Anti-CCR5 mAb binding is decreased with BCD-treated cells.

(A) CCR5 epitopes recognized by mAbs, clones CTC5, 45502, 45523, 45531, and 45549, are diagrammed. (B) CEM-R5 cells were treated with BCD and subsequently analyzed by flow cytometric analysis as described in “Materials and methods.” The results for untreated cells are graphed as a filled peak, and BCD-treated cells are graphed as a solid line. (C) CEM-R5 cells treated with BCD were then reloaded with cholesterol and subsequently analyzed for binding of control IgG2a, anti-CD45, and anti-CCR5 mAbs, CTC5 and 45531. Results are expressed as MFI from one representative experiment of 3. (D) BCD-treated CEM-R5 cells were incubated with either CTC5 or 45531. Cells were subsequently washed, fixed, and permeabilized or simply treated with PBS, containing 2% FBS, and then stained with GAM-AF488. The percentage of positive is expressed on the graph. Negative control mouse IgG2a binding to nonpermeabilized untreated cells was gated as 2% positive. Crosshatched bars represent permeabilized cells and noncrosshatched bars represent nonpermeabilized cells.