JOURNAL OF VIROLOGY, Sept. 2007, p. 8933–8943
Copyright © 2007, American Society for Microbiology. All Rights Reserved.
Vol. 81, No. 17
Functionally Distinct Transmission of Human Immunodeficiency Virus
Type 1 Mediated by Immature and Mature Dendritic Cells?
Jian-Hua Wang, Alicia M. Janas, Wendy J. Olson,† and Li Wu*
Department of Microbiology and Molecular Genetics, Medical College of Wisconsin,
8701 Watertown Plank Road, Milwaukee, Wisconsin 53226
Received 24 April 2007/Accepted 3 June 2007
Dendritic cells (DCs) potently stimulate the transmission of human immunodeficiency virus type 1 (HIV-1)
to CD4?T cells. Immature DCs (iDCs) located in submucosal tissues can capture HIV-1 and migrate to
lymphoid tissues, where they become mature DCs (mDCs) for effective antigen presentation. DC maturation
promotes HIV-1 transmission; however, the underlying mechanisms remain unclear. Here we have compared
monocyte-derived iDCs and mDCs for their efficiencies and mechanisms of HIV-1 transmission. We have found
that mDCs significantly facilitate HIV-1 endocytosis and efficiently concentrate HIV-1 at virological synapses,
which contributes to mDC-enhanced viral transmission, at least in part. mDCs were more efficient than iDCs
in transferring HIV-1 to various types of target cells independently of C-type lectins, which partially accounted
for iDC-mediated HIV-1 transmission. Efficient HIV-1 trans-infection mediated by iDCs and mDCs required
contact between DCs and target cells. Moreover, rapid HIV-1 degradation occurred in both iDCs and mDCs,
which correlated with the lack of HIV-1 retention-mediated long-term viral transmission. Our results provide
new insights into the mechanisms underlying DC-mediated HIV-1 transmission, suggesting that HIV-1 exploits
mDCs to facilitate its dissemination within lymphoid tissues.
Dendritic cells (DCs) perform an essential role in the induc-
tion and regulation of the adaptive immune response (4). In
opposition to the immune function of DCs presenting pro-
cessed antigens, human immunodeficiency virus type 1 (HIV-1
[referred to subsequently as HIV]) hijacks DCs to promote
viral spread. DCs are proposed to be among the first cells that
encounter HIV at the mucosa and to play an important role in
HIV infection and dissemination (54). After capture or uptake
of HIV, immature DCs (iDCs) located in submucosal tissues
migrate to lymphoid tissues and become mature DCs (mDCs)
to potently present antigens to T cells. Interestingly, the trans-
mission efficiency of HIV is enhanced by DC maturation (11,
23, 27, 34, 42, 50), suggesting that mDCs efficiently facilitate
HIV transfer to activated CD4?T cells in lymphoid tissues.
Increased mDC–T-cell interactions may augment HIV transfer
to CD4?T cells (34, 42); however, the mechanisms underlying
mDC-enhanced HIV transmission remain elusive.
DCs can transmit HIV to CD4?T cells through trans-infec-
tion and cis-infection (reviewed in reference 54). Trans-infec-
tion mediated by DCs can occur by two pathways: HIV trans-
mission across the synaptic junctions or infectious/virological
synapses (2, 34, 48) and HIV transmission by immature mono-
cyte-derived DCs via an exocytic pathway that involves HIV-
associated exosomes (52). HIV cis-infection of DCs results in
de novo viral production and long-term transmission of HIV,
although viral replication in DCs is less efficient than that in
CD4?T cells (8, 23, 31, 38, 48). These mechanisms of HIV
transmission may coexist in vivo and contribute to viral dis-
semination; however, whether mDC-enhanced HIV transmis-
sion involves these pathways is unclear.
Initial observations suggested that a C-type lectin, DC-SIGN
(for “DC-specific intercellular adhesion molecule 3 [ICAM-3]-
grabbing nonintegrin”), facilitates DC-mediated HIV trans-
infection (22). Subsequent studies have indicated that DCs also
have DC-SIGN-independent mechanisms of HIV trans-infec-
tion of CD4?T cells (5, 6, 24–26, 46, 49, 53, 57). Other C-type
lectin molecules may be involved in DC-SIGN-independent
HIV transmission mediated by DCs (54). However, whether
mDC-enhanced HIV transmission is dependent on C-type
lectins remains to be examined.
Previous studies indicated that DC contact with CD4?T
cells is required for efficient HIV trans-infection (34, 42, 47).
HIV trafficking has been suggested to be important for DC-
mediated viral transmission (21, 29, 34, 52). Immature DCs can
internalize HIV to late endosomal compartments or multive-
sicular bodies (52), while mDCs sequester internalized HIV
into an endocytic compartment that is distinct from the con-
ventional multivesicular bodies (21). In contrast, a recent study
suggests that DC-mediated HIV trans-infection of CD4?T
cells primarily originates from virions bound on DC surfaces
(11). The discordance of these studies may result from differ-
ent approaches to DC generation, various stimuli of DC mat-
uration, and different HIV reporter systems. Nevertheless, the
role of HIV trafficking in iDC- and mDC-mediated viral trans-
mission remains to be defined.
Here we report functionally distinct HIV trans-infection of
CD4?T cells mediated by iDCs and mDCs. mDCs were more
efficient than iDCs in transmitting HIV to various types of
target cells independently of C-type lectins, which partially
accounts for iDC-mediated HIV transmission. Compared with
iDCs, mDCs significantly enhanced HIV endocytosis and effi-
* Corresponding author. Mailing address: Department of Microbi-
ology and Molecular Genetics, Medical College of Wisconsin, 8701
Watertown Plank Road, Milwaukee, WI 53226. Phone: (414) 456-
4075. Fax: (414) 456-6535. E-mail: firstname.lastname@example.org.
† Present address: University of Wisconsin—Milwaukee, Milwaukee,
?Published ahead of print on 13 June 2007.
ciently concentrated HIV at virological synapses, which likely
play a role in promoting viral transmission to CD4?T cells.
Our results suggest that HIV exploits mDCs to facilitate its
dissemination within lymphoid tissues.
MATERIALS AND METHODS
Cell culture. Peripheral blood lymphocytes (PBLs) and CD14?monocytes
were isolated from buffy coat units of healthy donors (provided by the Blood
Center of Wisconsin, Milwaukee, WI) as previously described (49, 53). iDCs
were generated from purified monocytes treated with granulocyte-macrophage
colony-stimulating factor and interleukin 4 (IL-4) for 6 days, as described pre-
viously (57). mDCs were generated by adding 10 ng/ml of lipopolysaccharide
(LPS) (Escherichia coli strain O55:B5; Sigma-Aldrich) to iDCs and cultured for
an additional 2 days. The monocyte-differentiated iDCs were more than 98.5%
pure by DC-SIGN, HLA-DR, CD11b, and CD11c staining but were negative for
CD3 and CD14. PBLs were activated with 5 ?g/ml of phytohemagglutinin
(Sigma-Aldrich) and cultured in the presence of 20 IU/ml of IL-2 (NIH AIDS
Research and Reference Reagent Program), as described previously (49). The
human embryonic kidney cell line HEK293T, the CD4?T-cell line Hut/CCR5,
the human B-cell line Raji/DC-SIGN, and the HIV indicator cell line
GHOST/R5 (kind gifts from Vineet KewalRamani, National Cancer Institute,
Frederick, MD) have been described previously (49, 57).
Flow cytometry. DCs (1 ? 105) were stained with specific monoclonal anti-
bodies (MAbs) or isotype-matched immunoglobulin G (IgG) controls, as previ-
ously described (55). Phycoerythrin-conjugated mouse anti-human MAbs (BD
Biosciences [unless specified]) against the following molecules (clone numbers
are given in parentheses) were used for staining: DC-SIGN (120507; R&D
Systems), CD3 (HIT3a), CD11b (VIM12), CD11c (BU15), CD14 (TU ¨K4),
HLA-DR (TU ¨36), CD80 (L307.4), CD86 (2331), and IgG isotype control MAbs.
When indicated, DCs were treated with 0.25 mg/ml of trypsin (without EDTA)
(Invitrogen) at room temperature for 4 min or with 0.25 mg/ml of pronase E
(P6911; Sigma-Aldrich) on ice for 10 min. DCs were subsequently neutralized
with culture medium and washed before staining for DC-SIGN. Stained cells
were analyzed with a FACSCalibur flow cytometer (Becton Dickinson).
HIV stocks. Single-cycle infectious HIV stocks were generated by calcium
phosphate cotransfections of HEK293T cells with pLai3?envLuc2 (58) (a kind
gift from Michael Emerman, Fred Hutchinson Cancer Research Center) and
expression plasmids for HIV envelope protein (Env) of JRFL (R5-tropic) or
HXB2 (X4-tropic), as described previously (57). The infectivity of the virus
stocks was evaluated by limiting dilution on GHOST/R5 cells (57). Aldrithiol-2
(AT-2)-inactivated R5-tropic HIV (Bal/Supt1-CCR5 cl30) was a kind gift from
Jeffery Lifson (AIDS Vaccine Program, SAIC, Frederick, MD).
HIV binding and internalization assays. iDCs and mDCs (7.5 ? 104) were
incubated separately with infectious HIV-Luc/JRFL or AT-2-inactivated HIV
(30 ng of p24) for 2 h at 37°C or 4°C. Cells were then washed intensively and
lysed for HIV Gag p24 quantification with an enzyme-linked immunosorbent
assay kit (PerkinElmer), as previously described (49). When indicated, HIV-
pulsed DCs were treated with 0.25 mg/ml of trypsin (without EDTA) at room
temperature for 4 min; cells were subsequently neutralized with culture medium
and washed before lysis for HIV p24 quantification.
HIV transmission and infection assays. HIV transmission and direct infection
assays using luciferase viruses were performed as previously described (57). Cell
lysates were analyzed for luciferase activity with a commercially available kit
(Promega). When indicated, iDCs and mDCs were preincubated with either 10
?g/ml of anti-DC-SIGN (cocktail of clones 120518, 120526, and 120612 [R&D
Systems]) at room temperature or 20 ?g/ml of mannan (Sigma-Aldrich) at 37°C
for 30 min prior to HIV incubation, as described previously (57). Transwell
culture plates (Costar) with inserts of polycarbonate membranes (pore size, 3
?m) were used to separate donor cells from target cells as described previously
(56). When indicated, HIV-pulsed DCs were treated either with 0.25 mg/ml of
trypsin (without EDTA) at room temperature for 4 min or with pronase E at
various concentrations on ice for 10 min. Cells were subsequently neutralized
with culture medium and washed before coculturing with Hut/CCR5 cells.
For HIV transmission assays using trafficking inhibitors, DCs (3 ? 105) were
incubated separately with 1,2-bis(2-aminophenoxy)ethane-N,N,N?,N?-tetraacetic
acid acetoxymethyl ester (BAPTA-AM) (25 ?M), ammonium chloride (NH4Cl)
(10 mM), monensin sodium (30 ?M), brefeldin A (3.6 ?M), and epoxomicin (0.2
?M) (all inhibitors were purchased from Sigma-Aldrich) at 37°C for 30 min and
then pulsed with HIV at 37°C for 2 h in the presence of the inhibitors before
coculture with Hut/CCR5 cells. DC viability after the inhibitor treatment was
examined with 7-amino-actinomycin D staining (Annexin V-PE apoptosis detec-
tion kit; BD Pharmingen) and flow cytometry.
Electron microscopy. For visualization of HIV trafficking, iDCs and mDCs
(6 ? 105) were incubated separately with AT-2-inactivated HIV (2 ?g of p24) at
37°C for 1.5 h. After a wash, DCs were cultured for 1 h and fixed and processed
for conventional transmission electron microscopy as previously described (49).
For HIV transmission from DCs to CD4?T cells, after incubation with AT-2-
inactivated HIV as described above, DCs were washed extensively and cocul-
tured with Hut/CCR5 cells (6 ? 105) for 1 h prior to fixation and sample
preparation. Cells were washed and processed for electron microscopy as previ-
ously described (49). For ruthenium red (RR) labeling of plasma membranes,
cells were washed with 0.1 M ice-cold sodium-cacodylate buffer and then fixed
with 1% glutaraldehyde in 0.1 M sodium-cacodylate buffer containing 0.05% RR
for 1 h on ice. After a wash, cells were postfixed in 1% osmium tetroxide
containing 0.05% RR for 1 h on ice. Thin sections were examined with a
transmission electron microscope (Hitachi H-600 or JEOL 2100 LaB6).
Statistical analyses. Statistical analyses were performed with the Wilcoxon
paired t test with Prism software or Dunnett’s multiple comparison test with the
mDCs enhance HIV transmission to different types of target
cells independently of C-type lectins. To better understand the
cell-cell interactions underlying mDC-enhanced HIV transmis-
sion, the efficiencies of HIV trans-infection mediated by iDCs
and mDCs were compared and the role of C-type lectins in
viral transmission was examined. Purified CD14?monocytes
were used to generate iDCs, and the maturation of iDCs was
achieved by LPS treatment (34). The phenotypes of iDCs and
mDCs were confirmed by immunostaining of cell surface
markers. As expected, iDCs and mDCs uniformly expressed
high levels of CD11c, and they were nearly negative for CD14
at day 7 of differentiation (Fig. 1A). HLA-DR and CD86
expression levels were significantly increased in mDCs relative
to iDCs, indicating efficient DC maturation, while the surface
expression of DC-SIGN was decreased in mDCs (Fig. 1A).
To quantify HIV transmission efficiency mediated by iDCs
and mDCs, a single-cycle luciferase reporter HIV was used.
The virus was pseudotyped separately with R5- or X4-tropic
HIV Env. Various types of target cells were used in HIV
transmission assays, including activated autologous PBLs,
the human CD4?T-cell line Hut/CCR5 (57), and the human
osteosarcoma cell line GHOST/R5, and were engineered to
express HIV receptors (12). When DCs were pulsed with small
amounts of R5-tropic HIV and cocultured separately with dif-
ferent types of target cells, mDCs were 3-fold (P ? 0.05),
18-fold (P ? 0.001), and 8-fold (P ? 0.01) more effective than
iDCs in transmitting HIV infection to activated PBLs, Hut/
CCR5 cells, and GHOST/R5 cells, respectively (Fig. 1B). Sim-
ilar results were observed in independent experiments using
autologous DCs and PBLs derived from four different donors
and using HIV pseudotyped with different R5-tropic Env pro-
teins (data not shown).
Preincubation of iDCs with cocktails of DC-SIGN MAbs
reduced HIV transmission to various types of target cells by
33% to 54% (P ? 0.05) (Fig. 1B), a finding consistent with our
previous results (49, 57). Similarly, blockade of iDCs with
mannan, an inhibitor of mannose-binding C-type lectins, de-
creased HIV transmission by 22% to 56% (P ? 0.05). How-
ever, DC-SIGN MAbs and mannan had no effect on HIV
transmission mediated by mDCs (Fig. 1B). These results sug-
gest that mDC-enhanced HIV transmission is independent of
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