Intrathecal pathogenic anti–aquaporin‐4 antibodies in early neuromyelitis optica

Abstract

Objective-The serum of most neuromyelitis optica (NMO) patients contains autoantibodies (NMO-IgGs) directed against the aquaporin-4 (AQP4) water channel located on astrocyte foot processes in the perivessel and subpial areas of the brain. Our objectives were to determine the source of central nervous system (CNS) NMO-IgGs and their role in disease pathogenesis.Methods-Fluorescence activated cell sorting and single-cell reverse transcriptase PCR were used to identify overrepresented plasma cell immunoglobulin (Ig) sequences in the cerebrospinal fluid (CSF) of an NMO patient after a first clinical attack. Monoclonal recombinant antibodies (rAbs) were generated from the paired heavy and light chain sequences and tested for target specificity and Fc effector function. The effect of CSF rAbs on CNS immunopathology was investigated by delivering single rAbs to rats with experimental autoimmune encephalomyelitis (EAE).Results-Repertoire analysis revealed a dynamic, clonally expanded plasma cell population with features of an antigen-targeted response. Using multiple independent assays, 6 of 11 rAbs generated from CSF plasma cell clones specifically bound to AQP4. AQP4-specific rAbs recognized conformational epitopes and mediated both AQP4-directed antibody-dependent cellular cytotoxicity and complement-mediated lysis. When administered to rats with EAE, an AQP4-specific NMO CSF rAb induced NMO immunopathology: perivascular astrocyte depletion, myelinolysis and complement and Ig deposition.Interpretation-Molecular characterization of the CSF plasma cell repertoire in an early NMO patient demonstrates that AQP4-specfic Ig is synthesized intrathecally at disease onset and directly

CNS Aquaporin‐4‐specific B cells connect with multiple B‐cell compartments in neuromyelitis optica spectrum disorder

“…Single CSF mononuclear cells (MNCs) were prepared as described previously 1. Peripheral blood was collected in CPT tubes and mononuclear cells isolated according to the manufacturer's instructions (BD Vacutainer, CPT cell preparation tubes with sodium acetate) and cryopreserved at −80°C for later sample analysis.…”

“…CSF CD19 + CD138 + plasmablast heavy‐ (VH) and light‐chain (VL) variable region sequences were recovered by RT‐PCR and DNA sequencing as described previously 1, 7. Recombinant antibodies were produced in HEK293 cells (Invitrogen, Carlsbad, CA, R620‐07)7 and reactivity tested via immunofluorescence for binding to a permanent cell line expressing M23‐AQP4 8…”

“…Functional V(D)J sequences were assigned into clones based on identical nucleic acid complementarity determining region‐3 (CDR3) sequence length, VH variable gene segment, VH joining gene segment, and ≥ 75% CDR3 sequence identity at the nucleotide level. The stringency for CDR3 identity was chosen based on prior analysis of NMOSD CSF plasmablast clones1 that showed significant intraclonal CDR3 sequence variability. A similar approach was taken in the analysis of antibody‐secreting cell populations in systemic lupus erythematosus (SLE) 12.…”

“…B cells may play multiple roles in the pathogenesis of neuromyelitis optica spectrum disorders (NMOSD) 1. In 75% of NMOSD patients, autoreactive B cells produce antibodies against the aquaporin‐4 (AQP4) water channel (AQP4‐IgG) 2, 3.…”

“…In 75% of NMOSD patients, autoreactive B cells produce antibodies against the aquaporin‐4 (AQP4) water channel (AQP4‐IgG) 2, 3. In the central nervous system (CNS), AQP4 is highly expressed on astrocyte end‐feet, and AQP4‐IgG has been shown to initiate an inflammatory cascade that ultimately leads to demyelination and neuronal injury 1, 4, 5. However, the location(s) of initial antigen presentation and affinity maturation, as well as the composition of migratory AQP4‐reactive B cells remains largely unknown.…”