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Blood, Vol. 94 No. 1 (July 1), 1999:
pp. 62-73
By
From Telethon Institute for Gene Therapy (TIGET); AIDS
Immunopathogenesis Unit, DIBIT; and Laboratory of Separative
Techniques, Scientific Institute H.S. Raffaele, Milan, Italy.
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
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 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.
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 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 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 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.
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.
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-
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
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).
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.
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 thank Dr E. Zappone for providing clinical samples, Dr M. Salomoni
for experimental help, and G. Torriani for cell sorting.
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.
1.
Uchida N, Fleming WH, Alpern EJ, Weissman IL:
Heterogeneity of hematopoietic stem cells.
Curr Opin Immunol
5:177, 1993[Medline]
[Order article via Infotrieve]
2.
Aguila H, Akashi K, Domen J, Gandy K, Lagasse E, Mebius R, Morrison S, Shirizu J, Strober S, Uchida N, Wright D, Weissman I:
From stem cells to lymphocyte: Biology and transplantation.
Immunol Rev
157:13, 1997[Medline]
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TOP
ABSTRACT
INTRODUCTION
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.
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,
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.
), or an isotype-matched
control mouse IgG (
).
(TGF-
