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*
Department of Neurosciences, Lerner Research Institute,
Department of Neurology, Mellen Center for Multiple Sclerosis Research, and
Department of Biostatistics, Cleveland Clinic Foundation, Cleveland, OH 44195; and
§
Institute of Pathology, Case Western Reserve University, Cleveland, OH 44106
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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and IL-5 production. MS patients
(n = 22) responded to 4 times as many PLP peptides
as did healthy controls (n = 22). The epitopes
recognized in individual patients, up to 22 peptides, were scattered
throughout the PLP molecule, showing considerable heterogeneity among
MS patients. Frequency measurements showed that the number of PLP
peptide-specific IFN--producing cells averaged 11 times higher in MS
patients than in controls. PLP peptide-induced IL-5-producing T cells
occurred in very low frequencies in both MS patients and controls. This
first comprehensive assessment of the anti-PLP-Th1/Th2 response in
MS shows a greatly increased Th1 effector cell mass in MS patients.
Moreover, the highly IFN--polarized, IL-5-negative cytokine profile
of the PLP-reactive T cells suggests that these cells are committed Th1
cells. The essential absence of uncommitted Th0 cells producing both
cytokines may explain why therapeutic strategies that aim at the
induction of immune deviation show little efficacy in the established
disease. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, IL-2, TNF-
, and IL-12 (reviewed in Ref.
2). Proinflammatory cytokines are thought to be crucial
for the initiation and amplification of inflammatory brain lesions and
direct myelin damage in MS (3, 4, 5). The role of Th2-like
cytokines in MS is less clear. Regulatory cytokines such as IL-4, IL-5,
IL-10, and IL-13 may play a role in the resolution of relapses
(2), but in some situations they are associated with
exacerbation of autoimmune disease (6). It is likely that
both inflammatory and regulatory processes occur simultaneously in MS.
This may explain why proinflammatory and regulatory cytokines are often
up-regulated simultaneously in MS (7, 8, 9). Although the pathogenesis of MS is thought to involve autoreactivity directed against myelin Ags, such as MBP and PLP, the poor aqueous solubility and strong hydrophobicity of PLP have impeded studies with this molecule. Using intact PLP as an Ag, proliferation assays have not shown convincing or consistent responses in PBMC or cerebral spinal fluid of MS patients (10, 11). Even the detection of vaccine Ag-specific memory cells in immunized individuals is frequently a technical challenge due to the very low frequency of cells. New technological developments that are permitting better insights are the use of MHC tetramers and a new generation of computer-assisted ELISPOT assay optimized for single-cell resolution. Although highly sensitive, comprehensive tetramer analysis requires knowing both the Ag and the MHC restriction element and does not lend itself to systematic testing of peptide libraries. With increasing evidence for diverse autoimmune repertoires in MS resulting from determinant spreading (12, 13), such studies might be required for comprehensive assessment of the autoimmune T cell repertoire. An alternative method is by a pepscan approach in conjunction with ELISPOT analysis. The ELISPOT assay can be performed without knowing the MHC restriction element and is ideally suited for comprehensive peptide mapping and determinations of very low frequency Ag-specific T cells (range of 1:10,000 to 1:1,000,000) that are undetectable by other cytokine detection methods, such as intracytoplasmic staining (14). We reasoned that with the extensive MHC heterogeneity in humans, a comprehensive study is required in which all possible determinants are examined regardless of whether the peptide is dominant or cryptic following natural processing. To accomplish this goal, we used a PLP peptide library consisting of single-amino acid overlapping 9-mer peptides. Peptides of this length can bind directly on the cell surface to MHC class I and class II molecules of APC where they can stimulate peptide specific T cells. This systematic approach should reveal the total PLP-specific T cell pool, its fine specificity, and the overall clonal sizes of the PLP peptide reactive repertoire.
The ELISPOT assay demonstrates very high sensitivity and specificity
(14) and has been used for detection of anti-PLP
responses in mononuclear cells from cerebral spinal fluid and blood of
MS patients (15, 16). Findings from studies using a select
number of synthetic PLP peptide Ags in proliferation assays indicate
that T cell responses to a heterogeneous array of PLP determinants
occur in MS patients (12, 13, 17, 18). Similarly,
longitudinal analysis of selected PLP peptide responses in MS suggest
that chronic immune sensitization to myelin determinants leads to
acquired immunity to new self-Ags, known as determinant spreading
(12, 13, 19, 20). Whereas previous studies focused on the
response to whole PLP, using only IFN- as a readout (15, 16), we have examined reactivity using peptides spanning the
entire PLP molecule and we have expanded our analysis to include both
type 1 and type 2 cytokines.
In MS and EAE, the autoimmune disease state has been associated with
enhanced Th1 responses to self. Controversy still exists over the
degree of Th1 polarization in autoimmune disease as well as the
contribution of Th2 or Th0 responses to pathogenesis. To fully
understand the diversity of the autoimmune response, however, a
systematic study covering all possible determinants on the molecule is
still missing. The delineation of PLP-reactive memory cells as
proinflammatory Th1 or anti-inflammatory Th2 is essential to the
understanding of the role these cells play in the autoimmune process.
We sought to provide this missing information at single-cell
resolution, performing ELISPOT assays for the detection of PLP
peptide-reactive memory cells that produce the Th1 cytokine IFN- and
the Th2 cytokine IL-5.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The ELISPOT protocol used in this study was developed in our
laboratory (21, 22). ELISPOT plates (UNIFILTER, low volume
catalogue no. 7770-0052, Whatman, Clifton, NJ) were precoated with
cytokine capture Abs (specified below) in PBS (50 µl/well) and placed
at 4°C overnight. Study subject PBMC were isolated from heparinized
blood by Ficoll density gradient centrifugation, washed three times,
and set up in a preactivation culture with PLP peptides in 96-well
V-bottom tissue culture plates (Costar, Corning, NY) at 1.5 x
105 cells/well in 200 µl of complete medium.
PLP peptides were added to the wells at a final concentration of 7
µM. The cells were incubated for 24 h at 37°C in a 7%
CO2 incubator. Ab-coated ELISPOT plates
were washed three times with 100 µl/well sterile PBS. Plates were
blocked to prevent nonspecific binding of proteins using 100 µl/well
sterile PBS containing 1% BSA (fraction V) for 2 h at room
temperature and then washed again three times with 100 µl/well
sterile PBS. Cells from the preactivation cultures were carefully
resuspended, and half the cells (100 µl, 7.5 x
104 cells) were transferred to ELISPOT plates
that were precoated with IFN- capture Ab; the remaining cells were
transferred to plates coated with IL-5 capture Ab. The cells were
incubated for 24 h at 37°C in 7% CO2.
Cells were then washed away using three washes with 100 µl/well PBS
followed by three washes with 100 µl/well PBS/0.05% Tween. Secondary
biotinylated anti-cytokine Ab was diluted in PBS/0.05% Tween/1%
BSA, added at 50 µl/well, and incubated overnight at 4°C. Plates
were then washed three times with 100 µl/well PBS/Tween, and a 1/2000
dilution of streptavidin-HRP in PBS/Tween/BSA was added at 100
µl/well for 2 h at room temperature. Plates were washed three
times in 100 µl/well PBS, and spot color was developed by adding 100
µl/well AEC substrate diluted 1/30 in 0.1 M acetate buffer (pH 5.0)
containing a 1/2000 dilution of 30%
H2O2. Plates were observed
for spot development for a maximum of 1 h at room temperature and
then were washed three times with dH2O (200
µl/well) to stop the reaction. Plates were dried overnight at room
temperature, images of the wells were acquired and saved on compact
disc using an automated ImmunoSpot Series 1, and the spots were
enumerated on an ImmunoSpot Satellite analyzer (Cellular Technology
Ltd., Cleveland, OH) using software specifically designed for the
ELISPOT assay. Briefly, digitized images were analyzed for the presence
of areas in which color density exceeds background by a factor
calculated from comparing control wells to experimental wells. After
separating spots that touch or partially overlap, additional criteria
of spot size and circularity were applied to gate out noise caused by
spontaneous substrate precipitation and nonspecific Ab binding. Objects
that did not meet these criteria were ignored, and areas that met them
were recognized as spots, counted, and highlighted. Positive responses
were defined as two or more adjacent peptide-stimulated wells giving
responses greater than the mean ± 3 SD of unstimulated wells.
Abs and reagents
Primary or capture Abs were anti-IFN- (M-700A, Endogen,
Cambridge, MA) at a concentration of 4 µg/ml or anti-IL-5
(18551D, PharMingen, San Diego, CA) at a concentration of 5 µg/ml.
Secondary or detecting Abs were anti-IFN- (M-701, Endogen;
biotinylated according to the manufacturer’s directions) at a
concentration of 4 µg/ml or anti-IL-5 (18522D, PharMingen) at a
concentration of 1 µg/ml. We purchased streptavidin-HRP from Dako
(D0397, Carpenteria, CA) and 30%
H2O2 from Sigma (H-1009,
St. Louis, MO). The AEC substrate (A-5754, Sigma) stock solution was
prepared with 100 mg of AEC in 10 ml of dimethylformamide (Sigma), then
diluted 1/30 in 0.1 M acetate buffer (pH 5.0). The AEC/acetate buffer
solution was filtered through a 0.45-µm pore size filter before use
to remove colored precipitates. Tween-20 (polyoxyetylene 20-sorbitan
monolaurate) was purchased from Fisher (BP337–100, Pittsburgh, PA).
BSA (fraction V) was obtained from Sigma (A-1933). Ficoll-Paque plus
was purchased from Pharmacia (M-1440-03, Piscataway, NJ). Complete
medium consisted of RPMI 1640, 100 U/ml penicillin, 100 µg/ml
streptomycin, 2 mM L-glutamine, and 10% newborn bovine
serum (all from Life Technologies, Gaithersburg, MD).
Study subjects
Patients at the Mellen Center for Multiple Sclerosis Research at the Cleveland Clinic Foundation were invited to participate in this study. Healthy controls consisted of volunteer researchers from both Case Western Reserve University and the Cleveland Clinic Foundation. Research subjects signed an institutional review board-approved informed consent document to donate blood for this study. Patients (n = 22) were categorized as relapsing-remitting (n = 9), secondary progressive (n = 8), or primary progressive (n = 5). Patients had not received steroid treatment for a period of at least 3 mo before blood drawing. Some patients were using IFN-ß or glatiramer acetate as therapy. Patient and control groups were equally divided between males and females. Up to 40 ml of blood was obtained from study subjects by phlebotomy.
Peptides and Ags
Systematic mapping of determinants involving hundreds of
peptides was performed as previously described (23, 24).
The PLP peptides were synthesized in a fully automated fashion using
the pin method and have >96% purity; they were obtained from Chiron
Mimetopes (San Diego, CA). Nine-amino acid-long PLP peptides that span
the PLP molecule in single amino acid steps were used to stimulate PBMC
at a final concentration of 7 µM, which is optimal for pulsing of APC
(23, 24). Because the MHC class I groove is closed on both
ends (25), we chose to use nonamer peptides, which can
bind extracellularly to class I molecules, in addition to class II
molecules, bypassing intracellular processing (26).
Although class II molecules preferentially bind longer peptides,
nonamer peptides are also capable of binding MHC class II molecules and
stimulating CD4 T cells (22, 24). Recall Ags consisted of
tetanus toxoid (1/20 dilution), diphtheria toxoid (100 µg/ml), both
from Connaught Laboratories (Swiftwater, PA) as well as Candida
albicans extract (1/20 dilution; Bayer, Spokane, WA). As a
positive control we used the mitogen PHA (PHA-P; 2.5 µg/ml for
IFN- assays and 10 µg/ml for IL-5 assays; Sigma). We also tested
purified human MBP (10 and 100 µg/ml; provided by Dr. Henry
McFarland, National Institute of Neurological Disorders and Stroke,
National Institutes of Health, Bethesda, MD) as well as an MBP-PLP
fusion protein known as MP4, which consists of the 21.5-kDa isoform of
MBP fused to a genetically engineered form of PLP (
PLP4) containing
the four major hydrophilic regions of the PLP molecule (10 and 100
µg/ml; provided by Alexion Pharmaceuticals, New Haven, CT)
(27).
Statistical analysis
Statistical analysis was performed in collaboration with the Cleveland Clinic Biostatistics Department. Analysis of the number and magnitude of the PLP peptide response was performed using the Wilcoxon rank-sum test, comparing MS patients to healthy controls. Age was compared between the study groups using Student’s t test. The average age for MS patients in the study was significantly older than that in the controls, so we performed an analysis of covariance with rank data to adjust for possible age effects in the group comparisons. A p value < 0.05 was considered significant, and all tests were two-sided. All statistical analyses were performed using the SAS statistical software package (SAS Institute, Cary, NC).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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ELISPOT assays demonstrate a significantly elevated number
of stimulatory PLP peptides and a highly increased frequency of
PLP-peptide reactive cells in MS patients compared with controls
We tested PBL obtained from 22 MS patients and 22 healthy controls
for PLP-peptide-induced production of IFN- and IL-5 in ex vivo
ELISPOT assays. The mean age of MS patients was 49 ± 9 years (age
range, 32–70 years), and the mean age of controls was 40 ± 11
years (range, 24–67 years). The average Expanded Disability Status
Score (EDSS) of MS patients was 5.0 ± 2.4, with a range from 1 to 8.5.
Mean disease duration was 12 ± 7 years (range, 2–30 years; Table I
). The peptides used spanned the
entire PLP sequence in steps of single amino acids, as illustrated in
Fig. 1A. IFN- and IL-5
ELISPOT measurements were selected because in our previous work
(14) we showed, first, that such ELISPOT assays detect
Ag-specific memory T cells (naive T cells do not produce these
cytokines, and recall responses are not detected in unsensitized
individuals); and second, they measure the true frequency of
Ag-specific T cells in the low frequency range in the absence of
bystander reactions. We selected IL-5 over IL-4 and IL-10 for
measurement of Th2 memory/effector cells because IL-5 production is
confined to T cells, while IL-4 and IL-10 can be produced by APC, and
subsequently can give high backgrounds and bystander activity
(M. Tary-Lehmann, unpublished observations).
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-positive response, involving
three adjacent PLP peptides, is shown in Fig. 1
B. The
positive PLP 9-mer peptides demonstrated stimulation indexes above the
medium control value of 3.9, 10.6, and 17.6, respectively. Examples of
the actual ELISPOT wells from the medium control and the highest
stimulating peptide are shown (Fig. 1C).
Table II shows comparisons of PLP 9-mer
peptide-induced responses for each individual MS patient and healthy
control. For IFN- responses, 13 of the 22 MS patients (59%)
responded to PLP peptide clusters consisting of minimally two adjacent
peptides. In these patients the number of positive determinants varied
between two and 22, with seven of the patients responding to >10
determinants. In contrast, eight of the 22 controls (36%) showed
IFN- responses to PLP determinants. Of these, five responded to only
two determinants, and another three healthy donors responded to six to
nine peptide clusters. Thus, many more MS patients responded to PLP
than controls, and MS patients reacted to a significantly higher number
of determinants (Tables II and III).
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spots, and only two donors exhibited >100 total spots (Table II). In
contrast, the maximal cumulative spot number reached in a single MS
patients was 2110, and seven of the patients generated >100 spots. The
overall frequency of PLP peptide-specific T cells, therefore, was
very strongly increased in MS patients.
A summary of the combined group data comparing MS patients to controls
is shown in Table III. The total number of IFN--positive PLP
peptides was >4 times higher in MS patients than in healthy controls.
MS patients showed a significantly higher mean number of
IFN--positive PLP peptides compared with controls (6.5 ± 1.7
vs 1.5 ± 0.6; p = 0.022). The magnitude of the
IFN- response (i.e., the average number of single-cell spots in each
peptide-stimulated well) was >11 times higher in MS patients than in
controls (MS mean, 232.0 ± 105.3; control mean, 21.8 ±
9.7). Because the number of spots was extremely variable from one
person to another, with one patient having a total of 2110
IFN--positive cells and another having none, the magnitude of the
IFN- response approached, but did not reach, significance
(p = 0.06). Age-adjusted results indicate
similar findings.
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, PLP peptide-induced IL-5-secreting cells
occurred in very low frequencies. Although the total number of
IL-5-positive peptides and the magnitude of the IL-5 response were both
>3 times higher in MS patients than in controls, the comparison of
individual mean values showed that neither category was significantly
different between MS patients and healthy controls
(p = 0.58 and 0.52, respectively; Table III).
Thus, after stimulation with PLP peptides, only IFN-, and not IL-5,
responses showed a significant increase in MS patients, but not in
controls. MS patients responded to many more PLP epitopes and they do not demonstrate a skewed response to a limited set of peptides compared with healthy controls
We wanted to examine which regions of the PLP molecule gave
positive responses and to compare those epitopes between MS patients
and controls to see whether there were regions unique to either group
or if there were "hot spots" within PLP. To facilitate this
comparison, the sum of IFN--positive cells for all MS
patients and controls is plotted in Fig. 2. There were many more epitopes of PLP
recognized uniquely by MS patients. The recognition spanned the entire
PLP molecule from N-terminus to C-terminus in an extremely
heterogeneous pattern. Healthy controls did not demonstrate recognition
of unique PLP determinants, because virtually every PLP epitope
recognized by a control was also recognized by one or more MS patients.
Fig. 3 shows a similar histogram of the
sum of IL-5-positive cells for MS patients and controls. There are many
fewer PLP determinants that elicited any IL-5 response in either group.
The magnitude of the IL-5 responses was extremely low compared with
that of IFN- and generally did not show common epitopes between MS
patients and controls.
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Next, we studied the exact determinant regions recognized by the
PLP-specific T cells, because this information pertains to therapeutic
strategies designed to selectively inactivate autoreactive T cells, and
it is relevant for selecting the "right peptide(s)" for
tetramer-based immunodiagnostic approaches. Figs. 4 and 5
show the localization of the PLP determinants targeted in the
individual MS patients and controls. The plots are lined up vertically
to allow a more detailed comparison of common or unique PLP
determinants between MS patients and controls. The data show that the
stimulatory peptides were scattered over the entire PLP molecule, and
the patterns of peptide recognition were highly unique for the
individual patients. Overall, the IFN- responses (Fig. 4) were not
skewed to any set of peptides, but two regions appeared to be partially
shared: PLP133–152 (five patients reacted) and
PLP161–178 (seven patients reacted). Most other
shared determinants were observed in no more than four patients at a
time. This diversity of determinant recognition is consistent with the
multitude of different allelic MHC molecules expressed in the patients,
each having different peptide binding motifs. With regard to
determinant recognition (either the numbers of determinants or the
magnitude of the response), we did not observe any correlation with
disease state, amount of disability (EDSS), or disease duration.
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); only two healthy controls
and five MS patients gave positive IL-5 responses to PLP peptides. IL-5
responses in both groups were observed primarily in the N- and
C-terminal regions, with almost no reactivity in the center of the
molecule. The peptides showed no conservation between MS patients or
controls. We also observed that the frequency of IL-5-producing cells
in positive wells was very low compared with that in positive IFN-
wells (Fig. 3 and Tables II and III).
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Because the PLP peptide responses in MS patients were biased
toward Th1 recognition, we wanted to make sure that the overall immune
responsiveness in the patients did not reflect general Th1
hyper-responsiveness. Using the same IFN- and IL-5 ELISPOT approach
as a readout, we asked whether the MS patients and the controls respond
differently to recall Ags, mitogen, or myelin proteins. The recall Ags
consisted of vaccine Ags, environmental Ags, or mitogen. While these
third party responses were quite variable from subject to subject, they
did not show significant differences between patients and controls
(Fig. 6). IFN- responses to PHA were
too numerous to count and are not shown on the IFN- plot. The
IFN- responses to the MBP-PLP fusion protein, MP4, showed a strong
increase in MS patients compared with controls at the higher dose of
100 µg/ml (p = 0.045). Although we tested
responses to MBP, we did not observe significant IFN- or IL-5
differences between patients and controls (data not shown), suggesting
that the positive MP4 responses were due to recognition of the PLP
component of that fusion protein. Importantly, the MS patients showed
IL-5 responses to these recall Ags and to the mitogen with similar
frequencies as those in healthy controls, suggesting that there is no
overall Th1 bias in the patients.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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immune response to PLP compared with
healthy controls. This autoimmune response in MS involves recognition
of a greater diversity of PLP peptides as well as a >11-fold higher
number of cells responding to those PLP peptides. IL-5 responses were
not different between MS patients and controls. These differences were
not due to a generally increased capacity to produce IFN- in MS,
because the numbers of cells responding to recall Ags or mitogen did
not differ significantly between patients and controls. For
IFN--positive responses, epitopes recognized by controls were also
recognized by MS patients, but there were many more epitopes of PLP
recognized uniquely by MS patients, and the recognition spanned the
entire PLP molecule from N-terminus to C-terminus in an extremely
heterogeneous pattern. Although we cannot tell whether these
PLP-specific cells are the primary cells or whether they have been
secondarily engaged after determinant spreading, once they are primed
it is likely that they participate in the autoimmune process. This idea
was introduced by Mor and Cohen (28) in rodent
experimental allergic encephalomyelitis; while only one determinant of
MBP was immunodominant, 11 of 16 peptides were capable of causing
histological or clinical EAE. After processing and presentation of
native Ag, determinant display may not be a reliable measure of the
pathogenic T cell repertoire. This might explain why recall responses
with the MBP-PLP fusion protein were much weaker than those induced by
the peptides. By providing every conceivable PLP determinant for the T
cells to recognize, our single-amino acid pepscan strategy aimed to
monitor the entire PLP-specific repertoire. With analogy to the study
by Mor and Cohen (28), while not all of the primed
PLP-peptide specific T cells will contribute to the disease process,
one can predict that many of these T cells do contribute to the
disease. Due to the multitude of determinants targeted in most
patients, our data suggest that the autoimmune repertoire shows an
unexpectedly highly diversity with respect to determinant specificity
and, by analogy, TCR usage.
Multiple studies have examined lymphocyte responses to PLP with varying
degrees of success due in part to the poor aqueous solubility and
strong hydrophobicity of the PLP molecule and to the techniques used to
measure reactivity. Studies that failed to find reactivity used whole
PLP and measured reactivity in proliferation assays (10, 11). Limiting dilution analysis and proliferation assays now are
thought to be less sensitive methods for the detection of clonally
expanded memory cell populations than more recent techniques, such
as single-cell cytokine ELISPOT assays and tetramer analysis. Studies
that found significantly increased PLP responses in MS compared with
controls used single-cell techniques and IFN- output as a measure of
reactivity (15, 16). Many researchers have examined T cell
lines and clones specific for PLP; however, there is always the
potential for in vitro skewing of the cytokine responses by virtue of
the culture methods used to obtain lines and clones. Additional studies
have found a very strong association with whole PLP stimulation and
IFN- and IL-4 mRNA expression in MS (29, 30, 31). An
increasing number of researchers have taken to using synthetic peptides
of PLP and have had much more success in detecting PLP-specific
responses in both cerebral spinal fluid and peripheral blood (17, 18, 32, 33, 34, 35, 36). This study confirms the previously observed
increased PLP responses in MS compared with controls, but, in addition,
we performed the first comprehensive epitope mapping using PLP 9 mers
spanning the entire PLP molecule examining both type 1 and type 2
cytokine responses in MS patients and healthy controls.
While the data presented here show a clear difference in the IFN-
response to PLP peptides in MS patients, it is worth noting that the
role of IFN- in EAE and MS remains controversial. Relapses in EAE
initially were shown to correlate with increased expression of IFN-
(37, 38). Intrathecal injections of IFN- induced
inflammation in Lewis rats (39), and in transgenic mouse
models, organ-specific expression of IFN- led to autoimmune diseases
resembling diabetes, myasthenia gravis, and uveitis
(40, 41, 42). Likewise, the potentially damaging role of
IFN- in MS has been demonstrated in several studies. IFN- was
shown to induce oligodendrocyte death by apoptosis (5).
Systemic administration of IFN- to MS patients was shown to worsen
the disease (43). Beck et al. reported increased IFN-
production preceding MS exacerbations (44), and
IFN--expressing cells were found to be present in MS lesions
(45). However, several conflicting observations about the
role of IFN- have appeared. Administering anti-IFN- mAb to
genetically EAE-resistant mice induced an EAE-susceptible phenotype
(46, 47). Intraventricular injection of IFN- prevented
EAE in rats (48), and IFN- was not necessary for the
induction of EAE using IFN- knockout mice back-crossed to an
EAE-susceptible strain (49). Similarly, in knockout
animals, lack of IFN- converted an otherwise EAE-resistant mouse to
become susceptible to disease (50). In MS, the ELISPOT
assay has been used to demonstrate that MS patients have increased
numbers of mononuclear cells in blood and cerebral spinal fluid,
secreting IFN- in response to stimulation with myelin proteins and
viral Ags (15, 16, 51). Paradoxically, regulatory
cytokines such as IL-4 and TGF-ß were shown to be simultaneously
up-regulated with IFN- in MS (29, 30, 31). Thus, in both
EAE and MS, cytokine interactions are much more complex than originally
proposed and, as such, do not support a rigid classification of MS into
the Th1/Th2 model. Instead, certain cytokines, such as IFN-, IL-4,
IL-5, and IL-10, may play an important role in the pathogenesis of
disease, and their relative roles may change over the course of T
cell-mediated disease.
In the current study we observed that the number and distribution of
IFN--positive PLP epitopes was quite heterogeneous even though there
were two common regions recognized by both MS patients and controls:
PLP133–152 and
PLP161–178. These common regions appear
different from most other published immunodominant regions (17, 18, 32, 52, 53). There are several possible explanations for
this difference. First, the HLA type of the study subjects determines
which peptides are able to bind and stimulate T cells. Without HLA
typing of our study subjects, it is impossible to compare the commonly
recognized regions of PLP between our own patients and controls or
between subjects in our study and those in other published studies.
Second, by using the ELISPOT technique to detect these PLP epitopes, it
is possible that the sensitivity of the assay technique may allow us to
observe more or different regions of PLP recognition than proliferation
assays or techniques that rely on growing out T cell clones. Third, our
epitope-mapping technique using overlapping 9-mer peptides may skew the
responses we observed compared with studies that have used longer
peptides. Because the MHC class I groove is closed on both ends
(25), we chose to use nonamer peptides, which can bind
extracellularly to class I molecules, bypassing intracellular
processing (26). These nonamer peptides are also capable
of binding MHC class II molecules (22, 24), but might
stimulate primarily high affinity CD4 cells only. Recent evidence in a
humanized MHC model of EAE demonstrated equivalent T cell responses
between 9-mer and 20-mer peptides derived from an encephalitogenic
myelin protein (T. Forsthuber, Cleveland, OH, personal communication).
As mentioned previously, the techniques that were used in the past
favored the detection of CD4 cells in MS patients, while our detection
system might have a CD8 bias. We did not want to limit ourselves to
only one class of response; however, a rigorous analysis of the HLA
restriction elements for each study subject and for every positive
peptide that was recognized in this analysis (208 peptides) was beyond
the scope of the present study.
Several points have emerged that highlight PLP as a unique, potential autoimmune target in MS. In contrast to the relatively restricted reactivity to MBP in MS patients, which appears to be directed primarily to PLP83–106 and PLP142–170 (reviewed in Ref. 1), reactivity to PLP can be observed for virtually the entire molecule, as we show here and others have shown previously (12, 19, 54). Also, for PLP, the determinants recognized vary considerably between patients, and many patients respond to more than one peptide. Unlike MBP, no single immunodominant region of PLP has been identified. Add to this the compelling evidence for epitope spreading within PLP in MS with progression of disease (12, 54), and it becomes very difficult to determine whether immunodominant epitopes exist. Perhaps, as Tuohy et al. (54) suggest, they exist only in the temporal context of an "epitope du jour." This argues that a cross-sectional analysis such as the present study, performed at one moment in time, may not accurately represent PLP reactivity in MS. We have examined patients with different types of MS, and each of them is at a potentially different stage of disease compared with the others. Consequently, the PLP peptide reactivity we observed may be highly specific for that particular patient, for that moment in time. In addition, other myelin proteins implicated in MS autoreactivity, such as MBP and MOG, may also play a role in progression; however, the current epitope-mapping technique we have used here did not permit simultaneous analysis of all other potential autoantigens. Experiments designed to address longitudinal cytokine responses to myelin determinants in a more narrowly defined MS population are currently in progress.
When studied at single-cell resolution, it is striking how purely Th1
(IFN-+ and IL-5-) the
anti-PLP peptide responses are. Although it is still controversial
whether Th2 cells are pathogenic, there are virtually no PLP-specific
IL-5-producing Th2 cells in these patients. The PLP-specific cells also
do not appear to be Th0, which should coexpress IFN- and IL-5 at
comparable frequencies. This observation might be important for
therapeutic considerations. The cytokine differentiation of Th0 cells
can be redirected toward a putatively nonpathogenic class (Th2/Th3),
while the Th1 cells are committed. This might help explain why oral and
other Ag therapies work well for prevention, but less well for the
treatment of established disease.
As we observed, PLP-reactive T cells could be detected in healthy controls; however, those frequencies were much lower than those in MS patients. While some controls gave strong responses to individual peptides, MS patients showed reactivity to more determinants, consistent with a role for epitope spreading, which has been implicated in disease progression (12, 13, 22, 54, 55). Thus, the clinical presentation of MS may not represent loss of self tolerance, so much as the strength of the autoimmune response that is engaged. These data may suggest the existence of a pathogenic threshold, as has been observed in EAE, where a minimum number of neuroantigen-reactive cells is required to achieve disease. The processes that lead to violation of self tolerance, such as the priming of autoreactive T cells by cross-reactive infections, might occur in patients and healthy controls alike. Thus, quantitative differences in clonal sizes may influence the development of clinical MS.
Here we show the feasibility of quantification of the full PLP-specific T cell repertoire at single-cell resolution, overcoming the limitations of MHC diversity and determinant diversity that are inherent for humans and for the autoimmune process. The data affirm the autoimmune hypothesis by suggesting that disease vs health is a quantitative and not necessarily qualitative matter and show strong Th1 polarization, which suggests that therapies designed at redirecting cytokine expression might be difficult. The ability to comprehensively measure the overall effector cell mass to an autoantigen, that is, directly measuring the frequency of cells specific for hundreds of determinants, should be valuable for clinical trials that are aimed at modifying this effector cell pool. Thus, it should be possible to determine the effect of therapy on the magnitude, fine specificity, and effector cell class as measured by cytokine expression. The ability to perform such measurements, using a high throughput technique with automated analysis, may revolutionize clinical trials that to date have been unable to obtain direct information on how the autoantigen-specific effector cells are affected by autoantigen treatment.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Clara Pelfrey, Department of Neurosciences, NC30, Cleveland Clinic Foundation Lerner Research Institute, 9500 Euclid Avenue, Cleveland, OH 44195.
3 Abbreviations used in this paper: MS, multiple sclerosis; AEC, 3-amino-9-ethylcarbazole; EAE, experimental autoimmune encephalomyelitis; ELISPOT, enzyme-linked immunospot assay; MBP, myelin basic protein; MP4, MBP-PLP fusion protein; MOG, myelin oligodendrocyte glycoprotein; PLP, proteolipid protein; EDSS, Expanded Disability Status Score.
Received for publication March 15, 2000. Accepted for publication May 16, 2000.
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) and MS patients
(MS; 
) were tested simultaneously with PLP peptides. The numbers
tested are in parentheses.