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IMMUNOBIOLOGY
The C-type lectin surface receptor DCIR acts as a new attachment factor for
HIV-1 in dendritic cells and contributes to trans- and cis-infection pathways
Alexandra A. Lambert,1 Caroline Gilbert,2 Manon Richard,3 André D. Beaulieu,3 and Michel J. Tremblay1
1Centre de Recherche en Infectiologie, 2Centre de Recherche en Rhumatologie et Immunologie, and 3Laboratoire de recherche sur l’Arthrite et l’Inflammation,
Centre Hospitalier de l’Université Laval, and Faculté de Médecine, Université Laval, Québec, QC
The dynamic interplay between dendritic
cells (DCs) and human immunodeficiency
virus type-1 (HIV-1) is thought to result in
viral dissemination and evasion of antiviral immunity. Although initial observations suggested that the C-type lectin
receptor (CLR) DC-SIGN was responsible
for the trans-infection function of the virus, subsequent studies demonstrated
that trans-infection of CD4ⴙ T cells with
HIV-1 can also occur through DC-SIGN–
independent mechanisms. We demon-
strate that a cell surface molecule designated DCIR (for DC immunoreceptor), a
member of a recently described family of
DC-expressing CLRs, can participate in
the capture of HIV-1 and promote infection in trans and in cis of autologous
CD4ⴙ T cells from human immature monocyte-derived DCs. The contribution of
DCIR to these processes was revealed
using DCIR-specific siRNAs and a polyclonal antibody specific for the carbohydrate recognition domain of DCIR. Data
from transfection experiments indicated
that DCIR acts as a ligand for HIV-1 and is
involved in events leading to productive
virus infection. Finally, we show that the
neck domain of DCIR is important for the
DCIR-mediated effect on virus binding
and infection. These results point to a
possible role for DCIR in HIV-1 pathogenesis by supporting the productive infection of DCs and promoting virus propagation. (Blood. 2008;112:1299-1307)
Introduction
For HIV-1 infection to establish, the virus must be first transferred
and disseminated from the virus entry sites at the mucosal surfaces
to T-cell zones in secondary lymphoid organs, where the virus can
productively infect CD4⫹ T cells. Several hypotheses have been
proposed to explain how the virus can cross the mucosal epithelium. For example, it has been postulated that HIV-1 may enter the
body through microlesions or can cross the epithelium barrier by
different processes such as infection and/or trancytosis of epithelial
cells or infection of dendritic cells (DCs) using both DC-specific
ICAM3-grabbing nonintegrin (DC-SIGN)–dependent and –independent mechanisms.1,2 Given that cell-free virions do not efficiently
cross genital epithelial cells, it has been proposed that HIV-1 uses
primarily DCs to penetrate the mucosal epithelium,1,3 and this cell
type has been proposed to be the first target in the genital mucosa.4
There is now clear evidence that HIV-1 establishes a complex
interplay with DCs. It has been reported that HIV-1 transfer from
DCs toward CD4⫹ T lymphocytes takes place in a biphasic mode.5
In the initial transfer phase (ie, early transfer or trans infectious
mode), the virus located within endosomal compartments in DCs is
rapidly relocated at the DC/T-cell contact zone. This local concentration of virus between the 2 cell types is referred to as the
virologic synapse. This is followed by a second phase (ie, late
transfer or cis infectious mode), which is dependent on productive
infection of DCs and eventual transfer of progeny virus to
surrounding CD4⫹ T cells. Although involvement of DCs in HIV-1
pathogenesis was discovered very soon after the identification of
the virus and several aspects of this complex interplay have been
elucidated, there are still fundamental questions that remain to be
answered about the multifaceted interactions between HIV-1 and
DCs.6 The first crucial event in HIV-1 entry and cis infection of
DCs is the binding of the external envelope glycoprotein gp120 to
cell surface receptor CD4 and coreceptor (eg, CCR5), a step that
results in the formation of a fusion pore between viral and cellular
membranes. Recent findings suggest that HIV-1 internalization
within DCs is dependent on the association between gp120 and
different C-type lectin receptors (CLRs) such as DC-SIGN, mannose receptor (MR) (also known as CD206), langerin (CD207), and
syndecan-3.2,7,8 In contrast to the sequence of events initiated by
gp120 binding to CD4 and a 7-transmembrane coreceptor, early
interactions between gp120 and CLRs will not trigger sufficient
conformational changes in the virus envelope spikes to mediate
fusion of viral and cellular membranes. Instead, these receptors are
thought to facilitate attachment of virions to the cell surface
resulting in capture and transmission of HIV-1 to CD4⫹ T cells in
an efficient trans infectious mode (reviewed in Turville et al9).
Based on previous data, it is expected that a viral entity that is
bound to CLRs will be taken up within endolysosomal vacuoles
and protected from degradation while remaining in an infectious
state for about 2 days, which is the time required for DCs to migrate
to lymph nodes where HIV-1 transmission to CD4⫹ T cells occurs
through the virologic synapse.5,10,11
It recently became obvious that DC-SIGN, MR, langerin, and
syndecan-3 are not the only receptors on DCs involved in HIV-1
capture and transfer.12,13 Therefore, in an attempt to shed light on
the ability of other members of the CLR family to act as attachment
factors for HIV-1 on the surface of DCs, we focused our attention
on the recently described DC immunoreceptor (DCIR). This cell
surface component has been identified as a prototypic DCassociated CLR that is, as DC-SIGN, down-regulated upon maturation of DCs.14 DCIR, also called C-type lectin superfamily 6
Submitted January 28, 2008; accepted May 23, 2008. Prepublished online as
Blood First Edition paper, June 9, 2008; DOI 10.1182/blood-2008-01-136473.
payment. Therefore, and solely to indicate this fact, this article is hereby
marked ‘‘advertisement’’ in accordance with 18 USC section 1734.
The publication costs of this article were defrayed in part by page charge
© 2008 by The American Society of Hematology
BLOOD, 15 AUGUST 2008 䡠 VOLUME 112, NUMBER 4
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BLOOD, 15 AUGUST 2008 䡠 VOLUME 112, NUMBER 4
LAMBERT et al
(CLECSF6)15 and LLIR,16 is a novel type II transmembrane
molecule of the CLR family containing a consensus intracellular
immunoreceptor tyrosine–based inhibitory motif (ITIM),17 an
extracellular region that carries a membrane-distal carbohydrate
recognition domain (CRD),18 and a neck domain that, by analogy
with DC-SIGN, is probably responsible for its oligomerization.19
Additional studies indicated that the neck region of DCIR consists
of 23–amino acid repeats. Four different forms of DCIR mRNAs
have been cloned that are thought to result from alternative
splicing. The longest form was detected in several cell types and
tissues,14,15 whereas a form missing the neck domain was cloned
from neutrophils.15 Finally, 2 alternatively spliced transmembrane
deletion variants were found in DCs.16 DCIR expression is
decreased following maturation of DCs induced by various stimuli
such as CD40 ligand, lipopolysaccharide, and TNF-␣.14 However,
no ligand of DCIR has yet been identified and the in vivo function
of this receptor remains elusive. Given that DCIR is expressed on
cell types known to harbor HIV-1 (eg, DCs and macrophages) and
considering that this CLR shares several features with DC-SIGN,
we investigated the contribution of DCIR as a putative attachment
factor for HIV-1. Importantly, it has been shown that DCIR is
expressed at high levels on various antigen-presenting cells (APCs)
such as B cells, monocytes, and myeloid DCs, whereas Langerhans
cells express low surface levels of DCIR.14 Recently, DCIR was
also detected on the surface of plasmacytoid DCs.20 Altogether,
these observations provide physiological significance to studies
aimed at defining the possible role played by DCIR in interactions
between DCs and HIV-1.
In this report, we demonstrate for the first time that DCIR can
participate in DC-mediated capture and transmission of HIV-1. The
role played by DCIR in virus transfer from immature monocytederived DCs to autologous CD4⫹ T cells was established using
2 distinct but complementary strategies, namely gene silencing and
blocking experiments with a specific antibody. The DCIR-mediated
transmission of HIV-1 was due to an intracellular storage of intact
virions (ie, trans-infection pathway) and also de novo virus
production by DCs (ie, cis-infection pathway). We provide
evidence that the neck domain of DCIR plays a key role in the
process of virus capture and transfer mediated by this CLR.
Together, these results indicate that DCIR serves as a portal for
HIV-1 infection on DCs.
Methods
Reagents
Recombinant human interleukin-2 (rhIL-2) and efavirenz (EFV) were
obtained from the AIDS Repository Reagent Program (Germantown, MD).
IL-4 was purchased from R&D systems (Minneapolis, MN), whereas
granulocyte macrophage–colony-stimulating factor (GM-CSF) was a generous gift from Cangene (Winnipeg, MB). The culture medium consisted of
RPMI-1640 supplemented with 10% fetal bovine serum (FBS), penicillin G
(100 U/mL), streptomycin (100 U/mL), primocine (Amaxa Biosystems,
Gaithersburg, MD), and glutamine (2 mM).
Antibodies
The phycoerythrin (PE)–labeled anti-DCIR monoclonal antibody (Ab)
(clone 216110) was purchased from R&D Systems. A polyclonal anti-DCIR
was produced in rabbits following immunization with a peptide called 27P4
corresponding to the COOH-terminal domain of DCIR and more precisely
to a region of DCIR spanning amino acids 223 to 237 (ie, LGPQRSVCEMMKIHL).17 The polyclonal anti-DCIR was purified using mAbTrap protein
G affinity columns according to the manufacturer’s instructions (Amersham
Pharmacia Biotech, Piscataway, NJ). PE-conjugated donkey anti–rabbit
immunoglobulin G (IgG) was purchased from Jackson ImmunoResearch
Laboratories (West Grove, PA). The FITC-tagged anti–DC-SIGN monoclonal Ab (clone eB-h209) was purchased from eBioscience (San Diego, CA).
Hybridomas producing 183-H12-5C and 31-90-25, 2 Abs recognizing
different epitopes of the HIV-1 major viral core protein p24, were supplied
by the AIDS Repository Reagent Program and ATCC (Manassas, VA),
respectively. Abs obtained from these hybridoma cell lines were also
purified using mAbTrap protein G affinity columns.
Cells
DCs were generated from purified human monocytes (ie, CD14⫹ cells).
Briefly, peripheral blood was collected from healthy donors with informed
consent obtained in accordance with the Declaration of Helsinki, and
peripheral blood mononuclear cells (PBMCs) were prepared by centrifugation on a Ficoll-Hypaque density gradient. Next, CD14⫹ cells were isolated
from fresh PBMCs using a monocyte positive selection kit according to the
manufacturer’s instructions (MACS CD14 micro beads; StemCell Technologies, Vancouver, BC) as described previously.21 To generate immature
monocyte-derived dendritic cells (IM-MDDCs), purified monocytes were
cultured in complete culture medium that was supplemented every other
day with GM-CSF (1000 U/mL) and IL-4 (200 U/mL) for 7 days. The
percentage of cells expressing the surface markers CD3 and CD19 was
evaluated to assess contamination with T and B cells, respectively.
Experiments were performed with cell preparations that contained a
minimal amount of contaminants (ie, DC: purity ⬎ 95%; CD4⫹ T cells:
purity ⬎ 98%). Autologous CD4⫹ T cells were isolated using a negative
selection kit according to the manufacturer’s instructions (StemCell Technologies). These cells were activated with phytohemagglutinin L (PHA-L;
1 ␮g/mL) and maintained in complete culture medium supplemented with
rhIL-2 (30 U/mL) at a density of 2 ⫻ 106 cells/mL. Raji-CD4 is a B-cell
line carrying the Epstein-Barr virus that has been rendered susceptible to
HIV-1 infection by stable transfection with a cDNA encoding human CD4.
These cells were cultured in RPMI-1640 medium supplemented with 10%
FBS along with 1 mg/mL of the selective agent G418 (GIBCO-BRL,
Gaithersburg, MD). Human embryonic kidney 293T cells were cultured in
Dulbecco modified Eagle medium supplemented with 10% FBS.
Flow cytometric analysis
Cell surface expression of DCIR and DC-SIGN was monitored by flow
cytometric analyses (Epics ELITE ESP; Coulter Electronics, Burlington,
ON). Before staining, cells were incubated for 15 minutes at 4°C with 10%
pooled human sera to block nonspecific binding sites and washed once with
phosphate-buffered saline (PBS) supplemented with 0.5% bovine serum
albumin (BSA). Next, cells were incubated for 45 minutes at 4°C with
anti-DCIR (0.25 ␮g) or anti–DC-SIGN (0.5 ␮g) monoclonal Ab and then
washed twice with PBS and 0.5% BSA. Nonspecific staining was determined using an isotype-matched irrelevant control Ab. After 2 final washes
with PBS, cells were fixed in 2% paraformaldehyde and analyzed.
Production of virus stocks
Virions were initially produced upon transient transfection of human
embryonic kidney 293T cells as previously described. The infectious
molecular clones used in this study included pNL4-3balenv (R5-tropic) and
pNL4-3 (X4-tropic). The pNL4-3balenv vector was generated by replacing
the env gene of the T-tropic HIV-1 strain, NL4-3, with that of the
macrophage-tropic HIV-1 Bal strain, thus resulting in an infectious
molecular clone with macrophage-tropic properties (provided by R. Pomerantz, Thomas Jefferson University, Philadelphia, PA).22 Stocks of NL43balenv were made upon acute infection of PBMCs that were stimulated
initially with PHA-L and maintained in culture medium containing rhIL-2
(used in Figures 3-5). The virus-containing supernatants were harvested at
day 7 after infection, filtered through a 0.22-␮m cellulose acetate syringe
filter, ultracentrifugated, and normalized for virion content using a sensitive
in-house double-antibody sandwich enzyme-linked immunosorbent assay
(ELISA) specific for the viral p24 protein. In this test, the 183-H12-5C and
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BLOOD, 15 AUGUST 2008 䡠 VOLUME 112, NUMBER 4
DCIR AS A SURFACE RECEPTOR FOR HIV-1
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31-90-25 Abs are used in combination to quantify p24 levels.23 Preparations
of NL4-3 were produced by infecting Raji-CD4 (used in Figures 6,7).
Briefly, Raji-CD4 cells (5 ⫻ 106 cells) were inoculated with NL4-3 (at a
ratio of 1 ng p24 per 105 cells) for 2 hours at 37°C. Cells were then washed
extensively to eliminate uninternalized virions and maintained in culture for
6 days.
Nucleofection
The Amaxa cell line nucleofector Kit V (Amaxa, Cologne, Germany) was
used to achieve transient transfection of DCIR into Raji-CD4 (program
M-13). Briefly, Raji-CD4 cells (10 ⫻ 106 cells) were nucleofected with
pBK-CMV-DCIR or pBK-CMV-DCIR⌬neck (2.5 ␮g for each plasmid per
10 ⫻ 106 cells). These vectors were constructed by subcloning the cDNA
coding for full-length human DCIR or a neck-deleted version of DCIR (ie,
DCIR⌬neck) into the pBK-CMV vector (Stratagene, La Jolla, CA) as a
KpnI-Xho fragment. These plasmids were purified using an EndoFree
plasmid maxi kit (Qiagen, Mississauga, ON) to obtain DNA of high quality
for nucleofection. Raji-CD4 cells were incubated for 5 hours in RPMI-1640
medium supplemented with 20% FBS prior to nucleofection.
Gene silencing of DCIR with siRNAs
Small interfering RNAs (siRNAs) either specific (ie, 5⬘-ATTTAGGTGGTCTGTCA-3⬘) or nonspecific for DCIR (ie, 5⬘-AATTCTCCGAAGGTGTCACGT-3⬘) were obtained from Qiagen and dissolved in an appropriate
buffer. The studied siRNAs were subsequently tested in IM-MDDCs as
previously described.21 Controls consisted of cells treated with either
oligofectamine alone or oligofectamine and a nonspecific siRNA. Forty
hours following transfection, a virus transfer test was carried out. The
efficiency of DCIR silencing with the tested siRNA was monitored by flow
cytometry.
HIV-1 transfer assay
IM-MDDCs (105 cells in 100 ␮L) transfected either with the listed siRNAs
or preincubated with the polyclonal anti-DCIR (10 ␮g/1.5 ⫻ 105 cells)
were exposed to HIV-1 (10 ng p24) for 60 minutes at 37°C. Next, the
virus-cell mixture was washed 3 times with PBS to remove unadsorbed
virions. In some experiments, cells were also treated with the anti–HIV-1
drug EFV (50 ␮M). Finally, IM-MDDCs were cocultured with autologous
activated CD4⫹ T cells at a 1:3 ratio in complete RPMI-1640 medium
supplemented with IL-2 (30 U/mL) in 96-well plates in a final volume of
200 ␮L. Virus production was estimated by measuring p24 levels in
cell-free culture supernatants.
HIV-1 infection of IM-MDDCs
IM-MDDCs (2 ⫻ 105 cells in a final volume of 100 ␮L) either transfected
with the studied siRNAs or preincubated with the polyclonal anti-DCIR
were exposed to HIV-1 (20 ng p24) for 2 hours at 37°C. After 3 washes with
PBS, cells were maintained in complete RPMI-1640 culture medium
supplemented with GM-CSF and IL-4 in 96-well plates in a final volume of
200 ␮L. Every 3 days and for a period lasting 9 days, half of the medium
was removed and kept frozen at ⫺20°C until assayed. Virus production was
estimated by measuring p24 levels in culture supernatants by ELISA. Note
that in all experiments using the polyclonal anti-DCIR, DCs were pretreated
with 10% of pooled human sera to avoid nonspecific reactivity with Fc
receptors.
Virus binding and infection assays in Raji-CD4
The role played by DCIR as a putative attachment factor for HIV-1 was
assessed using a virus binding test. In brief, Raji-CD4 cells, either negative
(ie, parental cell line) or positive for DCIR (3 ⫻ 106 cells), were incubated
with NL4-3 (300 ng p24) for 60 minutes at 37°C. Next, the virus-cell
mixture was washed 3 times with PBS to remove unbound virus and
resuspended in PBS containing 1% BSA. The p24 content was determined
by our homemade assay. Susceptibility of the studied Raji-CD4 cells to
HIV-1 infection was assessed by initially exposing DCIR-negative and
Figure 1. Expression of DCIR in IM-MDDCs. Purified monocytes were cultured with
GM-CSF and IL-4 for 7 days to derive IM-MDDCs. DCIR expression was determined
by flow cytometric analysis after staining with a commercial PE-conjugated anti-DCIR
monoclonal Ab. Expression of DCIR is shown as a dotted line, whereas the
continuous line represents staining obtained with an isotype-matched irrelevant
control Ab. Results shown are representative of 7 independent experiments.
DCIR-positive Raji-CD4 (1.5 ⫻ 105 cells) to NL4-3 (1.5 ng p24) for
2 hours at 37°C. Thereafter, cells were washed 3 times with PBS to remove
nonspecifically bound virions and maintained in culture in 48-well plates in
a final volume of 400 ␮L. Every 3 days after infection and for a period
lasting 9 days, half of the medium was removed from each well and kept
frozen at ⫺20°C until assayed. Virus production was estimated by
measuring p24 levels in cell-free culture supernatants.
Statistical analysis
Statistical analyses were carried out according to the methods outlined by
Zar.24 Means were compared using either the Student t test or a single-factor
ANOVA followed by Dunnett multiple comparison when more than
2 means were considered. P values of less than .05 were deemed
statistically significant. Calculations were performed with the GraphPad
Prism software (GraphPad Software, La Jolla, CA).
Results
DC-mediated transmission of HIV-1 is modulated by DCIR
A previous study has demonstrated that DCIR is expressed by all
circulating CD14⫹ monocytes, in DCs derived from CD34⫹ cord
blood progenitors, as well as on the surface of DCs generated in
vitro upon culturing blood monocytes with GM-CSF and IL-4 (ie,
immature monocyte-derived DCs [IM-MDDCs]).14 This last observation is of high importance because IM-MDDCs are routinely
used as an experimental cell system to study characteristics of
mucosal myeloid DCs and to define the complexity of interactions
between DCs and HIV-1.12 To confirm that DCIR expression is
maintained in IM-MDDCs, cell surface expression of DCIR was
determined by immunofluorescence staining and flow cytometric
analysis. Data depicted in Figure 1 indicate that DCIR is strongly
expressed after culturing purified monocytes for 7 days with
GM-CSF and IL-4, a treatment known to induce differentiation of
monocytes into IM-MDDCs.
We then asked whether DCIR was involved in HIV-1 uptake by
IM-MDDCs and eventual transfer to autologous CD4⫹ T lymphocytes. To this end, we used 2 different experimental strategies,
namely RNA interference (ie, small interfering RNA or siRNA) to
reduce DCIR expression in IM-MDDCs and a polyclonal Ab
specific for the COOH-terminal end of DCIR. For the virus transfer
assay, IM-MDDCs were intentionally exposed to low doses of
virions to better reflect the events occurring during mucosal
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LAMBERT et al
BLOOD, 15 AUGUST 2008 䡠 VOLUME 112, NUMBER 4
made when using another DCIR-specific siRNA (data not shown).
To eliminate the possibility that the DCIR-mediated effect on
HIV-1 transfer is due to a reduced ability of IM-MDDCs to interact
and activate CD4⫹ T cells, which in turn can modulate the virus
transmission process, cellular proliferation was estimated in our
coculture experiments. Data from a colorimetric method (ie, MTS
cell proliferation assay) indicate that IM-MDDCs transfected either
with the DCIR-specific siRNA or nonspecific control siRNA
display a comparable capacity to induce proliferation of autologous
CD4⫹ T cells (data not shown).
DCIR is involved in trans- and cis-infection pathways
Figure 2. Decreased DCIR expression in IM-MDDCs by siRNA treatment.
IM-MDDCs were either left untreated (control), transfected with a control siRNA, or
transfected with a DCIR-specific siRNA. After 40 hours, flow cytometry analysis of
DCIR was performed using a combination of PE-labeled anti-DCIR and isotypematched control Ab. Data shown correspond to a single experiment representative of
7 independent experiments.
transmission in vivo. Moreover, all virus stocks used in transmission assays were produced upon acute infection of primary human
cells (ie, PBMCs) to more closely parallel the natural microenvironment.25 These cells were pulsed with R5-tropic virions (ie,
NL4-3Balenv) for 60 minutes at 37°C, before being cocultured
with autologous activated CD4⫹ T cells. The extent of virus
transferred to CD4⫹ T cells was determined by measuring the
major core viral protein p24 in culture supernatants. We initially
used RNA interference to reduce DCIR expression in IM-MDDCs
and analyzed the possible effect on HIV-1 transfer. An approximately 30% decrease in surface expression of DCIR in IMMDDCs was observed following the use of DCIR-specific siRNA
(Figure 2). It should be noted that DC-SIGN expression was not
affected by DCIR-specific siRNA, and cell viability was minimally
affected upon siRNA treatment (data not shown). Interestingly,
transfer of HIV-1 was reduced significantly when IM-MDDCs
were transfected with DCIR-specific siRNA but not with a
nonspecific control siRNA (Figure 3). Similar observations were
Figure 3. Involvement of DCIR in HIV-1 transmission by IM-MDDCs. IM-MDDCs
(1 ⫻ 106 cells) were treated with oligofectamine and then either left untreated
(control) or exposed to a nonspecific siRNA and a DCIR-specific siRNA. Next, cells
(1 ⫻ 105) were pulsed with NL4-3balenv (10 ng p24) for 60 minutes at 37°C. After
3 washes with PBS to eliminate unbound virus, IM-MDDCs were cocultured with
autologous CD4⫹ T cells at a 1:3 ratio. Cell-free culture supernatants were collected
at day 2 following initiation of the coculture and analyzed for the p24 content. Data
shown represent the means (⫹ SD) of triplicate samples from 3 combined independent experiments. Asterisks denote statistically significant data (***P ⬍ .001).
Previously published data demonstrated that HIV-1 is transferred
from DCs to CD4⫹ T cells through both trans- and cis-infection
pathways.5 Experiments were therefore carried out to define
whether DCIR can contribute to HIV-1 dissemination via transand/or cis-infection processes. To solve this issue, virus transfer
studies were performed using IM-MDDCs that were initially
transfected with the DCIR-specific siRNA and also treated with
EFV, a nonnucleoside reverse transcriptase inhibitor. This antiretroviral compound hampers the late transfer (ie, de novo virus
production by IM-MDDCs or cis-infection pathway) without
affecting the early transfer mode (ie, trans-infection pathway).
Results illustrated in Figure 4A demonstrate that the use of the
DCIR-specific siRNA led to a reduction in virus transfer corresponding to an average decrease of 30% in absence of EFV compared
with an average diminution of 40% in cells treated with EFV. Such
a small increment in the reduction of HIV-1 transfer by EFV
suggests that DCIR contributes primarily to the early transfer phase
(ie, trans-infection pathway) with a small effect on the late transfer
(ie, cis-infection pathway). The possible involvement of DCIR in
the cis-infection mode was investigated further because of the
limited effect of EFV on HIV-1 propagation. To this end, IMMDDCs treated with the DCIR-specific siRNA were first pulsed
with HIV-1 and cultured for 4 days before initiating a coculture
with autologous CD4⫹ T cells. This experimental setup was
prompted by the previous demonstration that, in IM-MDDCs, most
endosome-associated virus, which is responsible for the early
transfer, is degraded within 24 to 48 hours.5,26 The connection
between DCIR and the cis-infection pathway was confirmed using
this experimental strategy (Figure 4B). Moreover, the use of the
DCIR-specific siRNA was found to decrease de novo virus
production in IM-MDDCs (Figure 4C), which represents additional
evidence that HIV-1 can use DCIR to achieve productive infection
of this cell type.
Next, HIV-1 transmission by IM-MDDCs was evaluated in the
presence of a polyclonal Ab targeting the single CRD in the
extracellular COOH-terminal end of DCIR. The specificity of this
Ab was monitored by flow cytometry using 293T cells transiently
transfected with mammalian expression vectors coding for DCIR
and DC-SIGN (data not shown). A decrease in virus replication was
observed when IM-MDDCs were acutely infected with HIV-1 in
the presence of polyclonal anti-DCIR (Figure 5 left panels). In
addition, pretreatment of IM-MDDCs with anti-DCIR before
pulsing with HIV-1 and coculture with autologous CD4⫹ T cells
induced a significant decrease in HIV-1 transfer, compared with
transmission in the presence of a control Ab (Figure 5 right panels).
These results corroborate those obtained with DCIR-specific
siRNAs and indicate that DCIR can act as a receptor for HIV-1 on
the surface of IM-MDDCs. Together, these results demonstrate that
DCIR is involved in both trans- and cis-infection pathways
mediated by DCs.
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BLOOD, 15 AUGUST 2008 䡠 VOLUME 112, NUMBER 4
DCIR AS A SURFACE RECEPTOR FOR HIV-1
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Figure 4. DCIR affects both trans- and cis-infection
pathways. (A) IM-MDDCs (1 ⫻ 106 cells) were treated
with oligofectamine and then either left untreated (control) or exposed to a nonspecific siRNA and a DCIRspecific siRNA. Next, cells were either left untreated or
treated with EFV. IM-MDDCs (2 ⫻ 105 cells) were pulsed
with NL4–3balenv (20 ng p24) for 60 minutes at 37°C.
After 3 washes with PBS to eliminate unbound virus,
IM-MDDCs were cocultured with autologous CD4⫹ T cells
at a 1:3 ratio. Cell-free culture supernatants were collected at day 2 following initiation of the coculture and
analyzed for the p24 content. Data shown represent the
means plus or minus SD of triplicate samples from 2
different donors and are representative of 4 separate
donors. Asterisks denote statistically significant data
(*P ⬍ .05; **P ⬍ .01). (B) IM-MDDCs were treated with
oligofectamine and then either left untreated (control) or
exposed to a nonspecific siRNA and a DCIR-specific
siRNA. Cells (2 ⫻ 105) were pulsed with NL4-3balenv
(20 ng p24) for 60 minutes at 37°C. After 3 washes with
PBS to eliminate unbound virus, IM-MDDCs were maintained in culture for 4 days. Finally, IM-MDDCs were
cocultured with autologous CD4⫹ T cells at a 1:3 ratio.
Cell-free culture supernatants were collected at different
time points (ie, 2, 4, and 6 days) and analyzed for p24
contents. Data shown represent the means plus or minus
SD of triplicate samples from 2 different donors and are
representative of 3 separate donors. Asterisks denote
statistically significant data (*P ⬍ .05). (C) IM-MDDCs
were treated with oligofectamine and then either left
untreated (control) or exposed to a nonspecific siRNA
and a DCIR-specific siRNA. Cells (2 ⫻ 105) were pulsed
with NL4–3balenv (20 ng p24) for 2 hours at 37°C. After
3 washes with PBS to eliminate unbound virus, IMMDDCs were maintained in complete culture medium
supplemented with GM-CSF and IL-4. Cell-free culture
supernatants were collected at the indicated time points
and analyzed for the p24 content. Data shown correspond to the means plus or minus SD of triplicate
samples from 2 different donors and are representative
of 4 separate donors. Asterisks denote statistically significant data (*P ⬍ .05; **P ⬍ .01).
DCIR-mediated virus capture and infection requires the neck
domain of DCIR
Previous studies have used the Epstein-Barr virus (EBV) genomecarrying B-cell line Raji as a model system to reveal the role of
DC-SIGN in HIV-1 capture and transmission.27 To delineate the
contribution of DCIR in HIV-1 capture and transfer, our next
experiments were carried out using Raji-CD4, a Raji derivative that
stably expresses CD4 and is highly susceptible to infection with
X4-tropic strains of HIV-1. Transient expression of DCIR was
achieved in Raji-CD4 using nucleofection (Figure 6A), an electroporation-based method that is thought to target plasmid DNA
directly to the cell nucleus. As illustrated in Figure 6B, binding of
HIV-1 was increased in the presence of cell surface DCIR in
Raji-CD4. In addition, virus production was enhanced in DCIRexpressing Raji-CD4 compared with the DCIR-negative parental
cell line (Figure 6C).
DCIR carries a neck region that is composed of a variable
number of 23-amino acid tandem repeats. Previous observations
suggest that DC-SIGN, which is closely related to DCIR, forms
tetramers stabilized by the neck domain.19,28 The importance of the
neck portion in the observed phenomenon was addressed using a
neck-deleted DCIR mutant (ie, DCIR⌬neck). Nucleofection of
Raji-CD4 with an expression vector coding for DCIR⌬neck
resulted in an efficient surface expression of the mutated DCIR
(Figure 7A). Attachment of HIV-1 particles was not modulated
following expression of DCIR⌬neck on the surface of Raji-CD4
cells (Figure 7B). In contrast to what was seen in cells expressing
wild-type DICR (Figure 6C), virus replication was not increased in
the presence of DCIR⌬neck on the cell surface of Raji-CD4
(Figure 7C). However, expression of the neck-deleted DCIR
mutant led to a reduction in virus replication, which might be due to
a reduced cell viability (data not shown). Data from these studies
indicate that the neck region of DCIR plays a crucial role in its
interaction with HIV-1.
Discussion
Attachment of HIV-1 particles to host cells constitutes a critical
event in the infection process and is attributed mainly to the
interaction of virus-encoded envelope glycoproteins (ie, gp120)
with cell surface structures acting as receptors (eg, CD4 and a
7-transmembrane coreceptor). These interactions elicit conformational changes in gp120 that induce fusion of viral and cellular
membranes and entry of the viral core within the cell cytoplasm. It
has been shown that initial binding of HIV-1 to DCs also involves
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1304
LAMBERT et al
BLOOD, 15 AUGUST 2008 䡠 VOLUME 112, NUMBER 4
Figure 5. HIV-1 replication in IM-MDDCs is reduced
by anti-DCIR. (Left panels) IM-MDDCs (2 ⫻ 105 cells)
were initially treated with either a control Ab or a polyclonal anti-DCIR (10 ␮g per 1.5 ⫻ 105 cells). Next, cells
were extensively washed to eliminate excess Ab and
pulsed with NL4-3balenv (20 ng p24) for 2 hours at 37°C.
After 3 washes with PBS to eliminate unbound virus,
IM-MDDCs were maintained in complete culture medium
supplemented with GM-CSF and IL-4. Cell-free culture
supernatants were collected at the indicated time points
and analyzed for the p24 content. Results obtained at 3
days after infection are displayed in the small inserts.
Data shown represent the means plus or minus SD of
triplicate samples from 2 different donors and are representative of 3 separate donors. Asterisks denote statistically significant data (*P ⬍ .05; **P ⬍ .01; ***P ⬍ .001).
(Right panels) IM-MDDCs (1 ⫻ 105 cells) were first treated
with either a control Ab or a polyclonal anti-DCIR (10 ␮g
per 1.5 ⫻ 105 cells). Cells were next washed to eliminate
excess Ab and pulsed with NL4-3balenv (10 ng p24) for
2 hours at 37°C. After 3 washes with PBS to eliminate
unbound virus, IM-MDDCs were cocultured with autologous CD4⫹ T cells at a 1:3 ratio. Cell-free culture
supernatants were collected at day 2 following initiation
of the coculture and analyzed for the p24 content. Data
shown represent the means plus or minus SD of triplicate
samples from 2 distinct donors. Asterisks denote statistically significant data (**P ⬍ .01).
interactions between gp120 and other cell surface components such
as CLRs, which can lead to virus internalization within endosomal
compartments. At least 4 distinct CLRs can associate with gp120,
namely DC-SIGN, langerin, MR, and a yet to be identified
carbohydrate- and calcium-dependent trypsin-resistant CLR (reviewed in Turville et al12,13). Interestingly, langerin, as opposed to
the 3 other CLRs, was found to reduce HIV-1 transmission, thus
suggesting that virus capture is not directly leading to a more
efficient transfer process.29 The direct involvement of DC-SIGN in
the efficient capture and transmission of HIV-1 has recently been
put into question.30,31 Indeed, several studies have demonstrated
that DCs can participate in HIV-1 trans-infection of CD4⫹ T cells
via DC-SIGN–independent pathways.32–34 It is now thought that no
unique CLR is fully responsible for HIV-1 attachment to all DC
subsets and virus dissemination by DCs.2 Therefore, the quest for
other receptors on the surface of DCs that can result in virus capture
and contribute to HIV-1 dissemination is still a timely subject.
Here we provide evidence that DCIR, a recently described CLR
that is considered to be a DC-expressed activating immunoreceptor, can serve as an HIV-1 attachment factor on the surface of DCs.
The functional role played by DCIR in HIV-1 trans-infection of
CD4⫹ T lymphocytes by DCs was established through the use of
specific siRNAs and a polyclonal Ab. Additional studies indicated
that DCIR-mediated virus transfer to CD4⫹ T cells also involves de
novo replication of HIV-1 in DCs (ie, the second phase of the
transfer process). We provide evidence that the DCIR-mediated
effect on virus propagation is not linked with a diminished capacity
of DCs to cause proliferation of CD4⫹ T cells. Moreover, our
studies suggest that DCIR-mediated HIV-1 attachment and subsequent transmission require the neck domain of DCIR. Recently, it
has been demonstrated that DC-SIGN blocks HIV-1 budding by
inducing internalization of gp120.35 Given that DCIR is promoting
HIV-1 capture and transfer, it is thus unlikely that virus budding is
affected by this CLR at least under the tested experimental
conditions.
Based on observations made in the present work and
previous published reports, 3 distinct and not mutually exclusive
hypotheses can be proposed to explain how the interaction
between HIV-1 and DCIR can contribute to DC-mediated virus
capture and dissemination. First, it can be postulated that the
binding of HIV-1 particles to cellular DCIR might favor the
association between gp120 and a sufficient number of CD4 and
coreceptor molecules to trigger formation of a fusion pore. This
route of virus entry is known to be the most efficient infection
process with the fastest kinetics (reviewed in Clapham and
McKnight36). This hypothesis is supported by the DCIRdependent increase in HIV-1 binding and infection when using
Raji-CD4 cells as targets. Given that gp120 is one of the most
heavily glycosylated proteins in nature (reviewed in Vigerust
and Shepherd37) and considering that DC-SIGN–dependent
HIV-1 capture relies on interactions between the CRD of
DC-SIGN and gp120,7,38 we propose that an association between
the CRD of DICR and gp120 is most likely responsible for the
capacity of DCIR to act as an attachment factor for HIV-1.
Studies are currently under way to address this issue using a
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BLOOD, 15 AUGUST 2008 䡠 VOLUME 112, NUMBER 4
DCIR AS A SURFACE RECEPTOR FOR HIV-1
1305
Figure 6. DCIR expression in Raji-CD4 increases
HIV-1 binding and virus production. (A) Raji-CD4 cells
were nucleofected with either an empty control vector
(left panel) or a mammalian expression vector coding for
human DCIR (right panel). Five hours later, flow cytometric analysis of DCIR was performed using a combination
of PE-labeled anti-DCIR Ab (dotted lines) and a control
Ab (continuous lines). Data shown correspond to a single
experiment representative of 7 independent experiments. (B) Parental (DCIR-negative) and DCIR-expressing Raji-CD4 (3 ⫻ 106 cells) were exposed to NL4–3
(300 ng p24) for 60 minutes at 37°C. After 3 washes with
PBS to remove unabsorbed virus, cell-associated virus
was quantified by measuring the p24 content. Data
shown correspond to the means plus or minus SD of
triplicate samples from 7 combined independent experiments. The asterisk denotes statistically significant data
(*P ⬍ .05). (C) Parental (DCIR-negative) and DCIRexpressing Raji-CD4 (1.5 ⫻ 105 cells) were exposed to
NL4–3 (1.5 ng p24) for 2 hours at 37°C. After 3 washes
with PBS to remove excess virus, cells were maintained
in culture. Cell-free culture supernatants were collected
at the indicated time points and assayed for the p24
content. Data shown correspond to the means plus or
minus SD of triplicate samples from 3 combined independent experiments. The asterisk denotes statistically significant data (*P ⬍ .05).
Fc-gp120 chimera39 along with appropriate anti-gp120 antibodies.5,9,40 However, such studies are compromised by the fact that
DCs and Raji B cells do naturally express high surface levels of
Fc receptors and blocking agents might affect some basic
functions of DCs and, consequently, the interplay with HIV-1.
The involvement of the CRD domain of DCIR in virus capture is
illustrated by the reduced HIV-1 binding and transfer in the
presence of a polyclonal Ab that is specific for the single CRD in
the extracellular COOH-terminal end of DCIR. Experiments
conducted with a neck-deficient DCIR mutant (ie, DCIR⌬neck)
suggest that this domain is important to permit the interaction
between HIV-1 and DCIR. Based on the previous demonstration
that the neck region of CLRs drives multimerization,19,41 it is
possible that this domain is required for efficient recognition of
multivalent ligands such as gp120-covered HIV-1 particles. The
importance of the oligomerization status in ligand binding
efficiency is underscored by the asialoglycoprotein receptor,42
another CLR that is closely related to DCIR. The second
hypothesis refers to a DCIR-mediated internalization mode
where such endocytosed HIV-1 particles would be spared from
the rapid and extensive degradation that is normally seen in the
endolysosomal apparatus. DCIR bears an intracellular ITIM and
is considered to be the first reported DC-expressed, ITIMbearing receptor of the CLR family.43 It is possible that
engagement of DICR by HIV-1 might lead to activation of ITIM
and allow recruitment of SHP-1 and SHP-2, which can act as
adaptor proteins independently of their catalytic activity, by
virtue of their tandem SH2 domains.44 It is of interest to note
that poliovirus entry has recently been reported to occur through
receptor-induced activation of the protein tyrosine phosphatase
SHP-2.45 Assuming that DCIR allows HIV-1 to gain access to
nondegradative endosomal organelles, this might allow the virus
to persist for a sufficiently long time to be transported from
mucosal surfaces to the T-cell compartment in lymphoid tissues,
as it has been proposed for DC-SIGN.46,47 Moreover, storage in
endosomes could lead to fusion between viral and endosomal
membranes and productive infection, as reported in macrophages.48 Finally, it can be proposed that the interaction between
HIV-1 and DCIR will initiate intracellular biochemical events
that can reverse to some extent the various blocks responsible
for the limited virus infection seen in DCs compared with CD4⫹
T cells (reviewed in Piguet and Steinman49). The DCIRmediated signal transduction events might favor the recruitment of second messengers that might in turn promote reverse
transcription or counter the limitation in virus entry, as described recently by Pion et al.50 For example, possible recruitment of SHP-1 or SHP-2 upon engagement of DCIR could
release tyrosine kinases such as Lyn and/or Syk, which have been
shown to be implicated in the cis-infection pathway in DCs.21
In summary, although no natural ligand of DCIR has yet been
described, we demonstrate that DCIR modulates the early and late
infection-dependent phases of HIV-1 propagation to CD4⫹ T cells
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1306
BLOOD, 15 AUGUST 2008 䡠 VOLUME 112, NUMBER 4
LAMBERT et al
Figure 7. The neck domain of DCIR is important for
interaction with HIV-1. (A) Raji-CD4 cells were nucleofected with either an empty control vector (left panel) or a
mammalian expression vector coding for a neck-deleted
DCIR mutant (ie, DCIR⌬neck) (right panel). Five hours
later, flow cytometric analysis of DCIR was performed
using a combination of PE-labeled anti-DCIR monoclonal
Ab (dotted lines) and a control Ab (continuous lines).
Data shown correspond to a single experiment representative of 5 combined independent experiments. (B) Parental (DCIR-negative) and DCIR⌬neck-expressing RajiCD4 (3 ⫻ 106 cells) were exposed to NL4-3 (300 ng p24)
for 60 minutes at 37°C. After 3 washes with PBS to
remove unabsorbed virus, cell-associated virus was
quantified by measuring the p24 content. Data shown
correspond to the means plus or minus SD of triplicate
samples from 5 combined independent experiments.
(C) Parental (DCIR-negative) and DCIR⌬neck-expressing Raji-CD4 (1.5 ⫻ 105 cells) were exposed to NL4-3
(1.5 ng p24) for 2 hours at 37°C. After 3 washes with PBS
to remove excess virus, cells were maintained in culture.
Cell-free culture supernatants were collected at the
indicated time points and assayed for the p24 content.
Data shown correspond to the means plus or minus SD
of triplicate samples and are representative of 5 combined independent experiments. The asterisk denotes
statistically significant data (*P ⬍ .05).
by interacting with the virus itself. The relative importance of
DCIR in HIV-1 pathogenesis is still unknown, but it is expected to
be nonnegligible considering that DCIR is expressed on immune
cells, and more particularly on APCs (ie, various DC subsets,
monocytes, macrophages, and B cells), and can be modulated by
some proinflammatory agents.14,15 DCIR could also play a role in
mounting a virus-specific immune response. A more complete
understanding of the precise contribution of DCIR to the virus life
cycle or in directing specific immunity is needed because it might
lead to the development of alternative strategies to treat infected
individuals.
This work was supported by operating grants to M.J.T. from the
Canadian Institutes of Health Research (CIHR) (Ottawa, ON)
under the HIV/AIDS research program (MOP-79542 and HET85519). A.L. holds a Doctoral Award from the CIHR HIV/AIDS
research program. C.G was the recipient of a Fellowship Award
from the CIHR HIV/AIDS Research Program, and M.J.T. holds the
Canada Research Chair (Ottawa, ON) in Human ImmunoRetrovirology (Tier 1 level).
Authorship
Acknowledgments
The authors thank Sylvie Méthot for her excellent technical
assistance in writing this paper and also Marc Bergeron for
statistical analysis. We appreciate the excellent technical contribution of Maurice Dufour, Odette Simard, and Caroline Côté. We
express our gratitude to Corinne Barat for critical and constructive
comments for this study and Dave Bélanger for his involvement in
the construction of DCIR expression vectors.
Contribution: A.A.L. performed research, analyzed data, and wrote
the paper; C.G. and M.J.T. designed research, analyzed data, and
wrote the paper; M.R. and A.D.B. contributed vital new reagents.
Conflict-of-interest disclosure: The authors declare no competing financial interests.
Correspondence: Michel J. Tremblay, Laboratoire d’ImmunoRétrovirologie Humaine, Centre de Recherche en Infectiologie,
RC709, Centre Hospitalier de l’Université Laval, 2705 boul,
Laurier, Québec (QC), Canada, G1V 4G2; e-mail: michel.j.
[email protected].
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BLOOD, 15 AUGUST 2008 䡠 VOLUME 112, NUMBER 4
DCIR AS A SURFACE RECEPTOR FOR HIV-1
1307
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2008 112: 1299-1307
doi:10.1182/blood-2008-01-136473 originally published
online June 9, 2008
The C-type lectin surface receptor DCIR acts as a new attachment factor
for HIV-1 in dendritic cells and contributes to trans- and cis-infection
pathways
Alexandra A. Lambert, Caroline Gilbert, Manon Richard, André D. Beaulieu and Michel J. Tremblay
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