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Departments of
* Neurology and
Transplantationimmunology and Immunehematology, University of Tübingen, Tübingen, Germany;
Institute of Pathology, Case Western Reserve University, Cleveland, OH 44106; and
EMC Microcollections, Tübingen, Germany
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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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0.01% of the protein content of the myelin
sheath and is exposed on its outer surface (6). It has an
extracellular part including aa 1–122 with an Ig-like domain, a
transmembrane part, and an intracellular part comprising aa 123–218
(Fig. 7
; Refs. 9, 10, 11). Up to now, T cell responses against
the extracellular part of MOG have been mainly looked at. Using the
ELISPOT methodology, Wallström et al. (12)
demonstrated increased IFN-
-secreting cells in HLA-DR2(15) positive
MS patients compared with controls toward several MOG peptides among
which MOG 63–87 was immunodominant. Others investigated T cell
responses to MOG of non-HLA-stratified MS patients and controls
indicating increased T cell reactivity to MOG in MS patients
(13, 14, 15). Also, MOG-specific B cell responses are
up-regulated in MS, and MOG-specific Abs contribute to lesion formation
(16, 17). Because MOG can be considered an important
autoantigen in MS and a potential target for therapy, we investigated T
cell responses by ELISPOT assay for IFN--secreting cells to peptides
of the complete human MOG 1–218 sequence. We used the ELISPOT assay to
detect IFN--secreting cells, because this is more sensitive than
proliferation as assessed by [3H]TdR-uptake,
and adds a functional outread. We demonstrate a previously unknown
dominant T cell epitope that is recognized in context of HLA-DR
molecules by CD4+ T cells within the
transmembrane and intracellular sequence 146–154 of MOG in MS patients
and controls.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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MS patients and controls were stratified for HLA-DRB1*1501. This resulted in four groups with 11 MS patients and 10 controls being HLA-DRB1*1501 positive, and 7 MS patients and 10 controls being HLA-DRB1*1501 negative. A total of 13 patients had a relapsing-remitting MS disease course with an average disease duration at a sample collection of 1 year and 6 mo (mean expanded disability status scale (EDSS) 2.0). Four patients had a secondary progressive disease course at sample collection with an average disease duration of 8 years (mean EDSS 5.0), and one patient had a primary progressive disease course with a duration of 7 years at sample collection (EDSS 6.5). Sixteen of 18 patients had not been treated at all or had not received any immunosuppressive or immunomodulatory treatment for at least 1 year before sample collection. Two of 18 patients had not received immunosuppressive or immunomodulatory treatment for 1 mo before sample collection. All patients were admitted to Department of Neurology of the University of Tubingen (Tubingen, Germany) due to acute relapse (relapsing remitting disease course) or fast worsening of neurological deficits (primary or secondary progressive disease course). The mean age of MS patients was 34 ± 9 years and of controls 38 ± 11 years, with a male to female ratio of 50:50% in MS patients and 55:45% in controls. The age distribution and female:male ratio was equal in the HLA-DRB1*1501 positive and negative groups. The studies had been approved by the ethical review board of the University of Tübingen.
HLA-typing
Genomic DNA for HLA-genotyping was prepared by the QIAampBlood kit (Qiagen, Hilden, Germany). Low resolution pregenotyping was performed for the 116 major HLA-DRB1, -DRB3, -DRB4, -DRB5, and 29 HLA-DQB alleles, and HLA-DR or HLA-DQ subtyping was done specifically for the pretyped HLA-DRB1/DQB1 alleles by group-specific amplification and subsequent direct sequencing in patients and controls.
Cloning and bacterial expression of recombinant human (rh)MOG 1–125
The cDNA of rhMOG was obtained by reverse transcription of total RNA from a human glioma cell line. RNA was prepared with TRIzol reagent (Life Technologies, Gaithersburg, MD) and first strand cDNA was synthesized using Moloney murine leukemia virus reverse transcriptase (Promega, Madison, WI) and the gene-specific primer R785. The amplified PCR product that contains the whole coding sequence of MOG mRNA was separated by agarose gel electrophoresis and purified with a QIAEXII-kit (Qiagen). The cDNA fragment was cloned into Bluescript II KS+ vector (Stratagene, La Jolla, CA). The DNA sequence encoding the extracellular domain of the mature protein (including four N-terminal amino acids of the transmembrane domain) was PCR-amplified and subcloned in pQ60 (Qiagen). The Escherichia coli-expressed His-tagged fusion protein (rhMOG 1–125) was purified under denaturing conditions by metal chelate affinity chromatography on Ni-NTA agarose columns (Qiagen) according to the manufacturer’s guidelines.
Synthesis and analysis of peptides
The 16 aa-long peptides (Table I)
and the N-acetylated C-amidated 9 aa-long peptides (Table II) were prepared by solid phase peptide
synthesis using F-moc/tBu chemistry. The peptides were purified by
preparative HPLC (Abimed, Langenfeld, Germany). The identity of the
purified peptides was confirmed by electrospray mass spectrometry. The
purity of peptides was >95% as determined by analytical HPLC
(Abimed). MBP 85–99 (ENPVVHFFKNIVTPR) and influenza A peptide
(YRNLVWFIKKNTRYP) (18) were synthesized and purified in
the same way. The peptides were used at a concentration of 10 µg/ml
in all experiments. This concentration had given the highest number of
spots in ELISPOT analyses in pilot experiments (data not shown).
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PHA was purchased from Sigma-Aldrich (Deisenhofen, Germany) and used in a concentration of 3 µg/ml in all experiments. This concentration had given optimal results in preceding experiments.
Purification of PBMC
PBMCs were isolated from heparinized blood samples on Lymphoprep density gradients (Nyegaard, Oslo, Norway; 200 x g, 25 min, room temperature). Cells were frozen at a density of 5 x 106 cells/ml in freezing medium containing 40% FCS Gold (PAA, Linz, Austria), 10% DMSO (Serva, München, Germany), and 50% complete medium (CM) consisting of RPMI 1640 (Life Technology, Eggenstein, Germany) supplemented with 2 mM glutamine (Life Technology), 100 U/ml of penicillin, 100 µg/ml of streptomycin (Biochrom, Berlin, Germany), and 3% heat-inactivated human AB serum in liquid nitrogen. The cells were recovered by thawing in a 37°C water bath until the cells reached the melting point and then by adding 1 ml CM per 5 x 106 cells three times every 5 min at room temperature. Thereafter, the cells were washed three times in CM and resuspended at a density of 106/ml in CM. The viability was 90–95%.
ELISPOT analysis for IFN--secreting cells and restriction
analysis
Ninety six-well nitrocellulose plates (Millipore, Molsheim,
France) were coated with 10 µg/ml capture mAb 1-D1K (Mabtech,
Stockholm, Sweden) overnight at 4°C. After washing, the membrane was
blocked with culture medium containing 10% FCS (FCS Gold, PAA) for
1 h. A total of 2 x 105 PBMCs/well
were cultured for 40 h in 37°C, 5% CO2.
For each Ag or mitogen, triplicates were used. Each plate contained a
positive and negative control. All peptides and mitogens were tested
for one patient or control at the same time point with exactly the same
procedures. After 40 h, the cells were discarded and the membranes
were thoroughly washed by immersing the plates six times in PBS. To
visualize areas of the membrane that had bound secreted IFN-,
biotinylated detector mAb 7-6B-1 (1 µg/ml, Mabtech) was added for
3 h and staining performed with avidin-biotin peroxidase complex
(Vectastatin Elite ABC kit; Vector Laboratories, Burlingame, CA) and
chromogen solution containing carbazole (Sigma-Aldrich). Areas of the
membrane where a specific color reaction had occurred appeared as dark
brown-red spots and were both counted by an automated ELISPOT counter
(Autoimmun-Diagnostika, Albstadt, Germany) and manually
cross-checked. The average number of spots in triplicates secreted
after exposure with Ag or mitogen were expressed as numbers of
IFN--secreting cells per 2 x 105 cells
added initially to the wells. For the restriction analysis, the HLA
typing Abs Genox with the specificity anti-HLA-DQ and/or Tü
36 with the specificity anti-HLA-DR were added at a concentration
of 10 µg/ml to the cultures. These concentrations of Abs had given
optimal results in pilot experiments (data not shown).
Enrichment of IFN--secreting cells and FACS analyses
Enrichment of cells secreting IFN- after contact with Ag was
performed with magnetic cell separation as described by the
manufacturer (IFN- secretion assay; Miltenyi Biotec, Bergisch
Gladbach, Germany). PBMC from MS patients and controls were incubated
with no Ag, MOG 146–154 (10 µg/ml), or as a positive control
staphylococcal enterotoxin B (Sigma-Aldrich, 10 µg/ml) for
12 h at a concentration of 5 x 106
cells/ml at 37°C, 5% CO2. Thereafter, cells
were exposed to a bivalent Ab to IFN- and leukocyte surface Ag
(Miltenyi Biotec) at 4°C resulting in an activity matrix for IFN-
on the cell surface. Subsequently, cells were reinduced to secrete
IFN- at 37°C, 5% CO2. Secreted IFN- is
captured by the activity matrix on the cell surface. Next, cells were
incubated with a PE-labeled Ab to IFN- (Miltenyi Biotec). Finally,
cells with bound IFN- on their cell surface were separated with a
PE-specific microbead (Miltenyi Biotec) in a magnetic field. Cells were
analyzed for expression of IFN- (mouse PE-labeled anti-human
IFN-; Miltenyi Biotec) and CD4 (mouse FITC-labeled anti-human
CD4; BD Biosciences, Lincoln Park, NJ) and/or CD8 (mouse
FITC-labeled anti-human CD8) expression following standard
procedures by FACS (BD Biosciences).
Peptide binding assay
Relative affinities of MOG peptides for purified HLA-DRB1*1501 and HLA-DRB1*0401 molecules were measured by an inhibition ELISA based on a dissociation-enhanced lanthanide fluoroimmunoassay (Wallac, Turku, Finland). In the inhibition ELISA, HLA-DR (50 nM) molecules were incubated with fixed amounts of respective tracer peptides (10–50 nM) in the presence of a range of dilutions of the unlabeled MOG-peptides (10-fold dilutions between 1 nM and 100 µM). The binding buffer consisted of a carbonate buffer titrated to pH 5 containing 2 mM EDTA, 0.01% azide, 0.1 mM PMSF, and 0.1% Nonidet P-40 (Boehringer Mannheim, Indianapolis, IN). After an incubation of 48 h at 37°C, the peptide-MHC complexes were transferred to Ab-coated (L243) ELISA plates (FluoroNunc; Nunc, Roskilde, Denmark) to remove the excess of nonbound peptides. Europium-labeled streptavidin (Wallac) was added to the plates and incubated for 1 h at room temperature. Finally, the plates were treated with an enhancement solution (Wallac), which releases chelated europium from streptavidin and forms a highly fluorescent solution that can be measured by using a dissociation-enhanced lanthanide fluoroimmunoassay fluorometer (Wallac). The peptide concentration yielding 50% inhibition of binding of the tracer peptide (IC50) was determined by plotting the percentage of inhibition vs the concentration of added MOG peptide. Peptides were tested in two to three independent experiments.
Statistical analysis
Repeated measures ANOVA on signed ranks were used with the Dunnett’s test (Sigma Stat; Jandel Scientific, San Rafael, CA). The Friedman repeated measures analysis of variance on ranks (repeated measures ANOVA on signed ranks) is a parametric test that compares effects of a series of different experimental treatments on a single group. Each subject’s responses are ranked from smallest to largest without regard to other subjects, then the rank sums for the subjects are compared. Dunnett’s test is the analog of the Student-Newman-Keuls test for the case of multiple comparisons against a single control group. It is conducted similarly to the Bonferroni t test, but with a more sophisticated mathematical model of the way the error accumulates to derive the associated table of critical values for hypothesis testing. This test is less conservative than the Bonferroni test, and is only available for multiple comparisons vs a control. This analysis was performed independently for each of the four groups (DRB1*1501 positive MS patients, DRB1*1501 positive controls, DRB1*1501 negative MS patients, DRB1*1501 negative controls). As controls, the individual background reactivities were used (T cell responses without Ag).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We stimulated PBMCs from MS patients and controls with 52
overlapping peptides covering the complete human MOG sequence (Table I
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or with the extracellular rhMOG 1–125 (mature peptide, aa 1–125) and
enumerated IFN--secreting cells with
the ELISPOT assay (Figs. 1 and 2). The T
cell responses as assessed by IFN- secretion toward peptides of the
extracellular part of MOG were weak and heterogeneous. HLA-DRB1*1501
positive controls showed responses to peptide MOG 81–96
(p < 0.05) (Fig. 2), while MS patients and
HLA-DRB1*1501 negative controls reacted weakly (NS). Some controls
reacted with MOG 73–88 (NS), but not MS patients (NS). Only a few
individuals had IFN--producing cells that reacted with rhMOG 1–125
(NS). Background values for nonstimulated cultures did not differ
significantly between MS patients and controls (NS) (data not
shown).
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Compared with the extracellular part of MOG, more reactive T cells
were present to the transmembrane and intracellular part of MOG in MS
patients and controls (Table I, Figs. 1 and 2). DRB1*1501 positive
controls reacted strongly to several peptides predominantly in the
region MOG 125–160 (peptides MOG 125–140, p < 0.05;
MOG 129–144, p < 0.05; MOG 133–148,
p < 0.05; MOG 137–152, p < 0.05; MOG
141–156, p < 0.05; and MOG 145–160,
p < 0.05), region MOG 169–188 (peptide MOG 169–184,
p < 0.05; MOG 173–188, p < 0.05),
peptide MOG 181–196 (p < 0.05), peptide MOG
189–204 (p < 0.05), and peptide MOG 201–216
(p < 0.05). In contrast, HLA-DRB1*1501
positive patients showed a focused response toward MOG region 141–160
(peptides MOG 141–156, p < 0.05; MOG 145–160,
p < 0.05). HLA-DRB1*1501 negative controls showed
responses to MOG peptide 145–160 (p < 0.05),
MOG region 169–188 (peptides MOG 169–184, p < 0.05;
MOG 173–188, p < 0.05), MOG peptide 189–204
(p < 0.05), and MOG peptide 201–216
(p < 0.05). Like HLA-DRB1*1501 positive MS
patients, HLA-DRB1*1501 negative patients had a focused response to MOG
region 141–160 (peptides MOG 141–156, p < 0.05; MOG
145–160, p < 0.05). MS patients and controls showed
quantitatively dominant reactivities to MOG region 141–160 (peptides
MOG 141–156 and MOG 145–160) with up to 423 IFN--secreting cells
per 2 x 105 PBMC (0.2%) (Fig. 1, individual quantitative data shown for one HLA-DRB1*1501 positive
patient). All investigated subjects showed T cell responses to PHA of
at least 1000 spots per 2 x 105 PBMC (data
not shown).
MOG 146–154 is the dominant T cell determinant
We analyzed the region within MOG 141–160 more in detail with
overlapping nine amino acid-long N-acetylated C-amidated
peptides (Table II, Fig. 3). The
N-acetylated nine amino acid-long peptides bind to MHC class
II molecules, but not to MHC class I molecules due to the
N-acetyl group. This analysis revealed a dominant T cell
response toward peptide MOG 146–154 in HLA-DRB1*1501 positive MS
patients and controls (both p < 0.05). There were
broader T cell reactivities in HLA-DRB1*1501 negative patients
(peptides MOG 144–152, p < 0.05; MOG 145–153,
p < 0.05; and MOG 146–154, p < 0.05)
and controls (peptides MOG 144–152, p < 0.05; MOG
145–153, p < 0.05; MOG 146–154, p <
0.05; and MOG 148–156, p < 0.05), indicating that the
T cell epitopes might be slightly shifted within MOG 141–160 depending
on the HLA molecules present. To exclude an influence of disulfide
bonds or posttranslational modifications of cysteine (C), we
investigated the T cell responses with a modified peptide containing
valine (V) instead of cysteine (C) in position 148
(Ac-LVFLCLQYR-NH2
Ac-LVFLVLQYR-NH2).
There was no decreased reactivity to this peptide in MS patients or
controls (data not shown).
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To exclude a generally decreased immune reactivity in MS patients,
we measured T cell reactivities to an influenza A peptide
(YRNLVWFIKKNTRYP) (18), with ELISPOT assay for IFN-
secreting cells. MS patients and controls reacted similarly to this
peptide arguing against a generally compromised immune status in MS
patients (NS) (data not shown).
Phenotypic analysis of MOG 146–154 reactive cells
To determine the phenotype of IFN--secreting MOG 146–154
reactive T cells, we performed enrichment of these cells and subsequent
FACS analysis for expression of CD4 or CD8 and IFN-. MOG
146–154-specific T cells expressing IFN- were enriched by the
IFN- enrichment assay in three MS patients (two HLA-DRB1*1501
positive, one HLA-DRB1*1501 negative) and one control (HLA-DRB1*1501
positive). The MOG 146–154-reactive IFN--secreting T cells were of
the CD4 phenotype (Fig. 4).
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The restriction pattern of MOG 146–154 reactive T cells was
assessed in a HLA-DRB1*1501 positive and a negative MS patient and a
HLA-DRB1*1501 positive control. The T cell response was HLA-DR
restricted in the investigated HLA-DRB1*0701 DRB1*15011 DQB1*02x
DQB1*0602 MS patient and in the HLA-DRB1*1103 DRB1*15011 DQB1*0301
DQB1*0602 control as well as in a HLA-DRB1*0701 DRB1*0407 DQB1*02x
DQB1*0301 MS patient, because only anti-HLA-DR Abs (Tü 36)
resulted in decreased T cell responses as compared with addition of
anti-HLA-DQ Abs (Genox) (Fig. 5).
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We assessed binding affinities of MOG 141–156, MOG 145–160, the
complete set of single stepped MOG peptides covering the region MOG
141–160, MBP 85–99, and influenza A peptide to purified HLA-DRB1*1501
and HLA-DRB1*0401 molecules. MOG 141–156 and MOG 145–160, as well as
influenza A peptide and MBP 85–99 bound to both alleles. The strength
of the binding for these peptides differed in both alleles with
HLA-DRB1*1501 binding all four peptides with a higher affinity as
indicated by a lower IC50 and a higher
1/IC50 (Fig. 6).
Large differences emerged in the binding patterns of the shorter
N-acetylated C-amidated nine amino acid-long stepped MOG peptides.
HLA-DRB1*1501 molecules bound all these peptides with low to high
affinity, depending on the peptide sequence (Fig. 6). In contrast,
HLA-DRB1*0401 only bound peptides MOG 144–152, MOG 145–153, and MOG
146–154 with a weak affinity (Fig. 6). Within the set of overlapping
single stepped nine amino acid-long MOG peptides, intermediate to high
affinity binding values were obtained for MOG 145–153, MOG 146–154,
MOG 148–156, MOG 150–158, and MOG 151–159 for HLA-DRB1*1501
molecules. The highest affinity values for HLA-DRB1*0401 molecules,
representing weak affinities, were obtained for MOG 144–152, MOG
145–153, and MOG 146–154. Taken together, this data indicates
allele-specific differences in the binding of MOG peptides to
HLA-DRB1*1501 and HLA-DRB1*0401 molecules with more peptides binding
with a higher affinity to HLA-DRB1*1501 molecules. MOG 146–154 had
similar to slightly better affinity than MBP 85–99 for HLA-DRB1*1501
or DRB1*0401 molecules. MBP 85–99 had been shown to be the major
HLA-DRB1*1501-restricted T cell epitope in MS patients, but is also
recognized by T cells in context with several other HLA-DR molecules
(19, 20, 21, 22, 23).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In animal models with marmosets, rats, and mice, the extracellular part
of MOG leads to a very MS-like disease (7, 24, 25, 26) (Fig. 7). In addition, Amor et al.
(25) investigated the encephalitogenic potential of
peptides derived from the transmembrane and intracellular part of MOG
in Biozzi AB/H and SJL mice. Our study is the first systematic study
investigating T cell responses in MS patients and controls to peptides
of the complete MOG sequence. Interestingly, besides the T cell
responses to MOG 81–96, only minor T cell responses to the
extracellular part of MOG were detectable. In contrast, several
stretches of the transmembrane/intracellular domain of MOG proved to be
highly immunogenic. We defined an immunodominant T cell epitope
encompassing MOG 146–154 with N-acetylated and C-amidated
nine amino acid-long peptides that can bind to MHC class II molecules.
There was no major HLA-guided influence on the selection of T cells
arguing for promiscuous presentation of this MOG peptide on MHC class
II molecules. In the tested MS patients and controls, the MOG
146–154-specific T cell response was HLA-DR restricted. We assessed in
vitro binding affinities of MOG peptides spanning MOG sequence 141–160
to two purified HLA-DR molecules. Both HLA-DRB1*1501 as well as
HLA-DRB1*0401 molecules bound MOG 146–154 to a varying degree allowing
presentation of this peptide to T cells. Interestingly, HLA-DRB1*1501
molecules bound more of the nine amino acid-long MOG peptides within
the sequence MOG 142–160 compared with HLA-DRB1*0401 molecules. Both
alleles bound MOG 146–154 slightly better than MBP 85–99 that is the
major HLA-DRB1*1501-restricted MBP stretch in MS patients
(19, 20, 21, 22, 23).
Our results of promiscuous recognition of MOG 146–154 by T cells in context of several MHC class II molecules are partly in line with T cell reactivity to MBP in MS patients and controls. The immunodominant MBP 84–102 and MBP 87–106 peptides and their core sequence MBP 89–99 are recognized in the context of several MHC molecules in man as well as in mice and rats (19, 20, 21, 22, 23, 27). This MBP stretch induces experimental autoimmune encephalomyelitis (EAE) in HLA-DR2 transgenic mice (28). In future studies, our laboratories will assess the immunogenic and encephalitogenic potential of intracellular MOG determinants in animal models.
Epitope mapping studies in humans with regard to myelin-Ags have shown
conflicting results as far as reactivity patterns of T cell responses
in diseased individuals compared with controls are concerned. Some
studies have shown that there is an increased frequency of autoreactive
T cells from blood in diseased individuals and that T cell reactivity
is increased in MS patients, while others recorded negative findings in
regard with disease-associated differences in blood (12, 23, 29). Especially IFN--secreting cells have been shown to be
elevated in diseased individuals in blood and cerebrospinal fluid
(30). Our study is partly contradictory to these
observations because controls showed even more T cell reactivities in
blood ex vivo compared with MS patients. Importantly, we did not find a
generally compromised immune status in regard to PBMC reactivity in MS
patients that would have explained this finding, because T cells from
blood reacted similar to an influenza A peptide in MS patients and
controls (18).
We did not observe the same reactivity profile to the extracellular
part of MOG as Wallström et al. (12) had
demonstrated with MOG 63–87 being immunodominant. Reasons might lie in
differences in the genetic background of the patients and the preceding
Ag exposure with effects on the T cell repertoire in the Swedish
compared with the German MS population (31). Additionally,
compared with the explicit strength of the T cell responses to MOG
81–96 in some MS patients and controls and intracellular MOG in our
study, in this preceding study the T cell responses as assessed by
IFN- ELISPOT were very weak with 3–4 spots in mean per 1 x
105 PBMC to the immunodominant MOG 63–87. In
this study, beside MOG 63–87 also MOG 76–100 was slightly more
recognized by MS patients compared with controls (1–2 spots per 1
x 105 PBMC) (12). The peptide MOG
79–96 has been shown to induce severe disease in DBA/1
(H-2q) mice (32) and mild disease in
LEW.1AV1 (RT1av1) and LEW.1N
(RT1n) rats (26). Interestingly, we
did not find T cell responses in humans to peptides MOG 89–104, MOG
93–108, MOG 97–108, and MOG 97–112. These peptides contain the major
encephalitogenic MOG stretches MOG 92–106 in SJL/J
(H-2s) mice (25), MOG 97–108 in
HLA-DRB1*0401 transgenic mice (33), and MOG 91–108 in
different inbred rat strains, bearing the RT1a,
RT1av1, and RT1n
haplotypes, respectively (26). Nor did we detect strong T
cell responses to peptides MOG 33–48 and MOG 37–52 that contain the
encephalitogenic MOG determinant MOG 35–55 of C57BL/6
(H-2b) and NOD/LT (H-2g7)
mice (6). In Biozzi AB/H (H-2dq1)
mice, MOG 134–148 induced mild signs of EAE in one of five mice
(25). The same authors did not detect encephalitogenic
sequences in Biozzi AB/H (H-2dq1) and SJL
(H-2s) mice to overlapping peptides covering MOG
141–218 (25). The data underscores the need to evaluate
encephalitogenic MOG responses in humanized animal models additionally
to inbred mouse and rat strains (34).
In MOG-induced EAE in LEW.1N rats, we have recently shown that MHC class II-regulated CNS autoaggression and T cell responses in peripheral lymphoid tissues are dissociated (26). The major encephalitogenic MOG 91–108 peptide in LEW.1N rats did not induce a detectable proliferative response and T1 or T2 T cell response in lymph nodes or spleen ex vivo after active immunization with MOG 91–108 or MOG 1–125. Instead, in the target tissue, the CNS, strong cellular, and cytokine responses were present after immunization with this peptide (26). In contrast, there were several determinants within MOG 1–125 that did raise strong T cell responses in peripheral lymphoid tissue, but did not induce disease (26). These data point to the need to investigate disease-associated cellular responses within the target organ (35). Unfortunately, investigations of the intra-CNS immune response in humans are difficult to perform because the availability of cerebrospinal fluid is limited and there are a number of ethical problems.
There was a focusing of the T cell reactivity patterns toward MOG
141–160 in MS patients. The decreased number of T cell determinants in
MS patients could reflect alteration of the T cell repertoire by
preceding Ag exposure after damage of the blood brain barrier leading
to a state of peripheral T cell tolerance to certain MOG determinants
(36). Moreover, migration of encephalitogenic T cells in
MS patients to the target organ and reduction of the size of the T cell
pool in blood reactive with MOG cannot be excluded at present (3, 37). Such a scenario would argue for a pathogenic role of the T
cell reactivities that are absent or reduced in the blood of MS
patients compared with controls in the disease process and again forces
investigations at the target organ site. Alternatively, other
functional differences might exist between the MOG reactive T cells
secreting IFN- of MS patients and controls. Moreover, regulatory
mechanisms could be up-regulated in MS patients after establishment of
disease reducing the reactivity profile of T cells to MOG
determinants in blood and lymphoid organs like up-regulation of TGF-
(38) or increased NK cell reactivity
(39).
Our data indicate that during thymic selection, MOG 146–154
cross-reactive T cells must be positively selected on several HLA class
II molecules and not deleted. In EAE, it has been recently shown that
intrathymic expression of myelin components can lead to tolerance
(40). Compared with other proteins of the myelin sheath,
the expression of MOG is low because it comprises only 0.01% of
the protein content of the myelin sheath (6). To date, no
expression of human MOG on non-CNS tissue including the thymus has been
reported, but it is not excluded that MOG is expressed at a very low
level in the thymus. Potentially, this absent or low level expression
would allow escape of MOG-specific T cells from negative selection in
the thymus (40). The extracellular part of MOG shows
homology to butyrophilins and B7 family members that are expressed in
the thymus (41). In rats, butyrophilins have been shown to
be cross-reactive with the extracellular part of MOG (42).
In contrast, structural similarities between self-proteins and the
transmembrane or intracellular domain of MOG have not been described to
the same degree as to the extracellular part of MOG. As a consequence,
central tolerance mechanisms might result in more effective negative
selection of T cells reactive to the extracellular part of MOG compared
with the intracellular part. This would explain the high number of
IFN--secreting cells against the intracellular part of MOG present
in the periphery.
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Robert Weissert, Department of Neurology, University of Tübingen, Hoppe-Seyler-Strasse 3, 72076 Tübingen, Germany. E-mail address: robert.weissert{at}uni-tuebingen.de
3 Abbreviations used in this paper: MS, multiple sclerosis; CM, complete medium; EAE, experimental autoimmune encephalomyelitis; EDSS, expanded disability status scale; MBP, myelin basic protein; MOG, myelin oligodendrocyte glycoprotein; rhMOG, recombinant human MOG.
Received for publication June 18, 2001. Accepted for publication April 22, 2002.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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signaling in T cells leads to spontaneous T cell differentiation and autoimmune disease. Immunity 12:171.[Medline]
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  B. Greve, R. Simonenko, Z. Illes, A. Peterfalvi, N. Hamdi, M. P Mycko, K. W Selmaj, C. Rozsa, K. Rajczy, P. Bauer, et al. Multiple sclerosis and the CTLA4 autoimmunity polymorphism CT60: no association in patients from Germany, Hungary and Poland Multiple Sclerosis, March 1, 2008; 14(2): 153 - 158. [Abstract] [PDF]
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 T. Burster, A. Beck, E. Tolosa, P. Schnorrer, R. Weissert, M. Reich, M. Kraus, H. Kalbacher, H.-U. Haring, E. Weber, et al. Differential Processing of Autoantigens in Lysosomes from Human Monocyte-Derived and Peripheral Blood Dendritic Cells J. Immunol., November 1, 2005; 175(9): 5940 - 5949. [Abstract] [Full Text] [PDF]
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 I. M. Pomeroy, P. M. Matthews, J. A. Frank, E. K. Jordan, and M. M. Esiri Demyelinated neocortical lesions in marmoset autoimmune encephalomyelitis mimic those in multiple sclerosis Brain, November 1, 2005; 128(11): 2713 - 2721. [Abstract] [Full Text] [PDF]
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 M. M. Herrmann, S. Gaertner, C. Stadelmann, J. van den Brandt, R. Boscke, W. Budach, H. M. Reichardt, and R. Weissert Tolerance induction by bone marrow transplantation in a multiple sclerosis model Blood, September 1, 2005; 106(5): 1875 - 1883. [Abstract] [Full Text] [PDF]
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