Figure 2: Overexpression of FOXO1 in CAR T cells promotes a memory phenotype, antitumor activity, and metabolic fitness during chronic stimulation.

a, Schematic depicting engineering of truncated NGFR-only (tNGFR), TCF1/tNGFR- (TCF1_OE), and FOXO1/tNGFR- (FOXO1_OE) overexpressing CAR T cells and magnetic isolation of tNGFR positive cells for downstream analyses. b, Flow cytometric analysis confirming transcription factor overexpression in TCF1_OE (red) and FOXO1_OE (blue) compared to tNGFR (black) (left), and CD19.28ζ CAR expression across groups compared to untransduced cells (Mock, gray) (right). c-f, tNGFR-purified CD8⁺ CD19.BBζ CAR T cells overexpressing FOXO1 (blue), TCF1 (red), or tNGFR (black) were repeatedly challenged with Nalm6 at a 1:4 effector:target ratio. c, CD8⁺ T cell expansion measured 72h after each challenge (n = 2 donors). d, IL-2 and IFNγ secretion after the fourth tumor challenge (mean ± s.d. of 2–3 wells from 1 representative donor, n = 2 donors). e,f, Flow cytometric analysis of memory- and exhaustion-associated markers at baseline and 7 days after the third tumor challenge. Contour plots show a representative donor and bar graphs show mean ± s.e.m. of MFI normalized to tNGFR levels within each donor (n = 2–4 donors from 3 independent experiments). g, CAR T cell exhaustion model^15,24 whereby T cells express a high-affinity GD2-targeting CAR (HA.28ζ) that promotes antigen-independent tonic CAR signaling for 15 days. h, Day 15 IL-2 and IFNγ secretion from HA.28ζ CAR T cells overexpressing tNGFR (black), TCF1 (red), or FOXO1 (blue) in response to 143B osteosarcoma cells. Plots show mean ± s.d. of 3 wells from 1 representative donor (n = 4 donors) i-k, Seahorse analysis was performed on tNGFR-purified HA.28ζ CAR T cells overexpressing tNGFR (black), TCF1 (red), or FOXO1 (blue) (n = 2 donors). i, Oxygen consumption rate (OCR) before and after treatment with oligomycin (Oligo), FCCP, and rotenone and antimycin (R+A). Plot shows mean ± s.d. from 11 technical replicates from one representative donor. j, Ratio of OCR to extracellular acidification rate (ECAR). k, Spare respiratory capacity. j,k, Bar graphs show mean ± s.d. of three representative time points within each donor. d, 1-way ANOVA with Dunnett multiple comparisons test. f,h-k, 2-way ANOVA with Dunnett’s multiple comparisons test or Mixed-effects model with Dunnett’s multiple comparisons test. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.