Gene expression and epigenetic architecture of PRDM1-knockout CAR-T cells. (A-F) The CD19-targeting CAR-T cells with or without PRDM1 knockout were repeatedly stimulated with NALM-6 3 times and analyzed for gene expression profiles by RNA-sequencing (A-C) (n = 3 different donors) or epigenetic profiles by ATAC-sequencing (D-F) (n = 2 different donors). The data shown are unsupervised hierarchical clustering (A), principal component analysis (B) of differentially expressed genes (FDR < 0.01), and gene set enrichment analysis between control and PRDM1-knockout CAR-T cells using genes upregulated in T cells with an early memory phenotype (C) (nominal P values are shown). (D) Heatmap of differentially accessible regions between control and PRDM1-ablated CAR-T cells (FDR < 0.05). (E) Log2 fold change in read counts (y-axis) between control and PRDM1-knockout CAR-T cells were plotted against log2 average read counts of all samples (x-axis) for individual peak regions. Red dots denote differentially accessible sites at FDR < 0.05. (F) Chromatin accessibility tracks at the promoter regions of the indicated genes. (G-H) Correlation between RNA-seq and ATAC-seq results in the genes with differential expression and chromatin accessibility at promoter regions (FDR < 0.05). Only transcription factor-encoding genes are shown in H. (I) CAR-T cells with or without ectopic expression of TCF7 or PRDM1 knockout were restimulated with NALM-6 and analyzed for the indicated memory markers 4 days later. Mean fluorescence intensity calculated for each sample was shown (n = 4, one-way ANOVA with multiple comparisons test). (J) CAR-T cells transduced with constitutively active STAT1 (caSTAT1) and/or ablated with PRDM1 were analyzed for cytokine production upon restimulation with NALM-6 (n = 4, one-way ANOVA with multiple comparisons test). In (I-J), horizontal lines denote the mean values. NS, not significant.