Brain, Vol. 122, No. 11, 2089-2100,
November 1999
© 1999 Oxford University Press
Invited review |
Multiple sclerosis: B- and T-cell responses to the extracellular domain of the myelin oligodendrocyte glycoprotein
1 Department of Neuroimmunology, Max Planck Institute for Neurobiology, Martinsried, 2 Department of Neurology and Institute for Clinical Neuroimmunology, Klinikum Grosshadern, Ludwig Maximilians University, Munich, Germany
Correspondence to:
Dr C. Linington, Department of Neuroimmunology, Max Planck Institute for Neurobiology, Am Klopferspitz 18A, D-82152 Martinsried, Germany E-mail: lining{at}neuro.mpg.de
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Abstract |
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We report a comparative study of the B- and T-cell responses to the extracellular immunoglobulin (Ig)-like domain of human myelin–oligodendrocyte glycoprotein (MOGIgd) in the blood of patients with multiple sclerosis and healthy controls using a bacterial recombinant human protein (rhMOGIgd). The frequency of anti-rhMOGIgd-seropositive samples, as determined by Western blotting, was significantly higher in the multiple sclerosis group (54%) than in normal random controls (excluding laboratory workers exposed to MOG) (22%; P = 0.02). In contrast, there was no difference in rhMOGIgd-induced proliferation indices of peripheral blood T cells between patients and controls. To characterize the rhMOGIgd-reactive T-cell repertoire, we isolated a panel of MOG-specific CD4+ T-cell lines from multiple sclerosis patients and normal subjects, and these revealed a heterogeneous response with respect to epitope specificity, cytokine response, MHC (major histocompatibility complex) restriction and T-cell receptor Vß-chain usage. The majority of the T-cell lines recognized epitopes in the N-terminal region of MOG (amino acids 1–60). One epitope (represented by peptide 27–50) was exclusively recognized by T-cell lines from normal controls. Forty per cent of the MOG-specific T-cell lines analysed displayed a Th-2 or Th-0 cytokine profile and could therefore act as helper T cells in vivo.
multiple sclerosis; autoantibodies; myelin–oligodendrocyte glycoprotein (MOG); T lymphocytes
CD = cluster of differentiation; EAE = experimental autoimmune encephalomyelitis; FACS = fluorescence-activated cell sorter; HLA = human leucocyte antigen; IFN = interferon; Ig = immunoglobulin; IL = interleukin; mAb = monoclonal antibody; MBP = myelin basic protein; MHC = major histocompatibility complex; MOG = myelin–oligodendrocyte glycoprotein; MOGIgd = immunoglobulin-like domain of MOG; PBMC = peripheral blood-derived mononuclear cell; pCP = primary chronic progressive; rh = recombinant human; rr = recombinant rat; RR = relapsing–remitting; sCP = secondary chronic progressive; TCR = T-cell receptor
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Introduction |
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The pathogenesis of multiple sclerosis involves multiple genetic and environmental factors that interact to disrupt immunological self-tolerance to CNS myelin. In susceptible individuals this results in a chronic inflammatory response in the CNS associated with demyelination, axonal loss and ultimately the formation of gliotic scar tissue. Identification of the autoantigens and immune effector mechanism responsible for this pathology is essential for the future development of diagnostic and therapeutic strategies for multiple sclerosis. One of several candidate autoantigens is the myelin–oligodendrocyte glycoprotein (MOG) (Wekerle et al., 1994
), which was initially identified as the dominant target for autoantibody-mediated demyelination in experimental autoimmune encephalomyelitis (EAE) (Lebar et al., 1986).
MOG is a CNS myelin-specific, type I membrane protein encoded within the telomeric region of the major histocompatibility complex (MHC) distal to the MHC F locus (Gardinier et al., 1992; Pham-Dinh et al., 1995). The mature protein is preferentially incorporated into the outermost surface of the myelin sheath (Linington et al., 1988; Brunner et al., 1989), where a single immunoglobulin (Ig)-like domain (MOGIgd) is exposed to the extracellular environment (Kroepfl et al., 1996). This domain is unique in that it is the only protein structure known to induce both demyelinating autoantibody and encephalitogenic T-cell responses in EAE (Linington et al., 1993; Amor et al., 1994; Genain et al., 1996). Synergy between the immune effector mechanisms triggered by these responses results in a chronic/relapsing disease associated with extensive CNS demyelination in rats and primates (Linington and Lassmann, 1987; Adelmann et al., 1995; Johns et al., 1995; Genain et al., 1995; Genain and Hauser, 1997; Weissert et al., 1998; Storch et al., 1998b). Moreover, demyelination in these models is characterized by patterns of myelin vesiculation and immunoglobulin/neoC9 deposition that reproduce those seen in a subset of multiple sclerosis patients (Lucchinetti et al., 1996, 1998; Lassmann, 1998; Storch et al., 1998a, b; Genain et al., 1999; Raine et al., 1999).
Elevated autoreactive MOG-specific T-cell (Sun et al., 1991a; Kerlero de Rosbo et al., 1993, 1997; Wallström et al., 1998) and B-cell responses (Sun et al., 1991a; Xiao et al., 1991) in multiple sclerosis have been described previously by several groups. However, as this is also the case for the autoimmune response to other myelin proteins in multiple sclerosis, the relevance of these observations with respect to disease pathogenesis is uncertain. In the present study we investigated the B- and T-cell responses to human MOGIgd in the blood of patients with multiple sclerosis and of healthy controls, using a highly purified recombinant human protein preparation (rhMOGIgd) expressed in Escherichia coli.
We report the novel observation that although anti-MOGIgd autoantibodies are significantly more frequent in multiple sclerosis patients than in healthy controls, this is not accompanied by a corresponding proliferative T-cell response. However, analysis of MOGIgd-specific CD4+ T-cell lines by intracytoplasmic staining for interferon (IFN)-
and interleukin (IL)-4 revealed the presence of both Th-1 and Th-2 clones in the immune repertoire of both multiple sclerosis patients and healthy controls. Epitope mapping demonstrated that, although the specificity of this response was heterogeneous, there was a clustering of epitopes in the N-terminal region of the protein. The presence of MOGIgd-specific Th-1 and Th-2 T cells together with MOGIgd-specific autoantibodies has obvious implications for the future development of immunotherapies for multiple sclerosis.
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Material and methods |
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Cell culture
Peripheral blood-derived mononuclear cells (PBMC) were isolated from EDTA (ethylene diamine tetraacetic acid)-buffered blood on a discontinuous density gradient (Lymphoprep, Nycomed, Oslo, Norway) (Boyum, 1974
). PBMC were cultured in RPMI 1640 supplemented with 5% pooled human AB serum, 1% glutamine (0.2 M) and 1% penicillin/streptomycin (10 000 IU/ml penicillin/10 000 µg/ml streptomycin) and incubated at 37°C in 5% CO2 (standard protocol). IL-2 (kindly donated by M. Gately and J. Sepinwall, Hoffmann/La Roche, Nutley, NJ, USA) was added to primary cultures and T-cell lines at final concentrations of 7.5 and 30 U/ml, respectively. Murine L cells were cultured in RPMI 1640 supplemented with 10% FCS (foetal bovine serum), 1% glutamine (0.2 M) and 1% penicillin/streptomycin.
Patients and healthy controls
Peripheral blood was taken with informed consent every 2–3 weeks from five healthy controls and five patients with relapsing–remitting (RR) multiple sclerosis for isolation of MOG-reactive T-cell lines. All of these donors were typed for human leucocyte antigens (HLA) (Professor E. Albert, Laboratory for Immunogenetics, Ludwig Maximilians University, Munich, Germany). Comparative analysis of the peripheral T- and B-cell responses to rhMOGIgd was performed in 37 healthy controls and 37 multiple sclerosis patients [10 primary chronic progressive (pCP), 10 secondary chronic progressive (sCP), 12 RR patients and five patients with a first episode of neurological symptoms and laboratory and MRI features consistent with multiple sclerosis]. None of the patients had received either immunosuppressive or immunomodulatory treatment during the last 6 months, or corticosteroids during the last 6 weeks prior to blood sampling.
Antigens
Myelin basic protein (MBP) was purified from human brain according to standard protocols (Eylar et al., 1979). The purity of MBP was assessed by gel electrophoresis. Recombinant rat S100ß was expressed and purified as described elsewhere (Schmidt et al., 1997). rhMOGIgd (amino acids 1–125) was expressed in E. coli and purified by nickel-chelate affinity chromatography on chelating Sepharose Fast Flow (Pharmacia, Uppsala, Sweden) in 6 M urea (Brehm et al., 1999) followed by preparative SDS (sodium dodecyl sulphate) gel purification to remove endotoxin and other bacterial contaminants. Homogeneity was assessed by SDS–PAGE (polyacrylamide gel electrophoresis) followed by silver staining. Protein concentration was determined using Peterson's modification of the micro-Lowry method (Sigma-Aldrich, Deisenhofen, Germany). Antigens were stored at –20°C. The synthetic MOG peptides were purchased from Neosystem (Strasbourg, France) with the sequences shown in Table 1. The peptide purity was assessed by HPLC (high-pressure liquid chromatography) analysis and mass spectrometry.
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Isolation of MOG-reactive long term T-cell lines
PBMC (2 x 105/well) were cultured in 96-well round-bottom microtitre plates (Nunc, Roskilde, Denmark) and challenged with rhMOGIgd (15 µg/ml) or the set of MOG peptides (10 µg/ml each). Medium containing IL-2 (15 U/ml) was added to the cultures every 4–5 days. After 4 weeks, rhMOGIgd-reactive cells were identified using a modification of the split-well technique (Meinl et al., 1993
). Briefly, irradiated (50 Gy) autologous PBMC were used as antigen-presenting cells. Antigen-presenting cells (1 x 105/well) were pre- incubated with rhMOGIgd (15 µg/ml), a recombinant control protein (15 µg/ml) or without antigen as a negative control. Each well of the primary culture was then split into three wells containing the preincubated antigen-presenting cells. MOG reactivity was assessed microscopically for 3 days. Specific proliferative aggregates were further expanded in the presence of IL-2 (final concentration 30 U/ml), then restimulated every 16–21 days with either rhMOGIgd or MOG peptides using irradiated autologous PBMC as antigen- presenting cells and fed with medium containing IL-2 (60 U/ml) every 3–4 days.
Proliferation assays
Freshly isolated PBMC were cultured in 96-well microtitre plates (2 x 105/well) for 5 days. The following antigens were added at the beginning of the culture in triplicate: rhMOGIgd (15 µg/ml), MOG peptides (10 µg/ml each), MBP (20 µg/ml), recombinant control antigen [recombinant rat (rr) S100ß, 15 µg/ml], tetanus toxoid (2 µg/ml; Behringwerke, Marburg, Germany), purified protein derivative from tuberculin (2 µg/ml; Statens Institute, Copenhagen, Denmark) and phytohaemagglutinin (2 µg/ml; Sigma, Deisenhofen, Germany). [3H]Thymidine (0.2 µCi) was added during the last 18 h of culture. Cells were harvested and [3H]thymidine incorporation was measured using a direct ß-counter (Matrix TM 96 Direct Beta Counter, Packard, Frankfurt, Germany) that yielded ~20% of counts obtained by liquid scintillation counting. The criteria for a positive response were set at a stimulation index >3 with an absolute value of >1000 c.p.m. Antigen specificity of the T-cell lines was determined using irradiated (50 Gy) autologous PBMC (1 x 105/well) preincubated in the presence or absence of antigen in 96-well microtitre plates. T-cell line cells (1 x 105/well) were added to duplicate wells and [3H]thymidine (0.2 µCi) was added after 48 h. Cells were harvested after 66–68 h of culture and [3H]thymidine incorporation was measured as described above.
MHC restriction of MOGIgd-reactive T-cell lines
The MHC restriction of MOGIgd-specific T-cell lines was determined using blocking monoclonal antibodies (mAb) to HLA-DR (L243, ATCC, Rockville, Md., USA), HLA-DP (B7/21, Biodesign, Kennebunk, Me., USA) and HLA-DQ (TÜ169, Pharmingen, Hamburg, Germany). Anti-MHC class I mAb (W6/32, ATCC) was used as a control. Irradiated (50 Gy) autologous PBMC (1 x 105/well) were preincubated in the presence or absence of the above mAb (20–25 µg/ml final concentration) in duplicate wells for 30 min. T-cell line cells (1 x 105/well) were added, and after an incubation period of 30 min the MOG peptide recognized by the particular line (10 µg/ml) was added to the wells. Labelling and harvesting were performed as described for proliferation assays. To further define the restriction of the T-cell lines, T-cell line cells (1 x 105/well) were incubated with irradiated (40–60 Gy) human HLA-DR-transfected mouse fibroblasts (L cells; 2 x 104 cells/well) in the presence of peptide (10 µg/ml) for 68 h and proliferation was determined as described above.
Characterization of T-cell lines by flow cytometry
T-cell lines were phenotypically characterized by fluorescence-activated cell sorter (FACS) scanning (FACScan, Becton Dickinson, Heidelberg, Germany). Unlabelled mouse mAb (final concentration 1–2 µg/ml) to CD3, CD8 (DAKO, Hamburg, Germany) and CD4 (Becton Dickinson) and a mouse IgG1 isotype control (Becton Dickinson) were visualized with a FITC (fluorescein isothiocyanate)-labelled goat anti-mouse mAb (1–2 µg/ml; DAKO). The T-cell receptor Vß region expressed was analysed using mAb (1–2 µg/ml) recognizing the following subfamilies: Vß2, Vß7, Vß8, Vß9, Vß11, Vß12, Vß13.1, Vß13.6, Vß14, Vß16, Vß17, Vß18, Vß20, Vß21.3, Vß22, Vß23 (Immunotech, Marseille, France), Vß6.7, Vß7.1 (Labgen, Frankfurt, Germany), Vß3.1, Vß5.1, Vß5.2, Vß5.3 and Vß8.1 (T-Cell Diagnostics, Woburn, Mass., USA).
The intracellular cytokine profiles of the T-cell lines in response to stimulation were also characterized by FACS. MOGIgd-reactive T-cell line cells (1 x 105/well) were stimulated according to standard protocols [PMA (phorbol-12-myristate-13-acetate) 5 µg/ml, ionomycin 250 ng/ml] for 3 h, the last 2 h in the presence of monensin (2 nmol/ml). T cells were then washed, fixed, permeabilized and labelled for IFN- (FITC, 1 µg/ml; Pharmingen, Hamburg, Germany) and IL-4 [PE (phycoerythrin), 1 µg/ml; Pharmingen] or the appropriate isotype controls (negative control R-PE, 1 µg/ml, DAKO; negative control FITC, 1 µg/ml, Immunotech).
Immunoblotting
Antibody responses were determined using standard Western blot techniques with rhMOGIgd on Hybond-ECL (enhanced chemiluminescence) membranes (Amersham, Braunschweig, Germany). Previous studies have confirmed that this technique identifies both conformation-dependent and -independent antibody responses to MOGIgd (Brehm et al., 1999). After SDS–PAGE and blotting, nitrocellulose membranes were cut into 3- to 5-mm strips containing 1.5 µg MOGIgd per strip and then blocked with 5% low-fat milk powder in Tris-buffered saline containing 1% Tween 20 (milk-TBST). The strips were then incubated with plasma diluted 1 : 500 in 2% milk-TBST in mini-incubation trays (Bio-Rad, Munich, Germany) for 1 h at room temperature. Preliminary studies established that this plasma dilution provided the most discriminatory signal-to-noise ratio for the detection of the anti-MOGIgd response. A goat anti-human IgG + IgM [heavy and light (H and L) chains] coupled to horseradish peroxidase (Dianova, Hamburg, Germany) was used as secondary antibody (100 ng/ml in 2% milk-TBST) and incubated with washed strips for 45 min. The mouse monoclonal anti-MOG IgG1 antibody, purified from hybridoma 8–18C5 (Harlan Sera-Lab, Crawley Down, UK), was used as a positive control (5–50 µg/ml in 2% milk-TBST). Goat anti-mouse IgG + IgM (H and L) coupled to horseradish peroxidase (1 mg/ml; Dianova) was used at a dilution of between 1 : 4000 and 1 : 10 000 in 2% milk-TBST. Antibody binding was detected by enhanced chemiluminescence using Hyperfilm-ECL according to the manufacturer's instructions (Amersham). Immunoreactivity of the coded plasma samples was assessed by three independent observers.
Statistical analysis
ANOVA (analysis of variance) was performed using the SPSS statistics package for Windows for the stimulation indices. Western blot data were analysed with SPSS using non-parametric statistical (
2) tests.
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Results |
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Anti-MOGIgd antibody responses and rhMOGIgd-induced T-cell proliferation in multiple sclerosis patients and healthy controls
We investigated the B- and T-cell responses to MOG in 37 healthy controls and 37 multiple sclerosis patients (Table 2
). The proportion of anti-MOGIgd antibody-positive samples was significantly higher in the multiple sclerosis patient group (54%) than in the random controls (excluding laboratory workers exposed to MOG) (22%; P = 0.023). Anti-MOGIgd antibodies were also detected in three of five laboratory workers who regularly handled recombinant MOG preparations. Dividing the group of multiple sclerosis patients according to clinical course revealed that the highest proportion of anti-MOGIgd antibody-positive samples was found in the group with a first clinical manifestation (5/5; P < 0.01 compared with the random control group), followed by those with a RR course (8/12; P = 0.01) and with an sCP course (5/10, P = 0.08). Among multiple sclerosis patients with a pCP disease course, the number of anti-MOGIgd-antibody- seropositive donors (2/10) was not significantly different from that seen in the random control population (Table 2; P = 0.47).
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In contrast to the differences we observed in the anti-MOGIgd antibody response, we did not detect any significant difference between the multiple sclerosis patients and healthy controls with respect to the T-cell response in terms of the stimulation index obtained using either rhMOGIgd or synthetic MOGIgd-peptides. Furthermore, there was no difference between the proliferative response to rhMOGIgd, the recombinant control antigen (rrS100ß) or MBP in individual assays (Fig. 1
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Peptide fine specificity of MOG-reactive T-cell lines
In the absence of a proliferative response to either rhMOGIgd or the panel of MOGIgd-peptides in PBMC, we generated a panel of antigen-specific T-cell lines to further characterize the rhMOGIgd-specific T-cell response. The age, sex, clinical status and HLA type of the donors are listed in Table 3
. Table 4 shows the peptide specificity of the total of 27 MOG-reactive T-cell lines that were isolated from PBMC of five healthy controls and two patients with RR multiple sclerosis. The majority of the T-cell lines were isolated from cultures stimulated with rhMOGIgd (healthy controls: n = 16/20; multiple sclerosis: n = 5/7; indicated in Table 4 by the letter M after the donor's initials), but other MOG-specific T-cell lines were isolated from cultures stimulated with the pool of synthetic peptides (indicated by the letter P after the donor's initials). T-cell lines that did not exhibit a response to both the recombinant antigen and one or more peptides were discarded. Attempts to isolate MOG-reactive T-cell lines from three further RR multiple sclerosis patients, one of whom started immunomodulating treatment with IFN-ß during the investigation, were unsuccessful.
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All the T-cell lines, irrespective of whether they were selected with protein or peptide, were specific for MOG and did not respond to control recombinant antigen (Fig. 2
), and all but four of the lines exhibited a stimulation index >10 when confronted with the appropriate peptide (Table 4
). Epitope mapping revealed that the majority of the T-cell lines (21/27) responded to only one of the nine overlapping peptides. The remainder exhibited multiple specificities, which in some cases involved adjacent peptides, e.g. the response to peptides P3 and P4 (see Table 1) by the T-cell line CL-M1, suggesting that the response could be attributed to a single epitope within the overlapping amino acid sequence. In general there was no difference in the peptide specificity of the T-cell lines derived from healthy controls and of those derived from multiple sclerosis patients. The majority of T-cell lines recognized peptide epitopes present in the N-terminal sequence of MOG (amino acids 1–60). Intriguingly, despite the broad range of MHC haplotypes provided by this panel of donors (Table 3), no T-cell lines were isolated that responded to the peptide sequences 50–87 or 89–125. It was also noted that a response to peptide 3 (amino acids 27–50) was detected only in T-cell lines derived from the control population (Table 4).
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Cytokine production by MOG-reactive T-cell lines
In EAE induced in a non-human primate by sensitization with MOG, clinical disease can occur in association with a Th-2-like T-cell response, which may enhance the production of MOG-specific autoantibodies (Genain et al., 1996
). We therefore investigated the synthesis of IFN- and IL-4 by the MOGIgd-reactive T-cell lines for evidence of a similar cytokine pattern that may favour antibody production.
We were able to obtain sufficient cells for this study from 15 of the MOGIgd–reactive T-cell lines obtained from five of the donors. The T-cell lines were first expanded by several rounds of MOGIgd–specific restimulation followed by expansion in IL-2-containing medium. The T cells were then stimulated with PMA and ionomycin according to standard protocols to detect cytokine synthesis by intracellular FACS analysis. Only T-cell lines with >50% of T cells producing detectable amounts of IFN- and/or IL-4 after stimulation were taken into account. The T-cell lines were designated Th-1, Th-0 or Th-2 according to the predominant T-helper type (Table 5).
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The majority of the T-cell lines (60%) were of the Th-1 subset in that they synthesized only IFN-
. A further four T-cell lines consisted of a dominant population of Th-0 T cells co-expressing both IFN- and IL-4, whilst one line (CL-M36) contained similar percentages of Th-0 and Th-2 T cells. Only one T-cell line (ASt-M10C) expressed exclusively IL-4 and could therefore be assigned to the Th-2 T-cell subset. Interestingly, six out of 12 T-cell lines from the three anti-MOGIgd antibody-seropositive donors (HK, CL, ASt) produced significant amounts of IL-4 after stimulation (Th-0 or Th-2 subset responses), but no IL-4 production was seen in the three lines obtained from the seronegative donors (HW, TN).
MHC restriction and T-cell receptor Vß expression of MOG-reactive T-cell lines
FACScan identified all of the T-cell lines generated in this investigation as members of the CD4+, 
/ß TCR T-cell subset (data not presented). Restriction analysis of 14 T-cell lines using the anti-MHC antibody blocking assay demonstrated that the proliferation of six T-cell lines was inhibited by 
80% and in four other lines by ~50% by anti-HLA-DR mAb. No inhibition was seen in the presence of mAb to HLA-DP, -DQ or MHC class I, which was used as a control (Fig. 3). The proliferation of four T-cell lines could not be inhibited by any of the anti-MHC class II mAb that were available, but for two of these lines the response was shown to be restricted by DRB3*0202 (DR52) in the L-cell presentation assay.
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Analysis of surface TCR expression by these T-cell lines using the available panel of TCR Vß-specific mAb positively identified the surface expression of TCR Vß 8.1, 13.1, 17, 20 and 22 by eight T-cell lines. However, the sample size is too small to make any comment as to the possibility of preferential usage of any particular TCR Vß chain in the context of the MOG-specific T-cell response (Table 6
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Discussion |
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TopAbstractIntroductionMaterial and methodsResultsDiscussionReferences |
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In the present study we investigated the T- and B-cell responses to the extracellular Ig-like domain of human MOG (MOGIgd) in the blood of patients with multiple sclerosis and in healthy controls. The results demonstrate that MOGIgd-specific CD4+ helper T cells are represented in the immune repertoire of multiple sclerosis patients and normal subjects. Consistent with this observation, we found MOGIgd-reactive autoantibodies in a small proportion (22%) of random healthy controls. However, anti-MOG autoantibodies were significantly more frequent (54%) in multiple sclerosis patients.
A pathogenic role for CNS antigen-specific T cells in multiple sclerosis is suggested by findings in EAE studies, in which the adoptive transfer of T cells specific for several different antigens induces an inflammatory response in the CNS (Wekerle et al., 1994; Berger et al., 1997). The human T-cell response to autoantigens such as MBP, proteolipid protein and S100ß, which have been defined as being encephalitogenic in EAE, has been studied extensively (Martin et al., 1992; Hafler et al., 1996; Kondo et al., 1996; Greer et al., 1997; Schmidt et al., 1997; Stinissen et al., 1997; Trotter et al., 1997, 1998; Tuohy et al., 1997). In all cases, autoreactive T cells specific for these antigens are a component of the normal, healthy immune repertoire, an observation that can now be extended to include MOG. However, we were unable to confirm recent reports that MOG induces a specific proliferative T-cell response in primary PBMC cultures derived from multiple sclerosis patients but not from healthy controls (Kerlero de Rosbo et al., 1993, 1997).
Out of the entire panel of samples analysed, only one donor exhibited a stimulation index >3 in response to pooled synthetic MOG peptides. The sensitivity of primary proliferation assays is limited, however, and may be insufficient to detect the expansion of autoreactive T cells in PBMC cultures. Analysis of the frequency of T cells responding to myelin autoantigens by ELISpot suggests that this may indeed be the case, as this technique does detect increased T-cell responses to MOG in multiple sclerosis patients. However, this reflects a generalized enhancement of the myelin-specific autoimmunity rather than a selective response to MOG (Sun et al., 1991a, b; Fredrikson et al., 1992; Olsson et al., 1992; Wallström et al., 1998). It should also be noted that in this study we analysed only the response to the Ig domain of MOG and cannot exclude the possibility that immunodominant T-cell epitopes of MOG are located within the transmembrane or cytoplasmic domains of the protein. It is also important to consider that the `activation status' of the T cells at the time of isolation is unknown. Obviously, T cells anergic to MOG would not be detected in the current proliferation assay. Moreover, preactivated MOG-reactive T cells might undergo apoptosis when challenged with antigen in vitro. These mechanisms may explain why we were unable to isolate MOG-reactive T-cell lines from three of the multiple sclerosis patients (BM, MH and SS), one of whom (SS) was undergoing IFN-ß treatment.
Although no significant primary T-cell response to MOGIgd could be demonstrated, it was possible to select MOGIgd- reactive T-cell lines from the PBMC of both multiple sclerosis patients and healthy controls, giving us some insight into the nature of the MOG-specific T-cell repertoire. All the T-cell lines selected using recombinant MOGIgd in vitro recognized one or more of a panel of nine overlapping synthetic MOGIgd peptides, with a bias towards epitopes located within amino acid sequences 1–26 and 27–50, which together account for ~60% of the identified peptide specificities. Peptide 63–87, recently reported to be immunodominant in HLA-DR2-positive multiple sclerosis patients assessed by ELISpot (enzyme-linked immunoabsorbent spot assay) analysis of PBMC (Wallström et al., 1998), did not stimulate any of the MOGIgd-reactive T-cell lines (Table 4). This does not necessarily contradict the previous study (Wallström et al., 1998), since the number of T-cell lines from HLA-DR2-positive individuals analysed in our study was relatively small. Unfortunately, not all of the MOG/peptide-reactive T-cell lines could be fully characterized with respect to TCR Vß expression and MHC restriction, either because the necessary anti-TCR Vß mAb and human MHC class II-transfected L cells were unavailable, or because the number of these short-term T-cell lines was insufficient for all assays. Nevertheless, the T-cell lines were clearly heterogeneous with respect to all the parameters tested, and in this regard they resemble MBP-specific T-cell lines (Meinl et al., 1993).
In contrast to the absence of any factor that distinguished the MOGIgd-specific T-cell repertoire in multiple sclerosis from that seen in normal controls, we noted that the frequency of anti-MOGIgd antibody-seropositive donors was far higher in multiple sclerosis patients than in normal controls (Table 2). The high frequency of anti-MOG antibody responses in multiple sclerosis patients is consistent with a retrospective study that analysed a large panel of paired serum and CSF samples from multiple sclerosis patients and disease controls (Reindl et al., 1999). In the present study, fewer patients classified as having a primary progressive course of disease were seropositive for anti-MOG antibodies compared with patients having either first lapse/RR or an sCP course. This might support the hypothesis of a different pathogenetic background between these groups of patients (Thompson et al., 1997). Longitudinal studies are currently in progress to determine whether this actually reflects a loss of the anti-MOG response during the course of multiple sclerosis, or differentiates pathologically distinct subsets of progressive disease.
In the present study, the sample numbers are too small to determine whether or not the T-cell repertoire of anti-MOGIgd antibody-positive donors is associated with any specific cytokine profile. However, the three T-cell lines available from anti-MOGIgd antibody-negative donors (TN and HW) produced IFN- and no IL-4, whereas six T-cell lines obtained from three antibody-positive donors (multiple sclerosis patient HK and healthy controls CL and ASt) synthesized IL-4, either exclusively or together with IFN-. This observation is preliminary, but suggests a functional correlation between the presence or absence of anti-MOGIgd antibody and antigen-specific IL-4 synthesis. However, it will be necessary to generate a far larger set of MOGIgd-reactive T-cell lines from both anti-MOGIgd antibody-positive and -negative donors to confirm this hypothesis.
The functional significance of MOG-reactive T- and B-cell responses in the pathogenesis of multiple sclerosis is uncertain. In animal models, a MOG-specific Th-1 T-cell response can initiate an inflammatory response in the CNS (Linington et al., 1993; Amor et al., 1994; Kerlero de Rosbo et al., 1995), whereas anti-MOG autoantibodies are implicated in demyelination (Adelmann et al., 1995; Genain et al., 1995). Reports of receptor-mediated phagocytosis of myelin associated with the capping of Ig on the macrophage surface (Prineas and Graham, 1981), deposition of Ig and neoC9 on the myelin surface (Storch et al., 1998a) and the presence of MOG-specific autoantibodies in active multiple sclerosis lesions (Genain et al., 1999; Raine et al., 1999) are consistent with the hypothesis that demyelination, at least in a subset of patients, is antibody-mediated in multiple sclerosis (Lucchinetti et al., 1996).
It remains to be determined whether or not the presence of anti-MOG antibodies in multiple sclerosis is a primary or secondary response in disease pathogenesis. MOGIgd is highly immunogenic and will induce EAE at doses of >1 µg in the rat (Weissert et al., 1998). We were therefore perturbed to note that three of five laboratory workers routinely exposed to the protein were seropositive. We therefore strongly recommend that precautions should be taken to limit exposure to MOGIgd during its purification and use in the laboratory. In view of its immunogenicity, the anti-MOGIgd antibody response could be either a secondary event resulting from intermolecular determinant spreading during lesion formation, or it could be initiated by molecular mimicry with environmental agents prior to disease onset (Wucherpfennig and Strominger, 1995; Hemmer et al., 1997). The latter concept is supported by the observation that the anti-MOGIgd autoantibody response is not specific for multiple sclerosis, as 22% of the healthy controls were also seropositive. The factor(s) provoking this response are unknown, but the high percentage of responders in the control population indicates the involvement of a common environmental agent that affects a large population base.
Currently the anti-MOG antibody assay is qualitative and the development of a reliable anti-MOG antibody ELISA is a prerequisite to define the fine specificity and isotype usage of this antibody response in different subsets of patients and controls. This may eventually provide a serological marker to identify pathogenic anti-MOG antibody responses, allowing rational therapeutic choices to be made with respect to treatments that target the humoral arm of the immune response, such as plasmapheresis and intravenous Ig (Fazekas et al., 1997). Conversely, this would also provide a rational basis for the avoidance of therapeutic strategies that may favour B-cell activation (Hohlfeld, 1997). The harmful consequences of this were clearly seen in the marmoset model of MOG-induced EAE, in which high-dose soluble antigen treatment induced a high-titre antibody response that ultimately proved lethal to the treated animals (Genain et al., 1996).
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Acknowledgments |
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The authors thank M. Sölch for excellent technical assistance and Dr C. Farima and M. Kerschensteiner for helpful suggestions. This work was supported by the EC Biomed 2 program (contracts BMH4–97–2027 and BMH4-CT96–0893), Deutsche Forschungsgemeinschaft (SFB217, C13 and C14) and Hertiestiftung (GHS 2–339–95). R.-B.L. and C.G.H. hold postdoctoral fellowships from the Deutsche Forschungsgemeinschaft. The Institute for Clinical Neuroimmunology and C.L. are supported by the Hermann and Lilly Schilling Foundation.
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Received March 19, 1999. Revised May 5, 1999. Accepted May 15, 1999.
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