Antibodies to native myelin oligodendrocyte glycoprotein are serologic markers of early inflammation in multiple sclerosis

Abstract

Myelin oligodendrocyte glycoprotein (MOG) is an integral membrane protein expressed in CNS oligodendrocytes and outermost myelin lamellae. Anti-MOG Abs cause myelin destruction (demyelination) in animal models of multiple sclerosis (MS); however, such pathogenic Abs have not yet been characterized in humans. Here, a method that specifically detects IgG binding to human MOG in its native, membrane-embedded conformation on MOG-transfected mammalian cells was used to evaluate the significance of these auto Abs. Compared with healthy controls, native MOG-specific IgGs were most frequently found in serum of clinically isolated syndromes (P < 0.001) and relapsing-remitting MS (P < 0.01), only marginally in secondary progressive MS (P < 0.05), and not at all in primary progressive MS. We demonstrate that epitopes exposed in this cell-based assay are different from those exposed on the refolded, extracellular domain of human recombinant MOG tested by solid-phase ELISA. In marmoset monkeys induced to develop MS-like CNS inflammatory demyelination, IgG reactivity against the native membrane-bound MOG is always detected before clinical onset of disease (P < 0.0001), unlike that against other myelin constituents. We conclude that (i) epitopes displayed on native, glycosylated MOG expressed in vivo are early targets for pathogenic Abs; (ii) these Abs, which are not detected in solid-phase assays, might be the ones to play a pathogenic role in early MS with predominant inflammatory activity; and (iii) the cell-based assay provides a practical serologic marker for early detection of CNS autoimmune demyelination including its preclinical stage at least in the primate MS model.

Fig. 1.

Cell-based (hMOG_cme) assay. (A and B) Staining of MOG-transfected CHO cells with anti-MOG Ab 8-18C5 (0.5 μg/ml) and detection by FACS (A) and immunofluorescence (B). (B Inset) Negative control omitting primary Ab. (C) Positive control patient serum (RRMS 1158, 1:10) displaying a clear shift for MOG-transfected CHO cells (filled trace) when compared with ntCHO cells (open trace). (D) Mean BR (BR ± SEM) calculated as the Gmean from nine independent assays.

Fig. 2.

Binding characteristics of MOG-specific marmoset recombinant Fab fragments. Binding of monoclonal Fabs specific for rMOG₁₂₅ (0.5 μg/ml) by FACS against hMOG_cme. Background is represented by the binding to the ntCHO cells (open traces) and compared with the binding to MOG-transfected CHO cells (filled traces). Both Fabs M26 (A) and M3-31 (B) strongly recognize an epitope displayed on hMOG_cme, in contrast to Fabs M3-24 (C) and M3-8 (D).

Fig. 3.

Analysis of human serum IgG reactivity against hMOG_cme. BR normalized (BRN) is the Gmean value of IgG binding to MOG-transfected CHO cells divided by that of IgG binding to ntCHO cells, normalized to the value of the positive control tested in each plate. Horizontal bars = median. See text for details.

Fig. 4.

Time course of serum IgG directed against hMOG_cme in marmoset EAE. Results are from 11 EAE C. jacchus marmosets immunized with human white matter, 3 of which were killed before onset of clinical disease. First occurrence of serum IgG directed against hMOG_cme is compared with time of clinical onset of EAE in a Kaplan–Meier survival plot. Please see text and Table 1 for details.

Fig. 5.

Selective epitope presentation on hMOG_cme. (A) Binding to hMOG₁₂₅ by ELISA compared with binding to hMOG_cme by FACS in the CIS cohort (n = 36). By linear regression analysis, there is no correlation between the results of these two methods (P not significant, r² = 0.00023, Spearman r; straight line is the linear regression curve; dotted line indicates 95% confidence interval) even when clear serum reactivity is present in both assays. BRN, BR normalized. (B and C) Preabsorption of serum on either hMOG_cme (Left) or hMOG₁₂₅ (Right), followed by testing by FACS (B, hMOG_cme) or ELISA (C, hMOG₁₂₅). Preabsorption on hMOG₁₂₅ or hMOG_cme only altered the reactivity in the corresponding system of detection. See Materials and Methods for additional details.