Immunolocalization of HLE in HIV-1 permissive and nonpermissive clones. By TEM, HLE was found only on the cell surface of HIV-1 permissive clone 10 (A), and only in the intracytoplasmic compartment of unstimulated HIV-1 nonpermissive clone 17 (B). HLE was found on the cell surface and in the cytoplasm when clone 17 was stimulated by LPS (C). Homogeneous receptor density at equilibrium was achieved by exposing cells to α₁PI by culturing in medium containing 10% FBS prior to staining. Cells were prepared for TEM in 3 separate experiments, examined independently by 2 investigators, and representative images are presented. Each bar represents 0.1 μm. (D) HIV-1 permissive and nonpermissive subclones were gated for live cells and were analyzed for coreceptor levels using 3-color flow cytometry to detect PerCP-conjugated anti-CD4, PE-conjugated anti-CXCR4, and FITC-conjugated anti-HLE. Cells were first interacted with anti-CD4 and second with anti-HLE and anti-CXCR4 (▨). Alternatively, cells were first interacted with anti-HLE and second with anti-CD4 and anti-CXCR4 (▪). HLE was detected on the surface of nonpermissive clone 17 after stimulating with LBP and LPS to induce granule release. CD4, CXCR4, and HLE expression on clone 17 in the presence of LBP was indistinguishable from medium alone (data not shown). Relative fluorescence intensity (RFI) were calculated as receptor MFI minus isotype control MFI. Flow cytometric analysis was performed on 10 000 to 30 000 events in 4 separate experiments. Data represent one analysis.