Figure 2.

Mitochondrial dysfunction is observed in CD8⁺ but not CD4⁺ EMRA T cell subsets. (a) Representative flow cytometry plots and cumulative graphs of TMRE staining from middle‐aged donors showing membrane potential in CD45RA/CD27 T cell subsets directly ex vivo defined showing the percentage of cortactin‐positive (a) CD4⁺ and (b) CD8⁺ T cells analysed directly ex vivo. Data expressed as mean ± SEM of six donors. (b) Mitochondrial ROS measured using MitoSOX by flow cytometry in CD4⁺ and CD8⁺ EMRA T cells from middle‐aged donors. Data expressed as mean ± SEM of six donors. (c) Mitochondrial ROS production expressed as a ratio of mitochondrial mass. Calculated from data shown in Figures 1b and 2. (d) γH2AX expression as determined by flow cytometry in CD45RA/CD27‐defined T cell subsets directly ex vivo from middle‐aged donors; the graph shows the mean ± SEM for five donors. (e) Oxygen consumption rates (OCR) of the EMRA CD4⁺ and CD8⁺ T cell subsets from middle‐aged donors were measured following a 15‐min stimulation with 0.5 µg/ml anti‐CD3 and 5 ng/ml IL‐2; the cells were then subjected to a metabolic stress test using the indicated mitochondrial inhibitors. Data are representative of four independent experiments. (f) The basal OCR, extracellular acidification rate (ECAR) and spare respiratory capacity were measured following a 15‐min stimulation with 0.5 µg/ml anti‐CD3 and 5 ng/ml IL‐2. Graphs show the mean ± SEM for four donors. (g) ATP concentration in EMRA T cell subsets from middle‐aged donors, graphs show the mean ± SEM for five donors. p‐values were calculated using a t test. *p < .05, **p < .01, and ***p < .005