Dynamic Changes in Chromatin Accessibility Occur in CD8+ T Cells Responding to Viral Infection

Abstract

In response to acute infection, naive CD8⁺ T cells expand, differentiate into effector cells, and then contract to a long-lived pool of memory cells after pathogen clearance. During chronic infections or in tumors, CD8⁺ T cells acquire an "exhausted" phenotype. Here we present genome-wide comparisons of chromatin accessibility and gene expression from endogenous CD8⁺ T cells responding to acute and chronic viral infection using ATAC-seq and RNA-seq techniques. Acquisition of effector, memory, or exhausted phenotypes was associated with stable changes in chromatin accessibility away from the naive T cell state. Regions differentially accessible between functional subsets in vivo were enriched for binding sites of transcription factors known to regulate these subsets, including E2A, BATF, IRF4, T-bet, and TCF1. Exhaustion-specific accessible regions were enriched for consensus binding sites for NFAT and Nr4a family members, indicating that chronic stimulation confers a unique accessibility profile on exhausted cells.

Figure 1. High ATAC-seq signal in CD8⁺ T cells at conserved regions in promoters and distal regulatory elements

A) CD8⁺ T cell populations collected for ATAC-seq comparison. B) Mean ATAC-seq coverage at the 70kb Ifng locus with a scale of 0-1200 for all tracks. C) k-means clustered heat map of mean normalized counts or log₂ fold-change from global mean at all peaks. D) Pairwise euclidian distance comparison of asinh transformed ATAC-seq signal per peak for all populations using all peaks accessible in at least one cell type. Data in B,C,D are from mean of at least 2 independent samples, except for a single d35 KLRG1+ replicate. See also Figure S1.

Figure 2. Dynamic changes in chromatin accessibility occur in antigen specific effector and memory CD8⁺ T cells responding to acute viral infection

A–C) Scatterplots of mean ATAC-seq counts per peak comparing the indicated samples. D–F) Boxplots of ATAC-seq counts per peak from the indicated samples (labeled at bottom) at common or differentially-accessible regions from the comparison labeled above. Box indicates interquartile range with whiskers +/−1.5 times this range and outlier points. G–K) Mean ATAC-seq coverage at Il7r (G), Ccr7 (H), Gzma (I), Gzmk (J), Dmrta1 (K) loci with a scale of 0-1200 (left) or RNA-seq gene expression for the indicated genes (right). L,M) Venn diagrams illustrating intersection of differentially-accessible regions from pairwise comparisons of naive, effector, and memory CD8⁺ T cells characterizing regions “specific” to a subset (L) or “not” in a subset (M) with p values and odds ratios from Fisher's test comparisons. ATAC-seq data in A–K are from at least 2 independent replicates. RNA-seq data in G–K are mean of two independent replicates for RNA-seq. See also Figure S2.

Figure 3. Memory precursor effector cells are similar to short lived effector cells with a slight bias towards memory

A) Scatterplot of mean ATAC-seq counts per peak comparing the SLEC and MPEC. B) Boxplot of ATAC-seq counts per peak from the indicated samples (labeled at bottom) at common or differentially-accessible regions from the comparison labeled above. Box indicates interquartile range with whiskers +/−1.5 times this range and outlier points. C) Histograms of the log₂ fold-change between effector and memory cells at (top) or SLEC and MPEC (bottom) at regions differentially-accessible between effector and memory. D) Mean ATAC-seq coverage at Klrg1 and Aurkb loci with a scale of 0-1200. E) RNA-seq gene expression for Klrg1 and Aurkb. Data in A–D are from 3 independent replicates and E is mean of 2 independent replicates. See also Figure S3.

Figure 4. Chronic activation profile identified by comparison of viral antigen specific effector, memory, and exhausted CD8⁺ T cells

A,B) Scatterplots of mean ATAC-seq counts per peak comparing the indicated samples. C,D) Boxplots of ATAC-seq counts per peak from the indicated samples (labeled at bottom) at common or differentially-accessible regions from the comparison labeled above. Box indicates interquartile range with whiskers +/−1.5 times this range and outlier points. E–G) Mean ATAC-seq coverage at Havcr2 (E), Tox2 (F), and Satb1 (G) loci with a scale of 0-1200 (left) or RNA-seq gene expression for the indicated genes (right). H,I) Venn diagrams illustrating intersection of differentially-accessible regions from pairwise comparisons of effector, memory, and exhausted CD8⁺ T cells characterizing regions “specific” to a subset (H) or “not” in a subset (I) with p values and odds ratios from Fisher's test comparisons. ATAC-seq data in A–G are from at least 2 independent replicates. RNA-seq data in E–G are mean of two independent replicates. See also Figure S4.

Figure 5. Differentially-accessible regions in CD8⁺ T cells are associated with bhLH, bZIP, HMG, T-box, NR, and RHD family TFs

A) Two dimensional multidimensional scaling plot of ATAC-seq signal for all replicates of naive, effector, SLEC, MPEC, memory, and exhausted cells at 18,043 regions differentially-accessible regions identified from comparisons of naive, effector, memory, and exhausted cells. B) k-means clustered log₂ fold-change from mean ATAC-seq signal for all differentially-accessible regions identified from comparisons between naive, effector, SLEC, MPEC, memory, and exhausted CD8⁺ T cells. C) Enrichment of all known motifs within each cluster of differentially-accessible regions compared to all accessible regions in naive, effector, memory, and exhausted CD8⁺ T cells. All motifs with an enrichment log p-value less than −15 and found in 10% or more regions in at least one cluster are shown. D) Percent of each cluster of ATAC-seq peaks that overlap ChIP-seq peaks or the percent of all differentially-accessible regions in each cluster. The total number of ChIP-seq peaks for each TF and the fraction of these that overlap any of these differentially-accessible regions are shown below the plot. E) log₂ fold-change from mean RNA-seq counts per transcript are shown for all expressed TFs from families associated with each enriched motif. F) MeDIP-seq coverage compared to input for naive and effector CD8⁺ T cells 8 days after LCMV Arm5 infection. The top graph is for all accessible regions in CD8⁺ T cells, where each graph below is associated with the clusters indicated at left in panel B. ATAC-seq data in A and B are mean of at least 2 independent replicates and RNA-seq data in E are mean of 2 independent replicates. See also Figure S5.

Figure 6. Constitutively active NFAT partially recapitulates the chronic activation profile in vitro

A) Scatterplot of ATAC-seq counts per peak comparing in vitro cultured CD8⁺ T cells after transduction with retroviruses expressing the NFAT-CA-RIT mutant or left untransduced (Mock). B) Boxplots of ATAC-seq counts per peak in naive, effector, memory, and exhausted CD8⁺ T cells at common or differentially-accessible regions between Mock and NFAT-CA-RIT mutant expressing cells. Box indicates interquartile range with whiskers +/−1.5 times this range and outlier points. C) Scatter plot of NFAT-CA-RIT ChIP-seq coverage with log₂ fold-change ATAC-seq signal between Mock and NFAT-CA-RIT mutant expressing cells at regions with lower (top) or higher (bottom) ATAC-seq signal in exhausted compared to effector and memory CD8⁺ T cells. D) Mean ATAC-seq and NFAT ChIP-seq coverage at the Pdcd1 locus with a scale of 0-1200 for ATAC-seq tracks. E) Nr4 family member gene expression in CD8⁺ T cells over-expressing the NFAT-CA-RIT mutant or left untransduced (Mock) showing mean plus range. ATAC-seq data in A–D are from at least 2 independent replicates. See also Figure S6.