The present study demonstrates cell surface expression of both CXC chemokine receptor 4 (CXCR4) and CC chemokine receptor 5 (CCR5), major coreceptors for T cell-tropic and macrophage-tropic strains of HIV, respectively, on CD34+ progenitor cells derived from the peripheral blood. CD34+ progenitor cells were susceptible to infection by diverse strains of HIV, and infection could be sustained for prolonged periods in vitro. HIV entry into CD34+ progenitor cells could be modulated by soluble CD4, HIV gp120 third variable loop neutralizing mAb and the cognate ligands for the CXCR4 and CCR5 HIV coreceptors. This study suggests that a significant proportion of the circulating progenitor cell pool may serve as a reservoir for HIV that is capable of trafficking the virus to diverse anatomic compartments. Furthermore, the infection and ultimate destruction of these progenitor cells may explain in part the defective lymphopoiesis in certain HIV-infected individuals despite effective control of virus replication during highly active antiretroviral therapy.

The establishment of productive HIV infection in CD34+ progenitor cells and the ability of these cells to serve as an in vivo reservoir have been controversial. A number of studies in HIV-infected individuals have failed to detect productively infected CD34+ progenitor cells from bone marrow (1, 2, 3, 4, 5); however, other studies have shown that rare infection of CD34+ progenitor cells can occur (6, 7, 8) and may be more prevalent in a subset of HIV-infected patients with advanced disease (9). HIV infection in vitro has been reported in highly purified bone marrow-derived CD34+ cells (10) and in CD34+ progenitor cells that coexpress CD4 (11). In this regard, the CD4 molecule that binds with high affinity to HIV gp120 is expressed on a minor population of CD34+ progenitor cells (11, 12, 13, 14). Infection of host cells by HIV is determined by viral envelope binding to the CD4 molecule (15, 16) together with binding to a chemokine coreceptor such as CXCR42 or CCR5, which facilitate fusion and entry of T cell (T)-tropic and macrophage (M)-tropic HIV strains, respectively (17, 18, 19, 20, 21, 22, 23). T-tropic strains of HIV-1 have been shown to infect cultures of purified CD34+ progenitor cells in vitro, suggesting the presence of an HIV coreceptor similar or identical with CXCR4 (24, 25, 26, 27). Expression of CXCR4 and CCR5 mRNA in granulocyte CSF-mobilized progenitor cells has been recently reported (28); however, surface expression of CXCR4 and CCR5 chemokine receptors on CD34+ progenitor cells obtained from either medullary or extramedullary sites has not been demonstrated. Furthermore, susceptibility to HIV infection in peripheral blood-derived CD34+ progenitor cells has not been well characterized, nor has the role of these coreceptors in progenitor cell infection by HIV-1 been defined. Peripheral blood-derived CD34+ progenitor cells are capable of seeding extramedullary sites of lymphopoiesis (29, 30, 31) and, if infected, may serve to disseminate HIV into diverse anatomic sites. Defining the susceptibility to HIV infection specifically in peripheral blood-derived CD34+ progenitor cells is relevant not only to understanding the pathogenesis of HIV disease but also to addressing the potential for immune reconstitution. We therefore investigated the expression of the HIV coreceptors CXCR4 and CCR5 on peripheral blood-derived CD34+ progenitor cells and determined the susceptibility of these cells to infection by different strains of HIV.

Recombinant human IL-3 and IL-6 (Stem Cell Technologies, Vancouver, Canada) were used at a final concentration of 25 ng/ml. Recombinant human stem cell factor (Sigma, St. Louis, MO) was used at a final concentration of 100 ng/ml. RANTES and macrophage inflammatory protein-1β (MIP-1β; PeproTech, Rocky Hill, NJ) were used at a final concentration of 200 ng/ml. Stromal-derived factor-1α (SDF-1α) (a gift from Upstate Biotechnology, Lake Placid, NY) was used at 1 μg/ml.

Viral isolates used for infection of progenitor cells included pellet-purified HIV-1 strains NL4.3 (AIDS Research and Reference Reagent Program), IIIB (Advanced Biotechnologies, Columbia, MD), Ba-L (Advanced Biotechnologies), and MN (Advanced Biotechnologies) and JR-fl, JR-csf HIV-1 supernatants (AIDS Research and Reference Reagent Program). Infections were performed at a multiplicity of infection of 0.005 using viral particle count for pellet purified strains.

PBMC were obtained by Ficoll-Hypaque density centrifugation. T cells were depleted using neuraminidase (Sigma)-treated SRBC agglutination followed by a second Ficoll-Hypaque centrifugation. Monocytes and macrophages were depleted by overnight adherence on plastic flasks at 37°C in a 5% CO2 incubator. The remaining progenitor cell-enriched fraction was positively immunoselected for CD34+ cells by passage over a magnetic column (CD34 isolation kit, Miltenyi Biotech, Sunnyvale, CA). CD34+ cells were placed in DMEM/Iscove’s medium at a 1/1 ratio supplemented with 10% FCS, 5 μg/ml bovine insulin (Sigma), 0.8 mM sodium pyruvate (Life Technologies, Grand Island, NY), 2 μM glutamine, 7 mM HEPES (Life Technologies), 50 U/ml penicillin and 50 μg/ml streptomycin (Life Technologies), and cytokines, including recombinant human IL-3 and IL-6 (Stem Cell Technologies), each at a final concentration of 25 ng/ml and recombinant human stem cell factor (Sigma) at a final concentration of 100 ng/ml (complete medium). Cultures were maintained with biweekly 50% replenishment of complete medium and were incubated at 37°C in a fully humidified atmosphere containing 5% CO2.

Aliquots of cells were subjected to three-channel cytofluorometric analysis 12 to 24 h following the final progenitor cell purification to allow detachment of the CD34 mAb used for selection. The expression of cell surface Ag was determined by flow cytometry using a Coulter Elite FACS (Coulter, Hialeah, FL). The cells were washed, resuspended in PBS containing 10% FCS, and mixed with optimal concentrations of mAb. A panel of mAbs was used to assess progenitor cell purity; this panel included CD3 (clone HIT3a), CD4 (clone Q4120), CD8 (clone Leu 2a), CD14 (clone Leu M3), CD16 (clone Leu 11c), CD20 (clone Leu 16), CD33 (clone Leu M9), CD34 (clone HPCA-2), CD35 (clone E11), CD38 (clone Leu 17), CD45 (clone HLe-1), CD56 (clone Leu 19), CD57 (clone vc1.1/hnk), and CD90w (clone 5E10; Becton Dickinson, San Jose, CA). A range of 5 × 103 to 1 × 104 events were collected per sample. CD34+ progenitor cells stained with murine keyhole limpet hemocyanin mAb conjugated to IgG1 FITC and IgG1 phycoerythrin (PE) were used to set the positive gate.

Progenitor cell cultures were maintained up to 40 days in vitro following infection with different strains of HIV-1. Progenitor cell supernatants were harvested at serial intervals and assessed for HIV replication by RT assay (32, 33).

Aliquots of purified CD34+ progenitor cells (5–10 × 105 cells/tube) were pretreated for 30 min with a panel of chemokines or receptor antagonists in parallel sets that were subsequently infected with different strains of HIV-1 pretreated with RNase-free DNase (Boehringer Mannheim, Indianapolis, IN) or sCD4. Cells were incubated at 37°C in 5% CO2, and serial harvests were collected at 0, 8, and 18 h postinfection. Cells were washed three times with PBS and pelleted before freezing at −80°C. Pellets were thawed and resuspended in lysis buffer (0.6 mg/ml proteinase K (Life Technologies), 0.01% Triton-X-100 (Sigma), 0.1% SDS (Sigma), 25 mM Tris-HCl (pH 8.0), and 10 mM EDTA (Sigma)) and allowed to incubate at 56°C for 60 min, then at 94°C for 15 min. PCR amplifications were performed on 5 to 10 × 105 cells/lysate using HIV sense LTR primer M667 (5′-GGC-TAA-CTA-GGG-AAC-CCA-CTG) and HIV antisense LTR primer AA55 (5′-CTG-CTA-GAG-ATT-TTC-CAC-ACT-GAC) with standard cycling conditions (34). Amplified products were denatured at 94°C for 5 min, then hybridized at 56°C for 10 min with a 32P end-labeled internal LTR probe CG24 (5′-CTC-AAT-AAA-GCT-TGC-CTT-GAG-TGC). Samples were run on 10% nondenaturing polyacrylamide gels, and hybridized sequences were visualized using the STORM860 PhosphorImage system (Molecular Dynamics, Sunnyvale, CA) and were analyzed using the ImageQuant program (Molecular Dynamics). ACH-2 cells, which contain 1 HIV DNA copy/cell, were serially diluted to 1000, 100, 10, and 1 HIV-LTR copy/50λ lysate and were used to construct a standard curve for each PCR assay. Samples containing buffer only or uninfected mononuclear cells served as negative controls.

Bulk 3-day-old cultures of CD34+ progenitor cells submitted for immunofluorescence studies were preincubated in a buffer containing pooled human IgG and BSA for Fc receptor (FcR) blockade. CD34+ progenitor cells were plated at a density of 500 cells/mm2 onto 12-mm circular glass number 1 coverslips (Fisher Scientific, Pittsburgh, PA) coated with 25 μg/mm2 laminin (Life Technologies) and placed in a 37°C, 5% CO2 incubator overnight before staining. After cells were firmly adherent and morphologically distinct, the coverslips were rinsed, and the resident cells were blocked with 2% deionized BSA before staining with different mAbs at optimal concentrations. Abs used in this study include anti-CD34-FITC or PE mAb (clone HPCA-2, Becton Dickinson), anti-CCR5 mAb (clone 5C7, provided by Charles R. MacKay, Leukosite, Boston, MA), anti-CXCR4 mAb (clone 12G5, provided by James Hoxie, University of Pennsylvania, Philadelphia, PA). Secondary Abs included IgG Oregon green-514 (Molecular Probes, Eugene, OR). CCR5 mAb was FITC-conjugated (Boehringer Mannheim, Indianapolis, IN) before use in colocalization studies with CXCR4 mAb. Nonspecific fluorescence was determined by staining with an irrelevant murine isotype-specific control or a secondary mAb alone.

Laser scanning confocal fluorescence microscopy was performed using a Zeiss LSM 410 scanning laser confocal microscope system (Zeiss, Thornwood, NY) built around a Zeiss 135 Axiovert inverted scope fitted with an Omnicron argon/krypton dual gas laser set to emit laser lines at 488, 568, and/or 647 nm. Oregon green and FITC images were recorded using a 488-nm argon excitation and broad band pass emission filter of 515 to 525 nm. PE images were recorded with a 488/568-nm argon excitation and a broad band pass emission filter of 580 to 640 nm. Brightness and contrast were set on appropriate positive control samples to obtain a full 8-bit gray scale rendering of each image. Negative controls were subsequently recorded at these same settings. Images were obtained using either a Zeiss ×40 Achroplan 0.60 Korr Ph2 objective or a Zeiss ×63 oil/NA = 1.25 Neofluor objective. Image processing with signal filtering and digital contrast enhancement were performed using Zeiss LSM 410 (version 3.8) software.

Comparison of mean values was performed using the Mann-Whitney U test.

PBMC obtained from normal donors (n = 7) and enriched for CD34+ progenitor cells demonstrated surface expression of CXCR4 and CCR5 (Fig. 1, top panel). CXCR4 and CCR5 expression was found on 78 ± 13 and 62.5 ± 23%, respectively, of the CD34+ CD38+ progenitor cells derived from peripheral blood (Table I). CD34+ progenitor cells that coexpressed CD4 (19.3 ± 9.9%; data not shown) demonstrated a similar percentage expression of CXCR4 and CCR5 (Fig. 1, lower panel) as did CD34+ CD38+ progenitor cells.

FIGURE 1.

Phenotypic expression of CXCR4 and CCR5 on peripheral blood-derived CD34+ CD38+ and CD34+ CD4+ progenitor cells. Surface expression of CXCR4 and CCR5 receptors in CD34+ CD38+ progenitor cells (top panel) and CD34+ CD4+ progenitor cells (lower panel) as demonstrated by FACS in PBMC following Ficoll-Hypaque centrifugation and a single round of CD34-positive immunoselection. Further depletion was not performed for this analysis.

FIGURE 1.

Phenotypic expression of CXCR4 and CCR5 on peripheral blood-derived CD34+ CD38+ and CD34+ CD4+ progenitor cells. Surface expression of CXCR4 and CCR5 receptors in CD34+ CD38+ progenitor cells (top panel) and CD34+ CD4+ progenitor cells (lower panel) as demonstrated by FACS in PBMC following Ficoll-Hypaque centrifugation and a single round of CD34-positive immunoselection. Further depletion was not performed for this analysis.

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Table I.

HIV coreceptor expression on CD34+ progenitor cell subsets

Progenitor Cell Subset% Cells Positive for Coreceptor Expression
CXCR4CCR5
CD34+CD38+ 78 ± 13 62 ± 24 
CD34+CD4+ 75 ± 24 68 ± 33 
CD34+CD90w (Thy1)+ 94 ± 6 100 ± 0 
Progenitor Cell Subset% Cells Positive for Coreceptor Expression
CXCR4CCR5
CD34+CD38+ 78 ± 13 62 ± 24 
CD34+CD4+ 75 ± 24 68 ± 33 
CD34+CD90w (Thy1)+ 94 ± 6 100 ± 0 

The CD4 molecule is expressed on a minor population of CD34+ progenitor cells (Fig. 1) (11, 12, 13). Progenitor cells that lack lineage-specific markers and coexpress CD4 and CD90 (Thy-1) phenotypically define primitive totipotent and multipotent (prelymphoid) CD34+ progenitor subsets (4, 29, 31). CXCR4 and CCR5 expression was higher in CD34+ progenitor cells that coexpressed CD90 (Thy-1) than in CD34+ CD38+ lineage-committed progenitor cells. CXCR4 expression in the CD34+ CD90+ (Thy-1) subset was 96 ± 4% compared with 78 ± 13% among CD34+ CD38+ progenitor cells (p = 0.07) (Table I). CCR5 expression in the CD34+ CD90+ (Thy-1) subset was 100 ± 0% compared with 62 ± 23% among CD34+ CD38+ progenitor cells (p = 0.01; Table I).

The CD34 sialomucin receptor is one of several adhesins involved in the intra- and extramedullary homing of progenitor cells into distinct microenvironments (35, 36, 37). Chemokines function similarly in their ability to direct leukocyte migration (38). Confocal microscopy was performed to analyze the distribution of chemokine receptors among CD34+ progenitor cells. High power photomicrographs (Fig. 2) illustrate the discrete distribution of CD34 and chemokine receptor staining on an individual progenitor cell (Fig. 2, a–c). A progenitor cell stained with anti-CD34-PE (Fig. 2,a, red staining) and anti-CCR5 mAb-Oregon green (Fig. 2,b, green staining) demonstrates the expression of CCR5 localized to hemopoietic vesicles that were organized singly or in clusters at the plasma membrane (39, 40) (Fig. 2,c, arrow). CCR5 expression appeared diffusely colocalized, focally colocalized, or independent of CD34. Discrete areas of CD34 and CCR5 colocalization were visualized along ruffled folds of the cell membrane (Fig. 2,c, red plus green = gold). In contrast, a progenitor cell stained with anti-CD34-PE (Fig. 2,d, red staining) and anti-CXCR4 mAb-Oregon green (Fig. 2,e, green staining) demonstrates that CXCR4 expression was distributed basolaterally and exhibited less colocalization with CD34 (Fig. 2,f, red plus green = gold). Photomicrographs (Fig. 2, g, low power, and h, zoom 3) illustrate CXCR4 and CCR5 receptor expression among CD34+ progenitor cells. The majority of progenitor cells expressed both receptors (Fig. 2,g, visualized as CXCR4 (red), CCR5 (green), or both receptors colocalized (gold)); however, there was a heterogeneous distribution (Fig. 2 h) among subsets within the progenitor cell population appreciable as different densities of each coreceptor.

FIGURE 2.

Immunocytochemistry using laser scanning confocal microscopy. Photomicrographs of purified CD34+ progenitor cells taken with a Zeiss ×63 oil/NA1.25 Neofluor objective. a, Progenitor cell stained with anti-CD34-PE (red). b, The same progenitor cell stained with anti-CCR5 mAb (clone 5C7)-Oregon green (green). c, Colocalization of the CCR5 receptors (green) and CD34 sialomucin receptor (red) is visualized in gold (spectral overlay of red plus green = gold). The CCR5 chemokine receptor displays distinct hemopoietic vesicle caveolae-like morphology noted by the arrow. Zoom, ×8. d, Progenitor cell stained with anti-CD34-PE (red). e, Progenitor cell stained with anti-CXCR4 mAb (clone 12G5)-Oregon green (green). f, Colocalization of the CXCR4 receptors (green) and CD34 sialomucin receptor (red) is visualized in gold. Zoom, ×4. g, The majority of CD34+ progenitor cells express both CCR5 (green) and CXCR4 (red), which appear as gold when colocalized. h, The distribution of chemokine receptor expression is variable for each coreceptor among CD34+ progenitor cells. Zoom, ×3.

FIGURE 2.

Immunocytochemistry using laser scanning confocal microscopy. Photomicrographs of purified CD34+ progenitor cells taken with a Zeiss ×63 oil/NA1.25 Neofluor objective. a, Progenitor cell stained with anti-CD34-PE (red). b, The same progenitor cell stained with anti-CCR5 mAb (clone 5C7)-Oregon green (green). c, Colocalization of the CCR5 receptors (green) and CD34 sialomucin receptor (red) is visualized in gold (spectral overlay of red plus green = gold). The CCR5 chemokine receptor displays distinct hemopoietic vesicle caveolae-like morphology noted by the arrow. Zoom, ×8. d, Progenitor cell stained with anti-CD34-PE (red). e, Progenitor cell stained with anti-CXCR4 mAb (clone 12G5)-Oregon green (green). f, Colocalization of the CXCR4 receptors (green) and CD34 sialomucin receptor (red) is visualized in gold. Zoom, ×4. g, The majority of CD34+ progenitor cells express both CCR5 (green) and CXCR4 (red), which appear as gold when colocalized. h, The distribution of chemokine receptor expression is variable for each coreceptor among CD34+ progenitor cells. Zoom, ×3.

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Peripheral blood-derived CD34+ progenitor cells were cultured in the presence of M-tropic (BaL, JR-fl, JR-csf), T-tropic (NL4.3), T cell line-adapted (IIIB), or dual tropic (MN) HIV-1 viral strains and maintained in vitro for up to 40 days. FACS analysis performed on cells harvested after 1 wk of in vitro culture with different strains of HIV demonstrated that cultures exposed to virus maintained a lower mean percentage of CD34+-expressing cells compared with control cultures (data not shown).

Highly purified progenitor cells with <1% contamination by CD3+, CD16+, or CD57+ cells (n = 5) all demonstrated active HIV replication, as determined by mean RT activity (Fig. 3, A and B). RT activity could be detected as early as day 6 in both cell suspension and adherent cell supernatants (data not shown). RT activity was detected throughout the in vitro culture period (>30 days) for all HIV-1 strains. Similar results were obtained in five independent experiments.

FIGURE 3.

Peripheral blood-derived progenitor cells can be infected by diverse strains of HIV. Purified CD34+ progenitor cells were infected in parallel cultures with (A) a dual tropic strain (MN), a T cell laboratory-adapted strain (IIIB), or a T-tropic clone (NL4.3) of HIV or with (B) M-tropic (BaL, JR-fl, JR-csf) strains of HIV. Cell supernatants were harvested at serial intervals and assessed for HIV release by RT assay.

FIGURE 3.

Peripheral blood-derived progenitor cells can be infected by diverse strains of HIV. Purified CD34+ progenitor cells were infected in parallel cultures with (A) a dual tropic strain (MN), a T cell laboratory-adapted strain (IIIB), or a T-tropic clone (NL4.3) of HIV or with (B) M-tropic (BaL, JR-fl, JR-csf) strains of HIV. Cell supernatants were harvested at serial intervals and assessed for HIV release by RT assay.

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HIV entry is dependent not only on surface expression of CD4, but also on the expression of certain chemokine coreceptors, such as CXCR4 and CCR5 (17, 18, 19, 20, 21, 22, 23). The third variable loop (V3) of HIV gp120 facilitates coreceptor binding, potentially through multiple conformational domains (41, 42, 43). To address whether HIV-1 infection in progenitor cells was dependent on the V3 region of gp120, two different neutralizing V3 loop mAb were employed in neutralization studies. M-tropic (BaL) and several T-tropic HIV-1 strains were pretreated with either neutralizing mAb 178.1 or mAb F1.9B that bind to epitopes within the V3 loop, but that do not interfere with HIV gp120 binding to the CD4 receptor (44, 45, 46). The mAb 178.1 specifically binds the V3 loop of T-tropic strains of HIV and pretreatment with mAb 178.1 inhibits the infection of T tropic strains in mature CD4+ T cells (46). In contrast, mAb F1.9B has been shown to bind specifically to the V3 loop of M-tropic strains of HIV (44), and pretreatment with mAb F1.9B inhibits the infection of M-tropic strains in mature CD4+T cells. Mature CD4+ T cells pretreated with neutralizing mAb F1.9B and exposed to the M-tropic BaL strain demonstrated a >80% reduction in the level of early HIV-1 LTR transcripts compared with T cells exposed to BaL with no mAb pretreatment or with pretreatment with an isotype control mAb (Fig. 4). Pretreatment of BaL with neutralizing mAb 178.1 had no significant effect on the level of BaL early LTR transcripts in mature CD4+ T cells (data not shown). Mature CD4+ T cells exposed to the T-tropic NL4.3 strain pretreated with neutralizing mAb 178.1 demonstrated a >90% reduction in the level of early HIV-1 LTR transcripts compared with T cells exposed to NL4.3 receiving no mAb pretreatment or after pretreatment with an isotype control mAb (Fig. 4). Pretreatment of NL4.3 with neutralizing mAb F1.9B had no significant effect on the level of NL4.3 early LTR transcripts in mature CD4+ T cells (data not shown). Isotype-specific mAb were included to evaluate potential effects due to Fc binding on progenitor cells. Progenitor cells exposed to M-tropic BaL or T-tropic strains pretreated with neutralizing mAb F1.9B or mAb 178.1, respectively, demonstrated a significant reduction in the level of early HIV-1 LTR transcripts compared with CD34+ progenitor cells exposed to virus pretreated with an isotype control mAb or exposed to virus alone (Fig. 4). The observed decrease in early HIV LTR transcripts in the presence of isotype-specific mAb is consistent with the expression of Fc receptors on progenitor cells (29, 31).

FIGURE 4.

Neutralization of infection of CD34+ progenitor cells and mature CD4+ T cells by anti-HIV gp120 V3 loop mAb. Pretreatment of HIV by anti-gp120 V3 loop mAb reduced HIV-1 early viral transcripts in CD34+ progenitor cells and mature CD4+ T cells. F1.9B mAb reduced BaL entry and early LTR transcripts, and 178.1 mAb reduced NL4.3 entry and early LTR transcripts into progenitor cells and mature T cells.

FIGURE 4.

Neutralization of infection of CD34+ progenitor cells and mature CD4+ T cells by anti-HIV gp120 V3 loop mAb. Pretreatment of HIV by anti-gp120 V3 loop mAb reduced HIV-1 early viral transcripts in CD34+ progenitor cells and mature CD4+ T cells. F1.9B mAb reduced BaL entry and early LTR transcripts, and 178.1 mAb reduced NL4.3 entry and early LTR transcripts into progenitor cells and mature T cells.

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The V3 loop of HIV-1 gp-120 of HIV-1 is a key determinant for defining host cell tropism (41, 42, 43, 47, 48). Cells that express CD4, CXCR4, and CCR5 may be permissive for viral entry by diverse strains of HIV; however, a viral phenotypic preference may predominate in progenitor cells due to differences in CD4 receptor density, chemokine coreceptor expression, or the modulation of these receptors by their respective ligands. To address whether HIV infection of CD34+ progenitor cells required the presence of cell surface CD4 molecules, inhibition studies with sCD4 were performed. Pretreatment of M-tropic BaL and T-tropic NL4.3 HIV strains with sCD4 inhibited viral entry into CD34+ progenitor cells more efficiently compared with mature T cells (Fig. 5).

FIGURE 5.

HIV entry and early LTR transcripts in CD34+ progenitor cells can be modulated by CC chemokines and SDF-1 or inhibited with sCD4. Pretreatment of progenitor cells with the cognate ligand for each chemokine receptor reduced the level of early HIV-1 transcripts. The sCD4 reduced the level of early HIV-1 transcripts in CD34+ progenitor cells.

FIGURE 5.

HIV entry and early LTR transcripts in CD34+ progenitor cells can be modulated by CC chemokines and SDF-1 or inhibited with sCD4. Pretreatment of progenitor cells with the cognate ligand for each chemokine receptor reduced the level of early HIV-1 transcripts. The sCD4 reduced the level of early HIV-1 transcripts in CD34+ progenitor cells.

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To address whether T-tropic HIV-1 strains use the CXCR4 chemokine receptor as a coreceptor for viral entry into CD34+ progenitor cells, SDF-1α, the natural ligand for CXCR4, was added to progenitor cell cultures to prevent binding by HIV gp120 (49, 50, 51, 52). The mean channel CXCR4 density on progenitor cells is less than that on mature T cells (M. Ruiz, M. Ostrowski, and A. Kinter, unpublished observations); therefore, a suboptimal dose of SDF-1α was used. Progenitor cells treated with 1 μg/ml of SDF-1α before exposure to the T-tropic NL4.3 strain demonstrated a 70% reduction in the level of early LTR transcripts (Fig. 5) compared with that in cells not receiving SDF-1α pretreatment (Fig. 5). In contrast, treatment of mature T cells with 1 μg/ml SDF-1α resulted in only a 10% reduction in NL4.3 early LTR transcripts compared with that in untreated cells (Fig. 5).

Infection by HIV strains that use CCR5 as a coreceptor for viral entry may be inhibited in the presence of CCR5 agonists due to competition for receptor occupancy or receptor down-modulation (53). To address whether M-tropic HIV strains use the CCR5 chemokine receptor as a coreceptor for viral entry into progenitor cells, progenitor cells and mature CD4+ T cells derived from the same donor were pretreated with RANTES or MIP-1β (19, 23, 42). Mature T cells pretreated with RANTES demonstrated a 54% reduction in the level of early HIV-1 LTR transcripts compared with that in untreated controls when exposed to the M-tropic BaL strain; progenitor cells pretreated with RANTES and exposed to the M-tropic BaL strain demonstrated an 81% reduction in the level of early HIV-1 LTR transcripts compared with that in untreated controls (Fig. 5). Mature T cells pretreated with MIP-1β demonstrated an 89% reduction in the level of early HIV-1 LTR transcripts compared with that in untreated controls when exposed to the M-tropic BaL strain. Similarly, progenitor cells pretreated with MIP-1β and exposed to the M-tropic BaL strain demonstrated a 94% reduction in the level of early HIV-1 LTR transcripts compared with that in untreated controls (Fig. 5).

Different chemokine receptors may predominate in specific tissue microenvironments; however, the majority of HIV-1 isolates efficiently use CXCR4 and/or CCR5 as primary entry coreceptors; alternative chemokine receptors may be used less efficiently. The present study was designed to determine whether CD34+ progenitor cells express the same chemokine receptors that are known to facilitate the fusion and entry of HIV-1 into mature CD4+ T cells. The present study demonstrates the expression of chemokine receptors CXCR4 and CCR5, the primary T-tropic and M-tropic HIV-1 coreceptors, respectively, on peripheral blood derived-CD34+ progenitor cells analyzed immediately following isolation. The majority of these CD34+ progenitor cells expressed both CXCR4 and CCR5, although different patterns of coreceptor expression could be appreciated by FACS and confocal microscopy. Peripheral blood-derived CD34+ progenitor cells were capable of sustaining a prolonged productive infection by diverse strains of HIV-1. HIV entry into progenitor cells could be modulated by soluble CD4 and HIV gp120 V3 loop neutralizing mAb, demonstrating that infection was dependent on CD4, a coreceptor, and the V3 loop of the HIV envelope in a manner similar to HIV infection of mature mononuclear cells. The utilization of CXCR4 and CCR5 as entry cofactors by T-tropic and M-tropic HIV strains was demonstrated by a reduction of viral entry into progenitor cells in the presence of the cognate ligand for each chemokine coreceptor. These data suggest that the circulating CD34+ progenitor cell population may be infected in vivo and may serve as a dynamic reservoir for HIV that is capable of disseminating virus to diverse anatomic sites.

The susceptibility of host cells to infection by HIV is determined not only by surface expression of the CD4 molecule, but also by the surface expression of various chemokine receptors that are required for fusion and entry of virus into its target cells (15, 16, 17, 18, 19, 20, 21, 22, 23). Deichmann et al. (28) have recently demonstrated the presence of mRNA for both CXCR4 and CCR5 in granulocyte CSF-mobilized CD34+ progenitor cells; however, cell surface expression of CXCR4 and CCR5 on CD34+ progenitor cells has not been previously determined. The present study demonstrates cell surface expression of CXCR4 and CCR5 on peripheral blood-derived CD34+ progenitor cells. A lower percentage of CD34+ CD38+ lineage-committed cells express both coreceptors compared with the multipotent CD34+ CD90 (Thy-1)+ progenitor cells as determined by FACS analysis, suggesting that coreceptor expression may vary during differentiation. The high percentage of HIV coreceptor expression in the multipotent CD34+ CD90 (Thy-1)+ progenitor subset may result in an increased susceptibility to HIV infection. In this regard, select CD4+ subsets are altered in the bone marrow during the course of HIV infection; the proportion of nonlineage-committed primitive progenitor cells (CD34+ CD38 CD4+ CD90w+) is significantly decreased, while the proportion of lineage-committed (CD34+ CD38+ CD4+) progenitor cells is maintained (4). Direct infection of the primitive progenitor compartment, which represents a minor percentage (0.01%) of bone marrow cells, is difficult to detect. Depletion of primitive progenitors observed in later stages of HIV disease may represent a virally induced alteration in progenitor cell differentiation (54, 55, 56, 57, 58, 59, 60, 61, 62, 63) or may be due to exhaustion of prelymphoid progenitors that are mobilized to sites of extramedullary lymphopoiesis.

Morphologically, we observed that the distribution of each coreceptor on CD34+ progenitor cells was distinct, as demonstrated by confocal microscopy; this may indicate a differentiation-dependent or lineage-specific effect on expression. Colocalization of CXCR4 and CCR5 could be appreciated to varying degrees among the progenitor cell population. These data demonstrate that chemokine receptor expression may be dependent not only on the progenitor cell source as derived from the medullary (64) (M. Ruiz, unpublished observations) or vascular compartment, but also on the state of maturation and/or lineage commitment of the progenitor cells.

In this study, efficient viral entry occurred in mature CD4+ T cells and in CD34+ progenitor cells. The expression of a sufficient density of CD4 molecules on the host cell surface in addition to recruitable CXCR4 or CCR5 coreceptors are important variables that define the efficiency of viral fusion and entry into host cells. A minor population (range, 10–40%) of CD34+ progenitor cells express a low density of surface CD4 molecules, equivalent to 5% of the number of CD4 molecules found on mature T cells (this report and Refs. 11–13). A recent report (64) demonstrated that all CD34+ bone marrow progenitors express CD4 mRNA as well as CXCR4 and CCR5 mRNAs. The precise stoichiometry between CD4 and chemokine receptors that is necessary for the establishment of productive infection at the single cell level is not known. In this regard, it has been shown that infection of mature CD4+ T cells by M-tropic strains of HIV-1 is dependent on the density of CD4 molecules on the cell surface. Cells that express low or high amounts of CD4 are equally infectable when CCR5 concentrations are above threshold levels for maximal infection; however, infection becomes dependent on coreceptor expression levels when CD4 expression is low. Therefore, a high chemokine receptor density can compensate for low surface expression of CD4 in mediating HIV-1 infection. In contrast, infection of mature CD4+ T cells by dual tropic and T-tropic strains of HIV-1 is less dependent on the density of CD4 molecules on the surface of the cell (65). The selective expression of chemokine receptors and their local surface membrane association with CD4 receptors in CD34+ progenitor cells may influence the susceptibility of certain CD34+ subpopulations to HIV-1 infection in a strain-specific manner. In this study we have demonstrated that the majority of CD34+ progenitor cells derived from peripheral blood express both CXCR4 and CCR5 receptors and are susceptible to infection by M-tropic, dual tropic, T-tropic, and T cell laboratory-adapted strains of HIV and produced very high levels of virus (>10,000 cpm/μl mean RT activity). Unlike highly activated mature CD4+ T cells, CD34+ progenitor cells produced high levels of virus for a prolonged period of culture (>30 days), suggesting that progenitor cells may serve as a relatively stable source of HIV once a productive infection becomes established.

In the present study the primary targets for HIV infection appear to be the CD34+ CD4+ progenitor cells, since treatment with sCD4 effectively suppressed entry of all HIV-1 strains; however, the potential infection of CD34+ CD4 progenitor cells in vivo cannot be excluded. Abs directed against either the M- or T-tropic specific envelope V3 epitopes effectively inhibited HIV-1 infection in mature CD4+ T cells and in CD34+ progenitor cells, indicating that the interaction of these V3 epitopes with CD4/CCR5 or CD4/CXCR4 on progenitor cells is similar in both cell populations.

It has previously been demonstrated that the CCR5 ligands MIP-1α, MIP-1β, and RANTES, and the CXCR4 ligand SDF-1 potently suppress M- and T-tropic HIV entry and replication, respectively, in mature CD4+ T cells; however, the effects of chemokines on viral entry or replication in cells expressing low levels of CD4, as in dendritic cells or macrophages, is less evident (66). The effect of chemokines on HIV infection of CD34+ progenitor cells, which also express low levels of CD4, had not been previously delineated. HIV-1 entry and early replication events (early LTR transcripts) of the M-tropic BaL strain were sensitive to inhibition by the CCR5 ligands, RANTES and MIP-1β, in CD34+ progenitor cells and in mature CD4+ T cells. However, early replication events of the T-tropic NL4.3 strain in CD34+ progenitor cells appeared to be more sensitive to inhibition with low concentrations of the CXCR4 ligand, SDF-1, compared with mature CD4+ T cells. The basis of the increased sensitivity to SDF-1-mediated inhibition may be due to an altered stoichiometry between CD4 and CXCR4 receptors, due perhaps to the low density of CD4 molecules on CD34+ progenitor cells. Alternately, cell type-specific differences in the glycosylation of chemokine receptors may alter ligand or HIV binding characteristics (67).

Circulating CD34+ cells represent a distinct progenitor pool responsible for seeding extramedullary sites of hemopoiesis. Progenitor cell infection with HIV may effect long term functional consequences within extramedullary sites of lymphopoiesis. Cells within extramedullary sites of lymphopoiesis that express low levels of CD4, such as accessory cells and mesenchymal cells, are susceptible to infection by HIV-1 and, in turn, are capable of transmitting the virus to immature cells of the lymphoid and myeloid lineages (14, 68). Extramedullary lymphopoiesis is dependent on an intact mesenchymal environment that is functionally capable of accommodating immigrating multipotent progenitor cells originating from the bone marrow. The observed failure to normalize the CD4+ T cell count and the apparent failure to restore the TCR-Vβ repertoire in individuals with HIV, even those undergoing highly active antiretroviral therapy (69, 70), may be due to irreversible effects of HIV within the progenitor cell pool itself or within the progenitor cell microenvironment, either proximally within the bone marrow (4, 14) or distally within extramedullary sites of lymphopoiesis (5, 54, 69). Thus, infection of progenitor cells may have important consequences with regard to the potential for spontaneous immunologic reconstitution following adequate suppression of HIV replication by highly active antiretroviral therapy.

The sCD4 and other reagents were kindly provided by Raymond Sweet, SmithKline Beecham (King of Prussia, PA). mAb F1.9B was kindly provided by J. Robinson, Tulane University (New Orleans, LA). mAb 178.1 was kindly provided by J. Arthos, National Institute of Allergy and Infectious Diseases, National Institutes of Health. P. Walsh provided editorial assistance.

2

Abbreviations used in this paper: CXCR4, CXC chemokine receptor 4; CCR5, CC chemokine receptor 5; T-tropic, T cell-tropic; M-tropic, macrophage-tropic; MIP, macrophage inflammatory protein; SDF, stromal-derived factor; PE, phycoerythrin; LTR, long terminal repeat; V3, third variable loop; sCD4, soluble CD4.

1
von Laer, D., F. Hufert, T. Fenner, S. Schwander, M. Dietrich, H. Schmitz, P. Kern.
1990
. CD34+ hematopoietic progenitor cells are not a major reservoir of the human immunodeficiency virus.
Blood
76
:
1281
2
Molina, J. M., D. Scadden, M. Sakaguchi, B. Fuller, A. Woon, J. Groopman.
1990
. Lack of evidence for infection of or effect on growth of hematopoietic progenitor cells after in vivo or in vitro exposure to human immunodeficiency virus.
Blood
76
:
2476
3
Neal, T. F., H. K. Holland, C. M. Baum, F. Villinger, A. A. Ansari, R. Saral, J. Wingard, W. Fleming.
1995
. CD34+ progenitor cells from asymptomatic patients are not a major reservoir for human immunodeficiency virus-1.
Blood
86
:
1749
4
Marandin, A., A. Katz, E. Oksenhendler, M. Tulliez, F. Picard, W. Vainchenker, F. Louache.
1996
. Loss of primitive hematopoietic progenitors in patients with human immunodeficiency virus infection.
Blood
88
:
4568
5
Ruiz, M., A. P. Knutsen, S. T. Roodman.
1995
. Differentiation of bone marrow derived CD34+ stem cells from HIV+ hemophiliacs in cultured thymic epithelial fragments.
J. Allergy Clin. Immunol.
95
:
141
6
Davis, B., D. Schwartz, J. Marx, C. Johnson, J. M. Berry, J. Lyding, T. Merigan, A. Zander.
1991
. Absent or rare human immunodeficiency virus infection of bone marrow stem/progenitor cells in vivo.
J. Virol.
65
:
1985
7
Stutte, H. J., H. Muller, S. Falk, H. L. Schmidts.
1990
. Pathophysiological mechanisms of HIV-induced defects in haematopoiesis: pathology of the bone marrow.
Res. Virol.
141
:
195
8
Kaczmarski, R. S., F. Davison, E. Blair, S. Sutherland, J. Moxham, T. McManus, G. J. Mufti.
1992
. Detection of HIV in hematopoietic progenitors.
Br. J. Haematol.
82
:
764
9
Stanley, S. K., S. W. Kessler, J. S. Justement, S. M. Schnittman, J. J. Greenhouse, C. C. Brown, L. Musongela, K. Musey, B. Kapita, A. S. Fauci.
1992
. CD34+ bone marrow cells are infected with HIV in a subset of seropositive individuals.
J. Immunol.
149
:
689
10
Folks, T., S. Kessler, J. Orenstein, J. Justement, E. Jaffe, A. S. Fauci.
1988
. Infection and replication of HIV-1 in purified progenitor cells of normal human bone marrow.
Science
242
:
919
11
Zauli, G., G. Fulini, M. Vitale, M. C. Re, D. Gibellini, L. Zamai, G. Visani, P. Borgatti, S. Capitani, M. LaPlaca.
1994
. A subset of human CD34+ hematopoietic progenitors express low levels of CD4, the high affinity receptor for human immunodeficiency virus-type 1.
Blood
84
:
1896
12
Louache, F., N. Debili, A. Marandin, L. Culombel, W. Vainchenker.
1994
. Expression of CD4 by human hematopoietic progenitors.
Blood
84
:
3344
13
Muench, M., M. G. Roncarlo, R. Namikawa.
1997
. Phenotypic and functional evidence for the expression of CD4 by hematopoietic stem cells isolated from human fetal liver.
Blood
89
:
1364
14
Scadden, D., M. Zeira, A. Woon, Z. Wang, L. Schieve, K. Ikeuchi, B. Lim, J. Groopman.
1990
. Human immunodeficiency virus infection of human bone marrow stromal fibroblasts.
Blood
76
:
317
15
Dalgleish, A., P. Beverley, P. Clapham, D. Crawford, M. Greaves, R. Weiss.
1984
. The CD4 (T4) antigen is an essential component of the receptor for the AIDS retrovirus.
Nature
312
:
763
16
Madden, P. J., A. Dalgleish, J. McDougal, P. Clapham, R. Weiss, R. Axel.
1986
. The T4 gene encodes the AIDS virus receptor and is expressed in the immune system and the brain.
Cell
47
:
333
17
Feng, Y., C. C. Broder, P. E. Kennedy, E. A. Berger.
1996
. HIV-1 entry co-factor: functional cDNA cloning of a seven transmembrane G protein coupled receptor.
Science
272
:
872
18
Berson, J. F., D. Long, B. J. Doranz, J. Rucker, F. R. Jirik, R. W. Doms.
1996
. A seven transmembrane receptor involved in fusion and entry of T cell tropic HIV-1 strains.
J. Virol.
70
:
6288
19
Alkhatib, G., C. Combadiere, C. Broder, Y. Feng, P. Kennedy, P. Murphy, E. A. Berger.
1996
. CCCKR5: a RANTES, MIP-1α, MIP-1β receptor as a fusion cofactor for macrophage-tropic HIV-1.
Science
272
:
1955
20
Choe, H., Farzan M., Sun Y., Sullivan N., Rollins B., Ponath P. D., Wu L., Mackay C. R., LaRosa G., Newman W., et al
1996
. The β-chemokine receptors CCR3 and CCR5 facilitate infection by primary HIV-1 isolates.
Cell
85
:
1135
21
Doranz, B., J. Rucker, Y. Yi, R. Smyth, M. Samson, S. Peiper, M. Parmentier, R. G. Collman, R. W. Doms.
1996
. A dual-tropic primary HIV-1 isolate that uses fusin and the β-chemokine receptors CKR-5, CKR-3 and CKR-2b as fusion cofactors.
Cell
85
:
1149
22
Dragic, T., V. Litwin, G. P. Allaway, S. R. Martin, Y. Huang, K. A. Nagashima, C. Cayanan, P. J. Maddon, R. A. Koup, J. P. Moore, W. A. Paxton.
1996
. HIV-1 entry into CD4+ T cells is mediated by the chemokine receptor CC-CKR5.
Nature
381
:
667
23
Deng, H. K., Liu R., Ellmeier W., Choe S., Unutmaz D., Burkhart M., DiMarzio P., Marmon S., Sutton R. E., Hill C. M., et al
1996
. Identification of a major co-receptor for primary isolates of HIV-1.
Nature
381
:
661
24
Chelucci, C., H. J. Hassan, C. Locardi, D. Bulgarini, E. Pelosi, G. Mariani, U. Testa, M. Federico, M. Valtieri, C. Peschle.
1995
. In vitro human immunodeficiency virus-1 infection of purified hematopoietic progenitors in single cell culture.
Blood
85
:
1181
25
Steinberg, H., C. Crumpacker, P. Chatis.
1991
. In vitro suppression of normal human bone marrow progenitor cells by human immunodeficiency virus.
J. Virol.
65
:
1765
26
Cen, D., G. Zauli, R. Szarnicki, B. R. Davis.
1993
. Effect of different HIV-1 isolates on long term bone marrow hematopoiesis.
Br. J. Haematol.
85
:
596
27
Baiocchi, M., E. Olivetta, C. Chelucci, A. C. Santarcangelo, R. Bona, P. d’Aloja, U. Testa, N. Komatsu, P. Verani, M. Federico.
1997
. Human immunodeficiency virus (HIV)-resistant CD4+ UT-7 megakaryocytic human cell line becomes highly HIV-1 and HIV-2 susceptible upon CXCR4 transfection: induction of cell differentiation by HIV-1 infection.
Blood
89
:
2670
28
Deichmann, M., R. Kronenwett, R. Haas.
1997
. Expression of the human immunodeficiency virus type-1 coreceptors CXCR-4 (fusin, LESTR) and CKR-5 in CD34+ hematopoietic progenitor cells.
Blood
89
:
3522
29
Spits, H., L. Lanier, J. Phillips.
1995
. Development of human T and natural killer cells.
Blood
85
:
2654
30
Imhof, B. A., M. A. Deugnier, B. Bauvois, D. Dunon, J. P. Thiery.
1989
. Properties of pre-T cells and their chemotactic migration to the thymus. M. Kendall, and M. Ritter, eds.
Thymus Update
3
-20. Harwood Academic Publishers, New York.
31
Freshney, R. I., I. B. Pragnell, M. G. Freshney.
1994
.
Culture of Hematopoietic Cells
281
Wiley Liss, New York.
32
Goff, S., P. Traktman, D. Baltimore.
1981
. Isolation and properties of Moloney murine leukemia virus mutants: use of a rapid assay for release of virion reverse transcriptase.
J. Virol.
38
:
239
33
Willey, R. L., D. H. Smith, L. A. Lasky, R. S. Theodore.
1988
. In vitro mutagenesis identifies a region within the envelope gene of the human immunodeficiency virus that is critical for infectivity.
J. Virol.
62
:
139
34
Zack, J. A., S. J. Arrigo, S. R. Weitsman, A. S. Go, A. Haislip, I. S. Chen.
1990
. HIV-1 entry into quiescent primary lymphocytes molecular analysis reveals a labile, latent viral structure.
Cell
61
:
213
35
Livin, C. I..
1995
. Purification and expansion of human hematopoietic stem cells.
Leucocyte Typing V White Cell Differentiation Antigens, Vol. 1, sect. M10.10
869
-871. Oxford University Press, London.
36
Hardy, C. L., J. Mingrell.
1993
. Cellular interactions in hemopoietic progenitor-cell homing: a review.
Scanning Microsc.
7
:
333
37
Oxley, S., R. Sackstein.
1994
. Detection of an L-selectin ligand on a hematopoietic progenitor cell line.
Blood
84
:
3299
38
Rollins, B. J..
1997
. Chemokines.
Blood
90
:
909
39
Stenberg, P., T. Pestina, R. Barrie, C. Jackson.
1997
. The Src family kinases, Fgr, Fyn, Lck, and Lyn, colocalize with coated membranes in platelets.
Blood
89
:
2384
40
Simons, K., E. Ikonen.
1997
. Functional rafts in cell membranes.
Nature
387
:
569
41
Atchison, R., J. Gosling, F. Monteclaro, C. Franci, L. Digilio, I. Charo, M. Goldsmith.
1996
. Multiple extracellular elements of CCR5 and HIV-1 entry: dissociation from response to chemokines.
Science
274
:
1924
42
Rucker, J., Samson M., Doranz B., Libert F., Berson J., Yi Y., Smyth R., Collman R., Broder C., Vassart G., et al
1996
. Regions in β-chemokine receptors CCR5 and CCR2b that determine HIV-1 cofactor specificity.
Cell
87
:
437
43
Cocchi, F., A. DeVico, A. Garzino-Demo, A. Cara, R. Gallo, P. Lusso.
1996
. The V3 domain of the HIV-1 gp120 envelope glycoprotein is critical for chemokine-mediated blockade of infection.
Nat. Med.
2
:
1244
44
Trkola, A., T. Dragic, J. Arthos, J. M. Binley, W. C. Olson, G. P. Allaway, C. Cheng-Mayer, J. Robinson, P. J. Maddon, J. P. Moore.
1996
. CD4-dependent, antibody-sensitive interactions between HIV-1 and its co-receptor CCR-5.
Nature
384
:
184
45
Wu, L., Gerard N., Wyatt R., Choe H., Parolin C., Ruffing N., Borsetti A., Cardoso A. A., Desjardin E., Newman W., et al
1996
. CD4-induced interaction of primary HIV-1 gp120 glycoproteins with the chemokine receptor CCR-5.
Nature
384
:
179
46
Fouts, T. R., J. M. Binley, A. Trkola, J. E. Robinson, J. P. Moore.
1997
. Neutralization of the human immunodeficiency virus type 1 primary isolate JR-FL by human monoclonal antibodies correlates with antibody binding to the oligomeric form of the envelope glycoprotein complex.
J. Virol.
71
:
2779
47
Cheng-Mayer, C., M. Quiroga, J. W. Tung, D. Dina, J. A. Levy.
1990
. Viral determinants of human immunodeficiency virus type 1 T-cell or macrophage tropism, cytopathogenicity, and CD4 antigen modulation.
J. Virol.
64
:
4390
48
York-Higgins, D., C. Cheng-Mayer, D. Vauer, J. A. Levy, D. Dina.
1990
. Human immunodeficiency virus type 1 cellular host range, replication and cytopathicity are linked to the envelope region of the viral genome.
J. Virol.
64
:
4016
49
Bleul, C. C., M. Farzan, H. Choe, C. Parolin, I. Clark-Lewis, J. Sodroski, T. A. Springer.
1996
. The lymphocyte chemoattractant SDF-1 is a ligand for LESTR/fusin and blocks HIV-1 entry.
Nature
382
:
829
50
Oberlin, E., Amara A., Bachelerie F., Bessia C., Virelizier J.-L., Arenzana F., O. -Seisdedos, J. M. Schwartz, I. Heard, D. F. Clark-Lewis, D. F. Lagler, et al
1996
. The CXC chemokine SDF-1 is the ligand for LESTR/fusin and prevents infection by T cell line adapted HIV-1.
Nature
382
:
833
51
Nagasawa, T., S. Hirota, K. Tachibana, N. Takakura, S. I. Nishikawa, Y. Kitamura, N. Yoshida, H. Kikutani, T. Kishimoto.
1996
. Defects of B cell lymphopoiesis and bone marrow myelopoiesis in mice lacking the CXC chemokine PBSF/SDF-1.
Nature
382
:
635
52
Aiuti, A., I. Webb, C. Bleul, T. Springer, J. Gutierrez-Ramos.
1997
. The chemokine SDF-1 is a chemoattractant for human CD34+ hematopoietic progenitor cells and provides a new mechanism to explain the mobilization of CD34+ progenitors to peripheral blood.
J. Exp. Med.
185
:
111
53
Amara, A., S. L. Gall, O. Schwartz, J. Salamero, M. Montes, P. Loetscher, M. Baggiolini, J. L. Virelizier, F. Arenzana-Seisdedos.
1997
. HIV coreceptor downregulation as antiviral principle: SDF-1α-dependent internalization of the chemokine receptor CXCR4 contributes to inhibition of HIV replication.
J. Exp. Med.
186
:
139
54
Schnittman, S. M., S. M. Denning, J. J. Greenhouse, J. S. Justement, M. Baseler, J. Kurtzberg, B. F. Haynes, A. S. Fauci.
1990
. Evidence for susceptibility of intrathymic T-cell precursors and their progeny carrying T-cell antigen receptor phenotypes TCRαβ+ to human immunodeficiency virus infection: a mechanism for CD4 (T4) lymphocyte depletion.
Proc. Natl. Acad. Sci. USA
87
:
7727
55
Lunardi-Iskandar, Y., V. Georgoulias, M. Allouche, P. Meyer, J. C. Gluckman, M. Gentilini, C. Jasmin.
1985
. Abnormal in vitro proliferation and differentiation of T-colony forming cells in AIDS patients and clinically normal male homosexuals.
Clin. Exp. Immunol.
60
:
285
56
DeLuca, A., L. Teofili, A. Antinori, M. S. Iovino, P. Mencarini, E. Visconti.
1993
. Haematopoietic CD34+ progenitor cells are not infected by HIV-1 in vivo but show impaired clonogenesis.
Br. J. Haematol.
85
:
20
57
Zauli, G., B. R. Davis, M. C. Re, G. Visani, G. Furlini, M. LaPlaca.
1992
. Tat protein stimulates production of transforming growth factor-β by marrow macrophages: a potential mechanism for human immunodeficiency virus-1 induced hematopoietic suppression.
Blood
80
:
3036
58
Zauli, G., M. Vitale, D. Gibellini, S. Capitani.
1996
. Inhibition of purified CD34+ hematopoietic progenitor cells by human immunodeficiency virus-1 or gp-120 mediated by endogenous transforming growth factor β-1.
J. Exp. Med.
183
:
99
59
Zauli, G., M. C. Re, G. Visani.
1992
. Inhibitory effect of HIV-1 envelope glycoproteins gp120 and gp160 on the in vitro growth of enriched (CD34+) hematopoietic progenitor cells.
Arch. Virol.
122
:
271
60
Re, M. C., G. Zauli, D. Gibellini, G. Furlini, E. Ramazzotti, P. Monari.
1993
. Uninfected haematopoietic progenitors (CD34+) cells purified from the bone marrow of AIDS patients are committed to apoptotic cell death in culture.
AIDS
7
:
1049
61
Maciejewski, J. P., F. F. Weichold, N. S. Young.
1994
. HIV-1 suppression of hematopoiesis in vitro mediated by envelope glycoprotein and TNF-α.
J. Immunol.
153
:
4303
62
Furlini, G., M. Vignoli, E. Ramazzotti, M. C. Re, G. Visani, M. La Placa.
1996
. A concurrent human herpesvirus-6 infection renders two human hematopoietic progenitor (TF-1 and KG-1) cell lines susceptible to human immunodeficiency virus type-1.
Blood
87
:
4737
63
Bagnara, G. P., G. Zauli, M. Giovannini, M. C. Re, G. Furlini, M. La Placa.
1990
. Early loss of circulating hemopoietic progenitors in HIV-1 infected subjects.
Exp. Hematol.
18
:
426
64
Shen, H., T. Cheng, O. Yang, M. Tomasson, D. E. Golan, F. I. Preffer, A. D. Luster, D. T. Scadden. 1997. Expression of HIV-1 receptors is insufficient for infection of hematopoietic stem cells. Blood 90:(Suppl. 1):2567.
65
Platt, E. J., Wehrly K., Kuhmann S., Chesebro B., Kabat D..
1998
. Effects of CCR5 and CD4 cell surface concentrations on infections by macrophagetropic isolates of human immunodeficiency virus type 1.
J. Virol.
72
:
2855
66
Hart, D. N. J..
1997
. Dendritic cells: unique leukocyte populations which control the primary immune response.
Blood
90
:
3245
67
Fenouillet, E., R. Miquelis, R. Drillien.
1996
. Biological properties of recombinant HIV envelope synthesized in CHO glycosylation-mutant cell lines.
Virology
218
:
224
68
Savino, W. M., M. Dardenne, C. Marche.
1985
. Thymic epithelium in AIDS: an immunohistologic study.
Am. J. Pathol.
122
:
302
69
Autran, B., T. Carcelian, T. S. Li, C. Blanc, D. Mathez, R. Tubiana, C. Katlama, P. Debre, J. Leibowitch.
1997
. Positive effects of combined antiretroviral therapy on CD4+ T cell homeostasis and function in advanced HIV disease.
Science
277
:
112
70
Connors, M., J. A. Kovacs, S. Krevat, J. C. Gea-Banacloche, H. C. Lane.
1997
. HIV infection induces changes in CD4+ T-cell phenotype and depletions within the CD4+ T-cell repertoire that are not immediately restored by antiviral or immune-based therapies.
Nat. Med.
3
:
533