Clonally expanded plasma cells in the cerebrospinal fluid of MS patients produce myelin-specific antibodies.

Clonally expanded plasma cells (cePC) and their presumed products, oligoclonal immunoglobulin G bands (OCB), are characteristic findings in the cerebrospinal fluid (CSF) of patients with multiple sclerosis (MS). While cePC and OCB strongly suggest an involvement of B cell-dependent immune mechanisms in the pathogenesis of MS, their actual pathological relevance and target antigens remain unknown. To further understand the potential role played by cePC, we generated a panel of monoclonal antibodies (MS-mAb) from CSF-derived cePC from four patients with early or definite MS. Single-cell RT-PCR of correctly paired heavy and light chain immunoglobulin genes from individual cePC ensured the subsequent resurrection of their original antigen specificity. Immunofluorescence stainings of MS lesion tissue with MS-mAb revealed myelin reactivity in the cePC repertoire of all four patients and intracellular filament reactivity in one patient. While myelin staining by MS-mAb was only rarely detectable in non-MS CNS white matter tissue, it was greatly enhanced at the edge of demyelinating lesions in MS brain tissue. Our findings provide conclusive evidence for the presence of an antigen-driven B cell response in the CSF of MS patients directed against epitopes present in areas of myelin degradation.

Pathogen specificity and autoimmunity are distinct features of antigen-driven immune responses in neuroborreliosis.

Neuroborreliosis (NB) is a chronic infectious disease of the central nervous system (CNS) caused by a tick-borne spirochete, Borrelia burgdorferi. In addition to direct effects of the causative infectious agent, additional immunity-mediated mechanisms are thought to play a role in the CNS pathology of NB. In order to further understand the involvement of humoral immune mechanisms in NB, we dissected the intrathecal antibody responses down to the single-plasma-cell level. Starting with single-cell reverse transcription-PCR of fluorescence-activated cell sorter-sorted cerebrospinal fluid plasma cells from an NB patient, we identified expanded clones and resurrected the antigen specificity of their secreted antibodies through recombinant expression of the correctly paired immunoglobulin heavy- and light-chain genes as monoclonal antibodies (MAbs). As expected, we found specificity for the causative infectious agent, B. burgdorferi, among the clonally expanded plasma cell (cePC)-derived MAbs. However, from an independent cePC of the same patient, we could derive MAbs specific for human CNS myelin, without detectable cross-reactivity with B. burgdorferi antigens. While reactivity against B. burgdorferi is a known feature of humoral immune responses in NB, we show (i) that immune responses specific for self antigens may be a distinct feature of CNS infections independent of pathogen reactivity and (ii) that humoral autoimmunity in NB (since found in cePC) is the result of a truly antigen-driven immune response. Our findings indicate that in NB mechanisms may be at play that induce distinct immune responses specific for pathogen and self antigens independent from "molecular mimicry."

Ceruloplasmin carries the anionic glycan oligo/poly alpha2,8 deaminoneuraminic acid.

Oligo/poly alpha2,8 deaminoneuraminic acid (KDN), a unique posttranslational protein modification, was found on megalin and a not yet characterized 150 kDa glycoprotein. We purified this glycoprotein from rat testis and identified it as ceruloplasmin. Furthermore, immunoprecipitated ceruloplasmin from rat thymus, ovary, blood serum, and postnatal day 2 but not adult lung and brain was immunoreactive for oligo/poly alpha2,8 KDN. The immunoreactivity for oligo/poly alpha2,8 KDN on purified serum ceruloplasmin was abolished by N-glycosidase F treatment but not by beta-elimination, indicating that it is present on N-glycosidically linked oligosaccharides. However, the copper binding activity of ceruloplasmin was independent of the presence of the anionic glycan. By immunohistochemistry, ceruloplasmin was detectable in histologically defined regions in rat ovary, thymus, and spleen. Likewise, by RT-PCR, ceruloplasmin expression was found in various non-hepatic rat tissues and showed a developmentally regulated pattern. Thus, ceruloplasmin, in addition to megalin, represents a glycoprotein carrying oligo/poly alpha2,8 KDN.