(A) Schematic for ATAC-sequencing of P14 cells from the models described in Figure 2A and Figure 5C. T_Dysf cells were FACS-sorted on day 19 of chronic stimulation as the PD-1⁺Tim3⁺ (double-positive, “T_Dysf-DP”) and rested for 4 days. Rested T_Dysf-DP cells were sorted on day 23 as PD-1⁺Tim3^– (single-positive, “Rest-SP”) or PD-1⁺Tim3⁺ (double-positive, “Rest-DP”). (B) Heatmap showing differentially open chromatin regions (OCRs) between Acute-7d and T_Dysf-DP cells (day 19) plotted based on relative Z-score with example genes listed. Red indicates “open” and blue indicates “closed” regions. (C) PCA comparing chromatin accessibility profiles for in vitro Acute-7d or T_Dysf subsets to published signatures for in vivo memory or exhausted CD8⁺ T cells (23). (D) Representative snapshots of accessible chromatin peaks (mapped in Integrative Genomics Viewer, IGV) within in vitro P14 or in vivo naive, memory, progenitor, or exhausted CD8⁺ T cells (44) at gene loci for Ccr7, (E) Ifng, and (F) Pdcd1. (G) Alluvial plot tracking the number of chromatin regions from Acute-7d cells that became open/closed in T_Dysf-DP (day 19) compared with Acute-7d and regions from T_Dysf-DP that remained unchanged or became open/closed in rested T_Dysf subsets (day 23). (H) Venn diagram comparing transcription factor (TF) motifs enriched within closed OCRs in T_Dysf-DP cells and regions that became open in Rest-SP cells or (I) open OCRs in T_Dysf-DP cells and regions that became closed in Rest-SP cells. (J) Average % CpG methylation at Tcf7 and (K) Pdcd1 differentially methylated regions (DMRs) within naive or in vitro–generated P14 cells (day 19). N = 2 biological replicates, representative of 2 to 3 independent experiments. Statistical significance was determined by DESeq2 (B and G) or PCA (C) using Partek software. Comparisons in J and K were determined by repeated measures 1-way ANOVA with Tukey’s multiple comparisons. Adjusted P value **P < 0.01, ***P < 0.001, ****P < 0.0001.