Extended Data Fig. 2. CRISPR knockout of FOXO1 attenuates memory formation and promotes exhaustion in CD19.BBζ and HA.28ζ CAR T cells.

a–i, CRISPR–Cas9 gene editing of AAVS1 (AAVS1) or FOXO1 (FOXO1_KO) in CD19.BBζ CAR T cells. a, Schematic depicting generation of FOXO1_KO CAR T cells and downstream assays. b, Day 14 FOXO1_KO expansion normalized to AAVS1. Data show mean±s.e.m. of n = 3 donors. c–f, Flow cytometric analysis of memory- and exhaustion-associated markers on CD8⁺ (c,e) and CD4⁺ (d,f) CD19.BBζ cells. Histograms and contour plots show a representative donor and bar graphs show mean±s.e.m. of n = 3-6 donors. CD62L, IL-7Rα, TCF1, and CD39 histograms in c also appear in Fig. 1c. g, MFI of CD62L in FOXO1⁺ and FOXO1⁻ gated subpopulations of CD19.BBζ cells. h, Schematic showing CD62L_lo / FOXO1_KO cell negative selection strategy for RNA-seq experiments. i, GO term analyses showing curated lists of up- and downregulated processes in FOXO1_KO compared to AAVS1. Data show Benjamini–Hochberg-adjusted P value (n = 3 donors). j, Flow cytometric analysis of memory- and exhaustion-associated markers in day 15 HA.28ζ CAR T cells. Background-subtracted MFI is displayed. k, Cytokine secretion from day 15 HA.28ζ cells in response to Nalm6. Graphs show mean±s.d. of 3 technical replicates from one representative donor (n = 2 donors). Statistical comparisons were performed using paired two-tailed Student’s t-test (b,c,d,g), two-way ANOVA with Bonferroni’s test (e,f), two-tailed Student’s t-test (k) and one-sided hypergeometric test (i).

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