Soluble anti cd3e

Naïve CD4⁺ T cells were isolated from the spleen of WT or GPx1^−/− × Cat^−/− mice using a naive CD4⁺ T cell isolation kit (R&D Systems, Minneapolis, MN). For the induction of Th₁₇ cell differentiation, 1×10⁵ naïve CD4⁺ cells were stimulated with soluble anti-CD3e (1 µg/mL) and soluble anti-CD28 (1 µg/mL, e-Biosciences) in the presence of 2×10⁴ CD11c⁺ DCs for 24 hours, and were cultured further for 2.5 days in the presence of TGF-β1 (5 ng/mL) and IL-6 (20 ng/mL) purchased from R&D Systems. For intracellular cytokine staining, cells were re-stimulated for 4 hr with PMA (25 ng/mL) and ionomycin (250 ng/mL, Sigma) in the presence of a protein trapsport inhibitor containing monensin (BD Biosciences). Then, the cells were harvested and stained for intracellular IL-17A and IFN-γ using a commercial kit for fixation and permeabilization ((BD Biosciences), anti-mouse IL-17A-PE (eBioscience) and anti-mouse IFN-γ-FITC (eBioscience). For the induction of iTreg differentiation, 1×10⁵ naïve CD4⁺ cells were stimulated with plate-coated anti-CD3e (50 ng/well) and soluble anti-CD28 (1 µg/mL) in the presence of TGF-β1 (5 ng/ml) and human recombinant IL-2 (10 U/mL, BD Biosciences). After three days of culture, the cells were harvested for intranuclear staining for FoxP3, using a mouse regulatory T cell staining kit.

Spleens from mice were collected and cells were purified from single-cell suspensions using a CD4⁺ naive T-cell isolation kit (Stemcell Technologies, Vancouver, British Columbia, Canada) according to the manufacturer’s guidelines. Following this, purified CD4⁺ naive T cells (2  ×  10⁵) were added to 12-well plates which had been added with RPMI-1640 medium containing soluble anti-CD3e (0.5 μg/ml; eBioscience, Waltham, Massachusetts, U.S.A), soluble anti-CD28 (1.0 μg/ml; eBioscience), and IL-2 (20 ng/ml; eBioscience). The cells were incubated with BECs for 24 h. Then, the cells were harvested for flow cytometry.