W. decline in CD4+ T lymphocytes (5, 15, 19). It is still unclear what selective pressures collude to drive the outgrowth of CXCR4-using (X4) isolates. The switch from CCR5 to CXCR4 may be the result of immune selection, fluctuations in target cell populations, and conformational constraints imposed by the constructions of gp120 and the coreceptors. Understanding the details of gp120 relationships with CCR5 and CXCR4 will provide insight into the mechanism of coreceptor switching. Our previous studies demonstrated the stem of gp120 hypervariable loop 3 interacts with specific sulfotyrosines and negatively charged residues in the amino-terminal website (Nt) of CCR5. The hypervariable loop 3 crown may bind residues in the second extracellular loop (ECL2) (6, 7, 20, 21). In contrast, CXCR4 domains and residues involved in gp120 attachment possess proven much more difficult to identify (examined in research 11). CXCR4 mutagenesis suggests that the gp120 binding site is definitely variable and isolate specific, involving charged and tyrosine residues that are dispersed throughout the four extracellular domains (3, 4, 14, 16, 18, 22, 24). It is still unclear whether any particular amino acids or domains of CXCR4 are totally required for viral access and to what degree the gp120 of any given isolate is able to use different Rabbit Polyclonal to PRKY residues on this coreceptor. Additionally, it has been suggested that antigenically unique conformations of CXCR4 are present within the cell surface, perhaps only some having coreceptor function (1). In order to further elucidate the determinants of CXCR4 coreceptor function, a panel of seven self-employed anti-CXCR4 monoclonal antibodies (MAbs) was characterized for binding, epitope specificity, and HIV-1 access inhibition. Binding of MAbs 701, 708, 716, 717, 718, 12G5 (R&D Systems), and 4G10 (9) to murine L1.2 hybridoma cells (23) expressing high levels of human being CXCR4 was analyzed by flow cytometry (Fig. ?(Fig.1).1). None of the MAbs cross-reacted with murine CXCR4, as evidenced by minimal binding to parental L1.2 (data not shown). Furthermore, binding of WQ 2743 the seven MAbs to CXCR4+ L1.2 was not altered by the presence of saturating concentrations of an anti-mouse CXCR4 MAb (R&D Systems) (data not shown). All test MAbs exhibited related half-maximal concentrations of binding (EC50s) to CXCR4+ L1.2 cells, ranging between 0.17 and 0.45 g/ml (Fig. ?(Fig.1).1). However, maximal binding of the MAbs assorted and did not correlate with the EC50s. Saturating concentrations of MAbs 701, 717, and 718 generated the highest mean fluorescence intensities (MFI, 1,200), whereas MAbs 708, 12G5, and 4G10 yielded the lowest signals (MFI, 400). Saturating binding of MAb 716 was intermediate (MFI, 800). Anti-CXCR4 MAbs efficiently cross-competed with each other for binding to CXCR4, e.g., binding of MAb 716 to CXCR4+ L1.2 was inhibited strongly by MAbs 717, 718, and 701 and somewhat less by MAbs 708 and 12G5 (Fig. ?(Fig.1,1, bottom). The test MAbs consequently bind human being CXCR4 specifically and with high affinity and may identify subpopulations of coreceptor molecules on the basis of variations in saturating binding to CXCR4+ L1.2 cells. WQ 2743 MAbs with the highest saturating binding presumably identify the widest array of CXCR4 subpopulations. Open in a separate windows 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 imply of three self-employed experiments the standard WQ 2743 deviation. Nonlinear regression (curve match) was used to calculate the EC50 of each MAb. CXCR4+ L1.2 cells were incubated with biotinylated MAb 716 (1 g/ml) in the presence of unlabeled, isotype-matched, nonspecific murine IgG or anti-CXCR4 MAbs (10 g/ml). Binding of 716 was measured by circulation cytometry after labeling with PE-conjugated streptavidin. Percent binding of 716 was determined with the method [(MFI 716 + anti-CXCR4 MAb)/MFI 716 only] 100. Since differential patterns of anti-CXCR4 MAb binding to CXCR4+ cells have been reported (1), we compared the binding of test MAbs to four human being cell lines that naturally communicate CXCR4, including HeLa (CD4? endothelial cell collection), CEMx174 (CD4+ T-B cross cell collection), Jurkat (CD4+ T-cell collection), and PM1 (CD4+ T-cell collection). HeLa, CEMx174, and PM1 cells communicate similar levels of CXCR4, whereas Jurkat cells communicate fivefold higher levels of the coreceptor, as determined by relative MFI measured by circulation cytometry.