Klonale experimentelle autoimmune Enzephalomyelitis: Charakterisierung myelinspezifischer Antikörper in einem TH2-induzierten adoptiven Transfermodell

Die Multiple Sklerose ist in Europa und Nordamerika die häufigste Entmarkungserkrankung des zentralen Nervensystems. Sie ist neben Inflammation und Demyelinisierung auch durch neurodegenerative Prozesse gekennzeichnet. Hierbei spielen T-Zell-vermittelte Mechanismen eine wesentliche Rolle. Aufgrund d...

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Bibliographic Details
Main Author: Sieren, Michael
Contributors: Sommer, Norbert (Prod. Dr.) (Thesis advisor)
Format: Doctoral Thesis
Published: Philipps-Universität Marburg 2012
Online Access:PDF Full Text
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Multiple sclerosis (MS) is a chronic inflammatory demyelinating disease of the central nervous system. Clinical manifestation and progression of neurological deficits are highly heterogeneous and unpredictable. Clinical heterogeneity is reflected by four distinct histopathological patterns and defined patterns of cerebrospinal fluid pathology. T-cell mediated mechanisms seem to play a pivotal role in the course of disease. Furthermore humoral immunity seems to contribute essentially to inflammation in several subtypes of MS. Experimental autoimmune encephalomyelitis (EAE) is an animal model of MS. It can be either induced by active immunization with myelin components or by adoptive transfer of myelin specific CD4+ TH1-cells. A shift towards a TH2-response however has been considered to lead to an amelioration of the clinical course. Recent developments revealed that this TH1-TH2-paradigm is an oversimplification of the complex immune response during the course of EAE. Further T-cell subpopulations seem play a pivotal role in the pathogenesis of EAE. Adoptive Transfer of TH17-cells e.g. is a very efficient way of EAE-induction without prior in-vitro stimulation. In addition adoptive transfer of clonal proteolipidprotein (PLP) 139-151 specific TH2-cells to immunocompetent non-transgenic SJL-mice in a new model leads to induction of an EAE with a chronifying course of disease. Demyelinating lesions in the chronic phase of disease contain B-cells, plasma cells, macrophages and massive deposits of antibodies. Furthermore PLP139-151 specific antibodies as well as antibodies against other myelin components can be detected in the serum of the animals. The aim of this study is the further characterization of the antibody response in this new TH2-induced EAE model. After EAE-induction by adoptive transfer of the prior established PLP139-151 specific TH2 clone 3-3 spleen cells were obtained during the chronic phase of disease approximately 90 days after T-cell transfer. By fusion of these cells with cells of the Sp2 myeloma cell line antibody producing hybridoma cells were created. By cloning in limiting dilution technique followed by affinity chromatography of cell culture supernatants, monoclonal myelin specific antibodies were purified. For further characterization the so gained monoclonal antibodies were tested in myelin specific ELISAs and Western Blots, in immunohistochemistry on central nervous system (CNS) tissue samples and in adoptive transfer experiments. A distinctive feature of this model is that monoclonal antibodies were generated without prior active immunization with myelin antigens. This offers a completely new more physiologic approach to characterize T-cell-B-cell interactions and the role of antibodies in a new TH2-induced EAE-Modell. In the first part of the study selected monoclonal PLP139-151 specific antibodies of isotype IgG1 and IgG2b were purified and characterized as described before. PLP139-151 specifity could be shown in a peptide coated ELISA. Neither in Western Blots nor in immunohistochemistry with CNS tissue samples could reactivity against PLP or other myelin components be observed. Adoptive transfer of antibodies in SJL-mice pre-treated with Pertussis toxin could not induce clinical symptoms of EAE. In the second part the study therefore was extended to monoclonal antibodies specific for other myelin components. The monoclonal myelin oligodendroglia glycoprotein (MOG) 35-55 specific antibody 4E6-2 stained MOG in a Western Blot as well as myelin on paraffin embedded CNS tissue sections. The antibody was tested in adoptive co-transfer experiments in SJL-mice after prior EAE-induction by adoptive transfer of cells of the myelin specific TH1 clone cPLP 25. This experiment did not reveal a disease modifying effect of 4E6-2. For clarification of the role of antibody mediated mechanisms in our new TH2 mediated model of EAE in SJL-mice further characterization of these antibodies in regard to their effector functions like e.g. their ability to fix complement has to be carried out. Nevertheless the findings support the concept of pathogenetic heterogeneity of EAE and MS and might have an impact on the struggle for new therapeutic approaches.