Plate coated anti cd3

CD4 + CD25 -T cells (2 × 10 6 per milliliter) were incubated with plate-coated anti-CD3 (10 µg/mL, eBioscience) and soluble anti-CD28 (eBioscience). T H 0 cells were left without exogenous cytokines. 10 ng/mL IFN-γ respectively 30 ng/mL IL-4 were added to induce T h 1 or T h 2 priming, 5 ng/mL TGF-β was added to induce Treg differentiation and 30 ng/mL IL-6, 1 ng/mL TGF-β, and, 10 ng/mL IL-23 were added to induce T h 17 cells.

EG7 cells are derived from EL4 cells and express chicken ovalbumin (OVA) as a tumor antigen. MC38 is a solid tumor predominantly composed of immunosuppressive cell types, such as monocytic myeloid-derived suppressor cells (M-MDSCs; ref. 21) . Congenic CD45.1 þ SJL (8-10-week-old) recipient mice were injected subcutaneously with 5 Â 10 5 EG7 cells. CD8 þ T cells were isolated from spleen and lymph nodes (dissociated as described above) of WT OT-1 or Peli1-KO/OT-1 mice using the Magnisort mouse CD8 þ T cell enrichment kit (Thermo Fisher Scientific). Isolated CD8 þ T cells were stimulated with platecoated anti-CD3 (5 mg/mL, eBioscience), soluble anti-CD28 (2 mg/mL, Invitrogen), and OVA257-264 peptide (1 mg/mL, InvivoGen) in RPMI medium supplemented with IL2 (20 ng/mL, R&D Systems) for 48 hours. 5 Â 10 6 WT OT-1 or Peli1-KO OT-1 cells were intravenously transferred into tumor-bearing mice 10 days after tumor cell injection. Tumors were monitored for 23 days postinoculation, and mice were sacrificed on day 23 to collect draining lymph nodes (DLN) and TILs. Tumor size was determined by caliper measurements 3 times a week.