Fig. 4. Mitochondrial respiration, but not ATP production, promotes Akt-Foxo1 signaling in activated T cells.

(A) A schematic depicting electron transfer and disposition by cytosolic LDHA and mitochondrial electron transport chain (ETC) associated with NADH to NAD⁺ conversion. (B) Naïve Ldha^fl/fl (wild-type, WT) and Cd4^CreLdha^fl/fl (knockout, KO) CD8⁺ T cells were stimulated with anti-CD3, anti-CD28, and IL-2 for 3 days. The amounts of NADH and NAD⁺ were measured. The ratios of NAD⁺ over NADH are plotted. (C) Measurements and quantifications of oxygen consumption rate (OCR) and spared respiratory capacity (SRC) of day-3 activated WT and KO CD8⁺ T cells. Sequential chemical treatments are indicated as shown in the graph. Oligo: oligomycin; FCCP: Trifluoromethoxy carbonylcyanide phenylhydrazone; Ant: antimycin; Rot: rotenone. (n=5 per genotype, mean ± SD) (D) A schematic of mitochondrial ETC, proton distribution, and ATP synthetase activity under the indicated treatment conditions. (E) Day-3 activated WT and KO CD8⁺ T cells were collected, and incubated in RPMI-1640 medium in the absence or presence of oligomycin and/or FCCP. T cells were subsequently restimulated with biotinylated anti-CD3 and anti-CD28 through streptavidin crosslinking. Immunoblotting and normalized expression of p-Akt (T308) to Akt, and p-Foxo1 (T24) or p-Foxo1 (S256) to Foxo1. Data are representative of three independent experiments (B, C and E). Unpaired t tests for the measurements between the two groups (B and C): **p<0.01 and ****p<0.0001.