Flow cytometry

For flow cytometry analysis, restimulated lymphocytes were treated with 5 µg/mL brefeldin A (Biovision, San Francisco, CA, USA) for 3–4 h to block cytokine release. Splenic and LN lymphocytes were subjected to a viability stain using a LIVE/DEAD Fixable Blue Dead Cell Stain Kit, for UV excitation (ThermoFisher). Cells were then washed with Dulbecco’s PBS (Gibco, ThermoFisher) plus 10% fetal bovine serum (Atlanta Biologicals), and labeled with mAbs specific for TCR-β, CD4, CD8α, CD19, CD25, CXCR5 (clone L138D7), PD-1 (clone 29F.1A12), TGF-β (BioLegend, San Diego, CA), and CD39 (eBioscience, San Diego, CA). The mAb clones were the same as those previously described³⁹ unless indicated. Cells were then fixed and permeabilized using the True-Nuclear Transcription Factor Buffer Set (BioLegend) and labeled with mAbs specific for IFN-γ (clone XMG1.2), IL-10 (clone JES5-16F3), IL-17 (clone TC11-18H10.1), Bcl-6 (clone 7D1; BioLegend); IL-6 (clone MP5-20F3; BD Pharmingen, San Jose, CA); IL-21 (clone mhalx21), GM-CSF (clone MP1-22E9), and Foxp3 (clone FJK-16a; eBioscience). Fluorescence was acquired on a Fortessa flow cytometer (Becton Dickinson Franklin Lakes, NJ), using FACSDiva software (Becton Dickinson). All samples were analyzed using FlowJo software (BD Biosciences, Ashland, OR).