Figure 1. ATT-I enhances the killing efficiency of CD8⁺ T cells against tumor cells.

(A) A total of 594 natural small molecule compounds purified from traditional medicinal plants were tested for their toxicity on MC38 cells and T cells freshly isolated from C57BL/6 mice. Data are presented as mean of 3 independent experiments. (B) The 446 drugs with low toxicity from (A) were tested for their effects on the CD8⁺ T cell–mediated cytotoxicity. MC38-OVA cells expressing luciferase were cocultured with OT-I CD8⁺ T cells in the presence of each drug (5.0 μM) and the T cell–mediated cytotoxicity was measured by the luciferase assay. Difference (log₂): (log₂ [relative viability] > 1; P < 0.05). Relative viability = (tumor cell viability of treated group) / (tumor cell viability of control group). Data are presented as mean of 3 independent experiments. Statistical analysis was conducted using 1-way ANOVA. (C) Chemical structure of ATT-I. (D) The effect of ATT-I treatment on the CD8⁺ T cell killing of MC38-OVA cells was measured under different ratios of tumor cells versus T cells as indicated. Data are presented as mean ± SD of 3 independent experiments. Statistical analysis was conducted using 2-way ANOVA. (E) CD8⁺ T cell killing assays were conducted using coculture of MC38-OVA cells and OT-I CD8⁺ T cells pretreated with 5 μM of ATT-I (+) or vehicle control DMSO (–). Data are presented as mean ± SD of 3 independent experiments. (F) The levels of IFN-γ (left) and TNF-α (right) in the supernatants after coculture of OT-I T cells and MC38-OVA cells pretreated with ATT-I (+) or DMSO control (–) were determined by ELISA. Data are presented as mean ± SD of 3 independent experiments. Statistical analyses were conducted using 2-way ANOVA. **P < 0.01; ***P < 0.001; ****P < 0.0001.