© 2017 Nature America, Inc., part of Springer Nature. All rights reserved. equally equally to this work. San Diego, California, USA. California, USA. 1 investigate to analysis RT-PCR quantitative single-cell of use the work through has Published limitations to sought cells. these address individual among heterogeneity potential the masks which cells, of ory in the specification of terminal differentiation versus long-lived mem role a with pathways and factors transcription many identified have studies such infections, microbial of course the during of lymphocytes specification fate the of T lymphocytes regulation molecular the of standing clearance pathogen after tissues (T cells T localization L-selectin as such receptors and anatomical capacity their proliferative and their CCR7, (CD62L) cytokine and homing of expression (T cells T (T memory cells T memory central subsets, two into interleukin the (IL-7R) receptor of 7 expression low and KLRG1 receptor lectin-like tiated effector T cells are bycharacterized high expression of the killer memory cells that provide long-lived protection. Terminally differen minal cells effector that mediate acute host defense and self-renewing a to ter give rise naive T to lymphocytes responding infection, microbial response immune an During infection. lym microbial T to of phocytes responses the of hallmark a is fate cell of Heterogeneity and necessity of single-cell approaches. subsequently refined by epigenetic silencing of transcripts associated with memory lymphocytes, which highlights the power findings suggest a model for the of differentiation terminal effector cells initiated by an early burst of activity transcriptional and and identified previously unknown molecular determinants that controlled the fate of specification CD8 infection single-cell approach RNA-sequencing and analyzed individual CD8 immediate and durable protection. To elucidate the dynamic changes transcriptional that underlie this process, we applied a During microbial infection, responding CD8 John T Chang Bingfei Yu Boyko Kakaradov RNA sequencing CD8 Early and transcriptional epigenetic regulation of nature immunology nature Received 9 July 2016; accepted 13 January 2017; published online 20 February 2017; Department Department of Cellular and Molecular Medicine, University of California, San Diego, California, USA. Transcriptional profiling approaches have greatly advanced under 6 . However, most studies have been conducted on bulk populations RM in in vivo + 2 cells), do not circulate but instead remain in peripheral peripheral in remain instead but circulate not do cells), . . A third subset of memory cells, tissue-resident memory 3 , Ellen , J Ellen Wehrens 4 3 T cell revealed differentiation by single-cell , Division Division of Biological Sciences, University of California, San Diego, California, USA. 5 . Through comparison of the gene expression of CD8 . . Our analyses revealed a striking divergence transcriptional among cells that had undergone their first division 2 7 These These authors jointly directed this work. should Correspondence be addressed to G.W.Y. ( , 1 7 . Circulating memory T cells can be categorized categorized be can cells T memory Circulating . EM & Gene W Yeo 1 cells), distinguished by differences in their their in differences by distinguished cells), , 5

6 Department Department of Physiology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore. , , Janilyn Arsenio aDV A NCE ONLINE PUBLIC ONLINE NCE 3 . 3 , , Justine Lopez 1 , 4 , 5 , 7 2 CM , + 6 T lymphocytes into differentiate subsets heterogeneous that together provide , Christella , E Christella Widjaja cells) and effector effector and cells) A TION 2 , , Stephanie H Kim

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In contrast, induction of the memory program seemed to be associated differentiation. memory-cell with associated determinants molecular program was subsequently refined by selective epigenetic repression of the peak of the adaptive immune response. That initial transcriptional entiating along the terminal effector pathway and was extinguished by differ cells in upregulated markedly was regulators, chromatin with that have been linked to effector and memory cell differentiation, along tion and fate specification. The expression of many transcriptionfunc effector metabolism, regulation, factorscell-cycle including functions, of hundreds of genes encoding products involved in diverse biological revealed two distinct subpopulations distinguished by their expression ity among that lymphocytes had undergone their first division, which heterogene transcriptional andsubstantial Weunexpected observed wide changes in CD8 individual study here, we applied a scRNA-seq approach to analyze transcriptome- T biological processes, including development diverse of understanding the advanced substantially has that tool ful of pathways. molecular and genes discovery unknown as-yet the precluded analysis for differentiation in involved products encode to known already genes of pre-selection identified, been have differentiation during cells individual in expression gene infection bacterial to ing patterns of the gene-expression CD8 single + H T lymphocytes sequentially throughout the course of a viral Single-cell RNA(scRNA-seq) power a as emerged has sequencing Single-cell 17 17 subset of helper T cells doi:10.1038/ni.368 2 , 6 , , Zhaoren He 2 , Elina , I Elina Zuniga 2 4 Department Department of Medicine, University of California, San Diego, Institute Institute for Genomic Medicine, University of California, 8 [email protected] 1 , , Stefan Aigner 3 n vivo in 9 , , Ananda W Goldrath , , and innate immune responses + T cells as differentiated they 7 . Although dynamic changes in in changes dynamic Although . ) ) or J.T.C. ( + T lymphocytes. Our 6 1 + These These authors contributed , , Patrick J Metz 8 T lymphocytes respond T lymphocytes , , the pathogenicity of the [email protected] ticles e l c i rt A 3 ,

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© 2017 Nature America, Inc., part of Springer Nature. All rights reserved. Data are representative of two experiments. two of representative are Data with lower expression in Div1 in expression lower with Div1 cells, naive individual by Div1 in than cells Div1 in expression higher with genes indicate brackets row); per cell (one plot) ( cell. individual an represents symbol Each T and CD8 2 Figure previously to CD8 linked products the encoding at genes of subset Assessment a of cell. level per single-cell (TPM) reads million per transcript 1 analyses. in-depth further in duplicates of pairs 32 and samples sequencing unique 224 into separated libraries cell 288 single- we included measures, quality-control these undertaking ( effects batch of absence an and were on plates assessed separate sequencing to ensure reproducibility nature immunology nature b a We detected over 6,000 genes with a mean expression of at least least at of expression mean a with genes 6,000 over Wedetected + T cells (obtained as in in as (obtained T cells EM Div1 Div1 cells (green); inset, tSNE clustering analysis of division 1 cells in the plot below, identifying Div1 identifying below, plot the in 1 cells division of analysis clustering tSNE inset, (green); cells

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cells and Div1 and cells r b = 0.78 ( = 0.78 Generation and application of ‘early-state’ and ‘fate’ classifiers classifiers ‘fate’ and ‘early-state’ of application and Generation P , n d = 2.2 × 10 = 2.2 = 48) and T and = 48) ) Cross-validated scores of division 1 CD8 division of scores ) Cross-validated Day 4cells n Div = 34) n P = 36 cells) ( cells) = 36 1 = 4.8 × 10 = 4.8 ME M −6 ; TE black and purple dashed lines indicate the the indicate lines dashed purple and black Div1 EM ). Each symbol ( symbol Each ). b cells (orange shaded area, 95% confidence confidence 95% area, shaded (orange cells ) and fate classifier ( classifier fate ) and cells ( cells TE Memory effector Day −8 a ) and day 7 effector cells (yellow; (yellow; cells 7 effector day ) and MEM n ); Spearman correlation (monotonic), (monotonic), correlation Spearman ); 7 = 48) ( = 48) f cells (blue; (blue; cells Memory score based on

+ ‘fate’ classifier T lymphocytes ( T lymphocytes 0. 0. 0. 0. 0. 1. b d a 0 2 4 6 8 0 , c 0. Memory score distribution Memory score distribution c ) (all cells obtained as in as obtained cells ) (all n 0 0 1. 0. 0. 0. 0. 0. , 0. 0. 0. 0. 0. 1. e = 96 cells), which include include which cells), = 96 e 0 0 2 4 6 8 0 2 4 6 8 0 ) Application of early- of ) Application , + f 6 4 2 0 8 6 4 2 0 T lymphocytes as assessed assessed as T lymphocytes d ) represents an individual individual an ) represents Memory scorebasedon ) were trained; results results trained; ) were .2 early ‘state’classifier n = 24 cells) from from cells) = 24 ‘Fate’ classifier 0. + 'Early-state 0 4 T lymphocytes T lymphocytes Cells Cells classifier Effector Memor Div Div d ) on which which ) on 1 1 .6 TE ME 8 y M ' 0. 10 10 1 8

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lymphocytes isolated at all time points after infection (as in in (as infection after points time all at isolated lymphocytes ( patterns manner, the gene-expression in an unbiased to visualize, analysis clustering embedding’) borhood neigh stochastic (‘t-distributed tSNE unsupervised Weperformed 1 CD8 division among heterogeneity Molecular infection. microbial to response in differentiation CD8 individual by expression exhibited gene in heterogeneity substantial the captured analysis ( populations bulk of analyses by identical as interpreted been have otherwise would that point time same the from derived cells individual among observed including genes, many of expression heterogeneous example, For populations. bulk of analyses by discernable previously not were that expression others ( others port, ribosome biogenesis, cell division and mRNA processing, among molecular and cellular processes involving transcription, protein trans diverse in involved products encoding those for enrichment showed ( 1) (division division cell first their undergone had that lymphocytes analysis revealed two distinct subpopulations among single CD8 single among subpopulations distinct two revealed analysis T and clusters; separate own their naive the however,near cell point grouped a time from each few cells formed 7 day and 4 day at cells most ( population each within neity ( clusters distinct In this tSNE analysis, naive cells, T gone their first cell division exhibited considerable transcriptional transcriptional considerable exhibited division cell first their gone CD8 that demonstrated data these Div1 in than (ref. Div1 in expression similar transcription factors with associated memory lymphocytes either had ( such with associated including cells, previously not molecules encoding genes to lymphocytes with that memory factors ing have associated several previously been cells effector of reprogramming terminal and metabolic and signaling molecules previously associated with the differentiation expression of genes encoding transcription factors, cytokine receptors that the revealed analysis set of ogy genes upregulated by Div1 Div1 in Div1 in expression higher having genes 27 and cells Div1 in expression higher having genes 903 sion of 930 genes Div1 distinguished respectively. (‘MEM’), populations memory-cell and (‘TE’) populations effector-cell terminal to larities ‘Div1 subpopulations ( to to T be 1 most similar seemed cells division CD8 effector 7 day and 4 day to similar most be to seemed cells 1 division of lation to ensure that all sorted cells had been activated markers, other than by cell-surface high expression of of the activation expression marker CD44 phenotypic their by not and peak) CFSE second (the CFSE dye division-tracking the of dilution their of basis Fig. 2 Fig. Supplementary Table 2 Table Supplementary Fig. 2 Fig. Notably, our single-cell analysis also revealed patterns of gene gene of patterns revealed also analysis single-cell our Notably, In contrast, Div1 contrast, In expres the in differences that revealed analysis Gene-expression 1 5 CM a a ) and and ) TE Supplementary Table 3 Supplementary ). We emphasize that the division 1 cells were isolated on the on the isolated were 1 cells division We). the that emphasize ). We provisionally designated these two division 1 (Div1) (Div1) 1 division two these designated provisionally We ). cell populations ( populations cell cells ( cells aDV Tbx21 6 Tcf7 MEM , including including , A + Btg1 T lymphocytes, whereas the other subpopulation of subpopulation other the whereas lymphocytes, T NCE ONLINE PUBLIC ONLINE NCE Fig. 2 Fig. , , ) ( ) Fig. 2 Fig. cells ( cells Gzmb Fig. 2 Fig. MEM , which encodes an anti-proliferative molecule an anti-proliferative encodes , which Fig. 1b Fig. TE b a cells had higher expression of genes encod genes of expression higher had cells and and ’ and ‘Div1 and ’ , , Eomes ), suggestive of unique molecular homoge molecular unique of suggestive ), ). Notably, however, many genes encoding encoding genes many however, Notably, ). e Il7r Id3 ) or had higher expression in Div1 in expression higher had or ) TE Fig. 2 Fig. , c , , , , Supplementary Table 2 Table Supplementary cells and Div1 and cells ). Thus, our single-cell transcriptomic transcriptomic single-cell our Thus, ). S1pr1 Il7r + , , Supplementary Table 1 Table Supplementary ). Moreover, ). Div1 T lymphocytes throughout their their throughout lymphocytes T Id3 a CM , , + ). Strikingly, unsupervised tSNE tSNE unsupervised Strikingly, ). MEM and and Il2ra TE T lymphocytes that had under had that lymphocytes T A and and cells and T TION cells from Div1 Fig. 2 Fig. ’ on the basis of their simi their of basis the on ’ , , Foxo1 Klf2 Sell

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© 2017 Nature America, Inc., part of Springer Nature. All rights reserved. expression patterns of selected genes across inferred paths of differentiation for effector cells (orange), T (orange), cells effector for differentiation of paths inferred across genes selected of patterns expression T (yellow), 7 cells day (orange), CD8 all by row) per (one genes common 89 of Div1 in (as cells memory versus cells effector by differentially expressed (T T cells memory by expression their to relative Div1 4 Figure in as obtained cells (analyzing differentiation of stages intermediate subsequent, in cells of identity the predict systematically we could Wewhether to determine sought lations might represent cells that had to begun already diverge in fate. disparate gene-expression patterns suggested that these two subpopu had division first that their undergone cells of subpopulations distinct two that observation The differentiation of stages intermediate in cells of Identification lymphocytes. memory with associated previously products encoding many transcripts including discernible majority of differentially expressed genes upregulated by Div1 previously not was that heterogeneity nature immunology nature experiments. two of representative are Data trajectories). expression single-cell possible all from (bootstrapped interval confidence 95% areas, a c MEM TE Genes cells relative to their expression by Div1 by expression their to relative cells

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+ TTE/ TMEM T lymphocyte populations of naive cells (gray), Div1 (gray), cells naive of populations T lymphocyte –10 MEM MEM –5 15 10 0 5 –10 Zscan2 cells (left) and those expressed differentially by terminally differentiated effector T cells (T T cells effector differentiated terminally by differentially expressed those and (left) cells Btg1 Malat1 Fmo1 cells) (right) (all cells obtained as in in as obtained cells (all (right) cells) EM r = 0.78 ( = 0.78 –5 High cells (purple) (one cell per column); left margin (brackets), hierarchical clustering. ( clustering. hierarchical (brackets), margin left column); per cell (one (purple) cells Expression (logmeanratio) A 7 TION , with the vast vast the with , 0 Div1 Mcm3 Cbx3

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© 2017 Nature America, Inc., part of Springer Nature. All rights reserved. PRC2 complex) in single CD8 single in complex) PRC2 5 Figure studies published of results the given validation, functional and (H3K27me3) Lys27 at H3 histone of ylation trimeth mediating by repression gene mediates that PRC2 complex cells. We selected effector differentiating in repression epigenetic of possibility ( expression in division, decrease rapid a first by followed the at expression in increase substantial a exhibit to tended pathway terminal-effector-cell the along differentiating cells and ( differentiation of state pre-effector a or state pre-memory a either in being as classified cells 4 day in regulators putative these encoding genes of expression the of basis the on differentiation cell memory- or terminal-effector-cell of pathways infer to able were we points, time of at cells us identity the intermediate to enabled predict ( points time all across regulators putative these encoding genes the of expression single-cell clustering by patterns expression temporal ( specification fate cell respectively. differentiation, memory-cell or differentiation terminal-effector-cell of regulators encoding genes identify to cells cells and effector cells or common only to Div1 ( sets both ( cells ory mem versus cells effector by terminal differentially expressed genes Div1 versus Div1 by differentially expressed genes 930 of set the Among in as obtained cells (analyzing cells memory in expression Div1 in sion by Div1 differentially expressed genes of set the between commonality for searching by differentiation cell We next sought to previously regulators unknown of identify CD8 CD8 of regulators of Identification states. subsequent or preceding the on trained classifiers binary supervised of use the through predicted readily be 4 Tables ( states cell pre-memory or effector prediction pre-terminal the of for genes of sets non-overlapping largely used ( they cells intermediate of identity the on agreed classifier fate and classifier early-state the Notably, cells. 4 day same the lyze ana to ‘past’ the into deployed was cells, memory 40 day and cells s e l c i rt A  ( *** and ( symbol CD62L percent indicate (left) quadrants in Numbers left. at results of summary CD62L percent indicate (left) areas outlined to adjacent Numbers ( left. at results of summary right, (bottom); in (as ( each. in cells percent indicate (left) areas outlined to adjacent Numbers left. at results of summary right, margin); (left infection after CD45.1 gated ( TNF ( infection. after Ezh2 gated of Ezh2 of intensity fluorescence mean indicate (left) areas outlined to adjacent Numbers left. at results of summary right, IL-2R on (gated 1 cells division CD8 1 (Div1) division gated of vivo in T and cells b–d Fig. 4 Fig.

The analysis described above yielded 89 putative regulators of CD8 ) or four ( four ) or Supplementary Fig. 2 Fig. Supplementary fl/fl g h . Numbers adjacent to outlined areas indicate percent Ezh2 percent indicate areas outlined to adjacent . Numbers ), among CD45.1 among ), ; left) of gated IL-2R gated of ; left) c Cd4

d P Ezh2 ). Because application of the ‘future-past’ binary classifiers classifiers binary ‘future-past’ the of application Because ). Ezh2 regulates effector CD8 effector regulates Ezh2 and and – < 0.001 (Student’s two-tailed two-tailed (Student’s < 0.001 k EM Supplementary Table 6 Table Supplementary Fig. Fig. 4a +/+ ) represents an individual mouse; small horizontal lines indicate the mean (+ s.e.m.). NS, not significant ( significant not NS, s.e.m.). (+ mean the indicate lines horizontal small mouse; individual an ) represents MEM Ezh2 cells (key) (presented as in in as (presented (key) cells MEM fl/fl f + or or – (P14) CD8 (P14) 5 h f Cd4 ). Thus, intermediate states of differentiation could could differentiation of states intermediate Thus, ). , , Ezh2 g k cells ( cells cells and by terminal effector cells relative to their their to relative cells effector terminal by and cells , , which encodes a catalytic subunit of subunit the Polycomb a catalytic , encodes which ) Flow cytometry of cells as in in as cells of cytometry ) Flow ) or three ( three ) or b +/+ ). ). We common genes only to Div1 selected next fl/fl (WT) or or (WT) + Supplementary Table 7 Supplementary (donor) CD8 (donor) Cd4 Supplementary Table 2 Table Supplementary + cells as in in as cells α Cre i hi + ) mice per genotype in each ( each in genotype per ) mice ). We observed that genes expressed in in expressed genes that observed We ). + T lymphocytes (obtained as in in as (obtained T lymphocytes CD62L IL-2R α Ezh2 P14 (CD45.1 P14 lo or IL-2R or fl/fl + α + ), only 115 genes were shared by shared were genes 115 only ), lo T lymphocyte differentiation. ( differentiation. T lymphocyte e cells, and the mean fluorescence intensity (right) of intracellular IFN- intracellular of (right) intensity fluorescence mean the and cells, hi and IL-2R and Cd4 , analyzing staining of the DNA-intercalating dye 7-AAD and the apoptosis marker annexin V (AnnV) at 3 or 5 d 3 or at V (AnnV) annexin marker apoptosis the and 7-AAD dye DNA-intercalating the of staining , analyzing CD62L t + TE Fig. 1 Fig. -test). Data are representative of two experiments ( experiments two of representative are Data -test). T cell differentiation T cell α Cre cells relative to their expres to relative their cells j ) Flow cytometry (left) of division 1, day 4 and day 7 CD8 day 4 and day 1, division of (left) cytometry ) Flow hi (KO) P14 CD8 P14 (KO) + cells), day 4 cells, day 7 cells, T 7 cells, day 4 cells, day cells), ) ) CD8 c lo ). ( ). Fig. 4 Fig. or IL-2R or e 1 α MEM 7 d after infection, showing the frequency of cells (left) with intracellular expression of IFN- of expression intracellular with (left) cells of frequency the showing infection, 7 d after 6 ). We visualized their their We ). visualized b , for further analysis analysis further for , lo ) and the set of 834 834 of set the and ) ) Flow cytometry of gated CD8 gated of cytometry ) Flow + CD62L T cells, followed by infection of the host mice with LCMV-Armstrong and analysis of cells 7 d cells of analysis and LCMV-Armstrong with mice host the of infection by followed T cells, cells and memory d ); this raised the the raised this ); Supplementary Supplementary α f lo hi – CD62L + Fig. 3 Fig. i cells among cells as in in as cells among cells Fig. 1 Fig. , T cells in the spleen of wild-type (CD45.2 wild-type of spleen the in T cells k ), or are pooled from two independent experiments ( experiments independent two from pooled are or ), hi lo Fig. Fig. Fig. 4 Fig. TE cells among CD8 among cells cells (left) or Ezh2 or (left) cells b hi f ), yet yet ), ) separated as naive cells, Div1 cells, naive as ) separated cells cells cells (key), assessing Ezh2 expression. ( expression. Ezh2 assessing (key), cells a ) Expression of of ) Expression + + 1 TE T T ). ). d - - - -

by days 5 and 7 after infection than were control cells ( cells control were than infection after 7 and 5 days by CD8 Ezh2-deficient Armstrong. (CD45.2 (CD45.1 ( Ezh2-deficient cell–specific T the from Ezh2 ( level mRNA at the that leled paral level protein the at differentiation during expression Ezh2 of IL-2R IL-2R ‘pre-effector’ putative Ezh2 of expression ( their in protein pattern bimodal a exhibited division CD8 that observed we ing, find that with Consistent cells. effector terminal of differentiation Div1 ( CD4 of function and differentiation the in for Ezh2 role a critical suggesting inflammatory cytokines relative to that of control cells ( cells control of that to relative cytokines inflammatory 3b Fig. Supplementary T cells by chromatin immunoprecipitation followed by sequencing sequencing by followed immunoprecipitation chromatin by cells T (KLRG1 mapped we H3K27me3 peaks derived from the analysis analysis, of naive, terminal scRNA-seq effector our by detected genes on ~6,000 the Focusing cells. effector terminal of differentiation the promote catalyze repressive H3K27me3 marks on a set of key genes and thereby Div1 in Ezh2 of expression high the that hypothesized we repression, transcriptional mediates complex PRC2 the Because determinants memory-associated of silencing Epigenetic cells. effector terminal of role a ings suggested for critical Ezh2 in the regulating differentiation ( an in cells, like’ ‘memory- not but cells, ‘effector-like’ early affected ‘preferentially’ ( infection after 5 day by apoptosis increased with ated ( CD8 of Ezh2-deficient Notably, ( cells of the effector absence Fig. 5i Fig. Eed Supplementary Fig. 3c Fig. Supplementary CM lo We next generated Ezh2 IL-2R cells and T and cells + , , MEM lee ( alleles T lymphocytes (as in in (as T lymphocytes α Suz12 + e T cells. ( T cells. + lo , along with genes encoding other components of the PRC2 PRC2 the of components other encoding genes with along , – Ezh2 , assessed after 3 d of 3 d of after , assessed α hi T cells T hi + + k CD62L hi cells ( cells (right) among the CD8 the among (right) cells i. 5 Fig. ) recipient mice that we subsequently infected with LCMV- ) recipient infected mice that we subsequently CD8 ) IL-7R and and cells (bottom right) or IL-2R or right) (bottom cells aDV and and , , a Eed EM Ezh2 ), two experiments with four ( four with experiments two ), A Supplementary Figs. 3e Figs. Supplementary Fig. Fig. 5 17– k α hi NCE ONLINE PUBLIC ONLINE NCE b cells (above plots) among CD8 among plots) (above cells + ) Flow cytometry (left) of cells treated as in in as treated cells of (left) cytometry ) Flow Set , , in vitro in Ezh2 cls ( cells T lo ), with high expression and low expression in in expression low and expression high with ), cells, respectively cells, 1 Suz12 ) CD8 ) 9 fl/fl ), had higher expression in Div1 in expression higher had ), . MEM a Ezh2 + ) are deleted by Cre recombinase expressed expressed recombinase Cre by deleted are ) ), ), suggestive of a role for Ezh2 in regulating the fl/fl + T cells as in in as T cells T cells to undergo activation or proliferation or proliferation T activation to cells undergo ) and exhibited an impaired ability to secrete to secrete ability an impaired ) and exhibited cells, Div1 cells, and and + model of CD8 of model , + Cd4 Cd4 ) recipient mice given adoptive transfer of transfer adoptive given mice ) recipient a d fl/fl T cells and total memory (CD44 memory total and cells T ) that had undergone their first cell division division cell first their undergone had ) that ). However, Ezh2 deficiency was associ was deficiency Ezh2 However, ). upeetr Fg 3a Fig. Supplementary + in vitro in γ Cd4 Set T cells that had undergone their first first their undergone had that cells T promoter) and adoptively transferred transferred adoptively and promoter) d Cre ( Fig. 5 Fig. α ) Flow cytometry (left) of naive cells, cells, naive of (left) cytometry ) Flow f ) or TNF ( TNF ) or hi (which encode components of the the of components encode (which o cnrl ( control or ) + Cre CD62L T cells were much less abundant abundant less much were cells T TE e e culture with IL-2 (top) or IL-15 IL-15 or (top) IL-2 with culture + 7 d , P14 mice (in which ; right, summary of results at left. left. at results of summary ; right, cells, day 4 cells, day 7 cells, T 7 cells, day 4 cells, day cells, A T lymphocytes. ( T lymphocytes. ( j P α ). ). ). TION Fig. Fig. 5 Fig. Fig. 5 < 0.05); * < 0.05); lo + CD62L events. ( events. and and g + lo b ). ( ). T cell differentiation cell T cells and ‘pre-memory’ ‘pre-memory’ and cells

, e c c h ) or three ( three ) or ) ) was not due to a failure ). ). Moreover, the kinetics nature immunology nature 4 + ) Flow cytometry (left) of (left) cytometry ) Flow T lymphocytes (as in in (as T lymphocytes ). Together these find Together these ). hi cells (top left). Each Each left). (top cells Ezh2 e P ) Absolute number number ) Absolute ≥ 0.05, ** ≥ 0.05, ) into wild-type wild-type into ) c fl/fl i ) Flow cytometry cytometry ) Flow ) Flow cytometry cytometry ) Flow TE d TE ) mice in each each in ) mice cells than in in than cells Cd4 lox Fig. 5 Fig. cells would would cells Fig. 5 Fig. i ; right, ; right, Fig. 5f Fig. P-flanked P-flanked +/+ hi γ P ( < 0.01 < 0.01 ) CD8 ) h f ) P14 P14 ) ) ) or e ) and and ) and and 20 , CM g a , 2 ); ); ). ). + 1 - - - -

© 2017 Nature America, Inc., part of Springer Nature. All rights reserved. lower gene expression, whereas those in memory cells exhibited exhibited cells memory to relative such coverage coverage, in H3K27me3 in losses considerable those whereas expression, with gene correlated lower that coverage H3K27me3 in gains significant ited (ChIP-seq) nature immunology nature i h e d a + IL-15 Day Day + + IL-2 CD45.1 Ezh2 expression (TPM) + 6 10 20 30 40 50

5 3 CD8 T cells (×10 ) Ezh2 0 0 1 2 3 4 7-AAD 10 10 10 10 10 10 10 10 10 10 10 10 0 2 4 6 10 7-AAD 2 10 10 10 10 10 10 10 10 2 CD8 0 2 4 6 0 2 4 6 0 10 . The promoter regions in terminal effector cells exhib cells effector terminal in regions promoter The . 0 2 4 6 0 2 4 6 WT 10 AnnV 10 0 63.5 Annexin 55.7 2 87.5 Naive 0 1,131 73.4 10 10 10 *** 2 4 2 WT 10 WT 10 10 Naive V 4 6 KO 4 20.7 14.6 20.7 20.5 10

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+ MEM 2

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– *** AnnV 4 A 10 15 20 25

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lo hi hi lo 10 10,000 15,000

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WT 4

CD62L cells (%) CD62L cells (%) CD62L cells (%) 10 WT WT Day 100 Ezh2 0 20 40 60 20 40 60 80 ticles e l c i rt A 0 1 2 3 4 5 0 0 7 IL-2R IL-2R 10

*** T WT 1 WT and WT CM *** ** 10 * T α α * 2 * hi lo KO EM KO CD62L CD62L KO KO 9 10 KO KO ): ): among 3 10 lo hi 4  © 2017 Nature America, Inc., part of Springer Nature. All rights reserved. ( processes) biological other in involved products encoding genes of sets in abundance to their (relative apoptosis and Wnt signaling, receptor binding, phosphoinositide binding, protein cytoskeletal tion, regula transcriptional involving processes in involved products ing encod those for enrichment showed Ezh2 by targeted genes of set Table 10 Supplementary ( H3K27me3 by marked genes 261 and Ezh2 by bound CD8 differentiating cells. memory of that for than might be more for crucial of the cells effector differentiation terminal silencing that epigenetic suggested This were by marked H3K27me3. s e l c i rt A  ( above initially described as Figure 6 Figure of three experiments ( experiments three of * Fig. Div1 in than 4 cells day in genes) 219 (Gain; expression higher or genes) 1,492 (Loss; expression ( ( scRNA-seq by detected genes 6,000 the of TSS) the around kb +1 to kb −1 (from regions promoter proximal at (key) both or (PolII) II ( cells. naive in than (Mem) cells memory or (Eff) cells effector in expression lower with ( gray). axis; vertical (right coverage of proportion or black) axis; vertical (left regions covered of quantification as ( set data scRNA-seq the in detected genes 6,000 the ( fates. those into differentiate cells naive as (blue) losses or (red) gains coverage as (key) presented are results (right); cells memory versus cells naive in or (left) cells effector terminal versus cells naive in (TSS), site start transcription the from (columns) distances various at (rows) genes various ( cells. effector terminal of differentiation e Supplementary Table11 Supplementary P

) Frequency of genes with a change in TPM among those with lower lower with those among TPM in a change with genes of ) Frequency b < 0.01 and ** and < 0.01 We next investigated binding of Ezh2 and H3K27me3 marks in in marks H3K27me3 and Ezh2 of binding investigated next We Covered regions/200 bp a 1 Genes 1,200 1,600

), with (Ezh2 bound) or without (Unbound) binding of Ezh2 (key). (key). Ezh2 of binding (Unbound) without or bound) (Ezh2 with ), –1,000 Base pairsfromTSS 400 800

b –800 terminal effector 0 ) Peak H3K27me3 coverage around the transcription start site for for site start transcription the around coverage H3K27me3 ) Peak –600 Increased epigenetic repression of genes expressed during the the during expressed genes of repression epigenetic Increased Naive –400 Naive vs –200 d 0 ) ChIP analysis of the binding of Ezh2 or RNA polymerase polymerase RNA or Ezh2 of binding the of analysis ) ChIP Effector 200 400 P 600 < 0.001 (Fisher’s exact test). Data are representative representative are Data test). exact (Fisher’s < 0.001 800 Memory 1,000 a +

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1,000 - - with a critical role in differentiating memory CD8 memory in role differentiating a critical with as well as as such homing, and survival cell T control that encodes a branched-chain aminotransferase isoenzyme tion factors, such as as such factors, tion transcrip memory-associated encoding genes included targets Ezh2 the input DNA control ( of that than Ezh2 with association greater exhibited cells effector of differentiation the to not but cells memory of differentiation the to linked been have that products encoding genes many that revealed This analysis cells. of but cells memory not the differentiation during gets whose expression during decreased the differentiation of effector pathways. disparate these along progressing cells in Ezh2 of expression due in to cells, of part differential memory differentiation during of the cells but differentiation effector terminal not during the repressed selectively of were set being same that the genes possibility and distribution; bottom, of shifting no by indicated (as cells memory and top, by a rapid loss by day 4 after infection ( infection after 4 day by loss rapid a by followed division first the at increase early substantial a with by Ezh2, targeted genes effector memory-cell-associated of those differentiating resembled in cells genes these of patterns expression The Myc ( molecules ing ( including factors transcription encoding differentiation, those effector-cell with products associated encoding genes previously of patterns expression the assessed also cells. these in expression Ezh2 of levels distinct to owing resulted in part from the presence or of absence repression epigenetic in cells effector and terminal differentiating cells memory might have Thus, the unique expression patterns of memory-cell-associated Ezh2. genes by repression transcriptional H3K27me3-mediated for role a 12 Tables Supplementary such genes, as memory-cell-associated of number a including genes, of diminished mRNA many from expression and increased coverage with H3K27me3 associated was deficiency Ezh2 that observed we Il7r as such genes, memory-cell-associated for observed was expression T mature became cells the as expression increased or stable by followed division first a distinct expression pattern bycharacterized a modest increase at the revealed cells memory of in same genes differentiating those analysis contrast, In cells. effector of differentiation the in genes associated memory-cell- of these products for the role a possible suggested this ( decrease rapid a by followed division first the at increase like others, ing division; the first as ( T fate ‘decisions’cell as such signaling, terminal terminal effector cells (as indicated by shifting of distribution; of differentiation the to during more exhibit repression tended genes cell and memory-cell differentiation revealed that H3K27me3-marked terminal-effector- during genes unmarked and H3K27me3-marked Div1 in than cells 4 day in expression higher with those than binding Ezh2 Div1 in than cells 4 day in lower Fig. 7 Fig. We investigated that possibility by analyzing a subset of Ezh2 tar Ezh2 of subset a analyzing by possibility that Weinvestigated In parallel with our analysis of memory-cell-associated genes, we we genes, memory-cell-associated of analysis our with parallel In Foxo1 Eomes , , ), along inferred terminal-effector-cell or memory-cell pathways. or memory-cell ), along terminal-effector-cell inferred Lef1 b TE and and and and Supplementary Fig. 5a Fig. Supplementary and and , , cells ( cells Opa1 Tcf7 aDV Supplementary Table 10 Supplementary Tcf7 Supplementary Fig. 5b Fig. Supplementary Bcl2 A and and Il2ra , which encodes a regulator of mitochondrial fusion fusion mitochondrial of regulator a encodes which , NCE ONLINE PUBLIC ONLINE NCE Fig. 6 Fig. CM , underwent a rapid decrease in expression follow expression in decrease rapid a underwent , , that were not targeted by Ezh2 ( Ezh2 by targeted not were that , Smad2 Klf2 and and 23– cells or T or cells Tcf7 e 2 ). Comparison of the expression patterns of of patterns expression the of Comparison ). Fig. Fig. 7 5 ( and and ; metabolic regulators, such as such regulators, ; metabolic Akt1 , whose product has been linked to CD8 to linked been has product whose , Fig. 7e Fig. and and b Eomes; ) and metabolic regulators ( regulators metabolic and ) EM and 13 Eomes ) than during the differentiation of of differentiation the during than ) cells ( cells TE – h ), consistent with the proposal of of proposal the with consistent ), cells were more likely to exhibit exhibit to likely more were cells Supplementary TableSupplementary 10 , , A molecules that mediate TGF- mediate that molecules Supplementary Figs. 6 Figs. Supplementary TION , ). Several of these genes, such such genes, these of Several ). c Batf and Fig. 7 Fig. ). These findings raised the the raised findings These ). Fig. 7 Fig.

, , Id3 Irf4 nature immunology nature c ). A similar pattern of pattern similar A ). , underwent an , initial underwent i and and ); in contrast, these these contrast, in ); Klf2 + T lymphocytes Fig. 7 Fig. Tbx21 (refs. 27–29); 27–29); (refs. 2 6 Bcat1 ; ; and factors d Hif1a ). Finally, ). ), signal ), Fig. 7 Fig. Fig. 7 Fig. ). ). These Fig. Fig. 7 , which , which – 8 and and and and c 3 a a ); ); β + 0 - - - - , ,

© 2017 Nature America, Inc., part of Springer Nature. All rights reserved. expression patterns (as in in (as patterns expression CD8 to linked products ( (blue). H3K27me3 at H3K27me3 and Ezh2 of binding the of in as obtained cells in assessed (key), H3K27me3 by targeted) (Not marked not or (Targeted) marked T (effector, pathways differentiation inferred three the in products encoding genes of values) 7 Figure (ratio as in in as (ratio in Ezh2, by targeted genes cell-associated ( (right). regions or (left) peaks binding H3K27me3 of quantification as presented 2)), (1, each replicates biologic in genes individual for ( trajectories). expression single-cell possible all from (bootstrapped interval confidence 95% areas, shaded (key); cells nature immunology nature h d g c a Lef1 Bcl2 Klf2 Bach2 expression (TPM) expression (TPM) expression (TPM) expression (TPM) Ezh2 mediates the effector differentiation of CD8 of differentiation effector the mediates Ezh2 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 Naive 10 Naive Expression (TPM) 3 4 5 6 7 8 9 0 1 2 3 4 5 6 7 g H3K27me3 coverage (%) Not targeted Targeted Smad2 ) plotted against RNA expression (of all genes) in in genes) all (of expression RNA against ) plotted Eomes 0.5 1.5 1. 2. Bach2 –100 Foxo1 Tcf12 –60 –20 100 0 0 Tcf7 Klf2 20 60 Div1 Div1 ld3 Naive

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ll7r 0 0 4 8 Div Div1 Input Cre expression (TPM) Chr3 Chr9 f d 1 Naive 0 1 2 3 4 5 6 7 8 9 ) H3K27me3 coverage in in coverage ) H3K27me3 ), in inferred paths of differentiation for effector cells, T cells, effector for differentiation of paths inferred in ),

CD8 Day Day 4 Div1 4

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 © 2017 Nature America, Inc., part of Springer Nature. All rights reserved. el, s t a be son ht T that shown been has it as cells, T whether elucidate and also question might this into insight additional provide probably should T T differentiating when unknown remains it but subsets, memory circulating both of progenitor common a represent might understood distinct molecularly T that appreciated been has it Although T as in as differentiating well cells, memory in those from distinct were that cells effector differentiating in sion expres of patterns suggested genes individual of ‘trajectories’ the of Visualization cells. memory or cells effector terminal of ferentiation dif the for pathways infer thereby and points time intermediate at ‘future-past’ binary classifiers enabled us to predict the identity of cells early states differentiation of The cellular these subsets. application of ory cells, we that hypothesized Div1 genes. specific few a of expression the in alterations nuanced more with associated be might program upregulation of hundreds of of genes, induction whereas the memory considerable involving burst transcriptional profound a with ciated asso were cells effector terminal of differentiation the in steps liest lower expression. These dichotomous patterns suggested that the ear Div1 Div1 Div1 by expression Div1 versus cells Div1 by differentially expressed genes of patterns expression the Moreover, cells. effector terminal of differentiation the in impor role tant but transient a have might factors transcription associated Div1 in expression Div1 in upregulated substantially were cells memory and cells effector both of differentiation the to linked tors fac transcription encoding genes Unexpectedly, and differentiation. metabolism translation, transcription, regulation, cell-cycle spanned Div1 by Div1 by expression higher had genes these of (97%) ity ‘Div1 called pro we visionally which identified, expressed subpopulations two genes the by of differentially hundreds with division, first their undergone had that cells among divergence transcriptional infection striking a viral revealed a of course the throughout CD8 of antigen-specific scRNA-seq ing CD8 of molecular specification of the regulation into insights new unknown gain and previously determinants discover molecular to sought we study, present the In DISCUSSION program. memory the of of chromatin regulators, followed by subsequent epigenetic upregulation repression the as well as fates, memory and effector the that promote products encoding genes of upregulation the includes that ity activ by that is a cells initiated rapid burst of transcriptional effector terminal of differentiation the for Together model a suggested mechanisms. findings these alternative by occur might during differentiation genes these of regulation the that suggested H3K27me3 by marked or Ezh2 by targeted not were genes effector-cell-associated ( proc differentiation the memory of during ess levels lower much at expressed were genes same s e l c i rt A 1 origin clonal common 0 EM On On the with basis and of effector similarities mem their molecular

cells diverge in fate. Future studies with more precise time points MEM MEM MEM cells were by also expressed Div1 cells. In contrast, the genes with higher expression by by expression higher with genes the contrast, In cells. 33 cells and encoded products with diverse functions that that functions diverse with products encoded and cells , 3 4 . Our data suggest the possibility that Div1 that possibility the suggest data Our . MEM MEM TE 31 cells were unique, in that the genes with higher higher with genes in the that were unique, cells cells had largely undetectable expression in in expression undetectable largely had cells , 3 TE 3 5 2 . el, hc sgetd ht memory-cell- that suggested which cells, , the ontology of these cells remains poorly poorly remains cells these of ontology the , cells’ and ‘Div1 and cells’ Fig. Fig. RM + cells are derived from Div1 from derived are cells TE T lymphocyte fates by perform by fates lymphocyte T RM 7 and Div1 i ). The observation that these these that observation The ). el ad T and cells + T cells derived sequentially sequentially derived T cells MEM CM TE CM cells, albeit with albeit cells, slightly cells and T and cells TE cells’. The vast major vast cells’. The n vivo in cells versus T versus cells cells relative to their their to relative cells MEM cells represented CM . Our analyses analyses Our . el sae a share cells TE CM EM cells than than cells MEM cells and and cells cells are are cells EM cells. cells. cells cells MEM TE ------

sion in Div1 in sion Div1 by differentially expressed differentiation by searching for commonality between the set of genes here. study our the in observed cells 1 division in heterogeneity transcriptional the mediating in division asymmetric signature transcriptional like effector- an with cells daughter to rise give division, asymmetric of T cells deficient in the atypical protein kinase PKC, a central regulator reported been has that pathways signaling kinase phosphatidylinositol-3-OH and mTOR Myc, the of distribution mitotic asymmetric the with sistent Div1 in programming metabolic Div1 of expression increased the in IFN- and IL-2 cytokines the via signaling promote could IL-2R receptors mitosis during cells daughter to unequally determinants certain apportion to lymphocytes T activated enables that mechanism conserved arily an evolution division, be asymmetric could factor One contributing question. open an remains division cell first the following observed tion of differentiating lymphocytes and was associated with more more with associated was and lymphocytes differentiating subpopula of tion distinct a in occur to seemed program memory the transcriptional of induction contrast, In lymphocytes. associated memory with transcripts of silencing epigenetic by refined and burst transcriptional by profound a and rapid initiated cells effector minal plasticity. of degree certain a cells effector on bivalent (H3K4me3 poised, a in remain might genes memory-cell-associated repressed that possible remains it However, genes. motion effector-cell-associated by into set program terminal-effector-cell-differentiation the enforce to serve might genes memory-cell-associated of repression the that suggested findings These Ezh2. by targeted selectively were effector terminal cells, transcription factors associated with the alternative differentiating memory fate In manner. state–specific cell T a in Ezh2 by differentially targeted be might fates alternative promote that factors transcription that demonstrating by observations those marks marks and repressive H3K27me3 subsets and during cell differentiation in their among deposition of permissive H3K4me3 differences substantial demonstrated have and CD8 memory and effector naive, of landscapes epigenetic process this in modifications histone and methylation DNA of importance the ing show studies published with appreciated, increasingly been has fate pathway signaling Notch the of components encoding those as well as molecules, pro-apoptotic encoding genes of repression H3K27me3-mediated through cells T CD8 effector human of survival and polyfunctionality the regulate CD8 in important regulators of functionally ofthe differentiation identifying approaches computational and experimental our of success the cells effector of terminal differentiation that candidate, showed We one metabolism. chromatin energy and proliferation, transcription, of structure, regulation spanned functions determinants molecular whose candidate 89 yielded approach This sets. sub memory-cell in expression their to relative subsets effector-cell We sought to identify previously unknown candidate regulators of regulators We candidate unknown previously to sought identify divergence transcriptional substantial the control factors Which In summary, our data suggest a model for the of differentiation ter A role for epigenetic regulation of determination of the CD8 + TE T cells. Consistent with our findings, Ezh2 has been shown to to shown been has Ezh2 findings, our with Consistent cells. T cells. Increased expression of genes encoding mediators of of mediators encoding genes of expression Increased cells. 1 1 . Asymmetric segregation of factors such as the cytokine cytokine the as such factors of segregation Asymmetric . aDV TE 37– A 42– cells and those expressed differentially by terminal- by differentially expressed those and cells Ezh2 NCE ONLINE PUBLIC ONLINE NCE α 3 4 n IFN- and 9 4 . Finally, the demonstration that activated CD8 activated that demonstration the Finally, . . Published studies have investigated the overall overall the investigated have studies Published . + H3K27me3 , encodes a product with a critical role in the the in role critical a with product a encodes , 4 γ 4 1 drn mitosis during R 0 . supports the possibility of a role for for role a of possibility the supports Il2ra + ) ) state MEM TE cells, moreover, would be con be would moreover, cells, A , , TION cells relative to their expres their to relative cells Stat5a 45 45 in in vivo , 4 , 4 6

6 and might thereby confer . . Our study has extended and and nature immunology nature , which demonstrated demonstrated , which 7 , Tbx21 11 , 3 6 , for example, example, for , observed in in observed γ and result result and + T cells cells T + T cell + + ------

© 2017 Nature America, Inc., part of Springer Nature. All rights reserved. 16. 15. 14. 13. 12. 11. 10. 9. 8. 7. 6. 5. 4. 3. 2. 1. reprints/index.htm R The authors declare no competing interests.financial J.A., C.E.W., Z.H., J.T.C. and G.W.Y. wrote the manuscript. J.A., C.E.W., S.A., P.J.M., B.Y., J.L. and S.H.K. performed experiments; and B.K., E.I.Z., B.Y. A.W.G., J.T.C. and G.W.Y. experiments designed and analyzed data; B.K., Z.H. and G.W.Y. performed computational analysis; J.A., C.E.W., E.J.W., Medical Institute (J.T.C.). AI113923 to E.I.Z.; DK007202 for C.E.W. and P.J.M.) and the Howard Hughes MH107367 to G.W.Y.; AI072117 and AI096852 to A.W.G; AI081923 and (DK093507, OD008469, and AI095277 to J.T.C.; NS075449, HG004659 and captures and sequencing. Supported by the US National Institutes of Health Core and Institute for Genomic Medicine Genomics Center for single-cell reading of the manuscript, and the Sanford Consortium Stem Genomics Cell We thank members of the Chang and Yeo laboratories for discussions and critical online version of the pape Note: Any Supplementary Information and Source Data files are available in the t the in available are references, and codes accession statements of including Methods, and data availability any associated M studies. future in approaches CD8 T lymphocytethe of fate and underscorespecification the control power and together necessity ofmechanisms single-cell epigenetic and transcriptional linked closely that suggest Together our findings genes. specific few a of expression the in increases gradual nuanced, nature immunology nature C AUTH A he pape he eprints and permissions information is available online at at online available is information permissions and eprints ck O

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© 2017 Nature America, Inc., part of Springer Nature. All rights reserved. and H3K27me3, wild-type CD8 wild-type H3K27me3, and H3K27me3. and Ezh2 of analysis ChIP-Seq Center. (IGM) Medicine Genomics for Institute UCSD the at or System Sequencing 100 HiSeq2500 the on 100) (paired-end single-end sequenced were libraries cDNA Single-cell (Illumina). System Sequencing MiSeq and Scientific), Fisher (Thermo Fluorometer 3.0 Qubit Technologies), (Agilent Bioanalyzer 2100 the librar on cDNA performed were single-cell ies the of measures control Quality instruc (Illumina). manufacturer’s tions the to according generated were libraries (cDNA) DNA complementary XT single-cell Nextera Illumina facturer’s instructions. manu the to according performed was amplification whole-transcriptome (Clontech) SMARTerchemistry chip. on performed were RT-PCR and Cell lysis imaged. manually were chip on captured cells exclude single to Viable cells. included dead was (Invitrogen) stain Live/dead chip. on capture cell C the onto loaded were 10 × 2.5 isolation, cell After simultaneously. cells RNA-sequencing. of up single to 96 amplification transcriptome and amplification C transcriptome Single-cell d. 3 additional an (PeproTech) for ng/ml) (15 IL-15 or U/ml) (10 IL-2 with cultured then were CD8 the using isolated were cells T After 1 (500 ng/ml). d peptide with gp33-41 of P14 activated CD8 activation, differentiation. and culture Cell recipient ( into mice transfer adoptive before (CFSE) ester succinimidyl diacetate 10 × 2 division, CD8 of isolation the For mice. ent 5 × 10 CD8 of isolation the For infection. after d 42 at ent mice at 7 d after infection and splenocytes and lymph nodes were harvested from were recipi isolated of mouse per units Splenocytes LCMV-Armstrong. 10 × 2 with later d 1 infection intraperitoneal by followed CD45.2 wild-type congenic into transferred adoptively were infection. and transfer viral Adoptive cell FACSCanto or Biosciences). FACSAria II, (BD C6, Accuri an on analyzed were samples All antibodies. intracellular with staining before lized and permeabi Services) Microscopy in fixed (Electron 4% paraformaldehyde then and antibodies surface with stained were cells °C; 37 at h 6 for (Sigma) A brefeldin of presence the in (KAVYNFATM) peptide (GenScript) gp33-41 of detection IFN- H-2D generate (NIH TetramerH-2D Core Facility) was conjugated to (Prozyme) streptavidin-PE to Biotinylated Technology. Cell and Biolegend from purchased were Flow Mito and Kit Detection Apoptosis V Annexin dilution. at 1:20 used was and Pharmingen BD from purchased was (11/Ezh2) Ezh2 to Antibody dilution. 1:100 at used was and Technologies Life from purchased IFN- V IL-2R (1M7), CD44 (2F1), KLRG1 (MEL-14), CD62L from Biolegend and were used at 1:100 dilution: CD8 cytometry. and flow Antibodies analysis. from experi infection and or was were used no For excluded no ments, blinding animals randomization old. weeks 6–8 male, were mice Recipient old. weeks P14 with crossed were and C57BL/6J were Wild-type conditions. mice All pathogen-free Diego. specific in San housed and bred California, of University the of Guidelines Use Mice. MET ONLINE nature immunology nature vitro in 1 β Single-Cell Auto Prep System (Fluidigm) was used to perform whole- perform to used was (Fluidigm) System Prep Auto Single-Cell 8.1/8.2 (KL16-133.18), IL-7R (KL16-133.18), 8.1/8.2 Ezh2 γ All animal work was approved by the Institutional Animal Care and and Care Animal Institutional the by approved was work animal All 4 (XMG1.2), TNF (MP6-XT22). Antibody to Granzyme B (GB11) was was (GB11) B Granzyme to Antibody (MP6-XT22). TNF (XMG1.2), n P14 CD8 = 24) and were harvested 2 d following LCMV infection. LCMV following d 2 harvested were and 24) = with 5 5 with fl/fl mice were obtained from the Jackson Laboratory. Laboratory. Jackson the from obtained were mice b 6 gp33 tetramer for flow cytometry analysis. For intracellular intracellular For analysis. cytometry flow for tetramer gp33 + P14 CD8 P14 H µ γ T cells per mouse were adoptively into transferred 24 recipi g/ml plate-bound anti-CD3 (145-2C11) and anti-CD28 anti-CD28 and (145-2C11) anti-CD3 plate-bound g/ml and TNF, CD8 ODS 5 to 2 × 10 × 2 to 1 Cd4 Single-Cell Auto Prep mRNA Array IFC for single- for IFC Array mRNA Prep Auto Single-Cell + T cells were first labeled with carboxyfluorescein carboxyfluorescein with labeled first were cells T Cre 6 mice. Donor mice were male or female, 6–8 6–8 female, or male were mice Donor mice. P14 CD8 P14 α Antibodies to the following were purchased to were purchased the following Antibodies + + Splenocytes obtained from P14 mice were were mice P14 from obtained Splenocytes T cells (from (from cells T (A7R34), T-bet (4B10), IRF4 (IRF4.3E4), (IRF4.3E4), IRF4 (4B10), T-bet (A7R34), T cells were stimulated + T cells that had undergone their first cell cell first their undergone had that cells T + T Isolation Kit (Miltenyi). CD8 (Miltenyi). Kit Isolation T + T cells sorted by flow cytometry cytometry flow by sorted cells T 5 × 10 For ChIP-seq analysis of Ezh2 Ezh2 of analysis ChIP-seq For + T cells at 4 d after infection, infection, after d 4 at cells T n 3 P14 CD45.1 = 4 mice) were activated activated were mice) 4 = α (53-6.7), CD45.1 (A20), α (PC61), V (PC61), ex ex vivo b 5 gp33 monomer monomer gp33 plaque-forming plaque-forming + recipient mice, mice, recipient + Ezh2 CD8 with LCMV α 2 (B20.1), 2 (B20.1), fl/fl + + T cells cells T T cells The The mice mice + ------

mRNA-stranded cDNA libraries were generated and sequenced on an Illumina by cytometry. and flow into mice sorted recipient transferred were adoptively 5 × 10 RNA-seq. Bulk-cell instrument. HighSeq4000 Illumina an on nt SE) (50 a reads on bp 250–650 to size by selected PippinPrep and to (Sage instrument sequenced Science), a depth of 30 million pooled, was library each of fmol 200 sample. per fmoles 200–600 yielded Amplification controls). or (IgG IPs) cycles 17 (H3K27me3 cycles 14 for amplified was and used was cells 500,000 coverage comparison of versus cells, wild-type chromatin Ezh2-deficient from for For IgG H3K27me3 the IPs, controls. for and 17 cycles Ezh2 IPs, 14 cycles and H3K27me3 for input controls II IPs, for 12 cycles pol 10 cycles used PCR to according by(NEB), the amplification manufacturer’sLibrary instructions. input controls (1%) using the NEB Next ChIP Preparation Library Reagent Set Sequence-indexed libraries were prepared from immunoprecipitated DNATechnologies). andLife (026102, IgG normal rabbit and Millipore) (12-371, IgG Millipore), mouse anti-RNA polymerase II (05-623, Millipore), mouse normal (07-449, rabbit anti-H3K27me3 Santa (H-80, Biotechnology), Cruz anti-Ezh2 10 × 1 from 3 and chromatin cells (IP), immunoprecipitation each hydro-shearing For E220 Covaris instrument. a using bp 300 of size average an to sheared facturer’s In were prepared brief, nuclear and extracts instructions. chromatin manu the to according (Millipore) kit ChIP EZ-Magna the using performed cells. 4 × 10 dead to exclude cytometry by flow for 4 d sorted and were X Bio Cell) (37.51, results in a dimensionality reduction that is invariant to reflection to invariant is that reduction dimensionality a in results and practice standard is transformations of composition 0.75 This = theta parameters). and 30 = perplexity (with 2 = D in visualize to bhSNE applied then only and D = 10, to down principal-component dimensionality the standard reduce to (PCA) applied analysis first we greater Therefore, cells all TPM). over 1 than expression mean a had which of over for (6,000 signal genes gave 22,000 cell each for data RNA-sequencing total our contrast, (approximately) ~40 = D is dimension meas original the cytometry where urements single-cell of millions visualize to used successfully been has and problem, crowding so-called the solves which objective non-convex (bhSNE) algorithm approximation (tSNE) embedding sto neighborhood t-distributed the chastic applied we plot, scatter 2-dimensional a in subtypes CD8 among expression gene of diversity visualization. the visualize and EXPR matrix expression cell-heterogeneity cell-by-gene the of and dimensionality the reduce reduction Dimensionality EXPR gene cell each for value TPM the Finally, gene. given a of transcripts tified quan all for values expression the summing by columns) = genes 22425 = G rows, = cells 288 = (C matrix expression cell-by-gene a into combined were Next, analysis. the per (TPM) outputsmillion transcript of for Kallisto all cells genome (mm10) STAR(v2.4.1b) by aligned and correction; bias out (v0.42.1) Kallisto by fastqc (v0.10.1) with metrics quality for checked were: reads RNA-seq raw cell, each For quantification. expression robust and (QC) control quality on focusing CD8 256 from data pre-processing. data RNA-seq Single-cell set. each in number gene the on depending analyses, gene-ontology 5 from ranged The distribution. Poisson a follows gene each of count read the that assumption the by calculated were genes of read-count expressed all genes in for total each sample. Differentially to to a gene according names and 1,000,000 was summed normalized Kallisto parameter: -1 200 –s 20 – single. The read count of each transcript derived from using GENCODE GRCm38.p4Kallisto transcriptomewith processed were as samples the bulk reference,The instrument. with the HighSeq4000 following g 4 c,g was natural log-transformed to yield a normalized expression value: value: expression normalized a yield to log-transformed natural was P14 wild-type CD8 = ln(1 + TPM + ln(1 = 4 8 ; poly-A and adaptor-trimmed with cutadapt (v1.8.1) cutadapt with ; and adaptor-trimmed poly-A µ 6 CD8 g of antibody were used. Antibodies used were as follows: rabbit rabbit follows: as were used Antibodies used. were antibody of g −5 5 o 5 to 2 + with default parameters for quality control and downstream T cells were in crosslinked 1% formaldehyde and ChIP was + 5 o ioain f CD8 of isolation For T cells were processed with a bioinformatics pipeline pipeline bioinformatics a with processed were cells T 0 −6 to a reference transcriptome (Gencode vM3) (Gencode transcriptome reference a to c,g for selection of differentially expressed genes for for genes expressed differentially of selection for ) 5 . + 4 T cells ( . tSNE is an unsupervised technique based on a on based technique unsupervised an is tSNE . P value threshold was Bonferroni-corrected and and Bonferroni-corrected was threshold value n = 4) or Ezh2-deficient CD8 + Single-cell mRNA sequencing sequencing mRNA Single-cell T cells at 4 d after infection, infection, after d 4 at cells T 5 1 to the reference mouse mouse reference the to 5 3 + via its Barnes-Hut Barnes-Hut its via T cells of different different of cells T doi: 10.1038/ni.3688 + T cells ( 4 9 ; quantified ; quantified 5 55– 4 8 8 . After After . with n c 5 and and = 8) 8 To . In . 4 7 6 - - - - - ,

© 2017 Nature America, Inc., part of Springer Nature. All rights reserved. In contrast, the ‘effector memory’ and ‘central memory’ lineages start from from start lineages memory’ ‘central and memory’ ‘effector the contrast, In Div1 the through Specifically, the ‘effector’data RT-qPCR of lineage courses starts time from similar the with work naive published population, of basis andthe on progresses a priori were determined orders These lineage. in a ordered particular lations each from cells population 50 and constructed all trajectories through the replacement cross-product of popuwith sampled we brief, In lineage. each for courses time differentiation hypothetical constructed we lineages, memory stand the temporal dynamics of expression for key genes along the effector and Temporal paths. through inferred lineage expression trajectories predictions. score ‘memory-ness’ their in agreement seeming their with contrast to computed was classifiers two the for natures score GINI by their from all G were The genes = signature for selected genes 22,425 each classifier analysis. this for redundant is and score ‘memory-ness’ the minus 1 is scores plotted and were shown to correlate ( mediate day 4 CD8 inter unseen previously provided both were their they on subpopulations, trained respective were classifier early-state and classifier fate the both After (ref. 90% remaining the on training while set test held-out 10% each on was computed curve (ROC) by combining ating the predictions characteristic tive training sets and evaluated using tenfold cross-validation. A receiver oper set for another early state classifier. Both classifiers were provided their respec and Div1 Div1 into T cells of daughter segregation observed Using newly the T cells. (T trees randomized unseen cells. Each classifier constructed an independent ensemble of extremely previously of ‘effector-ness’ or ‘memory-ness‘ the predict to used be can also subpopula butabove) described gene expression as cell (such the differential tions examined T the distinguish only not that signatures gene-expression Div1 ( subtypes division first our as such samples small two between even distributions, cumulative empirical the between difference largest the finds mate normality; and the Kolmogorov-Smirnov two-sample (KS2) test test rank-sum MWU) as known also (MWW; Mann-Whitney-Wilcoxon the expression: of heterogeneity for dropout to due bimodal be does not conform to the usual negative binomial distribution expression gene single-cell Since EXPR. matrix expression log-transformed all pairs of T cell sub-populations from two non-overlapping sets of rows in the results. interpretable more give to information extra the utilize which which are blind to the cell type labels, we also applied methods two supervised dimensionality reduction (PCA and tSNE) and hierarchical clustering methods of analysis data. gene expression Supervised SP_PIR_KEYWORDS, GOTERM_MF_FAT, categories. target for chosen were UP_SEQ_FEATURE GOTERM_BP_FAT, used were and threshold default and background Default analysis. for DAVID analysis. (GO) Gene-ontology green). and (purple cells T memory the of that to than (yellow) cells 7 day all of centroid the to space) tSNE (in closer Div1 remaining The T T all all and of (purple) cells centroid overall the to space) tSNE (in closer were they because Div1 proposed the Specifically, cells). ter (T T of cells effector clusters differentiated the of minally centroids the to proximity their to according terplot ( plot tSNE our in dots) (red 1 T cells of division clusters distinct two observed we Since two-dimensional population. cell T resulting corresponding its of stage the the by colored on was plot scatter point each reduction, dimensionality doi: n = 36) and Div1 and 36) = CM For the cell type classifier, we trained two T binary to cell classifiers identify For differential gene expression analysis, we performed this analysis between 10.1038/ni.3688 and T MEM EM Fig. 2 Fig. subpopulations after the first division, we built a second training ) ) populations, we built a training set for a fate classifier for CD8 6 MEM a TE 4 + ), we re-colored those cells distinctly for the inset scat inset the for distinctly cells those re-colored we ), . Using the terminally differentiated effector and memory and memory effector differentiated . Using terminally the 6 T cells. Their predicted ‘memory-ness’ scores were scatter- subpopulation, then onto day 4, and finally to day 7. 7. day to finally and 4, day onto then subpopulation, 5 TE . The surprisingly small overlap in gene-expression sig gene-expression in overlap small surprisingly . The ( EM cells ( cells n = 24). = cells (green) than to that of all day 7 cells (yellow). (yellow). 7 cells day of all that to than (green) cells 6 1 Fig. 2 Fig. , we used two non-parametric statistical tests tests statistical non-parametric two used we , 6 2 Generated gene lists were uploaded to to uploaded were lists gene Generated , which relies on a large sample to approxi to sample large a on relies which , a Fig. Fig. 3 , inset) remained red because they were were they because red remained , inset) MEM TE f cells (inset) were re-colored blue blue re-colored were (inset) cells cells) and memory T cells (T T cells memory and cells) ). ). For each T cell, its ‘effector-ness’ In contrast to the unsupervised 59 , 6 0 and can even To under 6 3 , , which MEM 6 CM 5 TE TE 7 ). ). + ------.

considered significant. Data are presented as the ratio of H3K27me3-marked of H3K27me3-marked ratio as the are presented Data significant. considered were bp 600 than greater changes coverage ChIP TSS absolute and 0.5 than In covered. significantly considered over input was coverage 3× read and any with region derived, was region TSS each of intensity way, read the In similar a sample. each for obtained reads of for both the input reads that were in located TSS regions and the total number normalized region, TSS the in located were that reads total the of sum the by and effector cells, respectively.memory Reads intensity from around TSS ( TSS around coverage cell naive deducting by calculated regions were calculated by BEDtools Intersect expressed 6,000 ( genes the for (TSS) site start transcriptional the around changes coverage analyze To values). (‘fold’ 4.0 as threshold input over enrichment ‘Homer Poisson by with command called histone were findPeaks’-style peaks ChIP default. as options other all and Fastx; outReadsUnmapped 1; limitOutSJoneRead 1; outFilterMultimapScoreRange None; 10; options: outSAMunmapped the following outFilterMultimapNmax with STAR with genome (v2.4.1b) reference to mm10 were mapped infection CD8 on analysis. data H3K27me3-coverage it. around area shaded a as interval confidence 95% the and presents segment line a with solid sample population for each expression plot time-series package software Seaborn a by summarized visually were trajectories bootstrapped These respectively. cells, Div1 the through naive, considered significant. considered ( by ChIP fied identi H3K27me3, or Ezh2 by targeted genes of comparisons for used was ( groups two involving comparisons for used ( expression differential significantly ( classifiers of by score to prediction memory supervised the assess significance analysis. Statistical samples. single-cell all across 1 = TPM expression mean had genes all that ensure to calculated was Div1 in that to relative cells 4 day in or loss of gain either expression with genes among total genes or Ezh2-bound In H3K27me3. by ≥ in of or Ezh2 targets H3K27me3 Ezh2 or H3K27me3 negatively correlated with TPM changes. To be included as Fisher’s test exact was genes to whether targeted then byperformed determine twofold. than more changed expression gene bulk if decreased or increased as marked were genes naive), vs. memory naive, vs. (effector comparisons of bulk gene expression TPM was from calculated data. single-cell In the two sets on gene expression in memory and effector differentiation ( and Ezh2 of H3K27me3 impact the In to quantify order cells. or effector cells memory from TSS around coverage cell naive deducting by calculated then regions were by calculated BEDtools ( genes expressed 6000 the of TSS of coverage the get to order In default. as options mand using input as background with following options: size 100; and all other were peaks by identified using the HOMER software package ‘findPeaks’ com 0 L 1000; minDist 200; size options: input following ‘findPeaks’ using with command, as background HOMER by called were regions ChIP H3K27me3 0.01. iterNorm normGC; command and the with ChIP HOMER input into data were converted tag then directory limi Fastx; and options as other all default. 1; outReadsUnmapped tOutSJoneRead 1; outFilterMultimapScoreRange 10; outFilterMultimapNmax None; genome with STAR (v2.4.1b) analysis. data ChIP Ezh2 expression. decreased with genes total over genes Fig. 3 Fig. 100-bp TSS region covered by Ezh2 peaks or or peaks Ezh2 by covered region TSS 100-bp f Fig. 6a Fig. Fig. 6 Fig. ). Pearson correlation was used to determine the genes with the most most the with genes the determine to used was correlation Pearson ). + T cells sorted at 8 d (effector cells) and 60 d (memory cells) after after cells) (memory d 60 and cells) (effector d 8 at sorted cells T 6 8 ‘makeTagDirectory’ with the following options: keepOne; tbp 1; 1; tbp keepOne; options: following the with ‘makeTagDirectory’ a Fig. 6c Fig. , ), the overlap of peaks and the 20 bins of 100 bp around TSS TSS around bp 100 of bins 20 the and peaks of overlap the ), d ), the overlap of peaks and the 20 bins of 100 bp around TSS TSS around bp 100 of bins 20 the and peaks of overlap the ), Figure 6 Figure Pearson correlation and Spearman correlation were used correlation and Spearman Pearson correlation , e ). Differences with an associated an associated with Differences ). ; and all other options as default. Ezh2 and PolII ChIP ChIP PolII and Ezh2 default. as options other all and MEM All ChIP data were mapped to mm10 reference reference mm10 to mapped were data ChIP All e 5 , data are presented as a percentage of unbound unbound of percentage a as presented are data , subpopulation, ending with T with ending subpopulation, 1 with the following options: outSAMunmapped Figures 6 Figures 6 Figure 6 Figure Data from H3K27me3 ChIP performed performed ChIP H3K27me3 from Data 7 TE . . The changes in coverage ( Fig. 4 Fig. cells. In In cells. and b 6 c ). Student’s unpaired unpaired Student’s ). 7 , absolute TPM changes greater , greater TPM changes absolute . . The coverage change was then

Fig. 5d- Fig. 7 ≥ , genes were required to have , were required genes 6 Figure Figure 600-bp TSS region covered covered region TSS 600-bp P 6 nature immunology nature , which links the average average the links which , value cutoff as 0.1% and and 0.1% as cutoff value Fig. Fig. 6 k P ). Fisher’s exact test test exact Fisher’s ). 7 value of <0.05 were of <0.05 value , normalized TPM TPM normalized , Fig. Fig. 6c EM b ) ) was calculated cells and T and cells Fig. Fig. 6 , e ), ), artificial t -test was was -test a ) ) were CM - - -

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