Extended Data Fig. 4. Overexpression of FOXO1 induces a memory-like transcriptional program in CAR T cells.

a–g, Bulk RNA-seq analyses of day 15 tNGFR⁺ CD4⁺ HA.28ζ CAR T cells overexpressing tNGFR, TCF1_OE, or FOXO1_OE (n = 3 donors). a, PCA of CD4⁺ cells. b, PCA that includes CD4⁺ samples plotted in a and CD8⁺ samples plotted in Fig. 2a. c, Venn diagram showing the number of unique and shared DEGs in CD4⁺ TCF1_OE and FOXO1_OE cells compared to tNGFR cells (Bonferroni-adjusted P < 0.05 with abs(log₂FC)>0.5). d, Expression of memory- and exhaustion-associated genes. Centre line represents the mean counts per million. e, Heat map and hierarchical clustering of DEGs. Genes of interest are shown. Scale shows normalized z-scores for each DEG. f, GSVA using published human CD4⁺ regulatory T cell (T_reg) signatures^42,43. Centre line represents mean score. g, GO term analyses showing curated lists of top up- and downregulated processes in CD4⁺ FOXO1_OE and TCF1_OE cells versus tNGFR cells. Data show Benjamini–Hochberg-adjusted P. h, QIAGEN IPA of upregulated and downregulated TF pathways in FOXO1_OE cells versus tNGFR cells. Data show adjusted P. i–k, Bulk RNA-seq analyses of day 15 tNGFR⁺ CD8⁺ CD19.28ζ cells overexpressing tNGFR, TCF1_OE, or FOXO1_OE (n = 3 donors). i, PCA analysis. j, Venn diagram showing the number of unique and shared DEGs in TCF1_OE and FOXO1_OE cells compared to tNGFR cells (Bonferroni-adjusted P < 0.05 with log₂(fold change) < 0.5). k, Heat map and hierarchical clustering of DEGs. Genes of interest are shown. Scale shows normalized z-scores for each DEG. Statistical comparisons were performed using DESeq2 (c,d,e,j,k), repeated-measures one-way ANOVA with Tukey’s test (f) and one-sided hypergeometric test (g). NS, not significant.

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