Anti-CXCR4 monoclonal antibodies recognizing overlapping epitopes differ significantly in their ability to inhibit entry of human immunodeficiency virus type 1

Abstract

CXCR4 is one of two physiologically relevant human immunodeficiency type 1 (HIV-1) entry coreceptors. Studies of CXCR4 mutants have not clearly identified the determinants of coreceptor function and specificity. We therefore used a panel of monoclonal antibodies to further elucidate CXCR4 expression, structure, and function. Our findings show the existence of conformational subpopulations of CXCR4 that are in equilibrium on the cell surface but are not cell type specific as previously reported. HIV-1 X4 isolates can interact with multiple CXCR4 conformations in order to gain entry into target cells.

FIG. 1.

Binding of anti-CXCR4 MAbs to CXCR4⁺ L1.2 cells. CXCR4⁺ L1.2 cells (5 × 10⁵) were incubated with different concentrations of each anti-CXCR4 MAb in assay buffer (1% bovine serum albumin and 0.05% azide in Dulbecco's phosphate-buffered saline) at room temperature, and binding was measured by flow cytometry after labeling with phycoerythrin (PE)-conjugated goat anti-mouse immunoglobulin G (IgG; 1:100; Caltag). Each MFI is the mean of three independent experiments ± the standard deviation. Nonlinear regression (curve fit) was used to calculate the EC₅₀ of each MAb. CXCR4⁺ L1.2 cells were incubated with biotinylated MAb 716 (1 μg/ml) in the presence of unlabeled, isotype-matched, nonspecific murine IgG or anti-CXCR4 MAbs (10 μg/ml). Binding of 716 was measured by flow cytometry after labeling with PE-conjugated streptavidin. Percent binding of 716 was calculated with the formula [(MFI 716 + anti-CXCR4 MAb)/MFI 716 alone] × 100.

FIG. 2.

Binding of anti-CXCR4 MAbs to human cell lines. CEMx174, Jurkat, PM1, and HeLa cells were stained with a panel of anti-CXCR4 MAbs (1 μg/ml), and binding was measured by flow cytometry after labeling with PE-conjugated goat anti-mouse immunoglobulin G as described in the legend to Fig. 1. The MFIs obtained for Jurkat cells were divided by 5 so that the same y-axis scale could be used for all cell lines. Each result is the mean of three independent experiments ± the standard deviation.

FIG. 3.

Inhibition of anti-CXCR4 MAb binding by AMD3100. HeLa or CEMx174 cells (5 × 10⁵) were incubated with anti-CXCR4 MAbs (1 μg/ml) in the presence of AMD3100 (1 μM; Sigma). Binding of MAbs was measured by flow cytometry after labeling with PE-conjugated goat anti-mouse immunoglobulin G. Percent MAb binding was calculated with the formula (MFI in the presence of AMD3100/MFI in the absence of AMD3100) × 100. Each result represents the mean of three independent experiments ± the standard deviation.

FIG. 4.

Inhibition of HIV-1 pseudotype entry by anti-CXCR4 MAbs. CD4⁺ HeLa cells were infected with NLluc⁺ env⁻ pseudovirions (∼10⁵ relative light units/10⁴ cells) in the presence of anti-CXCR4 MAbs (10 μg/ml) or AMD3100 (1 μM). Luciferase activity was measured in cell lysates with the Promega luciferase assay at 48 h postinfection. Percent viral entry was calculated with the formula (RLU with inhibitor/RLU without inhibitor) × 100. Each result is the mean of four independent experiments ± the standard deviation.