Antibody Test Validation and Immunophenotypic Characterisation of B Cells in Myelin Oligodendrocyte Glycoprotein Antibody-Associated Disease

Abstract

Autoimmunity against myelin contributes to the pathogenesis of inflammatory central nervous system (CNS) demyelination. Myelin oligodendrocyte glycoprotein (MOG) has been a candidate autoantigen in demyelinating disorders. The detection of antibodies against MOG using live cell-based assays (CBAs) lay the foundation for the recognition of several demyelinating syndromes distinct from multiple sclerosis and aquaporin-4 antibody-associated neuromyelitis optica spectrum disorder. Myelin oligodendrocyte glycoprotein antibody-associated disease (MOGAD) now forms a novel disease entity underpinned by the presence of human MOG antibodies. In line with this, there exists a growing need for the diagnostic testing of MOG antibodies in a clinical pathology setting. In addition, reports exploring autoreactive MOG-specific B cells remain scarce and characterising the immunophenotype of B cells in MOGAD patients is further warranted.

In this body of work, the optimisation and streamlining of a flow cytometry live cell-based assay for clinical diagnostics was performed to provide a high-throughput, semi-quantitative, and objective measure of MOG antibody serostatus to meet diagnostic demand. Our findings showed that compared to a previously utilised in-house live CBA, the streamlined diagnostic live CBA showed high serostatus agreement (98%) in a cohort of 58 paediatric and 54 adult MOGAD patients and improved intra- and inter-assay precision. In addition, inter-laboratory validation demonstrated high serostatus agreement of the diagnostic live CBA across laboratories. From testing 1893 adult sera of patients with demyelinating syndromes, we defined a borderline range that identified patients required for retesting. Further, while 24 sera controls have been typically used for threshold calculation, this study validated that 13 controls can provide an equally robust positivity threshold for serostatus determination.

Additionally, this thesis details the design and application of a novel high-parameter flow cytometry panel for the immunophenotyping of B cell subsets and the detection of antigen-specific B cells. This study demonstrated the use of conventional gating and computational analyses of flow cytometry data for the identification of cell subsets. In a healthy donor, B cells specific for Epstein-Barr nuclear antigen 1 (EBNA1) exhibited a memory phenotype, with expression of IgG, and were detected at low frequencies in peripheral blood, accounting for up to 1.2% of total B cells. In contrast, MOG-specific B cells were detected in adult MOGAD patients (n = 9) and age-matched controls, predominantly represented a naïve subset, and were detected at a frequency of approximately 0.09% of total B cells. MOG-specific B cells were not associated with disease activity or serum MOG antibody titres.

Together, the key findings of this thesis demonstrate the translation of a robust, sensitive, and specific live CBA for MOG antibody diagnostics by optimising and validating improvements to an existing in-house assay to relieve the burden of testing and meet the increasing demand for MOG antibody tests. The design and use of a B cell immunophenotyping and antigen-specific detection panel for flow cytometry has provided an avenue for characterising the B cell immune profile in disease and detecting rare, autoreactive subsets in MOGAD. Overall, this body of work contributes to our knowledge of the humoral response to MOG and aims to improve the clinical diagnosis and outcomes for MOGAD patients.