title: Intrathecal somatic hypermutation of IgM in multiple sclerosis and neuroinflammation.
abstract: Intrathecal oligoclonal bands of the cerebrospinal fluid are considered the most important immunological biomarkers of multiple sclerosis. They typically consist of clonally expanded IgG antibodies that underwent affinity maturation during sustained stimulation by largely unknown antigens. In addition, ∼40% of patients with multiple sclerosis have oligoclonal bands that consist of expanded IgM antibodies. We investigated the molecular composition of IgM- and IgG-chains from cerebrospinal fluid of 12 patients with multiple sclerosis, seven patients with other neurological diseases, and eight healthy control subjects by high-throughput deep-sequencing and single-cell PCR. Further, we studied the expression of activation-induced cytidine deaminase, the key enzyme for affinity maturation of antibodies, in cerebrospinal fluid samples of 16 patients. From the cerebrospinal fluid of two multiple sclerosis patients we isolated single B cells and investigated the co-expression of antibody chains with activation-induced cytidine deaminase. In striking contrast to IgM-chains from peripheral blood, IgM-chains from cerebrospinal fluid of patients with multiple sclerosis or neuroborreliosis showed a high degree of somatic hypermutation. We found a high content of mutations that caused amino acid exchanges as compared to silent mutations. In addition, more mutations were found in the complementarity determining regions of the IgM-chains, which interact with yet unknown antigens, as compared to framework regions. Both observations provide evidence for antigen-driven affinity maturation. Furthermore, single B cells from the cerebrospinal fluid of patients with multiple sclerosis co-expressed somatically hypermutated IgM-chains and activation-induced cytidine deaminase, an enzyme that is crucial for somatic hypermutation and class switch recombination of antibodies and is normally expressed during activation of B cells in germinal centres. Clonal tracking of particular IgM(+) B cells allowed us to relate unmutated ancestor clones in blood to hypermutated offspring clones in CSF. Unexpectedly, however, we found no evidence for intrathecal isotype switching from IgM to IgG. Our data suggest that the intrathecal milieu sustains a germinal centre-like reaction with clonal expansion and extensive accumulation of somatic hypermutation in IgM-producing B cells.
authors: 1 Institute of Clinical Neuroimmunology, University Hospital Grosshadern, LMU Munich, D-81377 Munich, Germany 2 Unidad Mixta de Esclerosis Múltiple y Neurorregeneración, IIS Hospital La Fe - Universitat de València, 46026 Valencia, Spain.
authors: Eduardo
authors: E
authors: Beltrán
authors: 1 Institute of Clinical Neuroimmunology, University Hospital Grosshadern, LMU Munich, D-81377 Munich, Germany.
authors: Birgit
authors: B
authors: Obermeier
authors: 3 Department for Molecular Medicine, Max-Planck-Institute for Biochemistry, D-82152 Martinsried, Germany.
authors: Markus
authors: M
authors: Moser
authors: 4 Neurology Department, Clinic University Hospital, 46026 Valencia, Spain.
authors: Francisco
authors: F
authors: Coret
authors: 2 Unidad Mixta de Esclerosis Múltiple y Neurorregeneración, IIS Hospital La Fe - Universitat de València, 46026 Valencia, Spain.
authors: María
authors: M
authors: Simó-Castelló
authors: 2 Unidad Mixta de Esclerosis Múltiple y Neurorregeneración, IIS Hospital La Fe - Universitat de València, 46026 Valencia, Spain.
authors: Isabel
authors: I
authors: Boscá
authors: 2 Unidad Mixta de Esclerosis Múltiple y Neurorregeneración, IIS Hospital La Fe - Universitat de València, 46026 Valencia, Spain.
authors: Francisco
authors: F
authors: Pérez-Miralles
authors: 5 Department of Immunology, Hospital Ramón y Cajal, 28034 Madrid, Spain.
authors: Luisa M
authors: LM
authors: Villar
authors: 6 Department of Neurology University of Ulm, D-89081 Ulm, Germany.
authors: Makbule
authors: M
authors: Senel
authors: 6 Department of Neurology University of Ulm, D-89081 Ulm, Germany.
authors: Hayrettin
authors: H
authors: Tumani
authors: 1 Institute of Clinical Neuroimmunology, University Hospital Grosshadern, LMU Munich, D-81377 Munich, Germany 7 Munich Cluster for Systems Neurology (SyNergy), Adolf-Butenandt-Institut, Ludwig-Maximilians-Universität München, 80336 München, Germany.
authors: Reinhard
authors: R
authors: Hohlfeld
authors: 2 Unidad Mixta de Esclerosis Múltiple y Neurorregeneración, IIS Hospital La Fe - Universitat de València, 46026 Valencia, Spain.
authors: Bonaventura
authors: B
authors: Casanova
authors: 1 Institute of Clinical Neuroimmunology, University Hospital Grosshadern, LMU Munich, D-81377 Munich, Germany 7 Munich Cluster for Systems Neurology (SyNergy), Adolf-Butenandt-Institut, Ludwig-Maximilians-Universität München, 80336 München, Germany Klaus.Dornmair@med.uni-muenchen.de.
authors: Klaus
authors: K
authors: Dornmair
mesh_terms: D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D000595:Amino Acid Sequence; D061067:Antibodies, Monoclonal, Humanized / Q000627:therapeutic use; D001402:B-Lymphocytes / Q000276:immunology; D001483:Base Sequence; D049109:Cell Proliferation; D005260:Female; D006801:Humans; D007074:Immunoglobulin G / Q000134:cerebrospinal fluid / Q000235:genetics; D007075:Immunoglobulin M / Q000134:cerebrospinal fluid* / Q000235:genetics*; D007249:Inflammation / Q000235:genetics* / Q000276:immunology*; D008297:Male; D008875:Middle Aged; D008969:Molecular Sequence Data; D009103:Multiple Sclerosis / Q000235:genetics* / Q000276:immunology*; D000069442:Natalizumab; D059010:Single-Cell Analysis; D027041:Somatic Hypermutation, Immunoglobulin / Q000235:genetics*; D013116:Spinal Cord / Q000276:immunology* / Q000378:metabolism*; D055815:Young Adult
chemical_list: D061067:Antibodies, Monoclonal, Humanized; D007074:Immunoglobulin G; D007075:Immunoglobulin M; D000069442:Natalizumab
keywords: cerebrospinal fluid; immunoglobulin; multiple sclerosis; neuroborreliosis; oligoclonal bands