Expansion of Mouse CD8+ T Cells

Mouse spleens were collected and processed as described previously (14 (link)). CD8⁺ T cells were enriched from the splenocytes with a mouse CD8a⁺ T-Cell Isolation Kit (Miltenyi Biotec, Inc.). Then, the cells were expanded as described previously with modifications (22 (link)). Briefly, the cells were activated in 6-well plates (5 × 10⁶ cells/5 mL/well) coated with anti-CD3 (0.5 μg/mL, clone 2C11, Bio X Cell) and anti-CD28 (5 μg/mL, clone 37.51, Bio X Cell) for 20 hours in RPMI1640 medium containing 10% FBS and antibiotics and supplemented with 2 mmol/L L-glutamine, 50 μmol/L 2-mercaptoethanol, 1% insulin-transferrin-selenium (ITS-G, Thermo Fisher Scientific), 6 ng/mL mouse IL2 and 0.5 ng/mL mouse IL7 (ProSpec). After another 48 hours expansion, the cells were harvested and used for adoptive T-cell therapy in mice.