Anti tnfr1 pe

Spleens were harvested from untreated wild-type, TNFR1 KO and SPRET mice, and single cells were obtained by teasing the spleen apart by pressing it through a 100-μm cell strainer, eliminating clumps and debris. Cells were collected in 10 ml DPBS and were spin down (4°C, 7 min, 400× g). Cells were lysed with 1 ml of lysis buffer (8.3 g NH₄Cl, 1 g KHCO₃ and 200 μl 0.5 mol/l EDTA), washed and counted. A total of 1 × 10⁶ splenocytes were stained for 30 min at 4°C in the dark with the following anti-mouse antibodies: anti-TCRb-FITC, anti-CD19-PECy7, anti-CD11b-Pacific Blue, anti-Ly6C-APC, anti-Ly6G-AF700 (all from BD Biosciences) and anti-TNFR1-PE (BioLegend). Cells were washed and dissolved in FACS buffer. Fluorescent events were acquired using an LSR2 and analysed using FACSDiva software (BD Biosciences). Neutrophils were defined as SSC^highCD11b⁺Ly6G^highLy6C^high. After gating neutrophils (see Supplementary Fig S7), TNFR1⁺ cells were plotted in a histogram. The correct neutrophil population and TNFR1⁺ populations were gated using an FMO conditions without anti-Ly6G-AF700 and anti-TNFR1-PE antibodies, respectively. Analysis was performed using FlowJo software (Tree Star, Inc.)

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.