Il 1β

For cultivation under IL-17 polarizing conditions cells were plated at 2 million PBMC/well in a sterile flat-bottom 96 well plate in complete medium (RPMI 1640 with 10% fetal calf serum, 1% penicillin/streptomycin solution and 1.5% 1 M HEPES; all Thermo Fisher, Germany) and stimulated with 25 µl/ml T cell activator (Stemcell Technologies, Canada). Cytokines were added to achieve the following final concentrations: 12.5 ng/ml for IL-1β, 5 ng/ml for TGFβ, 25 ng/ml for IL-6 (all Stemcell Technologies, Canada) and 25 ng/ml for IL-23 (Miltenyi Biotec, Germany). Cells in the control condition were stimulated with T cell activator only. Cells were cultured for 7 days at 37 °C, counted on days 3, 5, 7 and analyzed by flow cytometry on day 3 and 7 after restimulation with PMA/ionomycin.

Naive CD4+ T cells from WT mice were extracted by magnetic bead negative selection using the EasySep™ mouse naive CD4+ T cell isolation kit (Catalog # 19765, Stem Cell Technology, Vancouver, Canada). Extracted Naive CD4+ T cells were cultured in 10% FBS + RPMI 1640 medium with 100 U/mL of penicillin and streptomycin and induced in plates cultured with 2 mg/mL anti-CD3 (05112–25–100, Biogems, NJ, USA) and 2 mg/mL anti-CD28 (10312–25–100, Biogems). The conditions for Th17 cell polarization in vitro: IL-6 (10 ng/mL, 78052, Stem Cell Technology), TGF-β1 (5 ng/mL, 100-21C-10, PeproTech, NJ, USA), IL-23 (25 ng/mL, 200-23-10, PeproTech), IL-1β (10 ng/mL, 78035, Stem Cell Technology). After naive CD4+ T cells were stimulated, cells were collected, and Th17 cells were quantified by flow cytometry.