HLA-B7-restricted EBV-specific CD8+ T cells are dysregulated in multiple sclerosis.
ABSTRACT It was hypothesized that the EBV-specific CD8(+) T cell response may be dysregulated in multiple sclerosis (MS) patients, possibly leading to a suboptimal control of this virus. To examine the CD8(+) T cell response in greater detail, we analyzed the HLA-A2-, HLA-B7-, and HLA-B8-restricted EBV- and CMV-specific CD8(+) T cell responses in a high number of MS patients and control subjects using tetramers. Content in cytolytic granules, as well as cytotoxic activity, of EBV- and CMV-specific CD8(+) T cells was assessed. We found that MS patients had a lower or a higher prevalence of HLA-A2 and HLA-B7, respectively. Using HLA class I tetramers in HLA-B7(+) MS patients, there was a higher prevalence of MS patients with HLA-B*0702/EBV(RPP)-specific CD8(+) T cells ex vivo. However, the magnitude of the HLA-B*0702/EBV(RPP)-specific and HLA-B*0702/CMV(TPR)-specific CD8(+) T cell response (i.e., the percentage of tetramer(+) CD8(+) T cells in a study subject harboring CD8(+) T cells specific for the given epitope) was lower in MS patients. No differences were found using other tetramers. After stimulation with the HLA-B*0702/EBV(RPP) peptide, the production of IL-2, perforin, and granzyme B and the cytotoxicity of HLA-B*0702/EBV(RPP)-specific CD8(+) T cells were decreased. Altogether, our findings suggest that the HLA-B*0702-restricted viral (in particular the EBV one)-specific CD8(+) T cell response is dysregulated in MS patients. This observation is particularly interesting knowing that the HLA-B7 allele is more frequently expressed in MS patients and considering that EBV is associated with MS.
- SourceAvailable from: direct-ms.org[show abstract] [hide abstract]
ABSTRACT: Although genetic susceptibility explains the clustering of multiple sclerosis (MS) cases within families and the sharp decline in risk with increasing genetic distance, it cannot fully explain the geographic variations in MS frequency and the changes in risk that occur with migration. Epidemiological data provide some support for the "hygiene hypothesis," but with the additional proviso for a key role of Epstein-Barr virus (EBV) in determining MS risk. We show that whereas EBV stands out as the only infectious agent that can explain many of the key features of MS epidemiology, by itself the link between EBV and MS cannot explain the decline in risk among migrants from high to low MS prevalence areas. This decline implies that either EBV strains in low-risk areas have less propensity to cause MS, or that other infectious or noninfectious factors modify the host response to EBV or otherwise contribute to determine MS risk. The role of infectious factors is discussed here; in a companion article, we will examine the possible role of noninfectious factors and provide evidence that high levels of vitamin D may have a protective role, particularly during adolescence. The primary purpose of these reviews is to identify clues to the causes of MS and to evaluate the possibility of primary prevention.Annals of Neurology 05/2007; 61(4):288-99. · 11.19 Impact Factor
- [show abstract] [hide abstract]
ABSTRACT: Multiple sclerosis (MS) is an autoimmune demyelinating disease and a common cause of neurological disability in young adults. The modest heritability of MS reflects complex genetic effects and multifaceted gene-environment interactions. The human leukocyte antigen (HLA) region is the strongest susceptibility locus for MS, but a genome-wide association study recently identified new susceptibility genes. Progress in high-throughput genotyping and sequencing technologies and a better understanding of the structural organization of the human genome, together with powerful brain-imaging techniques that refine the phenotype, suggest that the tools could finally exist to identify the full set of genes influencing the pathogenesis of MS.Nature Reviews Genetics 08/2008; 9(7):516-26. · 41.06 Impact Factor
- [show abstract] [hide abstract]
ABSTRACT: Multiple sclerosis (MS) is a presumed autoimmune disease of the central nervous system, shown to be associated with the HLA class II haplotype DRB1*15,DQB1*06. Carrying the HLA class II haplotype DRB1*15,DQB1*06 increases the risk of MS by 3.6. By adopting a polymerase chain reaction (PCR)-based typing technique for HLA class I and class II genes, 200 Swedish MS patients and 210 Swedish healthy controls were analysed for their HLA alleles. Additional HLA class I alleles that increase and decrease the genetic susceptibility to MS were identified. The HLA-A*0301 allele increases the risk of MS (odds ratio=2.1) independently of DRB1*15,DQB1*06. HLA-A*0201 decreases the overall risk (odds ratio= 0.52) and the presence of A*0201 reduces the risk of MS for DRB1*15,DQB1*06 carriers from 3.6 to 1.5. Our findings are the first to identify a major modulating effect of HLA class I alleles on the susceptibility to a human autoimmune disease; a phenomenon that has previously only been observed in animal models.Tissue Antigens 03/2000; 55(2):140-8. · 2.93 Impact Factor
The Journal of Immunology
HLA-B7–Restricted EBV-Specific CD8+T Cells Are
Dysregulated in Multiple Sclerosis
Samantha Jilek,* Myriam Schluep,†Alexandre Harari,*,‡Mathieu Canales,*
Andreas Lysandropoulos,†Anastasia Zekeridou,†Giuseppe Pantaleo,*,‡and
Renaud A. Du Pasquier*,†
It was hypothesized that the EBV-specific CD8+T cell response may be dysregulated in multiple sclerosis (MS) patients, possibly
leading to a suboptimal control of this virus. To examine the CD8+T cell response in greater detail, we analyzed the HLA-A2–,
HLA-B7–, and HLA-B8–restricted EBV- and CMV-specific CD8+T cell responses in a high number of MS patients and control
subjects using tetramers. Content in cytolytic granules, as well as cytotoxic activity, of EBV- and CMV-specific CD8+T cells was
assessed. We found that MS patients had a lower or a higher prevalence of HLA-A2 and HLA-B7, respectively. Using HLA class I
tetramers in HLA-B7+MS patients, there was a higher prevalence of MS patients with HLA-B*0702/EBVRPP-specific CD8+T cells
ex vivo. However, the magnitude of the HLA-B*0702/EBVRPP-specific and HLA-B*0702/CMVTPR-specific CD8+T cell response
(i.e., the percentage of tetramer+CD8+T cells in a study subject harboring CD8+T cells specific for the given epitope) was lower in
MS patients. No differences were found using other tetramers. After stimulation with the HLA-B*0702/EBVRPPpeptide, the
production of IL-2, perforin, and granzyme B and the cytotoxicity of HLA-B*0702/EBVRPP-specific CD8+T cells were decreased.
Altogether, our findings suggest that the HLA-B*0702–restricted viral (in particular the EBVone)-specific CD8+T cell response is
dysregulated in MS patients. This observation is particularly interesting knowing that the HLA-B7 allele is more frequently
expressed in MS patients and considering that EBV is associated with MS.
factors, sero-epidemiological studies revealed that the g-herpesvirus
EBV was associated with MS (5, 6). We and other investigators
demonstrated that EBV-specific cellular immune responses are in-
creased not only in the blood (7–10) but also in the cerebrospinal
fluid of MS patients (11). Interestingly, we showed that the increase
in EBV-specific CD8+T cell response was maximal at disease onset
but decreased with disease duration (9). Others investigators found
a decreased reactivity to EBV-infected B cells in MS patients,
which was not correlated with disease duration. However, in the
latter study, clinically isolated syndrome (CIS) patients were not
The Journal of Immunology, 2012, 188: 4671–4680.
nvironmental and genetic factors are both thought to be
Among the latter, HLA class I alleles were shown to be
A higher EBV viral load, leading to the exhaustion of Ag-
specific CD8+T cells, may explain why EBV-specific CD8+
T cell responses decrease with time. However, whether an in-
creased EBV viral load is present in MS patients remains con-
troversial (10, 13). There also may be an intrinsic dysregulation of
EBV-specific CD8+T cells in MS patients (14). Indeed, CD8+
T cells are central to controlling EBV during primary infection
(15). Thus, a dysregulated immune response to EBV may explain
why there is a suboptimal control of EBV that precedes the
clinical onset of MS (5, 6). Such a suboptimal control was hy-
pothesized by some investigators (14). In our work, we have seen
some hints for a dysregulated EBV-specific CD8+T cell response,
as reflected by a concomitant increased expression of mRNA
coding for IFN-g and IL-4 (16), a cytokine combination that was
reported to be a marker of suboptimal control of viruses (17).
Indeed, virus-specific CD8+T cells can be defined according to
their ability to secrete IFN-g, TNF-a, and IL-2 (17, 18). Poly-
functional CD8+T cells are associated with protective antiviral
infections with concomitant production of IFN-g, TNF-a, and
IL-2. They are able to proliferate following Ag-specific stimula-
tion, thanks to IL-2, and have cytotoxic functions (19, 20). In con-
trast, effector CD8+T cells that secrete only IFN-g are associated
with a lack of virus control (17).
Viral-specific cytotoxicity relies to a large extent on cytotoxic
granules. Cytotoxic CD8+T lymphocytes (CTL) contain perforin,
granzymes (Grm) (21). In humans, five granzymes are known:
GrmA, GrmB, GrmH, GrmK, and GrmM (21). CTL bind to target
cells and secrete cytotoxic granules, which lead to the death of the
target cells (22). It was recently shown in our laboratory that the
and GrmK of virus-specific CD8+T cells differed, depending on the
virus for which the CD8+T cells were specific, with perforin and
GrmBbeing the moststronglyassociatedwiththe cytotoxicfunction
*Division of Immunology and Allergy, Department of Medicine, University Hospital
of Lausanne, 1011 Lausanne, Switzerland;†Division of Neurology, Department of
Clinical Neurosciences, University Hospital of Lausanne, 1011 Lausanne, Switzer-
land; and‡Swiss Vaccine Research Institute, 1011 Lausanne, Switzerland
Received for publication October 27, 2011. Accepted for publication March 3, 2012.
This work was supported by grants from the Swiss National Foundation (PP00B-
106716, PP00P3-124893), the Swiss Society for Multiple Sclerosis, and the Biaggi
Foundation (to R.A.D.P.).
Address correspondence and reprint requests to Prof. Renaud A. Du Pasquier, Division
of Neurology, Department of Clinical Neurosciences, BH-10, University Hospital of
The online version of this article contains supplemental material.
Abbreviations used in this article: CIS, clinically isolated syndrome; CTL, cytotoxic
CD8+T lymphocyte; Grm, granzyme; MFI, mean fluorescence intensity; MS, mul-
tiple sclerosis; NIND, noninflammatory neurologic disease; PD-1, programmed cell
death 1; rhIL-2, recombinant human IL-2.
the perforin/granzyme pathway rather than by the Fas/Fas ligand
We previously demonstrated that MS patients exhibited a strong
EBV-specific CD8+T cell response in the early course of MS,
a response that decreased significantly with the progression of the
disease (9). In this study, we sought to determine whether EBV-
Materials and Methods
We performed HLA class I (A and B) typing on 439 consecutive subjects,
including 221 MS patients and 218 control subjects. The MS patient group
included 43 patients with CIS, 123 patients with remitting-relapsing MS, 28
patients with secondary-progressive MS, and 27 patients with primary-
progressive MS. Storage and analysis of clinical data from patients with
MS were performed using iMed Software (Merck Serono, Geneva, Swit-
zerland). At the time of enrollment, the diagnosisof MSwas made using the
2005 revised McDonald criteria (25). The control subject group included
74 patients with other inflammatory neurologic diseases, 100 patients with
noninflammatory neurologic diseases (NIND), and 44 healthy subjects.
This study was accepted by the review board of the University Hospital of
Lausanne, and all subjects gave their written informed consent.
None of the study subjects had received corticosteroids within the 3 mo
prior to enrollment. MS patients were considered relapsing if a relapse had
started ,4 wk prior to the blood draw. Thirty-four patients were relapsing
at the time of the assay.
HLA-A2 was shown to be a protective allele in MS, because it is less
common in MS patients than in the general population (26), in contrast to
HLA-B7, which is more prevalent in MS patients (27). Therefore, we were
particularly interested in studying the virus-specific CD8+T cell response
restricted by these two HLA class I alleles. HLA-B8 has not been asso-
ciated with MS and was used as a “control” allele. Thus, the studies de-
scribed hereafter were performed on MS patients or control subjects who
harbored at least one of these three HLA class I alleles.
Because the prevalence of HLA-A*02, HLA-B*07, and HLA-B*08
haplotypes, other than the common HLA-A*0201, HLA-B*0702, or HLA-
B*0801 haplotype, is very low in the Swiss population (28), all of the
study subjects had a two-digit HLA typing.
To determine the EBV and CMV status [the latter virus being used as
a control (9, 11)], EBVand CMV serologies were performed in those study
subjects who had at least one of the three HLA class I alleles of interest.
For EBV, plasma IgG titers were determined for EA, VCA, and EBNA-1,
markers of primo-infection or recent reactivation, viral latency and reac-
tivation, and viral latency only, respectively (Athena Multi-Lyte IgG test
System; Teomed, Greifensee, Switzerland) in 1:20 diluted plasmas, using
a Luminex S-100 (Luminex, Oosterhout, The Netherlands). CMV IgG
titers were assessed with the Enzygnost anti-CMV/IgG kit (Behring, Bern,
Switzerland) using a plasma dilution of 1:200. Data are expressed as AU/
ml. Samples were considered positive when the result was .119 AU/ml.
Isolation of PBMC
PBMC were isolated as previously described and frozen until use (9).
Plasma was harvested and kept at 280˚C until use (9).
Detection of EBV DNA was performed in plasma and PBMC using real-
time PCR (10). DNA was extracted from plasma and PBMC with the
QIAamp DNA Mini Kit (QIAGEN, Basel, Switzerland). The set of primers
and probe were located in the BamHI W fragment of EBV. The forward
primer was 59-GGACCACTGCCCCTGGTA-39, the reverse primer was 59-
TTTGTGTGGACTCCTGGG-39, and the probe was 6-FAM-59-TCCTG-
CAGCTATTTCTGGTCGCATCA-39-TAMRA. The real-time PCR reac-
tion was performed as previously described (29). Positive controls for EBV
DNA detection were B-lymphoblastoid cell lines derived from an MS
patient in our laboratory. All preparatory PCR steps, including DNA ex-
traction, were done in a separate room isolated from any post-PCR samples
to prevent contamination. Because of a shortage of PBMC in 10 study
subjects, the interval between the real-time PCR assay and the cellular
assays was 0.02 6 0.9 y for MS patients and 0 6 0.1 y for control subjects.
Peptides and tetramer staining
Eight- to ten-mer peptide epitopes were used for PBMC stimulation (Table
I). All peptides were purchased from JPT Peptide Technologies (Berlin,
Germany) and used at 1 mg/ml.
To determine which patients had a virus epitope-specific CD8+T cell
response, PBMC of the patients harboring at least one of the three HLA
class I of interest and with positive serology for EBV and/or CMV were
stained with the tetramers listed in Table I. Fresh PBMC were stained for
30 min at 4˚C with the PE-coupled tetramers. CD3–PerCP–Cy5.5 and
CD8-allophycocyanin Abs (both from Becton Dickinson, Franklin Lakes,
NJ) were added for 20 min at 4˚C. Cells were washed, and data were
acquired on a FACSCalibur flow cytometer (Becton Dickinson). An ex
vivo tetramer staining was considered as positive if $0.05% of CD3+CD8+
T cells were tetramer+and if the tetramer+cells were clearly demarcated
from the other cells.
Content in IFN-g, TNF-a, and IL-2 in EBV-specific CD8+
To determine the content in proinflammatory cytokines, freshly isolated
PBMC were stimulated for 18 h with one of the five peptides used in this
study (Table I) in complete medium containing 1 ml/ml GolgiPlug (Becton
Dickinson) and 0.5 mg/ml anti-CD28 Ab (Becton Dickinson). An intra-
cellular cytokine staining assay was performed, as previously described
(9), with the following Abs: CD3–allophycocyanin–Cy7, CD8-Pacific
blue, IFN-g–allophycocyanin, TNF-a–FITC, IL-2–PE (all from Becton
Dickinson), and CD4-ECD (Beckman Coulter). Dead cells were excluded
using the Aqua LIVE/DEAD stain kit (Invitrogen, Basel, Switzerland).
Data were acquired on a LSRII flow cytometer (Becton Dickinson). PBMC
stimulated with complete medium alone were used to determine the
background. The background values never exceeded 0.1% of CD3+CD8+
T cells, and they were subtracted from the stimulated sample values before
Ex vivo analysis of cytotoxic granule content and
differentiation markers in EBV- and CMV-specific CD8+
To examine the ex vivo content in perforin, GrmA, GrmB, and GrmK of
virus epitope-specific CD8+T cells, 1–2 million PBMC were incubated
with the EBV or CMV tetramers at 4˚C for 30 min, permeabilized with
Cytofix/Cytoperm (Becton Dickinson), and stained at room temperature
for 20 min with GrmK-FITC (Santa Cruz Biotechnology, Santa Cruz, CA),
GrmA-Pacific blue, perforin-allophycocyanin (both from BioLegend, San
Table I.EBV- and CMV-specific tetramers used in this study
VirusHLA restriction ProteinPositionPeptide TetramerReferencesObtained From
EBV HLA A*0201 BMLF1 (lytic)259–267 GLCTLVAML HLA-A2/EBVGLC
Bharadwaj et al. (52)Beckman Coulter,
Champagne et al. (31)
National Institutes of Health
Tetramer Core Facility,
Champagne et al. (31)
HLA-B*0702 EBNA-3A (latent) 379–387
Hill et al. (53)
Bogedain et al. (54)
Diamond et al. (55)
CMV HLA-B*0702pp65417–426 TPRVTGGGAM HLA-B7/CMVTPR
Wills et al. (56)
4672HLA-B7–RESTRICTED EBV-SPECIFIC RESPONSE IN MS
Diego, CA), GrmB-Alexa Fluor 700, CCR7–PE–Cy7, CD8–PerCP–Cy5.5
(all from Becton Dickinson), or CD57–PE–Cy5 (Abcam, Cambridge,
U.K.) (23). For programmed cell death 1 (PD-1) staining, PBMC were
incubated with the tetramer, as described above, and stained with CD3-
ECD (Beckman Coulter), CD8–allophycocyanin–H7 (Becton Dickinson),
and PD-1–FITC (Becton Dickinson). Cells were acquired on a LSRII
SORP flow cytometer (Becton Dickinson).
Analysis of cytotoxic granule content in EBV- and
CMV-specific CD8+T cells after short-term cultures
To examine the change in the cytotoxic granule content of the virus-specific
CD8+T cell response upon antigenic challenge, 2–4 million PBMC were
stimulated with peptides, according to the HLA class I typing of studied
subjects. Peptides were added at 1 mg/ml, and PBMC were cultured for
7 d in RPMI 1640 supplemented with 8% human serum (Institut de Bio-
technologies Jacques Boy, Reims, France) in the presence or absence of 20
U/ml recombinant human IL-2 (rhIL-2; Roche Diagnostics, Rotkreuz,
Switzerland). When rhIL-2 was added, it was done 48 h after the start of
the culture. After 7 d, cultured PBMC were washed and stained with the
corresponding tetramers, as described above. Data were acquired on an
LSRII flow cytometer (Becton Dickinson).
Functional CFSE CTL assay
antigenic challenge, 2–4 millions PBMC were stimulated with the EBV- or
CMV-specific HLA-B*0702–restricted peptides. After 7 d, cultured PBMC
were used as effector cells in a functional CFSE CTL assay. Presence of
EBV- and CMV-specific CTL was assessed using the functional CFSE-
based CTL assay, as previously described (11). Briefly, target cells were
prepared by staining autologous PBMC with 0.25 mM CFSE (Invitrogen)
and loading them for 3 h with the relevant Ag at 1 mg/ml (EBV or CMV
HLA-B*0702–restricted peptides) or an irrelevant HIV Clade C 15-mer
GagHPVHAGPIAPGQMREpeptide. After incubation, cells were washed and
used as targets in the CTL assay. Four thousand target cells/well were
incubated in U-bottom 96-well plates for 18 h in a total volume of 200 ml/
well. Importantly, the assay was normalized in terms of tetramer effectors,
where the percentage of tetramer+cells after culture was assessed, and the
final effectortetramer+:target ratios were 0:1, 0.1:1, 0.25:1, 0.5:1, 1:1, and
2:1. The wells were harvested, and a constant number of allophycocyanin-
fluorescent beads (Becton Dickinson) was added just before acquisition
on a FACSCalibur flow cytometer (Becton Dickinson) to normalize the
number of CFSE-bright cells to the number of beads in each tube. The
specific lysis was calculated according to the following formula: per-
centage specific lysis = 1 2 (number of CFSE-bright cells/number of beads
for a particular E:T ratio)/(number of CFSE-bright cells/number of beads
at 0:1 ratio) 3 100, where the 0:1 ratio represents no effector cells. The
background lysis in the presence of the irrelevant HIV peptide at different
E:T ratios was subtracted from the EBV- or CMV-specific lysis results.
Flow cytometer data were acquired using FlowJo (Tree Star, Ashland, OR)
and SPICE (developed by Mario Roederer, Vaccine Research Center,
National Institute of Allergy and Infectious Diseases, National Institutes of
Health) software programs. Analysis and presentation of distributions were
performed using SPICE version 5.1, downloaded at http://exon.niaid.nih.
Statistical analysis was performed with GraphPad Prism software
(GraphPad Software, San Diego, CA). The differences among the groups of
study subjects were tested using the Kruskal–Wallis test for nonnormally
distributed variables. The difference between two groups was tested using
the nonparametric Mann–Whitney ranked test, whereas paired samples
were tested with the nonparametric Wilcoxon-ranked test. Analyses of
frequencies were performed using the Fisher exact test. A p value , 0.05
was considered significant.
HLA-B7 is more prevalent in MS patients
Of the 439 consecutively enrolled study subjects, 307 (70%) were
HLA-A2+, HLA-B7+, and/or HLA-B8+. As found by other in-
vestigators (26, 27), the prevalence of HLA-A2 was lower in MS
patients (33%) compared with control patients/subjects (52%, p =
0.0002), whereas the prevalence of HLA-B7 was higher in MS
patients (32%) compared with control subjects (21%, p = 0.013).
The prevalence of HLA-B8 was identical in both MS patients
(18%) and controls (18%; Fig. 1).
Subsequent experiments were not performed in 91 of the 307
HLA-compatible study subjects because of a lack of available
PBMC or loss to follow-up. All 220 remaining study subjects were
infected by EBV, except for 1 NIND control subject. Ninety-nine
of the 220 (45%) were infected by CMV, including the NIND
subject who was negative for EBV. Clinical and paraclinical data
for these 220 study subjects are displayed in Table II.
Higher prevalence of HLA-B7/EBVRPP–specific CD8+T cells
in MS patients
We then assessed the prevalence of EBV- and CMV-specific CD8+
T cells ex vivo using HLA-A*0201, HLA-B*0702, and HLA-
B*0801/viral peptide epitope tetramers in the 220 study subjects
(Table II). A positive tetramer staining specific for at least one
viral epitope was found in 155/220 study subjects (70%). Inter-
estingly, we found that the prevalence of HLA-B7/EBVRPP–spe-
cific CD8+T cells was higher in HLA-B7+MS patients (43/59
[73%]) than in HLA-B7+control subjects (19/39 [49%], p =
0.019; Fig. 2A). In contrast, there was no significant difference
between these two categories of study subjects in terms of the
prevalence of CD8+T cells specific for the four other viral epit-
opes (Fig. 2).
Contrasting with their higher prevalence in MS patients com-
pared with control subjects, the magnitude of HLA-B7/EBVRPP-
specific CD8+T cells (i.e., the percentage of tetramer+CD8+
T cells in a study subject harboring CD8+T cells specific for
a given epitope) tended to be lower (p = 0.07; Fig. 2A). The
magnitude of HLA-B7/CMVTPRtetramer cells was significantly
lower in MS patients (p = 0.004; Fig. 2B). In contrast, there was
no difference between MS patients and control subjects in terms
of the magnitude of tetramer staining for HLA-A2/EBVGLC, HLA
A2/CMVNLV, or HLA-B8/EBVRAK(Fig. 2). Of note, nine MS
hort of 439 patients. The percentages indicate the number of study subjects
carrying the HLA allele of interest compared with the total amount of
subjects tested. *p , 0.05, ***p ,0.001. CTRL, Control subjects in-
cluding other neurologic diseases patients and healthy subjects; MS, MS
Prevalence of HLA-A2, HLA-B7, and HLA-B8 in our co-
The Journal of Immunology 4673
patients were on IFN-b treatment, including six who were re-
lapsing, suggesting that the immunomodulatory effect of IFN-b
was overruled by the inflammation related to the relapse and, thus,
reasonably ruling out a significant role for IFN-b in our results.
Production of IFN-g and IL-2 is decreased in MS patients after
Having found that the presentation of EBVRPP and CMVTPR
immunodominant epitopes by the HLA-B7 allele induced a dif-
ferent CD8+T cell response between MS patients and control
subjects, we looked at the effector functions of these viral peptide
epitope-specific CD8+T cells. Specifically, we determined the
ex vivo production of IFN-g, TNF-a, and IL-2 by the five viral
peptide epitope-specific CD8+T cells and found no differences
between MS patients and control subjects.
However, we were also interested in determining the amount of
cytokine secreted by viral peptide epitope-specific CD8+T cells. To
this end, we examined the mean fluorescence intensity (MFI),
which reflects the amount of cytokine produced by a single cell. We
found that HLA-B7–restricted EBVRPP-specific CD8+T cells of
MS patients exhibited a lower MFI for IFN-g and IL-2 than did
those of control subjects (p = 0.015 and p = 0.0003, respectively,
Fig. 3A). In contrast, there was no difference in MFI between these
two categories for both HLA-A2–restricted EBVGLC-specific CD8+
T cells and HLA-B8–restricted EBVRAK-specific CD8+T cells
For the CMV-specific CD8+T cell responses, there was no
difference in the MFI for HLA-A2–restricted CMVNLV-specific
CD8+T cells for all three cytokines, whereas there was a trend
for an increased secretion of IL-2 in HLA-B7–restricted
CMVTPR-specific CD8+T cells in MS patients (p = 0.053; Fig.
The content in cytolytic granules of EBV- and CMV-specific
CD8+T cells is normal in MS patients
To pursue our detailed analysis of this seemingly dysregulated
HLA-B7/EBVRPP–specific CD8+T cell response, we investigated
the ex vivo content in cytolytic granules (perforin, GrmB, GrmA,
and GrmK) of HLA-A2, HLA-B7, or HLA-B8 tetramer+CD8+
T cells (representative patient, Supplemental Fig. 1A). To perform
an in-depth characterization, a sufficiently high number of tetra-
mer+CD8+T cells is needed. Thus, we included 77 subjects who
had $0.05% CD3+CD8+tetramer+cells ex vivo out of 152 tet-
We found no major difference in the total content in perforin,
GrmB, GrmA, or GrmK in EBV- or CMV-specific CD8+T cells
between MS patients and control subjects (Supplemental Fig. 1B,
Furthermore, we analyzed the 16 populations of tetramer+cells
using SPICE software, as characterized by the 16 possible com-
binations of cytolytic granules. As previously shown in our lab-
oratory, the distribution of perforin, GrmB, GrmA, and GrmK
differed markedly between EBV- and CMV-specific CD8+
T cells, regardless of the category of study subjects (23); however,
we did not find any difference between MS and control subjects
(Supplemental Fig. 2A, 2B).
Virus-specific CD8+T cells of MS patients have similar
expression of CCR7, CD57, and PD-1 markers compared with
To determine whether the differences between MS patients and
control subjects in terms of HLA-B7–restricted viral-specificCD8+
T cell responses were due to differences in their pattern of dif-
ferentiation, we analyzed the cell surface expression of CCR7,
Clinical data for the 220 HLA-A2+, HLA-B7+, and/or HLA-B8+study subjects
Delay Between Disease Onset
and Study Entrance (y)a,b
TTT (Relapsing) (n)c
EBV Rate of Infection
CMV Rate of
Infection (n [%])
Staining (n [%])
MS patients (n = 112)
CIS (n = 28)
34 6 13
0.3 6 1.1
2 6 1
RR-MS (n = 59)
37 6 11
2.8 6 7.6
2 6 1
SP-MS (n = 10)
52 6 21
15.3 6 9.4
6 6 2.5
PP-MS (n = 15)
50 6 12
5.5 6 4.5
3 6 1
Controls (n = 108)
OIND (n = 37)
48 6 26
0.2 6 1.1
NIND (n = 46)
44 6 24
0.3 6 2.4
HC (n = 25)
36 6 17
aData represent the median 6 interquartile range.
bStudy entrance corresponds to the blood draw.
cNumber of MS patients with immunomodulatory treatment: nine were on IFN-b, and five were on natalizumab.
dNumber of subjects carrying the HLA-A2, HLA-B7, and/or HLA-B8 allele, respectively.
EDSS, expanded disability status scale; HC, healthy subjects; OIND, other inflammatory neurologic disease; PP-MS, primary progressive MS; RR-MS, relapsing–remitting MS; SP-MS, secondary-progressive MS; TTT, treatment.
4674HLA-B7–RESTRICTED EBV-SPECIFIC RESPONSE IN MS
a marker of cell differentiation (31); CD57, a marker of senes-
cence (32); and PD-1, a marker of CD8+T cell proliferation and
viral exposure (33). As described previously (34), we found that
tetramer+cells were mostly CCR72for both viruses (97% for
EBV and 99% for CMV; Supplemental Fig. 3A), indicating
that they were either effector memory or effector CD8+T cells
(35). However, no difference was seen between MS and control
subjects for either EBV or CMV. Similarly, there was no differ-
ence in the expression of CD57, with the exception of HLA-B8/
EBVRAK CD8+T cells of MS patients, which expressed less
CD57 (Supplemental Fig. 3B). Finally, we found no difference in
the expression of PD-1 between the two groups (Supplemental
Fig. 3C). These data show that the decreased magnitude of the
HLA-B7–restricted viral-specific CD8+T cell response that we
observed in MS patients cannot be attributed to exhaustion or lack
of differentiation of these cells.
Upon Ag stimulation in short-term culture, the cytotoxic
phenotype of HLA-B7/EBVRPP–specific CD8+T cells of MS
patients is deficient
To further characterize the viral-specific CD8+T cell response
restricted by the different HLA class I, we set up a 1-wk stimu-
lation. We found that HLA-B7/EBVRPP–specific CD8+T cells of
MS patients expressed significantly less perforin (p = 0.011) and
GrmB (p = 0.022) than did those of control subjects (Fig. 4A).
Such a difference was not seen for GrmA or GrmK. Interestingly,
after the addition of rhIL-2, there was a significant increase in
perforin (65–84%, p = 0.027) and a strong trend for an increase in
GrmB (87–96%, p = 0.052) in MS patients. Thus, the difference
in perforin or GrmB content in HLA-B7/EBVRPP–specific CD8+
T cells between both categories vanished (Fig. 4A).
In contrast to HLA-B7/EBVRPP–specific CD8+T cells, we
found no difference with regard to cytotoxic granules content of
HLA-A2/EBVGLC–, HLA-B8/EBVRAK–, and HLA-B7/CMVTPR–
specific CD8+T cells between the two categories of study sub-
jects, either with or without exogenous rhIL-2 (Fig. 4).
In MS patients, HLA-B7/EBVRPP–specific CD8+T cell
cytotoxic activity is rescued by the addition of exogenous IL-2
In the next step, we attempted to determine whether the decrease in
perforin and GrmB in HLA-B7/EBVRPP–specific CD8+T cells of
MS patients affected their functional cytotoxicity. In a CFSE-
based CTL functional assay, HLA-B7/EBVRPP–specific CD8+
T cells of MS patients showed, in the absence of rhIL-2, a de-
creased cytotoxic activity compared with cultures with rhIL-2
(p = 0.034; Fig. 5A). Moreover, the strength of this HLA-B7/
EBVRPP–specific lysis was inversely proportional to the length of
MS duration: the longer the disease, the lower the EBV-specific
lysis (Fig. 5B, left panel). Adding exogenous rhIL-2 rescued the
cytotoxic capacity of the EBVRPP-specific CD8+T cells in MS
patients (Fig. 5B, right panel).
In contrast, there was no such rescue effect of rhIL-2 on the
cytotoxic activity in the control subjects (Fig. 5C). Contrasting
again with EBV, there was no difference in the cytotoxic activity
of HLA-B7/CMVTPR–specific CD8+T cells in the presence or
absence of exogenous rhIL-2, and there was no correlation with
disease duration (Fig. 5D, 5E). Similarly, there was no difference
for the control subjects (Fig. 5F).
Proliferation of HLA-B7/EBVRPP–specific CD8+T cells is not
deficient in MS patients
Compared with controls, HLA-B7/EBVRPP–specific CD8+T cells
from MS patients proliferated at the same rate without rhIL-2, and
they proliferated more when rhIL-2 was added (p = 0.04; Sup-
plemental Fig. 4A). We can infer from these data that the relative
lack of perforin and GrmB production by HLA-B7/EBVRPP–
specific CD8+T cells is not attributable to a deficit in the prolif-
eration of EBV-specific tetramer+cells. However, these data em-
phasize the high sensitivity of HLA-B7/EBVRPP–specific CD8+
T cells to rhIL-2. Indeed, upon addition of rhIL-2, the HLA-B7/
EBVRPP–specific CD8+T cells from MS patients proliferated
more than did the HLA-A2/EBVGLC– and HLA-B8/EBVRAK–
specific CD8+T cells (p = 0.004 and p = 0.02, respectively). No
subjects carrying the appropriate HLA allele. (A) EBV-specific CD3+CD8+T cells. (B) CMV-specific CD3+CD8+T cells. Horizontal lines represent the
median. *p , 0.05. CTRL, Control subjects; MS, MS patients; TET, tetramer.
Prevalence and magnitude of virus-specific HLA-A*0201, HLA-B*0702, or HLA-B*0801 tetramer+CD3+CD8+T cells among 220 study
The Journal of Immunology4675
such difference was seen for control subjects in either culture con-
ditions. Finally, proliferation of HLA-B7/CMVTPR–specific CD8+
T cells was comparable between MS patients and control subjects
under both culture conditions, although we acknowledge that the
number of control subjects was low (Supplemental Fig. 4B).
EBV DNA is not associated with deficient EBV-specific CD8+
T cell responses
To determine whether the deficit in HLA-B7/EBVRPP–specific
CD8+T cells in MS patients was associated with a lack of viral
control, we used real-time PCR to assess the presence of EBV
DNA in the plasma and PBMC of the 19 MS patients and 11
controls subjects who had been enrolled in the intracellular cy-
tokine staining and CFSE functional CTL assays. EBV DNA was
not detected in the plasma or in the PBMC of these 30 study
subjects (data not shown).
In this study, we first confirmed that MS patients express the HLA-
characteristic was shown to be due to linkage disequilibrium with
the HLA-DR2 (DRB1*1501-DQB1*0602) allele, which increases
the susceptibility to MS (3). However, our data suggest that HLA-
B7 may be instrumental in the pathogenesis of MS. Indeed, using
ex vivo tetramer staining, we show that, among HLA-B7+study
subjects, there is a significantly higher proportion of MS patients
who exhibit an HLA-B7/EBVRPP–specific CD8+T cell response
compared with control subjects. Such a differential CD8+T cell
response is not present for the EBV peptides restricted by HLA-
A2 or HLA-B8, nor is it present for HLA-A2– or HLA-B7–re-
stricted CMV peptide epitopes. However, the magnitude of the
CD8+T cell response in MS patients is significantly lower for the
HLA-B7/CMVTPRepitope and tends to be lower for the HLA-B7/
EBVRPPepitope. With regard to the CD8+T cell responses against
the latter, other investigators found either no difference between
MS patients and control subjects (37) or an increased magnitude
of HLA-B7/EBVRPP–specific CD8+T cells in MS patients (8), as
reflected by the secretion of IFN-g in an ELISPOT assay. How-
ever, a direct comparison is difficult, because the data on the
magnitude of tetramer+cells were obtained by gating on CD3+
and CD8+T cells, whereas the ELISPOT assays were performed
on total unfractionated PBMC.
Nevertheless, the discrepancy seen between the prevalence and
the magnitude (Fig. 2) raises the question of a dysregulation of
IFN-g and IL-2 is decreased in HLA-B7–
restricted EBVRPP-specific CD8+T cells.
PBMC were stimulated 18 h with EBV (A) or
CMV (B) peptide and stained for CD3, CD8,
IFN-g, TNF-a, and IL-2. Each symbol repre-
sents one study subject; horizontal lines repre-
sent the median. *p , 0.05, **p , 0.01. CTRL,
Control subjects; MS, MS patients.
In MS patients, the amount of
4676HLA-B7–RESTRICTED EBV-SPECIFIC RESPONSE IN MS
HLA-B7–restricted viral Ag presentation to CD8+T cells in MS
patients. Assessing in detail the ex vivo phenotype of viral-specific
CD8+T cells of MS patients and control subjects, we find a rela-
tive deficit in the secretion of IFN-g and primarily IL-2 in HLA-
B7–restricted EBVRPP-specific CD8+T cells but not in the four
other HLA-restricted viral peptide epitope-specific CD8+T cells.
Upon Ag stimulation in 1-wk cultures, without addition of ex-
ogenous rhIL-2, HLA-B7/EBVRPP–specific CD8+T cells of MS
patients exhibit a defect in their ability to upregulate perforin and
GrmB, which suggests that their memory HLA-B7/EBVRPP–spe-
cific CD8+T cells are less efficient. In agreement with previous
work showing a decreased secretion of IFN-g by EBV-specific
CD8+T cells with an increase in MS duration (9), we show in
this study that the HLA-B7/EBVRPP–specific CD8+T cell–medi-
ated cytotoxicity (but not HLA-B7/CMVTPR) also decreases with
It was proposed that CD8+T cells of MS patients may present an
intrinsic deficit that would lead to suboptimal control of EBV rep-
lication (12, 14). Supporting this theory, polymorphism of the per-
forin gene is increased in MS patients, a feature that could account
for a dysregulation of the cytotoxic activity of CD8+T cells (38).
Furthermore, an impaired function of CD8+T cells leading to a
decreased response against EBV, but not other viruses, does exist:
X-linked lymphoproliferative disease. This condition is due to
a dysfunction of the signaling lymphocytic activation molecule-
associated protein involved specifically in the response to EBV
(39). Affected patients exhibit a very high susceptibility to EBV
infection, often with a fatal outcome. Thus, the question arises
whether MS patients could be affected by a sort of attenuated X-
linked lymphoproliferative disease. Our data do not suggest that
there is a global or massive EBV-specific CD8+T cell deficiency in
MS patients. Indeed, we find no difference between MS patients
EBV-specific CD8+T cell response. Furthermore, we do not detect
EBV DNA either in the plasma or PBMC of MS patients or control
(10, 13), could be due to methodological differences. Furthermore,
the cellular assay time point, we may have missed a transient in-
crease in EBV viral load. Nevertheless, our data suggest that the
content. Content in perforin and GrmB in EBV-specific (A) or CMV-specific (B) tetramer+CD8+T cells after 1 wk of Ag stimulation in the absence or
presence of exogenous rhIL-2. Each symbol represents one subject; horizontal lines represent the median. *p , 0.05. CTRL, Control subjects; MS, MS
Upon Ag stimulation in short-term culture, HLA-B7/EBVRPP–specific CD8+T cells of MS patients exhibit a deficit in their cytotoxic granule
The Journal of Immunology 4677
impaired HLA-B7–restricted EBV-specific CD8+T cell responses
are not associated with an obvious increase in EBV viral load.
Therefore, basedonour findings, itis theinteraction between CD8+
T cells and the presentation of the EBVRPPepitope by the HLA-B7
allele that is suboptimal in MS patients. It was recently shown that
the HLA class I molecule is expressed at the same level in MS
patients and controls (40), suggesting that the dysregulation of the
HLA-B7/EBV–specific CD8+T cell response is not due to a quan-
titative deficit of this molecule on the surface of APCs. In fact, our
data suggest that a defective costimulation may explain this phe-
nomenon. Indeed, the content in perforin and GrmB, as well as the
cytotoxic function of HLA-B7/EBVRPP–specific CD8+T cells, is
fully restored if rhIL-2 is added to the 1-wk culture, suggesting that
theendogenous productionofIL-2 inMSpatientsisnotsufficientto
mount an optimal HLA-B7/EBVRPP–specific CD8+T cell response.
Therefore, we wondered whether such a need for exogenous rhIL-2
reflects anexhaustionofthesecells. Yet,exvivoHLA-B7/EBVRPP–
specific CD8+T cells of MS patients and control subjects are sim-
ilarly highly differentiated (35); do not express CD57, which is
associated with proliferation incompetence and replicative senes-
cence (32); and have low levels of PD-1, a marker of exhaustion
that is induced by antigenic stimulation (41). Furthermore, there is
greater proliferation of HLA-B7/EBVRPP–specific CD8+T cells in
fully able to respond to IL-2. Indeed, exogenous IL-2 was shown to
rescue the proliferative capacity of CD8+T cells (42). Thus, the
question is whether there is a deficiency in IL-2 in MS.
Specifically, ex vivo analysis of cytokine secretion by HLA-B7–
restricted EBVRPP-stimulated CD8+T cells shows that, although
∼50% of the CD8+T cells secrete IFN-g and IL-2 in both MS
patients and control subjects, the amount of IL-2 secreted by
a given HLA-B7–restricted EBVRPP-specific CD8+T cell is lower
in MS patients. We posit that a deficit in the synthesis of endog-
enous IL-2 by CD8+T cells explains the lower synthesis of per-
forin and GrmB and the lower cytotoxicity by HLA-B7/EBVRPP–
specific CD8+T cells during the rhIL-2–deprived 1-wk stimula-
tion. Contrasting to some degree with these data, it was shown
that MS patients have increased IL-2 concentrations in serum
and cerebrospinal fluid (43), whereas at the cellular level, produc-
tion of IL-2 varies with disease activity (44) and Ag-specific or
-aspecific stimulation (16, 45).
Interestingly, genome-wide association studies identified poly-
morphisms of IL2RA associated with several autoimmune dis-
eases, including MS (reviewed in Ref. 46). Indeed, two single-
nucleotide polymorphisms have been identified within the IL2RA
of MS patients (47–49). Further, functional studies demonstrated
that the diverse polymorphisms of the IL2RA gene contributed
independently to MS susceptibility (48). Because we did not look
at the IL2RA molecule in this study, we cannot rule out that this
mechanism is involved in the dysregulation of HLA-B7/EBVRPP–
specific CD8+T cells. Additional studies are warranted to examine
this particular aspect.
One can wonder whether the fact that the HLA-B7–restricted
EBVRPPis an epitope of a latent protein (EBNA3A) and the EBV
peptides restricted by HLA-A2 and HLA-B8 are epitopes of lytic
proteins (BMLF1 and BZLF1) may account for the observed
differences. Indeed, we cannot rule out that the CD8+T cell re-
sponse against latent peptide epitopes presented by other alleles
would have shown differences between MS patients and control
subjects. However, we do not think that our data can be solely
explained by this phenomenon. Indeed, the EBVRPPpeptide used
in this study is one of the most immunogenic of the EBV HLA-
B7–restricted peptides (50, 51). Second, in a recent work, we
showed that the cytotoxic activity of CD8+T cells against latent
peptides in general (also restricted by other HLA class I) was
greater than against lytic epitopes (11). Finally, the lower mag-
nitude of the HLA-B7/CMVTPR–specific CD8+T cell response, ex
vivo, in MS patients further points toward a deficient presentation
of viral peptide epitope by the HLA-B7 molecule.
In conclusion, our data suggest that, although there is a higher
proportion of HLA-B7+MS patients who harbor EBVRPP-specific
CD8+T cells, this trimolecular interaction is suboptimal. The fact
that IL-2 production is decreased in HLA-B7–restricted EBVRPP-
specific CD8+T cells and that the magnitude of the CD8+T cell
of HLA-B7/EBVRPP–specific CD8+T cells in the absence or presence of rhIL-2 in MS patients (A) or control subjects (C). (B) In the absence of rhIL-2,
HLA-B7/EBVRPP–specific CD8+T cell cytotoxicity is inversely correlated with MS duration (left panel); however, such a correlation is lost when cells are
cultured in the presence of rhIL-2 (right panel). Similar experiments were performed for HLA-B7/CMVTPR–specific CD8+T cells in MS patients (D) and
control subjects (F). (E) Absence of correlation of HLA-B7/CMVTPR–specific CD8+T cell cytotoxicity with MS disease duration. Each symbol represents
one subject; horizontal lines represent the median. *p , 0.05. CTRL, Control subjects; MS, MS patients; yr, years.
Upon stimulation with HLA-B7/EBVRPPpeptide, MS patients exhibit a deficit in CD8+cytotoxic activity. CFSE-based cytotoxic CTL assay
4678HLA-B7–RESTRICTED EBV-SPECIFIC RESPONSE IN MS
response against another HLA-B7–restricted viral peptide epitope
(CMVTPR) is also diminished in MS patients points toward a de-
ficient presentation of viral epitopes by this specific class I HLA.
This observation is particularly intriguing considering the fact that
HLA-B7 is more prevalent in MS patients, and EBV is associated
We thank K. Ellefsen-Lavoie and E. Medjitna for providing the HLA-B7/
EBVRPPand HLA-B7/CMVTPRtetramers and G. Le Goff and C. Cam-
piche for invaluable help in enrolling patients and obtaining blood samples.
We also are grateful to P. Meylan and S. Chappuis, who performed quan-
tification of virus-specific Abs. We thank the National Institutes of Health
Tetramer Core Facility at Emory University (Atlanta, GA) for providing
The authors have no financial conflicts of interest.
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4680 HLA-B7–RESTRICTED EBV-SPECIFIC RESPONSE IN MS