Asct2 overexpression on T cells enhances the function of CD8⁺ and CD4⁺ T cells cultured under low and high Gln concentrations. (A) Diagrams of retroviral vectors designed to overexpress Asct2 in T cells. (B) Transduction efficiency (Thy 1.1⁺) in CD8⁺ and CD4⁺ T cells assessed by flow cytometry. (C) Asct2 expression on the transduced T cells’ membrane measured by flow cytometry. (D) ³H-labeled Gln uptake by transduced CD4⁺ and CD8⁺ T cells. (E-F) Proliferation and IFN-γ secretion of Ctrl and Asct2-overexpressing CD4⁺ (E) and CD8⁺ (F) T cells activated with anti-CD3/CD28 beads under different Gln concentrations (0-2 mM). (G-J) Proliferation, IFN-γ secretion, and specific B16OVA lysis of Ctrl and CD8⁺ Asct2–OT-I T cells (G-H), and CD4⁺Asct2–OT-II T cells (I-J) under different Gln concentrations. (K-L) Seahorse metabolic assay was used to measure the OCR and the ECAR of Ctrl and Asct2-overexpressing CD8⁺ OT-I T cells (K), and CD4⁺ OT-II T cells (L). Data are representative of 2 to 3 independently repeated experiments. Data represent mean ± standard error of the mean (SEM) and were analyzed using 2-way analysis of variance (ANOVA) with Bonferroni multiple comparisons test (∗∗∗∗P < .0001; ∗∗∗P < .001; ∗∗P < .01; ∗P < .05). AntiA/Rote, antimycin/rotenone; APC, allophycocyanin; cpm, counts per minute; Ctrl, control; LTR, long termi; MFI, mean fluoresecence intensity; Oligom, oligomycin; unstim, unstimulated.