Effect of trypsin on CD4, MR, and DC-SIGN mAbs and b-gp120 binding to MDDCs.

(A) CD4 (Leu3a) (Ai), DC-SIGN (AZN-D2) (Aii), and MR (clone 19) (Aiii) staining before trypsinization. A total of 2 μg/mL of mAb to CD4, DC-SIGN, and MR was added as outlined in “Materials and methods.” The mAb binding was detected via goat antimouse FITC (1 μg/mL) (Becton Dickinson) and fluorescence measured as in Figure 1. Gray histograms represent antibody staining with open overlaid histogram staining by matching isotype controls. (B) CD4 (Leu3a) (Bi), DC-SIGN (AZN-D2) (Bii), and MR (clone 19) (Biii) staining after trypsinization. Cells were treated with 0.25% trypsin at 37°C for 5 minutes and subsequently washed in normal media before the addition of mAbs to CD4, DC-SIGN, and MR as in panel A. (C) The b-gp120 binding before trypsinization. (D) The b-gp120 binding to MDDCs after trypsinization: effect of inhibitors. Trypsinized cells were mock-treated (Di) or treated with excess mannan (5 mg/mL) (Dii), EGTA (5 mM) (Diii), or anti-MR (clones 19 and 3.29) (5 μg/mL) (Div) for 30 minutes at 4°C. The b-gp120 was added and detected as in Figure 1A. Gray histograms represent gp120 staining and open overlays matched negative controls (treatment without addition of b-gp120).