Fig. 5: TIM3-ProIL2V2 activates and expands TIM3⁻ CD8⁺T cells to overcome PD-1 resistance.

A–F MC38 tumor-bearing female C57BL/6 mice (n = 6/group) were treated with 10 μg TIM3-ProIL2V2 or TIM3-IL2V2-MMPs-Rα on day 16. After 3 days, mice were intraperitoneally injected with 50 μl of brefeldin A solution (BFA, 5 mg/ml). Six hours later, the mice were sacrificed and tumor tissues were collected, and FACS analysis was performed. The number of intratumoral total CD8⁺T cells/TIM3⁺ CD8⁺T cells and TIM3⁻CD8⁺T cells (A), IFN-γ⁺CD8⁺T cells (B), IFN-γ⁺TIM3⁻CD8⁺ T cells (C), and PD-1⁺TIM3⁻TCF1⁺CD8⁺ T cells (F) were measured by flow cytometric analysis. The percentage of IFN-γ⁺ (D) and Ki67⁺ (E) in TIM3⁻CD8⁺T cells were measured by flow cytometric analysis. G, H MC38-OVA tumor-bearing female C57BL/6 mice (n = 6/group) were treated with 10 μg TIM3-ProIL2V2 on day 16 after tumor inoculation. The mice were sacrificed and tumor tissues were collected 72 h after the first injection. The number of OVA-specific CD8⁺ T cells were measured by flow cytometric analysis. I–K MC38 tumor-bearing female CD45.2⁺ Havcr2^−/− mice (n = 6/group) were transferred with 2 × 10⁶ CD45.1⁺ Havcr2^wt/wt T cells on day 2 after tumor inoculation, followed by treatment with TIM3-ProIL2V2 or PBS on day 12 after tumor inoculation. The mice were sacrificed and tumor tissues were collected 72 h after TIM3-ProIL2V2 treatment. The number of intratumoral CD45.1⁻TIM3⁻CD8⁺ T cells (I) and IFN-γ⁺CD45.1⁻TIM3⁻CD8⁺ T cells (K) were measured by flow cytometric analysis. The percentage of Ki67⁺ (J) in CD45.1^-TIM3⁻CD8⁺ T cells were measured by flow cytometric analysis. L, M B16F10 bearing female C57BL/6 mice (n = 8/group) were i.p. treated with 25 μg TIM3-ProIL2V2 and/or 150 μg anti-PD-L1 twice on day 10 and 13 after tumor inoculation. Tumor volume (G) was measured as indicated and the mouse survival curve was shown in (H). N B16F10 bearing female C57BL/6 mice were treated with TIM3-ProIL2V2 and anti-PD-L1. 30 days after treatment, cured mice (n = 5/group) were re-challenged with 2 × 10⁶ B16F10 cells. Data are shown as mean ± SEM and representative of two to three independent experiments. P value was determined by unpaired two-tailed t tests (A–L, N). Statistical analysis of survival curve was compared using a log-rank test (M).