Dxflex system

The expression of exhausted and cytotoxic markers of T cell was analyzed by flow cytometry. After 5 days of co-culture, cells were suspended in PBS containing 2% FBS and incubated according to the manufacturer’s instructions with the following fluorochrome-labeled antibodies: anti-CD8-APC-A700 (#B49181, Beckman Coulter), anti-CD4-APC (#IM2468, Beckman Coulter), anti-TIGIT-PE-Cy7 (#372714, Biolegend), anti-CTLA-4-BV785 (#369624, Biolegend), anti-Tim-3-PE-Cy7 (#345052, Biolegend), anti-LAG-3-BV605 (#369324, Biolegend), anti-PD-1-BV510 (#367424, Biolegend), anti-Granzyme B-PE (#372208, Biolegend), anti-IFN-γ-FITC (#IM2716U, Beckman Coulter). Cells were stained with fluorochrome-conjugated antibodies for 30 min at room temperature in the dark. For intracellular staining, surface-stained cells were fixed and permeabilized using PerFix-nc Kit (#B31168, Beckman Coulter) according to the manufacturer's instructions. Flow cytometry analyses were performed on DxFlex system (Beckman Coulter) and data were analyzed using FlowJo software (v10.5.3).

NK cells were isolated as described above and incubated for 20 min at 4°C with antibodies against CD158a (Beckman Coulter, CA, USA), CD158b (Beckman Coulter), CD56 (Beckman Coulter), or CD16 (Beckman Coulter) or antibodies against LAG3 (Biolegend), TIGIT (Biolegend), CTLA4 (Biolegend), CD57 (Biolegend), NKG2A (Biolegend), CD107A (Biolegend), Nkp46 (Biolegend), or NKG2D (Biolegend). After membrane staining, the cells were fixed with Fixative Reagent (Beckman Coulter) at room temperature for 15 min, then permeabilized with 300 µl permeabilizing reagent (Beckman Coulter) to detect intracellular protein expression. Permeabilized cells were incubated for 15 min at room temperature with antibodies against Granzyme B (Biolegend), perforin (Beckman Coulter), or IFN‐γ (Beckman Coulter). Flow cytometry was conducted on a DxFlex system (Beckman Coulter), and data were analysed using FlowJo software (v10.5.3).