Chromatin states define tumour-specific T cell dysfunction and reprogramming

Abstract

Tumour-specific CD8 T cells in solid tumours are dysfunctional, allowing tumours to progress. The epigenetic regulation of T cell dysfunction and therapeutic reprogrammability (for example, to immune checkpoint blockade) is not well understood. Here we show that T cells in mouse tumours differentiate through two discrete chromatin states: a plastic dysfunctional state from which T cells can be rescued, and a fixed dysfunctional state in which the cells are resistant to reprogramming. We identified surface markers associated with each chromatin state that distinguished reprogrammable from non-reprogrammable PD1^hi dysfunctional T cells within heterogeneous T cell populations from tumours in mice; these surface markers were also expressed on human PD1^hi tumour-infiltrating CD8 T cells. Our study has important implications for cancer immunotherapy as we define key transcription factors and epigenetic programs underlying T cell dysfunction and surface markers that predict therapeutic reprogrammability.

Extended Data Figure 1. Phenotypic and functional characteristics of naïve TCR_TAG CD8 T cells differentiating to effector and memory T cells during acute Listeria infection

Naïve TCR_TAG (N; Thy1.1⁺) were transferred into B6 (Thy1.2⁺) mice, which were immunized with LmTAG one day later. At days 5, 7, and 60+ post LmTAG, effector (E5 and E7), and memory (M) T cells were isolated from spleens and assessed for phenotype and function. Flow cytometric analysis of CD44, CD62L, IL7Rα, TBET, and GZMB expression directly ex vivo (upper panel; inset numbers show MFI), and intracellular IFNγ and TNFα production and CD107 expression after 4-hour ex vivo TAG peptide stimulation (lower panel). Flow plots are gated on CD8⁺ Thy1.1⁺ cells. For cytokine production, in grey are shown no-peptide control cells. (n=8 total, with n=2 per cell state). Each symbol represents an individual mouse. Data show mean ± s.e.m; P values calculated using unpaired, two-tailed Student’s t-test. Data are representative of more than 4 independent experiments.

Extended Data Figure 2. Fragment length distribution plots of ATAC-seq samples

Plots are shown for all mouse (a) and human (b) CD8 T cell ATAC-seq samples displaying fragment length (bp; x-axis) and read counts (y-axis). (S1, S2, S3 = replicates per sample group).

Extended Data Figure 3. Epigenetic and transcriptional regulation of normal CD8 differentiation

a, ATAC-Seq data reveals massive chromatin remodeling during normal CD8 T cell differentiation. MA plot of Naïve (N) and Day 5 Effectors (E5) showing log₂ ratios of peak accessibility (E5/N) versus mean read counts for all atlas peaks. Significantly differentially accessible peaks are shown in red (FDR < 0.05). b, Epigenetic and transcriptional regulation of CD8 effector genes. ATAC-seq (left) and RNA-seq (right) signal profiles of Prf1 and Tnf in Naïve (N), Effectors (E5 and E7), and Memory (M) TCR_TAG during acute LmTAG infection. (c – d), Epigenetic and transcriptional regulation of early CD8 response genes in TCR_TAG during acute listeria infection. Published expression data from the Immunological Genome Project (JA Best et al, Nat Imm (2013); GSE 15907) were used; early-response genes increase in expression within the first 12–24 hours and late-response genes increase expression 24–48 hours after naïve T cells encounter LmOVA as determined by Best JA et al. c, Cumulative distribution function of peak accessibility changes between N and E5. Peaks associated with early-response genes show fewer changes in accessibility as compared to peaks associated with late-response genes. The black line shows all peaks accessible in N or E5, the red line shows peaks associated with early-response genes and the blue line shows peaks associated with late-response genes. d, ATAC-seq signal profiles (left) and RNA expression (right) of the early response genes Ldha (top) and Mki67 (bottom) in N, E5/E7, and M TCR_TAG during acute LmTAG infection (blue line; GSE 89309; current dataset Philip M et al.) overlaid with expression data from Best JA et al./Immunological Genome Project (red line).

Extended Data Figure 4. Chromatin peak accessibility changes during normal and dysfunctional CD8 T cell differentiation

a, Number of DESeq-determined chromatin peak accessibility changes during each transition during normal CD8 T cell differentiation (Listeria infection) (right) and CD8 T cell differentiation to dysfunction during tumorigenesis (left) broken down by |log₂FC|>2, |log₂FC|=1–2, and |log₂FC|<1. b, Chromatin accessibility peaks gained or lost during normal and dysfunctional CD8 T cell differentiation were mainly found in intergenic and intronic regions. Pie charts showing the proportions of reproducible ATAC-seq peaks in exonic, intronic, intergenic and promoter regions. (Left) Distribution for all peaks in the atlas. Green box – normal CD8 T cell differentiation during LmTAG immunization; distribution for common and variably accessible peaks in N, E5, E7, and M functional CD8 T cells. Blue box – differentiation to dysfunction in progressing tumors; distribution for common and variably accessible peaks in N, L5, L7, L14, L21, L28, L35, and L60+. Variable = significant change in at least one cell type comparison (FDR<0.05, |log₂FC|>1). Common = no change in any cell type comparison. c, Venn diagrams show the number of significantly-changed peaks during the transition from Naive (N) to D5-effectors (E5) TCR_TAG during acute listeria LmTAG infection versus N to L5 early malignant lesion-infiltrating TCR_TAG (FDR< 0.05, |log₂FC|>2) (Upper) Venn diagram shows opening peaks; (lower) Venn diagram shows closing peaks. d, Selected Biological process (BP) Gene Ontology (GO) terms enriched in peaks open in L5 relative to E5 as determined through GREAT analysis.

Extended Data Figure 5. NFATC1 targets become significantly more accessible during differentiation to dysfunction in early malignant lesions as compared to normal effector differentiation

a, Top 20 most-significantly enriched TF motifs in peaks opening (red) and closing (blue) between L5 and E5. b, Scatterplot comparing the changes in peak accessibility for all differentially-accessible peaks containing the NFATC1 motif during the transition from Naive (N) to D5-Effectors (E5) TCR_TAG during acute listeria LmTAG infection versus N to L5 in pre-malignant lesions (FDR< 0.05, |log₂FC|>1). Highlighted are NFATC1 target peaks associated with genes encoding negative regulatory transcription factors and inhibitory receptors. Some genes, e.g. Cblb and Klf4, had multiple NFATC1 target peaks, including peaks that decreased in accessibility. c, Genes with more accessible NFATC1 target peaks during differentiation to dysfunction in malignant lesions show increased expression levels. Gene expression for genes with peaks in sector 1 and sector 2, with increased and decreased accessibility in L5 vs E5 respectively. Heatmaps show RNA-Seq expression data (row-normalized) for differentially-expressed (P< 0.01, |log2FC| > 1) genes with NFATC1 target peaks contained in Sector 1 (red box) or Sector 2 (blue box) of scatterplot presented b. The majority of Sector 1 genes (195/223, 87%) revealed increased expression in dysfunctional TST as compared to E5, while the majority of Sector 2 genes (21/33, 63%) had decreased expression. Genes are clustered by row according to expression across the samples. Interestingly, while many genes in Sector 1 had transiently increased expression in L5 and L7 (red box, upper left), many genes increased in expression at later stages of tumorigenesis at L14 and beyond (red box, upper right). This suggests that NFATC1 activation of downstream targets (negative regulators of T cell function) may not only induce early dysfunction, but may cause or contribute to the transition from plastic to fixed dysfunction.

Extended Data Figure 6. Epigenetic and transcriptional changes during the L7 to L14 transition

a, Transcription factor footprinting in chromatin accessible regions. ATAC cut site distributions show footprints for CTCF, LEF1, NFATC1, and TCF7 in naïve CD8 T cells. Shown is the mean number of ATAC cut sites on the forward (red) or reverse (blue) strand 100bp up and downstream of the TF motif site, calculated for atlas peaks predicted by FIMO to be bound by the respective TF (p<10⁻⁴). b, TCF1 expression (MFI; mean fluorescence intensity). Each symbol represents individual mouse. Mean ± s.e.m. shown; *** P≤0.0001 (Student’s t-test). c, Selected Biological Processes (BP) (Gene Ontology [GO] terms) enriched in genes which significantly lost chromatin accessibility during the L7 to L14 transition as determined through GREAT analysis. d, Gain and losses of regulatory elements for top 50 most differentially expressed genes associated with TCR signaling during the L7 to L14 transition. Top 25 genes associated with TCR signaling with highest and lowest logFC gene expression changes are shown. Each gene is illustrated by a stack of diamonds, where each diamond represents a chromatin peak associated with the gene. Red diamonds denote peaks gained in the transition, blue diamonds denote peaks that were lost.

Extended Data Figure 7. Pharmacological targeting of NFAT and Wnt/β-catenin signaling prevents TST differentiation to the fixed dysfunctional state in vivo

a, Experimental scheme. Naïve TCR_TAG (Thy1.1⁺) were transferred into ASTxCre-ER^T2 (Thy1.2⁺) mice which were treated with tamoxifen (Tam) one day later. At days 2–9 mice were treated with the calcineurin inhibitor FK506 (2.5mg/kg/mouse) alone (FK506 treatment group; orange), or in combination with the GSK3β inhibitor TWS119 (0.75mg/mouse; days 5–8) (FK506 + TWS119 treatment group; green), or PBS/DMSO (control group; blue) as indicated. At day 10, TCR_TAG were isolated from livers and assessed for phenotype and function. b, Flow cytometric analysis of CD44, PD1, LAG3, TCF1, and EOMES expression of TCR_TAG. c, IFNγ and TNFα production of TCR_TAG isolated at day 10 (left panel; straight ex vivo), and post 3 days IL15 in vitro culture (right panel). Each symbol represents an individual mouse. Data show mean ± s.e.m; P values calculated using unpaired two-tailed t-test. d, Representative flow cytometric analysis of CD38 and CD101 expression of TCR_TAG (numbers indicate %); CD38, CD101 and CD5 expression. Each symbol represents an individual mouse. Data show mean ± s.e.m; P values calculated using unpaired two-tailed t-test. These data are representative of 2 independent experiments (with total n=10 for Exp#1; n=9, Exp#2).

Extended Data Figure 8. Epigenetic and expression dynamics of membrane proteins and transcription factors associated with T cell dysfunction

a, ATAC-Seq signal profile across the Cd38 loci with “state 2” uniquely accessible peaks highlighted in pink; activation-associated peaks highlighted in blue. b, Expression profiles of N, L5, L7, L14, and L60+ TCR_TAG for CD101 versus CD38, TCF versus PD1, and TCF1 versus CD38 by flow cytometric analysis. c, Expression of transcription factors and other proteins on tumor-specific TCR_TAG T cells over the course of tumorigenesis (MFI; mean fluorescence intensity). Each symbol represents an individual mouse. Data shows mean ± s.e.m. (bottom panel). Representative flow histogram overlays are shown. (d – f) TCR_OT1 TST in established B16-OVA tumors enter plastic and fixed dysfunctional states. d, Immunophenotype and cytokine production of TCR_OT1 re-isolated from established B16-OVA tumors 5 (D5) and 13 (D13) days post transfer. e, CD38, CD101 and CD5 expression on D5 and D13 TCR_OT-1. f, Cytokine production of D5 and D21 TCR_OT-1 after 3 days IL-15 in vitro culture. Each symbol represents individual mouse. Mean ± s.e.m. shown; *P=0.03, **P=0.002, ***P≤0.0003 (Student’s t-test).

Extended Data Figure 9. Chromatin state dynamics of memory TCR_TAG differentiating to the dysfunctional state in solid tumors

a, PD1 and LAG3 expression and cytokine production of memory TCR_TAG in liver tumors. Each symbol represents individual mouse. Mean ± s.e.m. shown; *P=0.03, **P=0.006, ***P<0.0001 (Student’s t-test); representative of 4 independent experiments. b, Numbers of ATAC-seq peaks significantly opening or closing (FDR < 0.05) during each transition as memory TCR_TAG differentiate to the dysfunctional state 7, 14, and 35 days post transfer into HCC-tumor bearing ASTxAlb:Cre mice with [(+); left] and without [(-); right] listeria LmTAG immunization; peaks opening (red), peaks closing (blue). c, Principal component analysis of peak accessibility during naïve TCR_TAG differentiation in acute infection (green), early tumorigenesis (blue), and memory TCR_TAG in established HCC (red). Circles = with LmTAG immunization; diamonds = no LmTAg immunization. d, Chromatin accessibility heatmap. Each row represents one of 11,698 selected peaks (differentially accessible between any sequential cell comparison; FDR <0.05, |log₂FC|>2). Shown are +/− 1kb from the peak summit (2kb total per region). e, ATAC-seq signal profiles of Pdcd1, Ctla4, Cd38, Tcf7, and Ifng genes of Naïve (N; grey), Memory (M; green), L7, L14, L35 (blue series), and ML7, ML14, and ML35 (red series) TCR_TAG. Pink boxes highlight peaks that become accessible in dysfunctional T cells compared to Naïve and Memory; blue boxes highlight peaks that become inaccessible in dysfunctional TCR_TAG compared to Naïve and Memory TCR_TAG. f, CD38, CD101, CD30L, and CD5 expression on ML7, ML14, ML21. Inset numbers show MFI.

Extended Data Figure 10. Chromatin states of human PD1^hi tumor-infiltrating CD8+ T cells and model for CD8 TST differentiation and dysfunction in tumors

a, Sorting scheme of peripheral blood lymphocytes for Naïve (N), Effector Memory (EM), Central Memory (CM) CD8 T cell populations (left), and PD1hi CD8 TIL from melanoma and NSCLC patients. b, Differentially accessible ATAC-seq peaks grouped by DESeq-defined differential accessibility pattern. Each column represents one biological replicate. Samples shown include CD45RA+ CD45RO- (Naïve; N; grey), CD45RA- CD45RO+, CD62L- (Effector Memory; EM; light green) and CD45RA- CD45RO+, CD62L+ (Central Memory; CM; dark green) peripheral blood CD8⁺ T cells from healthy donors, and CD45RA- CD45RO+, PD1^hi CD8+ T cells isolated and flow-sorted from human melanoma and lung tumors (PD1^hi TIL; blue). Open, accessible chromatin regions are presented in red; inaccessible chromatin regions are presented in blue. c, ATAC-seq signal profiles of SELL in N, EM and CM. Blue boxes highlight peaks that remain accessible in CM or become inaccessible in EM compared to N respectively. d, ATAC-seq signal profiles of IFNG, EGR2, CD5, CTLA4. Pink and blue boxes highlight peaks that become accessible or inaccessible in PD1^hi TIL compared to N or CM respectively. e, Model for tumor-specific CD8 T cell differentiation and dysfunction in tumors.

Figure 1. CD8 T cell chromatin state dynamics during acute infection

a, Experimental scheme. b, Number of chromatin peak accessibility changes during each transition (FDR<0.05). c, Chromatin accessibility heatmap grouped by differential accessibility patterns. Each row represents one of 8654 selected peaks (differentially accessible between at least one sequential cell comparison; FDR <0.05, |log₂FC|>2). d, (Left) K-means clustered (K = 6, row-normalized) RNA-Seq data for 1758 differentially expressed genes (|log₂FC|>1, FDR<0.05, base mean log2 expression≥10.) (Middle) Heatmap of differentially accessible peaks (FDR<0.05, |log₂FC|>1) presented as in (c) for genes in K-means clusters 1 and 3. (Right) ATAC-Seq signal profiles across Ifng and Gzma loci. Peaks present in all differentiation states highlighted in blue, activation-induced peaks in pink.

Figure 2. TST differentiate to dysfunctionality in developing tumors through discrete chromatin states

a, Experimental scheme. b, Immunophenotype and cytokine production (grey, no peptide control) (n=8 total, with n=2 per time point). Each symbol represents individual mouse. Representative of 5 independent experiments; mean ± s.e.m shown. *P=0.0002 (Student’s t-test); n.s. = not statistically significant. c, Number of peak changes during each transition (FDR<0.05). d, Principal component analysis (PCA) of peak accessibility in naïve TCR_TAG (N; grey) during normal differentiation (green) and during tumorigenesis (blue). Each symbol represents single biological replicate. e, Chromatin accessibility heatmap (15,275 differentially accessible peaks as in Fig. 1c). f, ATAC-Seq signal profiles across Pdcd1 and Ifng loci. Peaks uniquely lost (blue) or gained (pink) in TST.

Figure 3. Discrete chromatin states correlate with reprogrammability and surface protein expression profiles

a, (top) Cytokine production by L5, L12, and L17 after 3 days in vitro IL-15 culture (grey, no peptide control); (bottom) IFNγ production straight ex vivo (circles) or after 3–4 days IL-15 in vitro culture (triangles). Pooled from 3 experiments. b, Top 20 most-significantly enriched TF motifs in peaks opening (red) and closing (blue) between L7 and L14. c, RNA-Seq expression (row-normalized) for most differentially-expressed genes encoding membrane proteins. d, CD38, CD101, CD30L and CD5 expression. Representative of 3 independent experiments. e, Cytokine production by sorted CD38^lo/CD101^lo (blue) and CD38^hi/CD101^hi (red) L14 after 3 days IL-15 in vitro culture. Similar data obtained with sorted L10 in independent experiment. (a, d, e) Each symbol represents individual mouse. Mean ± s.e.m. shown; *P=0.005, **P=0.0005, ***P≤0.0001 (Student’s t-test).

Figure 4. Memory TST rapidly enter the fixed dysfunctional chromatin state in established tumors

a, Experimental scheme. b, Cytokine production of M isolated from liver tumors. c, PCA of peak accessibility in TCR_TAG during acute infection (green), tumorigenesis (blue), and memory TCR_TAG in established tumors (red). d, Chromatin accessibility heatmap showing M, M re-isolated at D35 from established HCC tumors (ML35), and naïve TCR_TAG isolated at day 35 (L35) from early malignant lesions (see Fig. 2). Each row represents one of 19,679 differentially accessible peaks as in Fig. 1c.

Figure 5. Human tumor-infiltrating PD1-high CD8 T cells enter a similar chromatin accessibility state as murine fixed dysfunctional TST

a, Peak accessibility PCA on human healthy donor PBL and PD1^hi TIL from melanoma and NSCLC tumors. b, For non-promoter peaks, normalized Spearman correlations of log₂FC calculated between human N and EM, CM or PD1^hi TIL versus log₂FC between murine N and E5, E7, M, and L5 to L60. P<10⁻¹⁶ for all comparisons between human PD1^hi TIL and mouse L14 - L60. c, ATAC-Seq signal profiles across human TCF7 and mouse Tcf7 gene loci; peaks ost in human PD1^hi TIL and mouse L21, L28, L35 highlighted in blue. d, CD38, CD101 and CD5 expression on human CM (green) and PD1^hi TIL (blue). Each symbol represents individual healthy donor or patient. Mean ± s.e.m. shown; *P=0.01, **P=0.006 (Student’s t-test).