Fig. 2. Genetic suppression of mitochondrial ATP production promotes glycolytic-transcriptional reprogramming of T cells.

a, b Analyzes of (a) oxygen consumption rate (OCR) and (b) glycolytic proton efflux rate (glycoPER) of WT and mPiC-deficient (Slc25a3^fl/flCd4^Cre) T cells at day 2 of culture using a Seahorse extracellular flux analyzer; means ± SEM of 7 mice. c Analysis of ATP concentrations in unstimulated and anti-CD3/CD28 activated WT and mPiC-deficient T cells; means ± SEM of 6 mice. d Relative contribution of glycolysis and mitochondrial respiration to cellular ATP production in anti-CD3/CD28 stimulated WT and mPiC-deficient T cells; means of 3 mice. e Network clustering of RNA sequencing data of significantly (p < 0.05) enriched gene expression signatures between CTL-differentiated WT and mPiC-deficient T cells. Down- and upregulated gene signatures in mPiC-deficient T cells are shown in blue and red, respectively. f, g Gene set enrichment analysis (GSEA) of (f) oxidative phosphorylation (KEGG pathway) and (g) effector versus exhausted T cells (GSE9650) gene signatures in differentiated WT and mPiC-deficient T cells after 6 days of culture. h Heatmap expression analysis of selected genes in WT and mPiC-deficient CTLs after 6 days in culture. i–k Differentiation of WT and mPiC-deficient T cells in vitro, means ± SEM of 5 mice. j, k Flow cytometric quantification of (j) exhaustion and (k) memory marker expression in WT and mPiC-deficient CTLs, means ± SEM of 5 mice. l Analysis of polyfunctional TNFα, IFNγ and IL-2 expression by WT and mPiC-deficient T cells after 6 days of culture and anti-CD3/CD28 restimulation; means ± SEM of 3 mice. m Analysis of apoptosis in resting and anti-CD3/CD28 stimulated T cells by flow cytometry; means ± SEM of 3 mice. Two-tailed unpaired Student’s t-test in (a–c) and (i–m).