Chronic activation of costimulatory domains directs distinct genomic activity. (A) Principal component analysis of bulk RNAseq of 19/28 and 19/BB cells on days 0, 6, and 15. (B) Heatmap of DEGs between day-15 19/28 cells and day-15 19/BB cells. (C) Volcano plot of day-15 DEGs. n = 1 donor for day-0 samples and n = 4 donors for days 6 and 15, all performed in technical triplicates. (D) Normalized transcript counts of key exhaustion markers over time. Significance determined using two-way ANOVA. (E-F) Gene set enrichment analysis of genes present in panel D >2 of 4 gene sets (E) and all 4 human TIL exhaustion gene sets (F). (G) Heatmap of day-15 sample expression of genes present in all 4 human TIL exhaustion gene sets. (H) Principal component analysis of ATAC sequencing of 19/28 and 19/BB cells at days 0, 6, and 15. n = 2 donors for all time points performed in technical triplicates. (I) Gene tracks at transcriptional start sites of PDCD1 and TOX2 reflecting chromatin accessibility. (J) Correlated transcript count and transcriptional start site accessibility for PDCD1 over time. (K) Overlap of genes with increased accessibility in exhausted T cells (as defined²²) and genes with increased accessibility in dysfunctional 19/28 or 19/BB cells. ∗∗∗∗P < .0001.