Mouse il 23

CD4⁺ T-cells from male Lewis rats were obtained from splenocytes and used in Th17 differentiation assays as previously described [23 (link)]. In brief, cells were incubated with the following rat-specific antibodies: CD8a (clone OX8; BioLegend), CD45RA (clone OX33; BioLegend), CD11b/c (clone OX42; BD Biosciences), CD25 (clone OX39; eBioscience), and erythrocytes (clone OX83; Cedarlane), followed by a washing step. Cells were incubated with anti-IgG coated microbeads (Miltenyi Biotec) and unwanted cells were magnetically depleted using a MACS column system according to the manufacturer`s instructions (Miltenyi Biotec). CD4<sup>+</sup> T-cells were stimulated with rat-specific anti-CD3 (pre-coated to the plated at 4 μg/ml, clone G4.18) and soluble anti-CD28 antibody (2 μg/ml, clone JJ319, BioXcell) together with recombinant human TGF-β1 (5 ng/ml, BioLegend), mouse IL-6 (20 ng/ml, BD Pharmingen), rat IL-1β (10 ng/ml, R&D Systems), and mouse IL-23 (10 ng/ml, BioLegend) in the presence of anti-rat IFN-γ antibody (5 μg/ml, BioLegend). After 72 hrs of incubation, the supernatants were harvested and IL-17A cytokine concentrations were quantified by ELISA according to the manufacturer`s specifications (eBioscience).

For detection of cytokines in serum and supernatants of cell cultures, commercial ELISA kits for human and mouse IL-17A (Biolegend) with mouse IL-23 (Biolegend), TNF-α, CCL5, CCL10, PGE₂ and TGF-β1 (all Senxiong Biotech, China) were used according to the manufacturers’ instructions. For all assays, optical density was determined on a plate reader (Thermo Fischer Scientific, USA) at an absorbency of 450 nm with wavelength correction at 540 nm for the optical imperfections on the plate.

Teff cells (3 × 10⁵ cells/well) were cultured in complete RPMI-1640 medium and stimulated with Dynabeads^® Mouse T-Activator CD3/CD28 (8 μl/well) for 72 h along with cytokines and neutralizing antibodies for the desired polarization as follows: human-IL-6 (100 ng/ml; PeproTech. INC.), mouse-IL-23 (10 ng/ml; Biolegend), human-TGF-β (5 ng/ml), anti-IL4 (10 μg/ml; Biolegend) and anti-IFN-γ (10 μg/ml; Biolegend) for Th17; mouse IL-2 (10 ng/ml), human-TGF-β (2 ng/ml), anti-IL4 (10 μg/ml) and anti-IFN-γ (10 μg/ml) for inducible Treg (iTreg) cell polarization. Cells were restimulated with PMA (50 ng/ml), ionomycin (1 μg/ml) and brefeldin A (1 μg/ml) for 6 h. Cells were harvested and stained with anti-IL-17 or anti-Foxp3 antibodies and analyzed by flow cytometry.