Comprehensive Phenotyping of Tumor-Infiltrating Lymphocytes

Cell viability was assessed by live labeling 0.1 × 10⁶ TILs with 7-AAD viability staining solution (Biolegend) (22 (link)). For purity, cells were stained with FITC anti-CD3 and PerCP anti-CD19 antibodies. For phenotyping, approximately 0.5 × 10⁶ TILs were stained for measuring the percentages of T_reg and T_EM cells using standard flow cytometry staining protocols (22 (link)). Briefly, for T_reg cells, TILs were labelled live with the following antibodies: FITC anti-CD3, PE anti-CD127, PerCP anti-CD8, APC anti- CD4, APC-Cy7 anti-CD25 and PB anti-CD45. For T_EM cells, TILs were labeled live with the following antibodies: FITC anti-CD45, PE anti-CD4, PerCP anti-CD8, PE-Cy7 anti-CCR7, APC anti-CD45RO, APC-Cy7 anti-CD3 and PB anti-CD45RA (all flow cytometry antibodies from BD Biosciences). Samples were fixed in 1% paraformaldehyde solution, read on a BD FACSCanto flow cytometer (BD Biosciences, San Jose, CA) and analyzed by FCS Express software (DeNovo Software,).