Fig. 2.

Activated CD4+ T-cell subsets incorporate glucose into the TCA cycle. a Oxidative phosphorylation profile of NV, EM and CM T- cells by measuring OCR before and following injections of oligomycin (0.75 μM), FCCP (1 μM) and antimycin A and rotenone (1 μM). Basal respiration (b), ATP-linked respiration (c), maximal respiration (d) and OCR/ECAR ratio (e) in NV, EM and CM T- cells. f Mitochondrial content of NV, EM and CM CD4+ T-cells measured using MitoTracker. g Representative images of NV, EM and CM T-cells stained with DRAQ5 (nucleus), cell mask orange (plasma membrane) and MitoTracker green (mitochondria) scale bar = 10 μm and h corresponding MitoTracker signal to area ratios. i Uniformly labelled ¹³C-glucose incorporation into T-cell metabolites via the TCA cycle in NV, EM and CM T-cells activated for 0.5 and 4 h. Relative abundance of ¹²C and ¹³C including citrate, α-ketoglutarate, succinate, fumarate and malate. j Relative abundance of ¹²C and ¹³C in non-essential amino acids glutamate and aspartate . Data are representative of either five independent experiments (a–e), four independent experiments (f), three experiments with <100 cells analysed as technical replicates (h) or three independent experiments (i, j). Statistical analysis was performed using a nonmatching one-way ANOVA with Tukey’s multiple comparison test (b–h). For non-parametric data, a Kruskal−Wallis test with Dunn’s multiple comparisons test was used. Data expressed as mean ± SEM; *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001