Quantification of CD 4 , CCR 5 , and CXCR 4 levels on lymphocyte subsets , dendritic cells , and differentially conditioned monocyte-derived macrophages

CCR5 and CXCR4 are the major HIV-1 coreceptors for R5 and X4 HIV-1 strains, respectively, and a threshold number of CD4 and chemokine receptor molecules is required to support virus infection. Therefore, we used a quantitative fluorescence-activated cell sorting assay to determine the number of CD4, CCR5, and CXCR4 antibody-binding sites (ABS) on various T cell lines, T cell subsets, peripheral blood dendritic cells (PBDC), and monocyte-derived macrophages by using four-color fluorescence-activated cell sorting analysis on fresh whole blood. Receptor levels varied dramatically among the various subsets examined and typically varied from 2to 5-fold between individuals. CCR5 was expressed at much higher levels in CD41yCD45RO1yCD62L-true memory cells compared with CD41yCD45RO1yCD62L1 cells. Fresh PBDC had the highest number of CCR5 ABS among the leukocyte subsets examined but had few CXCR4 ABS, affording a strategy for sort-purifying PBDC. In vitro maturation of PBDC resulted in median 3and 41-fold increases in CCR5 and CXCR4 ABS, respectively. We found that macrophage colony-stimulating factor caused the greatest up-regulation of both CCR5 and CXCR4 on macrophage maturation (from '5,000 to '50,000 ABS) whereas granulocytemacrophage colony-stimulating factor caused a marked decrease of CXCR4 (from '5,000 ABS to <500) while up-regulating CCR5 expression (from '5,000 to '20,000 ABS). Absolute ABS for CD4 and the major HIV-1 coreceptors serve as a more quantitative measure of cell surface expression, and we propose that this be used for future studies looking at the modulation of CD4 or chemokine receptor expression by cytokines, HIV-1 infection, or receptor polymorphisms. HIV-1 entry into cells requires sequential interactions between envelope (Env), CD4, and a coreceptor (1–3). Epidemiological and experimental evidence indicates that CD4 and coreceptor levels affect the efficiency of viral entry and that this may have consequences for the pathogenesis of HIV disease. Individuals homozygous for the D32-ccr5 allele have no surface expression of CCR5 and are highly protected against HIV-1 infection, whereas D32-ccr5 heterozygotes have lower CCR5 expression levels and progress to AIDS more slowly than individuals without this allele (reviewed in ref. 4). Individuals homozygous for a mutation in the SDF-1 gene also progress more slowly to clinical AIDS (5), perhaps because of increased expression of SDF-1 and modulation of CXCR4 expression. Indeed, in vitro studies have shown that CD4, CCR5, and CXCR4 expression levels impact the efficiency of viral entry (6–8). Chemokine receptor expression in both peripheral blood lymphocytes and monocyte-derived macrophages (MDM) is sensitive to cytokine-mediated modulation (reviewed in ref. 9). Because the presence of CD4 and either CCR5 andyor CXCR4 on specific leukocytes and MDMs designates these cells as potentially susceptible targets for viral infection, it is important to determine quantitatively the amount of CD4 and the major coreceptors present on various leukocyte and monocyte subpopulations to help clarify the roles these cells may play in the dynamics of viral replication in vivo and to rigorously address the effects of cytokines on coreceptor expression. In this report, we used a quantitative fluorescence-activated cell sorting (QFACS) assay that relies on a series of precalibrated beads that can bind a fixed number of mouse IgG molecules to determine the absolute number of CD4 and coreceptor molecules on the surface of numerous leukocyte subsets, MDMs, and peripheral blood dendritic cells (PBDC). By using this approach, we found great variation in chemokine receptor expression in T cell lines and lymphocyte subsets, in immature versus mature dendritic cells (DC), and in MDM depending on culture conditions. These results provide insight into the types of cells most susceptible to infection by R5 and X4 viruses and an understanding of the discrepancies in the literature regarding CD4 and coreceptor expression in cultured MDM. MATERIALS AND METHODS Cell Lines and Infection Studies. All cell lines were obtained from the American Type Culture Collection or the National Institutes of Health AIDS Reference and Reagent Program (GHOST cells). All cell lines were maintained according to the supplier’s recommendations. Pseudotyped luciferase reporter viruses were used for infection studies as described (10). Antibodies. Phycoerythrin-conjugated anti-CD4 (Q4120) was obtained from Sigma. Allophycocyanin-conjugated anti-CD4 (S3.5), anti-CD8 (3B5), anti-HLA-DR (TU36), FITC-conjugated anti-CD11c, and tricolor-conjugated anti-CD3, anti-CD14 (Tuk4), anti-CD16 (3G8), anti-CD19 (SJ35-C1), anti-CD45RA (MEM56), anti-CD45RO (UCHL1), anti-CD56 (NKI-nbl-1), anti-CD62L (DREG-56), anti-CD83 (HB15), and anti-HLA-DR (TU36) were obtained from Caltag (South San Francisco, CA). Cychrome-conjugated anti-CD26, phycoerythrin-conjugated anti-CCR5 (2D7), and anti-CXCR4 (12G5) were obtained from PharMingen. FITC-conjugated CD1a (B-B5) was obtained from BioSource International (Camarillo, CA). FACS Strategy. We used phycoerythrin (PE)and allophycocyanin (APC)-conjugated mAbs for quantification because they The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked ‘‘advertisement’’ in accordance with 18 U.S.C. §1734 solely to indicate this fact. PNAS is available online at www.pnas.org. This paper was submitted directly (Track II) to the Proceedings office. Abbreviations: ABS, antibody-binding sites; DC, dendritic cells; PBDC, peripheral blood DC; MDM, monocyte-derived macrophages; FACS, f luorescence-activated cell sorting; QFACS, quantitative FACS; PE, phycoerythrin; APC, allophycocyanin; PBMC, peripheral blood mononuclear cells; M-CSF, macrophage colony-stimulating factor; GM-CSF, granulocyte-macrophage CSF. §To whom reprint requests should be addressed at: Department of Pathology and Laboratory Medicine, 807 Abramson, 34th and Civic Center Boulevard, Philadelphia, PA 19104. e-mail: doms@mail.med. upenn.edu.