Flow cytometry

Cell surface molecular expression and intracellular cytokine production were evaluated using flow cytometry. FITC-conjugated anti-human CD8, IFN-γ, Ki67, or anti-mouse CD8, PE-conjugated anti-human Tim-3, T-bet, TGF-β1, or anti-mouse T-bet or GATA-3, PE/CY7-conjugated anti-human IL-10, TNF-α, CD62L, CD8, IFN-γ, IL-17A, or TGF-β1, APC-conjugated anti-human CTLA-4, IL-4, Foxp3, ROR-γt, or anti-mouse TNF-α or IL-10, Brilliant Violet 421-conjugated anti-human CD107, Ki67, IL-4, IL-10, GATA-3, IL-17A, or anti-mouse IFN-γ or IL-4, Pacific blue-conjugated human CD44 (Biolegend, USA) antibodies were used. For intracellular staining, cells were fixed and permeabilized using the Fix/Perm kit (Biolegend, USA). Flow cytometry was performed on a Beckman-Coulter CyAn ADP cytometer (Beckman-Coulter, USA) and analyzed with FlowJo software (Tree Star, Ashland, USA).