Isolation of tumor-infiltrating lymphocytes

Tumor-infiltrating cells were isolated from tumor tissue by density gradient centrifugation as described previously [18]. Briefly, H22 tumor tissues were minced and digested with 0.5 mg/ml collagenase IV (Sigma-Aldrich, St Louis, Missouri, USA) and 0.1 mg/ml DNase I (Roche, Basel, Switzerland) in RPMI-1640/5% fetal calf serum for 1 h at 37°C. The cell suspension was then filtered through a 70-mm nylon mesh, layered on a Percoll gradient (30–70%), and centrifuged for 20 min. The separated tumor-infiltrating lymphocyte fraction was then collected and washed twice before staining with the indicated cell surface markers.