Volume 45, Issue 6, Pages 1327-1340 (December 2016) Dynamic Changes in Chromatin Accessibility Occur in CD8+ T Cells Responding to Viral Infection  James P. Scott-Browne, Isaac F. López-Moyado, Sara Trifari, Victor Wong, Lukas Chavez, Anjana Rao, Renata M. Pereira  Immunity  Volume 45, Issue 6, Pages 1327-1340 (December 2016) DOI: 10.1016/j.immuni.2016.10.028 Copyright © 2016 Elsevier Inc. Terms and Conditions

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 70 kb Ifng locus with a scale of 0–1,200 for all tracks. (C) k-means clustered heatmap 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)–(D) are from mean of at least two independent samples, except for a single d35 KLRG1+ replicate. See also Figure S1. Immunity 2016 45, 1327-1340DOI: (10.1016/j.immuni.2016.10.028) Copyright © 2016 Elsevier Inc. Terms and Conditions

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), and Dmrta1 (K) loci with a scale of 0–1,200 (left) or RNA-seq gene expression for the indicated genes (right). (L and 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 two independent replicates. RNA-seq data in (G)–(K) are mean of two independent replicates for RNA-seq. See also Figure S2. Immunity 2016 45, 1327-1340DOI: (10.1016/j.immuni.2016.10.028) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 3 Memory Precursor Effector Cells Are Similar to Short-Lived Effector Cells with a Slight Bias toward Memory (A) Scatterplot of mean ATAC-seq counts per peak comparing the SLECs and MPECs. (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 (top) or SLECs and MPECs (bottom) at regions differentially accessible between effector and memory. (D) Mean ATAC-seq coverage at Klrg1 and Aurkb loci with a scale of 0–1,200. (E) RNA-seq gene expression for Klrg1 and Aurkb. Data in (A)–(D) are from three independent replicates and in (E) is mean of two independent replicates. See also Figure S3. Immunity 2016 45, 1327-1340DOI: (10.1016/j.immuni.2016.10.028) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 4 Chronic Activation Profile Identified by Comparison of Viral Antigen-Specific Effector, Memory, and Exhausted CD8+ T Cells (A and B) Scatterplots of mean ATAC-seq counts per peak comparing the indicated samples. (C and 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 0–1,200 (left) or RNA-seq gene expression for the indicated genes (right). (H and 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 two independent replicates. RNA-seq data in (E)–(G) are mean of two independent replicates. See also Figure S4. Immunity 2016 45, 1327-1340DOI: (10.1016/j.immuni.2016.10.028) Copyright © 2016 Elsevier Inc. Terms and Conditions

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 cells, effector cells, SLECs, MPECs, memory cells, 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 cells, effector cells, SLECs, MPECs, memory cells, and exhausted CD8+ T cells. (C) Enrichment of all known motifs within each cluster of differentially accessible regions compared to all differentially accessible regions in naive, effector, memory, and exhausted CD8+ T cells. All motifs with an enrichment log p value less than –35 and found in 10% or more regions in at least one cluster are shown. (D) Percent of ChIP-seq peaks overlapping differentially accessible regions in each cluster or percent of ATAC-seq peaks in each cluster (left column). 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 (B). ATAC-seq data in (A) and (B) are mean of at least two independent replicates and RNA-seq data in (E) are mean of two independent replicates. See also Figure S5. Immunity 2016 45, 1327-1340DOI: (10.1016/j.immuni.2016.10.028) Copyright © 2016 Elsevier Inc. Terms and Conditions

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 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) Scatterplot 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 0–1,200 for ATAC-seq tracks. (E) Nr4 family member gene expression in CD8+ T cells overexpressing the NFAT-CA-RIT mutant or left untransduced (Mock) showing mean plus range. ATAC-seq data in (A)–(D) are from at least two independent replicates. See also Figure S6. Immunity 2016 45, 1327-1340DOI: (10.1016/j.immuni.2016.10.028) Copyright © 2016 Elsevier Inc. Terms and Conditions