Multiparameter Flow Cytometry Analysis

MLN and spleen single-cell suspensions were prepared for flowcytometry analysis as previously described [61 (link)]. The cell suspensions were incubated with anti-mouse CD16/CD32 (Mouse BD Fc Block; BD Biosciences, Franklin Lake, NJ, USA) in PBS + 1% BSA for 15 min on ice to block unspecific binding sites. Afterwards, cells were stained with the following surface markers CD4-PerCp-Cy5.5, CD69-PE-Cy7, CXCR3-PE, CD25-AlexaFluor488, CD25-PE, (all purchased from eBioscience, San Diego, CA, USA) or T1ST2-FITC (MD Bioproducts, St. Paul, MN, USA) for 30 min on ice. Fixable Viability dye eFluor 780 (eBioscience) was used to exclude non-viable cells. Next, the cells were fixed and permeabilised with the Foxp3 Staining Buffer Set (eBioscience) according to the manufacturer’s protocol and then stained with the intracellular markers Foxp3-PE-Cy7, RORɣt-PE, IRF4-FITC, Tbet-eFluor660 or Gata3-eFluor660 (all purchased from eBioscience). Cells were measured on BD FACSCanto II flow cytometer, and results were analysed with FlowLogic software (Inivai Technologies, Mentone, Vic, Australia). The used gating strategy is shown in Figure S1.