Rm tnfα

Isolation of primary epidermal keratinocytes was performed as described earlier (23 (link)). Briefly, disinfected tail skins of adult mice were digested overnight at 4˚C with trypsin (0.25%; w/o CaCl₂, Gibco, Thermo Fisher Scientific, USA). Detached epidermal cells were seeded overnight at 32°C, 5% CO₂ in minimum essential medium with Earle`s Balanced Salt Solution medium (Lonza, Switzerland) containing 0.2 mM CaCl₂ (Merck, Germany). Subsequently 1x10⁶ isolated cells were cultured on fibronectin (Roche, Switzerland)/rat tail collagen I (Becton Dickinson, Corning, USA)-coated six well-culture plates in Keratinocyte Growth Medium 2 with supplement mix (PromoCell, Germany) to the confluence of 70–80%. Culture and stimulation conditions in Keratinocyte Growth Medium 2 (PromoCell, Germany) are indicated in Supplementary Figure 1. Times of stimulations are indicated in figure legends. Reagents: flagellin from Salmonella typhimurium (100 ng/ml, InvivoGen, France), rm IL-1α, rm IL-17A, rm IL-17F, and rm TNFα (100 ng/ml, BioLegend, USA), S100A8 (8 (link)). Possible endotoxin contaminations of S100A8 proteins were evaluated by a sensor chromogenic LAL endotoxin assay (GenScript, USA) and were < 2 pg/µg protein.

Naive CD4⁺ T cell isolation kit was purchased from Miltenyi Biotec (Cologne, Germany). Anti-CD3/CD28 coated beads were purchased from Gibco (New York, USA). RhIL-2, rhTGF-β, rmIL-6, and rmIL-12 were purchased from R&D Systems (Minneapolis, MN). Anti-TNFR1 PE, anti-TNFR2 PE, anti-Thy1.1 PercP/Cy5.5, anti-LAP APC, anti-PD-1 APC, anti-Nrp-1APC anti-GITR PE, anti- Ki-67 PE, anti-CD4 PercP/Cy5.5, anti-IFN-γ PE, anti-IL-17A APC, rmTNFα, anti-IL-4 antibody, anti-IFN-γ antibody, and CFSE were purchased from Biolegend (San Diego, CA). PMA, ionomycin, and cyclosporin A (CsA) were purchased from Calbiochem-EMD Millipore (Dormstadt, Germany). CFA was purchased from Sigma-Aldrich. Incomplete Freund's Adjuvant (IFA, Difco, MI, USA), killed Mycobacterium tuberculosis (strain H37Ra; Difco), and myelin oligodendrocyte glycoprotein (MOG, 35–55 peptide, (AnaSpec Inc, Fremont, CA) were also purchased.