Tumor inoculation and treatments

B16-OVA cells (2×10⁵) were injected subcutaneously into CD45.1 mice. At day 7, mice bearing tumor of a similar size were randomly divided into groups, sh-scrambled, sh-Rraga, sh-Rragb, sh-Rragc or sh-Rragd OT-I CD8⁺ T cells (5×10⁶) were injected intravenously, respectively. Mice were sacrificed after 14 days. MC38 cells (5×10⁵) were injected subcutaneously into Cd4^CreRragd^fl/fl or Cd4^Cre mice. MC38-OVA cells (5×10⁵) were injected subcutaneously into Rag1^−/− mice. Mice bearing tumor of a similar size were randomly divided into groups, Cd4^Cre Rragd^fl/fl OT-I CD8⁺ T cells (2×10⁶) or Cd4^Cre OT-I CD8⁺ T cells (2×10⁶), Cd4^Cre Rragd^fl/fl OT-I CD8⁺ T cells (1×10⁶) and Cd4^Cre OT-I CD45.1.2 CD8⁺ T cells (1×10⁶) (for co-transfer experiment), were injected intravenously. Mice were sacrificed at indicated days. Treatment of antiprogrammed cell death protein 1 (anti-PD-1) antibody (RMP1-14), or IgG isotype control (2A3) were given intraperitoneally at a dose of 100 µg per mouse on day 3 after tumor cell inoculation, then every 3 days for the duration of the experiment. LEU or PBS was given by intratumor injection at a dose of 70 mg/kg per mouse on day 3 after tumor inoculation, and every 2 days for the duration of the experiment. To isolate TILs, MC38 or MC38-OVA tumor were excised, minced and digested with 0.5 mg/mL collagenase IV (Sigma) and 200 IU/mL DNaseI (Sigma) for 30 min at 37°C, and then passed through 70 µm filters to remove undigested tumor tissues. TILs were then isolated by CD8 (TIL) MicroBeads (Miltenyi Biotec).