Supplementary Figure 1

Experimental design and quality-control metrics.

(a) Distribution of sequencing output (millions of reads per cell) from single-cell RNA-seq libraries for each cell population. Gaussian kernel smoothing was applied with bandwidth of 10. (b) Distribution of alignment quality (% of reads uniquely mapped) from single-cell RNA-seq libraries for each cell population. Gaussian kernel smoothing was applied with bandwidth of 10. (c) Eigen spectrum of

covariance for single-cell expression data matrix (sorted by % of variance explained for each principal component (PC)) justifying use of top 10 PCs for t-distributed Stochastic Neighborhood Embedding (tSNE). (d) Clustergram of pairwise distances between representative

triples of decoy plus duplicate samples from each cell population. Duplicate samples from the same single cell library (round braces) are closest to each other (dark red). Next closest (red) to them is a decoy sample of another single cell library from the same cell population (angled brackets). Duplicate and decoy samples form other cell populations are furthest away from each other (light red). (e) tSNE plot of each cell's raw expression counts (TPM) prior to log-transformation, colored by population. (f) Same tSNE plot as (e) but colored by plate (and sequencing batch) of each cell. Low batch effect was due to careful experimental design and random

assignment of cells from each population across at least 2 out of 3 plates.

Nature Immunology: doi:10.1038/ni.3688

Supplementary Figure 2

Temporal expression patterns of genes encoding members of the PRC2 complex.

Temporal expression patterns of Ezh2, Set, Eed, and Suz12 across inferred paths of differentiation for effector (orange), TCM (purple),

and TEM cells (green). Shaded areas around the lines indicate the 95% confidence interval bootstrapped from all possible single-cell expression trajectories.

Nature Immunology: doi:10.1038/ni.3688

Supplementary Figure 3

Ezh2-deficient CD8+ T lymphocytes undergo normal activation and proliferation.

(a) Expression of CD44 and CD62L by gated CD8+

cells from Ezh2fl/fl

Cd4+/+

(‘WT’) and Ezh2fl/fl

Cd4Cre

(‘KO’) P14 mice. (b) Proportion of

gated CD8+CD45.1

+ P14 WT and KO cells, analyzed at 2, 3, and 5 d post-infection following adoptive transfer into recipient mice

subsequently infected with LCMV. (c) Flow cytometry analysis of CD69 and CD44 by undivided (1st CFSE peak) WT and KO P14 CD8

+

T cells labeled with CFSE and adoptively transferred into recipient mice subsequently infected with LCMV and analyzed at 48 h post-infection. (d) Flow cytometry analysis of CFSE dilution by WT and KO CD8

+ T cells, as in (c), at 48 and 72 h post-infection with LCMV.

(e) Analysis of 7-AAD and Annexin V expression in gated 1st division (2

nd CFSE peak) IL-2R

hiCD62L

lo and IL-2R

loCD62L

hi WT and

KO P14 CD8+ T cells responding to LCMV infection in vivo, presented as flow cytometry analysis (left) and bar graphs (right). Lack of

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significant differences in apoptosis between Ezh2-deficient Division 1 ‘pre-effector’ IL-2RhiCD62L

lo and ‘pre-memory’ IL-2R

loCD62L

hi

cells may be due to the difficulty detecting subtle differences in apoptosis in vivo owing to rapid clearance of dying cells. * p < 0.05, *** p < 0.001 N.S. not significant (Student’s two-tailed t-test). Data are representative of two independent experiments with 4 mice in each group (a-e).

Nature Immunology: doi:10.1038/ni.3688

Supplementary Figure 4

Expression of effector-cell- and memory-cell-associated surface markers in wild-type and Ezh2-deficient CD8+ T lymphocytes.

Wild-type and Ezh2-deficient CD8+ T lymphocytes were adoptively transferred into recipient mice subsequently infected with LCMV and

analyzed at different times post-infection. Proportion of wild-type (‘WT’) and Ezh2-deficient (‘KO’) CD8+

T cells expressing CD44 and IL-

2R in (a) Division 1 cells and in (b) cells harvested at 4 d post-infection. (c) Proportion of WT and KO CD8+

T cells expressing CD44,

IL-2R , KLRG1, and IL-7R in cells harvested at 7 d post-infection. ** p < 0.01, N.S. not significant (Student’s two-tailed t-test). Data are

representative of 2 independent experiments with 3 mice in each group (a-c).

Nature Immunology: doi:10.1038/ni.3688

Supplementary Figure 5

Distribution of changes in the expression of H3K27me3-marked and unmarked genes during CD8+ T cell differentiation.

Histograms depicting the distribution of changes in expression, depicted as –log2 TPM ratio, in genes where TSS region is marked or unmarked by H3K27me3 during differentiation of (a) effector cells, (b) TCM cells, and (c) TEM cells. Significance was determined by the

Kolmogorov-Smirnov (KS) 2-sample test. P < 0.05 was considered significant.

Nature Immunology: doi:10.1038/ni.3688

Supplementary Figure 6

Bulk RNA-seq analysis of wild-type and Ezh2-deficient CD8+ T cells.

Wild-type (‘WT’) or Ezh2-deficient (‘KO’) CD8+ P14 T cells isolated 4 d following LCMV infection were analyzed using bulk RNA-seq. (a)

Normalized expression of H3K27me3-marked genes in WT and KO CD8+ T cells. (b) Distribution of changes in gene expression (Log2

TPM) gated on whether TSS region is marked or unmarked by H3K27me3 in WT and KO CD8+ T cells. (c, d) Gene Ontology analysis

of Ezh2-targeted genes (c) upregulated and (d) downregulated in KO CD8+ T cells compared to WT CD8

+ T cells. (e-g) Heatmaps

Nature Immunology: doi:10.1038/ni.3688

showing expression changes of (e) selected pro-apoptotic genes, (f) Ezh2-targeted memory-associated genes, and (g) Ezh2-

untargeted memory-associated genes in WT and KO CD8+ T cells. Two biological replicate samples, made from two individual pools of

n = 4 of each genotype, were utilized in the analysis. Significance was determined by the Kolmogorov-Smirnov (KS) 2-sample test (a,b). P < 0.05 was considered significant. *** p < 0.001.

Nature Immunology: doi:10.1038/ni.3688

Supplementary Figure 7

H3K27me3-coverage change of wild-type and Ezh2-deficient CD8+ T cells.

Wild-type (‘WT’) or Ezh2-deficient (‘KO’) CD8+ T cells isolated at 4 days following activation in vitro were subjected to H3K27me3

ChIP-seq analysis. Heatmaps show changes in H3K27me3 coverage of (a) Ezh2-targeted and (b) untargeted genes in KO CD8+ T cells

compared to WT cells.

Nature Immunology: doi:10.1038/ni.3688

Supplementary Figure 8

Ezh2 mediates the effector differentiation of CD8+ T lymphocytes through epigenetic repression.

(a) Normalized changes in H3K27me3 coverage, identified by ChIP-seq, of Ezh2-targeted genes (red) and untargeted genes (blue) in wild-type (‘WT’) and Ezh2-deficient (‘KO’) CD8

+ T cells isolated at 4 days following activation in vitro. (b) ChIP-seq analysis of

H3K27me3 binding at Eomes, Klf2, Foxo1, and Tcf7 loci in WT (red) and KO (blue) CD8+ T cells. Gray indicates input. Red or blue

arrows indicate H3K27me3 binding peaks in WT or KO cells, respectively.

Nature Immunology: doi:10.1038/ni.3688