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A Simple Cell-based Immunofluorescence Assay to Detect Autoantibody Against the N-Methyl-D-Aspartate NMDA Receptor in Blood
Published on: January 9, 2018
Human cerebrospinal fluid monoclonal N-methyl-D-aspartate receptor autoantibodies are sufficient for encephalitis
Jakob Kreye1, Nina K Wenke2, Mariya Chayka2
11German Center for Neurodegenerative Diseases (DZNE) Berlin, Germany 2Department of Neurology and Experimental Neurology, Charité, Universitätsmedizin Berlin, Germany.
Abstract:
SEE ZEKERIDOU AND LENNON DOI101093/AWW213 FOR A SCIENTIFIC COMMENTARY ON THIS ARTICLE: Anti-N-methyl-D-aspartate receptor (NMDAR) encephalitis is a recently discovered autoimmune syndrome associated with psychosis, dyskinesias, and seizures. Little is known about the cerebrospinal fluid autoantibody repertoire. Antibodies against the NR1 subunit of the NMDAR are thought to be pathogenic; however, direct proof is lacking as previous experiments could not distinguish the contribution of further anti-neuronal antibodies. Using single cell cloning of full-length immunoglobulin heavy and light chain genes, we generated a panel of recombinant monoclonal NR1 antibodies from cerebrospinal fluid memory B cells and antibody secreting cells of NMDAR encephalitis patients. Cells typically carried somatically mutated immunoglobulin genes and had undergone class-switching to immunoglobulin G, clonally expanded cells carried identical somatic hypermutation patterns. A fraction of NR1 antibodies were non-mutated, thus resembling 'naturally occurring antibodies' and indicating that tolerance induction against NMDAR was incomplete and somatic hypermutation not essential for functional antibodies. However, only a small percentage of cerebrospinal fluid-derived antibodies reacted against NR1. Instead, nearly all further antibodies bound specifically to diverse brain-expressed epitopes including neuronal surfaces, suggesting that a broad repertoire of antibody-secreting cells enrich in the central nervous system during encephalitis. Our functional data using primary hippocampal neurons indicate that human cerebrospinal fluid-derived monoclonal NR1 antibodies alone are sufficient to cause neuronal surface receptor downregulation and subsequent impairment of NMDAR-mediated currents, thus providing ultimate proof of antibody pathogenicity. The observed formation of immunological memory might be relevant for clinical relapses.
Insights
This study proves that antibodies targeting the N-methyl-D-aspartate receptor (NMDAR) directly cause autoimmune encephalitis by downregulating neuronal receptors. This finding offers crucial insights into the autoimmune disease mechanisms.
Area of Science:
- Neuroimmunology
- Autoimmune Diseases
- Molecular Neuroscience
Background:
- Anti-N-methyl-D-aspartate receptor (NMDAR) encephalitis is an autoimmune disorder linked to psychosis, movement disorders, and seizures.
- The specific autoantibodies involved and their pathogenic mechanisms in the cerebrospinal fluid remain poorly understood.
- Previous studies could not definitively prove the pathogenicity of NR1 subunit antibodies due to confounding factors from other anti-neuronal antibodies.
Purpose of the Study:
- To generate and characterize monoclonal antibodies against the NR1 subunit of NMDAR from patients with NMDAR encephalitis.
- To provide definitive proof of the pathogenicity of NR1 antibodies in causing neuronal dysfunction.
- To investigate the broader autoantibody repertoire in the cerebrospinal fluid during NMDAR encephalitis.
Main Methods:
- Single cell cloning of immunoglobulin genes from cerebrospinal fluid memory B cells and antibody-secreting cells of NMDAR encephalitis patients.
- Generation of recombinant monoclonal NR1 antibodies.
- Functional assays using primary hippocampal neurons to assess antibody effects on NMDAR function.
Main Results:
- Monoclonal NR1 antibodies derived from patient cerebrospinal fluid were generated, with some showing somatic mutations and others appearing unmutated.
- Nearly all cerebrospinal fluid-derived antibodies, not just NR1 antibodies, reacted with diverse brain-expressed epitopes, suggesting a broad immune response in the CNS.
- Human monoclonal NR1 antibodies alone were sufficient to downregulate NMDAR surface receptors and impair NMDAR-mediated currents in hippocampal neurons, proving pathogenicity.
Conclusions:
- This study provides the ultimate proof that antibodies against the NR1 subunit of NMDAR are pathogenic in autoimmune encephalitis.
- The findings highlight that a diverse range of autoantibodies may contribute to the disease pathology.
- The potential for immunological memory formation suggests a mechanism relevant to clinical relapses in NMDAR encephalitis.
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