Leukocyte activation cocktail

Peripheral blood mononuclear cells (PBMCs) were isolated using Ficoll density gradients. For the phenotypic analysis, PBMCs were stained with FITC-LAG-3, FITC-CD8, PE-2B4, PE-CD8, PE-CTLA4, PE-CF594-CD3, PE-CY7-CD4, BV421-PD-1, V450-CD8 (BD Biosciences, Franklin Lakes, NJ), and APC-Tim-3 (eBioscience, San Diego, CA, USA). For cytokine analysis, PBMCs were stimulated with a Leukocyte Activation Cocktail (eBioscience, San Diego, CA, USA), at 37° C for 4 h prior to intracellular staining using Pharmingen's staining protocol. Anti-human monoclonal antibodies against FITC-IFN-γ, PE-TNF-α (eBioscience), and PB-IL-2 (Biolegend, San Diego, CA, USA) were used. Corresponding isotype-matched controls were purchased from BD Biosciences and eBioscience. Data was acquired on a Gallios instrument (Beckman Coulter, Brea, CA, USA) and analyzed with FlowJo software (Ashland, OR, USA). The gating strategy for cytokines and exhaustion markers is shown in Supplementary Figure 1.

Peripheral blood monouclear cells (PBMCs) were stimulated with Leukocyte Activation Cocktail (eBioscience) at 37°C for 4 hours before intracellular staining using the PharMingen staining protocol. The cells were stained with PE‐CF594‐CD3, APC‐CD4, and V450‐CD8 monoclonal antibody (BD Biosciences) for 30 minutes at 4°C and then fixed and permeabilized using Cytofix/Cytoperm fixation/permeabilization solution (eBioscience) according to the manufacturer's instructions. The cells were stained with fluorescein isothiocyanate‐IFN‐γ, PE‐IL‐2, PE‐IL‐4, and PE‐CY7‐TNF‐α (eBioscience) for 45 minutes on ice and then washed. Corresponding isotype‐matched controls were purchased from BD Biosciences and eBioscience. Data were acquired on a Gallios instrument (Beckman Coulter, Brea, CA) and analyzed with FlowJo software (Ashland, OR).