Kinetics of CD4, DC-SIGN, and MR expression and gp120 binding during differentiation of monocytes to immature and mature MDDCs.

(A) CD4 and CLR expression and b-gp120 binding. Monocytes were stimulated to immature MDDCs as described in “Materials and methods.” Mature MDDCs were generated from day 6 to 8 by addition of 10 ng/mL TNF-α and expressed both maturation markers CD83 and CD86 by day 8 (> 70% +ve for both markers). The b-gp120, MAb to CD4 (leu3a), DC-SIGN (AZN-D2), and MR (clone 19) were added, incubated, and detected at days 0, 2, 4, 6, and 8 as outlined in Figures 1A and 4. The mean relative intensity of the isotype or negative control was subtracted from the mean fluorescent intensity for 10 000 cells. (B) HIV gp120 binding to CD4 and CLRs. At days 0, 2, 4, 6, and 8, cells were preincubated with either saturating levels of Leu3a (10 μg/mL) or mannan (5 mg/mL) and subsequently exposed to saturating levels of b-gp120 as outlined in Figures 1 and 2.