Immunofluorescence analyses of CCR5-stained fresh or permeabilized peripheral blood CD4⁺ T cells. (A) Specific detection of CCR5 molecules on and inside CD4⁺ T cells. CCR5 expression of nonpermeabilized (NP, top histograms) and permeabilized (P, bottom histograms) circulating CD4⁺ T cells from a WT/WT CCR5 (left histograms) and a Δ32/Δ32 (right histograms) donor were analyzed. For CCR5 extracellular detection, whole blood was stained with a phycoerythrin (PE)–conjugated anti-CD4 mAb and the anti-CCR5 mAb 2D7 conjugated to PE-cyanin-5 (PC5; open histogram) or with an isotype control (gray filled histogram). For CCR5 intracellular detection, peripheral blood mononuclear cells (PBMCs) were permeabilized using PBS containing 0.2% saponin as previously described.²  CCR5 expression was analyzed after gating for lymphocytes on the basis of forward and side scatter and then gating on CD4⁺ cells. The percentage of CD4⁺ T cells expressing CCR5 is indicated. (B) Detection of CCR5 molecules on and inside the Δ32/Δ32 CD4⁺ T cells transduced with CCR5. PBMC from a Δ32/Δ32 donor were transduced with an HIV-1–derived vector harboring the WT CCR5 gene (right histograms) or the LacZ gene (left histograms) as a negative control as previously described,⁷  permeabilized (P, bottom histograms) or not (NP, top histograms) 48 hours later, and processed as described in panel A. The percentage of CD4⁺ T cells expressing CCR5 is indicated. (C) Detection of intracellular CCR5 molecules by flow cytometry. PBMCs from a Δ32/Δ32 donor were transduced as described in panel B, incubated 48 hours later with 1 μg/mL 2D7, permeabilized (P, right histogram) or not (NP, left histogram), and processed as described in panel A. The percentage of CD4⁺ T cells expressing CCR5 is indicated. (D) Detection of intracellular CCR5 molecules by immunofluorescence microscopy. Wide-field immunofluorescence image of nonpermeabilized (NP) and permeabilized (P) PBMCs from a WT/WT CCR5 donor labeled using the 2D7 primary antibody (1 μg/mL) and a FITC-conjugated goat anti–mouse antibody. Cells were observed with a Leica DM6000 microscope. Scale bar, 3 μm. (E) Effect of brefeldin A on CCR5 staining of permeabilized (P) CD4⁺ T cells. PBMC from a WT/WT CCR5 donor were incubated (bottom histogram) or not (top histogram) for 24 hours with 10 μg/mL brefeldin A (BFA). Cells were then incubated with 1 μg/mL 2D7, permeabilized, and then processed as described in panel C. One representative experiment of 3 performed is shown. (F) Ligand-induced internalization of CCR5. PBMCs from a WT/WT CCR5 donor were incubated (bottom histograms) or not (top histograms) for 1 hour at 37°C with 500 ng/mL MIP-1β, and permeabilized (P, right histogram) or not (NP, left histogram). Nonpermeabilized cells were processed as described in panel A, and permeabilized cells as described in panel C. One representative experiment of 3 performed is shown.