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Journal of Virology, July 2001, p. 6710-6713, Vol. 75, No. 14
Department of Immunology, Imperial College
School of Medicine, Chelsea and Westminster Hospital, London SW10
9NH,1 and Flow Cytometry Department,
Imperial Cancer Research Fund, Lincolns Inn Field, London WC2
3PX,2 United Kingdom
Received 21 December 2000/Accepted 8 April 2001
Plasmacytoid dendritic cells (pcDC) and myeloid dendritic cells
(myDC) are shown to express CD4 and low levels of CCR5 and CXCR4, but
only myDC express DC SIGN, a C-type lectin that binds human
immunodeficiency virus but does not mediate virus entry. Both DC types
were more susceptible to infection with a macrophage than a
lymphotropic strain of human immunodeficiency virus type 1, but pcDC
were more readily infected than myDC.
Plasmacytoid dendritic cells (pcDC)
have only recently been recognized as a distinct population of blood
dendritic cells (DC) (6, 8, 9). pcDC, like myeloid DC
(myDC), stimulate strong T-cell-proliferative responses but differ in
other aspects of their biology. Blood myDC take up residence in the
peripheral tissues, where they intercept invading pathogens, and then
migrate to secondary lymphoid tissue, secrete interleukin-12, and
present processed antigens to T cells (1). By contrast,
pcDC migrate directly from blood to lymphoid tissue, where on
stimulation via CD40, they produce large amounts of The question of the susceptibility of DC to infection by human
immunodeficiency virus (HIV) has been controversial (2, 7). Some previous studies may have been unwittingly performed on
mixed populations of myDC and pcDC, and this could explain conflicting
findings. Here, we show that each DC population expresses the receptors
and coreceptors necessary for HIV infection but that pcDC are more
readily infected than myDC.
CD11c+ myDC and CD11c CD4, CCR5, and CXCR4 expression by DC was analyzed in uncultured
peripheral blood mononuclear cells (PBMC) by four-color
fluorescence-activated cell sorter (FACS) analysis. DC were identified
by the absence of labeling with phycoerythrin-conjugated antibodies
against CD3, CD14, CD16, and CD19 and strong labeling with
PerCp-conjugated anti HLA-DR (Becton Dickinson). myDC and pcDC were
differentiated by staining them with fluorescein
isothiocyanate-conjugated anti-CD11c (Dako). CD4, CXCR4, and CCR5
were detected by allophycocyanin-conjugated antibodies (Becton
Dickinson). RNA message for CD4, CCR5, CXCR4, DC SIGN (a C-type
lectin that binds HIV but does not mediate virus entry
[5]), and GAPDH (glyceraldehyde-3-phosphate
dehydrogenase) was detected in purified DC by reverse
transcription (RT)-PCR on DNase-treated RNA. cDNA was amplified by PCR
using the following primers: CD4 forward
(ATAAAGATTCTGGGAAATCAGGGCTCC) and CD4 reverse (TGCAACTTTCCTGTTTTCGCTTCAAGG) (product, 751 bp); CXCR4
forward (TGACTCCATGAAGGAACCCTG) and CXCR4
reverse (CTTGGCCTCTGACTGTTGCTG) (product, 381 bp); CCR5 forward (CCAAAAGCACATTGCCAAACG) and
CCR5 reverse (ACTTGAGTCCGTGTCACAAGCC) (product, 136 bp); DC
SIGN forward (ACAGAGGAGAGCCCAACAACG) and DC SIGN reverse
(AAGGGGGTGAAGTTCTGCTACG) (product, 202 bp); and GAPDH
forward (ATGGAGAAGGCTGGGGCTC) and GAPDH reverse
(AAGTTGTCATGGATGACCTTG) (product, 196 bp). Forty cycles of
PCR were performed by denaturation for 30 s at 94°C, annealing
for 1 min at 55°C, and then incubation for 2 min at 72°C.
Purified myDC and pcDC (105 cells) were infected with
DNase-treated lymphotropic, CXCR4-utilizing IIIB or
macrophage-tropic, CCR5-utilizing Ba-L (20 provirus-forming units
assayed in PM1 cells) strains of HIV type 1 (HIV-1). After 18 h,
they were washed three times and cultured for 7 to 9 days. DNA was
extracted, and the HIV provirus load was estimated by limiting-dilution
PCR using primers based on HIV-1 Pol, a technique shown to detect a
single provirus copy (11). The first-round primers were
forward, 5' CATGGGTACCAGCACAAAGG 3' and reverse, 5'
TCTACTTGTCCATGCATGGCTTC 3'. The second-round primers were
forward, 5' GGAGGAAATGAACAAGTAGATAAATTAGTCAG 3', and
reverse, 5' TCACTAGCCATTGCTCTCCAATT 3'. To test for release of infectious virus, supernatants were cocultured with PM1 cells for
5 h, and newly synthesized proviral DNA was measured by
limiting-dilution nested PCR.
myDC and pcDC were identified by FACS analysis (Fig. 1a and
b). CD4 was detected on both populations
but was more highly expressed on pcDC (Fig. 1c and d). After 48 h
of culture, surface expression of CD4 was downregulated on myDC but not
on pcDC (Fig. 1e and f). Data from one of five experiments to detect
chemokine expression on DC are shown, and for comparison, we also show
expression on T cells. Labeling for CXCR4 was very low on myDC but
higher on pcDC (Fig. 2a). There were low
levels of CCR5 on myDC and slightly higher levels on pcDC. CCR5 became
undetectable on the surfaces of both types of DC cultured for 48 h
whereas CXCR4 expression was little changed (Fig. 2b). Surprisingly,
mRNA for all three receptors was detectable in cultured as well as
fresh myDC and pcDC (Fig. 3). DC-SIGN
mRNA was detected in cultured and freshly isolated myDC but not in pcDC
(Fig. 3).
0022-538X/01/$04.00+0 DOI: 10.1128/JVI.75.14.6710-6713.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Plasmacytoid Dendritic Cells Are Highly Susceptible
to Human Immunodeficiency Virus Type 1 Infection and Release
Infectious Virus
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interferon and
thus may play an important role in innate antiviral immunity (3,
10).
pcDC were purified from
buffy coats as previously described (9). Purified DC
contained 2% or less contaminating T cells. Purified DC
(105) were cultured in 96-well plates with recombinant
granulocyte-macrophage colony-stimulating factor (100 ng/ml) for myDC
or interleukin-3 (10 ng/ml) for pcDC (cytokines were from R&D Systems
Europe Ltd). myDC and pcDC remained positive and negative,
respectively, for CD11c expression throughout a 7- to 9-day culture
period, and there was no evidence of expansion of a minor cell population.

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FIG. 1.
FACS analysis of DC for expression of CD4. (a)
Identification of DC in peripheral blood (R1); (b) myDC and pcDC are
differentiated by expression of CD11c; (c and d) expression of CD4 on
myDC (c) and pcDC (d) in freshly isolated PBMC. (e and f) CD4
expression on purified myDC (e) and pcDC (f) cultured for 2 days. On
all histograms, the left-hand curve represents staining with an isotype
control antibody.

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FIG. 2.
Expression of CXCR4 and CCR5. (a) FACS labeling for
CXCR4 and CCR5 on myDC, pcDC, and T cells in freshly isolated PBMC. (b)
Expression of CXCR4 and CCR5 on purified myDC and pcDC cultured for
48 h. The left-hand curve in each histogram represents staining
with the isotype control antibody.

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FIG. 3.
Detection of mRNA for CD4, CXCR4, CCR5, DC SIGN, and
GAPDH on cultured myDC and pcDC. Gel analysis of an RT-PCR to detect
mRNAs in purified DC cultured for 48 h. Lanes 1 to 5, myDC; lanes
6 to 10, pcDC; lanes 1 and 6, CD4; lanes 2 and 7, CCR5; lanes 3 and 8, CXCR4; lanes 4 and 9, DC SIGN; lanes 5 and 10, GAPDH.
HIV proviral DNA was detected in six of seven in vitro infection
experiments (Table 1). Experiment 5 shows
a higher proviral load in cultures infected with macrophage-tropic Ba-L
and also shows that virus grows more efficiently in pcDC than in myDC
(Fig. 4). This pattern was seen in all
experiments, although the differences were not always as great as those
observed in experiment 5. Nevertheless, in no experiment using DC
derived from the same donor was IIIB found to replicate to higher
levels than Ba-L nor were provirus loads ever observed to be higher in
myDC than in pcDC. Infectious virus, demonstrated by the ability of DC
supernatant to form provirus in PM1 cells, was detected in six of eight
DC cultures tested (experiments 2, 3, and 5), but the largest amounts
of virus were found in the pcDC cultures infected with Ba-L (Table 1).
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We show by FACS, analysis and RT-PCR that pcDC express CD4, CCR5, and CXCR4, the receptors required for infection by macrophage and lymphotropic strains of HIV-1. These receptors were also detected on myDC, but FACS analysis showed lower levels of expression, particularly for CD4 and CXCR4. On culture, surface expression of CXCR4 was little changed, whereas there was a loss of membrane CCR5 on both cell types and downregulation of CD4 on myDC. However, a positive signal for all three receptors on both populations was detected by RT-PCR on fresh and cultured cells. Whether these findings reflect inefficient transport to the cell surface, a high rate of membrane receptor turnover, or other possible explanations is not yet clear.
In vitro infection experiments with HIV-1 Ba-L confirmed that both cell types could be infected by HIV-1; however, the virus replicated more efficiently in pcDC. This may reflect lower CD4 expression by myDC. Alternatively, cellular entry into myDC may be blocked by attachment to DC SIGN. Lower provirus loads were detected in DC infected with the IIIB lymphotropic strain than in those infected with the Ba-L strain of HIV-1. For the myDC, this may be explained by poor expression of CXCR4; however, in pcDC, CCR5 and CXCR4 were expressed at similar levels. A further inconsistency of the data was that for both DC populations, in vitro culture resulted in downregulation of CCR5 but not of CXCR4. The findings may partly reflect a defective vpr gene, thought to facilitate infection of nondividing cells, in the IIIB strain of virus (4).
DC infected with 20 infectious units of virus had a high provirus copy number after 7 to 9 days, suggesting productive virus infection. Release of infectious virus was confirmed by culturing DC supernatants with PM1 cells and analyzing them for provirus formation. Infectious virus was released by both pcDC and myDC cultures. Low levels of contaminating T cells, 2% or less CD3+ cells, were present in purified DC preparations. Although it cannot be ruled out that some of the provirus detected is derived from T cells, this is unlikely to significantly distort the in vitro infection data for the following reasons: (i) the number of provirus copies after 9 days greatly exceeded the maximum number of T cells, (ii) significant replication in T cells would mask the differences between myDC and pcDC cultures, and (iii) autologous T cells do not support productive infection unless activated by exogenous stimuli.
The finding that pcDC are more readily infected than myDC may explain
previous inconsistencies and could reflect investigators working with
different populations of cells. Infection and loss of pcDC in vivo may
reduce levels of
interferon and result in higher virus loads and
disease progression.
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ACKNOWLEDGMENTS |
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This work was supported by the United Kingdom Medical Research Council.
HIV-1 virus stocks and the PM-1 cell line (originally donated by M. Reitz) were provided by the MRC Centralised Facility for AIDS Reagents at NIBSC, South Mimms, Herts., United Kingdom.
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FOOTNOTES |
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* Corresponding author. Mailing address: Department of Immunology, Imperial College School of Medicine, Chelsea and Westminster Hospital, 369 Fulham Rd., London SW10 9NH, United Kingdom. Phone: 44 20 8746 5934. Fax: 44 20 8746 5997. E-mail: s.patterson{at}ic.ac.uk.
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