aPC preincubation reduces mitochondrial respiration in CD4⁺CD25⁻ cells. (A-B) Independent experimental approaches to study mitochondrial metabolism in stimulated T cells: stimulation of human CD4⁺CD25⁻ cells by plate-bound αCD3 and αCD28 for 48 hours (A, top); stimulation of human CD4⁺CD25⁻ cells using an MLR (48 hours) (B, top). In both cases, T cells were preincubated with aPC (20 nM, 1 hour) before stimulation (plate-bound αCD3 and αCD28, or MLR). Representative line graph showing OCR (A-B; bottom) (A, n = 3; B, n = 5). (C) Seahorse analysis showing bioenergetics profile of CD4⁺CD25⁻ cells (OCR vs ECAR; energy graph) 48 hours after stimulation with αCD3 and αCD28 without (T) or with aPC preincubation (T + aPC). (D) Representative flow cytometry histogram (MFI, left) and bar graph with dot plot quantifying the percentage of tetramethylrhodamine ethyl ester (TMRE)-positive cells (flow cytometry, right) reflecting the mitochondrial membrane potential in human CD4⁺CD25⁻ cells preincubated without or with aPC (n = 4). The data are shown as the mean ± SEM, and statistical significance was determined by a 2-tailed Student t test. ∗∗∗P < .005. Images in panels A-B were created using BioRender.com.