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Neuromyelitis optica spectrum disorders (NMOSD) constitute rare autoimmune disorders of the CNS that are primarily characterized by severe inflammation of the spinal cord and optic nerve. Approximately 75% of NMOSD patients harbour circulating pathogenic autoantibodies targeting the aquaporin-4 water channel (AQP4). The source of these autoantibodies remains unclear, but parallels between NMOSD and other autoantibody-mediated diseases posit compromised B cell tolerance checkpoints as common underlying and contributing factors. Using a well established assay, we assessed tolerance fidelity by creating recombinant antibodies from B cell populations directly downstream of each checkpoint and testing them for polyreactivity and autoreactivity. We examined a total of 863 recombinant antibodies. Those derived from three anti-AQP4-IgG seropositive NMOSD patients (n = 130) were compared to 733 antibodies from 15 healthy donors. We found significantly higher frequencies of poly- and autoreactive new emigrant/transitional and mature naïve B cells in NMOSD patients compared to healthy donors (P-values < 0.003), thereby identifying defects in both central and peripheral B cell tolerance checkpoints in these patients. We next explored whether pathogenic NMOSD anti-AQP4 autoantibodies can originate from the pool of poly- and autoreactive clones that populate the naïve B cell compartment of NMOSD patients. Six human anti-AQP4 autoantibodies that acquired somatic mutations were reverted back to their unmutated germline precursors, which were tested for both binding to AQP4 and poly- or autoreactivity. While the affinity of mature autoantibodies against AQP4 ranged from modest to strong (Kd 15.2-559 nM), none of the germline revertants displayed any detectable binding to AQP4, revealing that somatic hypermutation is required for the generation of anti-AQP4 autoantibodies. However, two (33.3%) germline autoantibody revertants were polyreactive and four (66.7%) were autoreactive, suggesting that pathogenic anti-AQP4 autoantibodies can originate from the pool of autoreactive naïve B cells, which develops as a consequence of impaired early B cell tolerance checkpoints in NMOSD patients., (© The Author(s) (2019). Published by Oxford University Press on behalf of the Guarantors of Brain. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Background: Neuromyelitis optica (NMO) is a severe demyelinating disorder of the central nervous system (CNS) associated with the presence of an autoimmune antibody response (AQP4-IgG) against the water channel aquaporin-4 (AQP4). It remains unclear whether pathologic AQP4-IgG in the CNS is produced entirely by peripheral plasma cells or is generated in part by infiltrating B cells. To determine the overlap of AQP4-IgG idiotypes between the CNS and periphery, we compared the immunoglobulin G (IgG) transcriptome of cerebrospinal fluid (CSF) plasmablasts with the CSF and serum IgG proteomes in 7 AQP4-seropositive NMO patients following exacerbation., Methods: CSF variable region Ig heavy- (VH) and light-chain (VL) transcriptome libraries were generated for each patient from CSF plasmablasts by single cell sorting, reverse transcriptase polymerase chain reaction (RT-PCR), and DNA sequencing. Recombinant antibodies were generated from clonally expanded, paired VH and VL sequences and tested for AQP4-reactivity by cell-binding assay. CSF and serum IgG fractions were searched for sequences that matched their respective CSF IgG transcriptome. Matching peptides within the same patient's CSF and serum IgG proteomes were also identified., Results: In each NMO patient, we recovered CSF IgG VH and VL sequences that matched germline-mutated IgG protein sequences from the patient's CSF and serum IgG proteomes. Although a modest variation was observed between patients, the overlap between the transcriptome and proteome sequences was found primarily, but not exclusively, within the CSF. More than 50% of the CSF IgG transcriptome sequences were exclusively found in the CSF IgG proteome, whereas 28% were found in both the CSF and blood IgG proteome, and 18% were found exclusively in the blood proteome. A comparable distribution was noted when only AQP4-specific IgG clones were considered. Similarly, on average, only 50% of the CSF IgG proteome matched corresponding peptide sequences in the serum., Conclusions: During NMO exacerbations, a substantial fraction of the intrathecal Ig proteome is generated by an intrathecal B cell population composed of both novel and peripherally-derived clones. Intrathecal CSF B cell clones may contribute to NMO disease exacerbation and lesion formation and may be an important target for preventative therapies.

The CD19+ B-lymphocyte and CD138+ plasma cell repertoires in cerebrospinal fluid from four patients with monosymptomatic optic neuritis (ON) were analyzed by single-cell reverse transcriptase polymerase chain reaction. Amplified heavy (H)- and light (L)-chain antibody segments were sequenced and used to identify the rearranged germline and J segment of closest homology. Both the B-cell and plasma cell repertoires from ON cerebrospinal fluid demonstrated significant clonal expansion. Up to 75% of the amplified H- and L-chain sequences were contained in overrepresented populations and were somatically mutated, consistent with an antigen-targeted response. The relationship between clonal populations within the CD19+ B lymphocyte and CD138+ plasma cell populations suggests ongoing mutational pressure to refine antigen binding. Our observations demonstrate that an antigen-driven clonal B-lymphocyte and plasma cell response is prominent in the initial stages of central nervous system demyelination and suggest that detection of the disease-relevant antigens in ON may bear on the inciting antigens in chronic inflammatory disorders such as multiple sclerosis.