Antigenically Distinct Conformations of CXCR4

Equilibrium binding of anti-CXCR4 MAbs. (A) Jurkat CD4⁺ cells were stained with the indicated concentrations of CXCR4 MAbs 12G5, 1, 2, 8, 12, 16, 17, and 18 for 1 h followed by PE-coupled anti-mouse antibody and analyzed by flow cytometry as described in Materials and Methods. A representative experiment out of three done is shown. (B) Relative affinity for each antibody presented as normalized EC₅₀ (nEC₅₀). The EC₅₀ of each antibody was the concentration of antibody that gave a half-maximal GMCF value, based upon flow cytometric analysis using serial dilutions of the MAbs on Jurkat CD4⁺and SupT1 cells. The results are the mean and standard error of the mean obtained from three experiments with each cell type.

CXCR4 heterogeneity on lymphoid cell lines. The indicated cell lines were incubated with saturating levels of CXCR4 MAbs 12G5, 1, 2, 8, 12, 16, 17, and 18 for 1 h and then processed for flow cytometry. The cell lines are grouped into three panels (A, B, and C) for convenience. All results have been normalized to the GMCF value obtained for 12G5, which was set at 100%. The results are representative of five different experiments and are reported as the mean and standard error of the mean for each cell line.

CXCR4 heterogeneity on PBLs. Human PBLs treated for 4 days with IL-2 alone (A) or with IL-2–PHA (B) were stained with the panel of CXCR4 MAbs and analyzed by flow cytometry as described in Materials and Methods. Antibodies to CD8 and CD4 were also used to examine CXCR4 heterogeneity on these T-cell subsets. Tot., total. The results shown are the mean and standard error of the mean obtained for two or three donors in five different experiments.

CXCR4 heterogeneity on B cells. Fresh blood was stained with the panel of CXCR4 MAbs as described in Materials and Methods. All results have been normalized to the GMCF value obtained for 12G5, which was set at 100%. A representative experiment out of three done with eight donors each is shown. The mean value is indicated by a horizontal bar.

Reactivity of the CXCR4 MAbs to human, rat, and feline CXCR4. (A) Amino acid sequence comparison of the amino-terminal domain (N-term.) and the first, second, and third extracellular loops (ECL1, ECL2, and ECL3) of human, rat, and feline CXCR4. Human CXCR4 is used as the reference sequence, with different amino acids in the rat or feline sequence indicated in bold. A dash indicates amino acid identity, and a dot indicates a gap created to maximize the alignment. (B) 293T cells were transfected with human, rat, or feline CXCR4, and the reactivities of MAbs 12G5, 1, 2, 8, 12, 16, 17, 18, and 4G10 were analyzed by flow cytometry. The mean and standard error of the mean for one representative experiment out of three done in triplicate are shown.

Chemotaxis inhibition of T cells correlates with differential antibody reactivity. HS-Sultan cells were incubated with CXCR4 MAbs 12G5, 2, 1, 18, and 4G10. A fraction of the cells was analyzed by flow cytometry (white columns), and the rest was used for a chemotaxis assay (black columns). The mean and standard error of the mean for one representative experiment out of three done in triplicate are shown. The chemotaxis index is defined as the ratio of the number of cells that migrate under a given condition to the number of cells that migrate in the absence of the chemoattractant. NEG., chemotaxis index with no chemoattractant in the lower chamber; POS., chemotaxis in response to SDF-1α. The column labeled SDF is the chemotaxis control and shows the chemotaxis index when the concentration of SDF-1α in the upper chamber is equivalent to that in the lower chamber.

Sulfation and glycosylation do not influence antibody reactivity. 293T cells were transfected with CXCR4 or a CXCR4 construct lacking the first 12 (Δ12-CXCR4), 15 (Δ15-CXCR4), or 23 (Δ23-CXCR4) N-terminal amino acids. At 48 h later, the cells were stained with MAbs 12G5, 1, 2, and 18 and analyzed by flow cytometry. A representative experiment out of three done is shown.