Figure 3.

Bioenergetic modulation of T-cells by exogenous D-2HG. A) Mitochondrial respiration of T-cells cultured in the absence or presence of D-2HG for 72 h was analyzed in real-time using the Seahorse extracellular flux analyzer. Oxygen consumption rate (OCR) was recorded (Ai) and respiratory parameters were calculated (Aii-iii) after sequential addition of oligomycin, FCCP, and antimycin A/rotenone (n = 4). Likewise, extracellular acidification rate (ECAR) was assessed (Aiv) and glycolytic parameters were calculated (Av-vi) after sequential addition of glucose, oligomycin, and 2DG (n = 4). B) Intracellular concentrations of two representative intermediates of the TCA cycle (α-ketoglutarate: αKG, n = 3 and citrate, n = 5) were enzymatically determined. C) The expression of key metabolic genes was quantified by real-time PCR (n = 3-12). T-cells were either unstimulated (unstim, grey) or stimulated without (0 mM, black) or with (20 mM, orange) D-2HG. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001.