70 m nylon mesh

Blood samples were drawn from CT26 tumor-bearing mice on day 7 post-treatment. For each sample, 25 µL of fresh heparinized blood was incubated with the appropriate dilution of fluorescence-conjugated antibodies (see Flow cytometry section) for 30 min at 4°C in the dark. Tumors were harvested 7 days post-treatment, cut into small fragments, and digested in 1 mg/mL collagenase and 20 mg/mL deoxyribonuclease (DNase) (Sigma) in phosphate buffered saline for 45 min at room temperature. Subsequently, tumor-infiltrating lymphocytes (TILs) were filtered through 70 µm nylon mesh (Cell Treat), washed with 10 mL complete Roswell Park Memorial Institute media (RPMI), and collected by centrifugation (1500 rpm, 4 min). Pelleted cells were resuspended for staining and analysis by flow cytometry (see Flow cytometry section).

Peripheral blood (PBL) samples were drawn on day 7 post-treatment; 25 µL of fresh heparinized blood was incubated with fluorescence-conjugated antibodies (online supplemental table 2) for 30 min at 4°C in the dark. Tumors were harvested 3 or 7 days post-treatment, cut into small fragments, and digested in 1 mg/mL collagenase and 20 mg/mL DNase (Sigma) in serum-free RPMI-1640 for 30 min at room temperature (RT). TIL were filtered through 70 µm nylon mesh (Cell Treat), washed with 10 mL cRPMI, and collected by centrifugation (1500 rpm, 4 min). Pelleted cells were resuspended for staining and analysis by flow cytometry (see below). Lymph nodes (LNs) were harvested 7 days post-treatment and processed to obtain single-cell suspensions. Red blood cells were lysed with ACK buffer (Lonza) for 2 min at RT. Cells were then rinsed with cRPMI (ThermoFisher) and resuspended for antibody staining.