Fig. 3: RASA2 ablation improves functional T cell persistence through repeated cancer cell exposures.
From: RASA2 ablation in T cells boosts antigen sensitivity and long-term function
a, Schematic of experiment for modelling T cell persistence in vitro. RNP, ribonuclear protein. b, T cell viability and CD39 levels were measured by flow cytometry after each stimulation (n = 4 donors; mean ± s.e.m.). c, Expression of key genes in T cells by RNA-seq after the first and fifth stimulations (n = 3 donors, stimulated via CAR or TCR; mean ± s.e.m.; two-sided Wilcoxon test). d, GSEA of differentially expressed genes between T cells after first and fifth stimulation. Adjusted P-value by two-sided permutation test. e, Cancer cell growth in co-culture with TCR T cells after multiple stimulations. The line is the fitted mean for triplicates. f,g, Effector cytokine production after repeated stimulations, as measured by flow cytometry (f; n = 2 donors in triplicate; shape denotes donor) or by multiplex ELISA (g; n = 3 donors; technical duplicates as dots; lines show mean; two-sided Wilcoxon test). h, Oxygen consumption rate (OCR) trace of TCR T cells after repeated tumour stimulations. Arrows mark addition of oligomycin, FCCP and rotenone + antimycin A (R + A) (one donor in 6 technical replicates; mean ± s.d.) i, Oxygen consumption rate measured in mitochondrial stress test (n = 2 donors in 6 technical replicates; shape denotes donor; values normalized to a maximum of 1 for each donor). j, Cancer cell killing after 1 and 5 stimulations. The shaded area shows the 95% confidence interval for triplicates. k, Imaging of RFP+ A375 cells co-cultured with T cells exposed to repeated stimulations. Scale bar, 1 mm. l, Summary statistics for area under the growth curve of cancer cells over a range of effector T cell:target cell ratios (n = 7 donors; mean ± s.e.m.; two-sample Kolmogorov–Smirnov test). m, RASA2-KO CD19 CAR T cells maintained efficient killing after six previous stimulations. Data are representative of one of three donors. The shaded area shows the 95% confidence interval for triplicates. Statistical tests as indicated, *P < 0.05, **P < 0.01.
