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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 94 No. 1 (July 1), 1999:
pp. 62-73
Human CD34+ Cells Express CXCR4 and Its Ligand Stromal
Cell-Derived Factor-1. Implications for Infection by T-Cell Tropic
Human Immunodeficiency Virus
By
Alessandro Aiuti,
Lucia Turchetto,
Manuela Cota,
Arcadi Cipponi,
Andrea Brambilla,
Cinzia Arcelloni,
Rita Paroni,
Elisa Vicenzi,
Claudio Bordignon, and
Guido Poli
From Telethon Institute for Gene Therapy (TIGET); AIDS
Immunopathogenesis Unit, DIBIT; and Laboratory of Separative
Techniques, Scientific Institute H.S. Raffaele, Milan, Italy.
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ABSTRACT |
Human CD34+ hematopoietic progenitor cells obtained
from bone marrow (BM), umbilical cord blood (UCB), and mobilized
peripheral blood (MPB) were purified and investigated for the
expression of the chemokine receptor CXCR4 and its ligand, stromal
cell-derived factor-1 (SDF-1). CXCR4 was found present on the cell
surface of all CD34+ cells, although it was expressed at
lower density on MPB with respect to BM CD34+ cells.
Freshly isolated and in vitro-cultured CD34+ cells also
coexpressed SDF-1 mRNA, as determined by reverse
transcriptase-polymerase chain reaction (RT-PCR). Of interest,
CD34+/CD38+ committed progenitor cells,
unlike primitive CD34+/CD38 cells,
expressed SDF-1 mRNA. Supernatants from in vitro-cultured CD34+ cells contained substantial (3 to 8 ng/mL) amounts
of SDF-1 by enzyme-linked immunosorbent assay and induced migration of
CD34+ cells. Because CD34+ cells express
low levels of CD4, the primary receptor of the human immunodeficiency
virus (HIV), and CXCR4 is a coreceptor for T-cell tropic (X4) HIV
strains, we investigated the susceptibility of CD34+
cells to infection by this subset of viruses. Lack of productive infection was almost invariably observed as determined by a
conventional RT activity in culture supernatants and by real-time PCR
for HIV DNA in CD34+ cells exposed to both laboratory
adapted (LAI) and primary (BON) X4 T-cell tropic HIV-1 strain. Soluble
gp120 Env (sgp120) from X4 HIV-1 efficiently blocked binding of the
anti-CD4 Leu3a monoclonal antibody (MoAb) to either human
CD4+ T cells or CD34+ cells. In contrast,
sgp120 interfered with an anti-CXCR4 MoAb binding to human T
lymphocytes, but not to CD34+ cells. However, CXCR4 on
CD34+ cells was downregulated by SDF-1. These results
suggest that CXCR4 and its ligand SDF-1 expressed in
CD34+ progenitors may play an important role in
regulating the local and systemic trafficking of these cells. Moreover,
these findings suggest multiple and potentially synergistic mechanisms
at the basis of the resistance of CD34+ cells to X4 HIV
infection, including their ability to produce SDF-1, and the lack of
CXCR4 internalization following gp120 binding to CD4.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
HEMATOPOIETIC PROGENITOR CELLS normally
reside in the bone marrow (BM) in close contact with cells of the
stromal microenvironment that provide a rich milieu of cytokines,
extracellular matrix proteins, and adhesion molecules. Progenitor cells
are likely compartmentalized in different areas of the BM, based on
their degree of commitment and lineage differentiation.1
They home to and emigrate from the BM under experimental procedures
such as transplantation or peripheral blood (PB)
mobilization.2 However, little is known about the
mechanisms and molecules that regulate the homing, retention, and
emigration of progenitor cells in hematopoietic organs. By analogy with
mature leukocytes, these processes likely involve chemoattractant
molecules and their receptors, which are known to regulate the
trafficking of leukocytes under both physiological and pathological
conditions.3
It has been recently reported that human CD34+
hematopoietic progenitor cells are capable of responding to a gradient
of chemoattractant(s) produced by stromal cells.4 A
chemotactic factor was isolated and identified as stromal cell-derived
factor-1 (SDF-1), a CXC chemokine previously cloned from mouse BM
stromal cells.5 SDF-1 has been also shown to be a
low-potency, high-efficacy chemoattractant for human T lymphocytes and
monocytes.6 In addition, this chemokine is likely
constitutively expressed in a wide variety of tissues.5 The
receptor for SDF-1 was subsequently identified on human lymphocytes as
CXCR4 (previously named fusin, HUMSTR, or LESTR),7,8 a seven-transmembrane-domain protein member of the  chemokine receptor family. Before its identification as the receptor for SDF-1, CXCR4 was
shown to act, along with CD4, as a receptor for human immunodeficiency virus (HIV)-1 T-cell tropic strains,9 recently classified
as X4 viruses.10 Indeed, SDF-1 inhibits cell fusion and
infection by HIV strains with a syncytium-inducing (SI) phenotype
typically emerging during the late, symptomatic stages of the
disease.7,8
Recent studies have demonstrated the presence on CD34+
cells of both CD4 antigens (Ags),11,12 and of mRNA encoding
for CXCR4,13 raising the possibility that these cells may
be susceptible to HIV-1 infection by X4 viral strains. This issue of
CD34+ progenitor cell infection is of particular relevance
for understanding the frequent cytopenias, dysmyelopoiesis, and
impaired colony growth that affect HIV-1 patients in their advanced
stages14 as well as in the view of an anti-HIV gene therapy
approach of progenitor cells.15 Conflicting studies have
been reported regarding the susceptibility of human CD34+
cells to HIV-1 infection both in vivo and in vitro. However, most
studies indicate that progenitor cells are not a major viral reservoir
in HIV-1-infected individuals at different stages of disease.16-19 Susceptibility of human CD34+
cells to in vitro HIV-1 infection has been reported, although with
variable efficiency.21-25 However, no systematic attempts have been made thus far of comparing the susceptibility to HIV infection of progenitor cells obtained from different sources, such as
BM, umbilical cord blood (UCB), or mobilized PB (MPB). This would be of
particular relevance in the view of the different implications for
adult or in utero infection of progenitor cells as well as for the
proposed use of MPB CD34+ cells as targets for gene
therapy.26 In addition, a substantial heterogeneity exists
among progenitor cells obtained from different sources in terms of
their phenotypes,27 content in primitive and committed
populations,27-29 proliferative capacity,29-31
and cell-cycle status,32 all factors that may also affect
the ability of these cells to support HIV replication.
In the present study, we report the simultaneous expression of a
functional CXCR4 receptor and its ligand, SDF-1, on human BM, UCB, and
MPB CD34+ progenitor cells. Despite the expression of both
primary (CD4) and accessory (CXCR4) viral receptors, we almost
invariably observed lack of productive infection with X4 HIV-1 strains.
Our results suggest multiple and potentially synergistic mechanisms at
the basis of the resistance of CD34+ cells to X4 HIV-1 infection.
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MATERIALS AND METHODS |
Human CD34+ cell sources.
UCB was collected after normal deliveries according to institutional
guidelines for discarded material. BM was collected, after informed
consent, from normal healthy donors at the time of harvest for
allogeneic transplantation. Granulocyte colony-stimulating factor
(G-CSF) MPB was collected, after informed consent, by leukapheresis from normal healthy donors after subcutaneous injections of G-CSF (12 µg/kg) starting from day +4. Cyclophosphamide (CY)/G-CSF MPB was
collected from patients undergoing autologous BM transplantation for
multiple myeloma, breast cancer, or non-Hodgkin's lymphoma treated
with CY (7 g/m2, day 0) plus G-CSF (5 µg/kg) starting
from day +3. PB stem cells were collected on consecutive days at the
time of hematological recovery after the chemotherapy-induced leukopenia.
Mononuclear cell (MNC) and CD34+ cell purification.
MNCs were obtained after Ficoll (Lymphoprep; Nycomed Pharma, Oslo,
Norway) gradient separation from BM samples, UCB samples, G-CSF or
CY/G-CSF-mobilized apheresis products. CD34+
cells were purified by a two-round procedure using magnetic-cell sorting (MINIMACS; Milteny Biotech, Bergisch Gladbach, Germany) following the manufacturer's guidelines. This protocol consistently yielded CD34+ cells with a purity greater than 95%.
Flow cytometry and cell sorting.
For immunophenotyping, all cell stainings were performed in
phosphate-buffered saline (PBS) containing 1% bovine serum albumin (BSA) (Sigma, St Louis, MO), 0.1% sodium azide (PBS-FACS) in 100-µL volume for 30 minutes at +4°C. Staining of BM, UCB, and MBP MNC samples was performed as follows: 3 × 105 low-density
cells were incubated with either purified anti-CXCR4 12.G5 MoAb (5 µg/mL) (a kind gift of Dr J. Hoxie, University of Pennsylvania
Medical Center, Philadelphia) or mouse IgG2a control antibody (Ab).
After washing with PBS-FACS, cells were labeled with goat anti-mouse
phycoerythrin (PE)-conjugated polyclonal Ab (Southern Biotechnology,
Birmingham, AL), washed, and the free binding-site blocked by an excess
of mouse IgG (1 mg/mL; Sigma) for 10 minutes at room temperature.
Finally, cells were incubated with anti-CD34 fluorescein isothiocyanate
(FITC)-conjugated monoclonal Ab (MoAb) (Becton Dickinson, Mountain
View, CA) and anti-CD45 TC MoAb (Caltag, Burlingame, CA). The analysis
of CXCR4 expression was performed on low side scatter (SSC),
CD34+/CD45low gated cells, as
described.33 Fluorescence intensity of CXCR4 was
quantitated in terms of number of molecules of equivalent soluble
fluorochrome using Quantum-26 PE beads (Flow Cytometry Standards, San
Juan, PR). The fluorescence intensity (MESF, molecules of equivalent
soluble fluorochrome) of cells stained with the control MoAb were
subtracted from the CXCR4 values. Staining of freshly purified or in
vitro cultured CD34+ cells was performed as follows: 0.5 × 105 cells were labeled with biotinylated anti-CXCR4
(Pharmingen, San Diego, CA) or biotinylated mouse IgG2a (Caltag). After
washing, cells were incubated with anti-CD34 FITC MoAb, streptavidin TC (Caltag), and either anti-CD4 PE MoAb (Becton Dickinson) or mouse IgG1
PE, washed again, and then analyzed using a FACScan apparatus (Becton
Dickinson). All data were acquired in listmode with the Cell Quest
software (Becton Dickinson). To separate primitive (CD34+/CD38) from committed
(CD34+/CD38+) subpopulations of progenitor
cells, immunoaffinity-purified CD34+ cells were labeled
with anti-CD34 FITC MoAb (Becton Dickinson) and anti-CD38 PE MoAbs
(Coulter, Hialeah, FL). Cells were washed, resuspended in cold PBS
0.3% BSA at the concentration of 1 × 106/mL, and
sorted on a FACStarPLUS (Becton Dickinson). Nonspecific
background fluorescence was determined using cells stained with
isotype-matched control MoAbs. Sorted cells were collected in 10%
fetal calf serum (FCS) Iscove's Modified Dulbecco's Medium (IMDM),
whereas a fraction of the cells was restained and analyzed to verify
the purity of the sorted population. For cell sorting performed to
isolate the CD34+/CXCR4+/CD4+
subpopulation, cells were labeled with anti-CD34 FITC MoAb, anti-CD4 PE
MoAb, and biotinylated anti-CXCR4 MoAb, the latter revealed by
streptavidin TC. Sorted
CD34+/CXCR4+/CD4+ cells were
cultured 24 hours after cell sorting to release remaining bound MoAbs
and thereafter washed two times before infection.
Modulation of CD4/CXCR4 receptors.
Purified CD34+ cells or CD4+ T cells from UCB
(2.5 × 105 /test) were washed once with RPMI 0.2%
BSA 10 mmol/L HEPES and resuspended in 0.15 mL of the same medium
containing either: (1) HIV-1LAI/IIIB soluble (s) gp120 (5 µg/mL) (a kind gift of P. Lusso, DIBIT, Scientific Institute H.S.
Raffaele, Milan, Italy); this concentration of sgp120 Env was
previously found to be optimal for CD34+ cells binding
assays (A.A., unpublished observations, January 1998); (2) SDF-1 (1 mg/mL); (3) no ligands. Cells were incubated in continuous agitation
conditions for 2 hours at 4°C or 37°C and then washed once with
RPMI 0.2% BSA. Subsequently, the cells were incubated for 30 minutes
in PBS-FACS with anti-CD4 PE MoAb and anti-CXCR4 TC MoAb. Cells were
then analyzed by flow cytometry for the relative expression and
determination of the mean fluorescence intensity (MFI) of the CD4 and
CXCR4 Ags.
Ex vivo culture of CD34+ cells.
CD34+ cells (1 × 105/well) were seeded in
a 24-well plastic plate (Falcon; Becton Dickinson Labware, Lincoln
Park, NJ) in IMDM (GIBCO-BRL, Life Technology s.r.l., Milan, Italy)
containing 20% FCS or X-VIVO 20 serum-free medium (Biowhittaker,
Walkersville, MD) containing 2 mmol/L L-glutamin, 50 U/mL penicillin,
50 µg/mL streptomycin, in the presence of interleukin-3 (IL-3) (10 ng/mL), IL-6 (10 ng/mL), and stem cell factor (SCF) (10 ng/mL) (all
from R&D Systems, Minneapolis, MN), at a concentration previously
determined to be optimal for CD34+ cell expansion.
Colony-forming cell (CFC) assay.
Cells were seeded in duplicate cultures in 1 mL methylcellulose
assay-based media containing recombinant human (rHu) erythropoietin (2.5 U/mL) (Janssen-Cilag, Schaffausen, Switzerland), rHu SCF (10 ng/mL), rHuG-CSF (10 ng/mL) (Amgen, Thousand Oaks, CA), rHu granulocyte
macrophage (GM)-CSF (10 ng/mL) (Schering-Plough, Milan, Italy), rHu
IL-3 (10 ng/mL). Colonies were scored for colony-forming units
(CFU)-GM, CFU-MIX, and burst-forming units-erythroid
(BFU-E) after 2 weeks of incubation at 37°C, 5%
CO2.
Reverse transcription-polymerase chain reaction (RT-PCR) for
chemokines and chemokine receptor expression.
Aliquots (0.2 to 0.4 × 106 cells) of uninfected cells
were pelleted either immediately after purification or after 3, 6, and 11 days of culture by centrifuging at 12,000 rpm for 5 minutes at
4°C. Total RNA was extracted by the RNA-zol method (Duotech, Milan,
Italy) and resuspended in different volumes of water depending on cell
numbers. The same amount of RNA was reverse transcribed in the presence
of 1X RT buffer (GIBCO-BRL), 800 µmol/L of each dNTPs (Pharmacia
Biotech, Milan, Italy), 20 µg/mL random hexamers (Promega, Madison,
WI), 4 mmol/L dithiothreitol (GIBCO-BRL), 16 U RNA guard (Promega), and
400 U of murine-Moloney leukemia virus (M-MLV) RT (GIBCO-BRL). The
reaction mixture (50 µL) was incubated at 68°C for 5 minutes,
37°C for 60 minutes, heated at 94°C for 5 minutes, and cooled
on ice. Aliquots corresponding to 1/40 of the cDNA obtained were
amplified in the presence of 0.4 µmol/L primer pairs (PRIMM s.r.l.,
Milan, Italy), 200 µM dNTPs (Pharmacia), 1X PCR buffer, and 1.25 U of
AmpliTaq Gold DNA polymerase (Perkin-Elmer Corp, Norwalk, CN) in a
50-µL reaction mixture. The mRNA for the cellular housekeeping gene,
glyceraldehyde 3-phosphate dehydrogenase (GAPDH), was used as control.
The following primers were used for amplifying mRNAs for: GAPDH
sense,34 5'CCA TGG AGA AGG CTG GGG 3',
antisense 5'CAA AGT TGT CAT GGA TGA CC 3' (generating a
195-bp fragment); SDF-1 sense,35 5' ACG AAT TCG CGC
CAT GAA CGC CAA GGT CGT 3', antisense, 5'CAG GAT CCT GCA
AAC CTC AGG CCC GAT C 3' (kind gift of Dr S. Polo, DIBIT)
(generating a 451-bp length fragment); CXCR4 sense, 5' GCC AAC
GTC AGT GAG GCA GAT G 3'; antisense 5' GAG GAT GAC TGT GGT
CTT GAG G 3' (generating a 209-bp fragment); PCR products
were analyzed by electrophoresis in 2% agarose gel and visualized by
ethidium bromide staining.
SDF-1.
Murine SDF-1 was purified from MS-5 conditioned medium (kind gift of
Dr K. Itoh, Kyoto University, Kyoto, Japan)36
by affinity chromatography and high-performance liquid chromatography
(HPLC) according to a modification37 of a previous
protocol.6 Synthetic human SDF-1 was kindly donated by Iain
Clark-Lewis (Biomedical Research Centre, University of British
Columbia, Vancouver, Canada). The biochemical, antigenic, and
functional properties of human synthetic SDF-1 and native murine SDF-1
were comparable based on capillary electrophoreses, enzyme-linked
immunosorbent (ELISA), and chemotaxis assays (C. Arcelloni et al, submitted).
SDF-1 ELISA.
Supernatants of cells grown in X-VIVO 20 serum-free medium (50 µL),
or serial dilutions of SDF-1 in serum-free medium (from 500 ng/mL to 1 ng/mL) were incubated overnight in polycarbonate 96-well microtiter
plates (Nunc, Roskilde, Denmark). All samples were set up in duplicate.
The remaining binding sites were blocked by a 60-minute incubation with
PBS containing 2% BSA. Wells were then incubated for 90 minutes with a
goat polyclonal Ab (1 µg/mL) against human SDF-1 (R&D Systems). After
four washes, they were incubated for 90 minutes with a rabbit anti-goat
IgG polyclonal Ab conjugated to alkaline phosphatase (AP), affinity
isolated, adsorbed with human proteins (SIGMA, Milan, Italy) and washed again. The bound immunocomplexes were detected with 4-Nitrophenyl phosphate (1 mg/mL) (Boehringer Mannheim, Mannheim, Germany) in developing buffer pH 9.8 (Boehringer Mannheim) and the optical density
(OD) measured at 405 nm in a microplate reader. The
standard curve set up with SDF-1 allowed the detection of SDF-1 Ag in
serum-free supernatants ranging between 2 and 500 ng/mL.
Chemotaxis.
This assay was performed in duplicate using 5-µm pore polycarbonate
Transwell culture insert (Costar, Cambridge, MA). Immediately before
each assay, filters were rinsed in RPMI containing 0.3% human serum
albumin (SA). One hundred thousand CD34+ cells (in 100 µL) were loaded into each transwell filter. Filters were then
carefully transferred to another well containing human synthetic or
mouse native SDF-1 (at concentrations ranging from 1.5 to 0.01 µg/mL). After 3 hours, the upper chamber was carefully removed and
the cells in the bottom chamber resuspended and transferred to tubes
for determining the proportion of migrated CD34+ cells.
Cytofluorimetric analysis was performed on a FACScan for a constant,
predetermined period of time and counts obtained for each sample were
compared with flow cytometric counts obtained from control wells
containing 10% of the input population. Data were expressed as a
percentage of the input population or as the ratio of cells migrated to
SDF-1 to cells migrated to assay medium (chemotactic index).
HIV-1 infection and coreceptor usage assay.
HIV-1 LAI/IIIB is known to infect CD4+ cells
expressing CXCR4 (fusin).38 The viral stock was expanded on
phytohemagglutinin (PHA)-stimulated PB mononuclear cells (PBMC) and
titrated by an Mg2+-dependent RT activity
assay39 and by ID50 quantitation according to
the Reed and Muench formula.40 The primary isolate
BON was isolated from the plasma of an HIV-1+
individual onto a mixture of PHA-stimulated PBMC of two seronegative donors.39 The primary viral stock was aliquoted and
preserved at  80°C. The usage of HIV-1 coreceptors was
determined on U87 astrocytoma cell line permanently transfected with
human CD4 either alone or together with one of the following chemokine
receptor-expressing plasmids: CCR2b, CCR3, CCR5, or CXCR4, as
reported.41 By this assay, BON HIV-1 selectively infected
U87 cells coexpressing CD4 and CXCR4 (generous gift of Dr Dan Littman,
The Skirball Institute of Biomolecular Medicine, New York University
Medical Center, New York, NY), but not other coreceptors.
CD34+ cells (2.5 × 105/mL) were incubated
for 1 hour at 37°C with either HIV-1LAI/IIIB or BON at
the multiplicities of infection (m.o.i.) of 1, 0.1, and 0.01, respectively. Cells were washed twice in PBS and then seeded in a
48-well plate (Falcon; Becton Dickinson Labware) in duplicate cultures,
and maintained in IMDM medium supplemented with 20% FCS, IL-3 (10 ng/mL), IL-6 (10 ng/mL), and SCF (10 ng/mL) (complete medium)
throughout the period of infection (approximately 4 weeks). Culture
supernatants were harvested every 3 days, stored at 80°C,
and replaced with fresh complete medium.
HIV DNA copy number quantification by real-time PCR.
HIV-1LAI/IIIB was treated with 50 U/mL of RQ1 RNase-free Dnase (Boehringer) for 30 minutes at room
temperature (rt°) before infection. Cells (0.4 to 2 × 105) were harvested 1, 24, 48, 72, and 168 hours after infection, centrifuged at 12,000 rpm for 5 minutes, and the
pellets were stored at 80°C until DNA extraction. DNA
quantitation was performed simultaneously from all the stored samples.
Briefly, cell pellets were lysed by incubation at 50°C for 4 hours
with 200 µL of a buffer (0.25% Triton X, 0.25% sodium dodecyl
sulfate [SDS] in 1X Tris EDTA, TE) containing 600 µg/mL of
proteinase K; DNA extraction was performed by the phenol-chloroform
protocol followed by ethanol precipitation; DNA was resuspended in 10 µL of water. DNA proviral synthesis quantitation was performed by the
"real-time" quantitative PCR method.42-44 The
quantitation is based on the cleavage of fluorescent dye labeled probe
by the 5'-3' endonuclease activity of Taq DNA polymerase
during PCR and measurement of fluorescence intensity by the ABI Prism
7700 Sequence Detector System (PE Applied Biosystems, Foster City, CA).
A standard curve was generated by serial dilutions of the chronically
HIV-infected ACH-2 T-cell line (containing one proviral copy per
cell)45 in PBMC of a seronegative donor. Primer pair and
probe spanning gag were used: for 5'-ACA TCA AGC AGC CAT
GCA AAT-3'; rev 5'-ATC TGG CCT GGT GCA ATA GG-3';
probe 5'(FAM) CAT CAA TGA GGA AGC TGC AGA ATG GGA TAG A
(TAMRA)-3'. Amplification reactions (25 µL) contained 1X buffer
A, 2.5 mmol/L MgCl2, 200 µmol/L dATP, dCTP, and dGTP, 400 µmol/L dUTP, 0.625 U of AmpliTaq Gold, 0.25 U of AmpErase UNG (PE
Applied Biosystems), 0.25 µmol/L of each primer, and 75 nmol/L of
probe. The thermal cycling conditions were 50°C for 2 minutes,
95°C for 12 minutes, and 40 cycles of 95°C for 15 seconds and
65°C for 1 minute.
Statistics.
Results of experimental points from multiple experiments (n) were
expressed, as the mean ± SD, where applicable, or the range if n = 2. Significance levels were determined by two-sided Student's t-test analysis.
| |
RESULTS |
CXCR4 is expressed on human CD34+ progenitor cells
from different sources and acts as a receptor for SDF-1.
The anti-CXCR4 12.G5 MoAb46 was used to analyze the
expression of the receptor on CD34+/CD45dull
cells from various hematopoietic sources
(Fig 1A). On average, 39.2% ± 7% of
adult BM (n = 11), and 43.8% ± 5.8% of UCB (n = 7) expressed
CXCR4 on the cell surface. In comparison to BM, the proportion of
CXCR4+ expressing cells was reduced on CD34+
cells from Cy/G-CSF MPB (9.88 ± 6.8; n = 6; P < .005)
and, to a lesser extent, on CD34+ cells from G-CSF MPB
(30.3% ± 16%; n = 6). Furthermore, the MFI of CXCR4 expression
was considerably decreased both in G-CSF (1,068 ± 780, MESF) and
CY/G-CSF (784 ± 389) MPB CD34+ cells with respect to BM
CD34+ cells (2,280 ± 1,325, P < .05). To
confirm the specificity of the receptor-ligand interaction in human
CD34+ cells, we tested whether the anti-CXCR4 12.G5 MoAb,
previously shown to partially inhibit migration of lymphocytes and
CXCR4-transfected cells,47 could block SDF-1-mediated
chemotaxis of BM CD34+ cells. As shown in Fig 1B, when the
12.G5 MoAb (10 µg/mL) was added to CD34+ cells before
migration, the chemotactic response to optimal and suboptimal
concentrations of SDF-1 were reduced to approximately 50% and 10%,
respectively; no inhibition was observed in the presence of a control
Ab. A similar level of inhibition was obtained with CD34+
cells obtained from UCB or MPB. Thus, CXCR4 mediates SDF-1-induced chemotaxis of CD34+ cells of different origin.
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| Fig 1.
CXCR4 is expressed on CD34+cells and
mediates SDF-1-induced responses. (A) Flow cytometric analysis of
CXCR4 expression on CD34+ cells from bone marrow (BM),
umbilical cord blood (UCB), G-CSF mobilized peripheral blood (MPB)
cells, or cyclophosphamide plus G-CSF (CY/G-CSF) MPB. Low-density cells
were stained with CD34 FITC, CD45TC, and either a control mouse
antibody IgG2a (empty histograms) or anti-CXCR4 MoAb (filled
histograms), revealed by goat anti-mouse PE antibody, as described in
Materials and Methods. The analysis shows the staining of a gated
population of low SSC, CD34+, CD45dull cells.
(B) Inhibition of SDF-1-dependent chemotaxis by anti-CXCR4
antibody. Migration of BM CD34+ cells in response
to SDF-1 (1.5 and 0.3 µg/mL, respectively) and in the presence of 10 µg/mL of either 12.G5 anti-CXCR4 MoAb ( ), or an isotype-matched
control mouse IgG ( ).
|
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CD34+ cells express and secrete SDF-1.
Increasing evidence suggests that CD34+ progenitor cells
secrete, in an autocrine fashion, hematopoietic growth factors, such as
erythropoietin,48 IL-3, GM-CSF,49 and
transforming growth factor- (TGF-).50 Therefore, we
investigated whether CD34+ cells expressed the CXCR4 ligand
SDF-1 by means of RT-PCR. Total RNA was extracted from freshly purified
CD34+ cells obtained from BM, UCB, and MPB, reverse
transcribed, and amplified as described in Materials and Methods. SDF-1
mRNA transcripts of the expected length (451 bp) were present in all BM
CD34+ cells tested (n = 3), in all UCB samples (n = 4), and
in 2 of 3 MPB tested (Fig 2A, and data not
shown). No products were amplified in the absence of RT, thus excluding
contamination by genomic DNA (data not shown).
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| Fig 2.
RT-PCR analysis of the expression of SDF-1 and CXCR4 by
CD34+ cells. (A) Expression of CXCR4 and SDF-1 in freshly
isolated CD34+ cells from UCB, BM, and G-CSF MPB. The
RT-PCR was performed as indicated in Materials and Methods. GAPDH is
shown as a control. (B) RT-PCR analysis of CXCR4 and SDF-1 expression
in sorted CD34+/CD38 and
CD34+/CD38+ cells from UCB, BM, and MPB.
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The CD34+/CD38 immunophenotype defines a
rare, quiescent subpopulation of primitive progenitor cells that can be
functionally distinguished from committed
CD34+/CD38+ progenitor cells by sustained
clonogenicity in long-term culture,51 and the ability to
repopulate immunodeficient mice.52 Thus, CD34+
cells from UCB, BM, and MPB were sorted by flow cytometry into CD34+/CD38 and
CD34+/CD38+ cell subpopulations and analyzed by
RT-PCR for the expression of SDF-1 and CXCR4. As shown in Fig 2B, SDF-1
was found expressed by CD34+/CD38+ progenitor
cells, but not by CD34+/CD38 cells. In
contrast, both CD34+/CD38 and
CD34+/CD38+ subpopulations expressed CXCR4 by
RT-PCR (Fig 2B) and FACS analysis (data not shown). The kinetics of
both SDF-1 and CXCR4 expression were tested by RT-PCR in UCB
CD34+ cells grown in culture for up to 17 days in medium
containing IL-3, SCF, and IL-6. No substantial changes in chemokine or
chemokine receptor mRNA were observed over time (data not shown). To
confirm SDF-1 expression at the protein level, we set up a direct ELISA using an anti-SDF-1 polyclonal Ab that allowed us to detect the presence of SDF-1 Ag in serum-free medium with a lower detection limit
of 2 ng/mL. Cell supernatants were obtained from seven different CD34+ cell samples purified either from BM, UCB, and MPB
and cultured for 5 days in serum-free medium in the presence of IL-3,
SCF, and IL-6. The presence of SDF-1 was shown in five samples,
including sorted CD34+/CD38+ cells, in a range
variable between 3 and 8 ng/mL (Fig 3A).
SDF-1 Ag was detectable also in CD34+ cell supernatants
collected after 3 days of culture (data not shown). SDF-1 Ag was below
the detection level of this ELISA in two CD34+ cell
supernatants and in the supernatant of a polyclonal T-cell line.
Moreover, 50 to 100 ng/mL of SDF-1 Ag was detected in the supernatant
of MS-5 stromal cell line, which is known to constitutively produce
SDF-1 (Fig 3A).
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| Fig 3.
Secretion of SDF-1 by cultured CD34+ cells.
(A) Determination of SDF-1 Ag in CD34+ cell supernatant
by ELISA. CD34+ cells (2 × 105/mL) were
cultured for 5 days in serum-free medium in the presence of cytokines.
Cell supernatants were obtained from: different donors (A through G)
and sources (UCB, BM, MPB) of CD34+ cells; sorted MPB
CD34+/CD38+ cells; a human polyclonal
T-cell line (PBL); the mouse stromal cell line MS-5. Supernatants were
assayed for the presence of SDF-1 by an Ag capture ELISA, as indicated
in Materials and Methods. (B) Migration of CD34+ cells in
response to serum-free supernatant from UCB CD34+ cells
cultured in the presence of IL-3, SCF, and IL-6 for 4 days and 7 days,
respectively. Chemotaxis to serum-free medium containing cytokines
(assay medium) and to low doses of SDF-1 (10 ng/mL and 30 ng/mL) are
shown as controls. (C) Inhibition of chemotaxis induced by supernatant
from CD34+ cells. Chemotaxis of CD34+ cells
in response to 5 days supernatant from UCB CD34+ cells in
the presence of an anti-CXCR4 MoAb or of a control MoAb.
|
|
To further evaluate the secretion of SDF-1, cell supernatants from UCB
CD34+ cells cultured for 4 to 7 days were tested for their
ability to induce migration of freshly isolated CD34+ cells
in a chemotactic chamber (Fig 3B). Consistent with the production of
SDF-1 by CD34+ cells, their supernatant was able to induce
migration of CD34+ cells (Fig 3B), and the chemotactic
activity was inhibited by 45% when an anti-CXCR4 MoAb was added before
migration (Fig 3C).
Coexpression of the HIV-1 X4 strain receptors CXCR4 and CD4 on
CD34+ cells.
Because the simultaneous expression of functional CXCR4 and CD4
molecules on the cell surface should render CD34+ cells
susceptible to infections by X4 HIV strains, we investigated by FACS
analysis whether CD34+ cells coexpressed the two viral
receptors. In all samples tested, including UCB, BM, and G-CSF MPB, we
detected a significant proportion of CD34+ cells
coexpressing both molecules (Fig 4). UCB
cells showed the highest proportion of
CD34+/CD4+/CXCR4+ cells (31%, n = 4), in comparison to BM (20%, n = 3) and MPB (12.4%, n = 5). To
further confirm that this subpopulation contains hematopoietic
progenitor cells, CD34+ cells from UCB were sorted for
CXCR4 and CD4 coexpression and seeded in a standard methylcellulose
colony assay. We observed that both CXCR4+/CD4+
and CXCR4/CD4 cells yielded
comparable numbers of CFU-GM (110 and 104 colonies per 400 cells
plated, respectively), BFU-E (102 and 85), and CFU-MIX (12.5 and 13),
respectively (data not shown).
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| Fig 4.
Coexpression of HIV-1 X4 coreceptors on BM, UCB, and MPB
CD34+ cells. Flow cytometric analysis of purified
CD34+ cells labeled with isotype control PE/TC (A) or
anti-CD4 PE and anti-CXCR4 TC (B, bone marrow; C, umbilical cord blood;
D, G-CSF-mobilized peripheral blood).
|
|
CD34+ cells are resistant to X4
HIV-1LAI/IIIB infection.
We next investigated whether CD34+ cells derived either
from normal adult BM (n = 3), UCB (n = 4), or G-CSF MPB (n = 2) were susceptible to infection by X4 HIV-1. CD34+ cells were
isolated from PBMC cells by a two-round immunoaffinity procedure that
yielded cells with a purity greater than 95%. In all samples used for
HIV infection studies, the proportion of CD4+/CXCR4+ cells was greater than 15%. Cells
were infected for 1 hour at 37°C with HIV-1LAI/IIIB,
washed, and then cultured for up to 3 weeks in complete medium
containing IL-3, IL-6, and SCF (routinely used for ex vivo expansion
and gene transfer into progenitor cells). HIV-1 replication was
assessed by an Mg2+-dependent RT-assay on cell supernatant
every 3 to 4 days. We did not observe productive HIV infection of
CD34+ cells (Fig 5), except in
one experiment with UCB cells in which low levels of replication were
observed after 2 weeks of culture (Fig 5, exp. 1). In one additional
experiment, MPB CD34+ cells were sorted by flow cytometry
for coexpression of both CD4 and CXCR4 receptors (purity = 99.5%),
cultured for 24 hours before infection, and, again, no HIV-1 production
was observed (Fig 5, exp. 5). Experiments were performed also using the
primary X4 HIV isolate BON that similarly failed to replicate in BM
CD34+ cells (Fig 5, exp. 9). No substantial changes in the
levels of CXCR4 and SDF-1 mRNA were observed in CD34+ cells
exposed or unexposed to HIV, up to 11 days after incubation with the
virus (data not shown). FACS analysis confirmed the persistence of
CXCR4 on these cultured CD34+ cells. In addition, there
were no significant differences in the proportion of CD4+
cells in uninfected and HIV-1 infected CD34+ cells after 3 days of culture (with 16.9% v 19.5%, and 23.5% v
24.6% in uninfected v HIV-infected BM and UCB
CD34+ cells, respectively; data not shown). However, the
proportion of CD34+ cells bearing CD4 alone or both HIV-1
receptors decreased over time in CD34+ cells during culture
(Fig 6).
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| Fig 5.
CD34+ progenitor cells are not able to
productively sustain X4 HIV-1 infection. The UCB-, BM-, and MPB-derived
CD34+ cells were incubated for 1 hour with the HIV-1
LAI/IIIB strain of HIV-1 virus at the m.o.i. of 1. BM cells
were infected with BON primary isolate diluted 1/30 (exp. 9). In exp.
5, MPB CD34+ cells were sorted for the presence of both
CD4 and CXCR4 and cultured for 24 hours before infection, as indicated
in Materials and Methods. After infection, cells were washed twice and
plated in a 48-well plate. Culture supernatants were harvested every 3 days and the particles production was monitored by RT activity. A
kinetic of HIV-1 IIIB replication in PBMC is shown as control of the
HIV-1LAI/IIIB and BON infectivity.
|
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| Fig 6.
CXCR4 and CD4 expression during in vitro culture of
CD34+ cells. Flow cytometric analysis of CXCR4 and CD4
expression on UCB CD34+ cells cultured for 0, 5, and 8 days, respectively. Cells were stained with anti-CD34 FITC, anti-CD4
PE, and anti-CXCR4 TC and analyzed for HIV-1 coreceptor expression on
the subpopulation of cells that maintain the CD34 antigen during the
culture. Quadrant regions were set using isotype matched-control
antibodies. Similar results were obtained for uninfected and
HIV-1-infected cells.
|
|
To elucidate whether impaired HIV-1 infection in CD34+
cells was caused by inefficient entry of HIV into these cells, BM
CD34+ cells were infected with DNase-treated
HIV-1LAI/IIIB and harvested 1, 18, 48, 72, and 168 hours
after infection; cell lysates were subjected to HIV-1 DNA PCR to detect
proviral DNA synthesis. The levels of HIV-1 gag DNA in
CD34+ cells, although detectable, were consistently very
low (range, 1 to 25 copies per 105 cells) at the first
three time points and became undetectable (<1 copy) after 3 days of
culture (data not shown). By this method, productive HIV infection, as
determined by detection of RT activity in culture
supernatants,34 is associated with levels of proviral DNA
equal to or above approximately 1,000 copies of DNA/105
cells.34 Similar results were obtained with sorted MPB
CD34+ cells analyzed between 1 and 72 hours as well as with
CB CD34+ cells sorted for the simultaneous surface
expression of both CD4 and CXCR4 receptors before infection. These
results suggest that HIV-1 infection is curtailed in CD34+
cells at early stages of the viral life cycle, consistent with a poor
ability of the virus to enter CD34+ cells.
HIV-1 gp120 does not cause the formation of trimeric complexes with
CD4 and CXCR4 in CD34+ cells.
It has been recently shown that the binding of sgp120 to
CD4+ T cells leads to an interaction among CXCR4, CD4, and
gp120 that masks anti-CXCR4 epitopes and eventually induces the
cointernalization of these trimolecular complexes.53 To
investigate whether gp120Env was able to interact with CXCR4 on the
surface of CD34+/CD4+ cells, we tested the
ability of X4 HIV-1 sgp120 to interfere with MoAb binding to CD4 and
CXCR4. As expected, sgp120 bound efficiently to both human
CD4+ T cells and CD34+ cells, resulting in the
potent inhibition of the anti-CD4 Leu3A MoAb (recognizing an epitope of
CD4 required for HIV infection) staining
(Fig 7A). Binding of anti-CXCR4 12G5 MoAb
to CD4+ T cells was reduced by 20% at +4°C, and by
48% at 37°C, respectively, in the presence of prebound sgp120 (Fig
7B). In contrast, sgp120 did not interfere with the anti-CXCR4 MoAb
binding to CD34+ cells, either at +4°C or at 37°C
(Fig 7B). On the other hand, when cells were pretreated with the
natural ligand SDF-1, CXCR4 was downmodulated from the cell surface of
both CD4+ T cells (22% reduction of mean fluorescence
intensity at +4°C, 80% reduction at +37°C, respectively), and
CD34+ cells (70% reduction at +4°C, 100% at
+37°C, respectively) (Fig 7B). These results confirm that CXCR4
expressed on CD34+ cells can be downmodulated by its
ligand, as recently reported for other cell types including human
lymphocytes,54,55 but not by HIV-1 gp120/CD4 complexes.
Taken together, these observations suggest that the binding of gp120 to
CD4 expressed on human CD34+ cells, although efficient,
does not lead to the association with CXCR4 and consequent
internalization of the virus, providing a potential mechanism, in
addition to SDF-1 expression, for explaining the inefficient
susceptibility of these cells to X4 HIV-1 infection.
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| Fig 7.
Failure of sgp120/CD4 to induce CXCR4 downmodulation in
CD34+ progenitor cells. Purified CD34+
cells or CD4+ cells from UCB were incubated without
gp120, with sgp120 (5 µg/mL), or SDF-1 (1 µg/mL) for 2 hours at
either +4°C () or 37°C () in serum-free medium. Cells
were then washed once, labeled with anti-CD4 PE in combination with
anti-CXCR4 TC, and analyzed by flow cytometry. An aliquot of cells was
incubated with PE- and TC-conjugated isotype control-matched
antibodies. Columns represent the mean fluorescence intensity
for CD4 expression (A) and CXCR4 expression (B) on CD4+ T
cells (left) and CD34+ cells (right), respectively.
|
|
| |
DISCUSSION |
In the present study, we have observed that CD34+ precursor
cells obtained from BM, UBC, or MPB express the CXCR4 chemokine receptor. In addition, CD34+/CD38+ cells also
expressed and secreted SDF-1, the natural ligand of CXCR4. Because this
chemokine receptor serves, together with CD4, as coreceptor for T-cell
tropic HIV strains, we investigated the susceptibility of
CD34+ cells to this subset of viruses. No evidence of
productive X4 HIV infection was obtained, either with the
laboratory-adapted LAI/IIIB strain or with a primary X4 isolate (BON),
likely as a consequence of a very poor entry of the virus despite
cell-surface expression of both CD4 and CXCR4. This apparent
discrepancy is likely explained by multiple mechanisms. In addition to
the potential role of endogenous SDF-1 as coreceptor blocker, we
observed that sgp120 failed to downregulate CXCR4 from the surface of
CD34+ cells, in contrast to its effect on productively
infected adult CD4+ T cells, and despite the fact that the
chemokine receptor was susceptible to internalization after binding to
SDF-1.
We found that circulating CD34+ cells from individuals
treated with CY/G-CSF showed a considerable reduction both in the
proportion of cells expressing CXCR4 and in the cell-surface density of
the receptor in comparison to BM or UCB CD34+ cells. These
data are consistent with the diminished chemotactic responsiveness to
SDF-1 previously reported for CY/G-CSF MPB CD34+
cells.4 When G-CSF was administered alone, the reduction in the percentage of CXCR4-expressing cells was less pronounced, but the
receptor cell-surface density was still significantly lower in
comparison to BM CD34+ cells. These observations may help
to explain the exit of BM progenitor cells to the PB compartment under
experimental mobilization. It is possible that chemotherapy and/or
G-CSF induce a downregulation of the CXCR4 receptor on BM
CD34+ cells, which, in turn, may facilitate their exit from
the stromal microenvironment compartment into the blood sinusoids.
Alternatively, cells that already express low levels of CXCR4 may
preferentially leave the BM after mobilization. All these events may be
preceded, accompanied, or followed by changes either in the levels of
expression or in the function of adhesion receptors that have been
implicated in the mobilization process, such as 4 integrin,
L2, and c-kit.27,56,57 Our results suggest a role for
CXCR4 in regulating the exit of progenitors from the BM; however, they
do not exclude the possibility that MPB CD34+ cells may
have a reduced capacity to migrate in response to SDF-1 due to an
impaired cell migration or alteration in the signal transduction machinery.
The reduced expression of CXCR4 may render these cells less prone to
home back to the BM extravascular space when subsequently transplanted.
In this view, it is noteworthy that CY/G-CSF-mobilized mouse stem
cells show a reduced capacity to home to the BM with respect to their
normal BM counterpart.58 Yet, mouse mobilized cells do not
differ with respect to BM cells for in vitro clonogenic assays and
CFU-spleen activity.58 In addition, no evidence exists thus
far that this putative homing defect may affect the outcome of
autologous or allogeneic transplantation of MPB stem cells in humans,
because rapid neutrophil and platelet engraftment are routinely
obtained with these cell sources. Therefore, while the reduced
expression of CXCR4 may contribute to the mobilization of
CD34+ cells to the periphery, it remains to be established
whether it affects the homing potential of MPB stem cells during human cell transplantation.
Surprisingly enough, CD34+ cells, both freshly isolated and
in vitro-cultured, express SDF-1 mRNA by RT-PCR analysis. SDF-1 was
differentially expressed in progenitor cells, being produced by
committed (CD34+/CD38+), but not more
primitive, progenitor cells
(CD34+/CD38), whereas the CXCR4 receptor
was equally expressed on both cell subsets. In addition, when
CD34+ cells were cultured in vitro in the presence of
cytokines, SDF-1 production was demonstrated by ELISA, as well as by
the ability to induce the chemotaxis of other CD34+ cells
via the CXCR4 receptor. Because culture of CD34+ cells is
invariably associated with their differentiation, we cannot rule out
the possibility that SDF-1 detected in CD34+ cell
supernatant was produced also by their progeny of differentiated cells.
To our knowledge, this is the first report of a chemokine endogenously
produced by human CD34+ cells. The expression of SDF-1 by
CD34+ cells was, however, not totally unexpected because
this chemokine was previously found constitutively expressed in several
tissues. In addition, production of chemokines in an autocrine fashion, both under steady state and activated conditions, has been described for several blood cell types including monocytes (monocyte chemotactic protein-1 and macrophage inflammatory protein-1,
MIP-159,60), eosinophils (regulated upon activation
normal T-cell expressed and secreted, RANTES, and
eotaxin61,62), B cells (MIP-163), and
megakaryocytes (platelet factor-464). Moreover, the
endogenous production of the CC chemokines RANTES, MIP-1, and
MIP-1 by CD4+ T cells from both long-term nonprogressor
HIV-1-infected subjects and from some highly exposed but uninfected
individuals was shown to be responsible for the reduced infectability
of their PBMC to HIV strains using the CCR5 chemokine
receptor.65,66
We cannot formally exclude the possibility that
contaminating BM mesenchymal stromal cell precursors,67 or
endothelial cells expressing CD34,68 may contribute to
SDF-1 production in CD34+ cell cultures. However, this
event seems very unlikely, in that the frequency of these cells in the
BM is rare,67,69 and it is even more rare in
CB70 and PB samples.71 In addition, stromal cell precursors and endothelial cells do not express
CD38,69 whereas we observed that the expression of SDF-1
was restricted to the CD34+/CD38+ subset.
Finally, in light of the occasional event that some adherent cells
remained in the cultures of CD34+ cells, SDF-1 was found to
be expressed in the nonadherent cell population (data not shown).
The secretion of SDF-1 may allow progenitor cells to sense each other,
cluster together, and influence their respective migratory behavior
within the BM microenvironment. In support of this hypothesis, a recent
study of time-lapse microscopy showed that CD34+cells
exhibit in vitro a coordinated migratory pattern and the capacity to
aggregate together,72 two events that could be attributed to the endogenous production by CD34+ cells of a
chemotactic factor. Finally, the expression of SDF-1 by
CD34+ progenitors may represent a mechanism by which these
cells downregulate or desensitize their receptor, in response to
different stimuli.54
Our findings indicate that CD34+ cells derived from BM,
UCB, and MPB are not efficiently infected by X4 HIV-1 strains. HIV infection did not spread efficiently, as indicated both in terms of RT
activity and proviral DNA accumulation. The block in HIV-1 infection
seems to occur early in the viral cell cycle. This finding is in
agreement with earlier observations of BM-derived CD34+
cells21,23,73,74 and it is in contrast with
others.20 It should be noted, however, that in this latter
study incubation of CD34+ cells with HIV-1 was maintained
up to 24 hours before removal of the viral excess, allowing for a
prolonged contact of the virus with CD34+ cells undergoing
differentiation along the mononuclear phagocytic lineage.20
Endogenous SDF-1 production by CD34+ cells may compete with
X4 HIV-1 for binding to the coreceptor. In this regard, it has been
reported that the simple occupancy of HIV coreceptors by chemokines is
sufficient for inhibition of HIV infection, even in the absence of
G-protein signaling.75 Indeed, lymphocytic cell lines
transfected with native SDF-1 show a considerable reduction in their
susceptibility to HIV-1 infection, likely due to partial saturation of
the binding sites on CXCR4 caused by SDF-1 secretion.76
Although the concentrations of SDF-1 produced by CD34+ cell
cultures are substantially lower than those required to block in vitro
infection, its local accumulation at the cell surface may be sufficient
to interfere with HIV-1 and to prevent its entry and/or its spreading.
In addition, endogenous SDF-1 may downregulate CXCR4 expression by
inducing its endocytosis.54 However, the fact that CXCR4 is
rapidly recirculating from and to the plasma membrane,55
and that we have not observed a significant reduction of CXCR4
expression on cultured CD34+ cells, makes this mechanism
quite unlikely. An alternative explanation is that CXCR4 on
CD34+ cells may be in a conformation that does not allow
the binding of the gp120/CD4 complexes or that receptor internalization
upon gp120 binding may be reduced or impaired in CD34+
cells. In this regard, we have reported here that sgp120 failed to
downregulate CXCR4 from CD34+ cells, although it was fully
effective on activated PB-derived CD4+ T lymphocytes and it
interfered with the ability of an anti-CD4 MoAb to stain both
lymphocytes and CD34+ cells. Finally, the decreased
proportion of cells expressing both CD4 and CXCR4 during
CD34+ cell culture may have contributed to further reduce
the viral spreading.
Of interest, a mutation at the 3' untranslated region of SDF-1
has been recently described to delay the onset of acquired immunodeficiency syndrome (AIDS) symptoms of HIV-infected
individuals.77 Although the mechanism of the protective
role of SDF-1 has yet to be uncovered, it is likely that it may involve
regulation of endogenous levels of expression and/or of
tissue-specificity of this chemokine, therefore emphasizing the
importance for SDF-1 and CXCR4 in HIV infection.
In conclusion, this study provides novel elements relevant for both the
physiological trafficking and homing of CD34+ cells and for
their potential use in a gene therapy approach for HIV disease. The
ability of CD34+/CD38+ cells to produce SDF-1,
the ligand of a viral coreceptor, may represent an important
determinant in the natural resistance displayed by CD34+
cells to be infected by T-cell tropic HIV-1 strains.
| |
ACKNOWLEDGMENT |
We thank Dr E. Zappone for providing clinical samples, Dr M. Salomoni
for experimental help, and G. Torriani for cell sorting.
| |
FOOTNOTES |
Submitted July 9, 1998; accepted February 23, 1999.
A.A. and L.T. equally contributed to this study.
L.T. is the recipient of a fellowship of the Istituto Superiore di
Sanità, Rome, Italy. This research was funded by grants from
Telethon, EU-BIO4-CT95-0284, and by grants from the National Program
for AIDS Research, Istituto Superiore di Sanità, Rome, Italy.
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Address reprint requests to Alessandro Aiuti, MD, PhD, Telethon
Institute for Gene Therapy (TIGET), Scientific Institute H.S. Raffaele,
Via Olgettina 58, 20132, Milan, Italy; e-mail: aiuti{at}tigem.it.
| |
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TOP
ABSTRACT
INTRODUCTION