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(Journal of Leukocyte Biology. 2002;72:1063-1074.)
© 2002 by Society for Leukocyte Biology

Multiple determinants are involved in HIV coreceptor use as demonstrated by CCR4/CCL22 interaction in peripheral blood mononuclear cells (PBMCs)

Lokesh Agrawal, Zainab Vanhorn-Ali and Ghalib Alkhatib

Department of Microbiology and Immunology and Walther Oncology Center, Indiana University School of Medicine; and The Walther Cancer Institute, Indianapolis, IN

Correspondence: Ghalib Alkhatib, Department of Microbiology and Immunology and Walther Oncology Center, Indiana University School of Medicine, 635 Barnhill Drive, Room 420, Indianapolis, IN 46202. E-mail: galkhati{at}iupui.edu


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Although a number of chemokine receptors display coreceptor activities in vitro, chemokine receptor 5 (CCR5) and CXC chemokine receptor 4 (CXCR4) remain the major coreceptors used by the human immunodeficiency virus type 1 (HIV-1). In this study, we used an envelope-mediated fusion assay to demonstrate low CCR4 coreceptor activity with some primary HIV-1 and simian immunodeficiency virus-1 (mac316) isolates in vitro. The coreceptor activity was sensitive to CCR4-specific antibodies and to the CCR4-specific chemokine ligand macrophage-derived chemokine (MDC)/chemokine ligand 22 (CCL22). Treatment of peripheral blood mononuclear cells (PBMCs; which express high levels of CCR4) with CCL22 caused down-modulation of endogenous CCR4 but had no significant effect on HIV-1 entry, suggesting that CCR4 may not be used as an entry coreceptor. Despite expression of other minor coreceptors on PBMCs, CCR5 and CXCR4 are preferentially used by HIV-1 isolates, as shown by chemokine-inhibition data. To determine the factors involved in this selective use, we analyzed CCR4 coreceptor activity and compared it with CCR5 use in PBMCs. We used a quantitative fluorescence-activated cell-sorting assay to estimate the numbers of CCR4 and CCR5 antibody-binding sites (ABS) on PBMCs. Although CCR4 was found on a higher percentage of CD4(+) cells, CCR5 ABS was twofold greater than CCR4 ABS on CD4(+) cells. Confocal microscopy revealed strong cell-surface CD4/CCR5 but weak CD4/CCR4 colocalization in PBMCs. Binding studies demonstrated that soluble gp120 had greater affinity to CCR5 than CCR4. The results suggested that coreceptor density, colocalization with CD4, and affinity of the viral gp120 to the coreceptor may determine preferential coreceptor use by HIV-1.

Key Words: (MDC) • CCRA • coreceptors • HIV-1 • PBMCs • macrophage-derived chemokine


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Chemotactic cytokines (chemokines) are proinflammatory mediators that recruit various cell types to the inflammatory sites (reviewed in refs. [1 , 2 ]). The CC chemokines macrophage-inflamatory protein-1{alpha} (MIP-1{alpha})/chemokine ligand 3 (CCL3), MIP-1ß/CCL4, and regulated on activation, normal T expressed and secreted (RANTES)/CCL5, as well as the CXC chemokine stromal cell-derived factor-1 (SDF-1)/CXC chemokine ligand 12 (CXCL12), have all been shown to suppress human immunodeficiency virus type 1 (HIV-1) replication of R5 and X4 isolates [3 4 5 ]. Macrophage-derived chemokine (MDC)/CCL22, a member of the CC chemokine family, is produced and secreted by monocytes, dendritic cells [6 7 8 ], and natural killer cells [9 ]. The relevance of CCL22 to HIV-1 entry was suggested by Pal et al. [10 ], who demonstrated a broad antiviral activity by high-pressure liquid chromatography (HPLC)-purified fractions from supernatants of CD8(+) T lymphocytes. The anti-HIV-1 activity of CCL22 has been controversial [11 , 12 ]; however, recent studies have confirmed the antiviral effects of CCL22 against X4 and R5 HIV-1 infection of phytohemagglutinin (PHA)-activated PBMCs [7 , 13 ] or R5 infection of primary macrophages [14 ]. The chemokine receptor 4 (CCR4) is the only known functional receptor for CCL22 [15 ], and CCR4 mRNA is expressed predominantly in the thymus, spleen, and peripheral blood leukocytes, including T cells, basophils, monocytes [16 ], macrophages, and platelets [17 , 18 ]. The T cell-directed CC chemokine thymus and activation-regulated chemokine (TARC/CCL17) is also a specific biological ligand for CCR4 [19 ].

The CXC chemokine receptor 4 (CXCR4) and the CCR5 are the major coreceptors used by X4 and R5 HIV-1 isolates to gain entry into susceptible cells (reviewed in ref. [20 ]). Additional coreceptors including CCR4 have been demonstrated to support HIV-1 entry in vitro; however, their contribution to HIV disease in vivo remains unknown. Previous studies described CCR4 as an entry coreceptor [21 22 23 24 ], and others have not observed coreceptor activity for CCR4 [25 26 27 ]. These negative results probably resulted from poor transfection efficiency of CCR4 and inability to verify its surface expression. The recent development of monoclonal antibodies (mAb) against CCR4 made it possible to detect its abundant expression on PBMCs [28 ]. HIV-1 uses the major coreceptors for entry into PBMCs; however, the factors contributing to this selective coreceptor use are not entirely understood. The preferential use of the major coreceptors was revealed by chemokine-inhibition experiments previously reported by several groups [29 ]. In this study, we used MDC/CCL22 as well as CCR4-specific antibodies to examine whether CCR4 is used as an entry coreceptor in PBMCs in vitro.

Our previous studies demonstrated that CCL4, a chemokine ligand specific to CCR5, did not result in complete inhibition of envelope (Env) fusion [30 ], suggesting expression of other R5 coreceptors on primary cells. To examine the role of alternative coreceptors, functional cDNA screening was performed to isolate these putative coreceptors. We used a nonbiased cDNA screening strategy previously used to isolate CXCR4 [31 ] to isolate a cDNA clone encoding CCR4. The isolated cDNA showed coreceptor activity when challenged with HIV-1 Envs derived from primary isolates and the macrophage-tropic simian immunodeficiency virus (SIV)-mac316 Env. CCR4-mediated fusion activity was more sensitive to the truncated form of CCL22(3–69) than native CCL22(1–69), suggesting a more potent binding of the truncated form to CCR4. Despite the high levels of CCR4 expression on PBMCs, CCL22- and CCR4-specific antibodies had no significant effect on viral entry. However, a significant, donor-dependent CCL22 antiviral activity was consistently obtained in HIV-1 infectivity assays. The results argued against the use of CCR4 as a coreceptor for entry into PBMCs. Therefore, we wished to determine the factors that contribute to the preferential use of CCR5 as a coreceptor by HIV-1 isolates taking advantage of the high levels of cell-surface CCR4 on PBMCs [28 ].

We used a quantitative fluorescence-activated cell-sorting (QFACS) assay to measure antibody-binding sties (ABS) of CCR4, CCR5, and CXCR4 on PBMCs isolated from six different donors. Our data showed that high levels of CCR4 detected on PBMCs from all donors tested did not correlate with the observed CCR4 coreceptor activity and its inhibition by CCL22 in transfected murine cells. Confocal microscopy studies demonstrated efficient colocalization of CCR5 and CD4 in all PBMC samples tested compared with weak CCR4/CD4 colocalization. Binding studies using radiolabeled gp120 revealed that gp120 had greater affinity to CCR5 than CCR4. The data suggested that coreceptor density, colocalization with CD4, and gp120 affinity for CCR5 might contribute to the selective HIV-1 coreceptor use.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cells and viruses
HeLa cells and NIH 3T3 cells were purchased from the American Type Culture Collection (Manassas, VA). The 3T3.T4 cell line was obtained from the AIDS Research and Reference Reagent Program, Division of AIDS, NIAID, NIH (Rockville, MD). HeLa and 3T3.T4 cell lines were cultured in Dulbecco’s modified Eagle’s medium (Quality Biologicals, Gaithersburg, MD) containing 10% fetal bovine serum (FBS; HyClone, Logan, UT), 2 mM L-glutamine, and antibiotics. Recombinant vaccinia virus stocks were prepared by standard procedures [32 ]. The recombinant viruses used vCB-21R(pT7-LacZ), vTF7-3(T7 polymerase), vCB-3(CD4), vCB-16(Unc), vCB-43(BaL), vCB-41(LAV), vCB-28(JR-FL), vCB-39(ADA), vCB-74(SIVmac239), and vCB-75(SIVmac316) and were previously described [30 , 33 ].

PBMCs from normal donors were activated with PHA (10 µg/ml; Sigma Chemical Co., St. Louis, MO) and recombinant human interleukin-2 (rhIL-2; 100 U/ml; NIH AIDS Reagent Program) for 3 days. PBMCs were depleted of CD8(+) T cells by positive selection of cytotoxic cells by microbeads coated with antibodies to CD8 (Mitenyi Biotec, Auburn, CA). CD8(-) T cells were subsequently used in the fusion assay or in HIV-1 infection assays.

Chemokines, antibodies, and soluble proteins
All chemokines including CCL22(1–69) and CCL22(3–69) were purchased from PeproTech (Rocky Hill, NJ). CCL22(3–69) is a truncated form of CCL22(1–69) in which the first two amino acids are deleted. Polyclonal antibodies to CCR4 were raised in rabbits using a synthetic peptide corresponding to the first 15 amino acids of the N-terminus of CCR4. mAb to CCR4 were initially obtained from Dr. Lijun Wu (Millennium Inc., Cambridge, MA) and were subsequently purchased from PharMingen (San Diego, CA). Fluorescein isothiocyanate (FITC)-conjugated anti-CD4 and phycoerythrin (PE)-conjugated CXCR4 and CCR5 were purchased from R&D Systems (Minneapolis, MN). The isotype-matched antibody controls, immunoglobulin G (IgG)2a-PE, IgG1-PE, and IgG1-FITC, were all purchased from PharMingen. Soluble gp120 was obtained from the laboratory of Dr. Chris Broder. Soluble CD4 was obtained from the laboratory of Dr. Edward A. Berger (donated to Edward A. Berger by S. Johnson, Pharmacia Upjohn, Kalamazoo, MI).

Macrophage cDNA library, receptor expression plasmids, and sequencing
The human macrophage cDNA library (purchased from Invitrogen, San Diego, CA) was prepared in pCDNA3.1 using a unidirectional cloning strategy under the T-7 and cytomegalovirus (CMV) promoters. The cDNA library was used in a functional screening protocol to isolate a cDNA clone that conferred Env-fusion competence to murine cells. The screening method has been previously described [31 ]. The isolated cDNA was used to transfect (using DOTAP) NIH-3T3 cells, and expression of the cDNA clones was activated by infection with vTF7-3 (T7 RNA polymerase). The transfected cells were then infected with vCB-3 (CD4) to allow expression of the CD4 surface antigen. Effector HeLa cells were coinfected with vCB21R(lacZ) and vCB-43 (Ba-L), vEV-1 (89.6), or vCB-16 (Unc). Fusion activity, as a result of mixing the two cell populations, was detected by staining the fused cells with X-gal and counting blue cells. It is interesting that one of the cDNA clones gave a positive fusion signal with primary 89.6 Env but not with the R5 Ba-L. This cDNA clone was designated CCRX. Nucleotide sequencing of the cDNA fragment encoding CCRX indicated that it is identical to CCR4. The nucleotide sequence was confirmed by double-stranded DNA sequencing using the Sequenase DNA sequencing kit, Version 2.0 (USB, Cleveland, OH). The cDNA fragment encoding CCR4 was purified and cloned into pSC59 [34 ] under the control of a synthetic early/late vaccinia virus promoter and subsequently used to transfect 3T3.CD4 cells to determine its Env-mediated fusion specificity.

Env-mediated fusion and single-round infection assays
Coreceptor activity was determined by a vaccinia-based reporter gene assay quantitating Env-mediated cell fusion [35 ]. The cell fusion assay involves the analysis of fusion between two distinct cell populations, one expressing CD4 (endogenous or encoded by a recombinant virus) and the other expressing the indicated HIV-1 Env glycoprotein encoded by a recombinant vaccinia virus. Cell fusion is scored by a reporter gene activation assay in which the cytoplasm of one cell population expressing vaccinia virus-encoded T7 RNA polymerase and the cytoplasm of the other expressing lacZ gene linked to the T7 promoter; cell fusion activates ß-galactosidase. The cDNA fragments encoding the open reading frames of chemokine receptor CCR4 or CCR5 cloned in pSC59 [30 ] were used in subsequent fusion assays. Plasmids encoding CCR5 or CCR4 were transfected into NIH-3T3 cells using DOTAP lipofection (Boehringer Mannheim, Indianapolis, IN). After 4 h incubation with DOTAP at 37°C, cells were infected with vTF7-3 (encoding T7 RNA polymerase under the control of the vaccinia virus early/late synthetic promoter) and vCB-3 (encoding human CD4) at a multiplicity of infection of 10 pfu/cell for each virus. Control cells were transfected with the pCDNA3.1 or pSC59 vector alone. Effector HeLa cells were infected with vCB-21R (encoding ß-galactosidase under control of the T7 promoter) and recombinant vaccinia viruses encoding one of the following HIV-1 Envs: vCB-16 (uncleaved mutant of IIIB Env as a negative control), vCB-43 (encoding Ba-L), vCB-39 (encoding ADA), vCB-28 (encoding JR-FL), vCB-41 (encoding LAV), vSC60 (encoding IIIB), 89.6, vCB-74 (encoding SIV-mac239), or vCB-75 (encoding SIV-mac316). All cell populations (effectors and targets) were incubated overnight at 31°C to allow expression of the vaccinia-encoded proteins and were then washed and resuspended in Earle’s modified Eagle’s medium with 2.5% FBS. Duplicate samples containing 105 target cells expressing CD4 and 105 effector cells (expressing the indicated Env) were mixed in a 96-well microtiter plate and incubated at 37°C for 2 h. ß-Galactosidase activity, produced as a result of cell fusion activity between partner cells, was quantitated by colorimetric assay of detergent-treated cell lysates [34 ].

HIV-1 pseudotyped viral stocks were prepared as previously described [36 ]. Briefly, the HIV-1 genome used in this protocol includes the luciferase gene under the regulation of the SV40 promoter replacing the HIV-1 Env gene (HIV-Luc+Env-). Producer 293T cells were transfected with HIV-Luc+Env- and another plasmid DNA containing the R5 ADA HIV-1 Env. After 60–72 h, pseudotyped viral particles were harvested from the culture supernatants and titered by measuring the amount of p24 gag viral antigen.

To analyze the effect of chemokines on Env fusion activity, the CC chemokines CCL22(1–69), CCL22(3–69), CCL17, or CCL5 were added individually to target cells and incubated for 30–45 min at 37°C before mixing with Env-expressing HeLa cells or infection with the pseudotyped virus.

Productive HIV-1 infection assays
CD8(+) T cells were depleted from PBMCs that had been stimulated with PHA + rIL-2 for 3 days. Total PBMCs and CD8-depleted PBMC were used in the infectivity assay. Infection was performed in a 96-well plate (105 cells/well). Cells were incubated with chemokines CCL22(3–69) or CCL2 for 1 h, inoculated with ADA (105.5 IU/ml) or IIIB (104.5 IU/ml) for 3 h, and washed after virus adsorption, and fresh medium containing chemokines was added. Production of p24 was measured over the course of 12 days using a p24 enzyme-linked immunosorbent assay kit purchased from NCI-FCDRC (Frederick, MD). Monocyte chemoattractant protein-1 (MCP-1; R&D Systems) was used as negative control. CCL4 and CCL5 (PeproTech) and azidothymidine (AZT; 1 µM) were used as controls for inhibition of productive infection. Cell-containing aliquots were taken every 3 days and replaced with fresh medium containing fresh chemokines.

Cell-surface expression and QFACS
Human PBMCs stimulated with PHA + IL-2 were washed twice in FACS buffer (Hanks’ balanced saline solution supplemented with 0.5% FBS and 0.02% sodium azide) and were resuspended in 100 µl FACS buffer at 107/ml. Cells were then incubated with a 1:200 dilution of mAb raised against CCR4 [28 ] at 4°C for 30 min. Cells were then washed twice, resuspended in 100 µl ice-cold FACS buffer in the presence of PE-conjugated antimouse IgG (PharMingen), and incubated at 4°C for 30 min. Finally, cells were washed twice, resuspended in 500 µl ice-cold FACS buffer, and analyzed in a FACScan cytometer (Becton-Dickinson, San Jose, CA). QFACS was performed by converting the mean channel fluorescence into ABS by using a standardized microbeads kit purchased from Sigma Chemical Co. For quantitative analysis of ABS, cells were reacted with FITC-conjugated (anti-CD4) or PE-conjugated (anti-CCR4, anti-CCR5, and anti-CXCR4) antibodies. The conjugated antibodies were added at saturating amounts to 105 microbeads, and the mean fluorescence intensity (MFI) of the stained receptor was converted into ABS by coupling with the standard regression (once generated). ABS in each experiment was calculated as: ABS value of stained sample minus ABS value obtained with staining the cells with the isotype-matched control.

Confocal microscopy
PHA + IL-2-activated PBMCs were stained with an anti-CCR4-PE or anti-CCR5-PE mAb followed by anti-CD4-FITC in suspension. The cells were then fixed in 2% paraformaldehyde, and colocalization was performed on a glass-bottom microwell Petri dish (MatTek, Ashland, MA). Data were collected by a scanning confocal system (Bio-Rad, Hercules, CA) with a krypton/argon laser configured on an Axiovert microscope (Ziess, Oberkochen, Germany). Images were obtained with a 60x oil immersion objective lens.

Soluble gp120 binding assay
NIH-3T3 cells (2x106) were transfected with pcDNA3 vector, pcDNA3/CCR5, or pcDNA3/CCR4. Expression of CCR5 or CCR4 was activated by infection with vTF7-3 (vaccinia encoding T7 RNA polymerase). The cells were incubated in duplicates with 0.25 nM I125-labeled gp120 (89.6) at a specific activity of 2200 Ci/mmol plus varying concentrations of unlabeled gp120 and 100 nM sCD4 (NIH AIDS Reagent Program) in 20 µl binding buffer (50 mM HEPES with 0.5% bovine serum albumin, 1 mM CaCl2, and MgCl2, pH 7.2). After 1 h incubation at room temperature, cells were washed with binding buffer containing 0.5 M sodium chloride, and cell pellet-associated counts were measured in a {gamma} counter.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
CCR4 coreceptor activity in transfected murine cells
Functional screening of a human macrophage cDNA library was performed to isolate the cDNA clone encoding CCR4. We examined the ability of HIV-1 and SIV-1 Env-expressing cells to fuse with NIH3T3.T4 target cells transfected with empty vector (pSC59), CCR5, or CCR4. Cell-surface expression of CCR4 in transfected 3T3.T4 cells was verified by FACS analysis using an anti-CCR4 mAb (1G1 mAb; ref. [28 ] and data not shown). Considerable CCR4 coreceptor activity was obtained with primary HIV-1 (89.6, ADA, and MN) and SIV-1 Envs (Fig. 1 ). We show that the MN strain, known to use CXCR4, is also capable of using CCR5 and CCR4 as coreceptors in vitro. The MN was previously classified as a synctium-inducing (SI) isolate based on its infection of MT-2 cells that do not express CCR5. An interesting feature of CCR4 is its coreceptor activity with the macrophage-tropic SIV-mac316 and not with the T cell line-tropic SIV-mac239, which reached 50% of the activity obtained with CCR5 (Fig. 1) . These experiments demonstrated functional HIV-1 coreceptor activity of the isolated CCR4 cDNA clone in transfected murine cells.



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  		Figure 1. Coreceptor specificity of CCR4 for primary HIV-1 and SIV-1 isolates. NIH-3T3.T4 cells were transfected with plasmid DNA encoding CCR1, CXCR2, CCR4, or CCR5 under T7 promoter and coinfected with vTF7-3 (RNA polymerase) and vCB-3 (CD4). Separate populations of HeLa cells were coinfected with vCB-21R (lacZ under T7 promoter) and one of the indicated HIV-1 Envs and mixed with CD4/coreceptor targets. Production of ß-galactosidase was measured by a colorimetric assay to indicate the fusion index of each Env. Unc (nonfusogenic Env) is an uncleaved mutant of IIIB Env used to show the nonspecific background fusion signal. CCR1- and CXCR2-expressing cells were used as negative controls to show the specificity of CCR4 coreceptor activity. OD, Optical density.

 
Chemokine inhibition of CCR4 coreceptor activity
We examined the effect of CCL22, a chemokine ligand with high affinity for CCR4, on CCR4-mediated cell fusion with some of HIV-1 and SIV-1 Envs. CCR4 coreceptor activity was sensitive in a dose-dependent manner to increasing concentrations of CCL22(1–69) or CCL22(3–69) using ADA (Fig. 2A ), 89.6 (Fig. 2B) , and SIV-mac316 (Fig. 2C) . The same concentrations of CCL22 had no effect on CCR5 coreceptor activity (data not shown). CCL17 inhibited CCR4-mediated cell fusion activity at higher concentrations (Fig. 2A) , confirming the lower affinity of CCL17 to CCR4. The truncated CCL22(3–69; EC50=0.005 µg/ml) was at least 1000-fold more potent than CCL22(1–69; EC50=5 µg/ml). The relative potency of CCL22(3–69) depended on the Env used ranging from 1000 (89.6)- to 30,000 (SIV-mac316)-fold (Fig. 2B and 2C) . Complete inhibition of CCR4 coreceptor activity with ADA was readily obtained with CCL22(3–69) concentrations as low as 50 ng/ml (Fig. 2A) . These experiments demonstrated that CCL22(3–69) is a potent inhibitor of CCR4 coreceptor function in transfected murine cells and confirmed the specificity of CCL22 to CCR4-expressing cells.



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  		Figure 2. Chemokine inhibition of CCR4 coreceptor activity in transfected murine cells. The coreceptor-expressing cells (105 cells/well) were incubated with increasing amounts of the indicated chemokine for 1 h at 37°C and were then mixed with ADA (A), 89.6 (B), or SIV-mac316-expressing HeLa cells (C). After incubation at 37°C for 2.5 h, ß-galactosidase activity was measured in detergent-treated cell lysates. The background fusion values obtained with cells transfected with empty vectors are five for ADA, two for the 89.6, and 15 for SIV-mac316 Envs, respectively. CCL22(1–69) is the full-length CCL22/MDC, and CCL22(3–69) is the truncated form of CCL22 lacking the first two amino acids from the N-terminus. CCL17/TARC inhibition of CCR4 coreceptor activity (A) is consistent with the ligand specificity for CCR4. These experiments were repeated at least five times, producing similar results.

 
Cell-surface down-modulation of CCR4 by CCL22
To provide insight into the mechanism of blocking CCR4 coreceptor activity, we examined the ability of CCL22 to cause CCR4 internalization. Binding of ligands to G protein-coupled receptors results in reduced cell-surface expression of the receptor, a process known as receptor sequestration or down-modulation [37 ]. We investigated whether CCR4 internalization can explain CCL22 inhibition of CCR4 coreceptor activity. Figure 3 shows that CCR4 was efficiently down-modulated by CCL22(3–69) in transfected 3T3.T4 cells (Fig. 3A) . The efficient CCR4 down-modulation in the transfected murine cells excluded the possibility that CCL22(3–69) and 1G1 (anti-CCR4 mAb) competed for the same binding site. Both forms of CCL22 caused partial down-modulation ({approx}50%) of CCR4 at the highest concentration (5 µg/ml; Fig. 3B ). However, CCL22(3–69) was always more efficient at lower concentrations compared with CCL22(1–69) in transfected 3T3.T4 (Fig. 3A) or in PBMCs (Fig. 3B) . Treatment with CCL2 was used as a negative control for the specificity of CCR4 down-modulation by CCL22. A high concentration of CCL2 (5 µg/ml) had no significant effect on CCR4 surface expression, and CCL5 (a low-affinity CCR4 ligand) had a low ({approx}10%) down-modulation effect on CCR4 (Fig. 3A) . Treatment of PBMCs with SDF-1/CXCL12 or RANTES/CCL5 caused efficient (>90%) down-modulation of CXCR4 (Fig. 3C) and CCR5 (Fig. 3D) . The chemokines CCL22 and CCL2 that do not bind CXCR4 or CCR5 had no down-modulation effect (Fig. 3C and 3D) . The efficiency of CCR4 down-modulation in PBMCs was comparable in different individuals and was not donor-dependent (data not shown). These experiments demonstrated that CCL22 down-modulates CCR4 in transfected NIH-3T3 cells and that down-modulation of CCR4 on PBMCs confirmed interaction of CCL22 with CCR4 on these primary cells.



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  		Figure 3. Down-modulation of CCR4 by CCL22. CCR4-transfected 3T3.T4 cells (A) or PBMCs (B–D) stimulated with PHA + IL-2 for 3 days were incubated with the indicated chemokines for 1 h at 37°C. The samples were washed three times with PBS and stained with mAb to the different coreceptors (indicated at the upper left corner of each graph). The results shown are representative of at least three different experiments. C under the bars represents background staining obtained with the isotype-matched antibody control. None refers to control PBMCs that were not treated with chemokines.

 
Effect of CCL22(3–69) and CCR4-specific antibodies on HIV-1 entry into PBMCs
To study CCR4 coreceptor use, we examined the effect of CCL22(3–69) on HIV-1 Env-mediated fusion with human PBMCs isolated from six different donors. When total PBMCs were used, only one (donor #2) out of six PBMC donors showed {approx}10% inhibition of ADA-mediated fusion (Fig. 4A ). The first report describing CCL22 anti-HIV-1 activity used CD8-depleted PBMCs in the infectivity assays [10 ]. Therefore, we examined the effect of depleting the CD8+ T cell population on the response to CCL22 treatment. CD8 depletion had no effect on the response of PBMCs isolated from any of the six donors to CCL22 (data not shown). However, all PBMC samples responded well to CCL5/RANTES treatment, which resulted in more than 90% inhibition of ADA fusion (Fig. 4B) . CCL2/MCP-1 was used as a negative control in these experiments to show the specificity of chemokine inhibition. As previously reported [30 ], CCL2 had no significant effect on ADA-Env fusion (Fig. 4B) and was used as a negative control for the specificity of chemokine inhibition.



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  		Figure 4. CCL22 had no significant effect on HIV-1 Env fusion with PBMCs (A and B) or on pseudotyped HIV-1 infection (C). The chemokines were added to PBMCs and incubated for 1 h at 37°C before they were challenged with effector HeLa cells expressing ADA Env (A and B). The background fusion values obtained with cells expressing the control Unc were subtracted. Fusion activity is measured by the amount of ß-galactosidase produced as a result of cell fusion with the indicated Env. The results are representative of more than 15 different experiments using 15 different donors. The results of six donors are shown, as they were performed at the same time in the same experiment. In pseudotyped viral infection (C), PBMCs were treated with the indicated chemokines at a concentration of 1 µg/ml for 1 h prior to infection. At 72 h post-infection, cells were lysed, and cell lysates were analyzed for luciferase activity; viral entry was quantitated by the light units produced as a result of luciferase activity in this single-round infection assay. Values are reported as relative light units. (D and E) The effect of two CCR4-specific antibodies on CCR4 coreceptor activity in transfected cells (D) and PBMCs (E). The antibodies were added at 1:100 dilution to the target cells before challenging with Env-expressing cells. PBMCs isolated from donor #2 were used in this experiment, as it showed a slight response to CCL22. The broken line indicates the background value obtained with Unc.

 
The effect of CCL22 treatment on infection of PBMCs by luciferase reporter viruses pseudotyped with the R5 ADA envelope was analyzed. In this single-round infection assay, no significant CCL22 effect on viral entry was observed as measured by luciferase production (Fig. 4C) . A slight inhibition was observed in both assays using PBMCs from donor 2 (Figs. 4A and 4C) ; however, the percent inhibition was always lower than 12%. These experiments concluded that CCL22 did not significantly alter Env-mediated fusion or pseudotyped virus infection, two different assays that model the entry stage of the viral life cycle.

These results argued against CCR4 coreceptor use in PBMCs. To confirm this, we used two different antibodies to CCR4 to examine their effect on CCR4 coreceptor activity and Env fusion with PBMCs. When added to CCR4-transfected murine cells, the antibodies caused reduction in CCR4 coreceptor activity (Fig. 4D) . However, no effect was observed when these antibodies were added to PBMCs prior to challenge with HIV-1 89.6 Env-expressing cells (Fig. 4E) . The 89.6 was used because of the dual-tropic nature of this isolate. Antibodies to CCR5 or CXCR4 resulted in 50% reduction of Env fusion when added individually and in >90% reduction when added together. We did not observe augmentation of the blocking effect when CCR4 antibodies were added along with anti-CCR5 or anti-CXCR4 antibodies (Fig. 4E) . The results suggested that CCR4 is not used as a coreceptor on PBMCs.

Effect of CCL22(3–69) on productive HIV-1 infection
The chemokine effect on HIV-1 productive infection was examined by measuring the amount of p24 capsid protein produced in infected cells during 12–15 days of infection. PBMCs were stimulated with PHA/IL-2, used as a whole population or as CD8-depleted, and then were treated with CCL22(3–69) before virus adsorption. An inhibitory effect of 35–45% was always obtained with CCL22(3–69) treatment of PBMCs isolated from four out of five donors (Fig. 5B and 5D ). CCL22(3–69) resulted in a 35–40% reduction in ADA productive infection compared with >90% inhibition observed with CCL5/RANTES (Fig. 5A and 5B) . This lower inhibition by CCL22(3–69; compared with CCL5 inhibition) was not enhanced upon treatment with higher concentrations (10 µg/ml) of the chemokine (data not shown). The full length CCL22(1–69) was less potent in its antiviral activity compared with truncated CCL22(3–69). Figure 5B shows the CCL22(3–69) effect on p24 production in whole PBMCs (without CD8-depletion). There was no significant effect for CCL22(3–69) on X4 infection as demonstrated by IIIB infection (Fig. 5C) . Partial CCL22 inhibition of productive infection was observed in three out of four donors examined at the same time in the same experiment (Fig. 5D) . These results demonstrated partial but significant antiviral effect for CCL22 on R5 infection of PBMCs and confirmed that the observed effect is not dependent on CD8 depletion of PBMCs.



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  		Figure 5. CCL22 shows antiviral activity in HIV-1-productive infection assays. PBMCs (Donor #2) were CD8-depleted (A) using MACs beads (Miltenyi Biotec) or used as a whole (B). The PBMC samples were activated with PHA + IL-2 for 3 days before infection with ADA (A). CCL22(3–69) was added to activated cells at 1 µg/ml and incubated for 1 h before adding the virus innoculum. Cell-containing aliquots were removed every 3 days and replaced with fresh medium containing CCL22(3–69) for 12 days. AZT at 1 µM was used as a control for inhibition of ADA infection. The collected samples were analyzed for the amount of p24 present at each time point. The results were reproducible in at least four different experiments. Similar inhibition results were obtained with cells infected with the 89.6 HIV-1 isolate (not shown). (C) The effect of CCL22(3–69) on HIV-1-productive infection of PBMCs isolated from four different donors. Results of p24 production at day 6 post-infection are shown.

 
Quantitative analysis of coreceptor/CD4 expression pattern on PBMCs
As CCL4 is specific for CCR5, we reasoned that the observed efficient blocking activity by CCL4 (compared with CCL22) was a result of the preferential use of CCR5 as a coreceptor. We speculated that could be a result of higher expression levels of CCR5 on primary cells. To examine this possibility, we analyzed coreceptor expression quantitatively using QFACS and determined the percentage of CD4+ lymphocytes expressing the different coreceptors. As QFACS determines the number of ABS on a cell rather than the physical number of cell-surface molecules, we used mAb that efficiently stain CD4 and the coreceptors on PBMCs [38 ]. The mAb 1G1 against CCR4 is a recently described monoclonal that has been demonstrated to efficiently stain CCR4 on human PBMCs [28 ]. The quantitative assay uses Simply Cellular microbeads (Sigma Chemical Co.), which are manufactured with a known number of goat antimouse molecules on the surface. The microbeads are approximately the size of human peripheral blood lymphocytes. PBMC samples were processed in the same experiment so that washes and treatment times were identical. The MFI of microbeads stained with the mAb at saturating concentration was converted into ABS using a standard curve obtained by linear regression analysis of MFI against a known number of ABS on the four different sizes of the microbeads. This analysis was accomplished using the manufacturer’s QuickCal software (Sigma Chemical Co.), which permits convenient calibration of the flow cytometer by plotting the regression line of the ABS of the microbeads against the corresponding peak channels. ABS values of the unknowns were derived from this calibration curve.

In a two-color staining analysis of PHA/IL-2-activated PBMCs, CCR4 was found to be expressed on a large subset (22–56%) of CD4 lymphocytes in PHA/IL-2-activated PBMCs (Fig. 6 and Table 1 ). The FACS analysis revealed that CCR5 and CXCR4 were found to be expressed on 12% and 61.5% of CD4 lymphocytes, respectively (Fig. 6 and Table 1 ). CXCR4 was consistently expressed on a higher percentage of CD4(+) cells. These measurements confirmed previous observations by Lee et al. [38 ], who performed similar analyses on eight different PBMC samples. Expression levels of CCR4 in PBMCs were determined by estimating the CCR4 ABS compared with the ABS of the major coreceptors (Table 1) . The PBMC sample that responded to CCL22(3–69) had higher staining with CCR4 than other samples and had a higher estimate of CCR4 ABS than CCR5 ABS (sample #2 in Fig. 6 ). The results demonstrated that CXCR4 ABS is consistently higher than CCR5 or CCR4 ABS on all donors tested. Furthermore, the average of CCR5 ABS was higher than the average of CCR4 ABS, indicating that although CCR4 is abundantly expressed on primary cells, CCR5 ABS was, on the average, twofold higher than CCR4 ABS.



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  		Figure 6. Abundant expression of CCR4 on PBMCs from six different donors. Dot blots of CD4(+)-gated cells are shown with double-staining. A two-color staining protocol was used to assess expression of CCR4, CCR5, or CXCR4 and CD4 on PHA/IL-2-activated PBMCs isolated from six different individuals. The numbers at the top indicate the donor PBMC samples 1–6. Double-staining profiles of CXCR4/CD4, CCR4/CD4, and CCR5/CD4 for each donor are provided under each donor number. Coreceptor staining (PE-conjugated) is shown on the x-axis and CD4 staining (FITC-conjugated), on the y-axis in all plots. Quadrants were set according to the staining of control mAb.

 

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  		Table 1. Comparison of CXCR4, CCR4, CCR5, and CD4 ABS on PBMCs

 
Efficient CD4/CCR5 versus weak CD4/CCR4 colocalization
We speculated that HIV-1 selection of the coreceptor might be determined by the ability of a certain coreceptor to colocalize with CD4. The viral Env may preferentially use coreceptors found in close proximity with CD4. Evidence for CD4/CCR5 colocalization and physical association has been previously demonstrated [39 ]; however, colocalization of CD4 with the other minor coreceptors has never been investigated. We analyzed CCR4 colocalization with CD4 by confocal microscopy, where colocalization of two proteins can be demonstrated. To identify fluorescence colocalization, correlation maps were calculated by using a local statistical method. We consistently observed weaker colocalization of CD4 and CCR4 in all PBMC samples tested. Figure 7A shows a representative experiment. Colocalization of CCR5 and CD4 proteins was always more efficient, as demonstrated by the strong yellow (red-green colocalization) staining (Fig. 7A) . To determine whether CD4/CCR4 can colocalize in transfected murine cells, we performed the same confocal analysis on NIH-3T3 cells transiently expressing CCR5 or CCR4. The results indicated that CCR5/CD4 colocalization was pronounced and much stronger than CCR4/CD4 colocalization in murine 3T3 cells transiently expressing the coreceptors (Fig. 7B) . We reproduced these colocalization results using other mAb to human CD4 (OKT4, Ortho Diagnostic Systems, Raritan, NJ; T4-4 from NIH AIDS Reagent Program). The data suggested that colocalization with CD4 is an important factor that may contribute to the preferential use of CCR5 on PBMCs.



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  		Figure 7. Confocal microscopic analysis of coreceptor/CD4 colocalization. Activated PBMCs (A) or transfected NIH-3T3 cells (B) were stained with an anti-CCR4-PE or anti-CCR5-PE mAb followed by FITC-coupled anti-CD4 in suspension. The cells were then fixed in 2% paraformaldehyde, and colocalization was performed on a glass-bottom microwell petri dish (MatTek). Data were gathered by a scanning confocal system (Bio-Rad) with a krypton/argon laser configured on an Axiovert microscope (Ziess).

 
HIV-1 gp120 has higher affinity to CCR5
We used a technique based on previously published binding experiments using soluble gp120, in which CD4 was shown to significantly enhance the gp120 coreceptor-binding interaction [40 41 42 43 44 45 46 ]. Interaction of HIV-1 gp120 subunit with coreceptor-expressing cells was analyzed by comparing gp120 binding with CCR5 versus CCR4-expressing murine cells. NIH-3T3 cells were transfected with empty vector pCDNA3/CCR5 or pCDNA3/CCR4. Expression of the coreceptor was activated by infection with vTF7-3 (encoding T7 RNA polymerase). The ability of the purified gp120 subunit of HIV-1 Env to compete with I125-labeled gp120 derived from the primary 89.6 Env was studied in the presence of soluble CD4. Our choice of the soluble gp120 subunit was based on the fact that CCR4-expressing cells showed detectable coreceptor activity with the 89.6 Env (Fig. 1) . Radiolabeled gp120 bound to CCR5- or CCR4-expressing murine cells was competed out with cold gp120. The results demonstrated that radiolabeled gp120 bound 104 times more efficiently to CCR5-expressing cells (Fig. 8A ). This high signal of bound I125–gp120 was competed out with 1 nM cold gp120 added to CCR5-expressing cells. Despite the much lower signal of bound I125–gp120 on CCR4-expressing cells, a much higher concentration was needed to compete out 50% of the bound radioactivity (Fig. 8B) , whereas 10 ng/ml CCL22(3–69) could displace bound gp120 (Fig. 8C) . The results demonstrated that the 89.6 gp120 interaction with CCR5 is more efficient. The weaker affinity of gp120 to CCR4 (Fig. 8B) may explain the observed weak coreceptor activity (Fig. 1) .



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  		Figure 8. Binding of radiolabeled gp120 to CCR4 is lower than binding to CCR5. The binding analysis was performed in the presence of 100 nM sCD4. Binding of iodinated gp120 was determined in the presence of increasing concentrations of cold gp120 (A and B) or increasing amounts of CCL22 (C). QFACS analysis was used to determine expression levels of coreceptors. In this experiment, CCR4 ABS was 13,240 and CCR5 ABS was 13,560, excluding the possibility that the observed higher gp120 binding to CCR5 was a result of higher expression levels.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
An accepted model for viral entry suggests that the gp120 subunit of HIV-1 Env binds first to CD4, resulting in a conformational change that exposes the coreceptor interaction site in gp120. Interaction of gp120 with the coreceptor induces another conformational change that exposes the fusion peptide of gp41, initiating the process of membrane fusion. The effect of CD4 on gp120 binding to an HIV coreceptor (CXCR4) was first demonstrated by Lapham et al. [40 ]. The first experimental evidence for conformational changes in HIV-1 Env was reported by Jones et al. [47 ]. The HIV-1 entry model was supported by a number of studies demonstrating that sCD4 treatment of gp120 significantly enhanced the interaction of gp120 with CCR5 [41 42 43 44 45 46 ]. Chemokines are believed to block interaction of gp120 with the coreceptor, resulting in inhibition of viral entry (reviewed in refs. [48 , 49 ]). Previous studies regarding the effect of chemokines on HIV-1 infection concluded that CCR5 and CXCR4 are the major coreceptors used by HIV-1 [29 ]. The mechanism(s) of the preferential use of the major coreceptors, despite expression of other potential coreceptors, is not well understood. In this study, we analyzed the potential factors that contribute to the selective coreceptor use by HIV-1. We used CCR4 in our analysis for many reasons. First, CCR4 is abundantly expressed on human PBMCs. Second, a functionally isolated CCR4 showed appreciable coreceptor activity that was sensitive to the MDC/CCL2, a chemokine ligand that is specific for CCR4. Third, CCR4 coreceptor activity was sensitive to CCR4-specific antibodies, therefore providing an important tool to evaluate CCR4 coreceptor use in PBMCs.

Truncated CCL22 has been demonstrated to have broad antiviral activity, which includes R5, X4, and R5X4 isolates [10 , 14 ]. Although Pal and co-workers [50 ] demonstrated potent inhibition of HIV-1 infection by their CCL22 preparations, our data showed partial inhibition of ADA infection by CCL22(3–69) at 10 times the concentration used by Pal and co-workers [50 ] (10 µg/ml; data not shown). Furthermore, our results show that CCL22(3–69) had no effect on X4 infection. It is possible that the purified preparations used by Pal and co-workers [50 ] contained CCL22 molecules with different N-terminal truncations that were more potent in their antiviral effect. In this regard, a number of studies demonstrated that the NH2-terminal truncation affects the biological activity of chemokines [13 , 51 52 53 54 55 ]. Recent studies reported the isolation of a naturally occurring variant of the hemofiltrate CC chemokine 1, CCL14(9–74), which lacked the first eight amino acids of CCL14, providing evidence for the in vivo expression of a truncated chemokine [56 ]. Another possible factor contributing to the different observations may be the source of CCL22. Although Pal et al. [10 ] used HPLC-purified CCL22 isolated from supernatants of CD8(+)T cells, Cota et al. [14 ] used synthetic CCL22, which corresponded to the full-length CCL22. We used rCCL22(1–69) and CCL22(3–69; Peprotech) in our HIV-1 infection assays. Our results demonstrating partial inhibitory effect of CCL22(3–69) on HIV-1 infection of PBMCs are in agreement with results previously reported by Struyf et al. [7 ], who used the same source (Peprotech) of rCCL22(3–69).

CCL22 treatment of PBMCs had no significant effect on entry, as demonstrated in two different assays that model the entry step of viral infection. In contrast, CCL22 inhibitory effect on HIV-1-productive infection was consistently observed with many PBMC donors examined. These results provided further support for previous data that suggested a post-entry mechanism for the observed CCL22 antiviral effect [14 ].

Previous studies demonstrated that truncated CCL22(3–69) had reduced chemotactic potency for lymphocytes and suggested that it was a result of decreased binding to CCR4 [7 ]. In contrast, our analysis revealed that CCL22(3–69) was a more potent inhibitor of CCR4 coreceptor activity, which is consistent with increased binding. This discrepancy may be explained by the different assay systems used to evaluate CCL22 binding to CCR4. Our assay measures HIV-1 Env-mediated fusion activity that is dependent on Env binding to rCCR4 expressed on murine cells. Blocking this interaction by CCL22 resulted in the reduction of cell-fusion activity. In contrast, Struyf et al. [7 ] demonstrated reduced chemotactic activity for the truncated CCL22(3–69) using the HUT-78 T cell line (endogenous CCR4) in chemotaxis assay.

To understand the mechanism of coreceptor use, we compared CCR4 and CCR5 in terms of their densities on PBMCs, their abilities to colocalize with CD4, and their affinities to gp120. We demonstrated that CCL4, a chemokine ligand specific for CCR5, caused >90% blocking activity of HIV-1 entry, indicating the efficient use of CCR5 as a coreceptor. In contrast, CCL22/MDC had no significant effect on HIV-1 entry into PBMCs, implicating that CCR4 is not used as an entry coreceptor. Although CCR4 coreceptor activity was sensitive to CCR4-specific antibodies, treatment of PBMCs with these antibodies had no effect on HIV-1-Env fusion, suggesting that CCR4 is not used as a coreceptor.

Quantitative FACS analysis revealed that although CCR4 was expressed on a higher percentage of CD4+ lymphocytes, the CCR5 ABS was on the average twofold higher than CCR4 ABS. Unless there is a threshold effect, such a difference in the surface concentrations may not contribute to the dramatic difference in their coreceptor function. Because the CCR4 surface concentration is still relatively high, and it is known that at such concentrations CCR5 is a very efficient mediator of HIV entry, it is unlikely that the difference in the surface concentrations can be a major factor.

Consistent with the published data in the literature [39 ], we showed that CCR5 colocalizes with CD4 on activated PBMCs. However, we were unable to show significant CCR4/CD4 colocalization in any of the PBMC samples tested. It is possible that inability to associate with CD4 may explain why CCR4 is not efficiently used by HIV-1. The results are in agreement with previous literature that demonstrated strong CCR5/CD4 colocalization [39 ] in a HeLa-CD4 cell line transiently expressing CCR5 and in human macrophages [57 ]. Although Xiao et al. [39 ] were able to demonstrate that CD4/CCR5 colocalization translated into physical association by coimmunoprecipitation of CD4 and CCR5, Singer et al. [57 ] reported that CD4/CCR5 colocalization in human macrophages and T lymphocytes did not translate into physical association. Using immunogold electron microscopy (IEM), Singer et al. [57 ] showed that CCR5 and CD4 were clustered and closely apposed on micovilli of human macrophages and T cells. This discrepancy may be explained by the different experimental procedures used by Xiao et al. [39 ] and Singer et al. [57 ]. It is well known that IEM is not an appropriate technique to demonstrate protein-protein interaction in cases when the protein conformation is very sensitive to interactions with antibodies conjugated with gold particles. In particular, chemokine receptors have a relatively small extracellular portion (only several nm in diameter), which is comparable in size with the antibody-binding region, and are very conformationally flexible. Therefore, IEM can be used to detect single molecules of chemokine receptors but appears to be inappropriate to detect their interactions with other molecules unless special constructs are used, e.g., chimeric molecules, etc. In the study by Singer et al. [57 ], there was no positive control; e.g., in the presence of gp120, CD4 and CCR5 must associate and be detected. Singer et al. [57 ] had to use 13B8.2 CD4 mAb, as it was the only anti-CD4 antibody that intensely stained CD4-expressing HeLa cells following fixation. Xiao et al. [39 ] and we used similar CD4 mAb (OKT4 or T4-4), which gave similar CD4/CCR5 colocalization results using live cells.

Our analysis of gp120 binding to CCR4 or CCR5 indicated that the gp120 affinity to the coreceptor might play a critical role in coreceptor use. We found that gp120 affinity to CCR5 was 104 times higher than its affinity to CCR4. These results argue against the use of CCR4 as an entry coreceptor in PBMCs. The weak affinity of gp120 to CCR4 correlated with the observed weak coreceptor activity of CCR4 in transfected murine cells. Despite its lower affinity to CCR4, our results demonstrated for the first time that gp120 binding to CCR4 can occur in cells expressing CCR4 alone. Therefore, the failure to use CCR4 as a coreceptor in PBMCs may be explained by the presence of higher affinity coreceptors, coreceptor density, and colocalization with CD4. We believe that in the case of CCR4 versus CCR5, affinity to gp120 and colocalization with CD4 seem to determine the selective HIV coreceptor use in PBMCs.

Future studies should extend the analysis described in this paper to include the other minor coreceptors. Such studies will significantly contribute to our knowledge about HIV coreceptor use and the importance of the minor coreceptors in HIV disease.


   ACKNOWLEDGEMENTS
 
This research is supported by a grant from the American Foundation for AIDS Research (amfAR grant #02709-28-RG). The following reagents were obtained through the AIDS Research and Reference Reagent Program, Division of AIDS, NIAID, NIH (Rockville, MD): rhIL-2 from Dr. Maurice Gately, Hoffman-La Roch, Inc. (Nutley, NJ); VP1174 (recombinant vaccinia encoding MN envelope) from Virogenetics Corp. (Troy, NY); HIV-1 89.6 from Dr. Ronald Collman, University of Pennsylvania (Philadelphia); and HIV-1 ADA from Dr. Howard Gendelman, University of Nebraska Medical Center (Omaha). The authors thank Dr. Lujin Wu for providing 1G1 mAb against human CCR4; Edyta Vieth for excellent technical assistance; Arun Srivastiva and Johnny He for their comments on the manuscript; and Dr. Rebecca Chan for bleeding informed consent blood donors (Indiana University, Indianapolis, IRB Study #9812-05).

Received April 25, 2002; revised May 29, 2002; accepted May 30, 2002.


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