Facs canto 2 immunocytometry system

All antibodies were pre-titrated to determine appropriate working concentrations. Immunostaining with periridin-chlorophyll protein (PerCp)-Cy5.5-conjugated anti-CD3 (BD Biosciences, clone UCHT1), allophycocynanin (APC)-H7-conjugated anti-CD8 (BD Biosciences, clone SK1) and APC-conjugated anti-CD161 (BD Biosciences, clone DX12) in the phenotypic characterization of MAIT cell was performed according to the protocol set by the commercial manufacturer (BD Biosciences). Surface staining for specific receptors was performed using monoclonal antibodies (mAbs) directed against: FITC-conjugated PD-1 (clone MIH4), phycoerythrin (PE)-conjugated CCR6 (clone 11A9), PerCp-Cy5.5-conjugated CCR5 (clone 3A9) and PE-conjugated TCR Vα7.2 (clone 3C10) (all mAbs procured from BD Biosciences). Immunostained samples were washed twice prior to acquisition on a FACS Canto II Immunocytometry system (BD Biosciences) and analyzed using FACS Diva software. Data were analyzed using FlowJo (version 9.3.1 and version 10). Doublets were excluded based on FSC-H and FSC-A, lymphocytes were identified based on FSC and SSC, and dead cells were excluded based on BD Horizon Fixable Viability Stain 510 (BD Biosciences). CD3⁺ and CD8⁺ T lymphocytes were identified. Fluorescence minus one (FMO) controls was used for optimal gating.

For intracellular cytokine staining, the cells were stimulated with PMA (100 ng/ml) and ionomycin (0.67 μM) for 5 h at 37°C and 5% CO₂ prior to immunostaining. Brefeldin A (10 g/ml) was added for the last 4 h of stimulation. The immunostained samples were washed twice prior to acquisition on a FACS Canto II Immunocytometry system (BD Biosciences).

Following incubation with antigens, the cells were investigated for surface expression of co-inhibitory receptors. All antibodies were pretitrated to determine appropriate working concentrations. Cells were stained with Fixable Viability Stain (FVS510, BD Biosciences; clone R35-95) and incubated for 20 min. Monoclonal antibodies directed against CD3 (BD Biosciences clone UCHT1), CD4 (BD Biosciences clone SK7), CD8 (BD Biosciences clone SK1) and PD-1 (BD Biosciences clone MIH4), CTLA-4 (BD Biosciences clone BNI3) and TIM-3 (R and D Systems clone #344823) were added and incubated for 30 min. The samples were washed twice prior to acquisition on a FACSCanto II Immunocytometry system (BD Biosciences) and the data were analysed using FlowJo software version 10 (Ashland, OR, USA).