HLA class II transgenic mice authenticate restriction of myelin oligodendrocyte glycoprotein-specific immune response implicated in multiple sclerosis pathogenesis
Meenakshi Khare1,
Moses Rodriguez1,2 and
Chella S. David1
Departments of 1 Immunology and 2 Neurology, Mayo Clinic, Rochester, MN 55905, USA
Correspondence to: M. Rodriguez; E-mail: rodriguez.moses{at}mayo.edu
Transmitting editor: G. Hammerling
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Abstract
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Myelin oligodendrocyte glycoprotein (MOG) is a potential target antigen of the central nervous system (CNS), known to induce autoreactive T cell response and demyelinating anti-MOG antibodies in multiple sclerosis (MS) patients. Association of HLA class II genes with MS is well established. To better understand the role of HLA class II molecules in disease pathogenesis, we generated transgenic mice that express HLA-DR2, -DR3, -DR4, -DQB1*0601, -DQB1*0604 and -DQ8 without mouse class II (Aß0). We have for the first time characterized the T and B cell epitopes of human MOG restricted by different HLA class II molecules. Immunization with recombinant MOG (rMOG) generated a strong CD4+ T cell-mediated response in an HLA class II-restricted manner. Cytokine analysis revealed an increase in pro-inflammatory (IFN-
, IL-12 and IL-6) and anti-inflammatory (IL-10) cytokines. T cell autoreactivity to MOG was directed against peptides 120, 3150, 6180 and 91110, of which three are also immunodominant epitopes for MOG in MS. A strong B cell response to MOG was observed in all transgenic mice, and major B cell epitopes recognized were located within amino acids 130, 5180 and 101120 of human MOG, which consists of two epitopes reported in MS. Transgenic mice used in this study recognized the immunodominant MOG epitopes similar to HLA class II-restricted human T cells, and would therefore be valuable in elucidating the roles of HLA class II genes and autoantigens in MS.
Keywords: autoimmunity, experimental autoimmune encephalomyelitis, HLA class II, multiple sclerosis, myelin oligodendrocyte glycoprotein, transgenic/knockout mice
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Introduction
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Multiple sclerosis (MS) is a chronic inflammatory and demyelinating disease of the central nervous system (CNS), and is characterized by a prominent infiltration of macrophages and lymphocytes in CNS lesions and by demyelination. Experimental autoimmune encephalomyelitis (EAE), a model for MS, is mediated by CD4+ T cells and can be induced experimentally in susceptible strains of laboratory animals by immunization with CNS antigens such as myelin basic protein (MBP) or proteolipid protein (PLP) as well as by adoptive transfer of activated CD4+ T cells specific for myelin antigens in appropriate adjuvant (13). Recent studies demonstrated that a quantitatively minor myelin protein, myelin oligodendrocyte glycoprotein (MOG), is also strongly encephalogenic in mice (46), rats (7,8), marmosets (911) and rhesus monkeys (12), which is highly relevant to MS. About 50% of MS patients have peripheral blood mononuclear cells (PBMC) directed towards MOG rather than MBP or PLP (13,14). Therefore, MOG is now widely recognized as a important potential target antigen in MS pathogenesis.
MOG is a minor component of CNS myelin, which is exposed on the surface of the outermost lamellae of the myelin sheath (15). It is a 218-amino-acid glycoprotein with an extracellular Ig-like domain encompassing amino acids 1125, which is encoded within the distal region of MHC in humans, mice and rats (16,17), and therefore closely linked to gene loci associated with MS. MOG is the only identified myelin auto-antigen that triggers not only an encephalogenetic T cell response, but can also induce a pathogenic demyelinating auto-antibody response in rodents and primates. This is in contrast to other myelin antigens, such as PLP and MBP, which are encephalogenetic, but unable to induce a demyelinating antibody response. Synergy between the immune effector mechanisms triggered by these responses results in a chronic/relapsing disease associated with extensive antibody-dependent CNS demyelination in rats and primates (9,18,19), which reproduces the immunopathology found in MS (13,14,2022). An abnormal increase in the proliferative response against MOG has been reported in PBMC of patients with MS (14,20). It has recently been demonstrated that the autoimmune response to MOG in MS is directed against three main epitopes, amino acids 122, 3456 and 6496 (13). Anti-MOG antibodies are found in the serum and cerebrospinal fluid of MS patients (2123), and peptide specific anti-MOG antibody responses were observed against amino acids 126 and 6387 (24).
Susceptibility to MS, although multifactorial, is markedly influenced by the MHC/HLA class II genotype (25,26). The association of MS with the HLA class II haplotypes is heterogeneous in different populations. The strongest associations of MS are with haplotype HLA-DR2 (DRB1*1501, DRB5*0101 and DQB1*0602), detected in Caucasians, northern European populations as well as in Ashkenazi and non-Ashkenazi Jews, Turkish and Mexican Mestizo patients (2731). In contrast, HLA-DR3 (DRB1*0301-DQA1*0501-DQB1*0201) and -DR4 (DRB1*0405-DQA1*0501-DQB1*0301) haplotypes are associated with MS in the Sardinian population (32,33). In relative terms, the contribution of HLA genes to disease is in following order: HLA-DR2, -DR3 and -DR4. Although these associations have been known for some time, the molecular or biochemical basis for such HLA associations remains unclear. HLA class II molecules function at the level of both positive and negative selection for the T cell repertoire in thymus. HLA-DQ and -DR molecules also select and present peptides to CD4+ T cells in the periphery, and may play a role in MS through the presentation of myelin-specific peptides to pathogenic T cells. There are indications that different courses of MS are influenced by the HLA genotype (34,35). However, it is not clear yet how the presence of a specific HLA-DR or -DQ molecule contributes to increased disease susceptibility.
The identification of the immunodominant epitopes of target autoantigens, which are presented by different HLA molecules, is the key step towards delineating the role of MHC class II molecules in disease, as well as providing reagents for MS diagnosis and possible therapy. Such efforts are difficult in human patients due to the paucity of circulating autoreactive T cells in the peripheral blood and the difficulty in long-term maintenance of human T cell clones. In addition, the presence of multiple HLA molecules in each individual makes determining MHC restriction elements difficult. Currently available animal models do not fully reflect the responses seen in MS patients, because they lack human MHC molecules. To overcome these problems, we generated transgenic mice that express HLA-DR2, -DR3, -DR4, -DQB1*0601, -DQB1*0604 and -DQ8 molecules without endogenous class II molecules. Thus, the only functional class II molecules on antigen-presenting cells are the human class II molecules. The T cell repertoire in these mice is shaped by the human HLA class II molecules, such that all CD4+ T cells are restricted by human HLA class II molecules, thus making them a good model to study the role of HLA class II genes in human disease (36,37).
We studied the reactivity pattern and activation state of MOG-specific T cells in transgenic mice using T cell proliferation assays and IFN-
ELISPOT assays by using a panel of overlapping 20mer synthetic peptides covering the entire extracellular region of human MOG (amino acids 1170). We also defined the immunodominant as well as cryptic T and B cell epitopes for MOG, and demonstrated that these epitope are presented in an HLA class II-restricted manner. Moreover, several of these epitopes have also been found to elicit T and B cell responses in MS patients.
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Methods
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Transgenic mice
The production and characterization of transgenic mice expressing HLA-DR2 (DRA1*0101, DRB1*1502), -DR3 (DRA1*0101, DRB1*0301), -DR4 (DRA1*0101, DRB1*0401), -DQ6 (DQA1*0103, DQB1*0601 and DQA1*0103, DQB1*0604) and -DQ8 (DQA1*0301, DQB1*0302) genes have been described previously (3840). The transgenes for HLA class II transgenic mice were introduced initially into (B6 x SWR)F1 mice. These mice were then backcrossed to B10.M mice for several generations and carry B10 background, but are not fully congenic. The HLA transgenic mice were then mated to class II-deficient (Aß0) mice and intercrossed to generate these lines. Transgene negative littermates were used as controls. Thus, the different transgenic lines have similar background genes, but are not congenic. The presence of the transgenic ß and
chains as well as the Eß chain were first determined by PCR, and expression on the cell surface was determined by FACS analysis using the antibodies IVD12, L227, Y-17 and 14-4-4 (more details are included below). Mice were bred and maintained in the pathogen-free Immunogenetics Mouse Colony at the Mayo Clinic (Rochester, MN). All procedures performed on the mice were in accordance with the Mayo Institutional Animal Care and Use Committee.
Antigens
Recombinant MOG (rMOG) corresponding to the extracellular domain of rat MOG (amino acids 1125) was prepared as described previously (4). Briefly rMOG was expressed in Escherichia coli strain DH5
(generously provided by Dr C. Linington, Max Plank Institute, Germany) and purified by nickel chelate affinity chromatography on chelating Sepharose Fast Flow in 6 M urea by using a continuous imidazol gradient (40500 mM) followed by dialysis against acetate buffer (pH 3.0) and stored at 20°C. SDSPAGE and Western blot confirmed the purity of rMOG. The homology between human and rat MOG and human and mouse MOG is 96%. For fine mapping of MOG epitopes, a panel of 16 overlapping 20mer (overlap by 10 amino acids, Table 1) peptides encompassing the sequence of human MOG (amino acids 1170) was synthesized at the Peptide Core Facility of the Mayo Clinic (Rochester, MN) using an automated 430A peptide synthesizer (Applied Biosystems, Foster City, CA).
mAb
Culture supernatants from the cell lines producing mAb specific for HLA-DQ
chain (IVD 12), HLA-DR (L227), H2E-ß (Y-17), H2E-
(14-4-4), H2A-
(7-16-17), H2A-ß (25-5-16), CD4 (GK1.5) and CD8 (53.6.72) were prepared in our laboratory.
T cell proliferation assay
Mice were immunized with 200 µg of rMOG or individual peptide emulsified in complete Freunds adjuvant (CFA; Difco, Detroit, MI) at the base of the tail and in each hind limb footpad. Ten days post-injection mice were sacrificed. Draining lymph nodes (inguinal, poplitical and para-aortic) were removed and lymph node cells (LNC) were prepared for in vitro cultures in the presence or absence of relevant antigens as described previously (41). T cell proliferative responses were assessed using [3H]thymidine incorporation. Briefly, 1 x 106 cells/well were cultured in triplicate in 96-well flat-bottom tissue culture plates (Costar, Cambridge, MA) in 0.2 ml of RPMI supplemented with 5% horse serum, 2 mM L-glutamine, 100 µg/ml streptomycin and 100 U/ml penicillin in the presence of rMOG (20 µg/ml) or individual peptide (50 µg/ml), with concanavalin A or medium alone. After 48 h of culture plates were pulsed with 1 µCi [3H]thymidine and harvested 18 h thereafter for scintillation counting. The results were expressed as change (
) in c.p.m. calculated as
c.p.m. = (mean c.p.m. of triplicate antigen-containing cultures) (mean c.p.m. of triplicate medium-containing cultures) and as stimulation index (SI) = mean c.p.m. of antigen-containing cultures/mean c.p.m. of medium-containing cultures. For in vitro inhibition studies, specific mAb (20 µl of culture supernatant/well) were added to LNC challenged in vitro with rMOG.
Cytokine profile determination
For in vitro detection of cytokine production, LNC were cultured at a concentration of 5 x 106 cells/ml in 24-well plates (Costar, Cambridge, MA) with 20 µg/ml of rMOG or medium only at 37°C in the analogous culture medium with supplements that were used for proliferation assays. Supernatants were harvested after 72 h of culture and stored at 70°C. The concentrations of IL-4, IL-6, IL-10, IL-12p70 and IFN-
were measured by sandwich ELISA using matched antibody pairs and standards according to the guidelines of manufacturer (Genzyme Diagnostics, Cambridge, MA). Briefly, flat-bottom 96-well Immulon II ELISA plates (Dynatech, Chantilly, VA) were coated with anti-cytokine capture antibody in coating buffer and incubated overnight at 4°C. After blocking for 1 h, the plates were washed and 100 µl of each sample or standard dilution was added to the wells in duplicate for 2 h at room temperature. After washing, biotinylated detection antibodies were added to wells for 1 h. Horseradish peroxidase-conjugated streptavidin (Jackson ImmunoResearch, West Grove, PA) was used as a detection reagent followed by 3,3,5,5'-tetramethylbenzidine (Sigma, St Louis, MO) as substrate. Plates were monitored at 450 nm using a Model 3550 Bio-Rad ELISA reader. Cytokine concentrations were determined by comparing the OD of samples to the appropriate standard curves using the Microplate Manager software for the Macintosh computer (Bio-Rad, Hercules, CA).
IFN-
ELISPOT assay
IFN-
ELISPOT assay was performed according to the manufactures guidelines. Briefly, the ELISPOT plates (Millipore, Bedford, MA) were coated overnight with 10 µg/ml capture antibody for IFN-
(XMG1.2; eBioscience, San Diego, CA) diluted in PBS. The plates were washed with sterile PBS and blocked with complete RPMI 1640. Draining LNC from immunized mice were plated at a concentration of 2 x 105 cells/well in triplicates in medium alone or with 20 µg rMOG or with concanavalin A and incubated for 24 h at 37°C. Subsequently the cells were removed by washing and plates were incubated with biotinylated detecting antibody R4-6A2 (eBioscience) at 1 µg/ml for 2 h at room temperature followed by 30 min incubation with streptavidinhorseradish peroxidase diluted in 1:1000 in assay dilutent. A substrate solution containing 3-amino-9-ethylcarbazole (Sigma Aldrich, St Louis, MO; 4 µg/ml dissolved in 1 ml of dimethylformamide) in 14 ml of 0.1 M phosphate citrate buffer, pH 5.0, was used for developing plates. The plates were air-dried and spots were counted using a dissecting microscope (Leica, Deerfield, IL). The number of cytokine-secreting cells was calculated by subtracting the number of spots in control wells (medium only) from the number of spots in the presence of stimulation.
Serum ELISA
Mice were immunized with 200 µg of rMOG in CFA with 4 mg/ml of Mycobacterium tuberculosis s.c. in flanks. Mice were injected with pertussis toxin (200 ng/mouse) at day 0 and 2. Peripheral blood of mice was taken by tail bleeding, and after coagulation serum was obtained by centrifugation and stored at 20°C. For ELISA the 96-well Immulon ELISA plates were coated with 12.5 µg/ml of rMOG or synthetic peptides spanning the extracellular domain of MOG overnight at 4°C. The plates were washed with PBS containing 0.05% Tween 20 and blocked with 2% BSA in PBS at room temperature for 1 h. The plates were then incubated with 100 µl of diluted test serum overnight at 4°C. Specific binding was detected with alkaline phosphatase-conjugated goat anti-mouse IgG (Jackson ImmunoResearch). The reaction was visualized by using p-nitrophenylphosphate (Southern Biotechnology Associates, Birmingham, AL) as substrate and after 20 min the reaction was stopped with 1 N NaOH. The absorbance at 405 nm was read on an ELISA reader. Serum antibody levels were quantified by comparison to purified standards added to each plate.
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Results
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All transgenic mice showed a strong proliferative response with rMOG
Preliminary experiments involving priming and challenging transgenic mice with varying doses of rMOG revealed that an in vivo priming dose of 200 µg/mouse and an in vitro challenging dose of 20 µg/ml elicited the maximum [3H]thymidine incorporation (data not shown). Under these conditions all transgenic mice showed a strong proliferation response with rMOG (Fig. 1). No in vitro proliferation was detected in transgene-negative control mice that were also deficient in mouse class II MHC. Similar responses were observed when T cell reactivity was measured against rMOG in these transgenic mice using IFN-
ELISPOT assay (Fig. 2).

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Fig. 1. T cell proliferative responses against rMOG in HLA transgenic mice. Mice (three per group) were immunized with 200 µg of rMOG in CFA in the base of tails and hind footpads. Ten days later lymph node cells were harvested and cultured in presence of 20 µg/ml rMOG/well or medium alone. Proliferation was determined with [3H]thymidine incorporation and is expressed as the SI (see Methods). Data presented as the mean of triplicate cultures ± SD. All the transgenic mice showed strong proliferative response with rMOG. Using the same conditions no responses were found in transgene negative Aß0 mice.
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In vitro proliferative response of T cells with rMOG is mediated by CD4+ T cells and is restricted to HLA class II molecules
CD4+ T cells are known to be encephalogenetic in EAE models. Thus in order to verify that these are the cells responsible for the proliferation of T cells against rMOG in transgenic mice, anti-CD4 (GK1.5) or anti-CD8 (53-6.72) mAb were added to the cultures. As shown in Fig. 3(A and B), proliferation was inhibited with anti-CD4 mAb by 8095% in all transgenic mice. This would indicate that CD4+ T cells mediated the response. However, there was no significant inhibition of proliferation with anti-CD8 mAb, even though CD8+ T cells are also known to be encephalogenetic in some mouse models (42). The addition of L227 (anti-HLA-DR mAb) and IVD12 (anti-HLA-DQ mAb) resulted in the inhibition of proliferative response in DR and DQ transgenic mice respectively. No significant inhibition was observed in cultures containing control mAb specific for mouse H2A-
(7-16-17) and H2A-ß (25-5-16) (data not shown). Thus, in vitro responses in HLA transgenic mice to rMOG are mediated by CD4+ and HLA-restricted T cells.

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Fig. 3. CD4+ T cells mediate the in vitro response to rMOG and is HLA restricted. LNC from rMOG-immunized mice were challenged with rMOG in the presence of the indicated mAb. The data represents the percentage inhibition of [3H]thymidine incorporation in cultures with rMOG in three representative experiments. (A) HLA-DR mice. (B) HLA-DQ mice.
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Both Th1 and Th2 cytokines were generated following rMOG stimulation
It has been suggested that, in the development and progression of immune-mediated diseases such as EAE and MS, the secretion of particular cytokines by autoreactive T cells play an important role. To study the effect of HLA transgenes on cytokine profiles, culture supernatants from LNC of HLA-DR and DQ transgenic mice were assayed for the presence of IL-4, IL-6, IL10, IL-12p70 and IFN-
cytokines with or without rMOG. In vitro culture of LNC stimulated with rMOG showed a higher amount of IFN-
with rMOG in HLA-DR2, -DR4 and -DQB1*0604 mice, and a moderate amount of IFN-
in HLA-DR3, -DQB1*0601 and -DQ8 mice (Fig. 4). High amounts of IL-12 were generated in most transgenic mice in response to rMOG. IL-6 was expressed at high levels after treatment with rMOG of LNC from all transgenic mice. Minimal amounts of IL-4 were generated from rMOG-stimulated cultures in all mice except DQB1*0601 mice, which produced a higher amount of IL-4. Expression of IL-10 was high in all transgenic mice. Thus, both Th1 and Th2 cytokines were produced after rMOG stimulation, although the levels of Th1 cytokines were higher than Th2 cytokines in most of the mice.

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Fig. 4. Cytokine production by LNC of HLA transgenic mice in response to immunization with rMOG. Mice (three per group) were immunized with 200 µg of rMOG in CFA in the base of tail and hind footpads. Ten days later draining lymph node cells were harvested and cultured in the presence of rMOG or medium alone for 72 h. Pooled culture supernatants were then analyzed for the presence of cytokines using capture ELISA. Results are mean of cytokine concentration for one of two representative experiments.
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Human MOG T cell epitopes are recognized by HLA transgenic mice
Peptide specific mapping for autoantigens may provide a clue to the role of specific HLA molecules in T cell repertoire selection. To identify the T cell epitopes of human MOG, that are naturally processed and presented by HLA class II molecules, we immunized HLA transgenic mice with rMOG as described above. The draining LNC were challenged in vitro with either rMOG or synthetic overlapping peptides. HLA-DR2 mice responded to several peptides in the three different amino acids regions, 120, 3170 and 91110 (Fig. 5A). These regions contain the epitope 6387, which is found to be immunodominant in humans, and the epitopes 3555 and 92106, which are immunogenic and encephalitogenic in several strains of mice and rats. In DR3 mice the response was observed against four main regions of the MOG peptides, i.e.130, 4160, 71100 and 141160 (Fig. 5B). In contrast, peptides 3160 and 81110 were recognized by HLA-DR4 mice (Fig. 5C). All HLA-DQ transgenic mice responded to peptide 81100 (Fig. 5D, E and F). In addition DQB1*0601 mice showed responses to epitope 131150 (Fig. 5D), while DQB1*0604 mice showed a response to T cell epitopes throughout the extracellular domain of rMOG (Fig. 5E). DQ8 mice responded also to epitopes 6180 and 121160 (Fig. 5F).

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Fig. 5. T cell epitopes of human MOG recognized by HLA transgenic mice. Mice (three per group) were immunized with 200 µg of rMOG in CFA in the base of tail and hind footpads. Draining LNC from primed transgenic mice were challenged in vitro with 50 µg/ml of each of the overlapping peptides. Control samples were cultured in medium alone. Proliferation was determined with [3H]thymidine incorporation and represented as the means of SI for the triplicate cultures of three representative experiments. Under the same conditions, transgene-negative Aß0 were unresponsive to any of the peptide.
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To identify immunodominant as well as cryptic determinants, we also immunized mice with 200 µg of individual peptides and LNC were challenged in vitro with 50 µg/ml of the relevant peptide. The concentration of peptide for immunization and in vitro challenge were based on previous experiments. DR2 mice responded to several peptides in the N-terminal region of human MOG between amino acids 1 and 90 (Fig. 6A). HLA-DR3 mice recognized peptides in a set of three epitopes, 120, 4160 and 6180 (Fig. 6B). HLA-DRB1*0401 recognized not only all the peptides recognized by DR3 mice, but also peptides 91110 and 111130 (Fig. 6C). Epitopes 120, 6180 and 91110 were recognized by all HLA-DQ mice (Fig. 6DF). DQB1*0601 mice showed a unique response to epitope 91110 (Fig. 6D), whereas DQB1*0604 and DQ8 mice also responded against several other epitopes (Fig. 6E and F). We found that all six transgenic mice showed strong responses to epitopes 120, 3150 and 6180, suggesting that these epitopes are the common epitopes recognized by all HLA-DR and -DQ alleles.

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Fig. 6. In vitro responses of HLA transgenic mice to overlapping peptides of human MOG. Purified LNC from transgenic mice primed with 100 µg of each overlapping human MOG peptide were challenged in vitro with 50 µg/ml of same peptide or medium alone. Data represented as means of SI for triplicates in three representative experiments.
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These results also indicate that, although transgenic mice recognized T cell epitopes throughout the extracellular domain of MOG, they also recognized one or more of the immunodominant or encephalogenetic epitopes known for MS or EAE.
Anti-MOG antibody response and B cell epitope mapping in transgenic mice immunized with rMOG
Serum was collected by tail bleeding the mice weekly after 2 weeks of immunization. Anti-MOG antibody response was detected by capture ELISA using alkaline phosphatase-conjugated goat anti-mouse IgG. A high titer of anti-MOG IgG antibody was detected in all the transgenic mice (Fig. 7), whereas no positive anti-MOG antibody response was measured in Aß0 mice. Antibodies were detected at 2 weeks post-immunization, reached a plateau after 4 weeks post-immunization and were present after 12 weeks post-immunization (data not shown).

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Fig. 7. Antibody responses against rMOG in HLA transgenic mice. Transgenic mice were immunized s.c. in the flanks with 200 µg of rMOG in CFA. Sera were obtained from mice after 4 weeks post-immunization. Antibody directed against MOG was analyzed using ELISA as described in Methods. The serum dilutions were 1:200 for MOG-specific IgG. Non-specific binding to BSA was subtracted from all the values to calculate the mean absorbance (OD) ± SD. Strong responses were found in all transgenic mice.
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The epitope specificity of anti-MOG antibodies in transgenic mice was detected by analyzing sera from mice using a set of overlapping 20mer peptides, spanning the N-terminal extracellular part of MOG (amino acids 1170). The main reactivity of anti-MOG antibodies in all mice was directed against three separate regions, 130, 5180 and 101120, with the exception of DQB1*0604 mice, that only showed activity against epitope 81100 (Fig. 8AF). No antibody reactivity was detected in pre-immune sera or mice immunized with CFA only, indicating that the immune response was specific for MOG. We found no significant differences in epitope specificity of MOG peptides in relation to specific DR or DQ molecules. These results indicate that B cell epitopes for MOG are located within these three regions.

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Fig. 8. B cell epitope mapping of MOG in transgenic mice immunized with rMOG. Sera were obtained from mice 28 days after immunization and pooled. A panel of overlapping peptides spanning the extracellular region of MOG was used to measure specific reactivity by ELISA. Serum was diluted to 1:200 with PBS. Data is presented as means of two experiments. Sera from all mice reacted with peptides within amino acids 130, 5180 and 101130, except in DQB1*0604 mice, which showed reactivity to only peptide 81100.
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Discussion
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The most important finding in this study is the identification of several immunodominant T and B cell epitopes of human MOG restricted to HLA class II molecules. These epitopes are also found in MS patients, suggesting that our transgenic mice reflect human class II recognition of MOG epitopes. A unique advantage of this transgenic system is that human HLA class II genes restrict all the CD4+ T cells in these animals. Thus, these transgenic animals offer a model to study antigen-specific T and B cell responses in the context of only HLA class II genes. The expression of disease-associated HLA class II transgenes in Aß0 mice has been shown to confer susceptibility to experimentally induced autoimmune diseases, such as autoimmune thyroiditis (43) and arthritis (38). We demonstrate that HLA transgenic mice elicit a strong in vitro T cell response to rMOG, whereas no response was found in the transgene-negative class II knockout mice. Inhibition of MOG-specific proliferation by anti-CD4 antibody and anti-HLA class II (anti-DR and -DQ) antibody indicates that the T cell response in these mice is mediated by CD4+ T cells in an HLA class II-restricted manner. These results were further confirmed by ELISPOT assay. Preliminary studies in these mice showed that HLA-DR2 mice developed severe EAE associated with large inflammatory foci and extensive demyelination when injected with rMOG. Mild disease was seen in HLA-DR3 and -DR4 mice, whereas no disease was observed in HLA-DQ mice (manuscript in preparation).
Increased auto-reactive MOG-specific T cell (13,14,20,44) and B cell responses (20,45) in MS have been described previously. The approach of using HLA transgenic mice as a means to identify T cell epitopes may have the advantage of identifying a broader range of immunogenic T cell epitopes. Moreover, peptide-specific mapping for autoantigens provides a conclusive analysis for the role of specific HLA molecules in T cell repertoire selection in MS. Immunization and in vitro challenge of these mice with rMOG or MOG peptides showed that peptides 120, 3150, 6180 and 91110 induced a T cell response in most of the HLA transgenic mice. A recent study with HLA-DR4 mice showed that epitope 91110 was immunodominant as well as encephalogenic (46). These T cell epitopes are also immunodominant in rodents and marmosets (46,13). Primary responses to panels of overlapping synthetic peptides that span the extracellular domain of MOG in MS patients revealed immunodominant recognition of the epitopes 122, 3456 and 6496 (13). Another study showed that peptide 6387 induced the strongest response in a group of DR2+ MS patients (44). The specificity of rMOG-reactive T cell lines derived from rhesus monkeys with different MHC backgrounds identified peptides 420, 3550 and 94116 as the three major epitopes of MOG (12). In rodent models of MOG-induced EAE, these amino acid regions of MOG are encephalogenic and immunogenic in several strains of mice(46) and in Lewis rats (7) (Table 2).
In MOG-induced EAE, it is speculated that Th2 cytokines play a role in disease severity, recovery and remission (47,48), and that a shift from a Th1 to Th2-type immune response will exacerbate disease symptoms (10). In our studies we have found that both Th1 (IFN-
, IL-6, IL-12) and Th2 (IL-10) cytokines were elevated, consistent with several reports, demonstrating pro-inflammatory and anti-inflammatory cytokines in MOG-induced EAE (49). In most forms of EAE, the Th1 cells or Th1 cytokines promote disease, while Th2 cells or cytokines down-regulate disease. In MOG-induced EAE, however, both the cellular and the humoral response are essential for the complete manifestation of the disease, suggesting that Th2 cells or cytokines can play an important role in disease progression. Preliminary studies in our laboratory for disease induction in these transgenic mice suggested good correlation between the cytokine expression and disease similar to that found in MS patients (studies currently in progress).
The antibody response to MOG is likely to play an important role in MOG-induced EAE and is highly relevant in demyelinating models (9,10,50,51). In MS, anti-MOG antibody and anti-MOG antibody-secreting cells can be detected in peripheral blood and cerebrospinal fluid of patients (14,21). These observations, together with the direct visualization of anti-MOG Ig within MS lesions, have led to the speculation that a MOG-specific antibody response also plays a significant role in the pathogenesis of MS (50). However, the epitope specificity of MOG antibodies, in particular whether or not they recognize the pathogenic Ig domains, remains unknown. A recent study has demonstrated an association of MOG-specific antibodies with myelin debris in MS lesions, indicating that they may well be involved in lesion formation (50). In the present study we demonstrate that all transgenic mice showed a high titer of anti-MOG antibody to the extracellular Ig domain of MOG as early as 2 weeks after immunization. This high titer may be directly involved in disease pathogenesis.
Localization of MOG on the outer surface of the myelin sheath makes it a target for antibody-mediated demyelination in MS; therefore, it is critically important to identify those epitopes that may induce pathogenic response in vivo. We demonstrate with our MOG-immunized HLA transgenic mice that anti-MOG antibodies recognize epitopes within the amino acid sequences 130, 5180 and 101120 in the context of HLA class II molecules. Our data is consistent with previous reports in which MS patients had a heterogeneous epitope specificity with their MOG-specific antibody response. The most frequently recognized epitopes were located within amino acids sequences 126 and 6387 (52). It was important to find that all the transgenic mice except DQ6 (DQB1*0604) showed a response to these peptides, suggesting that these epitopes may have a significant role in pathogenesis of MS. Mapping studies established that the anti-MOG antibodies present in MOG-immunized marmosets exclusively recognized epitopes within amino acids 120, 2140 and 6180 of this protein (9, 10, 50), whereas in rhesus monkeys anti-MOG antibodies recognize epitopes 426, 2446 and 4466/5476 (12). Murine MOG-induced EAE is associated with an antibody response directed against multiple MOG peptides, in particular 125, 5071 and 98117(53) (Table 2). The association of a sustained anti-MOG Ig antibody response with MS is intriguing, but the demyelinating potential of this response is still unknown. Moreover, the pathogenic potential of anti-MOG antibodies may be critically dependent upon synergy with a T cell-mediated inflammatory response in the CNS. Circulating pathogenic anti-MOG antibodies are unable to induce demyelination by themselves. Only when permeability of the bloodbrain barrier is enhanced can antibody enter the CNS, bind to surface of the myelin sheath and initiate demyelination (53).
Association and linkage of certain HLA class II molecules with MS have been reported in several population-based studies (54), but the role of various class II genes in disease progression still remains controversial with regard to their role in long-term disability and disease phenotype. Strong linkage disequilibrium between the alleles of the susceptibility haplotypes makes it very difficult to determine whether the HLA-DR or -DQ gene is of primary importance. It is also possible that both HLA-DR and -DQ loci are important in the determination of disease risk. Identification of DR- and DQ-restricted epitopes in humans is difficult because of heterogeneity of the human population. Although studies using T cell clones, PBMC and serum from MS patients suggest potential immunodominant T and B epitopes, they do not provide a comprehensive picture of the specific class II-restricted epitopes. Our HLA-DR and -DQ mice provide a powerful model for exhaustive characterization and identification of epitopes of myelin antigens. Because humans have both HLA-DR and -DQ genes participating in the shaping of the T cell repertoire and disease pathogenesis, we are currently studying the immune response in double-transgenic mice.
In conclusion, we have identified the immunodominant MOG T and B cell epitopes restricted by HLA class II molecules implicated in predisposition to MS in humans. Studies are currently underway to determine whether any of the peptides constituting the immunodominant epitopes can induce disease in the transgenic mice. An HLA class II-restricted, MOG-induced disease model for MS greatly facilitate understanding the role HLA-DQ and -DR molecules in predisposition, onset, progression and severity of disease in humans.
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Acknowledgements
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We would like to thank Dr C. Linington (Max Plank Institute, Germany) for providing rMOG (amino acids 1125), Julie Hanson and her crew for outstanding mouse husbandry, Michelle Smart and Susan Demaray for tissue typing, and Dr P. D. Khare for helpful discussions. This research was supported by NIH program project grant NS 38468-02. The transgenic mice produced by support from NIH grant AI 14764.
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Abbreviations
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CFAcomplete Freunds adjuvant
CNScentral nervous system
EAEexperimental autoimmune encephalomyelitis
LNClymph node cells
MSmultiple sclerosis
MBPmyelin basic protein
MOGmyelin oligodendrocyte glycoprotein
PBMCperipheral blood mononuclear cell
PLPproteolipid protein
rMOGrecombinant rat MOG
SIstimulation index
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