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Cross-reactivity with myelin basic protein and human herpesvirus-6 in multiple sclerosis.

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Cross-Reactivity with Myelin Basic Protein
and Human Herpesvirus-6 in
Multiple Sclerosis
Maria V. Tejada-Simon, PhD,1 Ying C. Q. Zang, MD, PhD,1 Jian Hong, MD, PhD,1 Victor M. Rivera, MD,1
and Jingwu Z. Zhang, MD, PhD1,2
Viral infections are though to play an important role in the pathogenesis of multiple sclerosis (MS) potentially through
molecular mimicry. An identical sequence was found in both myelin basic protein (MBP, residues 96 –102), a candidate
autoantigen for MS, and human herpesvirus-6 (HHV-6 U24, residues 4 –10) that is a suspected viral agent associated
with MS. In this study, we showed that greater than 50% of T cells recognizing MBP93-105 cross-reacted with and could
be activated by a synthetic peptide corresponding to residues 1 to 13 of HHV-6 U24 in MS patients. The estimated
precursor frequency of these cross-reactive T cells recognizing both peptides, MBP93-105 and HHV-6 (U24)1-13, was
significantly elevated in MS patients compared with that in healthy controls. These cross-reactive CD4ⴙ T cells represented the same Th1 phenotype as that of monospecific T cells recognizing MBP93-105. There were increased antibody
titers for both peptide HHV-6 (U24)1-13 and peptide MBP93-105 in the same patients with MS compared with those in
healthy controls, suggesting B-cell sensitization to the antigens in MS patients. The study provides important evidence in
the understanding of the potential role of HHV-6 infection/reactivation in the activation of autoimmune reactivity to
MBP and its implication in the pathogenesis of MS.
Ann Neurol 2003;53:189 –197
Microbial infections, in particular viral infections, may
play an important role in the cause and pathogenesis of
multiple sclerosis (MS). Several viruses, including measles virus, Epstein–Barr virus, and most recently human
herpesvirus-6 (HHV-6), have been implicated as causative agents in MS based on epidemiological evidence,
geographic pattern, and abnormal immune response to
these viruses.1– 6 The potential pathological importance
of certain viral infections in MS is thought to involve
direct neurotropic properties of the virus, causing tissue
damage, and their ability to activate autoimmune responses directed at myelin tissue through the mechanism known as molecular mimicry.7–11 The later
mechanism is particularly relevant to MS because the
T-cell responses to candidate myelin antigens, such as
myelin basic protein (MBP), have been implicated in
the pathogenesis of MS.12 T cells recognizing MBP
have been shown to induce experimental autoimmune
encephalomyelitis, an animal model for MS,13,14 and
can be isolated from patients with MS and also from
healthy individuals.15–18 One of the important discrepancies between myelin-reactive T cells in MS patients
and those in normal individuals is the activation state
From the 1Multiple Sclerosis Research Unit, Department of Neurology and Baylor Multiple Sclerosis Center; and 2Department of
Immunology, Baylor College of Medicine, Houston, TX.
Received May 6, 2002, and in revised form Aug 27 and Sep 17.
Accepted for publication Sep 17, 2002.
of the T cells. MBP-reactive T cells are found to undergo in vivo activation and clonal expansion in patients with MS, as opposed to healthy individuals.17,19,20 It is speculated that these myelin-reactive T
cells may be activated in vivo by viral infections in patients with MS through molecular mimicry mechanism, which is supported by in vitro studies.21–23
HHV-6 has predominant tropism for CD4⫹ T
cells.24 It is the causative agent for infantile exanthem.
Common to other herpes viruses, latency of HHV-6 is
established after primary infection, and its genomic
material can be found in T cells of healthy adults.
HHV-6 infection can reactivate under certain conditions, such as in immunocompromised patients.25 Expression of HHV-6 virion proteins was found in oligodendrocytes obtained from patients with MS.26
More recent studies demonstrated higher titers of antibodies to HHV-6 and cell-free DNA of HHV-6 in
sera and cerebrospinal fluid of MS patients, suggesting
reactivation of HHV-6 in MS,27–33 whereas some
studies reported conflicting results.34 – 40 Interestingly,
a viral protein of both variant A and variant B of
HHV-6, designated U24, shares significant amino acid
Address correspondence to Dr Zhang, Department of Neurology,
Baylor College of Medicine, 6501 Fannin Street, NB302, Houston,
TX 77030.
© 2003 Wiley-Liss, Inc.
189
sequence homology with MBP. There is a stretch of
seven identical amino acids within residues 4 to 10 of
HHV-6 U24 antigen and residues 96 to 102 of human
MBP, raising the possibility that MBP-reactive T cells
may be susceptible to activation by the HHV-6 viral
antigen sharing the sequence homology.
This study was undertaken to examine the potential
role of HHV-6 in the activation of MBP-reactive T cells
through T-cell recognition of the shared sequence region
between HHV-6 and MBP. Two 13-mer peptides corresponding to residues 1 to 13 of HHV-6 U24 and residues 93 to 105 of MBP were synthesized to contain the
identical core sequence of seven amino acids with distinct flanking residues (MBP-IVTPRTPPPSQGK and
HHV6-MDRPRTPPPSYSE). An experimental system
was designed to differentiate cross-reactive T cells that
react with both peptides from monospecific T cells that
are committed to recognition of only one of the antigenic sequences. The study provides new evidence suggesting that a proportion of circulating MBP-reactive T
cells is susceptible to activation by the viral peptide derived from HHV-6 through molecular mimicry.
Subjects and Methods
Reagents
Two 13-mer peptides corresponding to MBP (residues 93–
105) and HHV-6 U24 (residues 1–13) were synthesized by
the peptide core laboratory at the M. D. Anderson Cancer
Center, Houston. The amino acid sequences of the MBP
peptide and the viral peptide were IVTPRTPPPSQGK and
MDRPRTPPPSYSE, respectively. The purity of the peptides
was greater than 95%. Media used for cell culture were
AIM-V serum-free medium (Gibco BRL, Grand Island, NY)
and RPMI-1640. Recombinant human IL-2 was purchased
from Boehringer-Mannheim (Indianapolis, IN).
Patients
Twelve MS patients were included in the study. All patients
were characterized as having relapsing-remitting or secondary
progressive MS for more than 2 years. They were not treated
with immunosuppressive agents or immunomodulatory
agents (eg, ␤-interferon (IFN) or glatiramer acetate) for at
least 3 months before the enrollment in the study and
throughout the study. Informed consent was obtained from
the patients after explaining the experimental procedures.
Eleven asymptomatic healthy volunteers were included as
control subjects.
Analysis of the Precursor Frequency of T Cells
Peripheral blood mononuclear cells (PBMCs) were seeded at
3 ⫻ 105 cells per well in 96-well U-bottomed plates in the
presence of the peptides of MBP93-105 or HHV-6 (U24)1-13
at 30␮g/ml or the whole MBP (40␮g/ml), respectively. A
total of 24 wells were plated for each antigen. Seven days
later, the cultures were tested for the reactivity to both peptides using medium alone as a control. To this end, each
culture was split into three identical aliquots and tested for
the reactivity to MBP and the indicated peptides in dupli-
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Annals of Neurology
Vol 53
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February 2003
cate. Two aliquots were tested for reactivity to peptide
MBP93-105 and peptide HHV-6 (U24)1-13 at the same concentration of 30␮g/ml, respectively, in the presence of irradiated (6,000 rads) autologous PBMCs (105 cells per well) as
a source of antigen-presenting cells. One aliquot served as a
control (medium alone in the presence of antigen-presenting
cells). The reactivity of the end cultures was measured after
72 hours by proliferation assays as described elsewhere.41 A
T-cell line was defined as reactive to a given peptide when
CPM were greater than 1,000 and exceeded counts per
minute (CPM) of the control (medium alone) and of the
other peptide by at least threefold.15,17,18,41 A cross-reactive
T-cell line was defined as CPM if both peptides exceeded
1,000 and the control CPM by at least three times. The
frequency of peptide-reactive T cells then was estimated by
dividing the number of positive wells by the total number of
PBMCs seeded in the initial culture.15,17,18,41
Generation of Peptide-Reactive T-cell Clones
The obtained T-cell lines were cloned under limiting dilution conditions in the presence of PHA (Sigma, St. Louis,
MO) and irradiated autologous PBMCs as accessory cells.41
In brief, T cells were plated at 0.3 cell per well under limiting dilution conditions and cultured with 105 irradiated autologous PBMCs and 5␮g/ml PHA. Cultures were fed with
fresh medium containing 50IU/ml recombinant IL-2 every 4
days. After approximately 10 to 12 days, growth-positive
wells became visible and were tested in proliferation assays
for proliferative responses to the peptides.
Analysis of T-cell Receptor V Gene Rearrangements
and DNA Sequencing
Total cellular RNA was extracted from each independent
T-cell clone using RNeasy mini kit (Qiagen, Chatsworth,
CA). T-cell receptor (TCR)–␤ chains were amplified by
polymerase chain reaction (PCR) with a set of V␤-specific
primers as described elsewhere.42,43 Briefly, RNA was reverse
transcribed to first-strand cDNA using an Oligo-dT primer
and the superscript preamplification system (Gibco, Gaithersburg, MD). cDNA was amplified in a standard PCR using a set of primers specific for 24 V␤ gene families in combination with C␤ primer, respectively. For each PCR
experiment, C␤ gene was amplified simultaneously to control the integrity of TCR cDNA. The amplification profile
used was 1 minute at 95°C for denaturation, 20 seconds at
56°C for annealing, 40 seconds at 72°C for extension in a
total of 35 cycles. The amplified PCR products were separated on a 1% agarose gel by electrophoresis and stained with
ethidium bromide. The purified PCR products were directly
sequenced with the T7 sequencing kit (Pharmacia, Uppsala,
Sweden).44
Cytokine Measurement
The cytokine profile of the resulting T-cell lines was determined quantitatively using enzyme-linked immunosorbent
assay (ELISA) kits (PharMingen, San Diego, CA). Microtiter
plates (96 wells, NUNC Maxisorp; Nunc, Naperville, IL)
were coated overnight at 4°C with 1␮g per well of a purified
mouse capturing monoclonal antibody to human cytokine
(IL-4, IL-10, tumor necrosis factor [TNF]–␣, ␥-IFN)
was added in duplicate wells. The rest of the procedure is the
same as that described above.
(PharMingen). Plates were washed and nonspecific binding
sites were saturated with 10% (wt/vol) fetal bovine serum
(FBS) for 1 hour and subsequently washed. Supernatants and
cytokine standards were diluted with phosphate-buffered saline (PBS) and added in duplicate wells. Plates were incubated at 37°C for 2 hours and subsequently washed with
PBS-T. Matched biotinylated detecting antibody were added
to each well and incubated at room temperature for 2 hours.
After washing, avidin-conjugated horseradish peroxidase was
added, and plates were incubated for 1 hour. 3,3⬘,5,5⬘Tetramethylbenzidine (Sigma) was used as a substrate for
color development. Optical density was measured at 450nm
using an ELISA reader (Bio-Rad Laboratories, Hercules,
CA), and cytokine concentrations were quantitated by Microplate computer software (Bio-Rad) using a double eightpoint standard curve.
Statistical Analysis
A Student’s t test for normally distributed variables and the
Mann–Whitney rank-sum test for nonnormally distributed
variables was used for data analysis. A p value of less than
0.05 was considered statistically significant.
Results
Estimated Precursor Frequency of T Cells Reactive to
the HHV-6 Peptide and the MBP Peptide in
Multiple Sclerosis Patients and Healthy Controls
A group of 12 patients with relapsing-remitting or secondary progressive MS was included in the study,
along with a control group of 11 healthy volunteers. As
reported previously,33 a higher incidence of serum cellfree HHV-6 viral DNA was detected in this group of
MS patients than that in healthy controls (Table 1).
We first examined the T-cell responses to two synthetic
peptides containing an identical core sequence of seven
amino acids by estimating the precursor frequency of
the T cells in PBMCs. Cultures were primed with peptide MBP93-105 or peptide HHV-6 (U24)1-13, respectively, and tested 7 days later for the specific reactivity
Detection of Titers and the Reactivity of Antibodies
to the Peptides
Antibody titers were determined in serum specimens by
ELISA. In brief, microtiter plates were coated overnight at
4°C with the peptides and a reference MBP peptide (residues
41–59), respectively, at 5␮g/ml in a carbonate buffer
(100mM, pH 9.5). Nonspecific binding sites were saturated
with FBS-PBS for 1 hour and washed subsequently with
PBS-T. Serum samples were prepared in serial dilutions (1:
50, 1:100, 1:500) with FBS-PBS, and 100␮l of each sample
Table 1. Clinical Characteristics and Demographic Information, Serum Cell-Free HHV-6 DNA and HLA Types of MS Patients
and Control Subjects
Subjects
Age (yr)
Gender
Type of
MS
Disease
Duration (yr)
EDSS
Serum Cell-Free
HHV-6 DNA
MS-1
MS-2
MS-3
MS-4
MS-5
MS-6
MS-7
MS-8
MS-9
MS-10
MS-11
MS-12
NS-1
NS-2
NS-3
NS-4
NS-5
NS-6
NS-7
NS-8
NS-9
NS-10
NS-11
44
38
46
47
45
60
51
57
46
41
31
50
47
49
35
32
46
60
33
34
43
46
30
M
F
F
F
F
F
F
F
F
F
F
F
M
F
M
F
F
F
F
F
F
F
F
RR
RR
RR
SP
RR
SP
SP
RR
RR
SP
RR
SP
—
—
—
—
—
—
—
—
—
—
—
5
2
4
7
4
11
13
6
6
13
4
28
—
—
—
—
—
—
—
—
—
—
—
3.0
1.0
3.0
7.0
1.5
6.6
3.5
1.5
1.5
6.0
1.0
7.0
—
—
—
—
—
—
—
—
—
—
—
⫺
⫹
⫹
⫹
⫹
⫺
⫹
⫹
⫹
⫹
⫹
⫹
⫺
⫺
⫺
⫹
⫹
⫹
⫺
⫹
⫹
⫺
⫹
HLA-DR Genotypes
B1*1501;
B1*0405;
B1*1501;
B1*1501;
B1*5101;
B1*1301;
B1*0103;
B1*1501;
B1*1501;
B1*1602;
B1*1602;
B1*1101;
B1*1501;
B1*0102;
B1*1503;
B1*0103;
B1*1101;
B1*1501;
B1*0103;
B1*1503;
B1*0101;
B1*0301;
B1*1501;
B1*1301;
B1*1301;
B1*0405;
B1*1502;
B1*0404;
B1*0802;
B1*0802
B1*0404;
B1*1104;
B1*0408;
B1*1303;
B1*0701;
B1*0701;
B1*0405;
B1*1301;
B1*1402;
B1*0701;
B1*0404;
B1*1104;
B1*1301;
B1*0405;
B1*0802;
B1*1402;
B3*0101;
B3*0101;
B5*0101;
B5*0101;
B5*0101;
B3*0101
B5*0101
B4*0103
B4*0103
B5*0102
B4*0131
B5*0101;
B3*0202;
B5*0202;
B3*0101;
B3*0202;
B5*0101;
B4*0131
B3*0101
B3*0101
B3*0202;
B5*0101;
B3*0202
B3*0101;
B4*0131
B3*0101
B3*0101
B4*0131
B5*0101
B4*0103
B5*0202
B4*0101
B4*0103
B4*0101
B4*0131
B5*0101
Cell-free viral DNA for HHV-6 was detected in serum specimens derived from the subjects using nested PCR and Southern hybridization with
specific primers and probes.33 The results are expressed as detectable (⫹) (⬎5 DNA copies/␮l) and undetectable (⫺).
MS ⫽ Multiple sclerosis; EDSS, expanded disability scales score. RR ⫽ relapsing-remitting MS; SP ⫽ secondary progressive MS; NS ⫽ normal
subject.
Tejada-Simon et al: Cross-Reactivity to MBP and HHV-6
191
to the two peptides in reference to a medium control.
According to the reactivity of the resulting T cells, they
were categorized as (1) MBP- or HHV-6 –reactive T
cells as they were primed in week 1 and exhibited specific reactivity only to the same peptide in week 2, and
as (2) cross-reactive T cells that were primed by either
the HHV-6 (U24)1-13 peptide or the MBP93-105 peptide in week 1 and displayed the reactivity to both peptides in week 2.
Fig 1. The T-cell responses to the peptides corresponding to
MBP93-105 and HHV-6 (U24)1-13 in multiple sclerosis (MS)
patients and healthy controls. The precursor frequency of T
cells recognizing two peptides encompassing MBP93-105 and
HHV-6 (U24)1-13 regions was estimated using a split-well
assay. Peripheral blood mononuclear cells obtained from 12
MS patients and 11 normal subjects (NS) were stimulated
with the indicated peptides or the whole myelin basic protein
(MBP) in culture (week 1), respectively, and were tested in
week 2 for the reactivity to the two peptides in proliferation
assays. Medium alone was included as a control. The precursor
frequency of T cells was estimated by dividing the number of
positive wells by the total number of cells seeded in the initial
culture. The circles represent the individual precursor frequency, and the bars indicate the mean precursor frequency of
the T cells within the group. The asterisks indicate statistically
significant differences (p ⬍ 0.05) between the MS group and
the control group.
As shown in Figure 1, although T cells recognizing
the whole MBP occurred at a similar estimated frequency (0.6 ⫻ 10⫺6 in PBMCs) in MS patients and
healthy subjects, the estimated frequency of T cells specific for the two peptides was significantly higher in
MS patients than in healthy controls (0.53 ⫻ 10⫺6 vs
0.19 ⫻ 10⫺6 for peptide MBP93-105 and 0.7 ⫻ 10⫺6
vs 0.32 ⫻ 10⫺6 for peptide HHV-6 [U24]1-13; p ⬍
0.05). The estimated frequency of the cross-reactive T
cells primed by peptide HHV-6 (U24)1-13 was 0.30 ⫻
10⫺6 in MS patients, which represented greater than
50% of all T cells recognizing peptide MBP93-105 in
the MS cohort (see Fig 1). T cells reactive to both peptides were also present but at significantly lower precursor frequency (0.17 ⫻ 10⫺6) in healthy controls.
Similarly, the cross-reactive T cells also were detected
in cultures primed with peptide MBP93-105 in MS patients as well as in control subjects (0.36 ⫻ 10⫺6 vs
0.09 ⫻ 10⫺6; p ⬍ 0.05). Consistent with these observations was the increased frequency of T cells that initially were primed with the whole MBP and reacted to
peptide HHV-6 (U24)1-13 in patients with MS compared with the control group (0.33 ⫻ 10⫺6 vs 0.05 ⫻
10⫺6), but the difference was not statistically significant ( p ⫽ 0.08). Furthermore, there was no correlation
between the detection of HHV-6 DNA and the specific T-cell frequencies. Taken together, the results suggest that a significant fraction of T cells recognizing the
93 to 105 region of MBP could be fully activated to
proliferate by peptide HHV-6 (U24)1-13 that shares the
sequence homology with the MBP peptide.
Cytokine Profile and Phenotypic Expression of the
Obtained T-cell Lines
We then assessed the cytokine profile of the resulting
peptide-specific T-cell lines and cross-reactive T-cell
lines obtained from MS patients (67 lines in total) and
control subjects (38 lines in total). As illustrated in Figure 2, overall the T-cell lines examined were predominantly of Th1 phenotype, producing predominantly
␥-IFN, various amounts of TNF-␣ but not IL-4 and
IL-10, regardless of their peptide specificity. However,
MS-derived T-cell lines specific for peptide MBP93-105
as well as the cross-reactive T-cell lines initially primed
by peptide MBP93-105 differed considerably in the production of ␥-IFN and TNF-␣ from those generated
from control subjects. In general, the cross-reactive
T-cell lines exhibited similar cytokine profile as those
specific for the same peptide by which the crossreactive T-cell lines initially were primed (see Fig 2B vs
A and D vs C). All T-cell lines obtained expressed the
CD4 phenotype as determined by flow cytometry (data
not shown).
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Reactivity Pattern and T-cell Receptor V Gene Usage
of Cross-reactive T-cell Clones
To confirm that the observed reactivity of the T cells
recognizing both peptides of HHV-6 (U24)1-13 and
MBP93-105 results from monoclonal T-cell populations,
we selectively cloned 24 T-cell lines and subsequently
characterized for their reactivity pattern to the peptides. As shown in Figure 3, the resulting 24 T-cell
clones could be categorized, according to their reactivity to the peptides, into monospecific T-cell clones and
cross-reactive T-cell clones. The reactivity of the T-cell
clones was consistent with that of the parental T-cell
lines from which the T-cell clones were generated. The
T-cell clones subsequently were expanded in culture in
an attempt to analyze the TCR V gene usage and
CDR3 sequence. Unfortunately, most of the clones
(18/24 clones) were lost during the process of expansion because of prolonged equipment/electric power
failure and closing of the facility as a result of severe
flood in the Houston area during the course of the
study, and only six clones survived. As shown in Table
2, the six clones (including five cross-reactive T-cell
clones) examined were found to express single TCR
BV genes and V-D-J junctional sequence, confirming
monoclonality of the T-cell clones.
Serum Antibody Titers and the Reactivity Pattern to
the Peptides of HHV-6 and Myelin Basic Protein
We further addressed whether the increased T-cell responses to both peptides of HHV-6 (U24)1-13 and
MBP93-105 were consistent with potential B-cell sensitization to the peptides in MS patients as compared
with healthy controls. To this end, serum specimens
freshly obtained from the same MS patients and the
control subjects were examined for specific antibody reactivity to the peptides by ELISA. A MBP41-59 and an
irrelevant peptide were used as references. As illustrated
in Figure 4, the results showed that the antibody titers
for both peptide HHV-6 (U24)1-13 and peptide
Fig 2. Cytokine concentrations produced by T cells after challenge with the peptides. (A) Cytokine profile of the T-cell lines
primed with peptide MBP93-105 and challenged with the same
peptide in week 2. (B) Cytokine profile of the cross-reactive
T-cell lines primed with peptide MBP93-105 and challenged
with peptide HHV-6 (U24)1-13 in week 2. (C) Cytokine profile of the T-cell lines primed with peptide HHV-6 (U24)1-13
and challenged with the same peptide in week 2. (D) Cytokine profile of cross-reactive T-cell lines primed with peptide
HHV-6 (U24)1-13 and challenged with peptide MBP93-105 in
week 2. Supernatants were collected from T-cell cultures 48
hours after challenge with the indicated peptides in week 2
(see legend to Fig 1) and assayed for the cytokine concentrations by ELISA. MBP ⫽ myelin basic protein; MS ⫽ multiple sclerosis; NS ⫽ normal subject; IFN ⫽ interferon;
TNF ⫽ tumor necrosis factor.
‹
Tejada-Simon et al: Cross-Reactivity to MBP and HHV-6
193
Fig 3. The reactivity pattern of the resulting T-cell clones The resulting T-cell clones were examined for their reactivity to the peptides of MBP93-105 and HHV-6 (U24)1-13 or a control peptide (T-cell receptor amino acid sequence: ASSENRASYNEQFFG) at a
concentration of 30␮g/ml in the presence of irradiated autologous antigen-presenting cells. Cell proliferation was measured by [ 3H]thymidine uptake assay and expressed as stimulation index (experimental CPM/CPM in medium alone) ⫾ standard deviation.
Cross-reactive T-cell lines are indicated as reactivity to the peptide used in primary stimulation/reactivity to the other peptide as in
the legend to Fig 1. (striped bars, top bar in each group of three bars) Control peptide; (white bars, middle) MBP93-105; (black
bars, bottom) HHV61-13; MBP ⫽ myelin basic protein.
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Table 2. T-Cell Receptor VDJ Region Sequence of the Cross-reactive T-Cell Clones
Clone
MS11C4-1
MS11B6-1
MS2E1-1
MS2A7-1
MS2-E3-1
MS9-A5-1
BV
V-D-J
BC
BV5
BV22
BV13
BV12
BV8
BV2
YLCASSLVRDSGYTFGSGTRLTVV
YFCASSENRASYNEQFFGPGTRLTVL
YFCASSLGRLINSPLHFGNGTRLTVT
YFCAISEDGNYGYTFGSGTRLTVV
YFCASSLRAGGYQYGYTFGSGTRLTVV
FYICSASLGMGDIQYFGAGTRLSVL
EDLNK
EDLKN
EDLNK
EDLNK
EDLNK
EDLKN
BV ⫽ Variable gene of beta-chain; V-D-J ⫽ Variable region-Diversity region-Joining region; BC ⫽ constant gene of beta chain.
MBP93-105 were elevated at the serum dilutions of 1 to
50 and 1 to 100 in MS patients as compared with
those detected in control subjects. However, the differ-
Fig 4. Serum IgG antibodies specific for the peptides corresponding to MBP93-105 and HHV-61-13 in multiple sclerosis
(MS) patients and healthy subjects. The titers of IgG antibodies to peptide MBP93-105 and peptide HHV-6 (U24)1-13 were
determined in serum specimens obtained from 12 MS patients
and 11 normal subjects (NS) by ELISA. Another peptide of
MBP (MBP41-59 ) and a control peptide (same as in Fig 3)
were used as references. The dotted lines represent two times of
background absorbance. The net absorbance was calculated as
mean experimental absorbance ⫺ mean background absorbance. Data are presented as mean net absorbance ⫾ standard deviation. MBP ⫽ myelin basic protein.
ences were not statistical significant probably because
of the small sample size.
Discussion
The observation that the precursor frequency of the
cross-reactive T cells is significantly elevated in MS patients suggests that these T cells are sensitized in vivo
in patients with MS. The possibility that the immune
system is sensitized to the peptides in MS patients is
further strengthened by the increase in antibody titers
for both peptides in serum specimens obtained from
the MS cohort. The findings are consistent with several
recent reports indicating the increased replication of
HHV-6 in MS patients as evident by more frequent
detection of elevated cell-free viral DNA of HHV-6 in
serum and cerebrospinal fluid specimens obtained from
MS patients. It is likely that active replication of
HHV-6 may be responsible for the observed sensitization of the immune system to the 93 to 105 region of
MBP in MS because peptide MBP93-105 shares an
identical sequence with peptide HHV-6 (U24)1-13.
Although the T-cell recognition of MBP in MS patients is relatively heterogeneous, some regions of MBP
have immunodominant properties, such as the 84 to
102/83 to 99 region and the 151 to 170 region of
MBP.15,17,18 The immunodominant properties of these
regions may be associated with their binding affinity to
HLA-DR (DRB1*1501) preferentially expressed in the
MS population. Valli and colleagues demonstrated that
peptides corresponding to both the 84 to 103 and the
144 to 163 regions have the highest binding affinity to
DRB1*1501.45 In this study, however, although the 93
to 105 region of MBP homologs to peptide HHV-6
(U24)1-13 has an overlap of four amino acids (PRTP)
with the 83 to 99 immunodominant region of MBP,
the PRTP sequence is not sufficient to form an individual T-cell epitope and is unlikely to represent the
immunodominant T-cell epitopes.21–23 Indeed, our additional experiments showed no cross-reactivity between the T-cell responses to the MBP83-99 peptide
and the MBP93-105 peptide when four independent
MBP83-99–specific T-cell clones and six MBP93-105–
specific T-cell clones were examined. However, the
Tejada-Simon et al: Cross-Reactivity to MBP and HHV-6
195
possibility exists that the increased T-cell sensitization
to MBP in MS patients initially may be triggered by
the HHV-6 antigen that shares the sequence homology
with MBP93-105 and later spread to other epitopes of
MBP by the mechanism known as epitope spreading.46
As the immunodominant properties of the T-cell
epitopes are preferentially associated with the HLA-DR
molecules expressed in MS patients regardless of the
sequence of stimulation events, the triggering
epitope(s) of MBP, such as MBP93-105 that does not
have high binding affinity,45 may not manifest itself as
an immunodominant epitope when the disease is established. It is somewhat contradictory that MBP93105–specific T cells derived from MS patients produce
fewer Th1 cytokines than those from healthy controls.
One possibility is that although patients studied here
did not receive any treatment 3 months before the
study, some T-cell lines were derived from patients
who had previous treatment with ␤-IFN (more than
5–9 months before the study), and their cytokine profile may be altered as a result. ␤-IFN has been shown
to have a regulatory effect on the cytokine profile of T
cells,47,48 and the effect may be sustained after the termination of the treatment.49
In this study, we have demonstrated that the T cells
reactive with the two peptides of MBP and HHV-6
can be categorized into three populations according to
their specificity for the peptides. Some T cells are
monospecific for either the MBP peptide or the viral
peptide, and others appear to recognize both peptides.
The findings suggest that the cross-reactive T cells recognizing both MBP and HHV-6 peptides represent a
significant subset of T cells with some degree of TCR
degeneracy. It appears that the recognition of the crossreactive T cells has a less-stringent requirement for the
flanking residues of the two peptides. In contrast, these
flanking residues are critical for the T-cell recognition
of monospecific T cells. Similar findings also have been
described for the T-cell recognition of the 83 to 99
peptide of MBP,20 –22 which demonstrated that the
different role of the central versus flanking residues, allowing some degree of promiscuity in T-cell recognition. In conclusion, the study described here provides
new evidence indicating the association between
HHV-6 and autoreactive immune responses to MBP,
which has particular importance and clinical relevance
because of the potential involvement of the two agents,
as a candidate myelin autoantigen and a suspected
causative agent, in the pathogenesis of MS.
The work was supported in part by the National Multiple Sclerosis
Society (RG-3150, Y.Z.), the Richardson Foundation (RFMS-2000
and RFMS-2001, J.Z.), the Methodist Foundation (J.Z.) and the
National Institutes of Health (MS36140, J.Z.).
196
Annals of Neurology
Vol 53
No 2
February 2003
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