Figure 2. Combined inhibition of glycolysis and glutaminolysis profoundly suppresses T cell responses.

(A) Glutaminase activity of CD4⁺ T cells cultured for 24 hrs in different conditions (anti-CD3, 2μg/ml; anti-CD28, 2μg/ml; DON, 5 μM; 2-DG, 0.6 mM; Metformin, 1 mM). Data are shown as mean ± SEM of three independent experiments. (B and C) Naïve WT C57BL/6 splenocytes labeled with eFluor 670 and stimulated with anti-CD3 in medium containing indicated metabolic inhibitors (DON, 5μM; 2-DG, 0.6mM; Metformin, 1mM). (B) 24-hour IFN-γ secretion to supernatants as interrogated by ELISA. Data are shown as mean ± SEM of three independent samples. (C) Proliferation of CD4⁺ and CD8⁺ T cells at 72h measured by dilution of eFluor 670. n.s., not significant, **p < 0.01, ***p < 0.001, ****p < 0.0001 (Student’s t test). Data are representative of at least two independent experiments. (D) ³H-acetate incorporation into lipids in preactivated and stimulated CD4+ T cells (anti-CD3, 1μg/ml; anti-CD28, 2μg/ml; cross-linking 0.75 μg/ml) with the presence of 2DG, Metformin, DON or in combination (2DG, 5mM; Metformin, 30mM; DON, 60μM). Data are shown as mean ± SEM of three independent samples. ***p < 0.001 (ANOVA). Data are representative of two independent experiments. (E) The phosphorylation state of the S6 ribosomal protein was measured in CD4⁺ T cells after 30 minutes stimulation (anti-CD3, 1μg/ml; anti-CD28, 2μg/ml; cross-linking 0.75 μg/ml) with the presence of Rapamycin, PP242 and metabolic inhibitors (Rapamycin, 1μM; pp242, 1μM; 2DG, 5mM, Metformin, 30mM; DON, 60μM). Data are representative of two independent experiments.