Quantifying Tumor-Specific CD8 T Cell Killing

UNSCC680AJ empty vector tumors were dissected from the mice and CD8 T cells were isolated from the tumors and spleens using the EasySep^™ Mouse CD8+ T Cell Isolation Kit (STEMCELL Technologies). UNSCC680AJ empty vector or B2m knockout cell lines were then co-cultured in vitro with the UNSCC680AJ empty vector tumor-infiltrating (or spleen) CD8 T cells for 24h at the effector/target (E/T) ratios of 0:1, 1:2, and 1:1. Cells were subsequently stained with anti-CD45-Pacific Blue (BioLegend), anti-CD3-APC-eFluor^® 780 (eBioscience), and anti-CD8-PE (BioLegend) at 4°C for 30 min. The cells were then washed, stained with a 7-AAD staining solution (BD Biosciences) for 30 min at 4°C, and analyzed by flow cytometry. Target cell killing by tumor-specific CD8 T cells was determined using the following formula adapted from previous reports (50 (link),51 (link)): Percentage of adjusted tumor cell death = (%7AAD⁺ CD45⁻ cells with effector T cells – %7AAD⁺ CD45⁻ cells without effector T cells). Spontaneous cell death was lower than 6 % in all experiments.