Flow cytometry
For flow cytometry, the cell surface was stained with the following antibodies specific for mouse proteins: anti-CD4, anti-CCR6, anti-CD25, anti-CTLA-4, anti-GITR, and anti-ICOS (BioLegend). Mouse Regulatory T Cell Staining Kit (eBioscience; San Diego, CA) was used to stain the transcription factors with anti-Foxp3 (Biolegend) and anti-RORγt antibodies (BD), anti- Blimp-1 (Biolegend) and anti-Helios (Biolegend), or intracellular cytokine staining with anti-IL-17A and anti-IL-10 (BioLegend). Data were acquired using the FACSVerseFlow Cytometer (Becton Dickinson; Mountain View, CA) or the SH800 Cell Sorter (Sony, Tokyo, Japan) and analyzed with FlowJo software (Tree Star; Ashland, OR).