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. 1997 Mar 4;94(5):1925-30.
doi: 10.1073/pnas.94.5.1925.

The HIV coreceptors CXCR4 and CCR5 are differentially expressed and regulated on human T lymphocytes

C C Bleul  1 , L Wu, J A Hoxie, T A Springer, C R Mackay
Affiliations

The HIV coreceptors CXCR4 and CCR5 are differentially expressed and regulated on human T lymphocytes

C C Bleul et al. Proc Natl Acad Sci U S A. .

Abstract

The chemokine receptors CXCR4 and CCR5 function as coreceptors for HIV-1 entry into CD4+ cells. During the early stages of HIV infection, viral isolates tend to use CCR5 for viral entry, while later isolates tend to use CXCR4. The pattern of expression of these chemokine receptors on T cell subsets and their regulation has important implications for AIDS pathogenesis and lymphocyte recirculation. A mAb to CXCR4, 12G5, showed partial inhibition of chemotaxis and calcium influx induced by SDF-1, the natural ligand of CXCR4. 12G5 stained predominantly the naive, unactivated CD26(low) CD45RA+ CD45R0- T lymphocyte subset of peripheral blood lymphocytes. In contrast, a mAb specific for CCR5, 5C7, stained CD26(high) CD45RA(low) CD45R0+ T lymphocytes, a subset thought to represent previously activated/memory cells. CXCR4 expression was rapidly up-regulated on peripheral blood mononuclear cells during phytohemagglutinin stimulation and interleukin 2 priming, and responsiveness to SDF-1 increased simultaneously. CCR5 expression, however, showed only a gradual increase over 12 days of culture with interleukin 2, while T cell activation with phytohemagglutinin was ineffective. Taken together, the data suggest distinct functions for the two receptors and their ligands in the migration of lymphocyte subsets through lymphoid and nonlymphoid tissues. Furthermore, the largely reciprocal expression of CXCR4 and CCR5 among peripheral blood T cells implies distinct susceptibility of T cell subsets to viral entry by T cell line-tropic versus macrophage-tropic strains during the course of HIV infection.

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Figures

Figure 1
Figure 1
The CXCR4-specific mAb 12G5 partially blocks SDF-1-induced cellular responses. (A) mAb 12G5 blocks the majority of SDF-1-induced lymphocyte chemotaxis. Freshly isolated PBMC were preincubated with the indicated concentrations of mAbs to CXCR4 (12G5) and CCR3 (7B11) for 15 min and subsequently added to the top chamber of bare filter Transwell inserts. SDF-1 at the optimal concentration of 1.5 μg/ml was added to the bottom chamber. Transmigrated cells were counted by flow cytometry scatter-gating on the lymphocytes. Results are shown as percentage of input. The experiment shown was representative of four independent experiments. (B) 12G5 partially blocks SDF-1-induced increases in intracellular calcium. CXCR4 stably transfected CHO cells (1C2) loaded with fura-2 AM were exposed to 1 μg/ml of full-length SDF-1α-(1–67) or truncated inactive SDF-1α-(6–67) (negative control) in the presence (lower reading) or absence (upper reading) of 10 μg/ml 12G5 mAb. Using the mAb at 50 μg/ml produced identical results.
Figure 2
Figure 2
The HIV coreceptors CXCR4 and CCR5 are expressed on distinct T lymphocyte subsets. (A) Three-color flow cytometry of freshly isolated PBMC shows expression on distinct T lymphocyte subsets. Two-dimensional contour plots show CXCR4 (12G5 mAb) and CCR5 (5C7 mAb) expression versus CD26, CD45RA, and CD45R0. (Top Left) Staining using irrelevant control antibodies. Lymphocytes were gated according to their forward and side scatter, and CD3-PerCP staining. (B) Two-dimensional contour plot shows expression of CXCR4 (12G5 mAb) versus CCR5 (biotinylated 3D8 mAb) on T cells. T cells were analyzed as in A. Percentages of cells in the respective quadrants are indicated.
Figure 3
Figure 3
CXCR4 and CCR5 are differentially regulated during PHA stimulation and IL-2 priming. (A) Expression of CXCR4 (squares) and CCR5 (triangles) changes during PHA stimulation (filled symbols) and IL-2 priming (open symbols). Immunofluorescent staining with 12G5 mAb to CXCR4 and 5C7 mAb to CCR5 is expressed for the entire population as number of molecules of equivalent soluble fluorochrome (MESF) from one representative experiment of three. Fluorescent intensity of the entire population for CCR5 decreased on day 3 as the CCR5-positive subset of T cells was markedly reduced. T cells were gated according to their forward and side scatter, and CD3-PerCP staining in A and C. (B) Chemotactic response to SDF-1 and MIP-1β correlates with expression of CXCR4 and CCR5, respectively. T cells migrated to control medium (dotted columns), SDF-1α-(1–67) (1.5 μg/ml) (filled columns), and MIP-1β (100 ng/ml) (open columns). Shown is chemotaxis of PBMC stimulated with PHA (Upper) or primed with IL-2 (Lower) for the indicated number of days. Bars indicate the range of duplicates. (C) CXCR4 is rapidly up-regulated during PHA stimulation and IL-2 priming. CXCR4 expression was measured in freshly isolated lymphocytes (thin line, Left), in day 3 PHA-stimulated T cell blast (thick line, Right), and in day 3 IL-2 stimulated T cells (thin line, Right). The filled bar graphs indicate staining with an unrelated control mAb.
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