CD4+ Tem cells exhibit a higher ECAR compared with CD4+ Tn or syngeneic CD4+ Tem in cGVHD. (A) Single-cell suspensions were generated from pooled harvested spleens of syngeneic (n = 5 mice) and allogeneic (n = 9 mice) HSCT recipients on day 14. The cells underwent positive selection using CD4 magnetic microbeads, followed by FACS. FACS-purified T cells were plated using CellTak at a seeding density of 100 000 live sorted cells per well and treated with oligomycin (1 µM), carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP; 0.5 µM), and antimycin A (AA)/Rotenone (1 µM) at indicated time points. The number of technical replicates equals n = 8 to 32 wells per subtype; data are representative of 2 independent experiments. (B) Basal glycolysis vs basal respiration. (C) Basal glycolysis based on the mean of the 2 time points before oligomycin injection. (D) Glycolytic capacity measured after FCCP addition (mean of 4 time points). (E) Basal respiration rate based on the mean of the 3 time points before oligomycin injection. All data are represented as mean + SEM, statistical testing using a 2-way ANOVA, *P < .05, **P < .01, ***P < .001. OCR, oxygen consumption rate.