plxdc1 + t Fig. 6 Fig. a Up Dnajb4 Nhsl1 -test; -test; vertical ) ) Change 4SP24 Mr –/– +/ –/– +/ Nlrc5 + + + 1 DP thymocytes and CD4 and thymocytes DP –

8SP24 Activation signature b H2D H2K -sufficient -sufficient mice ( E2f2 ). As expected, most genes upregulated in in upregulated genes most expected, As ). 0.1 –

8

b b

5

). ). Numbers in top corners indicate the number of signature genes of the upregulated (violet) and (cyan) downregulated Hmgb1

DP69 d

Relative expression Relative expression Fold change

0.5 0 1 1 0 + vs

DPsm 2 1 ISP Pgm1,2 + Nlrc5 DP thymocytes. Immediate earlygenes Dusp6

DPbl Pdk1,2 Jun Glycolysis +/+ + Pgam DPsm Srf DP thymocyte (left), whose products control key steps of the glycolytic and acid tricarboxylic (TCA) TCA ) ) and Hectd2 + Ndfip1 Nr4a2 Pdp1,2 CD8 Fos

DP69 1 + DP thymocytes, thymocytes, DP 10 Rpl3

+ Pgm2l1 Nlrc5 Nr4a1 My

a 4SP24 Supplementary TableSupplementary 4 int Egr2 Fosb Dusp5 ); ); colors indicate upregulated (violet) and (cyan) downregulated regulators transcriptional c Zfp36l2 d Hivep3 (intermedi Nfkbid

– Tnfrsf9 , , Cd2 ) Expression (maximum-normalized ) mean) Expression (maximum-normalized of eight transcripts regulated metabolism-related 8SP24 -deficient -deficient mice ( Rpl37 Cd53 Nfkb1 Itm2a Gadd45b Tagap Egr1 Nr4a3 3

– Cd69 0 415 ) ) that were induced (violet) or repressed (cyan), Glycogen 28 + Glu-1-P , , and and 100 Glycolysis Rpl38 Mitochondria b ) ) Gene probes remaining after of filtering-out genes whose expression changed - - PGM b , , with positive and negative selection. negative and positive with associated markers putative suggested profiles transcriptional the of versa (for example, thymocytes (for example, CD69 in expression higher had upregulated genes the of subset a However, thymocytes. of CD69 transcriptome to the a was contributor major selection example, (for CD69 Rpl39 P (–log10) Nlrc5 6 5 4 3 2 1 0 Acetyl-CoA ). ). ( TCA cycle 1,3-bPG Glu-6-P 2-PG 3-PG Pyr c Gl , , 0.01 + −/− ) ) Expression of MHC class I (MHCI) on various thymocyte subsets Rplp1 DP thymocytes had similar expression in CD4 in expression similar had thymocytes DP u ), ), detected by flow cytometry with anti–H2-K PGAM1 HK PDH Tcf12 Bach1 Mier1 advance online publication online advance Mxi1 Ets2 Mbtd1 Kdm3a , , PGM2L1 Cbfa2t2 Sin3b Cir1 Rps12 Cd53 Cbfa2t3 Nr1d2 PDK PDP in in vitro , , Glu-1,6-bP Nlrc5 , , 0.1 (inactive) PDH- Bcl2 + Klf7 Rps20 stimulation DP thymocytes than in intermediate CD4SP CD4SP intermediate in than thymocytes DP Zbtb20 and and , , P Nr4a1 Il7r , , Rps25 DP69 0.5

and H2-K1 e Fold change Relative expression Relative expression Relative expression , , +

Gadd45b 1 0 1 0 1 0 vsDPsm

2 1 , , Gimap3-Gimap4 Rps28 ), which confirmed that positive positive that confirmed which ),

ISP Structural ribosomalproteins Ribosome biogenesis , ,

DPbl Protein synthesis , , Zfpm Rps29

Pdcd1 e ) ) Expression (maximum- nature immunology nature

DPsm Nlrc5 Bcor 1 10 Carm Irf1 Tox2

, , DP69 b and Zfp827 or anti–H2-D Rrp15

). ). This divergence + 1 4SP24 Irf7 Bcl2l11 Id3 Id2 Zcchc12 – Kat2a Rbbp7 and + 8SP24 Runx3 CD8

+ – DP 100 Eef1b2 Rps6ka1 Zfp36l2 Rrs1 Fbl Nhp2 Rmrp Rpsa ) ) or vice int b (key) cells cells + . DP © 2013 Nature America, Inc. All rights reserved. stage of deletion by apoptosis. deletion of stage CD4 ( markers for these patterns CD4 the contrast, In by (encoded Helios and PD-1 NR4A1, of expression high with cells of proportion large ( fraction mocyte CD69 the for phenotypes distinct two discern to able strategy, (gating subpopulations H2-K1 in observed cells selected hall positively of mark transcriptional one was which of (upregulation I to class antibodies MHC of use combined The apoptosis. with associated was CD69 of subset a whether determine to cytometry ( experiments independent three of representative are Data counterparts. wild-type their and (Bim-KO) Bim or (MHC-KO) II class MHC I and class MHC in deficient doubly mice from ( (MHC-KO). II class MHC I and class MHC in deficient in (defined cells of subpopulations on CD69 and casp3) (Act caspase-3 of form cleaved activated, the of TCR and CD5 IL-7R, strategy, (gating in as lines (blue CD69 (CD4 CD69 in induced changes gene-expression ( models. TCR-transgenic in selection negative with associated reproducibly genes indicates red CD69 in left) (bottom <0.5 or right) (bottom >2 of change’ a ‘fold with genes total indicate corners in numbers plot; a volcano as presented (DPsm), thymocytes and axis) (horizontal expression 6 Figure nature immunology nature ( Fig. d a To test that hypothesis, we analyzed the activation of caspase-3 caspase-3 of activation the analyzed we hypothesis, that test To by flow markers of of those some We expression the explored thus P (–log10) 6 5 4 3 2 1 0 dull +

NR4A1 + 6 CD8 Bcl- DP thymocytes with greater (violet), similar (gray) or lower (red) upregulation in intermediate thymocytes thymocytes intermediate in upregulation (red) lower or (gray) similar (violet), greater with thymocytes DP ) and anti-CD69 subcategorized thymocytes into five distinct distinct five into thymocytes subcategorized anti-CD69 and ) e 458 CD8

(CD69 DPsm ). Notably, the CD4 the Notably, ). 0.01 Transcriptional and functional ‘footprints’ of clonal deletion in CD69 in deletion clonal of ‘footprints’ functional and Transcriptional 2 int ; vertical axis) versus small DP thymocytes (DPsm). Colors indicate genes induced over twofold in twofold over induced genes indicate Colors (DPsm). thymocytes DP small versus axis) ; vertical dull – MHC CD69 Fig. 3 Fig. Supplementary Fig. 7 Fig. Supplementary l – CD Fig. 6 Fig. β 4 and intracellular expression of Helios (IKZF2), NR4A1 and Bcl-2 by various cell subpopulations (key). ( (key). subpopulations cell various by Bcl-2 and NR4A1 (IKZF2), Helios of expression intracellular and hi PD-1 IL-7R Ikzf2 0. b + hi CD ). ( ). 1 CD8 MHCI 8

hi c c ) ). The CD4 The ). ) Expression of CD4 and CD8 on CD69 on CD8 and CD4 of ) Expression ) and little or no Bcl-2 or IL-7R ( IL-7R or Bcl-2 no or little and ) advance online publication online advance DP69 int Fold change Fig. 6 Fig. dull 0. subset had exactly opposite expression expression opposite exactly had subset − P 2 5 + c subset was enriched for cells at an early vsDPsm values ( values , CD8 d 1 ) or one experiment ( experiment one ) or d Supplementary Fig. 7 Fig. Supplementary ). These results suggested that the that suggested results ). These ). Numbers adjacent to outlined areas indicate percent cells in eachgate. ( eachgate. in cells percent indicate areas outlined to adjacent Numbers ). Helios TCR dull dull CD6 β t + population had a singularly singularly a had population -test; vertical axis) in CD69 in axis) vertical -test; DP thymocytes (DP69 thymocytes DP CD8 9 hi MHC 10 Gadd45b dull l + – Fig. 6 Fig. DP thymocytes relative to their expression in small DP; DP; small in expression their to relative thymocytes DP Bcl2l11 Nr4a1 Pdcd1 subset contained a contained subset CD f + ) Proportion of cells with activated caspase-3 among various thymocyte subpopulations (key) (key) subpopulations thymocyte various among caspase-3 activated with cells of ) Proportion CD4 CD4 e DP thymocytes thymocytes DP 5 478 b ). ; for example, example, for ; hi dull 100 + CD MHCI CD8 ). We were were We ). 8

int Fold change

dull + int Fig. 6 Fig. CD4 CD8 vs DPsm b 0. 0. + − 10 ; horizontal axis) and intermediate thymocytes thymocytes intermediate and axis) ; horizontal MHCI 1 2 1 5 − thy + MHC-KO DP thymocytes (DP69 thymocytes DP d ). ). − 0. - - (top) or CD69 or (top) e WT 1 their wild-type counterparts ( counterparts wild-type their in than many) as (25% mice Bim-deficient in and many) as (1–10% less abundant in mice doubly deficient in CD69 MHC class I and included MHC class lation CD4 II the in caspase-3 activated for positive Cells ( deletion clonal to sensitive be to known populations, SP immature with compared when 3%), even to (up caspase-3 activated for positive cells of frequency high of DP thymocytes. DP of repertoire polyclonal the in program deletional large an unexpectedly showed observations these Together ‘neglect’. by died that cells ( II and I class MHC of absence the CD4 the to contrast in that, CD69 of proportion stantial a sub contained also populations DP cell Small deletion. clonal true of characteristic is which manner, Bim-dependent and MHC- an in death cell underwent that mice unmanipulated in subset thymocyte + Act casp3 DP thymocytes. ( thymocytes. DP CD6 0.73 0.12 9 0. DPsm 5 hi MHCI 0.00 0.01 0.86 6. DP69 9 1 Fold change + ) and small DP small ) and − b + vsDPsm a (bottom) thymocytes thymocytes (bottom) ) Comparison of ) Comparison ) Change in ) Change 2 Nlrc5 1. 0.23 8 CD4 Cd − Gimap3 Id el ad CD69 and cells 5 Ikzf2 dull c Il7 2 , CD8 d r

Bcl2l1 ) from wild-type mice (WT) and mice doubly doubly mice and (WT) mice wild-type ) from 1. 0.01 62 5.5% − dull

1

Fig. Fig. 6e

Gimap4 dull cells positive for activated caspase-3 caspase-3 activated for positive cells i. 6 Fig. 1 10 Cd69

d Nr4a1 Gm5774 MHCI

Bcl2 ) Cell-surface expression of PD-1, PD-1, of expression ) Cell-surface CD8

Gadd45b Snord16a Fig. 6 Fig. Cd5 0.08 − , e Pdcd1 f

H2 Nr4a3 and and Tagap dull ). These results identified a DP ). identified results These CD4 3 -K 1 f

population, was larger in in larger was population, + hi + 396

f Act casp3 cells (%) CD8 0.05 ); this probably identified identified probably this ); cells, which were much much were which cells, 0. upeetr Fg 7 Fig. Supplementary e 5 5 0.49 ) Intracellular expression expression ) Intracellular int 94 CD4 c e c r u o s e r

WT CD8 MHC-KO dull CD6 CD69 0.11 CD8 0. 9 46 CD4 hi 3 – CD4 DPsm MHCI MHCI WT MHCI + dull dull Bim-KO CD8 93 41 – CD + – 9 0. 0. popu − 0.12 6 67 3 8 dull

). ).  - -

© 2013 Nature America, Inc. All rights reserved. another, another, by encoded molecules the of function the whereas complement, of action lytic the from cells protects and convertases C3 complement 8 Fig. mature 2; of change’threshold ‘fold from a (at counterparts ‘migration’ peripheral to cells T the during upregulation significant thymocytes had SP small mature the proliferating reflected of probably fraction which cycle, cell the to related were maturation a such of genes Most of level. downregulated the at evidence transcriptional the process little observed we ( Unexpectedly, cells T peripheral and thymic compared we scriptomes, tran their demarcate further and identities lineage reinforce could maturation differentiation. thymocyte during marks transcriptional few unexpectedly by distinguished were ages CD8 the for and Th-POK) well-described the by exemplified were these expression; different had regulators transcriptional encoding CD8 the in expression interferon- and ule- Crtam CD4 the in upregulated specifically were OX40) and CD40) receptor those, Among ( expression in difference a having genes few unexpectedly both for common were CD4 the changes the of most shift, transcriptome ( probes gene 2,600 than more of expression the in variations with differentiation, thymo cyte during observed changes largest the of one had event tion differentia That stage. SP the cells to the stage DP of the from fraction a differentiating only with population, CD69 heterogeneous a the was that indicated above presented results The CD4 of Differentiation ( regulators transcriptional putative or known 1,680 of in as lines (blue group per genes total indicate corner right bottom in in as (threshold differentiation (blue) CD8SP or (red) CD4SP during upregulation ‘preferential’ or (gray) similar with induced genes indicate Colors (DPsm). DP small versus axis) vertical (8SP24; thymocytes CD8SP mature and axis) horizontal (4SP24; thymocytes CD4SP mature in 7 Figure e c r u o s e r 1 in CD4 the was similar egress thymic after genes 0 Fold change a Egress from the thymus is coupled with a phase of functional functional of phase a with coupled is thymus the from Egress – 0.01

8SP24 vs DPsm 100 0.1 0. 10 5 1 2 0.01 a and and ). One of these genes was was genes these of One ).

Cd8a + Acquisition of CD4 of Acquisition Dntt Dapl1 lineage and the CD8 the and lineage Rorc Rag2 2 Arpp21 Cd226 3 + . To test the hypothesis that this maturation phase phase maturation this that hypothesis the test To . Cd8b1 lineage Ccr9 Cd40l Gata3 , is unknown in T cells. The upregulation of these these of upregulation The cells. T in unknown is , Aqp1 , which encode molecules associated with gran with associated molecules encode which , CD (which encodes the (which encodes ligand for the costimulatory Fig. 1 Fig. Tnfrsf4 γ 0.1 4 for the CD4 the for 8 -mediated cytotoxic functions cytotoxic -mediated 1 + 0 andCD + ( Plxnd1 + lineage. Correspondingly, very few genes genes few very Correspondingly, lineage. Gpr114 Fig. 7 Fig. and CD8 and + Eomes e and CD8 and Cxcr6 Gpr68 Klrd1 (which encodes the cell-survival factor factor cell-survival the encodes (which and and Cd7 Nkg7 8 + thymocytedifferentiation 4SP24 2 b + 0. Nrp1 lineage (approximately 93%), with with 93%), (approximately lineage Fold change ). Thus, the CD4 the Thus, ). 5 Cd4 H1f0 Fig. 7 Fig. Cd55 + + – + lineage and and lineage transcriptional identities during thymocyte differentiation. ( differentiation. thymocyte during identities transcriptional 1 lineages vs DPs Cd2 Itga Gata3 Crtam a , whose product deactivates deactivates product whose , e 8 ). Despite the scale of this this of scale the Despite ). 2 Runx3 m Il2r Cd226 Zbtb7b Ctsw a Nr4a Cd7 Zbtb16 + and CD8 1 + 8 Runx3 Supplementary Table 4 Supplementary + Il2rb 1 (which encodes encodes (which lineage, whereas whereas lineage, 10 Nlrc5 Ms4a4b . Only 12 genes genes 12 Only . and CD8 and Tnfrsf 7 9 Itgb3 Il6ra , had higher higher had , Ccr8 + and and Zbtb7b H2K1 4 DP subset subset DP Ly6a Art2b Cd40l Il7 + Itm2 Fig. 8 Fig. Fig. 7 Fig. Gimap4 lineages lineages r 2,480 Ccnd2 Gimap3 + Eomes a 91 81 Slfn line Fig. 3 Fig. 100 1 a a Igfbp4 ). ). ). ). S1pr1 - - - - -

b ). ( ). ). stimulation via the TCR and, unexpectedly, included lineage-specific via the lineage-specific TCR stimulation included and, unexpectedly, programs of CD4 ( cells most activation-induced changes were shared by CD4 profiling. Reminiscent of maturation-induced changes in thymocytes, gene-expression for anti-CD28 and anti-CD3 with them stimulated CD4 isolated also We similarity. T cells and CD8 NF- and MAPKAPKII S6, (Erk1/2, nodes downstream and SLP-76) and Lat CD4 phosphoryla in tion more slightly of finding our Despite points. time 15 at markers surface cell 7 of expression the and molecules signaling anti-CD28 and the assessed status phosphorylation of 15 intracellular and (anti-CD3) CD3 to antibody with cells node lymph of pensions cytometry mass multidimensional by cells T activation. during cascades post-translational in reflected the baseline transcriptomes might be amplified after activation and/or T cells and CD8 of functions CD4 distinct given the physiologically was unexpected, CD8 for activation), cell T of lator and cells; protein and kinases) mitogen-activated of regulator negative a functions: encodes (which signaling TCR with associated molecules ( before in thymocytes detected those lapped (159; ‘fold change’ rate,>2; false-discovery <10 CD4 peripheral of comparison direct profiling, thymocyte the with line CD4 of reinforcement no with consequences, transcriptional limited had subset. These demonstrated observations that egress from the thymus CD4 thymic the in cells T CD4 peripheral in ( Fig. 8 Fig. b b Fold change Therefore, we compared TCR signaling events in CD4 in events signaling TCR compared we Therefore, In cells? T peripheral in identity lineage defines ultimately What ) Comparison of the gene-expression changes in in changes gene-expression the of ) Comparison – 0.01

8SP24 vs DPsm 100 0. 0. 10 + + 1 5 1 2 0.01 or CD8 or CD8 or Fig. 8 Fig. a Rorc ). Finally, a small subset of genes selectively downregulated downregulated selectively genes of subset small a Finally, ). κ B) had very similar dynamics of phosphorylation in CD4 in phosphorylation of dynamics similar very had B) Dpp4 advance online publication online advance d + T cells, most key TCR-proximal nodes (CD3 nodes TCR-proximal key most cells, T ). Nonetheless, some divergence in the transcriptional transcriptional the in divergence some Nonetheless, ). Mapk11 + + lineage identity. lineage T cells identified few genes with different expression expression different with genes few identified cells T (which encodes CD26) and and CD26) encodes (which + + T cells. We reasoned that these modest differences in T cells ( + Myb cells and CD8 + a T cells reflected the inclusion of regulatory regulatory of inclusion the reflected cells T 0. ) Comparison of gene-expression changes induced induced changes gene-expression of ) Comparison (which encodes the kinase p38 kinase the encodes (which 1 Bcl6 Ets2 Fig. Fig. 8 Snai + Transcriptional regulators subset but not the lymph node CD4 node lymph the not but subset 3 Eomes 4SP2 c 0. ), which mirrored the transcriptional ), which mirrored the transcriptional + Fold change Runx1 2 5 Jun cells was evident as early as 1 h after + 4 or CD8 or – + 1 Runx3 vs DPs cells. Such limited divergence divergence limited Such cells. Gata3 Nr4a1 m

+ Rnf125 nature immunology nature Fig. Fig. 8 peripheral T cells and and cells T peripheral Myc 3 −4 Zbtb1 4 Id Foxp a . We stimulated sus Westimulated . Mllt3 ); ); most of these over , filtered for a list a list for , filtered 2 Fig. 5 Fig. b Nr4a3 10 3 6 (a positive regu positive (a ). A few encoded ). A few encoded + cells and CD8 Klf2 a Irf7 β ); numbers numbers ); Cd28 Zbtb7 ), for CD4 for ), + ζ or CD8 or , Zap70, Zap70, , b Chd7 , , 217 Trib2 4 8

100

+ + + + + + - - - -

© 2013 Nature America, Inc. All rights reserved. T cells increased over time ( time over increased cells T ( lineage each of tors regula of the master transcriptional genes encoding downregulation ( experiments independent four of representative CD4 the in regulation ‘preferential’ or (gray) in as lines Blue populations. cell unstimulated corresponding the in that to relative anti-CD28, and anti-CD3 with CD4 node lymph in induced changes expression ( control). (positive PMA ester phorbol the with stimulated cells (PMA), right far cells; unstimulated (0), left Far anti-CD28. CD8 ( regulators. transcriptional encoding genes indicate CD4 in expressed ( (T T cell regulatory indicates green counterparts; CD4 peripheral in twofold over by upregulated genes indicates Orange respectively). (8SP24), CD8SP CD8 and axis) (horizontal 8 Figure nature immunology nature efficient an for crucial is and cells dendritic attracts product whose b a d c ) Comparison of gene expression in lymph node CD4 node lymph in expression gene of ) Comparison h tasrpinl iegne f CD4 of divergence transcriptional The Fold change Fold change + – T cells (left) and CD4 and (left) T cells stim vs unstim T8Nve 100 T8Nve vs 8SP24 0.1 0.5 0.1 0.5 10 10 1 2 1 2

Definition of CD4 of Definition Gata3 Top2a Cd200r1 8 Zbtb7b 0.1 T Prolif signature periph migration Upreg upon re Ccna2 p-FAK p-RB p-PARP p-4EBP1 p-NF- I p-MAPKAPKII p-CREB p-S6 p-Erk1/2 p-SLP-76 p-c-CBL p-Lat p-Zap70 p-CD3 g κ 0.1 cell–related B Slamf1 + α Time (min):

(red) or CD8 or (red) Eomes κ ζ B Mki67

0.5 Zbtb7b Ccl3 + Rasgef

T cells (vertical axis) relative to that in their thymic parental populations (mature CD4SP thymocytes (4SP24) and mature mature and (4SP24) thymocytes CD4SP (mature populations parental thymic their in that to relative axis) (vertical T cells + 1 + Cxcr6 and CD8 and advance online publication online advance T cells (right), detected simultaneously by mass cytometry at multiple time points after crosslinking with anti-CD3 and and anti-CD3 with crosslinking after points time multiple at cytometry mass by simultaneously detected (right), T cells

0 , , Ets2 0.5 1 Fig. 8 Fig. + 2 Gata3 (blue) T cells; numbers in top left and bottom right corners indicate total corresponding gene probes; open circles circles open probes; gene corresponding total indicate corners right bottom and left top in numbers T cells; (blue)

h 0.5 Neto2 Runx3 Cxcr4

1 T4Nve vs4SP24 + Thymic export d , , transcriptional identities. ( identities. transcriptional 1 Fold change Nr4a1 ). One example of this was was this of example One ). Runx3 Rassf3

10 2 Ccr8 + Rras2 3 T cell (red) or CD8 or (red) T cell 2 F2rl1 Nr4a2 and and + Fam46a

reg 4 c T cells (horizontal axis) and CD8 and axis) (horizontal T cells – Nr4a3 + ). cell)–related signature genes; blue indicates proliferation (prolif) signature (blue lines as in in as lines (blue signature (prolif) proliferation indicates blue genes; signature cell)–related cls n CD8 and cells T 512 T8Nve Eomes c 5 25 Pde2a ) Heat maps of the phosphorylation (p-) of various signaling molecules (left margin) in lymph node node lymph in margin) (left molecules signaling various of (p-) phosphorylation the of maps ) Heat 100 + T cells (horizontal axis) and CD8 and axis) (horizontal T cells 6 Ust Gata3 Cd200r1 27 ; ; Zbtb7b 0.5 10

Runx3 8 Fig. 8 Fig. 0.1 Cd55

Mediandifference(Arcsinhrelativetotime0) 10 + Ly6c2 Ly6c1 T cell (blue) lineage (threshold as in in as (threshold lineage (blue) T cell a Crtam Dapl1 d Ccl4 Cd160 ) Comparison of gene-expression changes induced in lymph node CD4 node lymph in induced changes gene-expression of ) Comparison Ccr9 20 ). Xcl1 0.5

Stim vsunstimT4Nve 40 Plac8 Rasgef + - , Ccl3 Fold change

1 80

Snai3 Fosl1 by CD4 by ( regulated selectively also CD8 in involved directly (MIP-1 response cytotoxic 5 Racgap1 Eomes PMA 2 h Cd83 Xcl1 + T cells (vertical axis) stimulated for 1 h, 5 h or 24 h (above plots) plots) h (above 24 5 h 1 or h, for stimulated axis) (vertical T cells

0 b T8Nve expression (log )

α 10 Ptpla + 0.5 1 2 3 4 cells included included cells ) and and ) 10 + Tnfrsf4 T cells (vertical axis); colors indicate genes ‘preferentially’ ‘preferentially’ genes indicate colors axis); (vertical T cells Akr1c18 105 Plac8 Il2rb Cxcr6 Fasl

Utf1 1 Ikzf2 Itgae Snai3 1 Eomes Ccl4 2 Klrd1 Nkg7 Il2ra Ccr8 Crtam 1525 8 Il2

3 Cd8a 2 (MIP-1 39 . Other chemokine-encoding genes, such as . chemokine-encoding Other 100 + or CD8 or Fig. 5 Fig.

4 Epas1 Il18r1 Il2 Ccr9 Cd200r1 Il7r + T4Nve Il2rb 31 5 Zbtb7b Fig. 8 Fig. cell functions ( functions cell , , 0.1 Gata3 β a Fasl –2.35 Il2rb 2 ; numbers in corners as in in as corners in ; numbers Trat1 T4Nve expression(log Cd226 ), as well as genes encoding molecules molecules encoding genes as well as ), + 6 T cells relative to their thymic thymic their to relative T cells Peripheral Tcells Cd40l Gzm d Id2 Xcl1 a , , 8 Cd8b1 Zbtb32 ). Genes ‘preferentially’ expressed expressed ‘preferentially’ Genes ). ; colors indicate genes with similar similar with genes indicate ; colors Runx3 Tnfsf4 b Dapk1 0.5 10 Ctsw Prf1 20 1 (which encodes the ligand ligand the encodes (which Runx1 0 Tigit Itgae d CD4 3 Eomes Transcriptional regulators Genes withdifferentexpression ) Comparison of gene- of ) Comparison 24 Crtam Runx3

Cxcr6 40

2 CD8 Transcriptional regulator Genes withdifferentexpression Lrrc32 h 10 ) Il12rb2 80 Nkg7 e c r u o s e r Itgb3

and and PMA 2.35 Ccl3 Cd28 Itm2a Zbtb7b Zfp558 10 Trib Klrb1f Cd4 b + ). Data are are Data ). T cells T cells Gzmb 2 4 Il2ra Il2rb Il3 Tnfsf4 Tnfsf4 Racgap Tbx21 s Fig. 3 Fig. Cdkn1a ), were were ), 2873 Il2 54 77 Ccl3 100 1 b 11 ). ).

© 2013 Nature America, Inc. All rights reserved. take stock of the uncharted space across key transitions in thymocyte in stock of across take key space thymocyte transitions the uncharted and of selection, the ‘quasi-identity’ CD4 positive after reactivation cellular by followed cytes thymo DP cortical of shutdown deep the lineage, cell T the to ment commit of nature gradual apparently the events: differentiation of the appreciation that differently colors somewhat perspective unique a yielded has differentiation of their course entire the through tomes models. disease-relevant in perturbations of network origins of the identification the enable will differentiation thymocyte throughout regulation transcriptional of dynamic normal the of definition the Furthermore, differentiation. thymocyte early drive that factors transcription the of dependence ( Lef-1 factor transcription the encoding gene the of dysregulation by driven fact in is TCF-1 factor transcription the in deficiency by induced leukemia lymphoblastic acute cell T example, For networks. transcriptional of context the in stalls of how differentiation or identification of malignancies staging precise more allows subset thymocyte each of transcriptomes the of eases such leukemia, immunodeficiency and autoimmunity. Definition medulla. thymic the in tion selec to relative negative deletion wave early of this cortical quantify into take account the of dynamics that this process be will to needed more precisely studies Further deletion. undergoing DP of fraction a substantial highlighted which caspase-3, with activated thymocytes we were able to transition, this program ascribe to a subset of this CD69 through identified proteins of variety a tracking By interactions. TCR-peptide-MHC first their of consequence a subset, CD69 early the in deletion apoptotic with associated program a uncovered has profiling expression Our issue. a controversial been has setting polyclonal unmanipulated, an in stage this at occurs also selection negative substantial Whether selection. positive for gible H-Y TCR to leads efficiency a greater selection positive I–restricted class MHC the of expression transgenic with mice from stage in polyclonal settings been reported in mice with overexpression or deletion of thymocytes matureSP leads to smaller SP as thymocytes, well as a lower proportion of CD3 tion of c-Myc activity via overexpression of the inhibi c-Myc First, possibility.inhibitor this MAD1 of favor in are evidence of lines several of c-Myc investigated, remains stage to be at function this precise the the reactivation of cellular functions after positive and selection. Although stage DP small the at shutdown programmed the enforces that thatc-Myc ‘rheostat’ constitutes idea cellular the compatible with β ofdownregulation (via Myc of control Dual insights. new several provided has states ferentiation dif of thymocyte spectrum entire transcriptional of the analysis Our DISCUSSION functions. different very their of perception present the to contrast in is similarity tional transcrip close This stimulation. TCR to responses transcriptional through migration peripheral and was in reflected their and signaling the CD4 of transcriptomes the in similarity considerable the Therefore, and OX40) for e c r u o s e r 12 many and genes that candidate notable highlight were differentiation -selection checkpoint and -selection at DP–to–CD69the small In conclusion, our comprehensive resolution of dis cell T for reference important an here provided Wealso have complete a of expression The

expression at the transcriptional and post-transcriptional levels levels post-transcriptional and transcriptional the at expression + and CD8 and + lineages, acquired after positive selection, persisted persisted selection, positive after acquired lineages, Tnfrsf4 5 Mxi1 9 . Second, although no altered phenotype has has phenotype altered no although Second, . (which encodes OX40), among others. others. among OX40), encodes (which 53 + T and cells CD8 , 8 , , 3 Sin3b , Myc Lef1 α β and overexpression in DP thymocytes TCR renders DP thymocytes eli thymocytes DP renders TCR ) 8 4 , which emphasizes the inter the emphasizes which , Trpc4ap + T Wecells. were able to ) ) was apparent at the α β T cell transcrip + DP transition, 8 Myc 3 hi . MHCI at the DP + − DP DP DP hi ------

17. 16. 15. 14. 13. 12. 11. 10. M.M., M.M., T.K., D.G. and T.H. designed, did and analyzed some of the experiments and the Australian Research to,Council (LP110201169 T.H.). M.M.), the National Health and Medical Research Council (637353 to D.G.) and Health the (AI072073), Human Frontier ProgramScience (HFSP-LT000096 to for assistance with figure preparation. Supported by the US National Institutes of mice and antibodies; J. Moore and A. Kressler and for C. Laplace flow cytometry; We thank K. Hattori, and A. N. Ortiz-Lopez Asinovski for technical assistance with Note: codes. Accession the of in version available are references associated any and Methods M biology. cell T of view complete more a for facts known the with in unknowns’ ‘known the defined has our analysis Thus, characterized. CD4 in expression different with factors transcription (rare) the of all almost far, whereas thus analyzed been have transitions DP the through identified as-yet unexplored in T cells. Very few of the transcriptional regulators 9. 8. 7. 6. 5. 4. 3. 2. 1. reprints/index.ht R The authors declare no competing interests.financial directed the study, analyzed and interpreted results and wrote the manuscript. H.v.B. contributed resources and assisted with data analysis; and D.M. and C.B. M.H.S. designed, did and analyzed some of the experiments; G.P.N. and K.K. and wrote the manuscript; andR.C. J.E. helped with microarray data analysis; S.B. and AUTH A C eprints and permissions information is available online at at online available is information permissions and eprints ckn O

ethods α

MPETING FINANCIA MPETING iilw P, e, .. Buhan H & o Bemr H Pstv slcin of selection Positive H. Boehmer, von & H. Bluthmann, H.S., Teh, P., Kisielow, H.R. MacDonald, Zinkernagel, R.M., Callahan, G.N., Klein, J. & Dennert, G. Cytotoxic T cells learn specificity rsasy T. Kreslavsky, I. Prinz, J.A. Williams, and Commitment J.C. Zuniga-Pflucker, & H.T. Petrie, B.H., von, A., Bhandoola, Godfrey, D.I., Kennedy, J., Suda, T. & Zlotnik, A. A developmental pathway involving Rothenberg, E.V. T cell lineage commitment: identity and renunciation. of convergence a cell, T a becoming On Zuniga-Pflucker, J.C. & P.K.Thompson, models myths,debate: intense and fate Lineage J.H. Park, & S. Adoro, Singer,A., in cells T self-reactive of Selection K.A. Hogquist, & S.C. Jameson, Stritesky,G.L., self- learning tolerance: Central S.C. Jameson, & T.A. Baldwin, K.A., Hogquist, Notch. by fate cell T of Regulation E. Robey, thymocytes. of selection Positive M.J. Bevan, & K.A. Hogquist, S.C., Jameson, neglects useful, the selects P.Kisielow,thymus & The Teh,H.S. H., Boehmer, von Miller, J.F. The golden anniversary of the thymus. antigen-specific T cells in thymus by restricting MHCmolecules. byrestricting inthymus T cells antigen-specific products. gene II class forselfH-2 during diffentiation theinthymus. differentiation. 730–733 (1988). 730–733 thymus. adult Immunol. J. development: dysregulated CD28 costimulation can bypass the pre-TCR checkpoint. from. choose CD25 expression. to plenty precursors: and cell T intrathymic and extrathymic of potential developmental CD44 by defined thymocytes Immunol. J. mouse CD3 of adult subsets negative distinct functionally and phenotypically four 186 way.the alongNotch a up it kickfactors (2008). and mechanisms of CD4- versus CD8-lineage choice. thymus. the thymus. the in control (1999). Immunol. Rev. Annu. harmful. the destroys and useless the (2011). β

Supplementary information is available in the T T cell differentiation, and the challenge now is to integrate those owl O , 6649–6655 (2011). 6649–6655 , R R C the pape the t al. et edgments O advance online publication online advance NTRIBUTI

Annu. Rev. Immunol. Rev. Annu. m 175 150 Immunity iulzto o te alet tp of steps earliest the of Visualization Nat. Immunol. Nat. l t al. et t al. et . Immunity , 4199–4207 (2005). 4199–4207 , (1993). 4244–4252 , et al. et GEO: microarray data, data, microarray GEO: r + euae csiuain n h tyu i ciia fr cell T for critical is thymus the in costimulation Regulated

.

T cells versus CD8 versus cells T β 13

Nat. Rev. Immunol. Rev. Nat. Slcinidcd rlfrto i rqie for required is proliferation -Selection-induced Positive selection of CD4 of selection Positive Nature O 26 L

, 93–126 (1995). 93–126 , 37 NS INTERESTS , 678–689 (2007). 678–689 , , 840–853 (2012). 840–853 ,

7

336 , 995–1003 (2006). 995–1003 ,

30 , 471–473 (1988). 471–473 , , 95–114 (2012). 95–114 , Immunol. TodayImmunol. Semin. Immunol.Semin.

5 Nature , 772–782 (2005). 772–782 , Annu. Rev. Immunol. Rev. Annu. + GSE1590 T cells have already been been already have cells T Nat. Rev. Immunol. online version of the pape

+ thymocytes controlled by MHC by controlled thymocytes

Nat. Rev. Immunol. 271 nature immunology nature γ δ

cl dvlpet n the in development cell T 10 , 251–253, (1978).

http://www.nature.c , 57–61 (1989). 57–61 , 23 7 , 350–359 (2011).350–359 , . − CD4

17 − 11 Nature CD8

8 J. Immunol. , 283–295 , , 489–495 , 788–801 α β − online online r cell T triple- .

335 om/ ,

© 2013 Nature America, Inc. All rights reserved. 26. 25. 24. 23. 22. 21. 20. 19. 18. nature immunology nature 34. 33. 32. 31. 30. 29. 28. 27. 52. 51. 50. 49. 48. 47. 46. 45. 44. 43. 42. 41. 40. 39. 38. 37. 36. 35.

itn A. Liston, thymocyte of analysis expression Gene E.L. Reinherz, transcriptional & L.K. Clayton, I., of Schmitz, Characterization J. DeGregori, & D.L. Mann, D., DeRyckere, their assert cells T young maturation: Post-thymic D.W. Hendricks, & P.J. Fink, A.J. Leishman, M.S. Jordan, Teh, H.S. in development T-cell mature of Kinetics K. Shortman, & R. Scollay, M., Egerton, thymocyte-stromal of Dynamics E. Robey, & R.S. Lewis, N.R., P.,Bhakta, Bousso, Olenchock, B.A. TARPP,Karjalainen,K.&Kisielow, accompaniesA.C.Nairn, thatprotein novelJ., a S.V. Rakhilin, lineage the T-cell of specification Multilayered L. Li, & E.V.,J. Rothenberg, Zhang, P. Li, T.Ikawa, K. Narayan, S.C. Bendall, S. Luc, checkpoint commitment lineage cell T E.V.early Rothenberg, An & M. Leid, L., Li, J.A. Zhang, M. Mingueneau, deletion clonal of analysis Transcriptional K.A. Hogquist, & T.A. Baldwin, S. Zucchelli, Mick, V.E. Wong, G.W. checkpoints receptor-controlled T-cellrepertoire: the of Selection H. Boehmer, von A.P. Weng, B.N. Weber, K. Germar, Taghon,T. Y. Yashiro-Ohtani, S. González-Garcia, by expression gene lymphocyte-specific T Leder,P.of & induction B. Direct Reizis, M.L. Deftos, R. Guo, (2012). TCR of proliferation and T-celldevelopment. in leukemia/lymphoma. lymphoblastic differentiation. signaling. Notch to γ E2A. antagonizing leukemia. IL-7R lineage-specific T pathway. signaling Notch mammalian the thymocytes. (2008). kinase diacylglycerol by anergy. cell T of (2001). TCR gene rearrangement and thymocyte education. signaling. calmodulin-dependent fate. deletion. lineage. distinct continuum. hematopoietic (2011). human a across responses potential. lineage Bcl11b. factor transcription the on dependent (2012). identity. cell T establish control transcriptional and marking Med. Immunol. J. genetics. and selection positive (2004). polygenic trait characterized by defective induction of Bim. selection selection negative during regulation individuality. Immunity CD8 self-peptide. agonist an cells. T of (1988). phenotype CD4/CD8 the determine receptor thymus. the (2002). microscopy. two-photon by visualized interactions cell δ -selected pre-T cells in the adult mouse thymus. mouse adult the in cells pre-T -selected α

Immunol. Rev. Immunol. α 209 t al. et + cls r pstvl slce i te hms y gns self-peptides. agonist by thymus the in selected positively are cells T γ t al. et Science t al. et δ

Science in vivo in et al. et et al. , 623–637 (2012). 623–637 , 16 T cell subtypes. cell T J. Exp. Med. Exp. J. et al. t al. et et al. et et al. et al. et

t al. et ergamn o T el t ntrl ilrlk cls pn Bcl11b upon cells killer-like natural to cells T of Reprogramming Immunity Proc. Natl. Acad. Sci. USA Sci. Acad. Natl. Proc. et al. et 179 , 355–364 (2002). 355–364 , et al. et Nat. Rev. Immunol. Rev. Nat. et al. et t al. et h eris tyi T el rgntr ssan cl ad myeloid and cell B sustain progenitors cell T thymic earliest The Immunity t al. et yegsi cnrl f cl dvlpet n tmr suppression tumor and development cell T of control Synergistic t al. et An essential developmental checkpoint for production of the T cell T the of production for checkpoint developmental essential An Thymic major histocompatibility complex antigens and the t al. et Nature The regulated expression of a diverse set of genes during thymocyte t al. et t al. et Developmental and molecular characterization of emerging of characterization molecular and Developmental . eeaie rssac t tyi dlto i te O mue a mouse; NOD the in deletion thymic to resistance Generalized HES1 opposes a PTEN-dependent check on survival, differentiation,

et al. T-cell factor 1 is a gatekeeper for T-cell specification in response in T-cellspecification for gatekeeper a is 1 T-cellfactor et al. et , 837–844 (2007). 837–844 , Nat. Immunol. Nat.

329 -y i a iprat iet agt f oc1 n -el acute T-cell in Notch1 of target direct important an is c-Myc Int. Immunol. Int. in vivo in Intrathymic programming of effector fates in three molecularly three in fates effector of programming Intrathymic 329 Immunol. Nat. Notch1 signaling promotes the maturation of CD4 and CD8 SP CD8 and CD4 of maturation the promotes signaling Notch1 Thymic selection of CD4 of selection Thymic Genes Dev. Genes eetv cnrl oeac idcin n O mc: genomics mice: NOD in induction tolerance central Defective t al. et

Proc. Natl. Acad. Sci. USA Sci. Acad. Natl. Proc. igecl ms ctmty f ifrnil mue n drug and immune differential of cytometry mass Single-cell A critical role for TCF-1 in T-lineage specification and specification T-lineage in TCF-1 for role critical A 238 ntok f oto mdae b rgltr f calcium/ of regulator by mediated control of network A , 93–96 (2010). 93–96 , rcros f ucinl H cas - r ls II-restricted class or I- class MHC functional of Precursors Disruption of diacylglycerol metabolism impairs the induction Thymic negative selection is functional in NOD mice. NOD in functional is selection negative Thymic yai tasomtos f eoewd epigenetic genome-wide of transformations Dynamic

, 85–89 (2010). 85–89 , t al. et 13 476 Adv. Immunol. Adv.

206 α Nat. Immunol. Nat. 22 . , 150–168 (2010). 150–168 , r-C sgaig nciae Nth tasrpin by transcription Notch1 inactivates signaling Pre-TCR β α , 73–84 (2000). 73–84 , and

J. Immunol. J. slce mue thymocytes. mouse -selected ee xrsin n al hmn hmpiss and thymopoiesis human early in expression gene , 63–68 (2011). 63–68 , , 385–396 (2005). 385–396 , Nat. Immunol. Nat. , 779–791 (2009). 779–791 , advance online publication online advance S-ALdpnet oc1 inln controls signaling Notch1 CSL-MAML-dependent

23 ζ

. 15 7

Science 13 , 1665–1676 (2009). 1665–1676 , Proc. Natl. Acad. Sci. USA Sci. Acad. Natl. Proc. , 1174–1181 (2006). 1174–1181 , 11 , 1237–1248 (2003). 1237–1248 , Genes Dev. Genes , 412–419 (2012). 412–419 , in vivo in , 544–549 (2011). 544–549 ,

84

173 2

87

, 301–306 (2001). 301–306 , , 201–238 (2004). 201–238 , 306 Genes Dev. Genes

. , 5434–5444 (2004). 5434–5444 , 13 , 2579–2582 (1990). 2579–2582 , + J. Immunol. J. CD25 , 698–701 (2004). 698–701 , , 511–518 (2012). 511–518 ,

Science 20 108 Eur. J. Immunol. , 2096–2109 (2006). 2096–2109 , + Immunity , 20060–20065 (2011). 20060–20065 , regulatory T cells induced by induced cells T regulatory

16

Science 329

, 295–300 (2002). 295–300 , Blood Science 171 Immunity Nature , 89–93 (2010). 89–93 ,

, 802–811 (2003). 802–811 , 105 24 Cell

120 296 , 53–64 (2006). 53–64 ,

, 11909–11914 ,

31 332 335 149

1439–1448 , 1876–1880 ,

21 , 1141–1149 687–696 , 229–233 , 467–482 , , 817–830 α in vivo in β J. Exp. J. β T-cell - and - .

66. 65. 64. 63. 62. 61. 60. 59. 58. 57. 56. 55. 85. 84. 83. 82. 81. 80. 79. 78. 77. 76. 75. 74. 73. 72. 71. 70. 69. 68. 67. 54. 53.

oe, .. Zun, . custo o a ucinl cl rcpo during receptor cell T functional a of Acquisition Y. Zhuang, & M.E. Jones, selection during expression P.CD69 Kisielow, & B.H. von, M., of Dessing, W., Swat, selection negative and Positive K.A. Hogquist, & S.C. Jameson, T.K., Starr, P.A.Sharp, & C.B. Burge, G.X., Zheng, J.R., Neilson, miRNA of regulation Dynamic J. Lovén, Z. Nie, neutrophils. in mechanisms death cell of Peculiarities H.U. Simon, & B. Geering, B.M. Iritani, I. Maillard, M. Ciofani, complex. Mad(Mxi1)-Sin3 the by Repression R.A. DePinho, & N. Schreiber-Agus, Myc by transformation and regulation Transcriptional M. Eilers, & S. Adhikary, Tullai,J.W. S. Yu, system. immune the in XCR1 Rudolph, B., Hueber, A.O. & Evan, G.I. Reversible activation of c-Myc and in thymocytes XCL1 Y. Takahama, & Y. Lei, emigration before subsets thymocyte mature the of F.Expansion Vasseur, into & C. Pénit, Thpok-ing differentiation: lineage CD4–CD8 R. Bosselut, & L. Wang, and nectin with interact that Receptors M.J. Smyth, & D.M. Andrews, C.J., Chan, A. Liston, progenitor lineage-committed T a of Identification H. Boehmer, von & A. Krueger, of selection Negative M.M. Davis, & J.D. Altman, B.P., Trenchak, K.K., Baldwin, of deletion Clonal K.A. Hogquist, & M.S. T.A.,McCaughtry,Wilken,T.M., Baldwin, C. Viret, Surh, C.D. & Sprent, J. T-cell apoptosis detected J.P.Meerwijk, van P.Maliekal, reduce diabetes and Starvation R.A. Harris, & K.M. Popov, P., Wu, B., Huang, T.B.Meissner, G. Bain, R.R. Rivera, hi SH, rgt JB, ebr SA & oe MD Mc rti i stabilized is protein Myc M.D. Cole, & S.A. Gerber, J.B., Wright, S.H., Choi, Dose, M. lmhct dvlpet s nocd y E ad 2 tasrpin factors. transcription E2A and HEB by enforced is development lymphocyte T CD4 of maturation and cells. T (2007). development. cellular of stages ordered in expression (2012). cells. stem embryonic Differ. Death Cell (2002). by the Myc antagonist and transcriptional repressor Mad1. (2006). 2239–2245 vivo in differentiation. thymocyte during Bioessays proteins. cells. cancer in complex ligase (2010). E3 1236–1241 novel a of suppression by progression. n ucin n gnmc architecture. genomic (2007). and function in malignancy. and (2012). development cell T in roles opposing apoptosis and proliferation induces Oncogene and selection positive enhances Infect. periphery. the to nucleus. cancer. of immunotherapy and Immunol. immunosurveillance Opin. Curr. the in proteins nectin-like (2007). profiling. mRNA by analysis genomic genes: susceptibility blood. adult in development. thymic (1999). throughout occurs cells T medulla. the of involvement Med. no with cortex the in occur can thymocytes selection. negative intrathymic thymus. the in selection repertoire. family. the of members two synthase, glucose-1,6-bisphosphate kidney. and heart rat in phosphatase dehydrogenase Diabetes pyruvate of amount the genes. I class MHC cascade. MAPK Ras-ERK Id3. protein, Immunity

205

is absolute and independent of the pre-T cell receptor. cell pre-T the of independent and absolute is 14 J. Biol. Chem. Biol. J. et al. et t al. et Annu. Rev. Immunol. Rev. Annu.

et al. J. Immunol. J.

52 Nat. Rev. Mol. Cell Biol. Cell Mol. Rev. Nat.

t al. et

, 2575–2584 (2008). 2575–2584 , t al. et , 262–267 (2012). 262–267 , 27 20 et al. et t al. et 19 J. Exp. Med. Exp. J. et al. et et al. et t al. et , 1371–1376 (2003). 1371–1376 , et al. et et al. et c-Myc is a universal amplifier of expressed genes in lymphocytes and lymphocytes in genes expressed of amplifier universal a is c-Myc Blood , 860–870 (2007). 860–870 , , 808–818 (1998). 808–818 , et al. et , 1891–1900 (2000). 1891–1900 , h TF1 n LF1 rncito fcos ae oprtv and cooperative have factors transcription LEF-1 and TCF-1 The Immunity c-Myc mediates pre-TCR-induced proliferation but not developmental euain f h hlxlo-ei poen, 2 ad d, y the by Id3, and E2A proteins, helix-loop-helix the of Regulation t al. et Impairment of organ-specific T cell negative selection by diabetes by selection negative cell T organ-specific of Impairment Immunity rl fr cesblt t sl-etd-efMC opee in complexes self-peptide-self-MHC to accessibility for role A eiiig lbl ee xrsin analysis. expression gene global Revisiting The requirement for Notch signaling at the at signaling Notch for requirement The Immediate-early and delayed primary response genes are distinct are genes response primary delayed and Immediate-early Molecular identification of mammalian phosphopentomutase and phosphopentomutase mammalian of identification Molecular J. Immunol. J.

biaoy oe o coeaie inln b peTR n Notch and pre-TCR by signaling cooperative for role Obligatory Thymocyte selection is regulated by the helix-loop-helix inhibitor helix-loop-helix the by regulated is selection Thymocyte 18

Modulation of T-lymphocyte development, growth and cell size cell and growth development, T-lymphocyte of Modulation et al. et 108 Proc. Natl. Acad. Sci. USA Sci. Acad. Natl. Proc. L fml mme NR5 s tasrpinl euao of regulator transcriptional a is NLRC5 member family NLR , 1457–1469 (2011). 1457–1469 ,

183 24 282 Cell

+ , 2669–2677 (2006). 2669–2677 , Quantitative impact of thymic clonal deletion on the T cell T the on deletion clonal thymic of impact Quantitative

12 8 185 , 246–251 (2012). 246–251 ,

Nature + 26 Nat. Immunol. Nat. , 2903–2910 (2009). 2903–2910 , , 17–26 (2000). 17–26 , thymocytes.

, 31844–31851 (2007). 31844–31851 , 151 , 105–116 (2007). 105–116 , , 377–383 (1997). 377–383 ,

159 21 J. Immunol. J. , 68–79 (2012). 68–79 ,

372 , 139–176 (2003). 139–176 , J. Immunol. J.

, 4848–4856 (1997). 4848–4856 , 6 , 635–645 (2005). 635–645 , , 100–103 (1994). 100–103 , Eur. J. Immunol. J. Eur.

2 , 165–171 (2001). 165–171 , . il Chem. Biol. J.

166

172

in situ , 4429–4437 (2001). 4429–4437 , 107 , 5230–5239 (2004). 5230–5239 , . Immunol. J. , 13794–13799 (2010). 13794–13799 , during positive and negative ee Dev. Genes e c r u o s e r

EMBO J. Immunity 23 α

-D-phosphohexomutase β eoe Biol. Genome , 739–746 (1993). 739–746 , 282 -selection checkpoint -selection Cell J. Exp. Med. Exp. J.

23981–23995 ,

ee Dev. Genes 21 163 151

37 21 , 4820–4830 689–698 , 813–826 , 476–482 , 578–589 , Microbes

n vitro in 8 . Exp. J. R12 ,

203

24 13 . , ,

© 2013 Nature America, Inc. All rights reserved. of Massachusetts of Medical Massachusetts School, Worcester, USA. Massachusetts, University, Boston, USA. Massachusetts, Science Department, Brown University, Providence, Rhode Island, USA. Biomolecular Medicine, New York University School of Medicine, New York, New York, USA. Immunology, Washington University, St. Louis, Missouri, USA. of California San Francisco, San Francisco, California, USA. and Women’s Hospital, Boston, USA. Massachusetts, California, USA. 10 Kutlu Elpek J Derrick Rossi Michael Emmanuel Gundula Min- Adriana Francis Kim Daphne Koller The complete list of authors is as follows: Adam J Best e c r u o s e r 14 Division Division of Biological Sciences, University of California San Diego, La Jolla, California, USA.

L O Dustin rtiz- 12 L 25 14 Broad Broad Institute and Department of Biology, MIT, Cambridge, USA. Massachusetts, Gautier , , Angelique Bellemare-Pelletier , , Tata Nageswara Rao O 11 L 23 o opez , , Tal Shay , , Nidhi Malhotra 19 15 , , Susan A Shinton , Charlie , C Charlie Kim 16 1 , , Diane Mathis , 17 , Claudia , JakubzickClaudia 12 , , Aviv Regev 23 Program Program in Molecular Medicine, Children’s Hospital, Boston, USA. Massachusetts, 24 15 14 , , Katelyn Sylvia 14 1 20 , , , , Amy , , Christophe Benoist Joslin Joslin Diabetes Center, Boston, USA. Massachusetts, L , , Richard R Hardy ewis ewis 12 16 , , Nadia Cohen Icahn Icahn Medical Institute, Mount Sinai Hospital, New York, New York, USA. 18 W Department Department of Medicine, Boston University, Boston, USA. Massachusetts, L 16 25

25 agers L , , Gwendalyn J Randolph , Deepali Malhotra , Deepali Dana-Farber Dana-Farber Cancer Institute and Harvard Medical School, Boston, USA. Massachusetts, anier 22 Department Department of Biomedical Engineering, Howard Hughes Medical Institute, Boston 24 14 , , Joonsoo Kang 15 , , Tracy Heng 10 , , Jennifer Miller , , Jamie Knell 13 20 , , Patrick Brennan 1 , , David , , Natalie A Bezman 20 Fox Fox Chase Cancer Center, USA. Pennsylvania, Philadelphia, 11 1 25 Computer Computer Science Department, Stanford University, Stanford, L advance online publication online advance , , Jeffrey Ericson & Shannon Turley aidlaw 24 10 13 , , Taras Kreslavsky , , Ananda Goldrath Division Division of Rheumatology, Immunology and Allergy, Brigham 16 16 , , Brian Brown , 17 15 21 , , Paul Monach 13 Department Department of Microbiology & Immunology, University , , Jim Collins , , Michael Brenner 15 , , Joseph C Sun 1 , , Katherine Rothamel 24 25 16 Department Department of Pathology, University 25 , , Miriam Merad 22 10 18 , , Anne Fletcher , , Roi Gazit , , Vladimir Jojic , , David A Blair

17 nature immunology nature Department Department of Pathology & 19 15 13 Skirball Skirball Institute of , ,

23 ,

16 25 1 21 , 19 , 11 Computer Computer , , ,

© 2013 Nature America, Inc. All rights reserved. and resuspended in 1 1 in resuspended and were washed Cells Probes). Molecular DAPI (4,6-diamidino-2-phenylindole; in permeabilization/wash solution, then were stained for 10 min with 2.5 according to manufacturer’s were Cells (BD washed Biosciences). instructions reagent in fixation/permeabilization and fixed were washed then above), fied identi (antibodies proteins surface cell conjugatesto antibody with labeled RNA measurement. gated were cells electronically. out lineage-positive remaining the and ‘cocktail’ staining to the added was streptavidin Fluorochrome-conjugated cells. lineage-positive of depletion bead magnetic and (Dynal/Invitrogen) beads magnetic M-280 119; all from Biolegend), followed by incubation with streptavidin-conjugated (TER- anti-Ter-119 and (RB6-8C5) anti-Gr-1 (PK136), anti-NK1.1 (N418), anti-TCR (53-6.7), anti-CD8a (RM4-5), anti-CD4 biotinylated with suspensions of cell staining by cells lineage-negative for enriched were thymocytes total lations, are populations specific of available on the ImmGen website.sorting For the the isolation of DNfor thymocyte subpopu used combinations marker The (BD). II FACSAria and FACSAria a on sorted were Cells above). (identified ImmGen the to ( protocol sorting according ImmGen standardized prepared were cells sorting, cell For Star). (Tree software FlowJo with by analysis followed suspensions, cell of stained analysis rameter An LSR were II used. and FACSAria for were multipa used (BD) eBioscience IKZF2, NR4A1 and Bcl-2, Foxp3 fixation and permeabilization solutions from with °C 4 at of c-Myc, in Perm/Washdetection min diluted buffer. For antibodies intracellular 30 for stained then were Cells Pharmingen). (BD buffers and then Perm/Washwere and with fixed Cytofix/Cytoperm made permeable above), (identified markers surface to antibodies with labeled were cells ing, stain intracellular For above). (identified antibodies with °C 4 at min 20 for isolation. and cell cytometry Flow Biosciences). BD from all (J112-906; Ser811 and Ser807 at phosphorylated Rb to mAb and (K10-895.12-50) Ser529 at ylated NF- to mAb (N7-548), Ser236 and 235 Ser at S6 phosphorylated to mAb (47), Tyr700 at phosphorylated c-Cbl to mAb (J141-668.36.58), Tyr128 at phosphorylated SLP-76 to mAb (J96-1238.58.93), Tyr226 at ylated phosphor Lat mAb to at Tyr352 Tyr319 (17a), and phosphorylated Zap70 to mAb CD3 to (IM7), CD44 to and mAb to CD19 (1D3), mAb to TCR Signaling); Cell from all (236B4; Thr46 and Thr37 at phosphorylated 4EBP1 I to mAb Tyr397 (3283), at (27B7), FAK to phosphorylated antibody Thr334 polyclonal rabbit at phosphorylated MAPKAPKII to mAb (197G2), Tyr204 and Thr202 at phosphorylated Erk1/2 to mAb Biolegend); from all (53-7.3; as titrated and described prepared were of cytometry conjugates mass for Primary antibodies Signaling). following the Cell from all (54D2; RPS6 to mAb and (D84C12) c-Myc to mAb (5A1E), caspase-3 cleaved to mAb (12.14), NR4A1 mAb to (22.F6), mAb to IKZF2 (BCL/10C4), mAb and to Bcl-2 eBioscience); all 29F.1A12; from mAb Biolegend); to CD8 (anti-PD-1; CD279 to mAb (53-7.3), CD5 to mAb (H1.2F3), (Ter-119), mAb H-2K to Ter-119 to mAb (RB6-8C5), Gr-1 to mAb (PK136), -NK1.1 to mAb (N418), TCR to mAb (H57-597), mAb TCR to (RM4-5), mAb CD4 to (PC61), mAb CD25 to (IM7), CD44 to mAb (2B8), c-Kit to mAb (1D3), CD19 to (mAb) antibody monoclonal etry: Antibodies. described been have Bim or MHC in deficient Mice protocol. operating standard ImmGen to according arrival of week 1 within mice from obtained were Cells Committee). Use and Care Animal Institutional School Medical Harvard the of 02954 (protocol conditions pathogen–free specific in tained Mice. METHODS ONLINE doi: for Y pyronin scales on linear with an (BD) were LSRII acquired then 10 min, κ B 10.1038/ni.2590 α (L35A5), mAb to CREB phosphorylated at Ser133 (87G3) and mAb to to mAb and (87G3) Ser133 at phosphorylated CREB to mAb (L35A5), Male C57BL/6J mice 6 weeks of age (The Jackson Laboratory) were main β 8 el rp n srig SOP.pd sorting and prep Cell 7 (H57-597), anti-TCR (H57-597), : mAb to CD4 (RMA4-5), mAb to CD8 (53-6.7) and mAb and to CD5 mAb (53-6.7) to CD8 : mAb (RMA4-5), to CD4 The following monoclonal antibodies were used for were cytom used flow antibodies monoclonal The following For were pyronin thymocytes isolated Y freshly staining, µ g/ml pyronin Y (Sigma Aldrich) and incubated for for incubated and Aldrich) (Sigma Y pyronin g/ml b γ (AF6-88.5), mAb H-2D to (AF6-88.5), δ (GL3), mAb to CD11b (M1/70), mAb to CD11c CD11c to mAb (M1/70), CD11b to mAb (GL3), ζ α phosphorylated at Tyr142 (K25-407.69), mAb at Tyr142 (K25-407.69), phosphorylated (53-6.7) and (53-6.7) mAb to all CD127 from (A7R34; γ δ (GL3), anti-CD11b (M1/70), anti-CD11c anti-CD11c (M1/70), anti-CD11b (GL3), For surface staining, cells were incubated were incubated cells staining, For surface β (H57-597), mAb to CD25 (3C7), mAb f http://www.immgen.o ) with the appropriate antibodies antibodies appropriate the with ) b (KH95), mAb to CD69 mAb CD69 to (KH95), κ rg/Protocols/ B phosphor B µ 8 g/ml g/ml 6 . β ------

Microarray hybridization and hybridization analysis. Microarray described as analyzed and mouse or cells OP9-DL1 IRES-GFP with cultures encoding secondary in placed sorted, c-Myc–IRES–GFP, retrovirus with infected mice, transgenic Sca1 lineage-negative from ated transduction. retroviral and cultures OP9-DL1 13-acetate). 12-myristate 2%). (phorbol PMA concentration of 10 ng/ml addition the 3 after min fixed were controls (final PMA paraformaldehyde of addition the by fixed were 24 the of before °C addition 37 at min 2 for incubated were and Biolegend) (37.51; CD28 6 taining con medium in stimulated were cells node lymph total cytometry, mass by time various at collected points after bystimulation For cell sorting. events of analysis were phosphorylation and Dynal) 1:0.4; T-activator; (cells/beads, mouse anti-28 Dynabeads and anti-CD3 with coated beads with together CD8 and stimulation. Cell (Invitrogen). facturer’s instructions manu to according chemistry click by detection eU for processed then and above) identified antigens (antibodies for stained were extracellular collected, eU. Cells mM 5 of presence in °C 37 at incubation of h 2 during thymocytes isolated in freshly measured was activity Transcriptional and DAPI channels. were used for analysis of gene-ontology categories enrichment. TCR TCR enrichment. categories gene-ontology of analysis for used tool were visualization and analysis enrichment gene-ontology GOrilla and Discovery) Integrated and Visualization Annotation, for (Database base data DAVID The bioinformatics (ribosome). GO:0005840 and (translation) for values expression each probe GO:0006412 categories in the gene-ontology normalized maximum row of averaging by calculated was index ‘translation’ the Likewise, signature. proliferation the in probe each for values expression The histones. ‘Proliferation’ index was by calculated detect averaging of row that maximum normalized probes as well as (kinetochore), GO:0000776 and (spindle) GO:0005819 (centrosome), GO:0005813 process), (DNA metabolic all of lists of GO:0006259 cycle), (cell merging GO:0007049 categories in by probes gene-ontology the generated was signature’) (‘proliferation probes the HierarchicalClusteringImage module. The list of 2252 proliferation-related with visualized and distance row as distance Euclidian with GenePattern for module HierarchicalClustering the with clustered were clusters resulting the within Probes metric. as distance the were used distances Euclidian centered. probes 2088 the in and mean- were values log2-transformed Expression resulting in the analysis. included applied, was populations more the or of fourfold any expression between their changed and subset one least at in 120 SD.htm the 1.3 ExpressCluster tool ( DN2b, DN3a, DN3b, DN3b-DN4, ISP, DP blast and small DP populations with MPP, LT-HSC,ST-HSC, DN2a, ETP, ETP-DN2a, included that done set data a was on clustering K-means GenePattern. of module HeatMapImage the with created was map heat distance HierarchicalClusteringViewer Euclidian The the GenePattern. of module with visualized clustering and hierarchical distance, Euclidian with clustering, hierarchical for used was platform analysis genomic GenePattern the from module HierarchicalClustering The edu/Lab ( tool PCA Population The probes). all of 1.8% ing represent were probes (463 removed by criteria, analyses these from these all CV < the 1 accros interpopulation and <0.65 CV intra-population < 0.35 or >0.65, of (CV) variation of coef ficient intra-population an (with probes Noisy populations. analyzed across variation highest in the with 120 probes of 15% than the only including greater and subsets any values expression for filtered sets data mean-centered log on done were calculation distance Euclidian and analysis analysis. Bioinformatics procedures. operating standard ImmGen to according analysis microarray and RNA for processing used was l ). Prefiltering on the probes that had expression values greater than than greater values expression had that probes the on Prefiltering ). MembersPges/SD.htm + µ T cells, sorted naive CD4 naive sorted cells, T g/ml of biotinylated anti-CD3 biotinylated of g/ml µ g/ml streptavidin. At various times after crosslinking, cells cells crosslinking, after times various At streptavidin. g/ml For gene-expression profiling of activated CD4 activated of profiling gene-expression For 1 Hierarchical clustering, principal-component principal-component clustering, Hierarchical 4 http://cbdm.hms.harv . l ) was used for principal-component analysis. analysis. principal-component for used was ) + c-Kit α β + T cells and CD8 and cells T T cell ImmGen data set) were removed were removed set) data ImmGen T cell + precursors isolated from from isolated precursors An Affymetrix MoGene An 1.0 ST Affymetrix array ε (145-2C11; Biolegend) and anti- and Biolegend) (145-2C11; ard.edu/LabMembersPg nature immunology nature http://cbdm N cls ee gener were cells DN3 + T cells were cultured cultured were cells T 2 .hms.harvard. -transformed -transformed Vav-Bcl2 + T cells cells T 90 88 es/ , , 9 8 – 1 9 ------

© 2013 Nature America, Inc. All rights reserved. mass cytometry. mass multidimensional by obtained data of analysis for used was Cytobank zation on a CyTOF (DVS) as described H double-distilled in times three 1.6% in 1× and PBS), in washed then (paraformaldehyde solution intercalator above), then were washed and for then incubated 20 min at room temperature (identified antigens to intracellular of antibodies a mixture with temperature at min 20 for methanol 4 °C. After the methanol was washed off, in cells were for stained 45 min at room permeabilized and washed were then =, above) (identified markers to a with room surface of temperature mixture antibodies procedures to published according cytometry. mass by events phosphorylation of Analysis DAVIDof tool Annotation KEGG of pathway and genes genomes) (Kyoto encyclopedia the to mapped were clusters K-means from genes signaling–related nature immunology nature 88 , 8 9 8 . 7 2 . Post-background substraction and normali 0 before multidimensional mass cytometry cytometry mass multidimensional 0 before 8 7 . Fixed cells were incubated for 45 . were min at incubated cells Fixed 92 , 9 3 with the Functional Functional the with Cells were stained stained were Cells -

93. 92. 91. 90. 89. 88. 87. 86.

Kanehisa, M. Kanehisa, genomes. and genes of encyclopedia Kyoto KEGG: S. Goto, & M. Kanehisa, E. Eden, of lists ranked in motifs Discovering Z. Yakhini, & Yogev,S. D., Lipson, E., Eden, W.,Huang, paths tools: B.T.enrichment Sherman, Bioinformatics R.A. Lempicki, & of analysis integrative and Systematic R.A. Lempicki, B.T.W.,& Sherman, Huang, S.C. Bendall, P. Bouillet, epne, ekct hmotss ad o rcue autoimmunity. preclude to and homeostasis, leukocyte responses, data sets. data Res. Acids lists. gene ranked in sequences. DNA lists. gene large Res. of analysis functional comprehensive the toward resources. bioinformatics (2009). DAVID using lists gene large continuum. hematopoietic (2011). human a across responses (1999). 1735–1738

37 , 1–13 (2009). 1–13 , et al. et

Nucleic Acids Res. Acids Nucleic 28 t al. et et al. et , 27–30 (2000). 27–30 , t al. et GOrilla: a tool for discovery and visualization of enriched GO terms GO enriched of visualization and discovery for tool a GOrilla: PLOS Comput. Biol. Comput. PLOS raottc c- rltv Bm eurd o cran apoptotic certain for required Bim relative Bcl-2 Proapoptotic KEGG for integration and interpretation of large-scale molecular large-scale of interpretation and integration for KEGG igecl ms ctmty f ifrnil mue n drug and immune differential of cytometry mass Single-cell BMC Bioinformatics BMC

40 , D109–D114 (2012). D109–D114 ,

3 , e39 (2007). e39 ,

10 , 48 (2009). 48 , Science a. Protoc. Nat. doi: 10.1038/ni.2590

332 uli Acids Nucleic Science 687–696 ,

4 44–57 , Nucleic

286 , CORRIGENDA

Corrigendum: The transcriptional landscape of αβ T cell differentiation Michael Mingueneau, Taras Kreslavsky, Daniel Gray, Tracy Heng, Richard Cruse, Jeffrey Ericson, Sean Bendall, Matt Spitzer, Garry Nolan, Koichi Kobayashi, Harald von Boehmer, Diane Mathis, Christophe Benoist & the Immunological Genome Consortium Nat. Immunol.; doi:10.1038/ni.2590; corrected online 13 May 2013

In the version of this article initially published online, the eighth and ninth author names were incorrect. Those should be Matthew H. Spitzer and Garry P. Nolan. The error has been corrected for the print, PDF and HTML versions of this article. © 2013 Nature America, Inc. All rights reserved. America, Inc. © 2013 Nature npg