Asct2 overexpression on OT-I T cells enhances the efficacy of ACT immunotherapy. (A) Gln concentration measured in the TIF and sera of B16OVA-challenged mice. (B) Experimental design to evaluate antitumor activity of ACT. (C-E) Tumor growth measured over time (C-D) and OS of mice (E) after treatment with Asct2-CD8⁺ OT-I or with Ctrl CD8⁺ OT-I T cells (n = 9-11 mice per group). (F) Tumor weight measured at day 14 after treatment. (G) Number of Ag-specific T cells infiltrating the tumor measured by flow cytometry using SIINFEKL tetramers. (H) Percentage of expression of LAG3 and PD-1 measured in CD45.1⁺ tumor-infiltrating CD8⁺ OT-I and Asct2–OT-I T cells measured by flow cytometry. (I) Number of IFN-γ–producing Ag-specific T cells measured in the spleen of untreated mice or treated with Ctrl CD8⁺ OT-I or Asct2–OT-I T cells in response to SIINFEKL peptide. Data are representative of 2 independent experiments. Data represent mean ± SEM and were analyzed using Student t test, 2-way ANOVA, and 1-way ANOVA with Bonferroni multiple comparisons test (∗∗∗P < .001; ∗∗P < .01; ∗P < .05). MOS, median overall survival; PD-1, programmed cell death protein 1; SC, subcutaneous; TIF, tumor interstitial fluid; unstim, unstimulated.