Figure 1. CD28 costimulation endows T cells with latent mitochondrial respiratory capacity.

(See also Figure S1) Naïve CD8⁺ T cells were activated with bead- or plate-bound αCD3 (5 μg/ml), IL-2 (100U/ml), and ± soluble αCD28 (0.5 μg/ml). A) Extracellular acidification rate (ECAR) and oxygen consumption rate (OCR) were measured for 8 hr after activation with αCD3 beads in a Seahorse extracellular flux analyzer (EFA). Glycolytic reserve (GR) and spare respiratory capacity (SRC), after oligomycin (Oligo) and FCCP respectively, were quantified. B) 3 days after activation, cells were switched to IL-15 for 3 days to generate T_M cells and SRC was assessed [calculated as (OCR after FCCP – basal OCR) /basal OCR x 100%]. C) CD8⁺ T_M cells (CD44^hi/CD62L^hi) were sorted from spleens and lymph nodes of 15 week old uninfected age and sex-matched WT and CD80/86^−/− mice. OCR was assayed after consecutive injections of oligo, FCCP, and rotenone and antimycin (R+A). Graphs represented as mean ± SEM of three biological replicates. D) SRC of the graphs in panel c. was calculated. E) IL-15 T_M cells generated as in B were restimulated with αCD3/28 beads and IL-2. OCR was followed for 8 hours and maximal respiration was assessed by sequential treatment with oligo, FCCP, and R+A. F) IL-15 T_M cells were restimulated and intracellular IFN-γ was assessed by flow cytometry (% IFN-γ⁺ CD8⁺ T cells and MFI of IFN-γ is shown). G) T_E cells were switched to 10 mM (upper panels) or 0.3 mM glucose (acute glucose restriction, AGR) overnight (lower panels). Baseline ECAR was determined and the maximal respiration rate was measured after FCCP. H) SRC was determined as in panel A. Data shown as mean ±SEM, representative of ≥ 3 separate experiments. Statistical comparisons for two groups were calculated by using unpaired two-tailed Student’s t-test, * p < 0.05; ** < 0.01; *** < 0.001.