Multiparametric Flow Cytometry Analysis

Surface and intracellular staining for flow cytometry analysis were performed as described previously (23 (link), 26 (link)). The antibodies used for surface staining included APC-Cy7-anti-CD4, Pacific Blue-anti-CD8a, APC-anti-CD44, APC-anti-PD-1, PerCP-Cy5.5-anti-CD43, PE-Cy7-anti-CD62L, PE-anti-CTLA-4, PE-anti-LAG-3 (all from BD Biosciences), PE-anti-CD45.1, PE-anti-CD45.2, and PE-Cy7-anti-CD45.2 (all from BioLegend). For intracellular cytokine staining, cells were fixed and permeabilized using the Intracellular Fixation and Permeabilization Buffer Set (Invitrogen) and were stained with APC-anti-interferon (IFN)γ, PerCP-Cy5.5-anti-interleukin (IL)-2, FITC-anti-tumor necrosis factor (TNFα) (from BD Bioscience). For Ki67 staining, cells were fixed and permeabilized using the True-Nuclear Transcription Factor Buffer Set (Biolegend) and were stained with BV421-anti-Ki-67 (Biolegend). Freshly isolated cells were used for all assays and about 20,000–40,000 T cells were acquired for each sample using BD FACSCanto II flow cytometer. Data analysis was performed using FlowJo software (Tree Star, Ashland, OR, USA). Cell debris and dead cells were excluded from the analysis based on scatter signals and Fixable Viability Dye eFluor 506 (eBioscience).