Increased CD8+ T Cell Response to Epstein-Barr Virus Lytic Antigens in the Active Phase of Multiple Sclerosis
et al. (2013) Increased CD8+ T Cell Response to Epstein-Barr Virus Lytic Antigens in the Active Phase
of Multiple Sclerosis. PLoS Pathog 9(4): e1003220. doi:10.1371/journal.ppat.1003220
Increased CD8+ T Cell Response to Epstein-Barr Virus Lytic Antigens in the Active Phase of Multiple Sclerosis
Daniela F. Angelini 0
Barbara Serafini 0
Eleonora Piras 0
Martina Severa 0
Eliana M. Coccia 0
Barbara Rosicarelli 0
Serena Ruggieri 0
Claudio Gasperini 0
Fabio Buttari 0
Diego Centonze 0
Rosella Mechelli 0
Marco Salvetti 0
Giovanna Borsellino 0
Francesca Aloisi" 0
Luca Battistini" 0
Brian D. Evavold, Emory University, United States of America
0 1 Neuroimmunology Unit , Fondazione Santa Lucia, (I.R.C.C.S.), Rome , Italy , 2 Department of Cell Biology and Neuroscience, Istituto Superiore di Sanita` , Rome , Italy , 3 Department of Infectious, Parasitic and Immune-mediated Diseases, Istituto Superiore di Sanita` , Rome , Italy , 4 Department of Neurology and Psychiatry, Sapienza University of Rome , Rome , Italy , 5 Department of Neurosciences, S Camillo Forlanini Hospital , Rome , Italy , 6 Department of Neurosciences, University Tor Vergata , Rome , Italy , 7 Centre for Experimental Neurological Therapies, S. Andrea Hospital, Faculty of Medicine and Psychology, Sapienza University of Rome , Rome , Italy
It has long been known that multiple sclerosis (MS) is associated with an increased Epstein-Barr virus (EBV) seroprevalence and high immune reactivity to EBV and that infectious mononucleosis increases MS risk. This evidence led to postulate that EBV infection plays a role in MS etiopathogenesis, although the mechanisms are debated. This study was designed to assess the prevalence and magnitude of CD8+ T-cell responses to EBV latent (EBNA-3A, LMP-2A) and lytic (BZLF-1, BMLF-1) antigens in relapsing-remitting MS patients (n = 113) and healthy donors (HD) (n = 43) and to investigate whether the EBVspecific CD8+ T cell response correlates with disease activity, as defined by clinical evaluation and gadolinium-enhanced magnetic resonance imaging. Using HLA class I pentamers, lytic antigen-specific CD8+ T cell responses were detected in fewer untreated inactive MS patients than in active MS patients and HD while the frequency of CD8+ T cells specific for EBV lytic and latent antigens was higher in active and inactive MS patients, respectively. In contrast, the CD8+ T cell response to cytomegalovirus did not differ between HD and MS patients, irrespective of the disease phase. Marked differences in the prevalence of EBV-specific CD8+ T cell responses were observed in patients treated with interferon-b and natalizumab, two licensed drugs for relapsing-remitting MS. Longitudinal studies revealed expansion of CD8+ T cells specific for EBV lytic antigens during active disease in untreated MS patients but not in relapse-free, natalizumab-treated patients. Analysis of post-mortem MS brain samples showed expression of the EBV lytic protein BZLF-1 and interactions between cytotoxic CD8+ T cells and EBV lytically infected plasma cells in inflammatory white matter lesions and meninges. We therefore propose that inability to control EBV infection during inactive MS could set the stage for intracerebral viral reactivation and disease relapse.
Funding: The study was supported by Italian Multiple Sclerosis Foundation (to DC, MS, EMC, FA, LB), European FP6 NeuroproMiSe Integrated project (Contract:
LSHM-CT-2005-01863) (to FA, LB), Italian Ministry of Health, Ricerca Finalizzata 2007 - Strategic Project on Multiple Sclerosis (Contract: 107 to EMC, MS, FA, LB), and
Ricerca Finalizzata 2010-Giovani Ricercatori (to DC), the French Society for Research in Multiple Sclerosis (ARSEP) (to MS, LB). The funders had no role in study
design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: Marco Salvetti, MD, received lecture fees from Biogen-Dompe and research support from Bayer-Schering, Biogen-Dompe, Merck-Serono,
Sanofi-Aventis. Claudio Gasperini, MD, has served as a consultant for Merck Serono and Biogen Idec, and has received speaker honoraria from Teva, Merck Serono,
Bayer Shering and Biogec Idec. Diego Centonze, MD, is an Advisory Board member of Merck-Serono, Teva and Bayer Shering and received funding for travelling
and speaker honoraria or consultation fees from Merck Serono, Teva, Novartis, Bayer Shering, Sanofi-Aventis and Biogen Idec. He is also an external expert
consultant of the European Medicine Agency (EMA) and the principal investigator in clinical trials for Novartis, Merck Serono, Teva, Bayer Shering, Sanofi Aventis
and Biogen Idec. The other authors have no competing interests to report. This does not alter our adherence to all PLoS Pathogens policies on sharing data and
" FA and LB share senior authorship.
Multiple sclerosis (MS) is the most common chronic
inflammatory disease of the central nervous system (CNS) causing
demyelination, neurodegeneration and disability. In most cases,
MS is characterized by a relapsing-remitting course at onset which
eventually develops into a progressive form; more rarely MS
manifests as a primary progressive disease . Immunomodulating
and immunosuppressive drugs can reduce but not halt the disease
process. Both the etiology and pathogenic mechanisms of MS are
poorly understood. Genetic and environmental factors have been
implicated in MS development, but the identity of the antigens
(self or non-self) promoting chronic CNS inflammation remains
elusive . Several viruses have been linked to MS; however,
Esptein-Barr virus (EBV) shows the strongest association with the
disease . EBV is a B-lymphotropic DNA herpesvirus that
infects 9598% of individuals worldwide, establishes a life-long,
generally asymptomatic infection in B cells, and is the cause of
infectious mononucleosis and of several lymphatic and
nonlymphatic malignancies . EBV has also been implicated in
common autoimmune diseases, like systemic lupus erythematosus
and rheumatoid arthritis [7,8]. Numerous studies have consistently
There is general consensus that multiple sclerosis (MS) is
associated with Epstein-Barr virus (EBV) infection but the
mechanistic links are still debated. EBV is a B-lymphotropic
herpesvirus widespread in the human population and
normally contained as a persistent, asymptomatic infection
by immune surveillance. However, EBV can cause
infectious mononucleosis, is associated with numerous human
malignancies, and is implicated in some common
autoimmune diseases. While EBV infection alone cannot explain
MS development, it has been postulated that in
susceptible individuals alterations in the mechanisms regulating
the immune response to the virus may contribute to MS
pathogenesis. Here, we show that MS patients with
inactive disease exhibit a lower CD8+ T-cell response to
EBV when compared to healthy donors and active MS
patients while the latter have a higher frequency of CD8+ T
cells specific for EBV lytic antigens. Therapy with
interferon-b and natalizumab, two treatments for
relapsingremitting MS, was associated with marked changes in
the EBV specific CD8+ T cell response. We also
demonstrate that one of the EBV lytic antigens recognized by
CD8+ T cells expanding in the blood during active MS is
expressed in the inflamed MS brain. Our results support a
model of MS pathogenesis in which EBV infection and
reactivation in the CNS stimulates an immunopathological
response and suggest that antiviral or immunomodulatory
therapies aimed at restoring the host-EBV balance could
be beneficial to MS patients.
demonstrated a higher prevalence of EBV infection and higher
titers of antibodies to EBV antigens, in particular to EBV nuclear
antigen-1 (EBNA-1), in young and adult MS patients compared to
age-matched, healthy individuals . It has also been shown
that high titers of anti-EBNA-1 antibodies prior to MS onset 
or at the time of a clinically isolated syndrome  and a previous
history of infectious mononucleosis  increase the risk of
developing MS. Furthermore, MS patients have higher
frequencies of CD4+ T cells specific for EBNA-1 relatively to healthy,
seropositive individuals , while EBV-specific CD8+ T-cell
responses in MS have been reported to be increased or decreased
in different studies .
Although enhanced immune reactivity to EBV in MS suggests
perturbed EBV infection, it is debated whether and how this can
induce or influence the disease. EBV infection could contribute to
MS through multiple mechanisms, including molecular mimicry,
immortalization of autoantibody-producing B cell clones, and
immunopathology [3,24]. It has been shown that CD4+ T cells
from some MS patients cross-react with EBV and myelin antigens
but the relevance of this finding to disease pathogenesis is still
unclear [25,26]. EBV DNA load in the peripheral blood does not
differ significantly between MS patients and healthy donors (HD)
[16,18,23] and the possibility that a persistent EBV infection in the
CNS drives an immunopathological response that damages myelin
and neural cells is reasonable but remains controversial .
While several studies report absence of EBV in MS brain lesions
, we  and others  have shown that an
abnormally high proportion of B cells infiltrating the MS brain
are latently infected with EBV. We have also shown that ectopic
B-cell follicles present in the inflamed meninges of patients with
secondary progressive MS harbour EBV infected B cells and that
EBV can reactivate in plasma cells in immunologically active
white matter lesions and meningeal B-cell follicles .
If MS were the result of an immunopathological response
aimed at eliminating a persistent EBV infection in the CNS, a
positive correlation should be found between disease activity
assessed by magnetic resonance imaging (MRI) or clinical
progression and immune response to EBV. In support of this
hypothesis, it has been shown that serum levels of EBNA-1 IgG
positively correlate with gadolinium-enhancing MRI lesions
(characteristic of acute inflammation), lesion size and Expanded
Disability Status Scale (EDSS) in patients with MS  and in
patients with a clinically isolated syndrome who develop definite
MS . Another study reported higher disease activity on MRI
in a subgroup of relapsing-remitting MS patients with stable
levels of IgG specific for EBV early antigens expressed during
the lytic cycle . It has also been shown that CD8+ T-cell
responses toward pooled EBV latent and lytic antigens in the
blood of MS patients are high early in MS course and decrease
during disease progression suggesting a possible association with
more frequent episodes of CNS inflammation in early disease
phases . However, it is not known whether changes in the
CD8+ T cell response to individual EBV latent and/or lytic
antigens are associated with active and inactive MS phases. To
address this issue, we have used pentamer staining to
characterize the CD8+ T-cell response to EBV in the peripheral blood
of patients with relapsing-remitting MS, both untreated and
treated, and HLA-matched controls. Positivity to pentamer
staining was then correlated with disease activity and inactivity,
as assessed by clinical criteria and MRI of the brain. Our study
reveals a lower prevalence of the CD8+ T cell response to EBV
in inactive MS patients, a higher frequency of CD8+ T cells
specific for EBV lytic and latent antigens during active and
inactive disease, respectively, and marked changes in the
EBVspecific CD8+ T cell response during treatment with approved
disease-modifying drugs, such as interferon-b (IFN-b) and
natalizumab. By analyzing post-mortem MS brain tissue, we
demonstrate that the same EBV lytic antigen eliciting a higher
CD8+ T cell response in the peripheral blood during active MS
is expressed in inflammatory white matter lesions and meninges.
We also show interactions between CNS-infiltrating cytotoxic
CD8+ T cells and EBV lytically infected plasma cells, further
supporting the link between EBV reactivation, higher cytotoxic
immune responses to EBV lytic antigens and MS exacerbations.
The EBV-specific CD8+ T-cell response was studied with the
pentamer technology in 43 HD and 113 patients (79 untreated
and 34 treated) with relapsing-remitting MS who were selected
according to their HLA genotype (HLA-A*0201 and/or
HLAB*0801) (Table 1). The choice of pentamers was guided by the
high frequency of HLA-A2 family alleles in Caucasians, by
previous characterization of the immunodominant peptide
epitopes from EBV latent and lytic proteins that are most
frequently recognized by CD8+ T cells [38-40], and by the
possibility to evaluate the CD8+ T cell response to at least one
viral latent and one viral lytic protein in the same subject. We
therefore studied CD8+ T cell reactivities to the immunodominant
peptides of LMP-2A (EBV latent antigen) and BMLF-1 (EBV early
lytic antigen) restricted through the HLA-A*0201 allele, and of
EBNA-3A (EBV latent antigen) and BZLF-1 (EBV immediate
early lytic antigen) restricted through the HLA-B*0801 allele. As
control for anti-viral MHC class I restricted CD8+ T cell responses
we selected a HLA-A*0201 pentamer coupled with a peptide from
cytomegalovirus (CMV) pp65 protein.
HD (n = 43)
MS (n = 79) untreated
MS IFNb (n = 20)
MS NTZa (n = 14)
Prevalence and frequency of EBV-specific CD8+ T cells in
healthy donors and in untreated MS patients with active
or inactive disease
Freshly isolated PBMC from HD (n = 43) and untreated MS
patients (n = 79) were stained with the above mentioned
fluorochrome-labeled HLA-A*0201 and/or HLA-B*0801/viral peptide
epitope pentamers. We first examined the prevalence of
EBVspecific CD8+ T cell responses in our cohort, namely the
proportion of individuals with detectable pentamer+ CD8+ T
cells (Fig. S1). Positive pentamer staining specific for at least one
EBV epitope was found in a similar proportion of HD (39%) and
untreated MS patients (33%). The prevalence of CD8+ T cell
responses to EBV latent and lytic antigens was similar in HD and
untreated MS patients (Figure 1 A). In HLA-A2+ subjects where
both EBV- and CMV-specific CD8+ T cell responses could be
evaluated, no differences in the response to either virus were found
between HD (n = 34) and untreated MS patients (n = 45) (Figure
We then explored whether the prevalence of EBV-specific
CD8+ T-cell responses could be related to disease activity.
Untreated MS patients were subdivided in two groups consisting
of 31 active and 48 inactive patients, as defined by presence and
absence of clinical relapses and acute brain inflammation assessed
with gadolinium-enhanced MRI, respectively (Table 2). There
was a similar prevalence of latent and lytic antigen-specific CD8+
T cell responses in HD and active MS patients but a significantly
lower prevalence of lytic antigen-specific CD8+ T cell responses
in inactive MS patients than HD (19% versus 39%, p = 0.05).
Inactive MS patients also tended to have a lower prevalence of
latent and lytic antigen-specific CD8+ T cell responses when
compared with active MS patients (17% versus 35%, p = 0.1 and
19% versus 39%, p = 0.09, respectively) (Figure 1A). In contrast,
no differences were found among HD, inactive MS and active
MS patients in the CD8+ T cell response to CMV (Figure S2B).
These findings therefore indicated a weaker immune response
against EBV, particularly against lytic cycle antigens, in inactive
MS patients compared to HD and active MS patients. Of note,
39% of active MS patients, but only 15% of inactive MS patients
with a detectable EBV-specific CD8+ T cell response had a
disease duration longer than 8 years, suggesting a decay with time
of the EBV-specific immune response associated with inactive
We then evaluated the percentage of pentamer+ CD8+ T cells
within the circulating CD3+ CD8+ T cell population in the study
subjects with a detectable EBV-specific CD8+ T cell response.
Similarly to prevalence, the frequency of latent and lytic
antigenspecific CD8+ T cells did not differ significantly between HD
(n = 17) and total untreated MS patients (n = 26) (Figure 2 A). Also
the frequency of CD8+ T cells specific for EBV and CMV
antigens was similar in HLA-A2+ HD and total MS patients
(Figure S3A). Again, differences in the immune response to EBV
emerged only when patients were stratified according to disease
activity (Figure 2 BD). The frequency of latent antigen-specific
CD8+ T cells in inactive MS patients (1.862.6%, mean 6 SD)
tended to be higher than in HD (0.360.2%, mean 6 SD; p = 0.07)
and was significantly higher than in active MS patients
(0.2260.19%, mean 6 SD; p = 0.05) (cumulative data and
representative plots are shown in Figure 2 B and D, respectively).
This difference was mainly due to the fact that 6 out of 8 inactive
MS patients recognized EBNA-3A and 3 of these displayed a very
strong immune response against this EBV latent antigen (2.7, 2.8
and 7.6% of total circulating CD8+ T cells were
EBNA-3Aspecific). In contrast, the frequency of lytic antigen-specific CD8+
T cells in active MS patients (1.862.8%, mean 6 SD) was
significantly higher than in HD (0.3460.28%, mean 6 SD;
p = 0.03) and tended to be higher than in inactive MS patients
(0.360.2%, mean 6 SD, p = 0.1). The latter difference did not
reach statistical significance probably due to the low number of
inactive MS patients analyzed (n = 9). These findings therefore
indicated more frequent recognition of EBV latent and lytic
antigens during inactive and active MS phases, respectively. In
contrast, the frequencies of CMV-specific CD8+ T cells did not
differ significantly among HLA-A2+ HD, inactive MS and active
MS patients (Figure S3B, C). No correlation was found between
the frequency of EBV-specific CD8+ T cells and disease duration
in inactive and active MS patients (Figure S4).
Prevalence and frequency of EBV-specific CD8+ T cells of
MS patients treated with interferon-b and natalizumab
We then analyzed the EBV-specific CD8+ T cell response in
MS patients who were treated with IFN-b (n = 20) and
natalizumab (n = 14) (Table 1). IFN-b, the most frequently used
first-line treatment for relapsing-remitting MS, has antiviral and
immunoregulatory activities and reduces relapse frequency and
brain MRI activity in relapsing-remitting MS patients . The
monoclonal antibody natalizumab inhibits lymphocyte
extravasation into the CNS and is highly effective in suppressing clinical
relapses and disease activity in relapsing-remitting MS patients
who fail to respond to first-line therapies .
Among 20 MS patients treated with IFN-b for 1 to 11 years
(median = 4 years), 7 and 13 were in the active and inactive phase
of disease, respectively (Table 2). The prevalence of the CD8+ T
cell response to EBV latent and lytic antigens was similar in total
untreated and IFN-b-treated MS patients (Figure 1 A, B).
However, none of the 13 IFN-b-treated inactive patients had a
detectable CD8+ T cell response to EBV (Figure 1 B) and CD8+ T
cells for CMV were found only in 1 of 9 HLA-A02+ IFN-b-treated
inactive patients (data not shown), indicating that effective IFN-b
Figure 1. Prevalence of EBV-specific CD8+ T cell responses in HD and MS patients. (A) HLA-A*0201 and HLA-B*0801 pentamer+ CD8+ T
cells specific for peptides from EBV latent (left panel) and lytic (left) proteins were investigated in HLA matched HD (n = 43), total untreated MS
patients (n = 79) and the same patients subdivided into active (n = 31) and inactive (n = 48) MS patients (a-MS and i-MS, respectively) based on clinical
and MRI criteria, as shown in Table 2. (B) IFN-b (n = 20) and natalizumab (NTZ) (n = 14) treated patients were analyzed as whole population or
subdivided into active and inactive patients, as above. The numbers within or above the columns correspond to the percentages of individuals with
detectable EBV-pentamer+ CD8+ T cells (grey columns) among the total donors tested (white columns); p values were calculated with Pearsons
Active MS (n = 31)
Inactive MS (n = 48)
Active MS IFNb (n = 7)
Inactive MS IFNb (n = 13)
Inactive MS NTZ (n = 14)
therapy is associated with a general inhibition of the antiviral
response. In contrast, 57% (4/7) and 66% (4/6) of the
IFN-btreated MS patients with active disease had a detectable CD8+ T
cell response to EBV (Figure 1 B) and CMV (data not shown),
respectively. Moreover, the frequency of CD8+ T cells specific for
EBV latent, but not lytic, antigens was significantly higher in
IFNb-treated (0.860.6%, mean 6 SD) than in untreated (0.260.2%,
mean 6 SD ; p = 0.01) active MS patients (Figure 2 B, C).
After 8 to 16 months (median = 12 months) of treatment with
natalizumab, all 14 MS patients analyzed were in the inactive
phase of disease, both clinically and by MRI (Table 2).
Unexpectedly, nearly all patients in this group had a detectable
CD8+ T cell response to EBV latent and lytic antigens (87% and
93%, respectively) (Figure 1 B). This prevalence was significantly
higher than that found in HD and any other group of MS
patients (p,0.001). Nevertheless, the frequencies of latent and
lytic antigen-specific CD8+ T cells in natalizumab-treated MS
patients were similar to those found in untreated inactive MS
patients (Figure 2 B, C). As expected for patients in the inactive
phase of disease, the frequency of lytic antigen-specific CD8+ T
cells tended to be lower in natalizumab-treated patients than in
untreated active MS patients (p = 0.09) (Figure 2 C). The
frequency of CMV-specific CD8+ T cells did not differ among
HD, untreated and natalizumab-treated MS patients (data not
Longitudinal study of EBV-specific CD8+ T cell responses
We then asked whether changes in the EBV-specific CD8+ T
cell response during active and inactive MS phases could be
detected in longitudinal studies. Despite experiencing clinical
relapses, two patients (HLA-B08+ B2/B2-2 and HLA-A02+ A14)
in our cohort refused immunomodulatory therapy and agreed to
be monitored periodically for 27 and 7 months, respectively.
Patient B2/B2-2, who was clinically silent and was diagnosed MS
one year before our analysis started, displayed a very highy
frequency of EBNA-3A-specific CD8+ T cells (6% of circulating
CD8+ T cells) at the beginning of the observation period (month 0;
Figure 3 A). EBNA-3A-specific CD8+ T cells progressively
decreased during the subsequent months and became
undetectable between month 17 and 27. In parallel, the frequency of
BZLF-1 specific CD8+ T cells, which were undetectable at
previous time points, abruptly increased and reached a peak (11%
of total circulating CD8+ T cells) at month 21 in concomitance
with the presence of active MRI lesions. The percentage of
BZLF1-specific CD8+ T cells in the CD8+ T cell population then
declined to 2.5% in the subsequent 6 months, denoting marked
expansion and subsequent contraction of the immune response
toward this EBV lytic antigen (Figure 3 A).
In patient A14, who experienced frequent clinical relapses, the
frequency of CD8+ T cells specific for the EBV lytic antigen
BMLF-1 ranged between 0.56 and 0.71% during a clinical relapse
(months 0 and 3 of the observation period) and in the presence of
an active MRI scan, and dropped to 0.11% in the subsequent 4
months (Figure 3 A). During the same period, the frequency of
CD8+ T cells specific for the EBV latent antigen LMP-2A and for
CMV pp65 antigen remained low and stable (Figure 3 A).
Longitudinal analysis of EBV-specific CD8+ T-cell responses
was also performed in 2 HLA-B08+ MS patients (BTY5 and
BTY8) treated with natalizumab and monitored periodically for
15 and 12 months, respectively, starting at 1214 months after
therapy initiation. Both natalizumab-treated patients were in
the inactive phase of disease (according to clinical and MRI
evaluation) and had a detectable CD8+ T cell response to
EBNA-3A and BZLF-1 (Figure 3 B). However, while the
frequency of BZLF-1-specific CD8+ T cells was stable during
the whole observation period a steady rise in the CD8+ T cell
response to EBNA-3A was observed in both patients after 1518
months of therapy (Figure 3B). Three HD followed for 8 to 19
months did not show any significant variation in the frequency
of CD8+ T cells specific for EBV latent and lytic antigens
(Figure 3 C).
BZLF-1 expression in the inflamed MS brain
The increased frequency of CD8+ T cells specific for two EBV
lytic antigens (BZLF-1, BMLF-1) in the blood of MS patients with
an active MRI scan indirectly suggests a response to a previous or
concomitant viral reactivation in the brain. In search for a link
between immunological findings and brain inflammation, we
examined the expression of BZLF-1 mRNA and protein in
postmortem brain tissue from 7 patients who died in the secondary
progressive phase of MS and were characterized by a severe
clinical course and substantial brain inflammation. The MS brain
samples selected for this study contained immunologically active
(both active and chronic active) lesions in the white matter and
highly inflamed meninges with B-cell follicle-like structures that we
previously showed to be major EBV reservoirs .
In preliminary experiments aimed at evaluating the specificity
and binding of anti-BZLF-1 monoclonal antibody, we observed
BZLF-1 immunoreactivity in the nucleus of EBV transformed
B95-8 cells and in an EBV+ tonsil from a patient with infectious
mononucleosis (Figure S5). Conversely, no BZLF-1+ cells were
detected in sections of a non pathological human brain, of a brain
from a patient with tuberculous meningoencephalitis and of an
EBV-negative lymphoma (Figure S5). BZLF-1 immunoreactivity
was detected in brain samples of all MS cases analyzed using
immunohistochemical techniques (n = 5). Both isolated and small
groups of BZLF-1+ cells were present in the inflamed meninges, at
the periphery of B-cell follicle-like structures (Figure 4 AH) and in
diffuse inflammatory cell infiltrates (Figure 4 IK). BZLF-1+ cells
were also present in the perivascular cuffs of inflamed blood vessels
in active white matter lesions characterized by a high density of
intraparenchymal foamy macrophages (Figure 5), but not in
demyelinated, chronic active and inactive white matter lesions
(data not shown), thus linking EBV reactivation to acute
inflammation. Nearly all BZLF-1+ cells infiltrating the meninges
(Figure 4 C, D, K) and active WM lesions (Figure 5 D, E, G, H)
were identified as Ig-producing plasmablasts/plasma cells, which is
consistent with the knowledge that EBV reactivates upon B-cell
differentiation into plasma cells . At these sites the proportion
of Ig+ cells expressing BZLF-1 ranged between 1 and 10%.
BZLF1+ plasma cells were detected in the same infiltrated brain areas
where plasma cells expressing BFRF1, an EBV early lytic protein
induced by BZLF-1 , were also found (Figure 4 E, Figure 5 F).
Expression of BZLF-1 was also investigated using quantitative
real-time RT-PCR in 4 inflamed MS brain samples, 2 of which
had been analyzed by immunohistochemistry. No BZLF-1 RNA
was detected in whole MS brain sections (data not shown). This
negative result was expected given the paucity of EBV lytically
infected plasma cells relatively to the large and heterogeneous cell
population of the MS brain. To enrich for EBV infected cells and
increase the sensitivity of the technique, perivascular and
meningeal inflammatory cell infiltrates and the surrounding, non
infiltrated brain parenchymal regions were harvested from MS
brain sections using laser capture microdissection and analyzed
using pre-amplification, quantitative real time RT-PCR for
1. CD19 transcripts were also analyzed to optimally discriminate
between infiltrated and non infiltrated brain areas. BZLF-1
transcripts were detected in the perivascular cuffs isolated from
one active MS lesion and in 3 out of 4 meningeal B-cell follicles
but not in 3 chronic active lesions, 4 sparse meningeal infiltrates
and 7 non infiltrated parenchymal regions (Figure 6). A control
lymph node was negative for BZLF-1. Thus, both
immunohistochemical and RT-PCR findings corroborated BZLF-1 expression
and therefore shift to EBV lytic infection in immunologically active
white matter lesions and ectopic B-cell follicles.
Figure 3. Longitudinal monitoring of EBV-specific CD8 T cell responses in relapsing remitting MS. The frequencies of CD8+ T cells
specific for EBV lytic and latent antigens and CMV pp65 antigen were measured at different time points in 2 untreated MS patients (A), 2
natalizumabtreated MS patients (B) and 3 HD (C) using HLA-A*0201 and HLA-B*0801 pentamers. All patients were monitored clinically and with MRI at the times
indicated by the arrows. The untreated MS patients had gadolinium-enhancing MRI lesions 20 days (MSB2/B2-2) and 1 day (MSA14) before the peak
of the CD8+ T cell response to EBV lytic antigens while the 2 natalizumab-treated patients (MS BTY5 and MS BTY8) did not show any disease activity.
Percentages of pentamer+ cells within the CD3+ CD8+ population are given on the y axis; the numbers on the x-axis indicate months since the start
of the observation period (A, C) or of drug therapy (B).
EBV lytically infected cells in the MS brain are targeted by
a cytotoxic immune response
Then, we searched for interactions between cytotoxic CD8+ T
cells and EBV infected cells in the same MS brain samples in
which BZLF-1 protein and/or RNA were detected. We first
analyzed the presence and frequency of granzyme B-expressing
CD8+ T cells and their relationship to EBV litically infected cells.
We observed that most granzyme B+ cells in the MS brain
coexpressed CD8 and that the fraction of CD8+ T cells expressing
granzyme B ranged between 5 and 60% in the different MS cases
and brain areas analyzed, the highest values being detected in the
perivascular cuffs of active white matter lesions (Figure 7 A, B). In the
meninges, granzyme B+/CD8+ T cells were present in diffuse
meningeal infiltrates and at the periphery of B-cell follicle-like
structures, but were rarely seen inside these structures (Figure 7 C, D).
Given the nuclear localization of BZLF-1 and the relatively
small number of BZLF-1+ cells in the MS brain it was extremely
difficult to see contacts between granzyme B+ cells and lytically
infected cells using double immunofluorescence for BZLF-1 and
CD8 or granzyme B. We therefore stained MS brain sections for
the EBV lytic protein BFRF1 which has a perinuclear localization
and has been detected in a higher fraction of plasma cells (up to
50%) compared to BZLF-1 . We observed lymphoblastoid
CD8+ T cells adhering to or secreting granzyme B toward
BFRF1+ cells as well as contacts between CD8+ T cells and
BFRF1+ cells displaying granzyme B immunoreactivity on their
surface (Figure 7 EH). Such cytotoxic contacts were frequently
observed in sparse meningeal infiltrates and active white matter
lesions, but not inside ectopic B-cell follicle-like structures. Finally,
staining of MS brain sections for perforin and Ig allowed to
visualize perforin granules polarized toward Ig+ cells inside the
perivascular cuffs of active white matter lesions (Figure 7 I, J),
supporting further the idea that EBV harbouring cells might be the
target of a cytotoxic attack.
Altered control of EBV infection in individuals susceptible to
MS is suspected to play a role in the development of immune
dysfunction causing CNS pathology . Higher serum titers of
EBNA-1 IgG are associated with an increased risk of MS ,
increased conversion from a clinically isolated syndrome to
definite MS  and more severe disease activity and clinical
progression [16,36]. Virus-specific CD8+ T cell responses play an
essential role in the control of EBV infection  and have been
investigated in previous studies in MS using mainly IFN-c
ELISPOT analysis in PBMCs stimulated in vitro with EBV+
lymphoblastoid cells [20,22], viral lysates , individual  or
pooled  EBV lytic and latent peptides, and more recently using
MHC-peptide tetramer staining . Several studies have shown
that EBV-specific CD8+ T cell responses are significantly higher in
MS than in HD or in patients with other inflammatory
neurological diseases . However, lack of significant
differences between MS patients and controls [16,23] and even
reduced frequency of EBV-specific CD8+ T cells in MS patients
 have also been reported. Use of cryopreserved versus freshly
isolated PBMCs, analysis of patients with relapsing-remitting and
progressive MS courses, and lack of stratification of patients
Figure 5. Immunohistochemical detection of BZLF-1 in acute white matter lesions of the MS brain. (A) A large, B-cell enriched
perivascular immune infiltrate surrounding 3 blood vessels in an active white matter lesion of a MS case is visualized with anti-CD20 antibody. (B)
Immunostaining for myelin-oligodendrocyte glycoprotein (MOG) in a serial section reveals presence of myelin. (C) The same lesion shows massive
microglia/macrophage activation in the parenchyma. Sections shown in panels AC were counterstained with hematoxylin. (D, E) Double
immunofluorescence staining for Ig (green) and BZLF-1 (red) reveals the presence of several Ig+ plasmablasts/plasma cells co-expressing BZLF-1 in
the portion of the perivascular cuff marked with a frame in panel A. The upper and lower insets in E show 1 and 2 Ig+ cells co-expressing BZLF-1 at
high power magnification, respectively. (F) Double immunofluorescence staining for BFRF-1 (red) and Ig (green) shows presence of double labelled
BFRF-1+/Ig+ cells in the same area of the perivascular cuff stained for BZLF-1 in E. (G, H) Cells double stained for Ig (green) and BZLF-1 (red) in smaller
perivascular cuffs of the same active lesion shown in AC. Bars: 200 mm in A and C; 100 mm in B; 50 mm in D, E, G; 20 mm in F and H; 10 mm in the
insets in D.
according to disease activity may have hampered a clear
understanding of the possible link between EBV-specific CD8+
T cell responses and MS pathogenesis.
To obtain an accurate pattern of CD8+ T cell in vivo
specificities, we have used highly standardized flow cytometric
analysis with EBV-specific pentamers, that unequivocally identify
antigen-specific CD8+ T cells, on freshly isolated PBMCs obtained
from HD and relapsing-remitting MS patients. Importantly, both
untreated and treated MS patients were studied and disease
activity was evaluated in most patients with gadolinium-enhanced
MRI shortly before or at the time of blood collection. Such a
rigorous selection justifies the relatively small number of MS
The first main finding of this study is that differences in the
prevalence and magnitude of the CD8+ T cell response to certain
EBV latent and lytic proteins between MS patients and HD and
within the MS cohort emerge only when patients are stratified
according to disease activity and inactivity. By showing that fewer
inactive MS patients have a detectable CD8+ T cell response
against EBV lytic antigens compared with HD and active MS
patients and that the frequency of lytic antigen-specific CD8+ T
cells is higher in active MS patients than in HD and inactive MS
patients, we demonstrate for the first time that changes in the
immune control of EBV replication are associated with the active
and inactive phases of MS. This is corroborated by the
longitudinal study performed in two untreated MS patients
showing a peak in the frequency of CD8+ T cells to EBV lytic
antigens during active disease. Of the two EBV lytic antigens
analyzed, BZLF-1 is a transactivator expressed at the very
initiation of the lytic cycle and is involved in the induction of
early lytic proteins, including BMLF-1 . Thus, an increase in
BZLF-1- and BMLF-1-specific CD8+ T cells in concomitance
with acute brain inflammation on MRI strongly suggests an
attempt of the immune system to control intracerebral foci of EBV
replication. On the other hand, a logical explanation for the
decrease in lytic antigen-specific T cells associated with inactive
MS could be elimination of lytically infected cells brought about
by the strong cytotoxic response occurring in the active disease
phase. In this context, it is important to recall that EBV DNA load
in the blood of MS patients does not differ significantly from that
in healthy EBV carriers [16,18,23] indicating that fluctuations in
EBV-specific CD8+ T cell responses in MS patients do not result
in a generalized impairment of the immune control of EBV
infection. In contrast with the present findings, Jilek et al.  did
not observe differences in the prevalence and frequency of CD8+
T cell responses to BZLF-1 and BMLF-1 between MS patients and
control subjects. However, this study was not restricted to patients
with relapsing-remitting MS, did not distinguish between patients
with active and inactive disease and used cryopreserved PBMCs.
Importantly, in our study both the prevalence and magnitude of
the CD8+ T cell response to CMV were similar in HD and
untreated MS patients, irrespective of disease activity, indicating
that the differences observed in EBV-specific immunity are not the
consequence of a general activation of antiviral immune responses
driven by a still unknown MS-associated immune dysfunction.
Despite the fact that the prevalence of the CD8+ T cell response
to EBV latent antigens in inactive MS patients was similar to that
in HD and tended to be lower than in active MS patients, we
found that the magnitude of the CD8+ T cell response to
EBNA3A was higher in inactive MS patients than in HD and active MS
patients. Very high numbers of EBNA-3A-specific CD8+ T cells
were detected in half of the inactive MS patients harbouring this
immune reactivity (2.7 to 7.6% of the circulating CD8+ T cells
versus ,1% in HD and active MS patients). Furthermore,
longitudinal monitoring of a patient with a recent diagnosis of MS
showed substantial reduction of EBNA-3A-specific CD8+ T cells
just before the active disease phase and the rise of lytic
antigenspecific CD8+ T cells. It is known that EBNA-3A is expressed
shortly after EBV infection of B cells together with the whole set of
EBV latent proteins (EBNA-LP, 21, 22, 3A, 3B, and 23C,
LMP1, LMP-2A, LMP-2B) that are essential to drive infected B cells
into proliferation (latency III or growth program)  and elicit
strong T-cell responses . Most EBV-encoded latent antigens,
including EBNA-3A, are then sequentially downregulated as EBV
establishes a persistent infection in memory B cells (latency II and I
programmes) . Thus, the study of EBNA-3A-specific CD8+ T
cells suggests that at least in some inactive MS patients there is an
attempt by CD8+ T cells to control abnormal expansion of a
latently infected B-cell pool. A decrease in the immune response to
EBNA-3A could reflect a change in EBV latency programme and,
possibly, set the stage for switching to the lytic cycle. Of interest,
more abundant EBNA-3A-specific CD8+ T cells were detected
only in MS patients with a short disease duration (,5 years). A
decrease in immune reactivity toward EBNA-3A with disease
progression could be due to reduced antigenic stimulation or to
Tcell exhaustion which is known to occur during uncontrolled,
chronic viral infections . Relevant to this, we have shown that
most EBV latently infected B cells accumulating in the inflamed
MS brain during late-stage disease are memory B cells expressing
the latency II programme [32,33].
Based on the present findings, we propose that failure to fully
control EBV latent infection in an immune privileged site like the
CNS could lead to recrudescence of EBV reactivation. Exposure
to newly synthesized viral antigens would promote expansion of
lytic antigen-specific CD8+ T-cells targeting intracerebral viral
deposits and hence inducing the active phase of MS. Of relevance
for the present findings, it has been shown that after primary EBV
infection and during establishment of EBV persistence CD8+ T
cells specific for some EBV epitopes disappear from the circulation
after having upregulated Programmed Death-1 (PD-1) inhibitory
receptor, probably as a consequence of inadequate antigenic
stimulation . We are currently evaluating whether fluctuations
in EBV-specific CD8+ T cells in relapsing-remitting MS might be
associated with changes in PD-1 expression levels and T-cell
function (i.e., cytokine profile and cytotoxic activity). It would be
also interesting to compare the quality of the CD8+ T cell
response to EBV in MS with that in systemic lupus erythematosus,
an autoimmune disease associated with marked systemic EBV
dysregulation  and impaired cytotoxic immune response to the
The second main finding of this study is that treatment of
relapsing-remitting MS patients with IFN-b and natalizumab is
associated with marked changes in the CD8+ T cell response to
viral antigens. We have shown that CD8+ T cells specific for EBV
latent and lytic antigens and for CMV antigen were detectable in a
substantial fraction of the patients entering active disease despite
IFN-b treatment, but in none, except one, of the IFN-b-treated
patients with inactive disease. It is likely that such a strong
reduction in the CD8+ T cell response to both viruses might due to
the direct antiviral activity of the drug . Recently, Comabella
et al.  reported that clinically effective IFN-b therapy is
associated with downregulation of proliferative T cell responses to
EBNA-1 without significant changes in the CD8+ T cell response
against other (pooled) EBV antigens of the latent and lytic phase.
Discrepancies with the present study could be due to technical
issues, as discussed above. We have also shown that most
natalizumab-treated MS patients, all of which were in the inactive
phase of disease, had a detectable CD8+ T cell response to EBV.
Such high prevalences could be related to the fact that
natalizumab treatment causes a marked increase in lymphocyte
numbers in the blood due to interference with lymphocyte
extravasation and trafficking in lymphoid and non-lymphoid
tissues . Importantly, we found that in natalizumab-treated
MS patients the frequency of CD8+ T cells specific for EBV lytic
antigens was similar to that in HD and untreated inactive MS
patients and stable over time (up to 2227 months of therapy). It
therefore seems significant that the present analysis, though limited
to a relatively small number of donors, consistently showed no
expansion of EBV lytic antigen-specific CD8+ T cells during the
inactive phase of MS regardless of presence or absence of therapy.
In contrast, the frequency of EBNA-3A-specific CD8+ T cells,
which was comparable in untreated and natalizumab-treated
inactive MS patients within 816 months of therapy, progressively
increased during the second year of therapy in 2 longitudinally
monitored patients. These observations suggest dysregulation of
EBV latent infection upon prolonged treatment with natalizumab.
Although further studies are needed to clarify these aspects,
analysis of EBV-specific CD8+ T cell responses in MS patients
may help identify biomarkers useful for therapy monitoring and
shed light into the mechanisms underlying drug efficacy.
The third main finding of this study is that BZLF-1, one of the
two EBV lytic proteins recognized by CD8+ T cells expanding in
the blood of active MS patients, is expressed in post-mortem MS
brains with prominent immune infiltrates. The demonstration of
BZLF-1 protein and RNA in active white matter lesions, which
likely correspond to gadolinium-enhanced MRI lesions, and in the
inflamed meninges, where changes in water content cannot be
detected on MRI, lends support to the idea that acute brain
inflammation in MS is associated with switch to the viral lytic
cycle. In line with our previous results , we have also shown
that in the MS brain EBV reactivates in plasma cells and that the
latter can be found in close contact with lymphoblastoid CD8+ T
cells producing cytolotic enzymes. However, absence of CD8+
granzyme B+ T cells inside meningeal B-cell follicles, which
contain a high frequency of EBV latently infected cells ,
suggests that a local suppressive environment created by the virus
itself to elude immune control  could hamper virus clearance
from these structures. A cytotoxic attack toward EBV infected cells
in the MS brain is consistent with enrichment in EBV-specific
CD8+ T cells in the cerebrospinal fluid (CSF) of patients with early
MS , with increased CSF levels of granzymes during relapse in
relapsing-remitting MS patients , and with preferential
expansion of CD8+ T cells in MS brain lesions and CSF [55,56].
Defects in the control of viral infections are suspected to
promote the development of autoimmune diseases . Nearly all
of the genes whose variants have been associated with the risk of
developing MS are implicated in immune system function ,
making it plausible that in susceptible individuals subtle differences
in the regulation of the immune response might allow an EBV
infection to be established in the CNS and become the target of an
immunopathological response. Experimental studies suggest that
upon infection with persistent viruses establishment of
extralymphatic viral sanctuaries depends both on organ anatomy and
defective synergies between CD8+ T-cell- and antibody-mediated
immune responses . The present results do not answer the
question of whether EBV dysregulation is consequence or cause of
MS but disclose a link between EBV reactivation, antiviral
immune response and disease activity during the
relapsingremitting stage of MS. Such a scenario is consistent with the
results of randomized, double-blind, placebo-controlled clinical
and MRI studies of anti-herpesvirus therapy in relapsing-remitting
MS showing that anti-herpesvirus drugs inhibiting viral replication
have beneficial effects in subgroups of patients with higher
exacerbation rates and more severe disease activity [59,60].
Further work is needed to better understand whether and how an
altered balance between EBV and the host immune system
contributes to MS onset and verify the potential benefits of new
antiviral drugs in controlling MS .
Materials and Methods
All blood samples were obtained following acquisition of the
study participants written informed consent. The study protocol
was reviewed and approved by the local ethics committes of S.
Camillo Forlanini Hospital, Tor Vergata University, S. Andrea
Hospital, and Fondazione S. Lucia. Use of post-mortem human
brain material for the study purposes has been approved by the
ethics committee of the Istituto Superiore di Sanita`.
Patients and healthy donors
MS patients and HD were recruited between 2008 and 2012
from Tor Vergata University, S. Camillo Forlanini and S. Andrea
Hospitals in Rome. We enrolled 250 patients who were diagnosed
the relapsing-remitting form of MS according to the 2005 revised
McDonalds criteria . A neurologist (SR, CG, FB, DC, MS)
examined the patients, including assessment of the EDSS and
confirmation of clinical relapse or remission. Of the 250 enrolled
subjects, 113 MS patients were selected for this study according to
their HLA genotype (HLA-A*02101, B*0801) for which well
characterized EBV and CMV peptide antigens have been
described . Seventy-nine patients were free of therapy for
at least 3 months, 20 patients were treated with IFN-b
subcutaneously (12 with IFN-b 1a and 8 with IFN-b 1b) for 1
11 years (median = 4 years) and 14 MS patients were treated with
natalizumab for 816 months (median = 12 months). The control
subject group included 43 HD matched for sex and age and
selected for their HLA genotype (HLA-A*02101, B*0801). The
demographic and clinical characteristics of HD and MS patients
are summarized in Table 1. At the time of peripheral blood
collection 38 and 75 MS patients were in the active and inactive
phase of the disease, respectively, based on clinical assessment and
brain MRI (Table 2). Four MS patients (2 untreated and 2 treated
with natalizumab) and 3 HD were monitored longitudinally for 7
27 months and blood was drawn every 3 to 6 months.
Seventy-six % (60/79) of untreated patients and all
IFN-btreated patients were examined by brain MRI with gadolinium
enhancement on the same day or within 1 week before blood
collection; only in one case MRI was performed 3 weeks before
blood collection. All natalizumab-treated patients were monitored
with MRI every 6 months. Acquisition of brain MRI scans was
obtained in a single session. Conventional MRI scans were
acquired including the following sequences: Fast Fluid Attenuated
Inversion Recovery (FLAIR), T1 weighted images (T1-WI) before
and after gadolinium administration covering the whole brain.
The gadolinium enhanced T1-WI scans were obtained for all
patients 15 minutes after admnistration of gadolinium (0,1 mmol/
kg). MRI scans were classified as active if there was at least one
gadolinium enhancing lesion. As shown in Table 2, the majority
(86%) of active MS patients included in this study had both clinical
manifestations and an active MRI scan, while a minority showed
either clinical (6%) or MRI (8%) evidence of disease activity.
Conversely, all inactive patients exhibited neither clinical
manifestations nor disease activity on MRI.
Flow cytometry with antibodies and peptide/MHC
PBMCs were isolated on a Ficoll gradient (Ficoll-Paque PLUS,
GE Healthcare) and stained with pre-titrated antibodies. To
evaluate the CD8+ T cell response to EBV latent and lytic
antigens, PBMC from MS patients and HD were stained with
fluorochrome-labeled pentamers (ProImmune, Oxford, UK). The
analysis was conducted on freshly isolated PBMC with the
exclusion of dead cells, providing an accurate pattern of CD8+
T cell in vivo specificities. We analyzed CD8+ T cells specific for
two EBV lytic protein epitopes, the HLA-A*0201 restricted
epitope (GLCTLVAML) from BMLF-1 and the HLA-B*0801
restricted epitope (RAKFKQLL) from BZLF-1, and for two EBV
latent protein epitopes, the HLA-A*0201 restricted epitope
(CLGGLLTMV) from LMP-2A and the HLA-B*0801 restricted
epitope (FLRGRAYGL) from EBNA-3A. The CD8+ T cell
response to an HLA-A*0201 restricted immunodominant peptide
(NLVPMVATV) from pp65 of human cytomegalovirus (CMV)
was studied as a control for anti-viral MHC class I restricted CD8
One x 106 PBMCs were stained with 10 ml of PE
conjugatedpentamers alone, washed with PBS and then stained with anti
human CD3 APC Alexa e780 (eBioscience Inc., San Diego, CA)
and CD8 ECD (Beckman Coulter, Brea, CA). Cells were also
stained for dead cell exclusion (Fixable Dead Cell Stain Kits,
Invitrogen, Life Technologies, Paisley, UK). The samples were
acquired on a CyAN ADP cytometer (Beckman Coulter) and
analysed by FlowJo software (Tree Star, Ashland, OR).
Frequencies of pentamer+ cells below 0.02% of CD3+ T cells were
considered as background staining as indicated by the
manufacturer. An example of the gating strategy used to identify pentamer+
cells is shown in Figure S1.
Patients and tissues for immunohistochemical and
Thirteen cerebral tissue blocks from 7 MS cases (MS79, MS92,
MS121, MS154, MS180, MS234, MS342) who died in the
secondary progressive phase of MS and were characterized by
substantial brain inflammation were analyzed in this study. Tissues
were provided by the UK Multiple Sclerosis Tissue Bank at
Imperial College in London after collection via a prospective
donor program with fully informed consent. Based on the
available clinical documentation, all MS patients were in the
progressive phase of the disease, and no immunotherapy is
reported in the 6 months before death. Control tissues for BZLF-1
immunohistochemistry included fixed-frozen brain sections from
one control subject who died for cardiac failure (obtained from the
UK MS Tissue Bank), paraffin sections of a brain with tuberculous
meningo-encephalitis and of an EBV-negative brain B-cell
lymphoma (kindly provided by Dr R. Hoftberger, Clinical
Institute of Neurology, Wien), and paraffin sections of a tonsil
from a subject with active infectious mononucleosis (kindly
provided by Dr G. Niedobitek, Sana Klinikum Lichtenberg/
Eight brain tissue blocks (4 cm3 each; 1 snap frozen, 7 fixed
frozen) from 5 MS cases (MS92, MS121, MS154, MS180, MS342)
were used for immunohistochemical studies. Lesion inflammatory
activity was assessed as previously described . Five snap-frozen
brain tissue blocks from 4 MS cases (MS79, MS92, MS180,
MS342) were used to study BZLF-1 gene expression using
quantitative real-time RT-PCR. One snap-frozen control lymph
node was obtained from Dr Egidio Stigliano, Policlinico A.
Immunohistochemistry and immunofluorescence
Brain sections were stained using immunohistochemical and
single or double indirect immunofluorescence techniques.
Immunohistochemical detection of CD20, MHC class II antigen and
myelin-oligodendrocyte glycoprotein (MOG), and
immunofluorescence stainings for BFRF1, Ig-A,-G,-M, CD8 and perforin
alone or in different combinations were performed as previously
described . For BZLF-1 immunohistochemistry, deparaffinised
sections from infectious mononucleosis tonsil, cerebral B cell
lymphoma and brain with tuberculous meningo-encephalitis and
cryosections from PFA-fixed control brain were subjected to
antigen retrieval procedure in citrate buffer in microwave for 3
cycles of 3 min each before quenching of endogenous peroxidase
activity in PBS containing 0,1% H2O2. Sections were treated with
0.5% Triton X-100 in PBS for 10 minutes and incubated for
1 hour with normal serum 30%+0,25% Triton X-100 and then
overnight at 4uC with mouse monoclonal antibody (mAb) specific
for BZLF-1 protein (clone BZ-1, kindly provided by Dr J.
Middeldorp, VUMC, Amsterdam) diluted 1:50 in PBS containing
0,25% Triton X-100. Sections were then incubated with
biotinconjugated rabbit anti-mouse antibody (Jackson Immunoreaearch
Laboratories, West Grove, PA) for 1 hour at room temperature
(RT), ABC-peroxidase complex (Vector Laboratories, Burlingame,
CA) for 45 min, and AEC (DakoCytomation, Glostrup, Denmark)
or diaminobenzidine (Sigma, St Louis, MO) to reveal the immune
The EBV-producing B95-8 cells (marmoset B-cell line
transformed with EBV)  were used as positive control for BZLF-1
immunofluorescence staining. Paraformaldehyde-fixed frozen
brain sections from MS cases and one control case were air-dried
and post-fixed in 4% PFA for 5 minutes at RT or in iced acetone
for 10 minutes at 4uC. Sections were subjected to antigen
unmasking, permeabilization steps and block of unspecific binding
sites as described above and then incubated for 36 h at 4uC with
BZ-1 mAb (diluted 1:50 in PBS +0,1% Triton X-100). Antibody
binding was visualized using tetramethyl rhodamine
isothiocyanate (TRITC)-conjugated goat anti-mouse antibody (Jackson
Laboratories) containing 5% normal goat serum for 50 minutes
at RT. After washing, sections were sealed in DAPI-containing
medium or incubated further with FITC-conjugated rabbit
antihuman Ig-A-G-M (1:400; Dako Cytomation) for 1 hour at RT.
For double immunofluorescence for BZLF-1 and CD8, sections
were stained with BZ-1 mAb and anti-human CD8 rabbit
polyclonal antibody (1:50; Pierce, Thermo Fisher Scientific Inc.
Rockford, IL) followed by a mixture of Alexa Fluor
488conjugated goat anti-mouse and TRITC-conjugated goat
antirabbit secondary antibodies. Double and triple immunostainings
for CD8/granzyme B and CD8/granzyme B/BFRF1 were
performed by incubating PFA-fixed cryosections with a mixture
of anti-CD8 rabbit polyclonal antibody and anti-granzyme B mAb
(Dako), or anti-CD8 mAb, anti-BFRF1 rabbit polyclonal (1:800)
and anti-granzyme B goat polyclonal (1:50, Santa Cruz) antibodies
overnight at 4uC, and then with a mixture of donkey FITC
antimouse (Invitrogen, Eugene, OR), TRITC anti-rabbit and AMCA
anti-goat (Jackson Immunoresearch Lab) secondary antibodies.
Sections were sealed in ProLong Gold antifade reagent with
49,69diamidino-2-phenylindole (DAPI) (Invitrogen) or in Vectashield
(Vector Laboratories). Images were analysed and acquired with a
digital epifluorescence microscope (Leica Microsystem, Wetzlar,
Germany). Negative control stainings were performed using Ig
isotype controls and/or pre-immune sera.
Laser capture microdissection
Snap-frozen brain tissue blocks from 4 MS cases (MS79, MS92,
MS180, MS342) and control lymph node were used for laser
capture microdissection and subsequent RNA analysis. For each
tissue block, the integrity and quality of total RNA extracted with
the SV Total RNA Isolation System (Promega, Madison, WI) were
checked on ethidium bromide containing 1% agarose gels in
Trisborate/EDTA buffer. Ten to 20 serial brain sections for each MS
case and from control lymph-node were mounted on membrane
slides for laser capture microdissection (MMI AG, Glattbrugg,
Switzerland) and processed as described previously . Sections
before and after these series were stained for CD20 and Ig to
identify B cell- and plasma cell-containing regions in the inflamed
meninges and white matter lesions. Using a laser microdissector
SL Cut (MMI AG) equipped with a Nikon Eclipse TE2000-S
microscope, we isolated areas containing meningeal infiltrates and
B-cell follicles, lesioned grey matter, B cell-enriched perivascular
cuffs in white matter lesions, lesioned white matter surrounding
inflamed blood vessels, and normal-appearing white matter. The
same brain areas were cut in 3 to 10 serial sections and pooled in a
single cap. B cell follicles were isolated from the lymph-node. The
isolated tissue fragments were collected in 50 mL of lysis buffer
(PicoPure RNA isolation kit, Arcturus Engineering), incubated at
42Cu for 30 minutes and centrifuged at 8006 g for 2 minutes.
Lysates were stored at 280 Cu until use.
RNA isolation and quantitative real time RT-PCR
DNase-treated total RNA was extracted from 20-mm-thick brain
sections or microdissected areas from 4 MS brains and 1 control
lymph node, as previously described . RNA samples were
reverse-transcribed with oligo (dT) and random hexamers using
the Murine Leukemia Virus Reverse Transcriptase (Invitrogen
Life Technologies, Carlsbad, CA). PreAMP Master Mix Kit
(Applied Biosystems, Foster City, CA) was used to enrich for both
cellular and viral gene transcripts. The cDNAs obtained from
whole brain sections and microdissected brain and lymph node
samples were preamplified according to the manufacturers
instructions using 90 nmol/L of each primer in a mix containing
the same forward and reverse primers for GAPDH, CD19 and
BZLF-1 used for real time RT-PCR. Quantitative PCR assays
were performed in triplicate, as previously described . cDNA
from EBV-positive P3HR-1 cells and human primary B cells were
included in each run as positive controls for BZLF-1 and CD19
gene expression, respectively. Sample values were normalized by
calculating the relative quantity of each mRNA to that of GAPDH
using the formula 22DCt, where DCt represents the difference in
cycle threshold (Ct) between target mRNA and GAPDH mRNA.
The following primer pairs were used in this study: GAPDH_for
GCCTGCTTCACCACCTTCTTG ; BZLF-1_for
AACAGCTAGCAGACATTGGTG ; CD19_for AGAACCAGTACGGGAACGTG;
CD19_rev CTGCTCGGGTTTCCATAAGA .
Differences between categorical variables were evaluated by
Pearsons chi-squared test while differences between continuous
variables were analysed by unpaired t-test with 95% confidence
Figure S1 Flow cytometric analysis of EBV-specific
CD8+ T cells. Examples of flow cytometric profiles
demonstrating the gating strategy to identify live CD8+ T cells specific for one
of the EBV peptides (BZLF1) tested. The threshold for pentamer
positivity was set at .0.02% of CD3+ cells. The number in the
right panel indicates the percentage of pentamer+ cells within the
CD3+ T cell population.
Figure S2 Prevalence of EBV- and CMV- specific CD8+ T
cell responses in HLA-A2+ HD and MS patients. (A)
HLAA*0201 pentamer+ CD8+ T cells specific for latent (LMP-2A) and
lytic (BMLF-1) antigens (left panel) and for CMV antigen (pp65)
(right panel) were investigated in HLA-A2+ HD (n = 34) and MS
patients (n = 45). No differences were found in the percentages of
HD and MS patients with EBV- and CMV-specific CD8+ T cells
over the threshold for pentamer positivity. (B) When patients were
stratified according to disease activity (active MS = a-MS, n = 18;
inactive MS = i-MS, n = 27), the proportion of inactive MS patients
with a detectable EBV-specific CD8+ T cell response tended to be
lower than that of HD and active MS patients (left panel). In
contrast, no differences in the prevalence of CMV-specific CD8+ T
cell responses were found between HD, inactive MS and active MS
patients (right panel). The percentages of individuals with detectable
EBV or CMV pentamer+ CD8+ T cells (grey columns) among the
total donors tested (white columns) are shown; p values were
calculated with Pearsons chi-squared test.
Figure S3 Lack of differences in the magnitude of
EBVand CMV-specific CD8+ T cell responses between
HLAA2+ healthy donors and MS patients. (A) The frequencies of
CD8+ T cells specific for EBV latent (LMP-2A) and lytic
(BMLF1) antigens and for CMV antigen (pp65) were analyzed in
HLAA2+ HD (n = 17) and MS patients (n = 16) by staining with the
corresponding peptide/HLA-A*0201 pentamers. The percentages
of pentamer+ cells were calculated after gating on total
CD3+CD8+ T cells. No differences were found in the frequencies
of EBV- and CMV-specific CD8+ T cells between HD and total
MS patients. Bars represent the median 6 the minimum and
maximum value. (B) Similar frequencies of CMV-specific CD8+ T
cells were found in HD (n = 9), active MS (a-MS n = 6) and
inactive MS (i-MS n = 7) patients. Data in logarithmic scale and
mean values 6 SD are shown; p values are calculated with
unpaired t-test with 95% confidence intervals. (C) Examples of
flow cytometric profiles for pentamer+ CD8+ T cells specific for
CMV antigen in HD, active and inactive MS patients. The
numbers represent the percentages of pentamer+ cells within the
CD3+ CD8+ T-cell population.
Figure S4 Lack of correlation between frequency of
EBV-specific CD8+ T cells and MS disease duration.
Disease duration (x-axis) was correlated with the frequencies of
CD8+ T cells specific for the EBV latent and lytic antigens tested
(y-axis) in inactive MS (n = 13) (left panel) and active MS (n = 13)
(right panel) patients. Each symbol represents the individual
response to a different EBV antigen. No statistically significant
correlation was found between the frequency of EBV-specific
CD8+ T cells and disease duration in both patient groups
(Spearmans coefficient r).
Figure S5 Immunostaining for BZLF-1 protein in
control cells and tissues. A) EBV-producing B95-8 cells
[marmoset B-cell line transformed with EBV (Miller G. and A.
Lipman. Proc.Natl.Acad.Sci. USA 70: 190194, 1973)] were
induced for 48 h with 12-O-tetradecanoylphorbol-13-acetate
(20 ng/ml) and sodium butyrate (3 mM) to activate viral
replication, and used as positive control for BZLF-1
immunofluorescence staining. Many cells are positive for BZLF-1 (localized in
the nucleus, red staining); cell nuclei are visualized with DAPI
stain (blue). The inset shows a BZLF-1+ nucleus at high
magnification. B) Immunostaining for BZLF-1 in a tonsil from a
patient with infectious mononucleosis (nuclear brown signals); high
magnification of a BZLF-1+ cell is shown in the inset. Absence of
BZLF-1 immunostaining in brain sections from a control case,
died for cardiac failure (C), from a patient with tuberculous
meningoencephalitis (D) and in an EBV-negative cerebral B-cell
lymphoma (E). Bars: 200 mm in C-E; 50 mm in B; 20 mm in A and
inset in B; 10 mm in the inset in A.
All post-mortem MS brain samples were supplied by the UK MS Tissue
Bank (www.ukmstissuebank.imperial.ac.uk). The authors would like to
thank members of the UK MS Tissue Bank (R. Reynolds, G. Dveric, S.
Fordham, J. Steele) for the assistance in the collection of the material used
in this study. We further acknowledge Mrs Estella Sansonetti for graphical
Serafini, M. Salvetti, E.M. Coccia, G. Borsellino, F. Aloisi, L. Battistini.
Contributed reagents/materials/analysis tools: S. Ruggieri, C. Gasperini,
F. Buttari, D. Centonze, R. Mechelli, M. Salvetti. Performed the
experiments: D.F. Angelini, B. Serafini, E. Piras, M. Severa, B. Rosicarelli,
G. Borsellino. Wrote the paper: D.F. Angelini, G. Borsellino, F. Aloisi, L.
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