M-tropic HIV envelope induces migration of iDCs via CCR5. (A) The ability of monocyte-derived iDCs to migrate in response to various concentrations of HIV-1 M-tropic gp120 (0-50 nM) and the heat-inactivated (HI) control was analyzed using transwell migration assays. *P < .05 versus the heat-inactivated control. (B) Migration of iDCs in response to various clones of M-tropic gp120, YU2, ADA, and BaL (10 nM) was determined using transwell assays. *P < .05 versus the untreated control. (C) iDCs were pretreated with anti-CCR5 neutralizing antibodies (10 μg/mL) or RANTES (100 nM) for 1 hour at 37°C. The cells were then used in transwell migration assays in response to M-tropic gp120 (10 nM) and AT-2 HIV-1 (used at a final concentration of 3 μg p24/mL). *P < .05 versus the gp120-treated control. **P < .05 versus the AT-2 HIV-treated control. Data represent mean ± SD of 3 independent experiments. (D) iDCs were treated with tyrphostin (5 μM) or vehicle alone at 37°C for 1 hour and subsequently incubated with medium alone or M-gp120 (100 nM) at 37°C for different periods of time. After washing, cells were stained with control mouse IgG or monoclonal CCR5-FITC antibody. The percentage of cells positively stained with anti-CCR5 Ab was determined by flow cytometry. The graph shows a comparison of the percentage of internalization between the tyrphostin A9–treated and vehicle-treated iDCs stimulated with M-gp120. Results are representative of 3 separate experiments.