Immune activation suppresses initiation of lytic Epstein-Barr virus infection

Article (PDF Available)inCellular Microbiology 9(8):2055-69 · September 2007with24 Reads
DOI: 10.1111/j.1462-5822.2007.00937.x · Source: PubMed
Abstract
Primary infection with Epstein-Barr virus (EBV) is asymptomatic in children with immature immune systems but may manifest as infectious mononucleosis, a vigorous immune activation, in adolescents or adults with mature immune systems. Infectious mononucleosis and chronic immune activation are linked to increased risk for EBV-associated lymphoma. Here we show that EBV initiates progressive lytic infection by expression of BZLF-1 and the late lytic genes gp85 and gp350/220 in cord blood mononuclear cells (CBMC) but not in peripheral blood mononuclear cells (PBMC) from EBV-naive adults after EBV infection ex vivo. Lower levels of proinflammatory cytokines in CBMC, used to model a state of minimal immune activation and immature immunity, than in PBMC were associated with lytic EBV infection. Triggering the innate immunity specifically via Toll-like receptor-9 of B cells substantially suppressed BZLF-1 mRNA expression in acute EBV infection ex vivo and in anti-IgG-stimulated chronically latently EBV-infected Akata Burkitt lymphoma cells. This was mediated in part by IL-12 and IFN-gamma. These results identify immune activation as critical factor for the suppression of initiation of lytic EBV infection. We hypothesize that immune activation contributes to EBV-associated lymphomagenesis by suppressing lytic EBV and in turn promotes latent EBV with transformation potential.
Immune activation suppresses initiation of lytic
Epstein-Barr virus infection
Kristin Ladell,
1†‡
Marcus Dorner,
1‡
Ludwig Zauner,
1
Christoph Berger,
1
Franziska Zucol,
1
Michele Bernasconi,
1
Felix K. Niggli,
2
Roberto F. Speck
3
and David Nadal
1
*
1
Laboratory for Experimental Infectious Diseases and
Cancer Research of the Division of Infectious Diseases,
University Children’s Hospital of Zurich, 8032 Zurich,
Switzerland.
2
Division of Oncology, University Children’s Hospital of
Zurich, 8032 Zurich, Switzerland.
3
Division of Infectious Diseases and Hospital
Epidemiology, University Hospital of Zurich, 8091
Zurich, Switzerland.
Summary
Primary infection with Epstein-Barr virus (EBV) is
asymptomatic in children with immature immune
systems but may manifest as infectious mononucleo-
sis, a vigorous immune activation, in adolescents
or adults with mature immune systems. Infectious
mononucleosis and chronic immune activation
are linked to increased risk for EBV-associated
lymphoma. Here we show that EBV initiates progres-
sive lytic infection by expression of BZLF-1 and the
late lytic genes gp85 and gp350/220 in cord blood
mononuclear cells (CBMC) but not in peripheral blood
mononuclear cells (PBMC) from EBV-naive adults
after EBV infection ex vivo. Lower levels of proinflam-
matory cytokines in CBMC, used to model a state of
minimal immune activation and immature immunity,
than in PBMC were associated with lytic EBV
infection. Triggering the innate immunity specifically
via Toll-like receptor-9 of B cells substantially sup-
pressed BZLF-1 mRNA expression in acute EBV
infection ex vivo and in anti-IgG-stimulated chroni-
cally latently EBV-infected Akata Burkitt lymphoma
cells. This was mediated in part by IL-12 and IFN-g.
These results identify immune activation as critical
factor for the suppression of initiation of lytic EBV
infection. We hypothesize that immune activation
contributes to EBV-associated lymphomagenesis by
suppressing lytic EBV and in turn promotes latent
EBV with transformation potential.
Introduction
Epstein-Barr virus (EBV), a human B lymphotropic gam-
maherpesvirus, infects at least 90% of the world’s human
population. Different EBV latency gene programs allow
EBV to persist in the host in latently infected B cells.
Proliferation of the latently infected cells propagates EBV
to the daughter cells. Latent EBV may switch to its lytic
gene expression program, leading to EBV replication and
subsequent lysis of the infected cell (Cohen, 2000; Rick-
inson and Kieff, 2001; Thorley-Lawson, 2001; Thorley-
Lawson and Gross, 2004).
The vast majority of primary EBV infections occur in
infants and toddlers and are usually asymptomatic
(Biggar et al., 1978; Chan et al., 2001). By contrast,
primary EBV infection in adolescence or adulthood may
manifest as infectious mononucleosis (IM) (Biggar et al.,
1978), with fever and enlargement of tonsils, lymph
nodes, liver and spleen. This clinical presentation results
from the vigorous immune activation involving proinflam-
matory cytokines (Foss et al., 1994; Chan et al., 2001;
Rickinson and Kieff, 2001).
Epstein-Barr virus is also associated with B cell lym-
phoproliferative disorders, including Burkitt lymphoma,
Hodgkin lymphoma, and post-transplant lymphoprolifera-
tive disease harbouring latent EBV. Infection of B cells
with EBV in vitro in the absence of immune control is
associated with B cell proliferation and transformation,
indicating the oncogenic potential of EBV (Rickinson and
Kieff, 2001). Immunosuppression subsequent to organ
transplantation or secondary to infection with the human
immunodeficiency virus increases the risk of EBV-
associated lymphoproliferation (Cohen, 2000; Rickinson
and Kieff, 2001; Thorley-Lawson, 2001). Also immuno-
competent patients may develop Burkitt lymphomas and
Hodgkin lymphomas harbouring EBV.
Burkitt lymphoma harbouring EBV is mainly seen in
areas that are endemic for malaria leading to the specu-
lation that repeated immune activation by chronic malaria
or other infections is an important pathogenic factor for
this tumour (Rochford et al., 2005). Strikingly, young
Received 31 October, 2006; revised 21 February, 2007; accepted 28
February, 2007. *For correspondence. E-mail david.nadal@
kispi.unizh.ch; Tel. (+41) 44 2667562; Fax (+41) 44 2668072.
Present address: Department of Medicine, University of California,
San Francisco, CA 94110, USA.
These two authors contributed
equally.
Cellular Microbiology (2007) 9(8), 2055–2069 doi:10.1111/j.1462-5822.2007.00937.x
First published online 5 April 2007
© 2007 The Authors
Journal compilation © 2007 Blackwell Publishing Ltd
adults, experiencing IM and its vigorous immune activa-
tion to primary EBV infection, are at increased risk for
EBV-positive Hodgkin lymphoma (Hjalgrim et al., 2003).
Thus, immune activation seems to be a critical pathogenic
factor in EBV-associated lymphomagenesis. The impact
of activation via the innate immunity in this process is
largely unknown.
Toll-like receptors (TLRs) are key players in the innate
immunity. TLRs are transmembrane receptors related to
the TOLL protein of Drosophila (Hashimoto et al., 1988).
They are involved in the recognition of pathogens and
microbial products and activate antimicrobial effector path-
ways (Medzhitov, 2001). Among other TLRs, B cells
express TLR-9 (Hornung et al., 2002). TLR-9 sensors
unmethylated CpG (cytosine-guanosin) dinucleotides
within particular oligodeoxynucleotide sequences of micro-
organisms as well as the malaria pigment hemozoin
(Coban et al., 2005). While triggering TLR-9 increases
transformation rates of ex vivo EBV-infected B cells (Trag-
giai et al., 2004), its effect on the EBV gene expression
pattern is unknown.
Based on above-mentioned epidemiological and in vitro
Fig. 1. Epstein-Barr virus (EBV) expresses
lytic EBV mRNAs and proteins in cord blood
mononuclear cells (CBMC) but not in
EBV-seronegative peripheral blood
mononuclear cells (PBMC) infected ex vivo
with EBV.
A and B. mRNA expression of latent EBV
genes EBNA-1, EBNA-2, LMP-1 and LMP-2
in CBMC (A) and in PBMC (B).
C and D. mRNA expression of the
immediate-early lytic EBV gene BZLF-1 and
the late lytic EBV gene gp85 in CBMC (C)
and in PBMC (D).
E. Immunofluorescence of BZLF-1 protein
(green) in CBMC at 240 h post inoculation
with EBV.
F and G. Latent EBV infection rates in CBMC
(F) and PBMC (G).
H and I. Fraction of CD5
+
(H) and CD5
(I) B
cells in CBMC showing lytic EBV at 144 h
post inoculation with EBV.
mRNA expression was measured by real-time
PCR and normalized to the housekeeping
gene hydroxymethylbilane synthase (HMBS).
Values are expressed as means SD
induction of mRNA expression (fold) over
baseline mRNA expression. No transcription
was set to a value of 0.005 log
10
as
normalization to the expression of HMBS with
a cycle threshold value of 40 (i.e. no
transcription at cycle 40 of amplification) was
always below 0.01 log
10.
The dashed lines
indicate the lower limit of detection. Detection
of BZLF-1 was done by indirect
immunofluorescence staining in the nucleus
(blue) of approximately 1 in 10
6
mononuclear
cells in CBMC infected ex vivo with EBV.
Nuclear staining with DAPI. Scale bar: 15 mm.
The EBV infection rates were monitored by
flowcytometry using B95.8EBfaV-GFP, a
recombinant EBV encoding enhanced green
fluorescent protein, and a PE-labelled
anti-human CD19 antibody. Flow cytometry
detection of the late lytic EBV glycoprotein
gp350/220 after inoculation with B95.8 was
performed using a FITC-labelled anti-EBV
gp350/220 antibody, a PE-labelled anti-human
CD19 antibody and a Cy5-labelled anti-human
CD5 antibody to assess the susceptibility of B
cell subpopulations to lytic EBV infection.
2056 K. Ladell et al.
© 2007 The Authors
Journal compilation © 2007 Blackwell Publishing Ltd, Cellular Microbiology, 9, 2055–2069
observations, we hypothesized that immune activation
affects EBV gene expression. We tested our hypothesis
by activating cord blood mononuclear cells (CBMC) and
peripheral blood mononuclear cells (PBMC) from adults
acutely infected ex vivo with EBV, and chronically EBV-
infected Akata Burkitt lymphoma cells. The rationale to
use CBMC was its minimal immune activation and matu-
rity status compared with PBMC from adults (Bradley and
Cairo, 2005). We avoided bias from pre-existing EBV-
specific T-cell immunity by using primary cells only from
EBV-naive donors.
Results
Epstein-Barr virus expresses BZLF-1 and gp85 in
CBMC, but not in PBMC, after EBV infection ex vivo
We hypothesized that CBMC and adult PBMC, given
their different degrees of immune activation and matu-
ration, display distinct EBV gene expression patterns
after EBV infection. Using flow cytometry, we first veri-
fied that CBMC show a lower degree of immune activa-
tion than adult PBMC by assessing the proportion of
CD4
+
and CD8
+
cells expressing HLA-DR. Indeed, in
CBMC (n = 10 donors), the percentages of CD4
+
/HLA-
DR
+
and CD8
+
/HLA-DR
+
cells were 0.8 0.5 and
0.8 0.2, respectively, and in PBMC (n = 4 donors),
they were 11.5 0.4 and 20.5 3.6 respectively. Next,
to test our hypothesis, we quantified latent (EBNA-1,
EBNA-2, LMP-1 and LMP-2) and lytic (BZLF-1, the ini-
tiator of EBV lytic infection, and gp85, a late lytic gene)
EBV gene mRNA expression in CBMC and adult PBMC
after infection with EBV ex vivo. We used CBMC to
model a state of immature and less vigorous immune
responses than in adolescents or adults, and we used
PBMC from EBV-naive individuals to prevent potential
influences on EBV gene mRNA expression by pre-
existing EBV-specific immunity. We measured EBV gene
mRNA expression levels by real-time polymerase chain
reaction (PCR) and normalized to levels of the house-
keeping gene hydroxymethylbilane synthase (HMBS) at
0, 2, 24, 48, 72, 144 and 168 h after in vitro EBV inocu-
lation of CBMC or PBMC. Similar levels of the latent
genes EBNA-1, EBNA-2, LMP-1 and LMP-2 were
detected 24 h after EBV inoculation of CBMC or PBMC
(Fig. 1A and B), documenting successful infection with
EBV. Levels of EBNA-2 mRNA tended to be higher than
those of EBNA-1, and mRNA levels of these two genes
were higher than those of LMP-1, and LMP-2 both in
CBMC and PBMC. By contrast, significant mRNA
expression of the lytic genes BZLF-1 and gp85 was con-
sistently observed in CBMC (Fig. 1C) at and after 72 h
post inoculation of EBV, but was never seen in adult
PBMC (Fig. 1D). Accordingly, BZLF-1 protein was
detected by immunofluorescence in CBMC (Fig. 1E), but
not in PBMC (not shown). It is known that EBV lytic
cycle coincides with host shutoff mediated through
Fig. 1. cont.
Suppression of initiation of lytic EBV 2057
© 2007 The Authors
Journal compilation © 2007 Blackwell Publishing Ltd, Cellular Microbiology, 9, 2055–2069
mRNA degradation (Glaunsinger and Ganem, 2006). If
that concerns the housekeeping gene HMBS, we used
to normalize our data, this could lead to an overestima-
tion of the lytic EBV genes. We found that the cycle
threshold (Ct) values for HMBS mRNA expression in
CBMC (n = 6) following ex vivo infection with EBV were
rather constant in the first 72 h and showed a slight
decrease thereafter (not shown), indicating that the
abundance of HMBS mRNA expression is not diminish-
ing but rather increasing.
The fractions of B cells latently infected with EBV in
CBMC and PBMC following ex vivo infection are similar,
and CD5
+
and CD5
B cell subsets in CBMC are equally
susceptible to lytic EBV
We asked whether the difference in lytic EBV gene
expression between CBMC and PBMC was due to dif-
ferent EBV infection rates. Thus, we estimated the frac-
tions of latently EBV-infected B cells following ex vivo
infection using an enhanced green fluorescent protein
expressing B95.8 EBV, EBfaV-GFP (Speck and Long-
necker, 1999). In separate experiments we documented
that ex vivo infection with EBfaV-GFP resulted in quali-
tative and quantitative latent EBV gene expression pat-
terns in CBMC and PBMC similar to those observed
following ex vivo infection with B95.8 (M. Dorner et al.,
manuscript in preparation), suggesting that EBfaV-GFP
is a valid substitute of B95.8. The fraction of CD19+
B cells in CBMC (n = 3) and PBMC (n = 3) at baseline
was 11.6 2.4% and 7.3 2.3% respectively. The
overall EBV infection rates were similar in CBMC and
PBMC in the first 144 h after EBV inoculation ex vivo
when they reached around 1% in relation to all cells and
around 4–5% of CD19
+
B cells (Fig. 1F and G). To
assess the fractions of lytically infected cells we stained
the cells for the late lytic glycoprotein gp350/220 which
is expressed on the plasma membrane (Gong and Kieff,
1990) following ex vivo infection with 95.8 EBV. Using
flow cytometry we documented that the proportion of B
cells exhibiting lytic EBV infection peaked between 2
and 3% at 144 h post EBV inoculation in CBMC while no
cells expressing lytic EBV were found in PBMC (not
shown). The vast majority of B cells in CBMC (n = 3)
belonged to the CD5
+
B cell subset (74.17 8.15%),
whereas in adult PBMC the minority of B cells were
CD5
+
(28.81 11.16%). To evaluate whether the sus-
ceptibility of these B cell subsets in CBMC to lytic EBV
is different, we determined the numbers of CD5
+
and
CD5
B cells staining for gp350/220. Flow cytometry
showed that the numbers of CD5
+
B cells in CBMC
(n = 3) were 78 5% and that the proportions of CD5
+
and CD5
B cells expressing gp 350/220 in CBMC
(n = 3) were similar (11.5 3.2% vs. 12.8 3.8%) at
144 h following ex vivo infection (Fig. 1H and I), indicat-
ing comparable susceptibility to EBV lytic infection.
Thus, although the fractions of B cells infected with EBV
in CBMC and PBMC following ex vivo infection were
similar, CBMC exhibited lytic EBV infection whereas
PBMC did not. The expression of gp350/220 clearly indi-
cates that the lytic infection is not only initiated (Laichalk
and Thorley-Lawson, 2005) but is also fully executed in
CBMC. Furthermore, the difference between CBMC and
PBMC in lytic EBV gene expression cannot be attributed
to the higher content of CD5
+
cells in CBMC than in
PBMC, because CD5
+
and CD5
cells were equally sus-
ceptible to lytic EBV infection.
Cord blood mononuclear cells express lower levels of
IL-12 p35, IFN-g and IL-2 mRNA than PBMC at baseline
and in response to EBV
Because CBMC and adult PBMC represent immune cells
with dissimilar states of immune maturation with different
abilities to express cytokines, we asked whether the dis-
tinct EBV gene expression in CBMC and PBMC was
associated with differing cytokine gene expression. We
compared mRNA levels of proinflammatory cytokines in
CBMC and PBMC before and after EBV infection in vitro.
CBMC displayed lower mRNA levels of IL-12 p35
(P = 0.0001), IFN-g (P < 0.0075) and IL-2 (P < 0.001) than
PBMC at baseline (Fig. 2). mRNA levels of TNF-a, IL-1b,
IL-6 and IL-8 did not significantly differ between CBMC
and PBMC at baseline (not shown).
Inoculation with EBV led to increased mRNA levels of
IL-12 p35, IFN-g and IL-2 in CBMC and adult PBMC.
However, mRNA levels in CBMC never reached the
levels observed in adult PBMC, and significant differences
between CBMC and adult PBMC were also found after
inoculation with EBV (Fig. 3). Because host shutoff medi-
ated through mRNA degradation during EBV lytic gene
expression may result in false high positive cytokine mRNA
levels, we also measured IL-12, IFN-g and IL-2 at the
protein level. At 96 h after inoculation with EBV, protein
levels of IL-12 p40, IFN-g and IL-2 were 11.4 1.5 pg ml
-1
,
6.5 2.1 pg ml
-1
and 0.8 0.6 pg ml
-1
, respectively, in
CBMC supernatants (n = 4) versus 22.5 2.3 pg ml
-1
,
34.5 7.8 pg ml
-1
and 7.6 1.7 pg ml
-1
, respectively, in
PBMC supernatants (n = 4). Overall, mRNA levels of IL-12
p35, IFN-g and IL-2 were lower in CBMC than adult PBMC
before EBV infection ex vivo and both mRNA and protein
levels of these cytokines were also lower in CBMC than
adult PBMC after EBV infection ex vivo, suggesting that
differences in expression levels of these proinflammatory
cytokines may be due to maturational differences in cytok-
ine responses and the degree of immune activation
or maturation may influence the initiation of lytic EBV
infection.
2058 K. Ladell et al.
© 2007 The Authors
Journal compilation © 2007 Blackwell Publishing Ltd, Cellular Microbiology, 9, 2055–2069
The lower levels of IL-12, IFN-g and IL-2 in CBMC than
in PBMC in response to EBV are not associated with
higher levels of TGF-b or IL-10 mRNA
To explore whether lower levels of proinflammatory cytok-
ines in CBMC than PBMC after infection with EBV were
associated with higher mRNA levels of anti-inflammatory
cytokines in CBMC than in PBMC, we measured mRNA
expression of the anti-inflammatory cytokine genes TGF-b
and IL-10. Importantly, the assay we used to detect
human IL-10 is highly host specific and does not detect
EBV-encoded viral IL-10. At baseline, mRNA levels of
TGF-b were lower whereas levels of IL-10 were higher in
CBMC than in PBMC (Fig. 4). Following infection with
EBV, levels of TGF-b remained lower in CBMC compared
with in PBMC. By contrast, levels of IL-10 became signifi-
cantly lower in CBMC than in PBMC after EBV inoculation
in vitro (Fig. 4). These findings strongly indicate that the
lower levels of induction of IL-12, IFN-g and IL-2 in CBMC
than in PBMC after inoculation with EBV are not due to
higher expression of TGF-b or IL-10 mRNA in CBMC than
in PBMC.
To investigate the effects of IL-10 on lytic EBV infection,
we treated CBMC (n = 3) with recombinant human IL-10
at 1, 10, or 100 pg ml
-1
. Adding IL-10 did not result in
significant changes in BZLF-1 mRNA expression following
EBV infection ex vivo (not shown). Similarly, neutralizing
IL-10 in PBMC from EBV-seronegative adults (n = 3) with
anti-human IL-10 antibody at 1.0 U ml
-1
did not result in
BZLF-1 mRNA expression following EBV infection ex vivo
(not shown). Thus, IL-10 levels appear to have no effect
on EBV lytic gene expression patterns.
rIL-12 and rIFN-g decrease BZLF-1 mRNA expression in
CBMC during EBV infection in vitro
Next, we asked whether IL-12 or IFN-g have an effect on
BZLF-1 expression in CBMC after infection with EBV.
We infected CBMC with EBV ex vivo, treated with rIL-
12, rIFN-g or both, and measured mRNA expression
96 h after infection when BZLF-1 is detectable in all of
the EBV-infected untreated CBMC cultures (Fig. 5). In
CBMC treated with rIL-12 simultaneously with EBV
inoculation and then every 24 h, IL-12 p35 mRNA
expression was unchanged, whereas IFN-g mRNA
expression increased about 6.5-fold at 96 h, and BZLF-1
mRNA expression level was reduced by 50%, compared
with untreated CBMC (Fig. 5). As expected, treatment of
CBMC with rIL-12 also increased the IFN-g protein con-
centration in the cell-free supernatant of CBMC com-
pared with no treatment (1368 vs. 37 pg ml
-1
). This
higher increase in protein concentration compared with
the increase in mRNA expression may be explained by
either accumulation of transcribed protein in the super-
Fig. 2. CBMC express significantly lower mRNA levels of
proinflammatory cytokine genes than PBMC at baseline.
A. IL-12 p35.
B. IFN-g.
C. IL-2.
CBMC (n = 19) and PBMC (n = 20) were isolated by
density-gradient centrifugation. RNA was extracted from cell pellets
and treated with DNase to remove residual genomic DNA. The
mRNA was reverse transcribed into cDNA using an oligo-d(T)
15
primer. mRNA expression was measured by real-time PCR and
normalized to the housekeeping gene HMBS. Median values (solid
black line) of fold mRNA expression in relation to HMBS are shown
as box plots with whiskers that extend to the highest and lowest
values above and below the box. The dashed lines indicate the
lower limit of detection. * refers to CBMC versus PBMC.
Suppression of initiation of lytic EBV 2059
© 2007 The Authors
Journal compilation © 2007 Blackwell Publishing Ltd, Cellular Microbiology, 9, 2055–2069
natant or expressed mRNA being transcribed to protein
at higher rates, or both. Treatment of CBMC with rIFN-g,
simultaneously with EBV inoculation and then every
24 h, did not change IFN-g mRNA expression and did
not influence IL-12 p35 mRNA expression, but reduced
BZLF-1 mRNA expression by 50% compared with
untreated CBMC (Fig. 5). Finally, treatment with both
rIL-12 and rIFN-g simultaneously with EBV inoculation
and then every 24 h, did not change IL-12 p35 mRNA
expression, increased IFN-g mRNA expression around
17-fold, and resulted in a stronger suppression (sixfold)
of BZLF-1 mRNA expression than when treatment
included only one of both cytokines (Fig. 5). Conversely,
we treated adult PBMC infected with EBV ex vivo with
antibodies to IL-12 and IFN-g and could not provoke
BZLF-1 mRNA expression (not shown). Thus, substitu-
tion of the proinflammatory cytokines IL-12 and IFN-g
partially suppressed BZLF-1 mRNA expression in CBMC
infected with EBV ex vivo, indicating that the weaker
proinflammatory immune response in CBMC contributes
to the initiation of lytic EBV infection seen in CBMC. The
failure to provoke BZLF1 mRNA expression in acutely
infected PBMC with antibodies to IL-12 and IFN-g
together with their incomplete suppression of BZLF-1
mRNA expression suggests that these two cytokines are
Fig. 3. CBMC express significantly lower mRNA levels of
proinflammatory cytokine genes than PBMC from EBV-seronegative
adults following infection with EBV ex vivo.
A. IL-12 p35.
B. IFN-g.
C. IL-2.
mRNA expression was measured by real-time PCR and normalized
to the housekeeping gene HMBS. Results are means SD of
mRNA expression normalized to HMBS (fold) during 7 days of
culture. The dashed lines indicate the lower limit of detection. * or
** refers to CBMC (n = 3–8) versus PBMC (n = 3).
Fig. 4. CBMC express lower mRNA levels of anti-inflammatory
cytokine genes than PBMC from EBV-seronegative adults in
response ex vivo infection with EBV.
A. TGF-b.
B. IL-10.
mRNA expression was measured by real-time PCR and normalized
to the housekeeping gene HMBS. Results are means SD of
mRNA expression normalized to HMBS (fold) during 7 days of
culture. * refers to CBMC (n = 3–8) versus PBMC (n = 3).
2060 K. Ladell et al.
© 2007 The Authors
Journal compilation © 2007 Blackwell Publishing Ltd, Cellular Microbiology, 9, 2055–2069
not the only players suppressing the initiation of lytic
EBV infection.
CpG ODN 2006 suppress BZLF-1 mRNA expression in
CBMC infected with EBV in vitro
We next sought to test whether other means of immune
stimulation would lead to suppression of BZLF-1 expres-
sion in CBMC. To stimulate CBMC we used the unm-
ethylated CpG-containing ODN 2006 that triggers the
innate pathogen-associated molecular pattern recogni-
tion receptor TLR-9 which is also expressed in B cells
(Hornung et al., 2002) and exerts a proinflammatory
effect (Peng, 2005). We asked if stimulating CBMC with
CpG ODN 2006 alters EBV gene expression. Thus, we
cultured CBMC with or without CpG ODN 2006 and with
or without EBV for 96 h respectively. Treatment of
uninfected CBMC with CpG ODN 2006 resulted in a 2.6-
fold increase of TLR-9 mRNA expression versus no
treatment (Fig. 6A). EBV infection by itself led to 3.7-fold
increase in levels of TLR-9 mRNA expression in CBMC
over uninfected CBMC (Fig. 6B). The large number of
CpG motifs in the EBV DNA genome or a CpG-motif-
independent mechanism may explain the upregulation of
TLR-9 by EBV. Treatment of EBV-inoculated CBMC with
CpG ODN 2006 resulted in a further but not significant
increase of TLR-9 mRNA expression (Fig. 6B). As
expected, inoculation of CBMC with EBV resulted in
marked expression of BZLF-1 mRNA. By contrast, EBV-
infected CBMC cultures treated with CpG ODN 2006
exhibited a 5.8-fold lower BZLF-1 mRNA expression
(Fig. 6C). Although EBV itself induced TLR-9 mRNA
expression in CBMC, the induction did not suppress
BZLF-1 mRNA expression. Therefore, the reduction of
BZLF-1 mRNA expression in EBV-infected CBMC after
CpG ODN 2006 treatment did not seem to depend on
induction of TLR-9, but rather on the additional stimula-
tion by CpG ODN 2006 (e.g. increased TLR-9 signalling
mediated by CpG binding). Expression levels of latent
EBV gene mRNAs were not significantly different in
untreated or CpG ODN 2006-treated EBV-infected
CBMC (not shown).
Next, we asked whether triggering of other TLRs
present on B cells also results in suppression of lytic EBV.
Triggering of TLR-1/2, TLR-4, or TLR-7/8 on EBV-infected
CBMC did not result in significant suppression of BZLF-1
and gp85 mRNA expression and gp350/220 expression
compared with controls (Fig. 6D–F). These data suggest
that CpG ODN 2006 stimulation of TLR-9 on EBV-infected
CBMC is rather specific in inhibiting the mRNA and
protein expression of EBV genes involved in lytic infection
but has no effect on latent EBV gene mRNA expression.
This suppression of the initiation and completion of lytic
EBV infection in turn may support maintenance of EBV
latency.
Antibodies to IL-12 and IFN-g partially restore BZLF-1
mRNA expression in EBV-infected CBMC treated with
CpG ODN 2006
We next explored whether IL-12 and IFN-g contribute to
the suppression of BZLF-1 mRNA expression induced
after TLR-9 triggering by CpG ODN 2006. We added
antibodies to IL-12 and IFN-g to cultures of CBMC inocu-
lated with EBV and treated with CpG ODN 2006.
Indeed, treatment with anti-IL-12 and anti-IFN-g partially
restored expression of BZLF-1 mRNA in these CBMC
Fig. 5. rIL-12, rIFN-g, or both suppress the transcription of BZLF-1
in CBMC infected with EBV ex vivo. rIL-12, rIFN-g, or both were
added together with EBV and then every 24 h over 96 h to the
cultures. mRNA was measured by real-time PCR. Results shown
are from one representative experiment of six from CBMC from
different donors. RNA was extracted and analysed from four
different cell pellets per condition, except for the treatments with
rIFN-g (two cell pellets per condition). Means SD represent the
differences of mRNA expression between treated and untreated
samples after normalization to the housekeeping gene HMBS.
Suppression of initiation of lytic EBV 2061
© 2007 The Authors
Journal compilation © 2007 Blackwell Publishing Ltd, Cellular Microbiology, 9, 2055–2069
(Fig. 7A). Even though these antibodies exhibited little
effect on CpG ODN 2006-induced enhanced IL-12 p35
and IFN-g mRNA expression (Fig. 7B and D), the protein
levels of both cytokines were below the lower limit of
detection (Fig. 7C and E). These data at the protein
level excluded potentially misleading results due to host
shutoff mediated through mRNA degradation during EBV
lytic gene expression. Thus, our observations provide
evidence that part of the negative impact on the initiation
of lytic EBV infection in CBMC exhibited by CpG ODN
2006 through TLR-9 triggering is mediated by IL-12 and
IFN-g.
A key question is which cells are implicated in the
effects observed. Thus, we infected highly purified B cells
from CBMC and PBMC with EBV. Indeed, BZLF-1 was
expressed in B cells from CBMC but not from PBMC.
mRNA and protein levels for IL-12 and IFN-g were strik-
ingly lower in B cells from CBMC than from PBMC
(Fig. 7F–O). Moreover, we could reproduce the inhibitory
effects on BZFL-1 expression when triggering TLR-9
Fig. 6. CpG ODN 2006 specifically suppresses initiation and execution of lytic EBV in CBMC following ex vivo infection.
A. mRNA expression of TLR-9 in CBMC (n = 3) treated or not with CpG ODN 2006.
B. mRNA expression of TLR-9 in CBMC (n = 3) infected ex vivo with EBV and treated or not with CpG ODN 2006.
C. mRNA expression of BZLF-1 in CBMC (n = 3) infected ex vivo with EBV and treated or not with CpG ODN 2006.
D and E. mRNA expression of BZLF-1 (D) and EBV glycoprotein (gp) 85 (E) in CBMC (n = 3) that were stimulated with ligands of TLRs
present in B cells and infected ex vivo.
F. Expression of the lytic EBV glycoprotein gp350/220 in CBMC treated with ligands of TLRs present in B cells and infected ex vivo.
TLR ligands were added at 0 h and 90 h to 2 ¥ 10
6
CBMC (n = 3) infected ex vivo with EBV. Cells were collected at 96 h. Concentrations of
TLR ligands were 10 mgml
-1
for peptidoglycan (TLR-1/2), 3 mM for R-848 (TLR-7/8) and 1 mM for CpG ODN 2006 (TLR-9). RNA was extracted
from two cell pellets per condition, treated with DNase, and reverse-transcribed into cDNA with an oligo-dT15 primer. mRNA expression was
measured in duplicate by real-time PCR. Means SD of fold induction over resting normalized to the housekeeping gene HMBS.
Flowcytometry was performed using a FITC-anti-EBV gp350/220 antibody. Events shown are gated for CD19
+
B cells. One representative
experiment of three is shown.
2062 K. Ladell et al.
© 2007 The Authors
Journal compilation © 2007 Blackwell Publishing Ltd, Cellular Microbiology, 9, 2055–2069
similar as outlined above. TLR-9 triggering was associ-
ated with an increase in IL-12 and IFN-g at the mRNA as
well as protein level. Thus, the effects we observed when
triggering TLR-9 are rather direct than indirect.
CpG ODN 2006 suppresses induction of BZLF-1 mRNA
expression in Akata Burkitt lymphoma cells
The above experiments addressed the effect of immune
stimulation triggered by cytokines and TLR-9 on the
initiation of lytic EBV infection in cells exposed to acute
infection with EBV, but not in cells with chronic latent EBV
infection. Switching from latent to lytic EBV infection may
occur spontaneously or be provoked in EBV-transformed
cells by several agents in vitro (Kieff and Rickinson,
2001). Cells from the Burkitt lymphoma cell line Akata can
readily be provoked to switch from latent to lytic EBV
infection within hours by cross-linking their surface IgG
using anti-IgG antibodies. Thus, we asked whether trig-
gering of TLR-9 exhibits an effect on the induction of lytic
EBV infection in Akata cells, used as a surrogate for
Burkitt lymphoma cells. We first determined whether
Akata cells express TLR-9. Using quantitative PCR, we
demonstrated that Akata cells constitutively express
TLR-9 mRNA. Stimulation of Akata cells with CpG ODN
2006 did not increase TLR-9 mRNA expression (Fig. 8A).
This suggested that TLR-9 expression in the fully differ-
entiated Akata cells was maximal before treatment with
CpG ODN 2006 as opposed to CBMC which contain
naive B cells and showed an increase in TLR-9 mRNA
expression upon stimulation with CpG ODN 2006
(Fig. 6A). As expected, cross-linking of surface IgG after
treatment with anti-IgG provoked the expression of
BZLF-1 mRNA and thus the initiation of lytic EBV infection
(Fig. 8B). Treatment of Akata cells with CpG ODN 2006
before treatment with anti-IgG reduced BZLF-1 mRNA
expression provoked by surface IgG cross-linking by 50%
(Fig. 8B). By contrast, treatment with CpG ODN 2006
simultaneously or deferred to anti-IgG treatment had no
significant effect on the initiation of lytic EBV infection (not
shown); indicating that the signalling cascade initiated by
anti-IgG appears to be dominant to the intracellular
changes subsequent to triggering TLR-9. These data
suggest that triggering innate immunity via TLR-9 sup-
presses the initiation of lytic EBV infection in transformed
B cells with established EBV latency and that this sup-
pression is independent from other immune cells express-
ing TLR-9.
Discussion
Immune activation may be a critical factor in EBV-
associated lymphomagenesis. In this work, we examined
the effect of immune activation on EBV gene expression.
We found that (i) EBV expresses BZLF-1, the initiator of
lytic EBV infection, and the late lytic genes gp85 and
gp350/220 in CBMC, but not in adult PBMC infected ex
vivo with EBV, (ii) lower levels of proinflammatory cytok-
ines in CBMC than in adult PBMC are associated with
expression of lytic EBV genes and (iii) triggering of TLR-9
suppresses lytic gene expression in CBMC acutely
infected ex vivo with EBV and in anti-IgG-stimulated
chronically infected Akata Burkitt lymphoma cells. Our
findings, indeed, identify immune activation as critical
factor for the suppression of lytic EBV infection.
We used CBMC to model a state of minimal immune
activation compared with PBMC from adults. Importantly,
by using primary cells only from EBV-naive individuals,
we avoided bias from pre-existing EBV-specific T-cell
responses, which may be triggered by ex vivo EBV
infection. In CBMC, BZLF-1 and gp85 mRNA expression
and gp350/220 protein expression showed a sharp rise
after ex vivo EBV infection that persisted over the entire
observation time. In adult PBMC, no BZLF-1, gp85 or
gp350/220 expression was seen at all, although the frac-
tions of B cells infected with EBV following ex vivo infec-
tion were similar in CBMC and PBMC. The difference in
lytic EBV gene expression cannot be attributed to the
higher content of CD5
+
cells in CBMC than in PBMC,
because CD5
+
and CD5
cells exhibited lytic EBV equally.
By contrast, mRNA expression patterns of latent EBV
genes were similar in CBMC and PBMC. Extending data
published by Hunt et al. (1994) the proinflammatory cytok-
ines IL-12, IFN-g and IL-2 were lower in CBMC than in
PBMC before EBV infection. Furthermore, levels of these
cytokines in CBMC did not increase to the levels seen in
PBMC in response to EBV. Based on these data, we
hypothesized that the higher levels of proinflammatory
cytokines in PBMC may result in the suppression of
BZLF-1 expression (i.e. that differences in the status of
immune activation/maturation are responsible for the pro-
found difference in EBV gene expression between CBMC
and PBMC).
The main sources of IL-12 are monocytes and dendritic
cells (DCs) (Trinchieri, 2003). As mentioned above,
CBMC produce less IL-12 than PBMC (Hunt et al., 1994),
and DC derived from neonatal monocytes transcribe
much less IL-12 p35 than adult monocytes (Goriely et al.,
2001). IFN-g produced by natural killer (NK) cells (Biron
et al., 1999) may, in part, be responsible for the IFN-g
production in CBMC upon EBV encounter in vitro (Wilson
and Morgan, 2002). The frequency of NK cells in CBMC
and PBMC is similar, but NK cells in CBMC have an
immature function compared with NK cells in PBMC
(Nomura et al., 2001). To determine if the immune
activation/maturation deficiencies in IL-12 and IFN-g
production indeed enable BZLF-1 mRNA expression in
CBMC cultures, we added rIL-12 and rIFN-g to the CBMC
Suppression of initiation of lytic EBV 2063
© 2007 The Authors
Journal compilation © 2007 Blackwell Publishing Ltd, Cellular Microbiology, 9, 2055–2069
2064 K. Ladell et al.
© 2007 The Authors
Journal compilation © 2007 Blackwell Publishing Ltd, Cellular Microbiology, 9, 2055–2069
cultures infected ex vivo with EBV. Indeed, BZLF-1 mRNA
expression in CBMC decreased significantly, albeit not
completely, with rIL-12 and rIFN-g. The incomplete sup-
pression of BZLF-1 mRNA expression may be explained
by immature cytokine receptor signalling pathways in
CBMC (Marodi, 2002) or the need of additional stimuli
operative in the innate immune responses (Medzhitov,
2001). Thus, activation of the immune system results in
efficient suppression of the initiation of lytic EBV infection.
The anti-inflammatory cytokine TGF-b induces lytic
infection in EBV-transformed CBMC-derived cell lines
(Liang et al., 2002). Thus, we explored the possibility that
Fig. 7. BZLF-1 mRNA expression in CpG ODN 2006-treated CBMC infected ex vivo with EBV is dependent on IL-12 and IFN-g expressed in
B lymphocytes.
A. mRNA expression of BZLF-1 in CBMC infected ex vivo with EBV and treated or not with CpG ODN 2006 and with or without anti-IL12 plus
anti-IFN-g blocking antibodies.
B. mRNA expression of IL-12p35 in CBMC infected ex vivo with EBV and treated or not with CpG ODN 2006 and with or without anti-IL12
plus anti-IFN-g antibodies.
C. IL-12p40 in supernatants of CBMC infected ex vivo with EBV and treated or not with CpG ODN 2006 and with or without anti-IL12 plus
anti-IFN-g antibodies.
D. mRNA expression of IFN-g in CBMC infected ex vivo with EBV and treated or not with CpG ODN 2006 and with or without anti-IL12 plus
anti-IFN-g antibodies.
E. IFN-g in supernatants of CBMC infected ex vivo with EBV and treated or not with CpG ODN 2006 and with or without anti-IL12 plus
anti-IFN-g antibodies.
F. mRNA expression of BZLF-1 in CD19
+
B cells isolated from cord blood infected ex vivo with EBV.
G. mRNA expression of IL-12p35 in CD19
+
B cells isolated from cord blood infected ex vivo with EBV and treated or not with CpG ODN 2006.
H. IL-12p40 in supernatants of CD19
+
B cells isolated from cord blood infected ex vivo with EBV and treated or not with CpG ODN 2006.
I. mRNA expression of IFN-g in CD19
+
B cells isolated from cord blood infected ex vivo with EBV and treated or not with CpG ODN 2006.
J. IFN-g in supernatants of CD19
+
B cells isolated from cord blood infected ex vivo with EBV and treated or not with CpG ODN 2006.
K. mRNA expression of BZLF-1 in CD19
+
B cells isolated from peripheral blood infected ex vivo with EBV.
L. mRNA expression of IL-12p35 in CD19
+
B cells isolated from peripheral blood infected ex vivo with EBV and treated or not with CpG ODN
2006.
M. IL-12p40 in supernatants of CD19
+
B cells isolated from peripheral blood infected ex vivo with EBV and treated or not with CpG ODN
2006.
N. mRNA expression of IFN-g in CD19
+
B cells isolated from peripheral blood infected ex vivo with EBV and treated or not with CpG ODN
2006.
O. IFN-g in supernatants of CD19
+
B cells isolated from peripheral blood infected ex vivo with EBV and treated or not with CpG ODN 2006.
CpG ODN 2006 (1 mM) was added at 0 and 90 h to the EBV-containing culture medium of 2 ¥ 10
6
CBMC. Anti-IL-12 and anti-IFN-g antibodies
were given to the cultures 1 h before stimulation with CpG ODN 2006. Cells were collected at 96 h. RNA was extracted from two cell pellets
per condition, treated with DNase, and reverse-transcribed into cDNA with an oligo-dT15 primer. mRNA expression was measured in duplicate
by real-time PCR. Results are means SD of fold induction over resting normalized to the housekeeping gene HMBS. The dashed lines
indicate the lower limit of detection. One representative experiment of two is shown.
Fig. 8. CpG ODN 2006 suppresses induction of BZLF-1 mRNA expression in Akata Burkitt lymphoma cells provoked to switch to lytic EBV
infection.
A. Expression of TLR-9 mRNA before and after stimulation with CpG ODN 2006, anti-IgG, or both.
B. Expression of BZLF-1 mRNA before and after stimulation with CpG ODN 2006, anti-IgG, or both.
Akata cells (1.0 ¥ 10
6
each) were seeded and treated with or without CpG ODN 2006 (0.5 mM) After 6 h 0.1 mg ml
-1
polyclonal rabbit
anti-human IgG was added to the cultures. Cells were collected at 6 h after anti-IgG treatment. RNA was extracted from one cell pellet per
condition, treated with DNase, and reverse-transcribed into cDNA with an oligo-dT15 primer. mRNA expression was measured in duplicate by
real-time PCR. Results are means SD of fold induction over resting normalized to the housekeeping gene HMBS from three independent
experiments.
Suppression of initiation of lytic EBV 2065
© 2007 The Authors
Journal compilation © 2007 Blackwell Publishing Ltd, Cellular Microbiology, 9, 2055–2069
the lower levels of IL-12, IFN-g and IL-2 in CBMC than in
PBMC were coupled to higher mRNA levels of TGF-b.
However, TGF-b mRNA expression was lower in CBMC
than in PBMC irrespective of ex vivo EBV infection,
making a contribution of TGF-b to BZLF-1 expression in
CBMC highly unlikely. Another possible reason for the
lower IL-12, IFN-g and IL-2 levels in CBMC than in PBMC
could have been increased levels of the anti-inflammatory
cytokine IL-10 (Wang et al., 1994). The lower mRNA
levels of IL-10 in CBMC in response to EBV infection
compared with in PBMC, however, argued against IL-10
being responsible for the lower levels of proinflammatory
cytokines in CBMC. Notably, adding or blocking IL-10 had
no effect on lytic EBV gene expression patterns.
We wanted to verify our observation that activation of
the immune system results in suppression of BZLF-1 by
activating the innate immune response triggering TLR-9.
Indeed, triggering of innate immunity via TLR-9 with CpG
ODN 2006 resulted in suppression of BZLF-1 but not
latent EBV gene mRNA expression in acutely ex vivo
EBV-infected CBMC. Notably, triggering TLR-9 resulted in
higher transformation rates of B cells infected ex vivo with
EBV (Traggiai et al., 2004), but the effect of stimulating
TLR-9 of B cells on EBV gene expression was not
investigated. Thus, the molecular mechanism(s) resulting
in the more efficient transformation rate of B cells when
triggering TLR9 may be due to reduction of initiation of
lytic EBV infection and thereby reinforce maintenance of
EBV latency.
Our results seem to be in conflict with the findings of Liu
et al. (2005) and Lim et al. (2007). Liu et al. (2005)
reported that truncated thioredoxin (Trx80) inhibits B cell
growth in EBV infected CBMC through T cells activated by
monocyte derived IL-12. They assessed B cell transfor-
mation by EBV by measuring the thymidine incorporation
on the 12th day. Patterns of EBV latent and lytic gene
expression were not investigated. In contrast, our experi-
ments focusing on acute ex vivo EBV infection were
limited to 7 days. Through the use of isolated B cells in
selected experiments we showed that IL-12 derived from
B cells mediated suppression of lytic EBV. We did not
assess B cell transformation. Thus, the results of these
two studies cannot be directly compared due to the differ-
ent experimental settings used; they are not mutually
contradictory. Future experiments may resolve this
enigma. Furthermore, Lim et al. (2007), reported that
human plasmacytoid DCs regulated immune responses
to EBV in humanized NOD-SCID mice resulting in
delayed EBV-related mortality. From indirect proof using
an inhibitor for triggering TLR-9 they concluded that
TLR-9 in part mediated activation of plasmacytoid DCs
resulting in anti-EBV-active CD3
+
T cells. In this study,
PBMC from EBV-seropositive donors were used; thus, the
protective effect observed is most likely due to the boost-
ing effect of an adaptive EBV-specific cellular immune
response.
Next, we addressed the question whether TLR-9 trig-
gering affects on EBV in chronically infected cells. Chroni-
cally EBV-infected cells express latent EBV genes and
only very rarely lytic EBV genes. To assess the effects of
triggering of TLR-9 on lytic EBV in chronically EBV-
infected cells, we used Akata Burkitt lymphoma cells,
which undergo lytic EBV infection upon anti-IgG stimula-
tion (Kieff and Rickinson, 2001). Similarly to ex vivo
acutely infected B cells, TLR-9 triggering suppresses anti-
IgG-induced BZLF-1 expression in Akata cells. This result
also indicates that triggering TLR-9 directly affects the
EBV gene expression pattern and is not a consequence of
indirect effects due to stimulation of other cellular subsets.
Of note in this context, Plasmodium falciparum malaria
pigment hemozoin also stimulates TLR-9 (Coban et al.,
2005). Children in areas endemic for both EBV-positive
Burkitt lymphoma and malaria are dually infected with
EBV and malaria very early in life (Rochford et al., 2005).
We show that suppression of lytic EBV via TLRs on and in
B cells is specifically linked to triggering of TLR-9 and that
suppression of lytic EBV occurs following direct triggering
of TLR-9 in B cells. Thus, repeated activation of the innate
immunity via TLR-9 (e.g. due to chronic malaria infection)
may foster the propagation of latently EBV-infected cells
by suppressing lytic EBV infection and thus development
of Burkitt lymphoma.
We and others have documented increased plasma
EBV DNA levels in patients with IM or EBV-associated
lymphoproliferative diseases in immunocompetent and
immunodeficient patients (Berger et al., 2001; Ryan et al.,
2004) as well as in individuals with malaria (Moormann
et al., 2005; Donati et al., 2006). Plasma EBV DNA is
sensitive to DNase; this indicates that it is not encapsi-
dated and does not originate from lytic infection but rather
from dying latently infected cells (Ryan et al., 2004;
Donati et al., 2006). Moorman et al. also found elevated
EBV DNA blood levels in children with malaria and sug-
gested as likely reasons an increased frequency of
latently EBV-infected cells, indirectly due to polyclonal B
cell activation or due to suppression of EBV-specific
immunity, and that recurrent malaria infections affect
either the establishment or maintenance of EBV latency
(Moormann et al., 2005). Notably, no BZLF-1 transcription
is found in PBMC from IM patients (Tierney et al., 1994)
with high serum levels of IL-12, IFN-g and IL-2 (Corsi
et al., 2004). This is in line with our findings showing
IL-12- and IFN-g-mediated suppression of BZLF-1
expression. Our findings are further supported by the
observations that IFN-g blocks gammaherpesvirus reacti-
vation from latency (Steed et al., 2006) and nuclear factor
kB, activated downstream of TLR-9, inhibits gammaherp-
esvirus lytic replication (Brown et al., 2003). Thus, we
2066 K. Ladell et al.
© 2007 The Authors
Journal compilation © 2007 Blackwell Publishing Ltd, Cellular Microbiology, 9, 2055–2069
hypothesize that in states of increased immune activation
propagation of EBV is due to promotion of latent rather
than of lytic EBV infection. Because control of EBV infec-
tion may substantially differ between tissue compartments
(Hislop et al., 2005; Donati et al., 2006), in states of
immune activation or cellular immune compromise lytic
EBV infection may be confined to tissues at mucosal
surfaces with excretion of EBV particles.
Experimental procedures
Isolation of mononuclear cells and cell culture
Cord blood mononuclear cells and PBMC from healthy adult
EBV-seronegative donors were obtained from heparinized blood
by Ficoll-Hypaque (Amersham Biosciences Europe GmbH,
Otelfingen, Switzerland) gradient centrifugation. Cells were
washed with phosphate-buffered saline (Gibco, Invitrogen Life
Sciences, Basel, Switzerland). The EBV-producing cell lines
B95.8 (Miller and Lipman, 1973) and B95.8EBfaV-GFP (Speck
and Longnecker, 1999), CBMC, PBMC and Akata (Takada, 1984)
cells were cultured in RPMI 1640 supplemented with Hepes
buffer,
L-glutamine, 10% fetal bovine serum, 1 mM sodium pyru-
vate, 1 mM non-essential amino acids, 100 U ml
-1
penicillin and
100 mgml
-1
streptomycin sulphate (medium and supplements
from Gibco). Informed consent was obtained from subjects or
parents before the study. The institutional ethics committee
approved the collection and use of clinical material.
Isolation of B cells from CBMC and PBMC
B cells were isolated from CBMC or PBMC by the use of mag-
netic beads (Miltenyi Biotech, Bergisch-Gladbach, Germany)
according to the manufacturer’s instructions. Purity of isolated B
cells was determined by flow cytometry using anti-human CD19,
and anti-human CD3 antibodies for the detection of B cells and
eventually remaining T cells. The purity of each separation was
above 97%.
Epstein-Barr virus infections ex vivo
After resting overnight, CBMC or PBMC (1 ¥ 10
7
cells) were
infected with supernatants from B95.8 cells or B95.8EBfaV-GFP
(1 ¥ 10
6
ml
-1
) harvested on day 4 after splitting and filtered using
a 0.45 mm sterile filter (Millipore, Cork, Ireland). The cell-free
supernatants contained approximately 7 log
10
EBV copies ml
-1
,
as evaluated by real-time PCR for EBV DNA (Berger et al., 2001).
Infections were performed as described (Tosato, 1991). Briefly,
cells were centrifuged, resuspended in 2.5 ml of RPMI and 2.5 ml
of B95.8 supernatant, and incubated in 50 ml conical Falcon
tubes (BD Biosciences, Basel, Switzerland) at 37°C in a water
bath for 2 h. Subsequently, 5 ml of RPMI 1640 were added, and
1 ml aliquots (1 ¥ 10
6
cells ml
-1
) were seeded into 24-well plates
(BD Biosciences). Cell pellets were centrifuged at 300 g, frozen
on dry ice, and stored at -80°C.
Assessment of EBV and cytokine gene transcription
RNA extractions were performed with the RNA Easy Extraction
kit (Qiagen, Basel, Switzerland), according to the supplier’s
instructions. RNA was treated with DNase [DNAfree; Ambion
(Europe), Huntington, Cambridgeshire, UK] for removal of
residual DNA. RNA (1 mg) was reverse transcribed in a total
volume of 20 ml with oligo-dT15 primer (Microsynth, Balgach,
Switzerland) using Omniscript Reverse Transcription kit
(Qiagen). RNase inhibitor (10 units) (RNasin plus, Promega,
Catalys AG, Wallisellen, Switzerland) was added to each 20 ml
reaction. Real-time PCR (TaqMan) for human IL-2, IL-12 p35,
IFN-g,IL-1b, IL-6, IL-8, IL-10, TGF-b, TNF-a genes, EBV nuclear
antigen (EBNA)-1, EBNA-2, latent membrane protein (LMP)-1,
LMP-2, BamHI Z fragment (BZLF)-1, glycoprotein (gp) 85
(C. Berger, et al. submitted), and the housekeeping gene,
hydroxymethylbilane synthase (HMBS), were performed accord-
ing to the supplier’s instructions (Applied Biosystems, Foster City,
CA, USA) and as described (Bonanomi et al., 2003). The assays
were cDNA specific: either the forward or reverse primer or the
probe was designed to span exon-exon junctions. Specificity
(DNA/cDNA) was tested using RNA before and after DNase
treatment and cDNA with or without prior DNase treatment. The
assay for human IL-10 is highly specific and does not detect viral
IL-10 (data not shown). All reactions were performed in duplicate.
Each 15 ml reaction contained a mix of the 2¥ ABI-TaqMan
Master Mix (Applied Biosystems), primers (Microsynth) at
300 nM each, the probe (Biosearch Technologies, Novato, CA,
USA) at 200 nM, and 1 ml of cDNA template. Ct values obtained
for HMBS were used for normalization. Both positive (amplified
cDNA sequences of the selected cytokines or EBV genes tested)
and negative controls (no template) were included on every plate.
Immunofluorescence
Cord blood mononuclear cells were washed in PBS, transferred
to coated slides in a Cytospin 3 centrifuge (Shandon, Histocom,
Zug, Switzerland), air dried, fixed with acetone at 4°C, and stored
at -20°C. After thawing and before staining, the cells were
blocked with 5% goat serum in PBS, incubated with the anti-
BZLF-1 antibody (1:40; Clone BZ.1, DakoCytomation, Zug, Swit-
zerland), followed by the secondary goat anti-mouse IgG
antibody labelled with the green fluorescent Alexa Fluor 488 dye
(Molecular Probes-Invitrogen, Basel, Switzerland). Nuclei were
stained with 4,6 diamidino-2-phenylindole (DAPI; Vector Labora-
tories, Burlingame, CA, USA). Analysis was carried out with the
Zeiss AXIOSKOP 2 Mot Plus microscope, the Plan Neofluar
20 ¥/0.50 Ph2 objective, the Fluoarc Lamp, the AxioCam MR and
the AxioVision 3.1 software (all from Carl Zeiss AG, Oberkochen,
Germany). Adobe Photoshop 6.0 was used to magnify the region
of interest.
Flowcytometric analyses to determine T-cell activation
or fractions of EBV-infected B cells
The cell pellets were resuspended and washed in staining buffer
(PBS with 5% FBS and 0.1% sodium azide but without Ca
+2
or
Mg
+2
). Cells were double stained with an FITC-labelled and a
PE-labelled mouse anti-human monoclonal antibody (all from
BD Biosciences, if not stated otherwise) at 4°C in the dark for
30 min. As isotype controls, FITC-conjugated anti-mouse IgG
1
(FITC-IgG
1
) with PE-conjugated anti-mouse IgG
1
(PE-IgG
1
) and
FITC-IgG
1
with PE-HLA-ABC were used. Activated T cells were
evaluated with FITC-anti-HLA-DR and either PE-anti-CD4 or
Suppression of initiation of lytic EBV 2067
© 2007 The Authors
Journal compilation © 2007 Blackwell Publishing Ltd, Cellular Microbiology, 9, 2055–2069
PE-anti-CD8. B cells were evaluated with PE-anti-CD19 and
Cy5-anti-CD5. Detection of the late lytic EBV glycoprotein gp350/
220 was performed using a FITC-labelled anti-EBV gp350/220
antibody (Milan Analytica, La Roche, Switzerland).The samples
were analysed using a FACSCalibur (BD Biosciences) equipped
with 488 nm and 635 nm lasers for double colour analysis.
Events (10 000 per lymphocyte gate) were recorded and analy-
sed with the Cell Quest software (BD Biosciences).
Assessment of cytokine levels
Samples were analysed using multiplex bead analysis that uses
microspheres as the solid support for immunoassays (Chen
et al., 1999). Cytokine levels were measured according to the
manufacturer’s instructions (Upstate Biotechnology UK, Bucking-
ham, UK).
Stimulation of CBMC with rIFN-g, rIL-12, or IL-10 and
inhibition of IL-12 or IFN-g by addition of anti-IL-12,
anti-IFN-g, or IL-10 antibodies
Cord blood mononuclear cells (1 ¥ 10
6
ml
-1
) were infected with
EBV as described above, but with or without addition of
20 ng ml
-1
rIL-12, or 10 ng ml
-1
rIFN-g, or both (both from R&D
Systems, Abingdon, UK), or 1, 10, or 100 pg ml
-1
IL 10 (Pepro-
tech EC, London, UK). RIL-12, rIFN-g, or both, or IL-10 were
added in 24 h intervals to the cells. CBMC or PBMC (1 ¥ 10
6
ml
-1
)
were infected with EBV with or without 100 ng anti-IL-12, 1 mg
anti-IFN-g antibodies (both from R&D Systems), or anti-IL-10
antibodies (Biolegend, San Diego, USA).
Stimulation of CBMC, PBMC, or Akata cells with
ligands to TLRs
Cells (3 or 5 ¥ 10
6
cells ml
-1
) were left uninfected or infected with
EBV and were stimulated with TLR ligands added at 0 h and
90 h. Concentrations of TLR ligands were 10 mgml
-1
for pepti-
doglycan (TLR1/2), 20 mgml
-1
for lipopolysaccharide (TLR4),
3 mM for R-848 (TLR7/8) and 0.5 mM for CpG ODN 2006 (TLR9)
(InvivoGen, San Diego, CA, USA). The cells were kept in culture
for a total of 96 h.
Initiation of lytic EBV infection in Akata Burkitt
lymphoma cells
Akata cells were split to a concentration of 1 ¥ 10
6
cells ml
-1
24 h
before stimulation. Cells (1 ¥ 10
6
ml
-1
) were stimulated with
0.1 mg ml
-1
polyclonal rabbit anti-human IgG (Dako, Zug, Switzer-
land) and suspended in fresh RPMI 1640. After 6 h, stimulated
cells were collected for RNA isolation.
Statistical analyses
The Mann–Whitney U-test (two-tailed) was used for comparison
of differences between groups. The level of statistical signifi-
cance was set at P < 0.05.
Acknowledgements
We thank Dr Mohammad Shams and the staff from the obstetrics
ward, Hirslanden Hospital, Zurich, Switzerland, for facilitating the
collection of cord blood. We also thank Rahel Byland, Roger
Lauener, Gregory Melroe, and Erika Schlaepfer for their helpful
comments on the manuscript. This work was supported by the
Swiss Bridge Foundation, the Cancer League of the Kanton of
Zurich, and the Edoardo R. Giuseppe and Christina Sassella
Foundation.
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    • "Elevated serum levels of IL-6 have also been observed 1–3 years prior to the onset of AIDS-NHL, thus supporting the role of IL-6-driven B-cell stimulation in the development of these lym- phomas [45] . TLR9 substantially suppresses the expression of BZLF- 1, by histone modification in acute EBV infection ex vivo and in latently BL cells in vitro, suggesting that immune activation can also promote EBV-driven lymphomagenesis by suppressing the viral lytic cycle [46]. In addition, HIV-infected patients with high levels of EBV present higher levels of pro-inflammatory cytokines (IL-6, IL-10, TNFα ) and PAMPs (LPS, 16S rDNA) than patients with low EBV levels. "
    [Show abstract] [Hide abstract] ABSTRACT: Post-transplant lymphoproliferative disorders (PTLDs) represent the most severe complication of both solid organ and hematopoietic stem cell transplantation. The Epstein-Barr Virus (EBV) is a main driver of PTLD, particularly those occurring early after transplantation. EBV-driven malignancies are associated with selective expression of latent viral proteins, but uncontrolled lytic replication may favor early phases of cell transformation. Besides immunodepression, persistent immune activation and chronic inflammation play an important role in both virus reactivation and expansion of EBV-infected B cells. EBV-induced immortalization requires the expression of telomerase. TERT, the rate-limiting component of the telomerase complex, is central in the switch from the lytic to the latent viral program, and TERT inhibition induces the EBV lytic cycle and cell death. Immunotherapy and combination of EBV lytic cycle inducers with antiviral drugs are promising strategies to improve the treatment of PTLD patients. This review is aimed at providing an update on the intriguing association between EBV and PTLD, mainly focusing on cases arising after kidney and liver transplantation, which account for the vast majority of transplants. Copyright © 2015. Published by Elsevier Ireland Ltd.
    Full-text · Article · Aug 2015
    • "Gene expression was determined by quantitative reverse transcription polymerase chain reaction (RT-qPCR) using specific primers and probes for the housekeeping gene HMBS, the noncoding EBV encoded RNA EBER1, the latency associated EBV genes EBNA1, EBNA2, LMP1 and LMP2A and for the two genes related to the lytic replication cycle of EBV, BZLF1 and BXLF2, as described earlier535455 . Additionally, to detect BZLF1 gene expression in rM81 infected cells we used a different forward primer for the BZLF1 (5'-CAC GAC GTA CAA GGA AAC-3') and LMP1 (5'-TGG AGG CCT TGG TCT ACT CCT-3') primer/probe set, which we termed aBZLF1 and aLMP1, respectively, due to the sequence homology to the Akata EBV strain. "
    [Show abstract] [Hide abstract] ABSTRACT: The Epstein-Barr virus (EBV) is transmitted from host-to-host via saliva and is associated with epithelial malignancies including nasopharyngeal carcinoma (NPC) and some forms of gastric carcinoma (GC). Nevertheless, EBV does not transform epithelial cells in vitro where it is rapidly lost from infected primary epithelial cells or epithelial tumor cells. Long-term infection by EBV, however, can be established in hTERT-immortalized nasopharyngeal epithelial cells. Here, we hypothesized that increased telomerase activity in epithelial cells enhances their susceptibility to infection by EBV. Using HONE-1, AGS and HEK293 cells we generated epithelial model cell lines with increased or suppressed telomerase activity by stable ectopic expression of hTERT or of a catalytically inactive, dominant negative hTERT mutant. Infection experiments with recombinant prototypic EBV (rB95.8), recombinant NPC EBV (rM81) with increased epithelial cell tropism compared to B95.8, or recombinant B95.8 EBV with BZLF1-knockout that is not able to undergo lytic replication, revealed that infection frequencies positively correlate with telomerase activity in AGS cells but also partly depend on the cellular background. AGS cells with increased telomerase activity showed increased expression mainly of latent EBV genes, suggesting that increased telomerase activity directly acts on the EBV infection of epithelial cells by facilitating latent EBV gene expression early upon virus inoculation. Thus, our results indicate that infection of epithelial cells by EBV is a very selective process involving, among others, telomerase activity and cellular background to allow for optimized host-to-host transmission via saliva.
    Full-text · Article · Apr 2015
    • "These findings strongly suggest that immune reconstitution without reduction in HIV-1 viremia may increase B cell stimulation and the number of EBV-infected cells; this may be important for new therapeutic strategies against HIV-1 to implement immune reconstitution and/or to alleviate adverse drug effects. HIV-1 infected patients, treated with ART in combination with IL-2 to improve immunological reconstitution, showed a decrease in EBV plasmaviremia and citoviremia when treated with lowintermittent IL-2 doses, but presented an increase in EBV in both cells and plasma when treated with high doses of IL-2 (Burighel Nef Activation of B cells and induction of hypergammaglobulinemia through ferritin produced via Nef-mediated activation of NF-κB Swingler et al. (2008) Suppression of CD40-dependent immunoglobulin class switching Qiao et al. (2006) Tat Binding to Rb2/p130 and induction of cell cycle genes; modulation of cell cycle and increased proliferative capability of EBV + B cells Lazzi et al. (2002); Colombrino et al. (2004) PAMPs * LPS Engagement and activation of TLR4; induction of pro-inflammatory cytokines Brenchley et al. (2006) 16S rDNA, CpG DNA Engagement and activation of TLR9; suppression of EBV lytic cycle through inhibition of BZLF expression Ladell et al. (2007); Zauner and Nadal (2012) DAMPsBaiocchi and Caligiuri, 1994). Conversely, high doses of IL-2, by binding low-affinity receptors expressed on natural killer (NK) cells, may enhance several pro-inflammatory cytokines, and consequently B cell stimulation and activation (Jacobson et al., 1996; Malnati et al., 2002;Table 1). "
    [Show abstract] [Hide abstract] ABSTRACT: Epstein-Barr virus (EBV) is a ubiquitous human γ-herpes virus which establishes a life-long asymptomatic infection in immunocompetent hosts. In human immunodeficiency virus type 1 (HIV-1) infected patients, the impaired immunosurveillance against EBV may favor the development of EBV-related diseases, ranging from lymphoproliferative disorders to B cell non-Hodgkin's lymphomas (NHL). Antiretroviral therapy (ART) has significantly modified the natural course of HIV-1 infection, resulting in decreased HIV-1 plasmaviremia, increased CD4 lymphocytes, and decreased opportunistic infections, indicating a restoration of immune functions. However, the impact of ART appears to be less favorable on EBV-related malignancies than on other AIDS-defining tumors, such as Kaposi's sarcoma, and NHL remains the most common cancer during the ART era. EBV-driven tumors are associated with selective expression of latent oncogenic proteins, but uncontrolled lytic cycle with virus replication and/or reactivation may favor cell transformation, at least in the early phases. Several host's factors may promote EBV reactivation and replication; besides immunodepression, inflammation/chronic immune stimulation may play an important role. Microbial pathogen-associated molecular patterns and endogenous damage-associated molecular patterns, through Toll-like receptors, activate the immune system and may promote EBV reactivation and/or polyclonal expansion of EBV-infected cells. A body of evidence suggests that chronic immune stimulation is a hallmark of HIV-1 pathogenesis and may persist even in ART-treated patients. This review focuses on lymphomagenesis driven by EBV both in the context of the natural history of HIV-1 infection and in ART-treated patients. Understanding the mechanisms involved in the expansion of EBV-infected cells is a premise for the identification of prognostic markers of EBV-associated malignancies.
    Full-text · Article · Oct 2013
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