Antibody blockade of Dectin-2 suppresses house dust mite-induced Th2 cytokine production in dendritic cell- and monocyte-depleted peripheral blood mononuclear cell co-cultures from asthma patients

摘要

Binding of anti-Dectin-2 monoclonal antibodies to Dectin-2, ( ) The binding curve of monoclonal antibodies (MoAbs) to recombinant Dectin-2.Fc, as measured by ELISA. Microtiter plates were coated with 1 μg/mL of Dectin-2.Fc. Thereafter, MoAbs were added to the wells at a series of concentrations. A secondary goat anti-mouse MoAbs-conjugated horseradish peroxidase (HSP) was added, and colorimetric detection was performed using tetramethylbenzidine (TMB) substrate. After adding the stop solution, absorbance was measured at 450 nm using a spectrophotometer. The MoAbs 6A4G7 and 17A1D10 bound to Dectin-2.Fc in a dose-dependent manner, whereas the control MoAb 13C3G4 did not. HEK 293 T cells were transduced with pTagRFP/Dectin-2 or an empty vector. The binding of 6A4G7 MoAb (top) and 17A1D10 MoAb (bottom) to HEK 293 T cells transfected with Dectin-2 (open histogram) or empty vector (shaded histogram) was determined by flow cytometry. Data are representative of two independent experiments. Anti-Dectin-2 MoAb 6A4G7 could bind to Dectin-2 on mouse bone marrow-derived DCs (BMDCs). BMDCs were cultured from C57BL/6 (wild-type, WT) and FcεRI γ-chain knockout (KO) mice, which does not express Dectin-2 on the cell surface. BMDCs were stained with anti-Dectin-2 MoAbs (1 μg/10 cells) at 4 °C for 30 min. After washing with PBS, cells were incubated with fluorochrome-conjugated anti-mouse IgG at 4 °C for 30 min. After washing with PBS, cells were fixed with 1% paraformaldehyde. Binding was analyzed by flow cytometry