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*
Department of Medicine, University of Queensland, Brisbane, Australia;
Department of Neurology and
Division of Radiology, Royal Brisbane Hospital, Brisbane, Australia; and
§
Queensland Institute of Medical Research, Brisbane, Australia
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The aims of the present study were to determine whether the peripheral blood T cell response to the 184–209 region of PLP changes over time in healthy subjects and patients with relapsing-remitting MS and to determine whether changes in blood T cell reactivity correlate with disease activity. Limiting dilution analysis is a useful, reliable method to quantify T cell responses to Ags in individual subjects over time (7, 8). We performed monthly limiting dilution analysis to quantify the frequencies of circulating T cells capable of proliferating in response to PLP41–58, PLP184–199, PLP190–209, whole myelin basic protein (MBP), MBP82–100 (the immunodominant region of MBP (9, 10)) and to a control Ag, tetanus toxoid. Disease activity in the patients with MS was monitored by monthly clinical assessment and by gadolinium-enhanced magnetic resonance imaging (MRI) of the brain.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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This study was approved by the Research Ethics Committee of the
Royal Brisbane Hospital and by the Medical Research Ethics Committee of
the University of Queensland. All subjects gave informed consent to
participate in the study. The subjects of this study consisted of five
patients with clinically definite MS (11) of the
relapsing-remitting type and four healthy volunteers. The sex, age,
duration of MS, and HLA haplotypes of the subjects are shown in Table I
. All subjects had been previously
vaccinated with tetanus toxoid, but none was vaccinated during the
course of the study or within 6 mo before commencement of the study.
None of the patients with MS was receiving any immunosuppressant,
immunomodulatory, or corticosteroid therapy at the time of commencement
of the study, but three were commenced on IFN-ß-1b (8 x
106 U s.c. every second day) late in the study, and two
received one course of i.v. methylprednisolone therapy (500 mg daily
for 5 days) for a relapse. Once a month, at the time of blood
collection, each patient with MS was assessed clinically by
the same investigator (R.D.H.). A clinical relapse was defined as an
episode of new or worsening neurological symptoms and new or
worsening neurological signs without any other explanation (such as
fever) and lasting for at least 24 h.
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Genomic DNA was prepared from whole blood or from EBV-transformed lymphoblastoid cell lines from each subject. HLA-DR typing was performed using Dynal (Dynal, Carlton South, Victoria, Australia) sequence-specific primer sets according to the manufacturer’s instructions. HLA-DQ typing was conducted at the Department of Immunology, Westmead Hospital (Sydney, Australia). For HLA-DQ typing, DNA was initially ampli- fied by PCR using generic primer pairs. HLA-DQA1 and -DQB1 alleles were determined by RFLP analysis of the PCR products.
Antigens
PLP41–58 (GTEKLIETYFSKNYQDYE), PLP184–199 (QSIAFPSKTSASIGSL), and PLP190–209 (SKTSASIGSLCADARMYGVL) were synthesized by Auspep (Melbourne, Australia) according to the human sequence (12). They were >90% pure by HPLC analysis. PLP184–199 and PLP190–209 are moderately hydrophobic, and were dissolved at a concentration of 5 mg/ml in 0.2 M acetic acid before dilution in tissue culture medium for limiting dilution assays. Human MBP was extracted from human brain according to the method of Deibler et al. (13). MBP82–100 (DENPVVHFFKNIVTPRTPP), numbered according to the human sequence, was synthesized by Auspep. Tetanus toxoid was a gift from CSL (Melbourne, Australia). PLP41–58, whole MBP, MBP82–100, and tetanus toxoid were dissolved in water.
Limiting dilution assays
Heparinized blood was collected by venepuncture from each subject at monthly intervals. PBMC were separated from the blood by centrifugation through Histopaque (Sigma, St. Louis, MO) and washed twice. For the limiting dilution assays, we used 96-well round-bottom microtiter plates (Nunc, Roskilde, Denmark) containing 200 µl/well RPMl 1640 supplemented with 10% heat-inactivated pooled human serum, 2 mM L-glutamine, and 10 mM HEPES buffer. The PBMC were added to the wells at four different concentrations, 100,000, 50,000, 10,000, and 5,000 cells/well. Twenty-four wells were prepared at each cell concentration for each Ag. To each of the wells, 50,000 autologous irradiated PBMC were added as APC. The Ags were added at the following concentrations: PLP41–58, PLP184–199, PLP190–209 and MBP82–100 at 20 µg/ml; whole MBP at 30 µg/ml; and tetanus toxoid at 10 limes flocculation U (LF)/ml. The cultures were incubated for 6 days, with 0.5 µCi [3H]thymidine being added during the last 18 h. Cultures were harvested, and [3H]thymidine uptake was measured in cpm in a ß-plate counter (LKB Instruments, Gaithersburg, MD). Wells were considered positive if the cpm exceeded twice the mean cpm for control wells (at the same concentration of responder PBMC) not containing Ag and if it exceeded the control mean cpm + 3 SD. The number of negative wells for each Ag at each cell concentration was recorded and used to determine the frequency of T cells capable of proliferating in response to the specific Ag. To calculate the frequencies and the 95% confidence intervals, we used a computer program written by Dr C. Schmidt (Queensland Institute of Medical Research, Brisbane, Australia) that used the maximum likelihood estimation method (14).
Magnetic resonance imaging
Four of the patients with MS had monthly MRI brain scans at the
time of blood collection. The scans were performed with a 1.5 T Siemens
System (Siemens, Erlangen, Germany). Scout images were taken to confirm
the similar positioning of all subjects, and axial scanning was
conducted parallel to the plane defined by the anterior and posterior
commissures on a preliminary midline sagittal image. The slice
thickness was 5 mm in the first half of the study and 3 mm in the
second half. T1-weighted imaging was performed
before and 5 min after the i.v. administration of dimeglumine
gadopentetate (gadolinium, 0.1 mmol/kg). All of the scans were assessed
by the same investigator (P.D.M.), who was unaware of the results of
the clinical assessment and the limiting dilution assays. This
investigator determined the total number of gadolinium-enhancing
lesions 
2 mm in diameter and the number of new enhancing lesions
since the previous scan.
Statistical analysis
The means of the T cell frequencies for each Ag in healthy
subjects and patients with MS were compared by Student’s t
test after assessing for differences between variances. A surge in T
cell frequency was defined as a frequency 2 SD above the mean
frequency for that Ag in healthy subjects. The percentages of time
points at which surges occurred for each Ag in healthy subjects and
patients with MS were compared by the 
2 test
with Yates’ correction. To determine whether the T cell frequencies
correlated with the number of gadolinium-enhancing MRI lesions in
individual patients, we used linear regression analysis.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The frequencies of peripheral blood T cells capable of
proliferating in response to the tested specific Ags measured at
monthly intervals are illustrated in detail for one of the healthy
subjects in Fig. 1 and are summarized for
all four healthy subjects in Fig. 2. It
is clear that in each healthy subject the T cell frequencies for at
least one myelin Ag fluctuated over time. From the total set of
measurements in the four healthy subjects, we calculated the mean and
SD of the T cell frequency for each Ag and defined a surge in the T
cell frequency as a frequency 2 SD above the mean (Table II). Surges of high frequencies of T
cells for each Ag occurred in at least one of the healthy subjects and,
in the case of MBP82–100, in three of these
subjects (Fig. 2 and Table III). The high
frequencies of T cells reactive to tetanus toxoid in two of the
subjects (KC and LA in Fig. 2) were not explained by recent
vaccination.
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The frequencies of peripheral blood T cells capable of
proliferating in response to the tested specific Ags measured at
monthly intervals are summarized for all five MS patients in Fig. 2
and
are illustrated in detail for each of the five patients in Fig. 3 and
Figs. 5–8. Surges of high
frequencies of T cells reactive to PLP190–209
occurred significantly more often in MS patients than in healthy
subjects (Fig. 2 and Table III). These surges occurred particularly
often in one MS patient (MS 48) (Figs. 2 and 5), but even when this
patient was excluded the surges occurred more often in the MS patients
than in the healthy subjects. Surges of high frequencies of T cells
reactive to the overlapping PLP184–199 peptide
also occurred more often in patients with MS but the difference was not
statistically significant. When the results for the two overlapping PLP
peptides were considered together, surges of high frequencies of T
cells reactive to the 184–209 region of PLP were found in 32.8% of
the time points analyzed in the MS patients compared with 12.0% of the
time points in the healthy subjects (p = 0.016,
Table III). Surges of high frequencies of T cells reactive to
PLP184–199 and/or
PLP190–209 occurred in all five patients with MS
(Fig. 2). The fluctuating nature of the T cell response is emphasized
by the fact that the patient (MS 35) who, of these five patients, had
had the highest level of T cell reactivity to
PLP190–209 (stimulation index, 8.2) in our
previously published cross-sectional study (3) was the
only patient who did not have one or more surges of a high frequency of
T cells reactive to this peptide in the present longitudinal study
(Figs. 2 and 6). Surges of high T cell frequencies for MBP, but not
MBP82–100, occurred more often in patients with
MS than in healthy subjects, but the difference was not significant
(Fig. 2 and Table III). The above differences between MS patients and
healthy subjects were not altered by excluding the measurements made
after the commencement of IFN-ß-1b late in the course of the study in
three patients. The higher and more variable T cell reactivity to the
184–209 region of PLP in MS patients is also reflected in higher mean
T cell frequencies and variances for PLP184–199
and PLP190–209 (Table II).
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In the patients with MS the frequencies of T cells reactive to
PLP184–199 or PLP190–209
partly correlated with disease activity as assessed by clinical
relapses or the number of gadolinium-enhancing lesions on MRI brain
scans (
Figs. 3–8). In patient MS 42, there was a surge in the
frequency of T cells reactive to PLP184–199 just
before the first relapse shown in Fig. 3; the T cell frequencies just
before the second relapse are unknown because blood was not collected
in the preceding month. High frequencies of T cells reactive to
PLP184–199, PLP190–209,
or MBP also preceded or occurred concurrently with gadolinium-enhancing
MRI brain lesions (Fig. 3). Furthermore, there was a significant
correlation between the frequency of T cells reactive to
PLP184–199 and the total number of
gadolinium-enhancing MRI brain lesions in this patient (Fig. 4). In patient MS 48, a clinical relapse
was preceded by a surge in the frequency of T cells reactive to
PLP190–209, although other surges of this
reactivity were not associated with relapses (Fig. 5). Some of the surges in the frequencies
of these T cells in this patient were accompanied by increases in the
frequencies of T cells reactive to other Ags, including tetanus toxoid,
particularly following the i.v. administration of methylprednisolone
for the relapse. This posttreatment effect may represent a rebound
phenomenon following general immunosuppression by methylprednisolone.
One of the surges in the frequency of T cells reactive to
PLP190–209 (March 1998, Fig. 5) was accompanied
by the development of gadolinium-enhancing MRI brain lesions, but
there was no significant correlation between the number of
gadolinium-enhancing MRI brain lesions and the frequency of T cells
reactive to PLP184–199 or to
PLP190–209 in this patient. Treatment with
IFN-ß-1b in patients MS 42 and MS 48 suppressed the development of
gadolinium-enhancing MRI brain lesions (Figs. 3 and 5), as has been
previously reported (15). This was associated with low
frequencies of T cells reactive to PLP184–199 or
to PLP190–209 in patient MS 42 but not in
patient MS 48. In the other three MS patients, one relapse was preceded
by a surge in the frequency of T cells reactive to
PLP184–199 and to whole MBP (MS 35, Fig. 6), but three other relapses were not
preceded by an increase in T cell reactivity to any of the Ags tested
(Figs. 6 and 7). Two of these patients
(MS 6 and MS 45) had no gadolinium-enhancing MRI brain lesions
throughout the study despite surges in the frequencies of T cells
reactive to the 184–209 region of PLP (Figs. 7 and 8). The other patient (MS 35) did not
undergo MRI scanning.
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When the T cell frequencies of healthy subjects and MS patients
for each Ag were plotted against time, it became evident that there was
a temporal clustering of surges in the frequency of T cells reactive to
MBP82–100 in March–April 1998 (Fig. 9). During this period, two healthy
subjects and one patient with MS had a surge in this reactivity, and in
June 1998 a third healthy subject had such a surge. This temporal
clustering raises the possibility of the action of an environmental
factor such as infection by a virus or bacterium. No symptoms of
infection were detected in these subjects at the time, but it is
possible that these may have been missed. A clustering of surges of
reactivity to whole MBP was observed in June–July 1997. During this
time, three patients with MS had surges in this reactivity (data not
shown); blood was not collected from the healthy subjects at these time
points. There was no definite temporal clustering of the surges in T
cell reactivity to any of the three PLP peptides.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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It has been known for some time that healthy subjects possess T cells capable of reacting to self Ags, but the present study has revealed how the frequencies of circulating autoreactive T cells fluctuate over time. We found that surges of high T cell reactivity to all five myelin Ags tested occurred in healthy subjects. These surges in the frequencies of circulating autoreactive T cells indicate recent activation and expansion of these T cell populations. The temporal clustering of the surges in the frequencies of T cells reactive to MBP82–100 or MBP raises the possibility that these T cells were activated through cross-reactivity (molecular mimicry) after infection by a virus or bacterium. Recent evidence indicates that T cells are much more cross-reactive than previously thought (18) and that viral and bacterial peptides can activate myelin-reactive human T cells (19, 20). Vandenbark et al. (21) have reported temporal clustering of increases in T cell reactivity to whole MBP in patients with MS. They did not find such increases in healthy subjects but did not study the latter as often as the MS patients. It would be expected that the activated myelin-reactive T cells would enter the CNS in the healthy subjects, as activated T cells of any specificity, including autoreactive T cells, enter the normal CNS parenchyma (22, 23). The fate of myelin-reactive T cells in the CNS in healthy subjects is unknown, but it has recently been suggested that they may be eliminated by apoptosis (24), as occurs in rats recovering from experimental autoimmune encephalomyelitis (25, 26). We also observed some fluctuations in the frequencies of T cells reactive to tetanus toxoid in healthy subjects. Some variation in the frequency of T cells reactive to tetanus toxoid in healthy subjects over time has been previously observed (8) and may reflect activation through cross-reactivity or general up-regulation of the immune system after infection.
Some of the surges in T cell reactivity to myelin Ags in patients with MS may also be driven by cross-reactivity after viral or bacterial infection. Other surges may result from the activation of autoreactive T cells after the release of myelin Ags from the CNS during attacks of MS. Some previously activated myelin-reactive T cells may be reactivated nonspecifically by a general up-regulation of the immune system after infection. The latter mechanism may account for those surges of high frequencies of myelin-reactive T cells that occurred concurrently with high frequencies of T cells reactive to tetanus toxoid in the present study. As in the case of healthy subjects, it would be expected that the circulating activated myelin-reactive T cells would enter the CNS in MS patients. It has been hypothesized that, in contrast to the situation in healthy subjects, these T cells may fail to undergo apoptosis in the CNS and may thus be able to induce CNS demyelination with subsequent release of myelin Ags (24). The released myelin Ags may reactivate the originally aggressive autoreactive T cells and activate T cells specific for other myelin Ags, leading to the perpetuation and amplification of the autoimmune attack on the CNS.
In the present study, we found evidence that T cells reactive to PLP184–209 may contribute to the pathogenesis of MS. Some of the clinical relapses were preceded by surges in the frequency of circulating T cells reactive to PLP184–209, and in one patient there was a significant correlation between the frequency of circulating T cells reactive to PLP184–199 and the total number of gadolinium-enhancing MRI brain lesions. Gadolinium enhancement indicates a breakdown of the blood-brain barrier and correlates with a marked perivascular accumulation of inflammatory cells (27). It often precedes other MRI abnormalities and clinical evidence of a new lesion (28), although a recent study suggests that some lesions may commence without a preceding phase of gadolinium enhancement (29). We suggest that some of the gadolinium-enhancing lesions and relapses in our patients developed as a consequence of the entry of T cells reactive to PLP184–209 into the CNS following an increase of these T cells in the circulation. However, some of the surges of T cell reactivity to PLP184–209 were not associated with either a clinical relapse or the development of gadolinium-enhancing MRI brain lesions. The lack of a constant association with clinical relapse is not surprising, given that clinical relapses are insensitive indicators of disease activity compared with gadolinium-enhanced MRI (30). Even with the use of gadolinium-enhanced brain MRI as in the present study, new lesions would have been missed if they occurred in the spinal cord or optic nerve, if they were small brain lesions, or if they developed without preceding gadolinium enhancement. Our study has also shown that some clinical relapses are not associated with a surge in T cell reactivity to PLP184–209, PLP41–58, MBP82–100, or whole MBP. These relapses may have been induced by T cells reactive to other myelin Ags, such as other epitopes of PLP, epitopes of myelin/oligodendrocyte glycoprotein, or epitopes of the recently described oligodendrocyte-specific protein (31) or myelin-associated oligodendrocytic basic protein (32), but reactivity to these Ags was not determined in the present study.
Given the abundance of MBP in the CNS it is noteworthy that in contrast to the frequent surges of high T cell reactivity to PLP184–209, which contains the immunodominant PLP region, surges of high T cell reactivity to the immunodominant MBP peptide, MBP82–100, were infrequent in MS patients. There are several possible explanations for this, including: more effective processing of PLP epitopes than MBP epitopes from the native proteins released from CNS demyelinating lesions; higher binding of PLP epitopes to MHC molecules and thus greater presentation to T cells; higher levels of Abs to PLP epitopes with resultant greater uptake of these epitopes by specific B cells and greater presentation by B cells to T cells; and apoptotic deletion of MBP-reactive T cells, but not PLP-reactive T cells, in the peripheral nervous system, which contains MBP but not PLP, although there may be a failure of apoptosis of all autoreactive T cells in the CNS (24).
We have shown that the frequencies of circulating autoreactive T cells fluctuate over time in both healthy subjects and patients with MS. We also found that surges of high T cell reactivity to the encephalitogenic 184–209 region of PLP occur more often in MS patients than in healthy subjects. T cells reactive to this region of PLP may contribute to the development of some of the demyelinating lesions in the CNS in MS.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Michael P. Pender, Department of Medicine, Clinical Sciences Building, Royal Brisbane Hospital, Brisbane, Queensland 4029, Australia.
3 Abbreviations used in this paper: MS, multiple sclerosis; PLP, proteolipid protein; MBP, myelin basic protein; MRI, magnetic resonance imaging.
Received for publication April 24, 2000. Accepted for publication July 31, 2000.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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ß T lymphocytes in the nervous system in experimental autoimmune encephalomyelitis: its possible implications for recovery and acquired tolerance. J. Autoimmun. 5:401.[Medline]
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, numbers of new gadolinium-enhancing
lesions. IFN-ß, IFN-ß-1b, 8 x 106 U s.c. every
second day for the remainder of the study.
