The JI
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     
 

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Sun, P.
Right arrow Articles by Burgess, T.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Sun, P.
Right arrow Articles by Burgess, T.
The Journal of Immunology, 2006, 177: 6497-6503.
Copyright © 2006 by The American Association of Immunologists, Inc.

CD40 Ligand Enhances Dengue Viral Infection of Dendritic Cells: A Possible Mechanism for T Cell-Mediated Immunopathology1,2

Peifang Sun*,¶, Christina M. Celluzzi§, Mary Marovich{ddagger},¶, Hemavathy Subramanian*,¶, Michael Eller, Susana Widjaja{dagger}, Dupeh Palmer{ddagger}, Kevin Porter*,¶, Wellington Sun{ddagger} and Timothy Burgess3,*,{dagger},¶

* Naval Medical Research Center, Silver Spring, MD 20910; {dagger} Naval Medical Research Unit 2, Jakarta, Indonesia; {ddagger} Walter Reed Army Institute of Research, Silver Spring, MD 20910; § University of Maryland School of Medicine, Baltimore, MD 21201; and Henry M. Jackson Foundation, Rockville, MD 20852


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
We have previously shown that dengue virus (DV) productively infects immature human dendritic cells (DCs) through binding to cell surface DC-specific ICAM-3-grabbing nonintegrin molecules. Infected DCs are apoptotic, refractory to TNF-{alpha} stimulation, inhibited from undergoing maturation, and unable to stimulate T cells. In this study, we show that maturation of infected DCs could be restored by a strong stimulus, CD40L. Addition of CD40L significantly reduced apoptosis of DCs, promoted IL-12 production, and greatly elevated the IFN-{gamma} response of T cells, but yet did not restore T cell proliferation in MLR. Increased viral infection of DCs was also observed; however, increased infection did not appear to be mediated by DC-specific ICAM-3-grabbing nonintegrin, but rather was regulated by decreased production of IFN-{alpha} and decreased apoptotic death of infected DCs. Because CD40L is highly expressed on activated memory (but not naive) T cells, the observation that CD40L signaling results in enhanced DV infection of DC suggests a possible T cell-dependent mechanism for the immune-mediated enhancement of disease severity associated with some secondary dengue infections.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Dengue viruses (DV)4 are single positive-stranded RNA flaviviruses consisting of four serologically distinct serotypes, DV-1, -2, -3, and -4. DV infections are self-limited and viremia usually resolves within a period of a few days (1). Infection results in clinical manifestations that range from mild dengue fever (DF) to a variety of symptoms of severe pathology characterized by capillary leakage known as dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS). Cases of DHF/DSS are commonly associated with heterologous secondary infection, and are thought to be responsible for 200,000–500,000 deaths each year (2).

Several studies demonstrated that dendritic cells (DCs) are more permissive to DV infection compared with monocytes and macrophages (3, 4). DC-specific ICAM-3-grabbing nonintegrin (DC-SIGN), a cell surface C-type lectin expressed on DCs, plays a critical role in DV binding and infection (5, 6). Inflammatory cytokines that induce DC maturation, such as TNF-{alpha} and IFN-{alpha} (7), can down-regulate DC-SIGN expression and thus render DCs less permissive to infection. Our previous study (8) showed that although DV exposure induces TNF-{alpha} production and maturation of bystander (uninfected) cells, it renders the infected DCs refractory to TNF-{alpha} stimulation, thereby blocking their maturation. Such effects on DC maturation are commonly observed among a number of DC-targeting viruses including HSV, Ebola and Lassa viruses, CMV, and HIV (9, 10, 11, 12, 13, 14). Maturation is essential for DC to become potent APCs. Therefore, interference with this process may be detrimental to immune responses and lead to viral immune invasion. We found that DV-exposed DCs diminish T cell proliferation in a MLR. Clinical studies have shown that DV infection causes APC dysfunction, which may account for the loss of mitogenic T cell proliferation and reduced blood lymphocyte counts observed in acutely infected patients (15, 16).

CD40/CD40L, members of the TNF and TNFR superfamily, play a critical role in modulating immune responses. CD40L is expressed mainly on activated T cells, while its receptor CD40 is expressed on a variety of accessory cells, including B cells, DCs, monocytes, macrophages, mast cells, fibroblasts, follicular DCs, and endothelial cells. In vivo, CD40/CD40L plays an important role in T cell priming, memory formation, and induction of a Th1 immune response (17). Using blocking Abs and murine gene knockout models, CD40/CD40L interaction was shown to be required for protective immunity in several parasitic, bacterial, and viral disease systems (17). In vitro ligation of CD40 on the surface of DCs by soluble CD40L or CD40L-transfected L cells (CD40L+-L cells) stimulates DCs to increase expression of costimulatory and MHC molecules, produce IL-12 and -15, and prevent apoptosis (18, 19, 20). Because of these properties, CD40L has been used to generate mature DCs as APCs to expand CD8+ CTL specific for viral and bacterial Ags (18, 21, 22, 23).

CD40L-induced signaling can be interrupted by viruses. Measles virus (MV) suppresses CD40L-induced DC maturation and production of proinflammatory cytokines, IL-12, and IL-1{alpha}beta (9, 24). MV replication is significantly higher in DCs treated with CD40L compared with untreated ones, resulting in increased DC apoptosis. Impaired T cell responses and T cell death in DC-T cell cocultures correlate with enhanced DC infection. HIV also shows more rigorous replication in DCs in the presence of CD40L signaling (25). Extensive deletion of T cells during infection is proposed to be a potential mechanism for systemic immunosuppression caused by MV and HIV infection. Therefore, CD40L, as it enhances viral replication and causes more T cell apoptosis, may play a role in viral immunopathogenesis.

In dengue, DHF and DSS are almost exclusively found in secondary infection (26, 27), when memory/effector T cells, elicited by primary infection, are already present (28). T cell responses, particularly inflammatory cytokine responses, have been suggested by Ennis and colleagues (27) to be "double-edged," by reducing viral replication in most circumstances but also mediating disease severity in others. In this study, we examined the effect of CD40L on DV infection of DC, maturation of DC, and activation of T cells. We found that CD40L enhanced DV infection of DC. CD40L restored DC maturation and IL-12 production. We also found that CD40L significantly elevated IFN-{gamma} response in T cells cocultured with DV-exposed DC; however, T cell proliferation did not correlate with IFN-{gamma} response. Together these results suggest a potential mechanism of T cell-mediated immunopathogenesis through CD40-CD40L costimulation.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Virus preparation

DV-2 (strain 16803, originally isolated by S. Halstead et al., and maintained at the Walter Reed Army Institute of Research) was grown and propagated in mycoplasma-free Vero cell lines. The viral titer was determined by limiting dilution plaque assays on Vero cells. The presence of contaminating LPS in the virus stock and culture supernatants was evaluated by the Limulus Amebocyte Lysate test (BioWhittaker). Mycoplasma contamination of the virus stocks and culture supernatants was evaluated by the Mycoplasma Rapid Detection System (Gen-Probe). All virus stocks and culture supernatants used in the present study were free from LPS and mycoplasma.

Generation of DCs

PBMCs were isolated from whole blood units obtained from normal, healthy, seronegative consenting deidentified donors at the American Red Cross. All cell cultures were conducted in complete medium consisting of RPMI 1640, 1% L-glutamine, 1% penicillin/streptomycin, 1% essential amino acids, and 10% heat-inactivated FBS (all from Invitrogen Life Technologies). DCs were generated according to a protocol described elsewhere (4). Briefly, monocytes were obtained by adhering PBMCs in Primaria tissue culture dishes (100 x 20 mm, Falcon; BD Labware) at 5 x 107 cells/dish for 2 h, followed by vigorous washing to remove nonadherent cells. The nonadherent cells were cryopreserved for T cell isolation, whereas the adherent cells were further cultured in the presence of recombinant cytokines, 100 ng/ml recombinant human (rh) GM-CSF (Leukine; Immunex) and 50 ng/ml rhIL-4 (R&D Systems). Cultures were maintained in a 37°C, 5% CO2 humidified incubator. On day 3, half of the medium was replaced with fresh medium supplemented with cytokines. At day 5, cells were harvested by gentle pipetting and placed into fresh medium and cytokines, and seeded into 6-well plates (Corning). At day 7, cells were identified to be typical immature DC as indicated by the presence of characteristic cell surface markers (CD11chigh, CD1high, CD14 negative, CD40 positive, CD83 negative, CD80, and CD86int). DCs generated using this procedure had ≥95% purity.

Infection of DCs with DV

Immature DCs were pulsed with DV-2 at various multiplicities of infection (MOIs) for 2 h at 37°C in a 5% CO2 atmosphere. Cells were washed twice to remove cell-free virus and cultured further in complete medium at a density of 0.5 x 106 cells/ml in 12-well plates without cytokine. In some experiments, irradiated L cells transfected to express CD40L (CD40L+-L cells) were added to DCs at ratio of 1:5 or 1:10 of DC:CD40L+-L. DCs and culture supernatants were harvested at indicated culture periods (24 or 48 h) for assay analyses.

CD40L+-L cells and CD40L blocking Ab

The CD40L-transfected L cell line (CD40L+-L cell) was provided by Dr. A. Granelli-Piperno (Rockefeller University, New York, NY) and anti-CD40L mAb (M90) was provided by Amgen. CD40L+-L were gently treated with 2 mM EDTA and irradiated at 10,000 rad using a cobalt irradiator. For blocking CD40-CD40L interaction, mAb M90 was incubated with irradiated CD40L L cells for 1 h at 4°C before coculture with DCs. Mouse IgG1 mAb (clone MOPC) (BD Pharmingen) was included as an irrelevant isotype control Ab.

DC surface marker phenotyping

For measurement of cell surface expression of costimulatory and HLA molecules, DCs were stained with PE-conjugated mAbs specific for: CD40 and CD83 (Coulter Immunotech), CD80 (clone L307.4), CD86 (clone IT2.2), HLA-A,B,C (clone G46-2.6), and HLA-DR (clone G46-6) (BD Pharmingen). Isotype-matched PE-labeled controls, mouse IgG1 (clone MOPC-21C), and mouse IgG2b (clone 27-35) (BD Pharmingen) were included in each experiment.

Detection of virus intracellularly and in cell supernatants

Surface marker-labeled DCs were fixed and permeabilized with Cytofix and CytoPerm (BD Pharmingen) according to the manufacturer’s recommendation, then stained with FITC-2H2, a mAb specific for dengue complex premembrane expressed intracellularly during viral replication. Fluorescently labeled samples were acquired on a FACScan flow cytometer (BD Pharmingen) with CellQuest software. During sample acquisition, infected cells were analyzed separately from uninfected ones by drawing distinctive gates on 2H2+ and 2H2– subsets. Virus titers produced by infected cells in culture supernatants were determined by plaque assay on Vero cells.

Analyses of cell apoptosis

The viability of DV-exposed DCs in the presence or absence of CD40L was compared at 24 and 48 h. Cells were stained with propidium iodide (PI) and FITC-conjugated annexin V using the Annexin VFITC Apoptosis Detection kit I (BD Pharmingen). Nuclear matrix protein release was measured by an ELISA kit according to the manufacturer’s instruction (BD Pharmingen).

Cytokine production in culture supernatants

IFN-{alpha} was measured by ELISA according to the manufacturer’s recommendations (PBL Biomedical Laboratories). Samples were read on a SpectraMax 340 plate reader (Molecular Devices). All other cytokines were quantified by cytometric bead array (CBA) with inflammatory or Th1/Th2 CBA kits on a FACScan using CBA software (BD Pharmingen).

T lymphocyte stimulation

DCs were exposed for 24 h to live DV-2 at indicated MOI or with 10 µg/ml Staphylococcus aureus Cowan strain (Calbiochem). T cells were negatively selected from PBMCs by magnetic microbeads using a Pan-T cell isolation kit (Miltenyi Biotec), and cocultured with differently treated DCs (DCs unexposed or exposed to DV in the presence or absence of CD40L) in triplicate, in complete RPMI 1640 medium containing 5% of low endotoxin FBS (HyClone) for 5 days at DC:T ratios of 1:3. Proliferation was measured by adding 0.5 µCi [3H]thymidine/well for the last 18 h of culture. Radioisotope incorporation was assessed using a 1450 Microbeta Liquid Scintillation and Luminescence Counter (PerkinElmer Life Sciences). Supernatants of T cell and DC cocultures were collected at different time points and IFN-{gamma} was measured.

Statistical analysis

The paired t test was used to determine the significance of differences between experimental and control groups. A p value <0.05 was considered significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
DV infection in the presence of CD40L

To determine whether CD40L affected infection, DCs were exposed to DV at different MOIs in the presence or absence of CD40L+-L cells at ratios of 1:5 and 1:50 DC:L cells. FITC-2H2 staining revealed an elevated percentage (>2-fold) of DV-infected cells at 48 h in CD40L+-L cell-treated DCs compared with untreated cells (Fig. 1A). CD40L-treated cultures expressed significantly higher 2H2+ mean fluorescence intensity (MFI) compared with untreated cells (Fig. 1B), indicating the amount of intracellular premembrane expression among the infected cells was elevated by CD40L treatment. Infectious viruses secreted in the culture supernatants were quantified using a Vero cell plaque assay. An increase of >1 log PFUs in the supernatant of CD40L-treated cells was observed (Fig. 1C). These results indicate that CD40L significantly increases DV infection of DCs. Enhancement was dose dependent: the more CD40L provided, the greater the observed infection. Enhancement of infection could be blocked by addition of M90, a mAb specific to CD40L (Fig. 1D), but not by the isotype control Ab (data not shown).


Figure 1
View larger version (29K):
[in this window]
[in a new window]
  		FIGURE 1. CD40L enhancement of DV infection: immature DCs were pulsed with DV at MOI of 0.3 and 1 for 2 h. Cells pulsed with noninfected Vero cell supernatants were used as mock controls. DCs were washed to remove cell-free viruses and cultured in the presence or absence of CD40L+-L cells. Two concentrations of CD40L+-L cells were used: 1 L cell/50 DC (low) and 1 L cell/5 DC (high). At 48 h, DCs were collected to measure infection with 2H2-FITC by flow cytometry. Culture supernatants were harvested to determine the titer of infectious viruses in a Vero cell plaque assay. Data are presented as mean ± SEM (n = 3). The unpaired Student t test was performed to determine the significance of the difference between presence/absence of CD40L treatment: A, Percentage of 2H2+ cells (p = 0.029 and 0.016 at MOI of 0.3 and 1, respectively); B, MFI of 2H2+ cells (p = 0.0001 and 0.0019 at MOI of 0.3 and 1, respectively); C, PFU of culture supernatants (p = 0.035 and 0.043 at M0I of 0.3 and 1, respectively); D, Percentage of 2H2+ cells following addition of blocking Ab to CD40L.

 
Effect of CD40L on DC-SIGN expression

DC-SIGN is thought to mediate, in part, the infection of immature DC by DV (5, 6). We previously showed that levels of DC-SIGN expression positively correlated with infection of immature DCs (5). In this study, we examined whether there was a correlation between DC-SIGN expression and infection when cells were exposed to CD40L. In our experiments (data not shown), DV-infected and mock control DCs cocultured with or without CD40L+-L cells were stained with PE-conjugated anti-DC-SIGN mAb. DCs kept in GM-CSF and IL-4 were used as the control, because DC-SIGN expression is dependent on IL-4 (7). At 24 h, DC-SIGN expression levels were lower on both mock-control and DV-exposed DCs compared with DCs cultured in IL-4 and GM-CSF; however, the difference was not significant (p > 0.05). Addition of DV and CD40L caused a further decrease in DC-SIGN levels, but the difference was not significant (p > 0.5). There appeared to be no direct correlation between DC-SIGN expression and levels of enhanced dengue infection.

Influence of CD40L on DC cytokine production

We previously reported that DV-exposed DC produce inflammatory cytokines, TNF-{alpha} and IFN-{alpha}, but fail to produce IL-12p70 (8). These cytokines can affect DC maturation and polarization of T cell immune responses. We sought to determine whether CD40L stimulation modulates the production of inflammatory cytokines by DC in the context of DV infection, thereby affecting DC infectivity and function. As CD40L treatment alone promoted cytokine production, the t test was performed to determine whether the changes in cytokine response in the presence of both CD40L and virus were significantly different from cytokine responses with CD40L alone. We show that DCs produced TNF-{alpha} in response to DV infection (Fig. 2A) as reported previously (8), and that CD40L further augmented this response. CD40L alone was able to promote TNF-{alpha} production in the absence of viral Ag, but this response was significantly higher in the presence of DV (p < 0.05). By contrast, CD40L treatment negatively affected the IFN-{alpha} response (Fig. 2B). IFN-{alpha} was not induced by CD40L alone. It was observed in DV-infected DCs at 48 h, but when CD40L was added to infected cells, the response was markedly reduced (p < 0.05).


Figure 2
View larger version (13K):
[in this window]
[in a new window]
  		FIGURE 2. TNF-{alpha} (A) and IL-12p70 (C) production of DV-infected cells is enhanced by CD40L, but IFN-{alpha} (B) response is decreased. DCs pulsed with or without DV were cultured in the presence or absence of CD40L+-L cells. Supernatants of cell cultures were harvested at 24 and 48 h. Cytokine production was measured by CBA and presented as mean ± SEM (n = 3). As CD40L alone was able to promote cytokine production, the Student t test was performed to determine whether the changes in cytokine responses in the presence of CD40L and virus differed significantly from CD40L alone. At 24 and 48 h, respectively, p = 0.021 and 0.041 for TNF-{alpha}, 0.052 and 0.011 for IL-12p70, and 0.009 for IFN-{alpha}.

 
The lack of IL-12p70 production by DCs after DV exposure that we and others previously reported was restored by CD40L stimulation. Fig. 2C shows that IL-12p70 was nearly undetectable (<6.3 pg/ml) in infected cells without CD40L stimulation, but when CD40L was added, the levels increased to >30 pg/ml by 24 h and >60 pg/ml by 48 h (Fig. 2C). CD40L alone also induced IL-12p70; however, the levels were significantly higher in response to DV infection (p < 0.05).

Decreased DC apoptosis in the presence of CD40L

CD40-CD40L costimulation is known to enhance DC survival through activation of antiapoptosis molecular pathways. We showed previously that DV-infected DCs are prone to apoptosis (8). Because apoptotic elimination of infected cells can serve as a mechanism to control infection (29), we examined the level of DV-induced apoptosis in the presence and absence of CD40L stimulation to determine whether the increased infection seen with CD40L treatment was due to CD40L-mediated "rescue" of DV-infected cells. FACS analysis showed annexin V/PI staining was lower in CD40L-treated DV-infected and uninfected DCs compared with untreated DCs (Fig. 3A and the table). Nuclear matrix proteins released in the supernatant were also lower in CD40L-treated cultures compared with untreated cultures (Fig. 3B). These data indicate that CD40L signaling is able to decrease cell apoptosis in DCs exposed to DV.


Figure 3
View larger version (33K):
[in this window]
[in a new window]
  		FIGURE 3. DC apoptosis: CD40L improves survival of DV-infected DCs. DCs pulsed with or without DV were cultured in the presence or absence of CD40L+-L cells. A, Cells were harvested at 24 h, and stained with annexin V and PI. Cell apoptosis was measure by FACS. B, Cell supernatants were harvested at 48 h to measure nuclear matrix protein release. Data are presented as mean ± SEM (n = 3).

 
Up-regulation of costimulation markers on DCs by CD40L

Exposure of DCs to DV induces maturation of bystander cells, but not infected cells. Unlike bystander cells, infected DCs are unable to undergo maturation in response to exogenous TNF-{alpha} treatment (8). In the current study, we show that following DV exposure, CD40L stimulation enhanced expression of costimulatory molecules on DCs. Fig. 4 shows that expression of CD80, CD83, and CD86 on the mock-control DCs as well as the DV-exposed DCs (including both the 2H2+ infected and the 2H2– bystander cells). The paired Student t tests indicted that without the CD40L stimulation, only the 2H2– cells had significant up-regulation of the three markers (p values not shown), confirming the reported observation (8). Significant up-regulation of all three markers were shown following CD40L treatment on the mock-control cells and the infected cells (tabled p values showing the differences between the CD40L untreated and treated). The up-regulation of the markers was not obvious on bystander cells after CD40L treatment, because bystander cells were already showing phenotypic maturation in the absence of CD40L treatment. Levels of expression of these molecules in CD40L treated cells were similar among 2H2+, 2H2–, and mock-infected controls, indicating that infected cells proceed to maturation with a similar magnitude as the bystander cells in the presence of DV infection.


Figure 4
View larger version (19K):
[in this window]
[in a new window]
  		FIGURE 4. CD40L enhances expression of costimulatory markers on DCs: DCs pulsed with or without (mock) DV were cultured in the presence or absence of CD40L+-L cells. Cells were harvested at 48 h, and stained with PE-labeled CD83, CD80, and CD86. Expression of CD83, CD80, and CD86 on infected (2H2+) and noninfected (2H2–) cells was measured by FACS analysis and shown as MFI. Data presented as mean ± SEM (n = 3). To show the effect of CD40L for DC phenotype maturation, the paired Student t test was performed to compare the difference between the CD40L-treated and the CD40L-untreated data groups. Values of p are shown in the table.

 
T cell proliferation

As reported previously, DV-exposed DCs have a reduced capacity to induce T cell proliferation in a MLR, likely due to the abrogated DC maturation induced by viral infection (8). Fig. 4 shows that with CD40L stimulation, infected DCs undergo phenotypic maturation just as bystander cells (2H2–) and viral-unexposed cells (mock control). As shown in Fig. 5, CD40L-treated and untreated DV-exposed and -unexposed DCs were used to stimulate MLR. The overall level of T cell proliferation increased with CD40L-treated DCs compared with nontreated ones; however, the DV-exposed DCs (at MOI 3 and 1) did not reach the response levels of mock-infected DCs.


Figure 5
View larger version (14K):
[in this window]
[in a new window]
  		FIGURE 5. CD40L improves T cell stimulation and enhances IFN-{gamma} production in DV-infected DCs. DCs pulsed with or without (mock) DV were cultured in the presence or absence of CD40L+-L cells. Cells were harvested at 24 h and washed. T cells were added to the DCs at a DC:T ratio of 1:3. Cell proliferation was measured by [3H]thymidine incorporation at day 6. Stimulation index = cpm of T cells + DC/cpm of T cells (n = 3).

 
CD40L induces Th1 type cytokine production

We showed in Fig. 3 that CD40L costimulation allows IL-12p70 production in response to DV infection. To evaluate the T cell cytokine response induced by CD40L-treated DCs, IFN-{gamma} production in MLR was measured. Surprisingly, although there was suppression of T cell proliferation, DV-infected DCs up-regulated IFN-{gamma} production (from 33.8 to 92.7 pg/ml at 72 h), indicating induction of a Th1 type of T cell response to DV infection (Fig. 6A). This response was greatly enhanced by CD40L treatment of DCs (Fig. 6B).


Figure 6
View larger version (14K):
[in this window]
[in a new window]
  		FIGURE 6. DV-exposed DCs induce IFN-{gamma} response in T cells, which is enhanced further by CD40L stimulation: DCs pulsed with or without DV were cultured in the presence (B) or absence of CD40L+-L cells (A). Cells were harvested at 24 h and washed. T cells were added to the DCs at DC:T ratio of 1:3. IFN-{gamma} was measured from culture supernatants and data presented as mean ± SEM (n = 3).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
DHF and DSS represent severe clinical manifestations of DV infection. Although the mechanism controlling development of DHF and DSS is complicated, one consistent observation is that the increased risk of DHF is associated with a secondary heterologous DV infection (27). Ab-dependent enhancement (ADE), increased viral infection caused by subneutralizing Abs, has been used to explain how pre-existing suboptimal DV-specific Abs increase DV infection. ADE has been demonstrated in vivo in infants born to DV seropositive mothers (30) and in vitro in peripheral leukocytes (26) as well as the monocytic cell line K562 (4). ADE, however, cannot be demonstrated with immature DCs (4). DCs are permissible to DV, possibly serving as primary targets for early infection. Enhancing infection of DCs via mechanisms similar to ADE could impact the course of disease in dengue infection.

This study is the first to show the enhancement of DV replication in DCs in the presence of CD40-CD40L signaling. DC-SIGN is a possible binding receptor for DV, and infectivity correlates with DC-SIGN expression levels on the cell surface (5). IL-4 is critical to the expression of DC-SIGN on immature DCs derived from monocytes (7). IFN-{alpha}beta and TNF-{alpha} can interfere with the IL-4-dependent Stat6 and JAK2/JAK3 gene transcription pathways (31), and thus can negatively regulate DC-SIGN expression (7, 32) during differentiation of monocytes. Our monocyte-derived DCs were generated in GM-CSF and IL-4 for ~6 days. At this time, DC-SIGN expression varied within individuals, from 100 to 900 MFI (data not shown). Consistent with other studies (32), we report production of IFN-{alpha} and TNF-{alpha} from DV-exposed DCs. CD40L treatment further increased TNF-{alpha} production, but decreased IFN-{alpha} response. The inflammatory cytokine milieu may down-regulate DC-SIGN expression among cells exposed to DV and CD40L. In fact, we did observe less DC-SIGN expression after DCs were exposed to DV. Not surprisingly, DC-SIGN levels were somewhat lower upon exposing DCs to CD40L (although the differences were not significant). In light of our previous work showing that infectivity of DCs with DV correlates directly with DC-SIGN expression (5), these results demonstrate that the increased infectivity observed with CD40L signaling is not related to changes in DC-SIGN expression induced by CD40L. We found that CD40L treatment decreases IFN-{alpha} secretion by DCs, and that effect correlates with increased DV infection, suggesting a potential mechanism whereby decreased IFN-{alpha} production in DV-exposed DCs receiving CD40L treatment may be responsible for reducing antiviral activity against dengue replication and survival. Diamond et al. (33) and others have shown that IFN-{alpha} inhibits DV infection and replication, while Garcia-Sarstre and colleagues (34, 35) and Jones et al. (36) have reported that DV inhibits IFN-{alpha} signaling. Interestingly, Ho et al. (37) found that DC are refractory to the viral inhibitory action of IFN-{alpha} but not IFN-{gamma} following DV infection. We are continuing to explore the role of the IFN response in the context of DV infection, in particular with respect to different strains of DV, and a future manuscript is forthcoming. In addition to the effects of type 1 IFN, apoptosis can serve as a mechanism to eliminate dengue-infected cells, thereby controlling virus spread (29). The decreased DC apoptosis in the presence of CD40L signaling demonstrated in this study may serve as another explanation for the observed enhancement of DV infection.

Before our study, rapid (within 2 h) increase in expression of HIV p24 and gag transgenes in DCs exposed to vCP205 (a canarypox vector inserted with g24 and gag genes) via CD40-CD40L signaling was observed (Dr. M. Marovich, unpublished observations). Unlike DV, MV and HIV, Canarypox is replication incompetent (38). Others have demonstrated an increase in transgenic protein expression in DCs transfected with naked DNA by engaging CD40 on cell surface (39), and suggested that the increased levels of NF-{kappa}B, AP-1, and cAMP-responsive element, are essential to elevated gene transcription (39). It is possible that different mechanisms, including the cytokine milieu, apoptosis, and signal transduction, may act in concert in different systems to promote viral replication or viral protein expression.

Infected DC, unlike bystanders (DV-exposed cells that remained uninfected following exposure), are incapable of undergoing maturation in response to DV (8). In this study, a significant phenotypic maturation of infected DCs following CD40L stimulation was observed. Expression of each individual maturation marker, CD80, CD83, and CD86, on mock-control, DV-infected, and bystander DCs reached nearly the same level. We previously reported that addition of TNF-{alpha} to DV-exposed DCs only increased maturation of bystander cells, but not infected cells (8). Therefore, maturation of infected cells may not due to the increased production of TNF-{alpha} elicited by CD40L. The NF-{kappa}B complex is required for DC maturation (40). Recently, O’Sullivan et al. (40) reported striking differences between TNF-{alpha} and CD40L for induction of gene transcription of NF-{kappa}B and NF-{kappa}B complex: TNF-{alpha} induces a rapid transcription quickly followed by attenuation, whereas CD40L induces a slower, higher, and sustained transcription. This suggests that CD40L, compared with TNF-{alpha}, delivers a more potent DC maturation signal under conditions tested in this study. This also may explain why CD40L and TNF-{alpha} have unequal effects on DV-infected DCs.

IL-12 is not produced by DV-exposed DCs (8). CD40L is a potent signal, which restored DC IL-12 response to DV. IL-12p70 was induced by CD40L-treated DCs and was further increased by DV exposure. TLR3, a pattern recognition molecule specific for dsDNA and a synthetic analog of dsRNA, poly(I:C), is located intracellularly in monocyte-derived immature DCs (41). Activating DCs through TLR3 gives rise to IFN-{alpha}beta and IL-12 (42). Because DV replication causes accumulation of dsRNA, it is logical that DV should induce both cytokines. However, without extra stimuli, DV infection of DCs induces only IFN-{alpha} but not IL-12 (8), suggesting the possible pathway of TLR-3-mediated IL-12 production is hindered. DC-SIGN has been proposed to play a negative role in DC stimulation (43, 44, 45). Bacterial Ags that bind and are internalized through DC-SIGN are capable of diverting an IL-12 response to an IL-10 response (46). Our data suggest that DV may act through similar mechanism. In the presence of CD40L, the IL-12 response to DV is restored. Libraty et al. (47) observed restoration of IL-12 response to DV in the presence of exogenous IFN-{gamma}. This may indicate that DV is capable of eliciting a proinflammatory response if proper help from T cells is provided, and may also suggest that the inhibitory signal from DC-SIGN can be overcome by costimulation.

We previously demonstrated a suppressive effect of DV-exposed DCs on T cell proliferation and suggested that the APCs comprised proportionally of immature infected cells are impaired in their ability to stimulate allogeneic T cell proliferation (8). Here, we show that CD40L-treated DCs increased T cell proliferation in MLR. As significant phenotypic maturation was achieved by CD40L stimulation on all DC subpopulations, including those infected by DV; overall, increased T cell proliferation may be related to better DC-T cell costimulation. However, CD40L-induced phenotypic maturation of DV-exposed DCs did not fully restore T cell proliferation, suggesting DV-exposed DCs are still less functional than unexposed cells. Our data tend to support the in vivo observation showing that stimulation of functionally impaired accessory cells from dengue patients by engaging cell surface CD28 cannot restore mitogenic T cell proliferation (15). This suggests that more factors, other than costimulation, are involved in impairing APC function and diminishing T cell activity. With MV and HIV, infection can be transmitted from infected DCs to T cells through syncytia formation, resulting in a profound T cell apoptosis of not only infected, but also uninfected cells (9, 48). DV-induced apoptosis may also explain why some patients with DF and DHF have lower lymphocyte and monocyte counts in their peripheral blood (16, 27). Currently, we are studying issues related to activation-induced cell death using this in vitro DC model.

We showed a marked enhancement of IFN-{gamma} from T cells in response to DV-exposed DCs by CD40-CD40L signaling. Like other inflammatory cytokines, IFN-{gamma} is suggested to contribute to protective immunity due to its antiviral effects (49), and it is generally thought to correlate with long-term immunological memory.

Primary infection with dengue leads to long-term immunity against the homologous serotype. Severe disease emerges during secondary infections with heterologous serotypes. During a secondary infection, memory T cells generated by a primary infection (28) are likely to be activated by the infecting second strain of virus through cross-reacting epitopes. These cells may be activated even more vigorously upon virus re-exposure than naive T cells because they bear memory phenotypes. However, as suggested by others (27, 50), variant peptides from a heterologous virus can act as partial agonists to alter or weaken the function of these cross-reactive T cells. It has been demonstrated that the HLA-A11-restricted NS3 peptide-specific CD8+ T cells appear to bind weakly to peptides of the current serotype, but more strongly to the peptide variants presumably from the previously encountered virus. A high frequency of these T cells do not give IFN-{gamma} ELISPOT responses and display an apoptotic phenotype (51) during the acute phase of secondary infection. These cells are also found more abundantly than those of current strain-specific cells. In a separate study, Rothman and colleagues (52) showed that a variant T cell epitope of NS3 from DV-2 was able to weaken IFN-{gamma} responses of effector CD8+ T cells generated from a previous DV-3 infection. These studies suggest that secondary infections may activate cross-reactive T cells to express CD40L without proper antiviral function. We hypothesize that these T cells will not inhibit viral replication, but enhance infection through CD40L. In fact, higher viral titers during acute infection are associated with high risk of DHF (53), suggesting enhancement of infection is critical to altering the disease course.

In conclusion, we have demonstrated that CD40L enhances DV infection in DCs possibly through regulation of IFN-{alpha} secretion and apoptosis. CD40L stimulation restored IL-12 production from DV-exposed DCs and markedly enhanced IFN-{gamma} response from T cells stimulated with DV-infected DC. However, it was not sufficient to restore T cell proliferation from the suppression caused by DV-infected cells.


   Acknowledgments
 
T. Burgess is a military service member. This work was prepared as part of his official duties.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by U.S. Navy Work Unit Number 61102A.870.S.A0015 and by U.S. Army Work Unit DAMD17-02-0005. Back

2 The views expressed in this article are those of the authors and do not necessarily reflect the official policy or position of the Department of the Navy, Department of Defense, nor the U.S. government. Back

3 Address correspondence and reprint requests to Dr. Timothy Burgess, Naval Medical Research Unit 2, FPO AP 96520-8132. E-mail address: burgess{at}namru2.org Back

4 Abbreviations used in this paper: DV, dengue virus; DF, dengue fever; DHF, dengue hemorrhagic fever; DSS, dengue shock syndrome; DC, dendritic cell; MV, measles virus; rh, recombinant human; MOI, multiplicity of infection; PI, propidium iodide; CBA, cytometric bead array; MFI, mean fluorescence intensity; ADE, Ab-dependent enhancement; DC-SIGN, DC-specific ICAM-3-grabbing nonintegrin. Back

Received for publication November 1, 2005. Accepted for publication July 27, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

  1. Burke, D. S., A. Nisalak, D. E. Johnson, R. M. Scott. 1988. A prospective study of dengue infections in Bangkok. Am. J. Trop. Med. Hyg. 38: 172-180. [Abstract/Free Full Text]
  2. Halstead, S. B.. 1988. Pathogenesis of dengue: challenges to molecular biology. Science 239: 476-481. [Abstract/Free Full Text]
  3. Wu, S. J., G. Grouard-Vogel, W. Sun, J. R. Mascola, E. Brachtel, R. Putvatana, M. K. Louder, L. Filgueira, M. A. Marovich, H. K. Wong, et al 2000. Human skin Langerhans cells are targets of dengue virus infection. Nat. Med. 6: 816-820. [Medline]
  4. Marovich, M., G. Grouard-Vogel, M. Louder, M. Eller, W. Sun, S. J. Wu, R. Putvatana, G. Murphy, B. Tassaneetrithep, T. Burgess, et al 2001. Human dendritic cells as targets of dengue virus infection. J. Investig. Dermatol. Symp. Proc. 6: 219-224. [Medline]
  5. Tassaneetrithep, B., T. H. Burgess, A. Granelli-Piperno, C. Trumpfheller, J. Finke, W. Sun, M. A. Eller, K. Pattanapanyasat, S. Sarasombath, D. L. Birx, et al 2003. DC-SIGN (CD209) mediates dengue virus infection of human dendritic cells. J. Exp. Med. 197: 823-829. [Abstract/Free Full Text]
  6. Navarro-Sanchez, E., R. Altmeyer, A. Amara, O. Schwartz, F. Fieschi, J. L. Virelizier, F. Arenzana-Seisdedos, P. Despres. 2003. Dendritic-cell-specific ICAM3-grabbing non-integrin is essential for the productive infection of human dendritic cells by mosquito-cell-derived dengue viruses. EMBO Rep. 4: 723-728. [Medline]
  7. Relloso, M., A. Puig-Kroger, O. M. Pello, J. L. Rodriguez-Fernandez, G. de la Rosa, N. Longo, J. Navarro, M. A. Munoz-Fernandez, P. Sanchez-Mateos, A. L. Corbi. 2002. DC-SIGN (CD209) expression is IL-4 dependent and is negatively regulated by IFN, TGF-beta, and anti-inflammatory agents. J. Immunol. 168: 2634-2643. [Abstract/Free Full Text]
  8. Palmer, D. R., P. Sun, C. Celluzzi, J. Bisbing, S. Pang, W. Sun, M. A. Marovich, T. Burgess. 2005. Differential effects of dengue virus on infected and bystander dendritic cells. J. Virol. 79: 2432-2439. [Abstract/Free Full Text]
  9. Fugier-Vivier, I., C. Servet-Delprat, P. Rivailler, M. C. Rissoan, Y. J. Liu, C. Rabourdin-Combe. 1997. Measles virus suppresses cell-mediated immunity by interfering with the survival and functions of dendritic and T cells. J. Exp. Med. 186: 813-823. [Abstract/Free Full Text]
  10. Mahanty, S., K. Hutchinson, S. Agarwal, M. McRae, P. E. Rollin, B. Pulendran. 2003. Cutting edge: impairment of dendritic cells and adaptive immunity by Ebola and Lassa viruses. J. Immunol. 170: 2797-2801. [Abstract/Free Full Text]
  11. Granelli-Piperno, A., A. Golebiowska, C. Trumpfheller, F. P. Siegal, R. M. Steinman. 2004. HIV-1-infected monocyte-derived dendritic cells do not undergo maturation but can elicit IL-10 production and T cell regulation. Proc. Natl. Acad. Sci. USA 101: 7669-7674. [Abstract/Free Full Text]
  12. Kawamura, T., H. Gatanaga, D. L. Borris, M. Connors, H. Mitsuya, A. Blauvelt. 2003. Decreased stimulation of CD4+ T cell proliferation and IL-2 production by highly enriched populations of HIV-infected dendritic cells. J. Immunol. 170: 4260-4266. [Abstract/Free Full Text]
  13. Pollara, G., K. Speidel, L. Samady, M. Rajpopat, Y. McGrath, J. Ledermann, R. S. Coffin, D. R. Katz, B. Chain. 2003. Herpes simplex virus infection of dendritic cells: balance among activation, inhibition, and immunity. J. Infect. Dis. 187: 165-178. [Medline]
  14. Varani, S., G. Frascaroli, M. Homman-Loudiyi, S. Feld, M. P. Landini, C. Soderberg-Naucler. 2005. Human cytomegalovirus inhibits the migration of immature dendritic cells by down-regulating cell-surface CCR1 and CCR5. J. Leukocyte Biol. 77: 219-228. [Abstract/Free Full Text]
  15. Hermans, I. F., J. D. Silk, U. Gileadi, M. Salio, B. Mathew, G. Ritter, R. Schmidt, A. L. Harris, L. Old, V. Cerundolo. 2003. NKT cells enhance CD4+ and CD8+ T cell responses to soluble antigen in vivo through direct interaction with dendritic cells. J. Immunol. 171: 5140-5147. [Abstract/Free Full Text]
  16. Green, S., S. Pichyangkul, D. W. Vaughn, S. Kalayanarooj, S. Nimmannitya, A. Nisalak, I. Kurane, A. L. Rothman, F. A. Ennis. 1999. Early CD69 expression on peripheral blood lymphocytes from children with dengue hemorrhagic fever. J. Infect. Dis. 180: 1429-1435. [Medline]
  17. Grewal, I. S., R. A. Flavell. 1998. CD40 and CD154 in cell-mediated immunity. Annu. Rev. Immunol. 16: 111-135. [Medline]
  18. Yu, Q., J. X. Gu, C. Kovacs, J. Freedman, E. K. Thomas, M. A. Ostrowski. 2003. Cooperation of TNF family members CD40 ligand, receptor activator of NF-{kappa}B ligand, and TNF-{alpha} in the activation of dendritic cells and the expansion of viral specific CD8+ T cell memory responses in HIV-1-infected and HIV-1-uninfected individuals. J. Immunol. 170: 1797-1805. [Abstract/Free Full Text]
  19. Brossart, P., A. Zobywalski, F. Grunebach, L. Behnke, G. Stuhler, V. L. Reichardt, L. Kanz, W. Brugger. 2000. Tumor necrosis factor {alpha} and CD40 ligand antagonize the inhibitory effects of interleukin 10 on T-cell stimulatory capacity of dendritic cells. Cancer Res. 60: 4485-4492. [Abstract/Free Full Text]
  20. Cella, M., D. Scheidegger, K. Palmer-Lehmann, P. Lane, A. Lanzavecchia, G. Alber. 1996. Ligation of CD40 on dendritic cells triggers production of high levels of interleukin-12 and enhances T cell stimulatory capacity: T-T help via APC activation. J. Exp. Med. 184: 747-752. [Abstract/Free Full Text]
  21. Steinman, R. M., M. Pope. 2002. Exploiting dendritic cells to improve vaccine efficacy. J. Clin. Invest. 109: 1519-1526. [Medline]
  22. Samten, B., B. Wizel, H. Shams, S. E. Weis, P. Klucar, S. Wu, R. Vankayalapati, E. K. Thomas, S. Okada, A. M. Krensky, P. F. Barnes. 2003. CD40 ligand trimer enhances the response of CD8+ T cells to Mycobacterium tuberculosis. J. Immunol. 170: 3180-3186. [Abstract/Free Full Text]
  23. Ostrowski, M. A., S. J. Justement, L. Ehler, S. B. Mizell, S. Lui, J. Mican, B. D. Walker, E. K. Thomas, R. Seder, A. S. Fauci. 2000. The role of CD4+ T cell help and CD40 ligand in the in vitro expansion of HIV-1-specific memory cytotoxic CD8+ T cell responses. J. Immunol. 165: 6133-6141. [Abstract/Free Full Text]
  24. Servet-Delprat, C., P. O. Vidalain, H. Bausinger, S. Manie, F. Le Deist, O. Azocar, D. Hanau, A. Fischer, C. Rabourdin-Combe. 2000. Measles virus induces abnormal differentiation of CD40 ligand-activated human dendritic cells. J. Immunol. 164: 1753-1760. [Abstract/Free Full Text]
  25. Kawamura, T., M. Qualbani, E. K. Thomas, J. M. Orenstein, A. Blauvelt. 2001. Low levels of productive HIV infection in Langerhans cell-like dendritic cells differentiated in the presence of TGF-beta1 and increased viral replication with CD40 ligand-induced maturation. Eur. J. Immunol. 31: 360-368. [Medline]
  26. Halstead, S. B., E. J. O’Rourke. 1977. Dengue viruses and mononuclear phagocytes. I. Infection enhancement by non-neutralizing antibody. J. Exp. Med. 146: 201-217. [Abstract/Free Full Text]
  27. Pichyangkul, S., T. P. Endy, S. Kalayanarooj, A. Nisalak, K. Yongvanitchit, S. Green, A. L. Rothman, F. A. Ennis, D. H. Libraty. 2003. A blunted blood plasmacytoid dendritic cell response to an acute systemic viral infection is associated with increased disease severity. J. Immunol. 171: 5571-5578. [Abstract/Free Full Text]
  28. Sierra, B., G. Garcia, A. B. Perez, L. Morier, R. Rodriguez, M. Alvarez, M. G. Guzman. 2002. Long-term memory cellular immune response to dengue virus after a natural primary infection. Int. J. Infect. Dis. 6: 125-128. [Medline]
  29. Clem, R. J.. 2005. The role of apoptosis in defense against baculovirus infection in insects. Curr. Top. Microbiol. Immunol. 289: 113-129. [Medline]
  30. Kliks, S. C., S. Nimmanitya, A. Nisalak, D. S. Burke. 1988. Evidence that maternal dengue antibodies are important in the development of dengue hemorrhagic fever in infants. Am. J. Trop. Med. Hyg. 38: 411-419. [Abstract/Free Full Text]
  31. Dickensheets, H. L., R. P. Donnelly. 1999. Inhibition of IL-4-inducible gene expression in human monocytes by type I and type II interferons. J. Leukocyte Biol. 65: 307-312. [Abstract]
  32. Puig-Kroger, A., D. Serrano-Gomez, E. Caparros, A. Dominguez-Soto, M. Relloso, M. Colmenares, L. Martinez-Munoz, N. Longo, N. Sanchez-Sanchez, M. Rincon, et al 2004. Regulated expression of the pathogen receptor dendritic cell-specific intercellular adhesion molecule 3 (ICAM-3)-grabbing nonintegrin in THP-1 human leukemic cells, monocytes, and macrophages. J. Biol. Chem. 279: 25680-25688. [Abstract/Free Full Text]
  33. Diamond, M. S., T. G. Roberts, D. Edgil, B. Lu, J. Ernst, E. Harris. 2000. Modulation of Dengue virus infection in human cells by {alpha}, beta, and {gamma} interferons. J. Virol. 74: 4957-4966. [Abstract/Free Full Text]
  34. Munoz-Jordan, J. L., G. G. Sanchez-Burgos, M. Laurent-Rolle, A. Garcia-Sastre. 2003. Inhibition of interferon signaling by dengue virus. Proc. Natl. Acad. Sci. USA 100: 14333-14338. [Abstract/Free Full Text]
  35. Munoz-Jordan, J. L., M. Laurent-Rolle, J. Ashour, L. Martinez-Sobrido, M. Ashok, W. I. Lipkin, A. Garcia-Sastre. 2005. Inhibition of {alpha}/beta interferon signaling by the NS4B protein of flaviviruses. J. Virol. 79: 8004-8013. [Abstract/Free Full Text]
  36. Jones, M., A. Davidson, L. Hibbert, P. Gruenwald, J. Schlaak, S. Ball, G. R. Foster, M. Jacobs. 2005. Dengue virus inhibits {alpha} interferon signaling by reducing STAT2 expression. J. Virol. 79: 5414-5420. [Abstract/Free Full Text]
  37. Ho, L. J., J. J. Wang, M. F. Shaio, C. L. Kao, D. M. Chang, S. W. Han, J. H. Lai. 2001. Infection of human dendritic cells by dengue virus causes cell maturation and cytokine production. J. Immunol. 166: 1499-1506. [Abstract/Free Full Text]
  38. Ignatius, R., M. Marovich, E. Mehlhop, L. Villamide, K. Mahnke, W. I. Cox, F. Isdell, S. S. Frankel, J. R. Mascola, R. M. Steinman, M. Pope. 2000. Canarypox virus-induced maturation of dendritic cells is mediated by apoptotic cell death and tumor necrosis factor {alpha} secretion. J. Virol. 74: 11329-11338. [Abstract/Free Full Text]
  39. Larregina, A. T., A. E. Morelli, O. Tkacheva, G. Erdos, C. Donahue, S. C. Watkins, A. W. Thomson, L. D. Falo, Jr. 2004. Highly efficient expression of transgenic proteins by naked DNA-transfected dendritic cells through terminal differentiation. Blood 103: 811-819. [Abstract/Free Full Text]
  40. O’Sullivan, B. J., R. Thomas. 2002. CD40 ligation conditions dendritic cell antigen-presenting function through sustained activation of NF-{kappa}B. J. Immunol. 168: 5491-5498. [Abstract/Free Full Text]
  41. Matsumoto, M., K. Funami, M. Tanabe, H. Oshiumi, M. Shingai, Y. Seto, A. Yamamoto, T. Seya. 2003. Subcellular localization of Toll-like receptor 3 in human dendritic cells. J. Immunol. 171: 3154-3162. [Abstract/Free Full Text]
  42. Agrawal, S., A. Agrawal, B. Doughty, A. Gerwitz, J. Blenis, T. Van Dyke, B. Pulendran. 2003. Cutting edge: different Toll-like receptor agonists instruct dendritic cells to induce distinct Th responses via differential modulation of extracellular signal-regulated kinase-mitogen-activated protein kinase and c-Fos. J. Immunol. 171: 4984-4989. [Abstract/Free Full Text]
  43. Geijtenbeek, T. B., S. J. Van Vliet, E. A. Koppel, M. Sanchez-Hernandez, C. M. Vandenbroucke-Grauls, B. Appelmelk, Y. Van Kooyk. 2003. Mycobacteria target DC-SIGN to suppress dendritic cell function. J. Exp. Med. 197: 7-17. [Abstract/Free Full Text]
  44. Ludwig, I. S., A. N. Lekkerkerker, E. Depla, F. Bosman, R. J. Musters, S. Depraetere, Y. van Kooyk, T. B. Geijtenbeek. 2004. Hepatitis C virus targets DC-SIGN and L-SIGN to escape lysosomal degradation. J. Virol. 78: 8322-8332. [Abstract/Free Full Text]
  45. van Kooyk, Y., B. Appelmelk, T. B. Geijtenbeek. 2003. A fatal attraction: Mycobacterium tuberculosis and HIV-1 target DC-SIGN to escape immune surveillance. Trends Mol. Med. 9: 153-159. [Medline]
  46. Okano, M., A. R. Satoskar, K. Nishizaki, M. Abe, D. A. Harn, Jr. 1999. Induction of Th2 responses and IgE is largely due to carbohydrates functioning as adjuvants on Schistosoma mansoni egg antigens. J. Immunol. 163: 6712-6717. [Abstract/Free Full Text]
  47. Libraty, D. H., S. Pichyangkul, C. Ajariyakhajorn, T. P. Endy, F. A. Ennis. 2001. Human dendritic cells are activated by dengue virus infection: enhancement by {gamma} interferon and implications for disease pathogenesis. J. Virol. 75: 3501-3508. [Abstract/Free Full Text]
  48. Granelli-Piperno, A., V. Finkel, E. Delgado, R. M. Steinman. 1999. Virus replication begins in dendritic cells during the transmission of HIV-1 from mature dendritic cells to T cells. Curr. Biol. 9: 21-29. [Medline]
  49. Kakimi, K.. 2003. Immune-based novel therapies for chronic hepatitis C virus infection. Hum. Cell 16: 191-197. [Medline]
  50. Sloan-Lancaster, J., P. M. Allen. 1996. Altered peptide ligand-induced partial T cell activation: molecular mechanisms and role in T cell biology. Annu. Rev. Immunol. 14: 1-27. [Medline]
  51. Mongkolsapaya, J., W. Dejnirattisai, X. N. Xu, S. Vasanawathana, N. Tangthawornchaikul, A. Chairunsri, S. Sawasdivorn, T. Duangchinda, T. Dong, S. Rowland-Jones, et al 2003. Original antigenic sin and apoptosis in the pathogenesis of dengue hemorrhagic fever. Nat. Med. 9: 921-927. [Medline]
  52. Zivny, J., M. DeFronzo, W. Jarry, J. Jameson, J. Cruz, F. A. Ennis, A. L. Rothman. 1999. Partial agonist effect influences the CTL response to a heterologous dengue virus serotype. J. Immunol. 163: 2754-2760. [Abstract/Free Full Text]
  53. Vaughn, D. W., S. Green, S. Kalayanarooj, B. L. Innis, S. Nimmannitya, S. Suntayakorn, T. P. Endy, B. Raengsakulrach, A. L. Rothman, F. A. Ennis, A. Nisalak. 2000. Dengue viremia titer, antibody response pattern, and virus serotype correlate with disease severity. J. Infect. Dis. 181: 2-9. [Medline]




This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Sun, P.
Right arrow Articles by Burgess, T.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Sun, P.
Right arrow Articles by Burgess, T.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS