Jagged1

The cells were treated with or without 1 μg/ml Jagged-1 (R&D Systems, Minneapolis, MN, USA) for 72 h in the presence of anti-CD3/CD28 beads at a bead-to-cell ratio of 1:1 (Invitrogen, Carlsbad, CA, USA), 20 ng/ml IL-6, 10 ng/ml TGF-β, 20 ng/ml IL-23 (PeproTech, Rocky Hill, NJ, USA), 10 μg/ml anti-IFN-γ and 10 μg/ml anti-IL-4 (eBioscience, San Diego, CA, USA). The additional cells were treated with the equal volume of phosphate buffered saline (PBS) as a control. Six hours before the end of the treatment, the cells were stimulated with 50 ng/ml phorbol-12-myristate-13-acetate (PMA) (Sigma-Aldrich, St. Louis, MO, USA) plus 1 μg/ml of ionomycin (Alexis, Lausen, Switzerland) for the below experiments. Meanwhile, 10 μg/ml of GolgiStop (Becton Dickinson, Franklin Lakes, NJ, USA) was added to the cells. The cells were washed with PBS and stained with FITC-conjugated anti-mouse CD4 (0.125 μg per million cells) at 37°C for 20 min. The cells were washed, fixed, permeabilized with Fixation/Permeabilization Buffer and intracellular-stained with PE-conjugated anti-mouse IL-17 (0.05 μg per million cells) and FITC-conjugated anti-mouse IFN-γ (0.25 μg per million cells) (eBioscience, San Diego, CA, USA) for 30 min at 4°C, and analyzed with a flow cytometer (FACSCalibur, Becton Dickinson, Mountain View, CA, USA).