These results show that circulating high-titer antibodies to MOG are present in a substantial subset of pediatric-onset MS patients, in particular children with a very early disease onset. Stringent criteria establish their antigen specificity: the antibodies label MOG but not control transfectants, they can be affinity purified with recombinant MOG but not a structurally related control protein, and addition of soluble MOG inhibits antibody binding. Antibodies to MOG in pediatric-onset MS patients bound myelin in normal human white matter and myelinated axons in subpial grey matter. The majority of MOG-specific antibodies have the IgG1 subtype that can fix complement and bind to Fc receptors, indicating that they have the potential to damage myelin or oligodendrocytes if they gain access to the CNS. Such antibodies were rarely detected in pediatric controls or patients with adult-onset demyelinating conditions, including MS. In the pediatric patient population, their presence strongly correlated with age at disease onset and an initial ADEM-like presentation, but not with gender or ethnicity. We have begun analyzing autoreactive B cell responses in children with demyelinating events in greater detail. As part of this ongoing study, we have obtained serial serum samples from x children, 5 of whom were positive for antibodies to MOG. In 4 of the 5 cases, serum anti-MOG was detectable in follow-up samples, and persisted for 4 to 28 months after the initial clinic visit. Future prospective studies will examine the relationship between such an antibody response and clinical disease course, MRI features, response to treatment and prognosis.
Previous studies of autoantibodies in adult-onset MS have yielded conflicting results. While some have found antibodies to MOG in patients with MS or CIS (37
), others reported no difference in the frequency of anti-MOG between MS and other neurological diseases (39
) or healthy controls (42
). This controversy surrounding the presence of MOG antibodies in MS patients is likely attributable to differences in methodologies, and our flow cytometric approach therefore emphasized detection of antibodies to native, properly folded MOG protein.
Three other groups also generated MOG-expressing cells to examine the presence of autoantibodies in adult-onset MS patients. Haase et al. generated a stable MOG transfectant in LTK3−
cells and found that only one of 17 serum samples from adult MS patients labeled this transfectant, even though all patients as well as healthy control subjects had antibodies that detected linear MOG peptides in an ELISA (43
). Lavile et al. compared serum antibody staining of a MOG transfected CHO cell line to non-transfected CHO cells and concluded that IgG antibodies specific for native MOG were most frequently found in the serum of patients with CIS and relapsing-remitting MS (44
). However, the binding ratios of the MOG and control cells were less than two for all positive samples, even though high serum concentrations were used for staining (1:10 dilution, compared to the 1:50 dilution used here). We defined binding ratios of >5 as positive, and obtained binding ratios as high as 200.2 in pediatric MS samples.
In another study, Zhou et al. transduced a human glioblastoma cell line with a lentivirus encoding MOG and stained these cells at a serum dilution of 1:36 (45
). The MOG-specific antibody response was calculated by subtracting median fluorescence intensities obtained with MOG and control transfectants. The authors reported that 32% of adult-onset MS patients and 4% of control subjects had detectable antibodies, but the difference in MFI between the MOG and control cells was rather small (<50) for most sera. By comparison, we observed differences in MFI of 516 to 22,102 for the six representative examples shown in . We prefer to express the data as a binding ratio between the MOG and control transfectants rather than as a difference in MFI because the level of background differs greatly between sera. A sample with a high background and a small MOG-specific increase in binding can have a large change in MFI (for example, MFI values of 600 and 500 for MOG and GFP transfectants, respectively), while a sample with little background and the same MOG binding ratio would have a much smaller change in MFI (for example, 60 and 50). Although both analysis methods permit the identification of samples with high levels of MOG-specific antibodies, using MFI differences can result in the classification of samples with a high background as positive.
The pronounced therapeutic response to B cell depletion with Rituximab shows that B cells play a central role in the pathogenesis of adult-onset MS. It is likely that B cells contribute to disease progression not only as a source of autoantibodies, but also by presenting myelin-derived antigens to autoreactive T cells and driving inflammation through production of cytokines and chemokines. Antigen-specific B cells are highly effective as antigen presenting cells (46
), and their elimination could substantially decrease T cell priming and activation. The relative importance of these different mechanisms to the pathogenesis of MS remains unresolved. Also, the specificities of the involved B cells and their antibody products remain largely unknown.
In this study, we have analyzed the largest collection of adult-onset MS sera to date (n=254), and the results show differences in the frequency of MOG-positive cases between samples from MS centers in Canada (0/53, 0%), the US (5/129, 3.9%), and Switzerland (6/72, 8.3%). These apparent differences may be caused by the overall low frequency of antibodies to MOG in adult-onset MS, but differences in patient selection or genetic and environmental factors cannot be excluded. Sample handling may also be a contributing factor, but appears unlikely because we observed that MOG antibodies remain detectable after multiple freeze-thaw cycles.
Our findings support the conclusion that antibodies to MOG are rather uncommon among adult-onset MS patients, while the anti-MOG reactivity identified in a subset of pediatric MS cases is among the strongest autoantibody responses observed so far in MS. Using multimeric forms of folded and glycosylated MOG protein, we previously detected similar antibodies to MOG in a subset of ADEM patients (29
). In the current study of pediatric MS, the presence of MOG antibodies strongly correlated with an initial ADEM-like onset, although all children subsequently experienced two or more non-ADEM demyelinating events as required for a diagnosis of MS (17
). 50% of pediatric MS patients with an initial diagnosis of ADEM had antibodies to MOG, while 92% of MOG negative pediatric MS patients did not have an ADEM-like initial event. Prospective studies are required to further define the relationship between MOG antibodies and ADEM as well as pediatric MS.
Why have we only detected antibodies to MOG, but not MAG or OMG, which are also myelin surface antigens with large extracellular domains? Chronic inflammation can result in immune responses to multiple self-antigens, a phenomenon referred to as epitope spreading (48
). However, this should not be taken to mean that significant immune responses are mounted to every antigen in the target structure. Inherent properties of MOG may contribute to the generation of an antibody response in pediatric MS patients. MOG is a highly encephalitogenic protein in immunization-based animal models of MS and is the only known myelin component to induce pathogenic antibody and T cell responses (49
). MOG also has substantial sequence similarity with the milk protein butyrophilin, and antibody as well as T cell cross-reactivity between these antigens has been demonstrated (51
Why does the prevalence of these autoantibodies change with age at disease onset in pediatric populations? Several general and possibly interrelated factors may be involved: genetic susceptibility, the kinetics and magnitude of the autoimmune response, environmental factors, and the biology of myelination during childhood. In type 1 diabetes, children with particular combinations of MHC class II genes are more likely to become diabetic at a young age and thus appear to carry a higher genetic risk (53
). Similarly, children who develop MS at a young age may carry combinations of genes that raise susceptibility to MS to a higher level than in individuals who develop MS later in life. The frequency and functionality of MOG-specific T cells could also affect production of MOG antibodies. The importance of T cell – B cell collaboration was highlighted by recent studies which showed that genetically engineered mice with a high frequency of both MOG-specific T cells and B cells spontaneously develop severe CNS inflammation, while mice with only a high frequency of MOG specific B cells remain healthy (54
). Lastly, myelination is an ongoing process in children and adolescents, and local changes in the density and composition of myelin affect the distribution of MOG, which is exclusively found on mature myelin segments (57
Like adult-onset MS, pediatric MS appears to be a heterogeneous disease with complex biological mechanisms. Age at disease onset is an important factor in the specificity of the B cell response to myelin in pediatric MS patients, as MOG antibodies identify a subset of pediatric MS patients with a very early disease onset. Further investigation may define additional patient subgroups with other myelin antibody specificities.