Anti tigit pe cy7

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).

Th17–T_reg subsets (IL-17A⁺Foxp3^neg, IL-17A⁺Foxp3⁺, IL-17A^neg Foxp3^neg and IL-17A^neg Foxp3⁺) were stained with anti-neuropilin-1-PE-Cy7 (6 μg ml⁻¹, eBioscience, clone: 3DS304M), anti-Folr4-PE-Cy7 (6 μg ml⁻¹, eBioscience, clone: EBio12A5), anti-GARP-eFluor450 (6 μg ml⁻¹, eBioscience, clone: YGIC86), anti-ICOS-eFluor450 (6 μg ml⁻¹, eBioscience, clone: ISA-3), anti-Nrp-1-PE-Cy7 (6 μg ml⁻¹, eBioscience, clone: 3DS304M), anti-ST2-PerCP-Cy5.5 (6 μg ml⁻¹, Biolegend, clone: DIH9), anti-TIGIT-PE-Cy7 (6 μg ml⁻¹, Biolegend, clone: 1G9), anti-CD73-PerCP-Cy5.5 (6 μg ml⁻¹, Biolegend, clone: TY/11.8) and anti-Lag3-eFluor710 (6 μg ml⁻¹, eBioscience, clone: eBioC9B7W). The cells were fixed with Foxp3 Fix/Perm Buffer Set and stained intracellularly with anti-Helios-eFluor450 (6 μg ml⁻¹, eBioscience, clone: 22F6). Cells were analysed by flow cytometry (LSRFortessa, BD Biosciences). The fluorescence minus one controls were used to identify the gating boundaries.