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PBMC and BALF samples were incubated with Fc-receptor blocking reagent (BD Biosciences) at room temperature for 15 min and surface stained with the following fluorochrome-conjugated antibodies at 4°C for 40 min: CD45-APC (HI30, BioLegend), CD3-APC-cy7 (UCHT1, BioLegend), CD8-BV510 (SK1, BioLegend), CD4-BV421 (RPA-T4, BD Biosciences), CD161-PE (HP-3G10, BioLegend), CD161-BV421 (HP-3G10, BioLegend), Vα7.2-PE-cy7 (3C10, BioLegend), CD69-FITC (FN50, BioLegend), CD69-PE (FN50, BioLegend), and PD-1-BV421 (EH12.1, BD Biosciences). MAIT cells were identified as CD3⁺CD161^high Vα7.2⁺ cells.
For intracellular cytokine staining, PBMCs were cultured in RPMI 1640 supplemented with 10% FBS in the presence of phorbol 12-myristate 13-acetate (PMA) (25 ng/ml) and ionomycin (500 ng/ml) for 40 min followed by a 3.5-h incubation with brefeldin A in a 5% CO₂ incubator at 37°C. IL-17A- and/or IFN-γ-producing cells were identified by intracellular staining with anti-IL-17A-FITC (BL168, BioLegend) and anti-IFN-γ-FITC (4S.B3, BioLegend) for 45 min after surface staining and fixation/permeabilization. Fluorescence Minus One (FMO) was used as negative control; the gating strategy for surface markers and intracellular cytokines is shown in Supplemental Figure 1. Cells were acquired by flow cytometry using a FACSVerse (BD Biosciences) and analyzed with FlowJo software (TreeStar).