Presence of IVIgs on the surface and in the cytoplasm of DCs after maturation. (A) DCs were stimulated for 18 hours in the absence (left plot) or presence of biotinylated IVIgs (middle plot) or HSA (right plot). Using streptavidin-APC, binding of IVIg-biotin was detected on 92% (± 3%) of the surface of IVIg-DCs after maturation (N = 3). (B) DCs were stimulated for 18 hours in the absence (left plot) or presence of IVIgs (middle plot) or HSA (right plot). Rabbit anti–human IgG-FITC F(ab)2 was used to detect IVIg binding on the DC membrane. This showed equivalent results (N = 4). (C) To determine internalization of IVIgs during maturation, biotinylated IVIgs on the DC surface were detected by streptavidin-PerCP. Thereafter, residual-free biotinylated IVIgs on the cell surface were blocked using biotin-blocking reagent, DCs were fixed and permeabilized, and intracellular biotinylated IVIgs were detected using streptavidin-APC. We observed that IVIgs were present on the surface as well in the cytoplasm of the DCs (N = 3). Numbers on plots are percentage positive cells (± SD).
