Fig. 3: FOXO1 overexpression enhances the stemness and metabolic fitness of human CAR T cells.
From: FOXO1 enhances CAR T cell stemness, metabolic fitness and efficacy
Analysis of human CD8+CAR+ T cells expressing mCherry (ctrl), FOXO1, TCF7 or ID3. a, Proportion of CD62L+CD27+ or CD45RA+ CAR T cells. b, Proportion of CD8+CAR+ T cells with a CD45RA+CD62L+ phenotype. Left, representative staining (day 14 post-transduction). Right, time course analysis. Different shapes represent individual donors. c, Paired analysis from 12 individual experiments. d, t-Distributed stochastic neighbour embedding (t-SNE) plot of indicated cell surface markers. Max., maximum; min., minimum; WT, wild type. e,f, CD8+CAR+ T cells were analysed by RNA-seq before and after activation with MCF7 tumour cells. n = 3 biological replicates. e, Heat map depicting the 38 genes with highest differential expression in the G10 memory cluster identified by Sade-Feldmann et al.11. f, expression of glycolysis-related genes before and after co-culture with MCF7 tumour cells. g, Analysis of CAR T cell oxidative consumption by Seahorse MitoStress assay. Data represent paired analysis from n = 5 independent donors (left) or mean ± s.e.m. of n = 3 technical replicates of a representative donor (right). OCR, oxygen consumption rate. h–k, Control or FOXO1-expressing CAR T cells were left unstimulated or were stimulated for 16 h with MCF7 tumour cells and then analysed by single-cell RNA-seq (scRNA-seq). h,i, Uniform manifold approximation and projection (UMAP) plots, cell cluster composition and density plots showing expression of indicated genes of non-stimulated (h) and stimulated (i) CD8+CAR T cells. j, Visualization of gene signatures scores (SingleCellSignature) of memory, glycolysis and exhaustion gene sets in non-stimulated CD8+ CAR T cells. k, Gene set enrichment analysis for indicated pathways comparing FOXO1-expressing CAR T cells in cluster 1 to control CAR T cells within cluster 0 (non-stimulated clusters) or FOXO1-expressing CAR T cells in cluster 2 to control CAR T cells in cluster 5 (stimulated clusters). Two-sided, paired t-test (c,g).
