Rorγt apc

Flow cytometry of CD4⁺ T cells was performed as previously described (7 (link)). The following antibodies were used for T cell phenotyping: CD45 APC-Cy7 (Biolegend), CD4 APC (Biolegend), FoxP3 PE (Thermo Fisher/eBioscience), RORγt PerCP-Cy5.5 (BD Bioscience), and IL-17A-PE (Biolegend), and dead cells were excluded using Zombie NIR (Biolegend). To detect intracellular IL-17A, isolated lymphocytes were first restimulated with 5 ng/mL phorbal 12-myristate 13-acetate and 500 ng/mL ionomycin in the presence of monensin (Biolegend) for 3.5 h. FoxP3 and RORγt were analyzed in unstimulated cells. The Vβ chain repertoire analysis used the following antibodies: Vβ2 AF647 (Biolegend), Vβ3 BrilliantViolet 510 (BD), Vβ5.1/5.2 PE-Cy7 (Biolegend), Vβ6 BrilliantViolet 650 (BD), Vβ7 FITC (Biolegend), Vβ8.1/8.2 APC-Vio770 (Miltenyi), Vβ10b BrilliantViolet 711 (BD), Vβ11 BrilliantViolet 421 (BD), Vβ12 BrilliantViolet 480 (BD), Vβ13 PerCP-eFluor710 (ThermoFisher/eBioscience), Vβ14 biotin (ThermoFisher/eBioscience) with streptavidin Qdot800 (ThermoFisher), Vβ17a BrilliantViolet 605 (BD), CD45 BrilliantViolet 750 (BD), FoxP3 PE (ThermoFisher/eBioscience) RORγt APC (BD), and CD4 BrilliantViolet 570 (Biolegend). T cell phenotyping data and Vβ chain phenotyping data were acquired using an Aurora spectral cytometer (Cytek) and data were analyzed using SpectroFlo and FlowJo 10 software.