The epigenetic landscape of T cell exhaustion

Abstract

Exhausted T cells in cancer and chronic viral infection express distinctive patterns of genes, including sustained expression of programmed cell death protein 1 (PD-1). However, the regulation of gene expression in exhausted T cells is poorly understood. Here, we define the accessible chromatin landscape in exhausted CD8⁺ T cells and show that it is distinct from functional memory CD8⁺ T cells. Exhausted CD8⁺ T cells in humans and a mouse model of chronic viral infection acquire a state-specific epigenetic landscape organized into functional modules of enhancers. Genome editing shows that PD-1 expression is regulated in part by an exhaustion-specific enhancer that contains essential RAR, T-bet, and Sox3 motifs. Functional enhancer maps may offer targets for genome editing that alter gene expression preferentially in exhausted CD8⁺ T cells.

Fig. 1. CD8⁺ Tcell exhaustion is associated with extensive changes in accessible chromatin

(A) Representative ATAC-seq tracks at the Ccr7 and Ifng gene loci. (B) Developmental trajectory of new regions at each time point. (C) Overlap in ChARs between cell states. (D) Distribution of nondifferential (left) and differential (right) regions between acute and chronic CD8⁺ T cell states. TSS, transcription start site. (E) Correlation network of similarity between states measured by gene expression (left) and chromatin accessibility (right). Edge length corresponds to similarity (Spearman correlation).

Fig. 2. State-specific enhancers in CD8⁺ T cells form modules that map to functionally distinct classes of genes

(A) Heat map of peak intensity for all differentially accessible regions (rows) clustered by similarity across cell states (columns). Shown are normalized numbers of cut sites (supplementary methods), scaled linearly from row minimum (white) to maximum (purple). (B) Heat map showing row-normalized average mRNA expression of neighboring genes within each module in (A) in each cell state. Informative genes from each module are shown on right. (C) Heat map showing enrichment of Gene Ontology (GO) terms (rows) in each module (columns). P-values (hypergeometric test) presented as −log₁₀.

Fig. 3. High-resolution functional mapping of an exhaustion-specific enhancer identifies minimal sequences that regulate PD-1

(A) ATAC-seq tracks from CD8⁺ T cells, EL4 cell line, and regulatory CD4⁺ T cells (12). Arrowheads indicate individual ChARs. (B) Cell sorting gates (top) and corresponding genomic polymerase chain reaction amplification for the PD-1 enhancer region (bottom) showing proportion of wild-type (WT) or deleted (Del) alleles in EL4 cells transfected with control (left) or double-cut sgRNAs (right). Representative data shown from two replicates. (C) PD-1 expression of EL4 WT (light gray) or representative enhancer-deleted (red) single-cell clone out of 46 clones. (D) Normalized enrichment of sgRNAs (gray symbols) within PD-1–high and PD-1–low populations at locations shown (supplementary methods). Control nontargeting sgRNAs are pseudo mapped with 5-bp spacing. Red symbols correspond to the 21 sgRNAs with the largest effect within the −23.8 kb enhancer, for which isogenic cell lines were later produced. (E) Overlap of TF footprints and sgRNA activity within the −23.8 kb enhancer. TF footprints with binding probability >0.9 in chronic d27 are shown on top. Lines represent cut sites of top-scoring sgRNAs. Change in PD-1 mean fluorescence intensity (MFI) relative to control guide transfected populations for each sgRNA (red symbol, left axis); 10-bp running average of PD-1 MFI changes caused by sgRNA activity shown in black (right axis). (F) sgRNA cut sites within the SOX3, TBX21, and RAR motifs. (G) Log fold enrichment of predicted TF footprints in acute d27 versus chronic d27 CD8⁺ T cells (x axis) (see supplementary methods) are plotted against the corresponding P-value (hypergeometric).

Fig. 4. Exhaustion-specific epigenetic profiles in the mouse are conserved in antigen-specific exhausted human T cells in HIV-1 infection

(A) Representative ATAC-seq tracks from naïve, HIV-1 tetramer⁺, and CMV tetramer⁺ samples at the IFNG gene locus (top). Orthologous regions from five mouse cell states at the IFNG locus, based on mapping of mouse ChARs to the human genome (colored blocks, bottom). (B) Schematic diagram of mouse and human comparative analysis. (C) Heat map of average chromatin accessibility at regions orthologous to mouse naïve, memory, and exhaustion enhancers in human samples indicated. Color scale as in Fig. 2A. (D) PD-1 and CD39 expression measured by flow cytometry in HCV C63B tetramer⁺, HCV 174D tetramer⁺, and influenza (flu) matrix peptide (MP) tetramer⁺ populations from a single HCV-infected donor. (E) Viral sequences encoding C63B and 174D epitopes. (F) Heat map of average chromatin accessibility at regions orthologous to mouse naïve, memory, and exhaustion enhancers in human samples indicated from a single HCV-infected donor.