Investigating the origins of IgG1 pan-neurofascin antibodies in a severe subtype of autoimmune neuropathy

Thesis

IgG1 pan-neurofascin autoimmune nodopathy (pan-NF AN) is a rare and fulminant form of autoimmune neuropathy characterised by IgG1 antibodies targeting both neurofascin-155 (NF155) and neurofascin-186 (NF186), often resulting in severe sensorimotor and autonomic involvement, and poor responses to standard immunotherapies. While B-cell depletion therapy can lead to improvement, relapse and long-term disability remain common, underscoring the need to better understand the origins and regulation of the autoreactive B-cell responses in this disease.

In this thesis, I developed and applied a suite of immunological tools to dissect the neurofascin-specific B-cell response in pan-NF AN. A peripheral blood mononuclear cell (PBMC) culture system was optimised to induce robust immunoglobulin secretion, enabling the isolation, screening, and B-cell receptor (BCR) sequencing of 480 single CD27+IgD- memory B cells from a patient with severe pan-NF AN. Neurofascin reactivity was detected in 16 wells (3.3%); unexpectedly, IgM-secreting clones outnumbered IgG by approximately four to one. Recombinant monoclonal antibodies generated from three autoreactive clones confirmed stronger antigen binding when expressed as IgM compared to IgG1, despite identical variable regions. These results suggest that the multivalency of IgM may compensate for lower intrinsic affinity. Sequencing revealed minimal somatic hypermutation in the IgG-expressing autoreactive clones despite their memory surface phenotype, suggesting that autoreactive B cells may emerge via extrafollicular or germinal centre-independent pathways.

Evidence of dual IgM and IgG transcript expression in some autoreactive clones, together with the post hoc detection of serum IgM autoantibodies in all 13 IgG1 pan-NF AN patients screened, supports the hypothesis that IgM-class autoreactivity is a generalisable and previously unrecognised feature of the disease. To facilitate future investigation and clinical translation, biotinylated antigen “bait” tetramers were developed for NF155 and NF186, capable of binding patient autoantibodies and will enable future antigen-specific B-cell enrichment.

Together, these findings support a model in which both central and peripheral tolerance defects contribute to the generation of neurofascin-reactive B cells in IgG1 pan-NF AN, with early IgM responses giving rise to class-switched IgG via limited or non-canonical pathways. The characterisation of an IgM component has implications for diagnosis and therapy, providing a rationale for isotype-inclusive testing and antigen-specific therapeutic targeting of autoreactive B cells in pan-NF AN.

Authors: Johnson, CEB

• DOI: 10.5287/ora-zry9n6vrn
• Type of award: DPhil
• Level of award: Doctoral
• Awarding institution: University of Oxford
• Language: English
• Keywords: somatic hypermutation; single-cell B-cell culture; autoreactive B cells; autoimmune nodopathy; neurofascin-155; extrafollicular B-cell response; nodal/paranodal antibodies; B-cell tolerance; pan-neurofascin autoimmune nodopathy; chronic inflammatory demyelinating polyradiculoneuropathy; CD27+IgD- memory B cells; antigen tetramer; neurofascin-186; IgM autoantibodies; humoral autoimmunity; live cell-based assay; monoclonal antibody; guillain-barre syndrome; inflammatory neuropathy; B-cell receptor sequencing; memory B cells
• Subjects: Neuroimmunology; Medical sciences; Immunology; Neurosciences; Autoimmune diseases; Neurology; B cells