Rorγt

Staining was performed as previously described [7 (link),38 (link),41 (link)]. For intracellular staining of Foxp3, RORγt, and Helios, the isolated cells were stained with monoclonal antibodies (mAbs) against CD3e and CD4 (both from BioLegend, San Diego, CA, USA) and subsequently fixed with a True-Nuclear Transcription Factor Buffer Set (BioLegend). The cells were then stained with mAbs against Foxp3 (Thermo Fisher Scientific, Waltham, MA, USA), RORγt (BD Bioscience, Franklin Lakes, NJ, USA), and Helios (BioLegend), and then subjected to flow cytometry. For the intracellular cytokine staining, the LP cells were cultured for 5.5 h in a complete medium supplemented with 50 ng/mL PMA, 500 ng/mL ionomycin, and 5 μg/mL brefeldin A (Sigma, Burlington, MA, USA). The cells were harvested and stained with mAbs against cell surface antigens, followed by fixation in a fixation/permeabilization buffer (BioLegend). The fixed cells were subjected to intracellular staining, with mAbs, against IFNγ and TNFα (BioLegend). The stained samples were analyzed using a NovoCyte Flow Cytometer (ACEA Biosciences, San Diego, CA, USA) and the FlowJo software version 10 (TOMY Digital Biology, Tokyo, Japan).

Splenic and LN cells were stained for viability using a LIVE/DEAD Fixable Blue Dead Cell Stain Kit, for UV excitation (ThermoFisher). Cells were then washed with DPBS (Gibco) plus 10% FBS (Atlanta Biologicals) – and labeled with mAbs specific for TCR-β, CD4, CD25, CD44, Tigit, Tim-3, TGF-β (BioLegend), CD8α, CD39, PD-1, CXCR3 (eBioscience), CD62L, CD103, and CTLA-4 (Becton Dickinson [BD], Franklin Lakes, NJ) (Supplemental Table 1). Cells were then fixed and permeabilized using the True-Nuclear Transcription Factor Buffer Set (BioLegend, San Diego, CA) and labeled with mAbs specific for IFN-γ, IL-17, IL-4, IL-13 (BD), IL-10, Foxp3, Tbet, Gata-3 (eBioscience), TNF-α and RORγt (BioLegend) (Supplemental Table 1). Fluorescence was acquired on a BD Fortessa flow cytometer with BD FACSDiva software. All samples were analyzed using FlowJo software (BD).