Lag 3 clone c9b7w

Tumor tissue was mechanically dissociated and digested in 0.7 mg/mL Collagenase D (Roche) and 3 mg/mL DNase I (Roche) for 30–45 min to obtain single-cell suspension. For experiments looking at tumor-infiltrating leukocytes (TIL), TIL were isolated using Percoll gradient prior to staining. For experiments looking at B16RFP⁺ tumor cells, cells were stained immediately after digestion for 20 min. Cells were blocked with anti-CD16/32 (clone 93; Biolegend) prior to surface staining. Antibodies against the following antigens were used: CD11a (clone M17/4; ThermoFisher), CD8a (clone 53-6.7; BD Biosciences or Biolegend or eBioscience), CD45 (clone 30-F11; Invitrogen or eBioscience), CD45.1 (clone A20; Biolegend), CD3 (clone eBio500A2; eBioscience), CD45.2 (clone 104; eBioscience), CD90.1 (clone OX-7; BD Biosciences), CD127 (clone A7R34; eBioscience or clone SB/199; Biolegend), KLRG1 (clone 2F1/KLRG1; Biolegend or eBioscience), PD-1 (clone RMP1-30; eBioscience), NKG2A/C/E (clone 20d5; eBioscience), LAG3 (clone C9B7W; Biolegend), CD44 (clone IM7; eBioscience or Biolegend or BD Biosciences), Ly6C (clone HK1.4; Biolegend), Ly6G (clone 1A8; Biolegend), CD11b (M1/70; eBioscience or Biolegend), Qa-1^b (clone 6A8.6F10.1A6; Miltenyi Biotec), and PD-L1 (10F.9G2; Biolegend).

Several strategies were used to block the suspected binding of antibodies to Fcγ, including CD16/CD32 antibody (clone 93, Biolegend), CD16.2 antibody (clone 9E9, Biolegend), and mouse IgG (Solarbio). For antibody labeling, cells in the 200 µL ice-cold 1 × PBS were stained with CD11b (clone M1/70, BD Biosciences), CD45 (clone30-F11, Biolegend) and LAG-3 (clone C9B7W, Biolegend) or TIM-3 antibodies (clone RMT3-23, Biolegend) for 30 min on ice. Flow cytometry analysis was performed on a BD FACScanto. Data were analyzed using FlowJo software (TreeStar).