Binding of antibodies to DC-SIGN neck region triggers endocytosis. (A) CHO cells stably transfected with wild-type DC-SIGN or DC-SIGN lacking the neck (ΔRepeat) or lacking the CRD (ΔCRD) were incubated with anti-CRD (AZN-D1) or anti-neck (H200) antibody at 4°C, washed, and incubated for 10 or 20 minutes at 37°C to induce endocytosis. Cells were analyzed by flow cytometry, and the percentage of internalization was calculated. (B) Steady-state confocal microscopy image of immature DCs labeled with antineck (H200, green) and anti-CRD (AZN-D1, red) antibodies. Both antibodies label the same DC-SIGN molecules on one representative cell of several images from 2 experiments. Scale bar represents 5 μm. (C) Anti-neck and anti-CRD antibody internalization by immature DCs after 5 or 20 minutes at 37°C. Experiments were performed in triplicate, and 1 representative experiment of 3 is shown. Data represent mean ± SD.