Fig. 6: TIM3-Pro-IL2V2 could control cold tumor.

A, B 2 × 10⁶ stimulated CD45.1⁺OT-I⁺CD8⁺ T cells were adoptively transferred to B16F10-OVA bearing female C57BL/6 mice (n = 8/group) 10 days after tumor inoculation. 25 μg TIM3-ProIL2V2 was administrated on day 12 and 15 after tumor inoculation. Tumor volume (A) was measured as indicated and the mouse survival curve was shown in (B). C 30 days after treatment with OT-I⁺CD8⁺ T cells and TIM3-ProIL2V2, cured mice (n = 3/group) were re-challenged with 2 × 10⁶ B16F10-OVA cells. D–L B16F10-OVA bearing female C57BL/6 mice (n = 6/group) were treated with OT-I⁺CD8⁺ T cells with or without 25 μg TIM3-ProIL2V2 on day 12 and 15 after tumor inoculation. The mice were sacrificed and tumor tissues were collected 24 h after second injection. The percentage of total OVA-specific CD8⁺T cells (D), percentage of CD45.1⁺OT-I CD8⁺ T cells in total OVA-specific CD8⁺T cells (E), percentage of Ki67⁺ in CD45.1⁺OT-I CD8⁺ T cells (F), number and percentage of CD45.1⁺OT-I PD-1⁺TIM3⁻CD8⁺ T cells in CD45.1⁺OT-I CD8⁺ T cells (G and H), percentage of Ki67⁺ in CD45.1⁺OT-I PD-1⁺TIM3⁻CD8⁺ T cells (I), percentage of CD45.1⁻Tetramer⁺ PD-1⁺TIM3⁻CD8⁺ T cells in CD45.1⁻ Tetramer⁺ CD8⁺ T cells (J), percentage of total OVA-specific CD8⁺T cells in splenocyte CD8⁺ T cells (K) and in lymph node CD8⁺ T cells (L). M B16F10 bearing female C57BL/6 mice were treated with PBS (n = 6), 12 Gy Radiation (n = 8), 25 μg TIM3-ProIL2V2 (n = 6) or 12 Gy Radiation + 25 μg TIM3-ProIL2V2 (n = 8) on day 9 and 12 after inoculation. Tumor volume was measured as indicated. N–O B16F10 bearing female C57BL/6 mice (n = 8/group) were treated 12 Gy Radiation on day 9 and/or 25 μg TIM3-ProIL2V2 on day 9 and 12 after tumor inoculation. The mice were sacrificed and tumor tissues were collected 24 h after second injection. The number of CD8⁺ T cells (N) and percentage of IFN-γ⁺CD8⁺ T cells (O) were measured by FACS. 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, C–O). Statistical analysis of survival curve was compared using a log-rank test (B).