HIV-infected cells are major inducers of plasmacytoid dendritic cell interferon production, maturation, and migration.
ABSTRACT Plasmacytoid dendritic cells (PDC), natural type-1 interferon (IFN) producing cells, could play a role in the innate anti-HIV immune response. Previous reports indicated that PDC IFN production is induced by HIV. Our results show a more robust IFN induction when purified PDC (>95%) were exposed to HIV-infected cells. This effect was not observed with non-viable cells, DNA, and RNA extracted from infected cells, and viral proteins. The response was blocked by anti-CD4 and neutralizing anti-gp120 antibodies as well as soluble CD4. IFN induction by HIV-infected cells was also prevented by low-dose chloroquine, which inhibits endosomal acidification. PDC IFN release resulted in reduced HIV production by infected CD4+ cells, supporting an anti-HIV activity of PDC. Stimulated CD4+ cells induced PDC activation and maturation; markers for PDC migration (CCR7) were enhanced by HIV-infected CD4+ cells only. This latter finding could explain the decline in circulating PDC in HIV-infected individuals.
- SourceAvailable from: PubMed CentralFrontiers in Immunology 09/2014; 5:419.
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ABSTRACT: Plasmacytoid dendritic cells (pDC) expressing FcγRIIa are antigen-presenting cells able to link innate and adaptive immunity and producing various cytokines and chemokines. Although highly restricted, they are able to replicate HIV-1. We determined the activity of anti-HIV-1 neutralizing antibodies (NAb) and non-neutralizing inhibitory antibodies (NNIAb) on the infection of primary pDC by HIV-1 primary isolates and analyzed cytokines and chemokines production. Neutralization assay was performed with primary pDC in the presence of serial antibodies (Ab) concentrations. In parallel, we measured the release of cytokines and chemokines by ELISA and CBA Flex assay. We found that NAb, but not NNIAb, inhibit HIV-1 replication in pDC. This inhibitory activity was lower than that detected for myeloid dendritic cells (mDC) infection and independent of FcγRIIa expressed on pDC. Despite the complete protection, IFN-α production was detected in the supernatant of pDC treated with NAb VRC01, 4E10, PGT121, 10-1074, 10E8, or polyclonal IgG44 but not with NAb b12. Production of MIP-1α, MIP-1β, IL-6, and TNF-α by pDC was also maintained in the presence of 4E10, b12 and VRC01. These findings suggest that pDC can be protected from HIV-1 infection by both NAb and IFN-α release triggered by the innate immune response during infection.Scientific Reports 08/2014; 4:5845. · 5.08 Impact Factor
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ABSTRACT: Recognition of hepatitis C virus (HCV)-infected hepatocyes and interferon (IFN) induction are critical in antiviral immune response. We hypothesized that cell-cell contact between plasmacytoid dendritic cells (pDCs) and HCV-infected cells was required for IFN-α induction through the involvement of cell-surface molecules. Coculture of human peripheral blood mononuclear cells (PBMCs) with genotype 1a full-length (FL) HCV genomic replicon cells or genotype 2a Japanese fulminant hepatitis type 1 (JFH-1) virus-infected hepatoma cells (JFH-1), and not with uninfected hepatoma cells (Huh7.5), induced IFN-α production. Depletion of pDCs from PBMCs attenuated IFN-α release, and purified pDCs produced high levels of IFN-α after coculture with FL replicons or JFH-1-infected cells. IFN-α induction by HCV-containing hepatoma cells required viral replication, direct cell-cell contact with pDCs, and receptor-mediated endocytosis. We determined that the tetraspanin proteins, CD81 and CD9, and not other HCV entry receptors, were required for IFN-α induction in pDCs by HCV-infected hepatoma cells. Disruption of cholesterol-rich membrane microdomains, the localization site of CD81, or inhibition of the CD81 downstream molecule, Rac GTPase, inhibited IFN-α production. IFN-α induction involved HCV RNA and Toll-like receptor (TLR) 7. IFN-α production by HCV-infected hepatoma cells was decreased in pDCs from HCV-infected patients, compared to healthy controls. We found that preexposure of healthy PBMCs to HCV viral particles attenuated IFN-α induction by HCV-infected hepatoma cells or TLR ligands, and this inhibitory effect could be prevented by an anti-HCV envelope glycoprotein 2–blocking antibody. Conclusion: Our novel data show that recognition of HCV-infected hepatoma cells by pDCs involves CD81- and CD9-associated membrane microdomains and induces potent IFN-α production. (HEPATOLOGY 2013;58:940–949)Hepatology 09/2013; 58(3). · 11.19 Impact Factor
HIV-infected cells are major inducers of plasmacytoid dendritic cell
interferon production, maturation, and migration
Barbara Schmidt1, Brittany M. Ashlock, Hillary Foster, Sue H. Fujimura, Jay A. Levy*
Department of Medicine, Division Hematology/Oncology, University of California, San Francisco, CA 94143-0128, USA
Received 9 June 2005; returned to author for revision 16 July 2005; accepted 29 September 2005
Plasmacytoid dendritic cells (PDC), natural type-1 interferon (IFN) producing cells, could play a role in the innate anti-HIV immune response.
Previous reports indicated that PDC IFN production is induced by HIV. Our results show a more robust IFN induction when purified PDC (>95%)
were exposed to HIV-infected cells. This effect was not observed with non-viable cells, DNA, and RNA extracted from infected cells, and viral
proteins. The response was blocked by anti-CD4 and neutralizing anti-gp120 antibodies as well as soluble CD4. IFN induction by HIV-infected
cells was also prevented by low-dose chloroquine, which inhibits endosomal acidification. PDC IFN release resulted in reduced HIV production
by infected CD4+ cells, supporting an anti-HIVactivity of PDC. Stimulated CD4+ cells induced PDC activation and maturation; markers for PDC
migration (CCR7) were enhanced by HIV-infected CD4+ cells only. This latter finding could explain the decline in circulating PDC in HIV-
D 2005 Elsevier Inc. All rights reserved.
Keywords: HIV; Plasmacytoid dendritic cells; Interferon induction; TLR-9; CD4+ cells
During hematopoiesis, stem cells give rise to two major
types of dendritic cell (DC) precursors: myeloid (MDC, pre-
DC1) and lymphoid or the plasmacytoid dendritic cells (PDC,
pre-DC2) (Liu, 2001). The latter cells, also known as natural
interferon (IFN)-a producing cells (Fitzgerald-Bocarsly, 1993),
have been identified as the major source of type-1 IFN (Cella et
al., 1999; Fitzgerald-Bocarsly, 2002; Siegal et al., 1999). Upon
exposure to CD40 ligand (CD40L), PDC differentiate into
dendritic cells (DC-2) (Grouard et al., 1997), promoting naive
CD4+ T lymphocytes to produce IL-4, IL-5, and IL-10, and
directing the immune response into a T-helper (TH)-2 pheno-
type (Kadowaki et al., 2000; Rissoan et al., 1999). Upon
response to viral pathogens, PDC maturation drives a potent
TH-1 polarization (Cella et al., 2000). These observations link
PDC to innate and adaptive immunity (Kadowaki et al., 2000;
Rissoan et al., 1999).
A reduction in PDC numbers has been reported in HIV
primary infection (Pacanowski et al., 2001) as well as in
advanced stages of disease (Almeida et al., 2005; Chehimi et al.,
2002; Donaghy et al., 2001; Feldman et al., 2001; Finke et al.,
2004; Soumelis et al., 2001). PDC numbers have also been
shown to correlate directly with CD4+ cell numbers and
inversely with plasma HIV viral load (Barron et al., 2003;
Soumelis et al., 2001). These observations suggest an important
role of PDC in controlling HIV replication in infected
It is still unclear what mechanisms contribute to the decline
of circulating PDC in HIV infection. PDC, which express high
levels of surface CD4 as well as the chemokine receptors
CCR5 and CXCR4, can be infected by HIV (Donaghy et al.,
2003; Fong et al., 2002; Patterson et al., 2001; Schmidt et al.,
2004b; Yonezawa et al., 2003), but their viability is not affected
(Schmidt et al., 2004b; Yonezawa et al., 2003). Their decline
could be explained by enhanced migration of these cells to
secondary lymphatic tissue (Cyster, 1999). Evidence for this
hypothesis comes from the observation that PDC mature and
show an upregulation of the cell migration marker, CCR7
0042-6822/$ - see front matter D 2005 Elsevier Inc. All rights reserved.
* Corresponding author. Fax: +1 415 476 8365.
E-mail addresses: email@example.com (B. Schmidt),
firstname.lastname@example.org (J.A. Levy).
1Present address: Institute of Clinical and Molecular Virology, German
National Reference Centre for Retroviruses, Schlossgarten 4, 91054 Erlangen,
Virology 343 (2005) 256 – 266
(Fonteneau et al., 2004) concomitant with the release of IFN-a
after exposure to high concentrations of HIV (Fong et al.,
2002; Yonezawa et al., 2003).
The present study was conducted to characterize the
mechanism of IFN induction by HIV. In addition to the findings
by others (Fong et al., 2002, 2004; Yonezawa et al., 2003), we
have observed that virus-infected CD4+ cells induce a more
robust IFN production and maturation of PDC than virus alone.
Virus production by the infected CD4+ cells is subsequently
reduced. This induction is specially regulated since antibodies
to CD4 and gp120 as well as soluble CD4, but not antibodies to
CXCR4, block these effects on PDC. A similar result was
observed with antibodies to BDCA-2, and with chloroquine,
suggesting that endocytosis is required for PDC type 1 IFN
induction by HIV-infected cells. Moreover, the infected CD4+
cells readily induced high CCR7 expression in PDC. These
findings could explain changes in the levels of circulating PDC
in infected individuals and reflect the potential function of PDC
in controlling HIV replication.
Relative induction of PDC IFN production by HIV and
PDC have been demonstrated to produce type 1 interferons
in response to HIV (Fong et al., 2002; Yonezawa et al., 2003).
With purified PDC (>95%) in over 20 experiments, we noted a
limited release of IFN-a (up to 400 pg/ml) when 104PDC were
exposed to HIValone for 24 h and only when a very high input
of the X4-tropic SF33 isolate of HIV-1 (e.g. 10,000 TCID50)
was used. In contrast, a robust IFN response (mean, 1561.4 pg/
ml, 95% confidence level, 1174.6–1948.2 pg/ml) was readily
observed in over 30 separate assays with different PDC donors
when PDC (104cells) were cocultured with CD4+ cells acutely
infected with HIV-1 at a low MOI, 2 days prior to coculture
(data summarized from Schmidt et al., 2004a). This response of
PDC was shown to be directly dependent on the extent of HIV-
replication in the CD4+ cells (Fig. 1a). Similarly, variations in
the interferon induction by 2d-infected CD4+ cells were noted
and depended on the extent of viral replication in these cells.
The interferon production was not detected before 12 h of
coculture of the PDC with infected CD4+ cells, indicating a
time period needed for activating the interferon release (data
not shown). This effect on PDC was not a function of cell death
since the mock-infected cultures showed the same limited
extent of apoptosis as the infected cells as measured by annexin
V staining. Moreover, similar results were found using CD4+
cells infected with HIV-1 R5 and X4 isolates, as well as
cytopathic and noncytopathic HIV-2 isolates (e.g. UC-1)
(Evans et al., 1988) (data not shown).
Evaluating further this quantitative difference of free virus
and virus-infected cells on IFN production, we tested super-
natants of infected CD4+ cells and pelleted virus from these
fluids. IFN-a was only induced when a high viral input was
used (Fig. 1b). Because virus preparations can have cellular
contaminants that could influence the results (Trubey et al.,
2003), we filtered the HIV stock prior to inoculation onto PDC.
After this procedure, the extent of IFN induction by high-
titered virus was reduced by 70% (Fig. 1c). The amount of
IFN-a released by PDC in response to virus preparations was
substantially increased by using 40,000 instead of 10,000
PDC as well as removal of the supernatant after 48 h instead
of 24 h (Fig. 1d). Moreover, using an AT-2 inactivated virus,
substantial interferon induction (up to 4000 pg/ml) was
observed when 3 ? 104PDC, but not 1 ? 104cells, were
exposed to a large quantity of this virus (109RNA molecules/
ml). However, HIV-infected CD4+ cells induced substantially
higher IFN levels under the same conditions. Finally, separa-
tion of PDC and HIV-infected CD4+ cells by a transwell device
prevented the induction of IFN release (Fig. 1e).
We also determined directly the relative extent of IFN release
by PDC after exposure to free virus compared to virus-infected
cells. In these studies, CD4+ cells inoculated with a high
multiplicity of HIV infection (MOI 0.5) for only 6 h induced a
much greater IFN response than high-titered virus alone (Table
1). These results were observed even though the levels of virus
particles in the cell culture fluids, measured immediately before
and 24 h after PDC coculture, were very low (RTactivity, 10 ?
103cpm/ml) compared to the SF33 virus stock added directly to
the PDC (5418 ? 103cpm/ml). Because the CD4+ cells were
trypsinized 1 h after the initial virus inoculation, the IFN
virus (Tang and Levy, 1991). All of these findings indicate that
the HIV-infected cell, more than free virus, is a major inducer of
Effect of non-viable cells, nucleic acids, and viral proteins on
In evaluating the importance of viable cells in IFN
induction, we subjected PHA-stimulated, uninfected, or HIV-
infected CD4+ cells to repeated cycles of freezing and thawing
prior to coculture with PDC. In these experiments, non-viable
cells, infected or uninfected, did not induce IFN release by
PDC obtained from three different donors. Similarly, no IFN
induction was observed with uninfected CD4+ cells irradiated
with 700 to 3000 rad, resulting in up to 50% apoptotic cells.
Irradiation of HIV acutely infected CD4+ cells completely
abolished IFN induction, as confirmed in four separate
experiments. Similarly, IFN was not induced when PDC from
two different donors were challenged with DNA or RNA
extracted from infected or uninfected CD4+ cells. Finally, in
three separate experiments, viral components such as recom-
binant Env (monomeric gp120 from different HIV-1 isolates
and trimeric gp120 from HIV-1SF162), even when added in
combination with CD40L, as well as Gag (p24 and p55) and
Tat proteins elicited no type 1 IFN release by PDC (see
Methods for description of components).
Effect of interferon production by PDC on HIV-infected cells
Since the HIV-infected CD4+ cells induce IFN production
by PDC, we examined the antiviral effect of the released IFN on
B. Schmidt et al. / Virology 343 (2005) 256–266
the infected cells. The CD4+ T cells, infected by HIV-1SF33
(Fig. 2a), HIV-1SF162(Fig. 2b), or HIV-1SF2(Fig. 2c) for 2 days
and then cocultured with PDC readily induced IFN production
and these CD4+ cells showed a marked decrease in HIV
replication. This effect appeared to be a function of the extent of
virus replication in the CD4+ cells at the time of the assay. Virus
production in cells infected for only 6 h showed the highest
sensitivity to interferon release by PDC (80% suppression) (data
not shown). In support of this effect of IFN on HIV replication,
increasing amounts of recombinant IFN-a showed a dose-
Induction of interferon (IFN) production after exposure of plasmacytoid dendritic cells (PDC) to CD4+ cells infected with HIV-1 for 6 h prior to coculture or to
supernatant containing high-titered HIV-1
Parameters Exposure of PDC to
CD4+ cells infected with HIV-1SF33for only 6 h prior to cocultureSupernatant containing HIV-1SF33
Viral TCID50used for infection of
60,000 CD4+ cells
Viral TCID50added directly
RT prior to addition to PDC
RT 24 h after addition to PDC
IFN-a, 24 h after addition to PDC
CD4+ cells were trypsinized 1 h post-infection to remove virus attached to the cell surface (Tang and Levy, 1991).
104PDC were exposed to HIV-1SF33-infected CD4+ cells at a ratio of 1:6 (PDC/CD4) in 96-well flat-bottom plates.
RT, viral reverse transcriptase activity (cpm ? 103/ml); TCID50, tissue culture infectious dose 50%; n.a., not applicable.
Fig. 1. Comparison of interferon (IFN) release by plasmacytoid dendritic cells (PDC) after exposure to HIV-1 or HIV-1-infected cells. (a) IFN-a release by PDC
after exposure to herpes simplex virus (HSV) or PHA-stimulated CD4+ cells uninfected (uninf.) or infected (inf.) with HIV-1SF33for 6 h to 4 days prior to coculture
with PDC. To observe a dose-dependent effect, CD4+ cells were infected at a 10-fold lower MOI than usual. The reverse transcriptase (RT) activity was determined
in the fluids of the infected CD4+ cells (Hoffman et al., 1985) prior to coculture with PDC. Supernatants for IFN-a activity were harvested 24 h after exposure of
PDC to the different stimuli. This experiment is representative of three independent studies using different PDC donors. (b) Exposure of PDC to HIV-infected
CD4+ cells as well as the HIV-containing supernatants from these CD4+ cells and virus pelleted from the respective supernatants. Virus content was determined by
measuring particle-associated RT activity in the culture fluids of CD4+ T cells prior to PDC exposure. (c) IFN-a release by inoculation of PDC with fluid
containing 10,000 TCID50of HIV-1SF33pelleted from unfiltered and filtered fluids. Data represent three donors whose PDC responded to high-titered HIV-1SF33
with a limited IFN release. (d) IFN-a induction by 10,000 or 40,000 PDC exposed to fluids containing 10,000 TCID50of HIV-1SF33, with supernatants removed
after 24 h and 48 h of exposure. These data are representative of three separate experiments. (e) IFN release by PDC cultured with CD4+ cells infected (inf.) with
HIV-1SF33for 2–8 days without (w/o) (g) or with (w/t) (n) a transwell insert. Supernatants for IFN-a activity were harvested 24 h after PDC exposure. Wells with
no measurable IFN-a activity are labeled with an asterisk (*).
B. Schmidt et al. / Virology 343 (2005) 256–266
dependent reduction in virus production by HIV-1SF2-infected
CD4+ cells (Fig. 2d). Importantly, virus replication was
increased by adding IFN-a neutralizing antibodies, but not the
isotype control antibody, to cocultures of PDC and HIV-infected
CD4+ cells (Fig. 2e). These results demonstrate that the IFN-a
response of PDC to HIV-infected CD4+ cells can lead to
suppression of virus replication within the infected cells.
Features of cell:cell interaction involved in IFN release by
The mechanism by which HIV-infected CD4+ cells induce
IFN production was evaluated by a variety of procedures. HIV-
infected CD4+ T cells and PDC were cultured together with
anti-CD4 antibodies for 24 h. A dose-dependent reduction in
IFN induction was observed (Fig. 3a). As controls, the
antibodies to CD4 showed no effect on IFN induction by
HSV, CpG-A, and the synthetic Toll-like receptor (TLR)-7
agonist S-27609 (Doxsee et al., 2003). A similar effect in
preventing IFN production by PDC was noted when soluble
CD4 was added to the coculture of PDC with infected CD4+
cells (Fig. 3b). In contrast, HSV-induced IFN production was
not affected by soluble CD4 (data not shown). The soluble
CD4 and the anti-CD4 antibodies both reduced HIV-1SF33
infection of CD4+ cells by >95% (data not shown).
To determine if the CD4 receptor on the HIV-infected
CD4+ cells or on the PDC is involved in IFN induction, the
PDC were cocultured with pNL4-3-transfected 293 cells that
lack CD4 expression. The NL4-3 producing cells consistently
induced high levels of PDC IFN production, which could be
blocked by anti-CD4 and soluble CD4 (Fig. 3c). These
studies indicated the role of CD4 expression on PDC in IFN
In evaluating further the potential importance of the virus
envelope on PDC IFN induction, polyclonal and monoclo-
nal antibodies to gp120 and gp41 were added to the
coculture of PDC with HIV-1SF33-infected CD4+ cells. No
effect on IFN induction was observed (data not shown).
The anti-gp120 antibodies, however, reduced the ability of
HIV-1SF33 to infect CD4+ cells by only 20%, reflecting a
limited neutralizing activity. When neutralizing antibodies to
HIV-1SF2 were used in coculture with HIV-1SF2-infected
CD4+ cells, a marked reduction in IFN production was
observed (Fig. 3d). These antibodies block HIV-1SF2
infection of CD4+ cells by >90% (Levy et al., 1984).
These findings suggest that gp120 on the surface of the
virus in HIV-infected cells is directly involved in PDC IFN
We also examined whether an HIV interaction with its
chemokine coreceptor was needed for IFN release. Anti-
CXCR4 antibodies reduced the infection of CD4+ cells by
the X4-tropic virus HIV-1SF33 by 80% (data not shown).
However, these antibodies had no effect on IFN induction
by HIV-1SF33-infected cells (Fig. 3e).
Fig. 2. The effect of IFN release by PDC on HIVreplication in the HIV-infected CD4+ Tcells. CD4+ cells were infected (inf.) with (a) HIV-1SF33, (b) HIV-1SF162, and
(c) HIV-2SF2for 2 days and then exposed to PDC. Supernatants were harvested after another 24 h and assayed for virus replication (measured by reverse transcriptase
(RT) activity). The data for HIV-1SF33were obtained from three independent experiments. The experiments with HIV-1SF162and HIV-1SF2are each representative of
two separate studies.(d) Virus replicationby PHA-stimulated CD4+ cellsinfected (inf.) withHIV-1SF2for 2 daysin the presence of increasingamounts of recombinant
IFN-a. (e) IFN-a production (g) and virus replication measured by RTactivity (n) after coculture of PDC with 2d HIV-1SF33-infected CD4+ cells in the presence of
neutralizing antibodies to IFN-a or the corresponding isotype control antibody. This experiment is representative of three independent studies with X4 and R5 viruses.
B. Schmidt et al. / Virology 343 (2005) 256–266
Role of endocytosis in PDC IFN induction
In attempts to determine if some aspect of endocytosis is
involved in this induction of IFN, we examined the role of
the PDC surface marker BDCA2. BDCA2 is a novel type
II calcium-dependent lectin that has been reported to
mediate antigen capture and uptake (Dzionek et al.,
2000). Anti-BDCA2 antibodies blocked PDC IFN induction
by HIV-infected cells, CpG-A, and HSV, whereas IFN
induction by S-27609 remained unaffected (Fig. 4a). The
effect of anti-BDCA2 was observed at 100-fold lower
concentration compared to the isotype control antibody.
PDC were also treated with chloroquine, which blocks
TLR9-mediated signaling by inhibiting endosomal acidifica-
tion and maturation (Lee et al., 2003). This treatment
abolished IFN induction by HSV, CpG-A, and HIV-infected
cells at a 10-fold lower concentration than the IFN
induction by the synthetic TLR7 agonist (Fig. 4b).
Importantly, these concentrations were 5- to 10-fold lower
than those reported to inhibit HIV-1 replication in PBMC
(Rayne et al., 2004).
PDC maturation after exposure to different stimuli
In response to various stimuli, PDC can release interferon
and/or mature into DC. In evaluating this process, PDC
were exposed to HIV, uninfected and HIV-infected CD4+
cells, CD40L, HSV, CpG-A, and CpG-B. Expression of
surface markers for activation (CD80 and CD86), maturation
(CD83), and migration (CCR7) was determined after 24 and
48 h. Exposure to infectious HIV alone did not change
surface marker expression, even after 48 h (Fig. 5). In
contrast, all markers were upregulated within 24 h after
exposure of the PDC to HSV, CpG-B, and to a lesser extent
CpG-A. CpG-A readily induced IFN-a production by PDC,
whereas CpG-B is primarily an activator of B cells and, as
Fig. 3. Characterization of the PDC surface receptor interactions involved in the induction of IFN production. (a) Effect of sodium-azide free anti-CD4 (Leu3a) on
the induction of IFN-a by PDC after exposure to four different stimuli: CD4+ cells 2 days after infection by HIV-1SF33(n); irradiated herpes simplex virus type 1
(HSV-1) (g); CpG-A (0); and the synthetic TLR7 agonist S-27609 (N). Results are given as mean T 1 SD, representing at least three separate experiments using PDC
from different donors (control IFN-a levels for 2d HIV-1SF33-infected cells (1802 pg/ml), HSV (4377 pg/ml), CpG-A (4542 pg/ml), and S-27609 (2899 pg/ml). (b)
Effect of soluble (sol.) CD4 on the PDC IFN production during exposure to 2d HIV-1SF33-infected (inf.) CD4+ T cells. Results are representative of three separate
experiments. Bars are T1 SD. Soluble CD4 did not block PDC IFN induction by HSV (data not shown). (c) PDC IFN production after exposure to 293 cells
transfected (transf.) with medium (mock) or pNL4-3 in the absence and presence of anti-CD4 and soluble (sol.) CD4, as well as the supernatant (SN) from pNL4-3
transfected 293 cells. The mean RTactivity in the supernatant of the transfected 293 cells was 264 ? 103cpm/ml. Results are representative of three separate studies.
(d) Effect of HIV-1 gp120SF2antiserum (10 Al/well) on the PDC interferon production after coculture with CD4+ cells, 4 days after infection with HIV-1SF2(4d inf.).
(e) Effect of anti-CXCR4 antibodies on the PDC IFN production after exposure to uninfected (uninf.) CD4+ cells or CD4+ cells 2 days after infection (2d inf.) by
HIV-1SF33. The data are representative of two separate studies.
B. Schmidt et al. / Virology 343 (2005) 256–266
was shown by others (Rothenfusser et al., 2004), induced
maturation of PDC (Fig. 5).
The encounter of PDC with PHA-stimulated CD4+ cells
enhanced activation and maturation of autologous and heter-
ologous PDC after 24 h, whereas unstimulated CD4+ cells did
not (Fig. 5). In contrast, upregulation of CCR7 expression was
only observed with HIV-infected CD4+ cells, which similarly
enhanced PDC maturation and activation. This observation was
confirmed by studies with three other PDC donors (Table 2).
Innate immune responses are the first line of defense against
HIV infection and can be important in preventing transmission
or limiting the infection through antiviral activity (Levy, 2001).
One important member of the innate immune response, the
plasmacytoid dendritic cell (PDC), is the major producer of
type-1 IFN (Cella et al., 1999; Siegal et al., 1999). PDCs are
found at higher numbers in HIV-infected individuals who are
healthy, particularly long-term survivors of the infection
(Soumelis et al., 2001). Previous studies have suggested that
HIV itself can induce IFN production by cultured PDC (Fong
et al., 2002; Fonteneau et al., 2004; Yonezawa et al., 2003).
Our systematic approach exposing a low number of PDC to
different stimuli for 24 h indicates that the virus-infected cells
are better inducers of IFN-a production than HIV alone (Figs.
1a, b). This finding is supported by several observations: the
absence of substantial IFN production by cell-free virus
containing fluids (Figs. 1a, b, c), the abrogation of IFN
induction by HIV-infected cells using transwell inserts (Fig.
1e), and the lack of correlation of IFN release with high
particle-associated reverse transcriptase activity in cell culture
fluids vs. the lower viral levels associated with virus-infected
cells (Table 1).
Our findings with HIV alone contrast with previous reports
(Del Corno et al., 2005; Fong et al., 2002; Fonteneau et al.,
2004; Yonezawa et al., 2003) in which different approaches
were used. For example, the induction of IFN-a production by
HIV alone can be enhanced by exposing 40,000 PDC instead
of 10,000 PDC to the virus and measuring the cytokine after
48 h instead of 24 h (Fig. 1d). These experimental conditions
were used by Fong et al. (2002) and Yonezawa et al. (2003),
respectively. In a recent study, a very large number of PDC
(2.5 ? 105cells) were induced to release IFN after exposure to
different stimuli (Del Corno et al., 2005). Moreover, substan-
tial IFN induction by HIV alone was shown by Fonteneau
et al. (2004) after purifying virions by sucrose gradient
ultracentrifugation with a final 1000-fold concentration com-
pared to the original cell culture supernatant. Our results with
very high concentrations of AT-2 inactivated HIV-1 support
these findings. However, our studies were designed to
determine the relative importance of HIV vs. HIV-infected
cells in the induction of IFN production by PDC. With our
approach using low amounts of PDC and physiologically
relevant HIV concentrations, optimal IFN production was
found induced by HIV-infected cells.
Viability of the infected cells appeared to be necessary for
this effect on PDC, because IFN was not induced by apoptotic
uninfected cells nor by virus-infected cells killed by different
methods. In the evaluation of other cellular and viral
component(s) that could be important for this process, we
did not observe IFN induction by the viral envelope and
accessory proteins nor by DNA and RNA extracted from
virus-infected cells. In this regard, some earlier studies noted
IFN induction when PDC were exposed to the gp120 protein
derived from HIV-1IIIB (Ankel et al., 1994). This response
could be blocked by antibodies to the principal neutralizing
domains of HIV and the CD4 binding site, but not by
antibodies to domains in the V3 loop that prevented HIV
infection and cell fusion. This IFN induction was not observed
with gp120s derived from other HIV isolates. Thus, our
negative findings with recombinant gp120 proteins support
these latter observations.
The induction of IFN production was considerably reduced
by filtering viral stocks prior to PDC challenge (Fig. 1c). These
findings, in addition to the substantial PDC release of IFN after
exposure to HIV-infected cells, support a role of cell surface
molecules in this process. The IFN induction by HIV-infected
cells appears to be specially regulated since antibodies to CD4
(also Yonezawa et al., 2003), gp120, and soluble CD4 can
Fig. 4. Role of endocytosis in the induction of PDC IFN-a production. PDC were exposed to CD4+ cells 2 days after infection by HIV-1SF33(n); UV-irradiated
herpes simplex virus type 1 (HSV-1) (g); CpG-A (0); and the synthetic TLR7 agonist S-27609 (N). Effect of (a) anti-BDCA2 (control IFN-a levels for 2d HIV-1SF33-
infected cells (5037 pg/ml), HSV (2148 pg/ml), CpG-A (2625 pg/ml), and S-27609 (1648 pg/ml), and (b) chloroquine (control IFN-a levels for 2d HIV-1SF33-
infected cells (2301 pg/ml), HSV (4062 pg/ml), CpG-A (4498 pg/ml), and S-27609 (2900 pg/ml). All supernatants were removed 24 h after exposure of PDC to the
four different stimuli. Results are given as mean T 1 SD, representing at least three separate experiments using PDC from different donors.
B. Schmidt et al. / Virology 343 (2005) 256–266
block the effect on PDC (Figs. 3a, b, d). The results further
suggested that the CD4 receptor on PDC rather than HIV-
infected cells interacts with gp120 present on the surface of
HIV-infected cells (Fig. 3c). In addition, coreceptor binding of
viral particles to PDC and virus infection of PDC do not seem
to be required since antibodies to CXCR4 did not affect IFN
induction by HIV-1-infected cells (Fig. 3e). It cannot be
excluded, however, that only the initial binding step, i.e. the
attachment to the CD4 receptor, can be inhibited efficiently by
Fig. 5. Effect of different stimuli on the expression of PDC surface markers for cell activation (CD80, CD86), maturation (CD83), and migration (CCR7) after 24 h of
exposure. Results are compiled from four different experiments using freshly isolated PDC from the same donor. Data are representative of studies with four different
donors. Stimuli were IL-3 only, CD40 ligand (CD40L), CpG-B, HIV-1SF33(10,000 TCID50per experiment), uninfected (uninf.) unstimulated (unstim.) or PHA-
stimulated (stim.) CD4+ cells as well as HIV-1SF33-infected (inf.) stimulated CD4+ cells. For exposure of PDC to CpG-A, the respective percentages were 65
(CD80), 60 (CD83), 71 (CD86), and 43 (CCR7).
B. Schmidt et al. / Virology 343 (2005) 256–266
BDCA-2, a type II calcium-dependent lectin expressed on
PDC, mediates antigen capture and inhibits induction of type I
IFN (Arce et al., 2001; Dzionek et al., 2001). Our findings also
suggest a specific involvement of BDCA-2 in the process of
IFN induction by HIV-infected cells (Fig. 4a). Importantly, the
anti-BDCA-2 antibodies used did not affect IFN induction by
the TLR7 agonist at these concentrations, thus indicating that
IFN production was not generally blocked. The results could
indicate that BDCA2 has a role similar to that of DC-SIGN on
monocyte-derived dendritic cells (Kwon et al., 2002). It could
mediate antigen transfer to endocytotic vesicles in PDC.
This observation on BDCA-2 could have relevance to Toll-
like receptors (TLR), which are involved in the recognition of
non-self antigens in innate immunity. PDC preferentially
express TLR7 and TLR9 (Kadowaki et al., 2000); which are
located intracellularly (Krieg, 2002). TLR9 is activated via
unmethylated CpG DNA (Hemmi et al., 2000). The CpG
oligonucleotides are internalized into early endosomes and
subsequently transported to a tubular lysosomal compartment
where they encounter TLR9 redistributed from the endoplas-
mic reticulum (Latz et al., 2004); thus endocytosis is involved
in the function of TLR9.
The activity of TLR9 in comparison to TLR7 is blocked at
lower concentrations of compounds such as chloroquine that
prevent endosomal acidification and maturation (Lee et al.,
2003). Thus, our observations on the inhibitory effect of
chloroquine on the induction of IFN by HIV-infected cells (Fig.
4b) are relevant. They resemble findings by others showing a
chloroquine sensitivity of HSV and HSV-infected cells in their
induction of interferon production (Feldman et al., 1994;
Lebon, 1985). In this regard, recognition of HSV-2 by PDC has
been reported to be mediated by TLR9 in the mouse model
(Lund et al., 2003). Therefore, our results with chloroquine
(Fig. 4b) provide indirect evidence for a role of TLR9 on
human PDC in the IFN induction by HIV-infected cells.
However, further studies are required to establish direct
evidence of a role for specific TLRs on PDC in the IFN
induction by HIV-infected cells.
The IFN-a induction by PDC resulted in reduced HIV-1
replication in the infected CD4+ cells (Figs. 2a, b, c), which
was similarly reproduced using exogenous recombinant IFN-a
(Fig. 2d) and could be reversed by neutralizing antibodies to
IFN-a (Fig. 2e). These data indicate that PDC can have anti-
HIVactivity mediated by IFN-a production. Notably, this virus
suppression was observed using CD4+ cells which had been
infected with HIV 2 days prior to coculture with PDC. It is
likely that the PDC secreted IFN-a will have an even greater
antiviral effect on uninfected or recently infected CD4+ cells.
Another important finding from our studies was that the
expression of CCR7 associated with cell migration was more
readily enhanced by contact of PDC with HIV-infected CD4+
cells than CD4+ cells alone (Fig. 5 and Table 2). These results
suggest that PDC mature upon contact with CD40L expressed
by stimulated CD4+ cells, whereas trafficking of PDC to
secondary lymphatic tissue (via CCR7) is greatly enhanced by
contact with HIV-infected CD4+ cells. In HIV-infected
individuals, about 1 in 1000 to 10,000 circulating CD4+ cells
is HIV-infected. This number increases with progression to
disease, which enhances the chance of PDC encountering
these cells. The contact with HIV-infected cells will also occur
within secondary lymphatic tissue, which may contribute to
retention of PDC in these tissues. Thus, a reason for the
depletion of circulating PDC in late stages of HIV infection
(Soumelis et al., 2001) may be the transit to and maintenance
of PDC in the lymph nodes after contact with HIV-infected
cells. Understanding further the mechanism by which virus-
infected cells induce IFN production by PDC could thus be
helpful for the development of therapeutic approaches directed
at maintaining and increasing the number of PDC in HIV-
Isolation of PDC
Using Ficoll–Hypaque gradients (Sigma Diagnostics Inc.,
St. Louis, MO), peripheral blood mononuclear cells (PBMC)
were obtained from EDTA-containing blood of uninfected
volunteers or from buffy coats provided by the Blood Centers
of the Pacific (San Francisco, CA). This project received the
approval of the Committee for Human Research, University of
California, San Francisco, CA.
The PDC were purified from the PBMC using a BDCA-4
Cell Isolation Kit (Miltenyi Biotec, Auburn, CA) (Dzionek et
al., 2000). After two steps with LS and MS columns, these cells
were >95% pure as determined by flow cytometry. From a
buffy coat of 500 ? 106cells, about 5 ? 105PDC could be
recovered. The isolated PDC were cultivated in RPMI 1640
medium containing 10% heat-inactivated (56 -C, 30 min) fetal
bovine serum, 2 mM glutamine, 100 U/ml penicillin, and 100
Ag/ml streptomycin, supplemented with 20 ng/ml IL-3 (R&D
Systems, Minneapolis, MN). For these studies, the PDC were
generally plated at a density of 104cells/well in 96-well flat
bottom plates. For the FACS experiments, the PDC were plated
at a density of 105cells/well in 48-well flat bottom plates, and
for the transwell experiments at a density of 2.5 ? 104cells/
well in 24-well flat bottom plates.
Upregulation of CCR7 expression by PDC after exposure of these cells to
uninfected and HIV-infected CD4+ cells
Percentage of CCR7-expressing PDC after exposure of these cells to
Results of donor 01 are identical to those shown in Fig. 5.
PHA-stimulated CD4+ cells were infected by HIV-1SF33 2 days prior to
coculture. 105PDC were exposed to uninfected or HIV-infected CD4+ cells at a
ratio of 1:2 (PDC/CD4) in 48-well flat-bottom plates. After 24 h, the percentage
of CCR7-expressing BDCA4-positive cells was assessed using FACS analysis.
Mock = IL-3 containing medium alone.
B. Schmidt et al. / Virology 343 (2005) 256–266
CD4+ T cell isolation and infection
CD4+ T cells were isolated from the separated PBMC
using CD4 MicroBeads (Miltenyi Biotec, Auburn, CA) by
standard procedures (Mackewicz et al., 1991). The CD4+
cells were then cultured in the RPMI 1640 medium described
above, supplemented with 10 ng/ml human IL-2 (Roche
Diagnostics, Indianapolis, IN). They were stimulated with 3
Ag/ml of phytohemagglutinin (PHA) for 3 days, washed and
pretreated for 30 min with 2 Ag/ml of polybrene (Sigma
Chemicals, St. Louis, MO) prior to virus inoculation. The
cells were then infected with the SF33 isolate of HIV-1 at a
multiplicity of infection (MOI) of 0.0001 unless stated
otherwise. This virus is an X4 syncytium-inducing (SI)
chemokine-insensitive isolate (Mackewicz et al., 1996). Other
HIV-1 isolates used for these studies were SF162, SF128A, and
ME1, all NSI (R5) viruses, and ME46 and SF2, SI (X4)
viruses (Levy et al., 1985). ME1 and ME46 were obtained
from the AIDS Research and Reference Reagent Program,
NIH. The HIV-2UC2 isolate was also used (Castro et al.,
1990). After 1 h of infection, the cells were washed and
cultured at a concentration of 3 ? 106cells/ml for 6 h to 5
days prior to PDC coculture. Virus replication was assessed
by reverse transcriptase (RT) activity in the culture fluids
(Hoffman et al., 1985), prior to and after adding the HIV-
infected CD4+ cells to the PDC.
Virus stocks were generated by acutely infecting PHA-
stimulated CD4+ cells, then adding PBMC when high viral
production occurred. The supernatants were harvested at
peak viral replication, which usually occurred within 2–3
days of passage. The TCID50 was quantified in PBMC
stimulated by PHA and IL-2 as described (McDougal et al.,
Coculture with PDC
PDC were cocultured with HIV-infected or uninfected
CD4+ cells at a ratio of 1:6. For some experiments, PDC
were inoculated with 100 Al of supernatant from the HIV-
infected CD4+ cells (Fig. 1b) or pNL4-3-transfected 293 cells
(Fig. 3c) or with virus pelleted from these supernatants by
centrifugation at 12,000 rpm for 1 h. For other experiments,
viral particles adherent to the CD4+ cell surface were removed
by treating the cells with 0.15% trypsin (Tang and Levy, 1991)
1 h after virus inoculation. For the cell maturation experiments,
PDC were mixed with stimulated or unstimulated CD4+ cells
at a ratio of 1:2. Transwell inserts for 24-well flat bottom plates
were purchased from Corning Costar Corporation (Cambridge,
Transfection of 293 cells
293 cells were plated in 60 ? 15 mm tissue culture dishes
(Becton Dickinson Labware, Franklin Lakes, NJ). The cells
were transfected at 50% confluency using 3 Al of the
FuGENE 6 Transfection Reagent (Roche Diagnostics Corpo-
ration, Indianapolis, IN) with 1 Ag of DNA (pNL4–3). Virus
production was assessed by RT activity (Hoffman et al., 1985)
in the cell culture supernatants 48 h after transfection,
immediately before the 293 cells were cocultured with
Culture supernatants were generally harvested 24 h after
exposure of PDC to different stimuli unless indicated
otherwise. The supernatants were assayed for IFN-a activity
using a sandwich enzyme linked immunosorbent assay
(ELISA) (Biosource International, Camarillo, CA) according
to the manufacturer’s instructions. All samples were evalu-
ated in duplicate and diluted if appropriate. Each experiment
was performed at least twice with different PDC donors.
The following reagents were obtained from the National
Institutes of Health AIDS Research and Reference Reagent
Program (Bethesda, MD): HIV-1PB1MNantiserum, goat; HIV-
1 gp120SF2antiserum, goat; MAb (human) to HIV-1 gp120
(F105), MAb to HIV-1 gp120 (C2G12); MAb (human) to
HIV-1 gp41 (98-6); MAb to HIV-1 gp41 (F240); HIV-1IIIB
gp120; HIV-1SF2 gp120; HIV-1BaL gp120; HIV-1SF162
gp120; HIV-1 Tat; HIV-1SF2 p55 Gag, and recombinant
soluble CD4. Sodium-azide free antibodies to CD4 (Leu3a)
were kindly provided by Becton Dickinson (San Jose, CA).
Antibodies were generally used at 15 Ag/ml unless stated
otherwise, by preincubating PDC or HIV-infected CD4+
cells at 37 -C for 60 min. Proteins were added to PDC at
amounts of 0.2–1 Ag per well. CD40L (received from
Immunex Corp., Seattle, WA) was added at a concentration
of 0.5 ng/ml. The IFN-a antibody (cat. no. G026-501-568)
and the isotype control (cat. no. G027-501-568) were
obtained from the NIAID Research Resources; both anti-
bodies were resuspended in 0.5 ml of PBS and used at a
final dilution of 1:200. Monoclonal antibodies for FACS
analysis were obtained from BD Biosciences, San Jose, CA
(HLA-DR and CD4) and BD PharMingen, San Diego, CA
(CD80, CD86, and CCR7). Flow cytometry was performed
on a FACSort with CellQuest Software (BD Biosciences).
CpG-A (ODN 2336; provided by Coley Pharmaceutical
Group, Wellesley, MA) and CpG-B (5V-tcgtcgttttgtc-
gttttgtcgtT-3V, lower cases indicate phosphorothioate linkage,
synthesized by Invitrogen Corporation, Carlsbad, CA) were
used at final concentrations of 1 and 5 AM, respectively.
UV-irradiated HSV-1 was used at a final concentration of
106PFU/ml. Aldrithiol-2 inactivated HIV-1 (Arthur et al.,
1998) (isolate 97ZA012 from the NIH AIDS Research and
Reference Reagent Program) was used at a non-toxic
concentration of 109RNA copies/ml. Chloroquine (pur-
chased from Sigma-Aldrich CO, St. Louis, MO) was used at
0.05–5 AM. The synthetic TLR7 agonist S-27609, provided
by Richard Miller (3M Pharmaceuticals, St. Paul, UK), was
used at a concentration of 5 AM. Recombinant IFN-a was
obtained from Biosource.
B. Schmidt et al. / Virology 343 (2005) 256–266
Extraction of viral and cellular DNA and RNA
PHA-stimulated CD4+ cells were infected with HIV-1SF33
as described above and cultured for 3 days. Control uninfected
CD4+ cells were also cultured. The DNA or RNA was
extracted from 3 ? 106cells using a QIAamp DNA Mini Kit
(Qiagen, Valencia, CA) or the TRIzol Reagent (Invitrogen,
Carlsbad, CA), respectively. For the studies described, con-
centrations of 0.5–5 Ag/ml of the DNA obtained from infected
or uninfected cells were used.
Grant support: These studies were supported by the UCSF
California AIDS Research Center, a program of the UCSF
AIDS Research Institute and a fellowship to B. Schmidt from
the Max Kade Foundation.
The CXCR4 antibody (2G12) was provided by Jim Hoxie
(University of Pennsylvania, Philadelphia, PA), the trimeric
gp120 of HIV-1SF162by Susan Barnett (Chiron Corporation,
CA), CpG-A by Coley Pharmaceutical Group (Wellesley,
MA), the synthetic TLR7 agonist S-27609 by Richard Miller
(3M Pharmaceuticals, St. Paul, MN), the herpes simplex virus
(HSV-1) stock by Yong-Jun Liu (MD Anderson, Houston,
TX), and the aldrithiol-2-inactivated HIV stock by Haynes
Sheppard (California Department of Health Services, Rich-
mond, CA). Unconjugated antibodies to BDCA-2 and
BDCA-4 were kindly provided by Ju ¨rgen Schmitz (Miltenyi
Biotec, Bergisch-Gladbach, Germany). We thank Iain Scott
for his initial observations on PDCs. We thank Leyla Diaz
and Carl Mackewicz for helpful discussions, and Kaylynn
Peter and Ann Murai for assistance in the preparation of the
Almeida, M., Cordero, M., Almeida, J., Orfao, A., 2005. Different subsets of
peripheral blood dendritic cells show distinct phenotypic and functional
abnormalities in HIV-1 infection. AIDS 19, 261–271.
Ankel, H., Capobianchi, M.R., Castilletti, C., Dianzani, F., 1994. Interferon
induction by HIV glycoprotein 120: role of the V3 loop. Virology 205,
Arce, I., Roda-Navarro, P., Montoya, M.C., Hernanz-Falcon, P., Puig-Kroger,
A., Fernandez-Ruiz, E., 2001. Molecular and genomic characterization of
human DLEC, a novel member of the C-type lectin receptor gene family
preferentially expressed on monocyte-derived dendritic cells. Eur. J.
Immunol. 31, 2733–2740.
Arthur, L.O., Bess Jr., J.W., Chertova, E.N., Rossio, J.L., Esser, M.T.,
Benveniste, R.E., Henderson, L.E., Lifson, J.D., 1998. Chemical inacti-
vation of retroviral infectivity by targeting nucleocapsid protein zinc
fingers: a candidate SIV vaccine. AIDS Res. Hum. Retrovir. 14 (Suppl. 3),
Barron, M.A., Blyveis, N., Palmer, B.E., MaWhinney, S., Wilson, C.C., 2003.
Influence of plasma viremia on defects in number and immunophenotype of
blood dendritic cell subsets in human immunodeficiency virus 1-infected
individuals. J. Infect. Dis. 187, 26–37.
Castro, B.A., Barnett, S.W., Evans, L.A., Moreau, J., Odehouri, K., Levy, J.A.,
1990. Biologic heterogeneity of human immunodeficiency virus type 2
(HIV-2). Virology 178, 527–534.
Cella, M., Jarrossay, D., Facchetti, F., Alebardi, O., Nakajima, H., Lanzavec-
chia, A., Colonna, M., 1999. Plasmacytoid monocytes migrate to inflamed
lymph nodes and produce large amounts of type I interferon. Nat. Med. 5
Cella, M., Facchetti, F., Lanzavecchia, A., Colonna, M., 2000. Plasmacytoid
dendritic cells activated by influenza virus and CD40L drive a potent TH1
polarization. Nature Immunology 1, 305–310.
Chehimi, J., Campbell, D.E., Azzoni, L., Bacheller, D., Papasavvas, E., Jerandi,
G., Mounzer, K., Kostman, J., Trinchieri, G., Montaner, L.J., 2002.
Persistent decreases in blood plasmacytoid dendritic cell number and
function despite effective highly active antiretroviral therapy and increased
blood myeloid dendritic cells in HIV-infected individuals. J. Immunol. 168
Cyster, J.G., 1999. Chemokines and cell migration in secondary lymphoid
organs. Science 286, 2098–2102.
Del Corno, M., Gauzzi, M.C., Penna, G., Belardelli, F., Adorini, L., Gessani, S.,
2005. Human immunodeficiency virus type 1 gp120 and other activation
stimuli are highly effective in triggering alpha interferon and CC
chemokine production in circulating plasmacytoid but not myeloid
dendritic cells. J. Virol. 79 (19), 12597–12601.
Donaghy, H., Pozniak, A., Gazzard, B., Qazi, N., Gilmour, J., Gotch, F.,
Patterson, S., 2001. Loss of blood CD11c(+) myeloid and CD11c(?)
plasmacytoid dendritic cells in patients with HIV-1 infection correlates with
HIV-1 RNA virus load. Blood 98 (8), 2574–2576.
Donaghy, H., Gazzard, B., Gotch, F., Patterson, S., 2003. Dysfunction and
infection of freshly isolated blood myeloid and plasmacytoid dendritic cells
in patients infected with HIV-1. Blood 101, 4505–4511.
Doxsee, C.L., Riter, T.R., Reiter, M.J., Gibson, S.J., Vasilakos, J.P.,
Kedl, R.M., 2003. The immune response modifier and Toll-like
receptor 7 agonist S-27609 selectively induces Il-12 and TNF-alpha
production in CD11c+CD11b+CD8-dendritic cells. J. Immunol. 171
Dzionek, A., Fuchs, A., Schmidt, P., Cremer, S., Zysk, M., Miltenyi, S.,
Buck, D.W., Schmitz, J., 2000. BDCA-2, BDCA-3, and BDCA-4: three
markers for distinct subsets of dendritic cells in human peripheral blood.
J. Immunol. 165 (11), 6037–6046.
Dzionek, A., Sohma, Y., Nagafune, J., Cella, M., Colonna, M., Facchetti, F.,
Gu ¨nther, G., Johnston, I., Lanzavecchia, A., Nagasaka, T., Okada, T.,
Vermi, W., Winkels, G., Yamamoto, T., Zysk, M., Yamaguchi, Y., Schmitz,
J., 2001. BDCA-2, a novel plasmacytoid dendritic cell-specific type II
C-lectin mediates antigen capture and is a potent inhibitor on interferon
a/h induction. J. Exp. Med. 194, 1823–1934.
Evans, L.A., Moreau, J., Odehouri, K., Legg, H., Barboza, A., Cheng-Mayer,
C., Levy, J.A., 1988. Characterization of a noncytopathic HIV-2 strain with
unusual effects on CD4 expression. Science 240, 1522–1525.
Feldman, S.G., Ferraro, M., Zheng, H.M., Patel, N., Gould-Fogerite, S.,
Fitzgerald-Bocarsly, P., 1994. Viral induction of low frequency interferon-
alpha producing cells. Virology 204 (1), 1–7.
Feldman, S., Stein, D., Amrute, S., Denny, T., Garcia, Z., Kloser, P., Sun, Y.,
Megjugorac, N., Fitzgerald-Bocarsly, P., 2001. Decreased interferon-alpha
production in HIV-infected patients correlates with numerical and
functional deficiencies in circulating type 2 dendritic cell precursors. Clin.
Immunol. 101 (2), 201–210.
Finke, J.S., Shodell, M., Shah, K., Siegal, F.P., Steinman, R.M., 2004. Dendritic
cell numbers in the blood of HIV-1 infected patients before and after
changes in antiretroviral therapy. J. Clin. Immunol. 24 (6), 647–652.
Fitzgerald-Bocarsly, P., 1993. Human natural interferon-alpha producing cells.
Pharmacol. Ther. 60 (1), 39–62.
Fitzgerald-Bocarsly, P., 2002. Natural interferon-a producing cells: the
plasmacytoid dendritic cells. BioTechniques 33, 16–29.
Fong, L., Mengozzi, M., Abbey, N.W., Herdier, B.G., Engleman, E.G., 2002.
Productive infection of plasmacytoid dendritic cells with human immu-
nodeficiency virus 1 is triggered by CD40 ligation. J. Virol. 76 (21),
Fonteneau, J.F., Larsson, M., Beignon, A.S., McKenna, K., Dasilva, I., Amara,
A., Liu, Y.J., Lifson, J.D., Littman, D.R., Bhardwaj, N., 2004. Human
immunodeficiency virus type 1 activates plasmacytoid dendritic cells and
concomitantly induces the bystander maturation of myeloid dendritic cells.
J. Virol. 78, 5223–5232.
Grouard, G., Rissoan, M.C., Filgueira, L., Durand, I., Banchereau, J., Liu, Y.J.,
B. Schmidt et al. / Virology 343 (2005) 256–266
1997. The enigmatic plasmacytoid T cells develop into dendritic cells with
interleukin (IL)-3 and CD40-ligand. J. Exp. Med. 185 (6), 1101–1111.
Hemmi, H., Takeuchi, O., Kawai, T., Kaisho, T., Sato, S., Sanjo, H.,
Matsumoto, M., Hoshino, K., Wagner, H., Takeda, K., Akira, S., 2000.
A toll-like receptor recognizes bacterial DNA. Nature 408, 740.
Hoffman, A.D., Banapour, B., Levy, J.A., 1985. Characterization of the AIDS-
associated retrovirus reverse transcriptase and optimal conditions for its
detection in virions. Virology 147, 326–335.
Kadowaki, N., Antonenko, S., Lau, J.Y., Liu, Y.J., 2000. Natural interferon
alpha/beta-producing cells link innate and adaptive immunity. J. Exp. Med.
192 (2), 219–226.
Krieg, A.M., 2002. CpG motifs in bacterial DNA and their immune defects.
Annual Review of Immunology 20, 709–760.
Kwon, D.S., Gregorio, G., Bitton, N., Hendrickson, W.A., Littman, D.R., 2002.
DC-SIGN-mediated internalization of HIV is required for trans-enhance-
ment of T cell infection. Immunity 16 (1), 135–144.
Latz, E., Schoenemeyer, A., Visintin, A., Fitzgerald, K.A., Monks, B.G.,
Knetter, C.F., Lien, E., Nilsen, N.J., Espevik, T., Golenbock, D.T., 2004.
TLR9 signals after translocating from the ER to CpG DNA in the lysosome.
Nat. Immunol. 5, 190–198.
Lebon, P., 1985. Inhibition of herpes simplex virus type 1-induced interferon
synthesis by monoclonal antibodies against viral glycoprotein D and by
lysosomotropic drugs. J. Gen. Virol. 66, 2781–2786.
Lee, J., Chuang, T.H., Redecke, V., She, L., Pitha, P.M., Carson, D.A., Raz, E.,
Cottam, H.B., 2003. Molecular basis for the immunostimulatory activity of
guanine nucleoside analogs: activation of Toll-like receptor 7. Proc. Natl.
Acad. Sci. U.S.A. 100, 6646–6651.
Levy, J.A., 2001. The importance of the innate immune system in controlling
HIV infection and disease. Trends Immunol. 22, 312–316.
Levy, J.A., Hoffman, A.D., Kramer, S.M., Landis, J.A., Shimabukuro, J.M.,
Oshiro, L.S., 1984. Isolation of lymphocytopathic retroviruses from San
Francisco patients with AIDS. Science 225, 840–842.
Levy, J.A., Hollander, H., Shimabukuro, J., Mills, J., Kaminsky, L., 1985.
Isolation of AIDS-associated retroviruses from cerebrospinal fluid and brain
of patients with neurological symptoms. Lancet, 586–588.
Liu, Y.J., 2001. Dendritic cell subsets and lineages, and their functions in innate
and adaptive immunity. Cell 106 (3), 259–262.
Lund, J., Sata, A., Akira, S., Medzhitov, R., A., I., 2003. Toll-like receptor 9-
mediated recognition of Herpes simplex virus-2 by plasmacytoid dendritic
cells. J. Exp. Med. 198 (3), 513–520.
Mackewicz, C.E., Ortega, H.W., Levy, J.A., 1991. CD8+ cell anti-HIVactivity
correlates with the clinical state of the infected individual. J. Clin. Invest.
Mackewicz, C.E., Barker, E., Levy, J.A., 1996. Role of h-chemokines in
suppressing HIV replication. Science 274, 1393–1395.
McDougal, J.S., Cort, S.P., Kennedy, M.S., Cabridilla, C.D., Feorino, P.M.,
Francis, D.P., Hicks, D., Kalyanaraman, V.S., Martin, L.S., 1985.
Immunoassay for the detection and quantitation of infectious human
retrovirus, lymphadenopathy-associated virus (LAV). J. Immunol. Methods
Pacanowski, J., Kahi, S., Baillet, M., Lebon, P., Deveau, C., Goujard, C.,
Meyer, L., Oksenhendler, E., Sinet, M., Hosmalin, A., 2001. Reduced blood
CD123+ (lymphoid) and CD11c+ (myeloid) dendritic cell numbers in
primary HIV-1 infection. Blood 98 (10), 3016–3021.
Patterson, S., Rae, A., Hockey, N., Gilmour, J., Gotch, F., 2001. Plasmacytoid
dendritic cells are highly susceptible to human immunodeficiency virus
type 1 infection and release infectious virus. J. Virol. 75 (14), 6710–6713.
Rayne, F., Vendeville, A., Bonhoure, A., Beaumelle, B., 2004. The ability of
chloroquine to prevent tat-induced cytokine secretion by monocytes is
implicated in its in vivo anti-human immunodeficiency virus type 1 activity.
J. Virol. 78 (21), 12054–12057.
Rissoan, M.C., Soumelis, V., Kadowaki, N., Grouard, G., Briere, F., de Waal
Malefyt, R., Liu, Y.J., 1999. Reciprocal control of T helper cell and
dendritic cell differentiation. Science 283 (5405), 1183–1186.
Rothenfusser, S., Hornung, V., Ayyoub, M., Britsch, S., Towarowski, A., Krug,
A., Sarris, A., Lubenow, N., Speiser, D., Endres, S., Hartmann, G., 2004.
CpG-A and CpG-B oligonucleotides differentially enhance human peptide-
specific primary and memory CD8+ T-cell responses in vitro. Blood 103
Schmidt, B., Ashlock, B.M., Neipel, F., Levy, J.A., 2004a. Potential screening
assay for undetectable viruses based on their capacity to induce type 1
interferon production. J. Clin. Microbiol. 42 (9), 4300–4302.
Schmidt, B., Scott, I., Whitmore, R.G., Foster, H., Fujimura, S., Schmitz, J.,
Levy, J.A., 2004b. Low-level HIVinfection of plasmacytoid dendritic cells:
onset of cytopathic effects and cell death after PDC maturation. Virology
Siegal, F.P., Kadowaki, N., Shodell,M., Fitzgerald-Bocarsly, P.A., Shah, K., Ho,
S., Antonenko, S., Liu, Y.J., 1999. The nature of the principal type 1
Soumelis, V., Scott, I., Gheyas, F., Bouhour, D., Cozon, G., Cotte, L.,
Huang, L., Levy, J., Liu, Y.J., 2001. Depletion of circulating natural type
1 interferon-producing cells in HIV-infected AIDS patients. Blood 98,
Tang, S., Levy, J.A., 1991. Inactivation of HIV-1 by trypsin and its use in
demonstrating specific virus infection of cells. J. Virol. Methods 33, 39–46.
Trubey, C.M., Chertova, E., Coren, L.V., Hilburn, J.M., Hixson, C.V.,
Nagashima, K., Lifson, J.D., Ott, D.E., 2003. Quantitation of HLA Class
II protein incorporated into human immunodeficiency Type 1 virions
purified by Anti-CD45 immunoaffinity depletion of microvesicles. J. Virol.
77 (23), 12699–12709.
Yonezawa, A., Morita, R., Takaori-Kondo, A., Kadowaki, N., Kitawaki, T.,
Hori, T., Uchiyama, T., 2003. Natural alpha interferon-producing cells
respond to human immunodeficiency virus type 1 with alpha interferon
production and maturation into dendritic cells. J. Virol. 77 (6), 3777–3784.
B. Schmidt et al. / Virology 343 (2005) 256–266