Anti cd3 2c11

Ezh2 (Ezh2^fl/fl) mice were generated as described (20 (link)) and obtained from MMRRC repository. Cd4Cre was obtained from Taconic, and GzmbCre, OT-I, ROSA-26Sor^{tm39(CAG-hop/EYFP)}, and B6.SJL-PtpraJ mice from Jackson Laboratory. Ezh2^fl/fl mice were crossed with Cd4Cre to generate the Ezh2^fl/fl-Cd4Cre⁺ (Ezh2-c-KO) strain. Ezh2^fl/fl-Cd4Cre mice were further crossed with OT-I to generate Ezh2^fl/fl-Cd4Cre OT-I (Ezh2-c-KO OT-I) strain. Ezh2^fl/fl was also crossed with GzmbCre and Gt(ROSA)26Sor^{tm39(CAG-hop/EYFP)Hze} to create Ezh2^fl/flGzmbCre-YFP (Ezh2-g-KO) mice. All mice were maintained under specific pathogen free conditions at the animal facility of National Institute on Aging, and animal care was conducted in accordance with the guidelines of NIH.
CD8⁺ T cells were isolated from splenocytes or blood obtained from different murine strains. Naïve CD8⁺ T cells were defined as CD44⁻CD62L⁺ and purified using StemCell CD8⁺ Naïve T cell isolation kit with a final purity of more than 95%. Memory precursor and central memory CD8⁺ T cells were defined as CD127⁺KLRG1⁻ and CD44⁺CD62L⁺, respectively. Cells were cultured in RPMI-1640 with 10% FBS, 10 mM HEPES, 0.11 nM beta-metcaptoethanol, and 1× Pen/Strep/Glu from Thermo-Fisher. CD8⁺ T cell stimulations performed using plate coated anti-CD3 (2C11, 5 µg/ml) and soluble αCD28 (37.51, 1 µg/ml) (Biolegend).

For generating interfering targeting the Rraga, Rragb, Rragc or Rragd OT-I CD8⁺ T cells, retrovirus was produced via triple transfection of HEK-293T cells with a retroviral transfer vector (pmKO.1-GFP for short hairpin RNA (shRNA)-Rraga, shRNA-Rragb, shRNA-Rragc and shRNA-Rragd construction) and the packaging plasmid pCL-Eco at a 1:1 ratio by using a polyethyleneimine-based DNA transfection reagent (Sigma-Aldrich), as recommended by the manufacturer. The viral supernatant was collected 48 hours after transfection, filtered through a 0.45 μm filter and stored at −80°C. OT-I CD8⁺ T cells were activated with plate bounded anti-CD3 (2C11, 10 mg/mL) and anti-CD28 (37.51, 10 mg/mL; both from Biolegend) for 48 hours, and then transduced with retroviruses as described above, supplemented with polybrene (8 µg/mL, Sigma-Aldrich) and centrifuged at 2000 rpm and 32°C for 1 hour. The transduced CD8⁺ T cells were fed with fresh medium for 48 hours, and the knockdown efficiency was confirmed by real-time PCR before adoptive transferring.
For generating Slc3a2 knockdown cells, retrovirus were produced as described above. MC38 tumor cells were infected with the virus supernatant for 48 hours, and the supernatant was collected for CD8⁺ T cells culture. Knockdown efficiency was validated by flow cytometry.