Mass spectrometry screen of intracellular metabolites in CD4⁺ T-cell subsets early in cGVHD reveals increased aerobic glycolysis in allogeneic T_em cells. Representative example of the clinical score (A) and body weight (B) changes over time in the B10.D2 into BALB/c cGVHD model, n = 5 (syngeneic [syn]), n = 16 (allogeneic [allo]). (C) Schematic indicating the potential fate of pyruvate by being converted to either lactate (anaerobic/aerobic glycolysis), acetyl-CoA (TCA cycle), or alanine (transamination). (D-G) Intracellular concentrations of metabolites: lactate (D), citric acid (E), malic acid (F), and alanine (G). Single-cell suspensions were generated from pooled, freshly harvested spleens of syn (n = 19) and allo (n = 22) HSCT recipients on day 14. The cells underwent positive selection using CD4 microbeads. FACS-purified T-cell subsets were collected, and the enzyme activity was quenched with methanol. All samples were supplemented with an internal standard solution, and relative quantification was carried out using capillary electrophoresis time-of-flight mass spectrometry. Cells were pooled from 2 independent HSCTs. Each sample for MS analysis contained ∼3 million cells; n = 1 for syn/allo T_n samples, n = 2 for syngeneic T_em, and n = 3 for allogeneic T_em. Data are represented as mean + SEM, as appropriate. PDH, pyruvate dehydrogenase.