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IMMUNOBIOLOGY
From the Microbiology and Tumor Biology Center,
Karolinska Institutet, Stockholm, Sweden; the School of Biological
Sciences, University of Missouri, Kansas City; and the Department of
Pathology and Microbiology, School of Medical Sciences, University of
Bristol, United Kingdom.
Epstein-Barr virus (EBV) is a tumorigenic human herpesvirus that
persists for life in healthy immunocompetent carriers. The viral
strategies that prevent its clearance and allow reactivation in the
face of persistent immunity are not well understood. Here we
demonstrate that EBV infection of monocytes inhibits their development
into dendritic cells (DCs), leading to an abnormal cellular response to
granulocyte macrophage-colony-stimulating factor (GM-CSF) and
interleukin-4 (IL-4) and to apoptotic death. This proapoptotic activity
was not affected by UV inactivation and was neutralized by EBV
antibody-positive human sera, indicating that binding of the virus to
monocytes is sufficient to alter their response to the cytokines.
Experiments with the relevant blocking antibodies or with mutated EBV
strains lacking either the EBV envelope glycoprotein gp42 or gp85
demonstrated that interaction of the trimolecular
gp25-gp42-gp85 complex with the monocyte membrane is
required for the effect. Our data provide the first evidence that EBV
can prevent the development of DCs through a mechanism that appears to
bypass the requirement for viral gene expression, and they suggest a
new strategy for interference with the function of DCs during the
initiation and maintenance of virus-specific immune responses.
(Blood. 2002;99:3725-3734) Viruses that establish persistent infection develop
multiple strategies to evade host immune responses. Striking examples are provided by human herpesviruses that block antigen presentation and
produce homologues of cytokines and chemokines that can subvert inflammatory responses (reviewed in Ploegh1). EBV is an
oncogenic herpesvirus that infects most adults and persists for life in immunocompetent hosts. It is associated with several malignancies of
hematopoietic and nonhematopoietic origin. Several lines of evidence
indicate that major histocompatibility complex (MHC) class I-restricted
CD8+ cytotoxic T lymphocytes (CTLs) are critical for the
control of EBV infection. The symptomatic primary infection, infectious
mononucleosis, and the asymptomatic carrier-state are characterized by
vigorous CTL responses to viral proteins expressed in latently and
productively infected cells (reviewed in Rickinson and
Moss2). Lack of CTL responses correlates with uncontrolled
proliferation of EBV-carrying B-cell lymphomas in immunocompromised
patients, and these immunodeficiency-associated malignancies can be
prevented or even cured by adoptive transfer of EBV-specific
CTLs.3 Viral escape strategies that allow persistence and
reactivation in the face of this effective immunity are largely unknown.
DCs play a pivotal role in the initiation and maintenance of
immune responses because of their ability to capture exogenous antigens
and present them to T lymphocytes in the form of MHC class I-associated
peptides, a phenomenon known as cross-priming (reviewed in Banchereau
et al4). It is well established that interference with the
function of DCs regulates the life cycle and pathogenesis of many
viruses,5-11 but its contribution to EBV immunity is as
yet unexplored. Previous studies have focused on the ability of
EBV-transformed lymphoblastoid cell lines (LCLs) to reactivate
EBV-specific cytotoxic T-lymphocyte responses in vitro from autologous
peripheral blood.12 LCLs coexpress viral antigens and high
levels of MHC class I and class II adhesion and costimulatory
molecules, and it is generally assumed that this immunogenic phenotype
is responsible for the extensive expansion of reactive T cells that
characterize infectious mononucleosis. However, LCLs cannot activate
EBV-specific CD8+ T cells from naive donors. This is
consistent with a number of human and animal studies that demonstrated
a poor capacity of B cells to activate primary CTL
responses.13-16 In contrast to B cells, DCs are efficient
in triggering primary T-cell responses in vitro and in vivo. It has
been recently shown that monocyte-derived DCs can efficiently take up
EBV antigens from apoptotic or necrotic LCLs.17-19 These
antigens were processed and presented in association with MHC class I
molecules and were recognized by specific CTLs in vitro. Notably, the
EBV nuclear antigen (EBNA) 1, which is not presented to MHC class
I-restricted CTLs because of a cis-acting blockade of ubiquitin-proteasome-dependent
proteolysis,20,21 is still capable of inducing strong CTL
responses in HLA-A2 and -B35-positive individuals and requires
cross-priming for its presentation to CTLs.22 Thus,
specialized antigen-presenting cells are likely to be involved in the
induction of at least some EBV-specific responses in vivo.
Triggering of Th1-type immune responses and development of strong
cytotoxic activity usually require DCs derived from myeloid precursors,
such as blood monocytes.23 EBV can bind to monocytes, modulate cytokine production, and inhibit phagocytic activity of these
cells.24-28 Here we show that EBV infection prevents the development of DCs promoting the apoptosis of monocytes cultured in the
presence of granulocyte macrophage-colony-stimulating factor (GM-CSF)
and interleukin-4 (IL-4). Binding of the EBV envelope gp42-gp85-gp25
fusion complex to the monocyte surface appears to play a critical role
in the proapoptotic activity of the virus.
Preparation of blood dendritic cells
EBV infection
Analysis of cell surface marker expression and [3H]-thymidine incorporation assay Surface markers were detected using a panel of mAbs specific for HLA class I, HLA-DR, CD3, CD19, CD14, CD80, CD86, CD83, CD11C, and CD1a. Phycoerythrin and fluorescein isothiocyanate (FITC)-conjugated antibodies used for direct immunofluorescence and isotype-matched controls were described elsewhere.29 Fluorescence intensity was monitored with a FACScan flow cytometer (Becton Dickinson, San Jose, CA) using the CellQuest software.Stimulatory capacity of DC preparations was assessed by [3H]-thymidine incorporation assays. DCs were collected, irradiated with 40 Gy, and mixed with allogeneic PBMCs in complete medium at the indicated responder-stimulator ratios. The cell suspension was adjusted to a density of 3 × 105 cells/mL and was distributed in triplicate to 96-well, U-bottom microtiter plates (100µL/3 × 104 T cells/well). Cells were cultured in a CO2 incubator for 5 to 7 days and were pulsed with [3H]-thymidine (0.037 MBq/well) during the last 8 hours of incubation. Plates were then harvested to glass filters on a Tomtec harvester 96 (Orange, CT), and filters were counted on a Wallac 1450 Microbeta liquid scintillation counter (Wallac, Turku, Finland). Mutated viral strains, EBV-specific antibodies, and purified gp350/220 Recombinant viruses were constructed in the Akata strain by homologous recombination with appropriate DNA fragments in which the gp42 or gp85 open-reading frames were disrupted by the insertion of a neomycin resistance cassette (gp42) or a cassette expressing the neomycin resistance gene and a gene for green fluorescence protein (gp85), respectively.31,32 Wild-type and recombinant viruses were produced by inducing EBV lytic cycle in Akata cells with anti-human IgG as previously described.31 Viral stocks were equalized for EBV content, which was assessed by binding to Raji cells followed by indirect immunostaining with the gp350/220-specific mAb 2L10 and FACS analysis.A truncated EBV gp350 lacking the membrane anchor was produced in the mouse fibroblast cell line C127 using a bovine papilloma virus-based expression vector and purified as previously described.33 EBV antibody-positive and -negative human sera were obtained from healthy laboratory personnel and from patients with nasopharyngeal carcinoma. Antibody titers to the EBV nuclear antigens and viral capsid antigen were determined by anticomplement-enhanced immunofluorescence staining or indirect immunofluorescence, respectively. Mouse antibodies F-2-134 (specific to the EBV envelope glycoprotein gp42),35 E1D136 (reacting with the EBV gp85-gp25 complex),35 72A137 (specific to the EBV gp350/220 glycoprotein), and OKT338 (specific to CD3) were purified from culture supernatants of the corresponding hybridoma cell lines. Detection of apoptosis Cytospins of EBV-infected and control cells were fixed with freshly prepared paraformaldehyde (4% in PBS), and the cells were permeabilized (0.1% Triton X-100 in 0.1% sodium citrate) and stained with Hoechst 33258 (Sigma-Aldrich, Stockholm, Sweden) to detect apoptotic nuclei. The samples were examined with a Leitz-BMRB fluorescence microscope (Leica, Heidelberg, Germany). Images were captured with a Hamamatsu (Osaka, Japan) 4800 cooled CCD camera and were processed with Adobe Photoshop software. Efficiency of Annexin V binding to the surfaces of EBV-infected and control cells was measured using Annexin V-FITC Apoptosis Detection Kit I (PharMingen, San Diego, CA). Monoclonal antibodies specific to Bim (StressGen Biotechnologies, Victoria, BC, Canada), Bcl-2 (Zymed Laboratories, Carlton Court, CA), Bad and Bax (R&D Systems, Oxon, United Kingdom), and polyclonal rabbit anti-Bcl-XL and anti-caspase-3 antibodies (PharMingen) were used to analyze the expression of Bcl-2 family members and caspase-3 cleavage by immunoblotting.
EBV infection leads to apoptosis of developing DCs To determine whether EBV can interfere with the development of DCs, confluent monolayers of adherent mononuclear cells were infected with the prototype B95.8 virus and then cultured for 7 days in the presence of GM-CSF and IL-4. Cells expressing typical DC morphology and cell surface markers, including high levels of MHC class II and CD86, were recovered from the infected cultures, but a dramatic decrease in cell number was observed compared with uninfected controls (Table 1). Major differences were revealed on examination of the cultures by light microscopy and monitoring of cell recovery over time. Floating cells appeared in the uninfected cultures within 2 to 3 days; after that, the cultures appeared as single-cell suspensions, containing occasional loose clumps of large, viable, dendritic-shaped cells. The number of cells remained fairly constant during the first 7 days and started to decrease only after prolonged culture (Figure 1A). In the infected cultures, cell detachment had started already after 1 day, and the cultures were extensive formations of dense clumps containing dark and irregularly shaped cells. A significant decrease in cell viability began from days 3 to 5 (Figure 1A). Similar differences were observed when the effect of EBV infection was tested on purified CD14+ monocytes isolated by negative selection, and these cells were therefore used in subsequent experiments. To investigate whether the induction of apoptosis may explain the lower recovery caused by EBV infection, the cultures were examined by Hoechst staining. Typical apoptotic changes, including membrane ruffling and nuclear condensation, were detected in uninfected and EBV-infected cultures. However, the percentage of apoptotic cells was significantly higher in the infected cultures already at day 3 and steadily increased in parallel with the progressive drop of cell recovery (compare panels A and B in Figure 1). In independent experiments, comparable numbers of apoptotic cells were revealed by their capacity to bind Annexin V with a relatively high efficiency. The percentage of such cells also increased progressively from day 4 to day 6 in EBV-infected cultures, whereas it remained fairly constant in mock-infected populations (Figure 1C). Caspase-3 cleavage, a point of convergence for different apoptotic pathways,40 was monitored by immunoblotting of total cell lysates. Enzymatically active 20-kd and 17-kd forms of caspase-3 were revealed in cell lysates of EBV-infected DCs collected at day 4 and day 6 of culture but were less evident in lysates of mock-infected cells (Figure 1D). Consistent with the relatively slow kinetics of apoptotic death, prolonged exposure of filters was required to visualize these cleavage products. Induction of apoptosis was accompanied by strong up-regulation of Bim, a proapoptotic member of the Bcl-2 family (Figure 1E). In accordance with the notion that Bim induces apoptosis by binding to Bcl-2 and neutralizing its antiapoptotic activity,42,43 Bcl-2 expression was not affected or was only slightly increased in some experiments, resulting in a significant decrease of the Bcl-2-Bim ratio. Expression levels of Bcl-XL were low and appeared to be unchanged on EBV infection (data not shown). Expression of proapoptotic members of the Bcl-2 family, Bad and Bax, was, respectively, either not affected by EBV infection or not detectable in control or infected cells (data not shown).
The activity of apoptosis-inducing agents is often determined by the
stage of cell differentiation or activation. We asked, therefore,
whether the status of monocyte-DC differentiation affects the
sensitivity of cells to apoptotic death on EBV infection. First, cell
morphology and viability were monitored during culture in the absence
of cytokines. As illustrated by the representative experiment shown in
Figure 2A, EBV infection did not induce
apoptosis in monocytes. Indeed, the recovery of viable cells was
slightly higher in EBV-infected cultures than in uninfected controls.
This was partly explained by the early detachment of EBV-infected cells already noted in the cytokine-containing cultures. To analyze the
sensitivity of cells to EBV-induced apoptosis at different stages of
differentiation toward DC phenotype, cells cultured in the presence of
IL-4 and GM-CSF were infected with EBV at different time points, as
indicated in Figure 2B. In agreement with the data presented in Figure
1A, EBV infection at the initiation of the culture resulted in the loss
of approximately 70% of cells relative to uninfected controls. Cells
infected at day 3 or day 5 of culture also died in response to the
infection; however, the level of cell loss gradually decreased along
with lymphokine-induced differentiation. Thus, only 50% of cells were
lost in cultures infected at day 5. By day 7, virtually all cells
cultured in GM-CSF and IL-4 acquired an immature DC phenotype
characterized by the expression of CD86, CD80, and high levels of MHC
class I and II molecules (Figure 3A and
data not shown). In some cases, DCs that developed in EBV-infected
cultures showed an up-regulation of CD86, CD83, and more heterogeneous
expression of CD11c compared with mock-infected controls (Figure 3A and
data not shown). However, these changes were not observed with every DC
preparation and did not lead to any detectable differences in the
stimulatory capacity of these cells, as assessed by mixed-lymphocyte
reactions with allogeneic PBMCs (Figure 3B). At this stage, DCs in
control cultures appeared to be unresponsive to EBV, as assessed by
cell death (Figure 2B). These results indicated that apoptotic death is
triggered by EBV infection only in cells undergoing differentiation toward the DC phenotype and may reflect an abnormal cellular response to the differentiation-inducing lymphokines.
To explore this phenomenon further, cell morphology and viability were
compared in cultures containing either GM-CSF or IL-4 alone. A dramatic
difference was observed in the GM-CSF cultures. Although uninfected
cells formed monolayers of firmly adherent macrophages that remained
viable during the entire observation period, EBV-infected cells
detached rapidly and started to die at a rate only slightly slower than
that observed in the presence of GM-CSF and IL-4 (Figure
4A and not shown). Independently of EBV
infection, cells cultured in IL-4 alone died within 4 to 6 days, as
expected. However, a significantly higher percentage of small cells
showing morphologic signs of apoptosis was observed in the EBV-infected
cultures starting from day 2, and the recovery of viable cells was
significantly decreased by day 4 (Figure 4B). The effect of virus
infection on the response to cytokine stimulation was also confirmed by
analysis of surface marker expression. CD14 is rapidly down-regulated
upon culture of blood monocytes in GM-CSF and IL-4 because of the
differentiating activity of IL-4.44,45 This loss of
expression was significantly delayed in EBV-infected cultures, where
high levels of CD14 were still detected in most of the cells after 3 days (Figure 4C). Collectively, these findings demonstrate that EBV
infection blocks the differentiation of blood monocytes into DCs,
altering their response to GM-CSF and IL-4 and promoting apoptosis in
the presence of the cytokines.
Inhibition of DC development is independent of viral gene expression The finding that EBV infection inhibits the development of DCs from purified monocytes and the failure to reproduce the phenomenon by coculture of uninfected monocytes with infected mononuclear cells in a 2-chamber system (not shown) suggest that contact with the virus is required for the effect. The following sets of experiments were performed to confirm this observation. Monocytes were infected with B95.8 supernatants depleted of infectious virus by absorption on EBV receptor-positive Raji cells or with concentrated virus obtained by ultracentrifugation. The virus-depleted supernatant did not affect recovery, whereas the yield of DCs was further decreased by infection with concentrated virus (Figure 5A). To ensure the specificity of virus absorption, magnetic beads were conjugated with either the gp350-specific mAb 2L10 or the CD3-specific mAb OKT3 as a control. Incubation of B95.8 supernatant with OKT3 beads or beads with blocked binding sites resulted in the unspecific decrease of EBV titers by 30% to 50%, as assessed by EBNA staining or EBV binding assay (data not shown). Therefore, prolonged incubations were required to clearly reveal the inhibitory activity of the virus. Nevertheless, only absorption with 2L10 mAb, but not with OKT3 mAb-conjugated beads, significantly diminished the ability of the B95.8 supernatant to induce apoptosis in developing DCs, confirming that the inhibitory effect requires the presence of the virus (Figure 5B). In line with this conclusion, the activity of the virus was abolished by preincubation with sera from healthy EBV carriers or from patients with nasopharyngeal carcinoma that contained high titers of neutralizing antibodies, whereas sera from EBV antibody-negative donors had no effect (Figure 5C).
Recent reports suggest that EBV can replicate in monocytes, though with
low efficiency.26 We tested, therefore, whether viral gene
expression was required for the proapoptotic effect. Monocytes were
exposed to replication-competent or UV-inactivated virus, and cell
recovery was monitored in cultures containing GM-CSF and IL-4. As shown
in Figure 6, inactivation of the virus with doses of UV light sufficient to prevent the transformation of
freshly isolated B lymphocytes and the expression of EBNAs in the
EBV-negative Bjab cell line (not shown) did not affect its ability to
inhibit the development of DCs. Analysis of viral gene expression by
RT-PCR failed to detect transcripts of the latent viral genes
EBNA1, EBNA2, LMP1, and
LMP2a and the lytic gene BZLF1 in cells collected
at different times after infection (data not shown).
Effect of the virus requires engagement of the membrane-fusion complex The inhibitory activity of UV-inactivated virus suggests that binding of EBV to monocytes may be sufficient to alter their response to GM-CSF and IL-4. The major EBV envelope glycoprotein, gp350/220, was shown to interact with monocytes and to activate the expression of several cellular genes.27,28 We asked whether the effect of the virus could be reproduced by exposure to purified recombinant gp350. As shown in Figure 7A, culturing of monocytes in the presence of increasing concentrations of gp350 or on gp350-coated plates (not shown) had no effect on, or even slightly increased, the recovery of DCs. Hence, we turned to other components of the viral envelope. In addition to binding to CD21, infection of B lymphocytes requires the engagement of a trimolecular complex composed of the EBV glycoproteins gp42, gp85, and gp25.35,46,47 Gp42 interacts with the chain of HLA-DR, -DQ, and
-DP48,49 and promotes viral penetration, which is mediated
by the homologue of the herpesvirus membrane-fusion complex gH/gL,
gp85, and gp25. The gp85/gp25 heterodimer alone acts as an alternative
EBV ligand in CD21 , MHC class II-negative epithelial
cells by interacting with an as-yet-unidentified membrane
molecule.32,50 Preincubation of replication-competent
(Figure 7B) or UV-inactivated virus (not shown) with antibodies to
gp350/220 did not prevent cell death, further confirming that gp350/220
ligation is not involved in this viral effect. An isotype-matched
CD3-specific antibody was also inactive, whereas DCs were rescued by
preincubation with the mAb F-2-1 which blocks the interaction of gp42
with MHC class II51and E1D1which blocks penetration
mediated by gp85/2535,46 and inhibits binding to
epithelial cells in the absence of CD21.32 Involvement of
the tripartite complex was further confirmed using mutant EBV strains
deficient in the expression of the gp42 or gp85 proteins. Purified
CD14+ monocytes were infected with wild-type Akata virus,
gp42- or gp85-deficient virus, and B95.8 virus as a control and then
were cultured for 7 days in the presence of IL-4 and GM-CSF. The
wild-type Akata and B95.8 viruses exhibited a comparable ability to
suppress DC development, whereas the inhibitory activity of viruses
lacking either gp42 or gp85 was significantly impaired (Figure 7C).
Thus, binding of EBV to the proposed coreceptor expressed on monocytes appeared to be sufficient to modify their response to GM-CSF and IL-4
and to inhibit the development of DCs.
The data presented here provide the first demonstration that EBV infection inhibits the development of myeloid DCs from blood monocyte precursors. We show that EBV-infected monocytes die by apoptosis in the presence of GM-CSF and IL-4. This correlates with an aberrant response to both cytokines because the infected cells failed to differentiate into adherent macrophages in response to GM-CSF alone, and their death was accelerated in the presence of IL-4 compared with uninfected control cells. Viral interference with the cytokine effect is further illustrated by the early blockade of CD14 down-regulation (Figure 4), which is normally observed as a rapid response to IL-4. Strong up-regulation of Bim expression is likely to account for cell death induced by EBV infection and is also consistent with inability of IL-4 and GM-CSF to trigger survival signals known to prevent Bim up-regulation in other models.52,53 Interference with the function of DCs is a common feature of viral pathogenesis and is usually achieved by the expression of viral genes and virus replication in mature DCs or their precursors.6,9,10,54,55 We have found that the proapoptotic activity of EBV is not affected by UV inactivation, excluding a major contribution of de novo synthesis of viral proteins or mRNAs. Thus, EBV appears to be unique in its ability to suppress the development of DCs without the need for productive infection. Experiments with blocking antibodies and recombinant virus strains indicate that binding of EBV to monocytes through the viral membrane-fusion complex may be sufficient to trigger events that will eventually lead to cell death. Two components of the fusion complex, gp42 and the gp85-gp25 heterodimer, appear to be involved. Interestingly, the corresponding members of the human cytomegalovirus fusion complex were shown to activate signal transduction in infected cells but were not implicated in the induction of apoptosis.56,57 Purified gp42 has been shown to suppress lymphocyte proliferation in allogeneic mixed-lymphocyte cultures, but the mechanism of this effect has not been elucidated.49 Our data suggest that gp42 may exert its suppressive effect through the inhibition of DC development. The blocking activity of antibodies that interfere with the interaction of gp42 with HLA-DR and the impaired activity of the gp42-deficient virus indicate that ligation of HLA class II may be required for the effect. Indeed, HLA-DR mediates signals that may result in cell proliferation, cell-cell adhesion, or cell death. Triggering of MHC class II molecules with a subset of specific mAbs initiates apoptosis in DCs. Mature DCs are sensitive to HLA-DR-mediated apoptosis, whereas immature DCs and macrophages are less sensitive and monocytes appear to be resistant.58 In contrast, the most prominent proapoptotic activity of EBV is observed with the infection of monocytes at the early stages of their differentiation into DCs (Figure 2). Sensitivity of cells to the viral effect decreases along with monocyte progression to the DC phenotype, and the virus does not affect completely differentiated mature DCs. This suggests that gp42 mediates DC apoptosis through a mechanism that is distinct from antibody-mediated MHC class II ligation. The inhibitory effect of gp85-specific blocking antibodies and diminished proapoptotic activity of gp85-deficient virus are also consistent with an independent role of the gp85-gp25 heterodimer in the inhibition of DC development. If so, the interaction of gp42 with HLA class II may be required to bring the heterodimer in proximity with the cell membrane. In addition, other virion-associated components, such as tegument proteins that may be delivered to the cytosol after viral fusion, may also contribute to the induction of apoptosis. The mechanisms through which interaction with the EBV fusion complex
alters the response of monocytes to signals that usually promote
survival and differentiation remain unknown. Analysis of GM-CSF and
IL-4 receptor expression by staining with specific antibodies failed to
demonstrate significant changes in the infected cells (not shown), but
this does not exclude a receptor-mediated effect. Site-specific
phosphorylation of the IL-4 receptor by receptor-associated kinases
results in differential triggering of cell growth, gene activation,
differentiation, or resistance to apoptosis (reviewed in Nelms et
al59). Thus, EBV infection might affect the signaling
capacity of the receptors by altering their pattern of phosphorylation.
It is noteworthy that interaction of the most abundant EBV envelope
protein, gp350-gp220, with a CD21-like moiety on the monocyte
membrane, up-regulates tumor necrosis factor- Our findings indicate that EBV may interfere with the development and maintenance of specific immune responses. Prolonged contact between antigen-presenting DCs and naive T cells is required for the initiation of primary immune responses. According to different models of T-cell activation, the number of antigen-loaded DCs available to specific T cells represents a critical parameter for the induction of immunity, and whether the DC supply is efficient may largely determine the outcome of the immune response.61,62 At least a proportion of DCs develops directly from monocyte precursors.63 By inhibiting the differentiation of monocytes into mature DCs, EBV may temporarily halt the onset of immune responses during primary infection, creating a time window for efficient viral replication. This could permit the accumulation of a sufficiently large pool of virus-infected B lymphocytes and could allow their access to the memory B-cell compartment. Supporting this scenario, absolute monocytopenia was observed during the acute phase of infectious mononucleosis in the only reported case that was followed from the time of first encounter with the virus to the resolution of clinical symptoms.64 Notably, infectious mononucleosis represents a rare outcome of primary EBV infection. The clinical picture of the disease develops as a result of an abnormally intensive immune response and can be explained, in the context of the proposed model, as a failure of the virus to exert sufficiently strong suppression on the immune system. The usual success of this viral strategy may account for the generally asymptomatic course of primary EBV infection. Many viruses are known to act as potent activators of DC maturation, thereby promoting the development of specific antiviral immunity.65-68 However, several viruses block DC development or deregulate the process of DC maturation. Such activity has been reported for human herpes simplex10,69 and has been suggested for human cytomegalovirus,11 and it appears to be associated with the ability of herpesviruses to establish persistence in infected hosts. Constant viral shedding is a characteristic of healthy EBV carriers, who constitute most of the human population. DC activation by EBV virions could provide a constantly acting endogenous danger signal, resulting in a break of tolerance. It is tempting to propose that the inhibitory activity of EBV on DC development may represent an essential mechanism in the establishment of EBV persistence, working as a silencer for potentially deleterious immunologic consequences of viral reactivation. In agreement with this idea, analysis of surface marker expression did not reveal any significant phenotypic differences between control and EBV-infected cells surviving 7 days of culture in the presence of lymphokines. The 2 cell populations were also comparable in their functional activity, as determined by allogeneic mixed-lymphocyte reactions (Figure 3). Although we cannot exclude that some subtle differences between DCs developing in virus-infected and control cultures could be revealed on analysis of their capacity to stimulate or polarize antigen-specific T-cell responses, the available data indicate that neither exposure to the virus nor uptake of apoptotic material from EBV-infected, monocyte-derived cells results in DC activation. It should be noted, however, that in vivo the DC population is composed of phenotypically and functionally distinct subsets. Results presented in this study suggest that EBV could interact with and affect the development of DCs and DC precursors of different origins, and detailed characterization of such interactions should be forthcoming. EBV-infected memory B cells appear to be the primary site of virus reactivation in healthy virus carriers (reviewed in Masucci and Ernberg70). These cells are probably unable to boost immune responses because the presentation of antigen on resting B cells has a tolerizing effect on T lymphocytes.13 Their capacity to transfer the virus to adjacent monocytes may not be affected by the presence of neutralizing antibodies because of the ability of herpesviruses to spread by cell-to-cell contact in vivo. Notably, high titers of neutralizing antibodies do not protect against superinfection with EBV in immunocompromised patients.71 Thus, impairment of cross-priming by DCs may delay the reactivation of EBV-specific immune responses during persistent infection, allowing efficient spread to new hosts. The high capacity of developing DC to migrate from the periphery to the lymphatic tissue is well established. It is conceivable, therefore, that the death of EBV-infected developing DCs could also limit the transport of the virus to lymph nodes, where it can propagate through lytic replication and proliferation of latently infected cells.72 Finally, interference with the development of DCs may offer a new explanation for the role of virus replication in the pathogenesis of EBV-associated malignancies. Enhanced virus production has been shown to precede the outgrowth of EBV-associated large-cell lymphomas in immunosuppressed patients,73 and elevated titers of antibodies to antigens of the productive cycle correlate with increased risk and relapse of endemic Burkitt lymphoma and nasopharyngeal carcinoma.74,75 The significance of this prodromal burst of virus replication has remained obscure because EBV is strictly latent in the malignant cells. Our findings suggest that, by diminishing the activity of certain types of DCs, virus production could be an important factor in tumor progression.
Submitted June 28, 2001; accepted January 16, 2002.
Supported by grants from the Swedish Cancer Society, the Swedish Paediatric Cancer Foundation, the Swedish Foundation of Strategic Research, the Petrus and Augusta Hedlund Foundation, and the Karolinska Institutet, Stockholm, Sweden.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Victor Levitsky, Microbiology and Tumorbiology Center, Karolinska Institutet, Nobels väg 16, S-17177 Stockholm, Sweden; e-mail: victor.levitsky{at}mtc.ki.se.
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Abstract
Introduction
) or EBV-infected (EBV+) cultures was
analyzed by immunoblotting after the indicated periods of
time.
(TNF-
B family of transcription factors,