Age-dependent B cell autoimmunity to a myelin surface antigen in pediatric multiple sclerosis

Abstract

Multiple sclerosis (MS) typically manifests in early to mid adulthood, but there is increasing recognition of pediatric-onset MS, aided by improvements in imaging techniques. The immunological mechanisms of disease are largely unexplored in pediatric-onset MS, in part because studies have historically focused on adult-onset disease. We investigated autoantibodies to myelin surface Ags in a large cohort of pediatric MS cases by flow cytometric labeling of transfectants that expressed different myelin proteins. Although Abs to native myelin oligodendrocyte glycoprotein (MOG) were uncommon among adult-onset patients, a subset of pediatric patients had serum Abs that brightly labeled the MOG transfectant. Abs to two other myelin surface Ags were largely absent. Affinity purification of MOG Abs as well as competition of binding with soluble MOG documented their binding specificity. Such affinity purified Abs labeled myelin and glial cells in human CNS white matter as well as myelinated axons in gray matter. The prevalence of such autoantibodies was highest among patients with a very early onset of MS: 38.7% of patients less than 10 years of age at disease onset had MOG Abs, compared with 14.7% of patients in the 10- to 18-year age group. B cell autoimmunity to this myelin surface Ag is therefore most common in patients with a very early onset of MS.

Figure 1. Identification of autoantibodies to native MOG in pediatric MS patients

A. Brightness of fluorescent labeling with different positive pediatric MS sera. FACS data representing antibody binding to the MOG-GFP transfectant (open histograms) and the control GFP transfectant (shaded histograms) are shown for sera from six pediatric MS patients. Sera were incubated at a dilution of 1:50 with cells, and bound antibodies were detected using a biotinylated anti-human IgG and streptavidin-PE. For each sample, the ratio of the mean fluorescence intensity for the MOG and the control transfectant is shown in the top left corner. The six depicted examples represent the entire range of fluorescent intensities for positive sera, with binding ratios of approximately 5 to 200 for the MOG versus the control transfectant. B. Antibody titers measured by FACS analysis. Sera were incubated with MOG-GFP cells at the indicated dilutions. Antibodies to MOG were detectable in all positive sera at a dilution of 1:400 and many at a dilution of 1:800.

Figure 2. Autoantibodies to native MOG are more prevalent in pediatric than in adult-onset MS patients, and infrequent in other inflammatory demyelinating CNS diseases

A. Sera from pediatric (left) or adult individuals (right) were assayed for antibodies to MOG at a dilution of 1:50. The MOG/control binding ratio for each sample was calculated as described, and ratios greater than 5 were considered positive. Antibodies to MOG were significantly more common in pediatric than adult-onset MS patients (p=4.48×10⁻⁷) or control subjects and labeled the transfectant more brightly. B. The frequency of MOG antibodies differs between patient populations studied at MS centers in different countries. Pediatric and adult-onset MS sera were obtained from MS centers in a number of different countries. While anti-MOG was detectable in pediatric MS samples from all countries studied, the frequency of MOG antibodies in adults varied considerably between centers.

Figure 3. MOG antibodies are most prevalent in MS patients with a very early disease onset

A. Antibodies to MOG are most common in MS patients with a disease onset before 10 years of age. Pediatric-onset MS patients were ranked by age at the first clinical event. Although the majority of samples were from older patients (>10 years), the frequency of antibodies to MOG was significantly higher (p=0.090) in individuals under 10 years of age at the first clinical event. B. Analysis of MOG antibodies in pediatric and adult-onset MS patients based on age at disease onset. Antibodies to MOG were most common in the youngest pediatric-onset MS cases, while a correlation with age at diagnosis was not apparent among the infrequent adult-onset cases with MOG antibodies. C. Relationship between the presence of MOG antibodies and the time interval since diagnosis of MS. Antibodies were found in pediatric-onset MS patients with recent disease onset and established disease.

Figure 4. The majority of pediatric MS patients have MOG IgG1 antibodies, an IgG subtype that can fix complement and bind to Fc receptors

IgG subclass-specific secondary antibodies were used to detect the binding of serum antibodies to the MOG transfectant. In the example shown, only MOG-specific IgG1 antibodies were detected. IgG1 was the most common subtype among these pediatric MS patients, found in 8 of 12 tested sera. IgG2 and IgG3 were rarely found, and IgG4 was not detected in any sample.

Figure 5. Antibodies in pediatric MS sera are specific for MOG

A. A monoclonal antibody to MOG competes with serum antibodies for antigen binding. Sera and indicated amounts of the MOG-specific antibody 8–18C5 were incubated together with MOG-GFP cells for one hour. A human-specific secondary antibody was used to detect bound IgG. Addition of increasing amounts of 8–18C5 degreased the amount of serum antibody bound to cells. B. Affinity-purified antibodies specifically bind to MOG transfectants. Biotinylated recombinant MOG (rMOG, extracellular domain) or an Ig superfamily control protein (membrane-proximal Ig domain of CD80) were captured onto streptavidin beads for affinity isolation of MOG-specific antibodies from 5µL of serum. Unbound and eluted antibodies were used to stain MOG and control transfectants. Binding ratios obtained with purified antibodies from four sera are summarized in the table. Antibodies purified on MOG beads bound MOG on the cell surface, and addition of 25µg soluble recombinant MOG inhibited binding of eluted MOG-specific antibodies for all four sera tested.

Figure 6. Antibodies to MOG stain myelin in human brain

A. MOG protein and oligodendrocytes in the white matter were detected with monoclonal antibodies 8–18C5 and 14E, respectively. B. Immunocytochemistry with affinity-purified antibodies from pediatric MS patients. 200µg of biotinylated total serum IgG was purified on MOG or BSA coated beads, percentage used per staining reaction. MOG-specific antibodies from pediatric MS patients W52 and W24 labeled white matter in a myelin and oligodendrocyte-like pattern. No staining was seen with antibodies isolated on BSA control beads or antibodies from a control donor (D14). C. MOG-specific antibodies label myelinated axons in the subpial grey matter. Regions of grey matter in the same tissue sections as shown above were assessed for antibody binding. Myelinated axons were detectable with the monoclonal MOG antibody 8–18C5 and total IgG from a MOG-positive ADEM patient (R4), and glial cell bodies were identified with antibody 14E (not shown). Pediatric MS serum antibodies (patient W52) purified on MOG but not BSA beads also bound myelinated axons.

Figure 7. Antibodies to other myelin surface proteins in pediatric MS sera

A. Generation of transfectants that express other myelin proteins on the cell surface. Jurkat cells were transfected with vectors that drove expression of zsGreen as well as MOG, myelin-associated glycoprotein (MAG) or oligodendrocyte-myelin glycoprotein (OMG). Transfectants were cloned by single cell sorting and surface expression was verified using an antibody to the HA tag attached to the N-terminus of each protein. B. Examples of labeling of these transfectants with MOG-GFP positive pediatric MS sera. Samples were incubated at a 1:50 dilution with Jurkat cells transfected with HA-tagged antigens, and bound IgG was detected with biotinylated anti-human IgG and streptavidin-PE. C. Pediatric MS sera with MOG antibodies do not show broad anti-myelin reactivity. Antibody binding for 13 serum samples is shown as the median fluorescence intensity of PE. Although antibodies to MAG and OMG were detectable at low levels in a few sera, the labeling of MOG transfectants was considerably brighter in all cases. Antibodies to OMG and MAG were not detected in MOG negative pediatric MS sera or pediatric controls (not shown).