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IMMUNOBIOLOGY
Epstein-Barr virus inhibits the development of dendritic cells by promoting
apoptosis of their monocyte precursors in the presence of granulocyte
macrophage–colony-stimulating factor and interleukin-4
LiQi Li, Daorong Liu, Lindsey Hutt-Fletcher, Andrew Morgan, Maria G. Masucci, and Victor Levitsky
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 antibodypositive 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)
© 2002 by The American Society of Hematology
Introduction
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 carrierstate 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 immunodeficiencyassociated 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 crosspriming (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 EBVspecific 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 cisacting 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
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.
Reprints: Victor Levitsky, Microbiology and Tumorbiology Center, Karolinska
Institutet, Nobels väg 16, S-17177 Stockholm, Sweden; e-mail: victor.
[email protected].
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.
BLOOD, 15 MAY 2002 䡠 VOLUME 99, NUMBER 10
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.
© 2002 by The American Society of Hematology
3725
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3726
LI et al
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–colonystimulating 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.
Materials and methods
Preparation of blood dendritic cells
Blood DCs were generated from peripheral blood mononuclear cells
(PBMCs) as decribed.29 Mononuclear cells, isolated from the blood of
healthy donors by density gradient centrifugation, were resuspended at a
density of 1.5 to 2 ⫻ 106 cells/mL in culture medium (RPMI 1640 medium
supplemented with 10% heat-inactivated fetal calf serum, 2 mM Lglutamine, 100 U/mL penicillin, 100 ␮g/mL streptomycin) and were
allowed to adhere to the surface of a plastic flask for 2 hours at 37°C in a 5%
CO2 incubator. Confluent monolayers were washed 3 times in phosphatebuffered saline (PBS) to remove the nonadherent cells and then were
cultured for indicated times in medium supplemented with 1000 U/mL
recombinant GM-CSF and 1000 U/mL recombinant IL-4 (a kind gift of
Schering-Plough Research Institute, Kenilworth, NJ). Cytokines were
replenished by exchanging one third of the culture supernatant every third
day, at which time cell viability was also estimated by trypan blue dye
exclusion. Changes in cell morphology were monitored daily by visual
inspection using an inverted microscope. Where indicated, CD14⫹ monocytes were purified using the monocyte-negative isolation kit (Dynal AS,
Oslo, Norway) according to the manufacturer’s instructions. PBMCs
(10 ⫻ 106) were reacted with a mixture of antibodies specific for various T-,
B-, and NK-cell markers, and the positive cells were depleted by incubation
with antimouse antibody-conjugated Dynabeads, followed by capture on an
MPC-1 magnetic particle concentrator. The negatively selected population
contained more than 98% CD14⫹ cells as detected by staining and
fluorescence-activated cell sorter (FACS) analysis.
EBV infection
Infectious EBV was obtained from 10-day-old cell-free supernatant of the
virus producer B95.8 cell line. The virus was concentrated from filtered
supernatants (0.45 ␮M) by centrifugation at 45 000g for 90 minutes at 4°C.
Pellets were resuspended in PBS. B95.8 supernatants were depleted of
infectious virus either by 6 consecutive rounds of absorption on CD21⫹
Raji cells (1 ⫻ 108 cells in 20 mL B95.8 supernatant for 1 hour at 37°C).
Alternatively, virus absorption was performed on tosylactivated magnetic
beads (Dynabeads M-280; Dynal) conjugated with purified gp350-specific
monoclonal antibody (mAb) 2L10 (2 mg/mL beads). Beads treated with
blocking reagent or conjugated with CD3-specific mAb OKT3 were used as
controls. Antibody conjugation and blocking of active sites were performed
according to the manufacturer’s procedures. Virus titers were determined by
immortalization of freshly separated B lymphocytes and by expression of
the EBNAs in the EBV-negative B-lymphoma line Bjab. Anticomplementenhanced immunofluorescence staining was performed 48 hours after
infection using a previously characterized EBV antibody–positive human
serum. The virus was inactivated by exposure of B95.8 supernatant to UV
light (254 nm) for 5 minutes (UV source model UVG-11; Upland, CA) from
a distance of 10 cm, which resulted in a complete loss of transforming
activity and EBNA induction in Bjab cells. Confluent monolayers of
adherent PBMCs or purified CD14⫹ cells were infected by incubation for 2
hours at 37°C. The monolayers were then washed and cultured in medium
BLOOD, 15 MAY 2002 䡠 VOLUME 99, NUMBER 10
containing IL-4 and GM-CSF to induce DC development. Where indicated,
the virus preparations were preincubated for 30 minutes at room temperature with 1:20 dilution of the indicated human sera or 100 ␮g/mL purified
antibodies before they were used for the infection of monocytes. The
expression of EBV mRNA was detected by reverse transcription–
polymerase chain reaction (RT-PCR) using primers specific for the EBNA1,
EBNA2, LMP1, and BZLF1 messages, as previously described.30
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 (100␮L/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 antibodypositive 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
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BLOOD, 15 MAY 2002 䡠 VOLUME 99, NUMBER 10
INHIBITION OF DC DEVELOPMENT BY EBV
3727
(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.
Results
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).
Table 1. EBV infection inhibits the development of DCs from blood monocytes
Cell recovery (⫻ 106)
Experiment
Control
EBV
Cell loss, %
1
7.3
2.5
65.8
2
1.8
0.7
61.1
3
9.0
3.0
66.7
4
1.3
0.4
68.0
5
3.7
1.1
69.5
Mean ⫾ SD
—
—
66.2 ⫾ 3.2
Monolayers of uninfected and EBV-infected PBMCs were cultured for 7 days in
the presence of GM-CSF and IL-4. The yield of viable cells was evaluated by trypan
blue dye exclusion.
Figure 1. EBV infection induces apoptosis of monocytes cultured in GM-CSF and
IL-4. Monolayers of adherent PBMCs or purified CD14⫹ monocytes were infected with
supernatants from the EBV producer B95.8 cell line for 2 hours at 37°C and were cultured
in medium with or without GM-CSF and IL-4. Each culture condition was tested in triplicate
in every experiment. (A) Time kinetics of cell recovery in control (open symbols) and
EBV-infected cultures (closed symbols) containing GM-CSF and IL-4. Mean ⫾ SD of
triplicates. Results are from 1 of 20 representative experiments. (B) Cytospins of cells
cultured for 5 days in GM-CSF and IL-4 were examined by phase-contrast microscopy and
Hoechst staining. From day 3, the percentage of apoptotic cells was significantly higher in
EBV-infected cultures (gray bars) than in the uninfected control (white bars) and increased
in parallel with the decrease of cell recovery. Results are from 1 of 2 representative
experiments. (C) FACS analysis of FITC-conjugated Annexin V binding to EBV-infected
and control cells collected at day 4 or day 6 of culture. Gated populations of propidium
iodide-negative cells are shown. Values inside the histogram plots represent the percentage of gated cells in the M1 region. Results are from 1 of 2 representative experiments. (D)
Caspase-3 cleavage in EBV-infected cells. Total cell lysates of EBV peptide-specific
antigen-activated cytotoxic T-cell clone BK289 (lane 1), EBV-infected (lanes 2 and 4), and
control (lanes 3 and 5) monocytes collected at day 4 (lanes 2 and 3) or day 6 (lanes 4 and 5)
of culture with the lymphokines were analyzed by immunoblotting with caspase-3–specific
rabbit polyclonal antibody. The 20-kd and 17-kd products, corresponding to enzymatically
active forms of caspase-3 and generated by cleavage of 32-kd proenzyme, are easily
detectable in the T-cell lysate because of Fas triggering.39 The same products are seen in
the lysates of EBV-infected cells but not in control cells. The band of lower molecular weight
most likely represents the enzymatically inactive 12-kd small subunit of caspase-3.40,41
Identity of the fragment with the highest molecular weight remains uncertain. (E) Expression of Bim and Bcl-2 proteins in control (EBV⫺) or EBV-infected (EBV⫹) cultures was
analyzed by immunoblotting after the indicated periods of time.
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LI et al
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
BLOOD, 15 MAY 2002 䡠 VOLUME 99, NUMBER 10
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 lymphokineinduced 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 mixedlymphocyte 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
Figure 2. EBV infection selectively affects cells undergoing differentiation into
DCs. (A) Recovery of uninfected and EBV-infected cells cultured in the absence of
GM-CSF and IL-4. Results are from 1 of 4 representative experiments. (B) Complete
differentiation into immature DCs is associated with resistance to the inhibitory effect
of the virus. Cells cultured in the presence of GM-CSF and IL-4 were infected with
EBV at different days of culture. Cell recovery was monitored at indicated time points
after infection and was expressed as a percentage relative to uninfected controls to
compensate for cell loss always observed in DC cultures with prolonged incubation.
Mean values obtained from 3 independent experiments are shown.
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
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BLOOD, 15 MAY 2002 䡠 VOLUME 99, NUMBER 10
INHIBITION OF DC DEVELOPMENT BY EBV
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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
Figure 3. Phenotype and T-cell stimulatory capacity of DCs developing from
EBV- or mock-infected monocytes after 7 days of culture with GM-SCF and IL-4.
(A) Cells were recovered from mock (control) or EBV-infected cultures on day 7, and
surface expression of indicated markers was analyzed by immunostaining and FACS
analysis. (B) Uninfected (control), mock-infected (adsorbed EBV), or EBV-infected
monocytes were cultured for 7 days in the presence of the lymphokines, harvested,
irradiated, and used as stimulators at the indicated stimulator-responder ratios in
allogeneic mixed-lymphocyte cultures. [3H]-Thymidine incorporation by stimulated T
cells was measured after 7 days, as described in “Materials and methods.” In this
experiment, the loss of DCs in the EBV-infected cultures was approximately 65%
compared with the control cells. Results from 1 of 8 representative experiments
are shown.
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 mAbconjugated 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
Figure 4. Response of monocytes to GM-CSF and IL-4 is altered by EBV
infection. (A) Light microscopy images of purified monocytes cultured for 6 days in
medium containing 1000 U/mL recombinant GM-CSF. Most of the cells in the control
cultures adhered firmly to the plastic, whereas virtually all the EBV-infected cells
detached and formed tight, medium-sized clumps. Two of 10 representative cultures
initiated with monocytes from 2 donors are shown. (B) Cell recovery of purified
monocytes cultured with 1000 U/mL recombinant IL-4. Cells cultured in IL-4 alone
died within 5 to 6 days, as expected. The recovery of viable cells at day 4 was 5- to
10-fold lower in EBV-infected cultures than in uninfected controls. Results from 2
experiments performed in parallel are shown. (C) Surface expression of CD14 was
compared on control or EBV-infected cells by immunostaining and FACS analysis.
Down-regulation of CD14 was delayed in EBV-infected DCs. Results from 1 of 3
experiments are shown.
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LI et al
BLOOD, 15 MAY 2002 䡠 VOLUME 99, NUMBER 10
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 II51—and E1D1—which 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
Figure 5. Binding of the virus is required for the inhibition of DC development.
(A) The effect of the virus was abolished by adsorption on EBV receptor–positive cell
lines and was enhanced by concentration. EBV was concentrated from B95.8
supernatant by ultracentrifugation, and EBV-depleted supernatant was obtained by
adsorption on EBV receptor–positive Raji cells. Purified CD14⫹ monocytes were
infected with untreated, absorbed, or concentrated B95.8 supernatant for 2 hours at
37°C and then were cultured in the presence of GM-CSF and IL-4. Results are from 1
of 3 to 5 representative experiments performed with each type of virus preparation.
Mean ⫾ SD of triplicates. (B) Virus absorption was performed using magnetic beads
conjugated with 2L10 mAb. Beads conjugated with OKT3 mAb were used as control.
Results are from 1 of 3 representative experiments. (C) Effect of the virus was
neutralized by preincubation with EBV antibody–positive sera. B95.8 supernatant
was preincubated for 30 minutes at room temperature with 1:20 dilution of sera from
EBV antibody-negative (EBV⫺) or -positive (EBV⫹) healthy donors or from patients
with nasopharyngeal carcinoma before it was used for infection of purified monocytes. The percentage recovery relative to uninfected controls is shown. Mean ⫾ SD
of 4 experiments.
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
Figure 6. UV-inactivated EBV retains its inhibitory activity. Purified CD14⫹
monocytes were infected with replication-competent or inactivated virus. B95.8
supernatants were inactivated by exposure to a 254-nm UV light source for 5 minutes
from a distance of 10 cm. The dose corresponded to double the amount required to
abolish the transformation of freshly isolated B lymphocytes and the induction of
EBNA expression in the EBV-negative B-lymphoma line Bjab. The efficiency of
inactivation was confirmed by the failure to detect EBV-specific mRNAs by RT-PCR at
different times after infection (not shown). Results from 1 of 5 representative
experiments are shown. Mean ⫾ SD of triplicates.
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BLOOD, 15 MAY 2002 䡠 VOLUME 99, NUMBER 10
INHIBITION OF DC DEVELOPMENT BY EBV
3731
Figure 7. Engagement of the EBV membrane-fusion complex is required for inhibition of DC development. (A) Culture of monocytes in the presence of recombinant
gp350/220 did not inhibit the development of DCs. Purified monocytes were cultured for 7 days in medium containing GM-CSF, IL-4, and the indicated concentration of purified
recombinant gp350. Results are from 1 of 4 representative experiments. (B) CD14⫹ monocytes were infected with B95.8 virus or virus preincubated for 30 minutes at room
temperature with 100 ␮g/mL mAb, as indicated. The inhibitory activity of the virus was blocked by mAb F-2-1, which interferes with the interaction of gp42 with HLA class II, and
by E1D1, which prevents gp85–gp25-mediated membrane fusion and inhibits binding to epithelial cells in the absence of gp42. The 72A1 antibody to gp350 and OKT3 antibody
to CD3 had no effect. Results from 1 of 2 experiments giving comparable results are shown. (C) Recombinant EBV lacking gp42 or gp85 showed a significantly impaired
inhibitory activity compared to wild-type viruses derived from Akata or B95.8. Mean ⫾ SD of 3 experiments.
expression of the gp42 or gp85 proteins. Purified CD14⫹ monocytes were infected with wild-type Akata virus, gp42- or gp85deficient 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.
Discussion
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 antibodymediated MHC class II ligation. The inhibitory effect of gp85specific 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 receptormediated 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–␣ (TNF-␣) through activation of protein kinase C,
phosphatidylinositol 3 kinase, and the NF-␬B family of transcription factors,27,28 whereas exposure to intact virions inhibits TNF-␣
production.60 It remains to be seen whether specific combinations
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3732
BLOOD, 15 MAY 2002 䡠 VOLUME 99, NUMBER 10
LI et al
of kinases or phosphatases are recruited by binding of EBV through
the membrane-fusion complex.
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 virusinfected 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 EBVinfected 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 mixedlymphocyte reactions (Figure 3). Although we cannot exclude
that some subtle differences between DCs developing in virusinfected 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 EBVassociated 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.
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From www.bloodjournal.org by guest on July 8, 2017. For personal use only.
2002 99: 3725-3734
doi:10.1182/blood.V99.10.3725
Epstein-Barr virus inhibits the development of dendritic cells by promoting
apoptosis of their monocyte precursors in the presence of granulocyte m
acrophage−colony-stimulating factor and interleukin-4
LiQi Li, Daorong Liu, Lindsey Hutt-Fletcher, Andrew Morgan, Maria G. Masucci and Victor Levitsky
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