Extended Data Fig. 6. Nuclear-restricted FOXO1 promotes a memory-like phenotype but impairs effector function.
a, Schematic showing a mutated variant of FOXO1 that contains three amino acid substitutions (T24A, S256A, and S319A) which restrict nuclear export (FOXO13A). b, Analysis of soluble and chromatin-bound FOXO1 fractions isolated from tNGFR+ non-CAR T cells that were activated with Dynabeads for 24 h prior to cell collection. Western blots (left) and bar graph (right) representing the ratio of chromatin-bound to soluble FOXO1 normalized to mock T cells are shown for 1 representative donor (n = 2 donors). c, FOXO1 expression in CD19.28ζ and HA.28ζ CAR T cells from 1 representative donor (n = 5 donors). d, CD62L and IL-7Rα expression in CD19.28ζ and HA.28ζ CAR T cells from 1 representative donor (n = 3 donors). e, TCF1 and LEF1 expression in CD19.28ζ CAR T cells from 1 representative donor (n = 3 donors). f,g, RNA-seq on HA.28ζ CAR T cells. tNGFR and FOXO1OE samples are also represented in Fig. 2 and Extended Data Fig. 4. f, PCA. g, GO term analyses showing curated lists of top up- and downregulated processes in FOXO13A vs FOXO1OE. Data show Benjamini–Hochberg-adjusted P value. h, Cytotoxicity of HA.28ζ cells against Nalm6 at a 1:1 E:T ratio. Data are normalized to t = 0 and show mean±s.d. from 1 representative donor (n = 3 donors). Statistics were performed at t = 96 h. i, Cytokine secretion from day 15 HA.28ζ CAR T cells in response to 143B cells. Plots show mean±s.d. of 3 wells from 1 representative donor (n = 3 donors). Statistical comparisons were performed using one-sided hypergeometric test (g), one-way ANOVA with Tukey’s test (h) or Dunnett’s test (i). E:T ratio, effector:target cell ratio.