Extended Data Fig. 8: Phenotype and transcriptional profile of healthy donor CAR T cells following FOXO1 overexpression.
From: FOXO1 enhances CAR T cell stemness, metabolic fitness and efficacy
Healthy donor T cells were transduced with mCherry (ctrl), FOXO1, TCF7 or ID3 and an anti-Lewis Y CAR. a, Paired data of indicated phenotypic markers in control and FOXO1 expressing CAR T cells, n = 5 (CCR7, CD39, PD1) or 4 (TIM3) independent donors. Statistics determined by paired two-sided t test,. b-e, g, CAR T cells were serially cocultured with OVCAR-3 or MCF7 tumor cells over 72 h after which point CAR T cell number (b), cytokine production (c) or cell surface phenotype (d-e, g) were determined. b, d, Data represents the mean ± SD of biological triplicates from a representative experiment of n = 4. Statistics determined by unpaired two-sided t test. c, e, Paired data of indicated markers in control and FOXO1 expressing CAR T cells, n = 5 independent donors (c) or n = 4 independent donors (e). e, Statistics determined by paired two-sided t test. f, g, Flow cytometry showing the expression of CD39 and TIM3 prior to (f, g) or after co-culture with MCF7 tumor cells (g), representative of 2 independent experiments. h-n. Gene expression of CD8+ CAR T cells (h-j, l-n) and CD4+ CAR T cells (k-l) was determined by RNA-Sequencing as per Fig. 3. Data indicative of biological triplicates. h, enrichment of genes associated with less differentiated T cells that correlate with improved responses to immune checkpoint blockade in FOXO1 but not TCF7 expressing CAR T cells. i-j, Expression of indicated immune checkpoints in control, TCF7 or FOXO1 expressing CD8+ CAR T cells k, Expression of genes associated with glycolysis. l, Volcano plot highlighting differentially expressed genes in control or FOXO1-expressing CAR T cells prior to and after tumor cell coculture. m, negative enrichment of genes associated with exhaustion in FOXO1-expressing but not TCF7-expressing CD8+ CAR T cells, gene set defined by39. n, Control or FOXO1-expressing CD8+ CAR T cells were assessed by RNA-Sequencing prior to stimulation (left), after 24 h activation with 0.1 μg/ml plate bound anti-Lewis Y (middle) or after 7 days recovery post stimulation with anti-Lewis Y (right). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Source data are provided in the Source Data file.
