Fig. 2. Overexpression of FOXO1, but not TCF1, induces transcriptional and epigenetic features of T cell memory.

a–l, Bulk RNA-seq (a–f) and ATAC-seq (g–l) in tNGFR⁺CD8⁺ HA.28ζ CAR T cells (n = 3 donors). a, RNA-seq PCA. b, Venn diagram showing unique and shared DEGs in TCF1_OE and FOXO1_OE compared with tNGFR (Bonferroni-adjusted P < 0.05 with abs(log₂(FC) > 0.5). c, GSVA of DEGs using naive (T_N), T_pex and exhausted (T_ex) T cell signatures⁵⁵. Centre line represents mean score. d, Heat map and hierarchical clustering of DEGs. Genes of interest are highlighted. The colour bar shows normalized z-scores for each DEG. e,f, GO term analyses showing curated lists of the top upregulated and downregulated processes in FOXO1_OE (e) and TCF1_OE (f) versus tNGFR (Benjamini–Hochberg-adjusted P). g, ATAC-seq PCA. h, Number of differentially accessible peaks compared with tNGFR (P < 0.05 with abs(log₂FC) > 0.5). i, GSVA of differentially accessible peaks using an early T_ex cell epigenetic signature⁹. Centre line represents mean score. j, Chromatin accessibility tracks for the IL7R, KLF3, TOX and FASLG loci, for one representative donor. k,l, Rank-ordered plots of differentially accessible transcription factor (TF)-binding motifs in FOXO1_OE (k) and TCF1_OE (l) versus tNGFR. ZF, zinc-finger. Statistical comparisons were performed using DESeq2 (b,d,h,k,l), one-sided hypergeometric test (e,f) and repeated-measures one-way ANOVA with Dunnett’s test (c,i).

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