Intracellular IFN-γ staining assay

For IFN-γ production assays, co-culture of Tils with autologous tumor was performed and the generation of IFN-γ producing cells in Tils were analyzed by flow cytometry. Tils co-cultured with autologous tumor for 6 h, and subsequently analyzed for IFN-γ [17]. Brefeldin A (Sigma-Aldrich) was added to the culture medium (10 μg/ml) after 2 h of autologous tumor added. The cells were then stained with anti-CD3-PE-Cy7 and anti-CD8-PerCp-Cy5.5 antibodies, fixed, permeabilized, and then labeled with anti-human IFN-γ-APC (eBioscience). Ten thousand events were collected during flow cytometric analysis.