100 μm and 40 μm cell strainers

Freshly resected colon tissues from patients with colon cancer and tonsil tissues from children with tonsillar hypertrophy were minced into small pieces in RPMI-1640 medium (Invitrogen, Carlsbad, CA, USA) supplemented with 1% fetal calf serum (Lonza, Basel, Switzerland) and were sequentially digested with collagenase D (1 mg/ml; Sigma-Aldrich, St. Louis, MO, USA) and DNase I (50 μg/ml; Sigma-Aldrich) at 37°C for 40 min and 30 min, respectively. The cell suspensions were then passed through 100-μm and 40-μm cell strainers (BD Biosciences, Franklin Lakes, NJ, USA) to remove debris. The cell suspensions were resuspended in RPMI 1640 medium (Invitrogen) supplemented with 10% fetal calf serum (FCS; Lonza) and 1% penicillin/streptomycin (Sigma-Aldrich) before isolation of mononuclear cells (MNCs) by centrifugation over a Ficoll–Hypaque density gradient centrifugation for 30 minutes at 24°C for further analysis.

Peripheral blood mononuclear cells (PBMCs) were obtained from healthy donors by density gradient centrifugation with Lymphoprep (AXIS-SHIED Rodelokka, Oslo, Norway). CD3⁺ and CD8⁺ lymphocytes were isolated by a magnetic-activated cell sorter (MACS) using CD3 Microbeads and a CD8 T cell isolation kit (MiltenyiBiotec), respectively. The cells were then labeled with anti-CD8, anti-CCR7, and anti-CD45RA (Ebiosciences) and sorted into naïve and memory CD8⁺ T cell subsets on a BD fluorescence activated cell sorting (FACS) Aria sorter (BD Biosciences). For extraction of TILs, tissue from lung tumor samples were minced and digested with collagenase (2.5 mg/ml collagenase I) at 37 °C for one hour. Cell suspension was then twice filtered through 100-μm and 40-μm cell strainers (BD Biosciences) to obtain single cells. TILs were isolated from single cells utilizing density gradient centrifugation with Lymphoprep and further purified using CD8 MicroBeads (MiltenyiBiotec).