Modulation of Dendritic Cell Cytokine Production

FACS-sorted mDCs (2×10⁵/well) were incubated with 20 ng/ml TSLP (R&D Systems, 1398-TS-010/CF) (12 (link)), 100 μg/ml endotoxin-free curdlan (FUJIFILM Wako Pure Chemical Corporation) (24 (link)), 20 μg/ml (133 nM) anti-human Dectin-1 monoclonal antibody (anti-hDectin-1 mAb, clone 15E2) (18 (link)), 133 nM Pam₃CSK₄ (Invitrogen) or 133 nM anti-hDectin-1-Pam₃CSK₄ conjugate (described below) for indicated time. Cytokine concentration in the supernatants was measured by a bead-based multiplex immunoassay (Bio-Rad) according to manufacturer’s instruction. In cytokine blocking experiments, 10 μg/ml anti-IL-10 (BioLegend, 506813), anti-IL-10R (BioLegend, 308817), isotype control rat IgG2a (BioLegend, 400543), anti-TNFα (R&D Systems, MAB210-100) or isotype control mouse IgG1 (BioLegend, 400166) was added into culture 1h or 3h before stimulation. In some experiments, 1 μg/ml recombinant human TNFα (R&D Systems, 210-TA/CF) or IL-10 (PeproTech, 200-10) was added into culture. For inhibition assay, 10 μM STAT3 inhibitor Stattic (25 (link)) (Sigma-Aldrich, S7947-25MG), STA-21 (26 (link)) (Cayman Chemical, 14996) or Syk inhibitor R406 (24 (link), 27 (link)) (Invivogen, inh-r406) was used to inhibit kinase activity as indicated. DMSO was used as vehicle control at 0.1%.