Antigen-specific depletion of autoreactive B lymphocytes in multiple sclerosis

Multiple sclerosis (MS) is an inflammatory demyelinating autoimmune disease of the central nervous system triggered by the body's own immune system reacting against self-antigens. Autoreactive B and T lymphocytes as well as activated macrophages and the bodies complement system contribute to, and maintain the inflammatory lesions in brain and spinal cord. For over 20 years, especially autoreactive T lymphocytes attracted major attention due to their predominant presence in acute inflammatory lesions, their potential to transfer an autoimmune disease from one organism to another (EAE mouse model) and the link between specific MHC class II alleles and specific autoimmune diseases like rheumatoid arthritis, multiple sclerosis and type I diabetes mellitus. Presently antibody-secreting B lymphocytes get more and more in the focus of autoimmune diseases researchers because of recent strong evidence of their central role in the induction and maintenance of inflammatory reactions. Their main pathologic potential is based on the secretion of autoreactive antibodies that are able to bind and opsonize autoantigens, followed by stimulation of macrophages and fixation of the body's complement system. Moreover, autoreactive B cells can strongly enhance the T cell response by presenting processed autoantigens in combination with MHC molecules on their surface. In this thesis, novel autoantigen-specific fusion proteins for the targeting and elimination on one hand and the staining of autoreactive B cell populations on the other hand were successfully generated. The membrane-bound B cell receptors of the target cell populations and their specificity to a unique antigen will serve as target for the recombinant fusion proteins. For staining purposes, the extracellular domain of the myelin oligodendrocyte glycoprotein (MOG) was genetically fused to the enhanced green fluorescent protein (eGFP). The protein was characterized by western blotting and ELISA using the supernatant of MOG-specific 8.18-C5 hybridoma cells. In flow cytometry, the eGFP-MOG fusion protein specifically stained the above mentioned 8.18-C5 cells and isolated splenocytes from transgenic IgHMOG mice via their surface B-cell receptors. For the targeting and specific elimination of autoreactive B cell populations, the autoantigens MOG and PLP (proteolipid protein) were recombinantly fused to the truncated form of Pseudomonas exotoxin A (ETA'). As a negative control for subsequent toxicity assays, both proteins were cloned without toxic moiety. The so-called 'autoimmunotoxins' MOG-ETA' and PLP-ETA', as well as their counterparts without toxin MOG and PLP, were efficiently expressed in bacteria. After their purification via IMAC and size exclusion chromatography (SEC), they all showed a high purity as analyzed by SDS-PAGE and subsequent western blotting using their antigen-specific hybridoma cell supernatants 8.18-C5 (anti-MOG) and F4-2D2 (anti-PLP). The binding analyses in western blot, ELISA and further on in flow cytometry analyses was successful revealing a high specificity of all expressed proteins. The binding of MOG-ETA' and MOG to isolated splenocytes from IgHMOG mice was additionally verified by FACS analysis. MOG-ETA' and PLP-ETA' were used for XTT viability assays and demonstrated a highly specific, either strong (MOG-ETA') or moderate (PLP-ETA') toxicity on their specific target hybridoma cell line. As the expressed autoantigens without toxin moiety revealed no toxicity upon incubation with the target cell line, the toxic effect of the autoimmunotoxins can attributed to functional release of ETA' into the cytosol, followed by inhibition of the protein synthesis and the apoptosis of the cell. Subsequent toxicity analyses were carried out using isolated splenocytes from transgenic IgHMOG mice. MOG-ETA' was incubated with MOG-reactive splenocytes in different protein concentrations and its depleting effect could be clearly confirmed by quantitative FACS analyses. Successive animal experiments were carried out using two different mouse models. First the toxic effect of MOG-ETA' was investigated in transgenic IgHMOG mice. The daily intravenous application of MOG-ETA' administered over a time period of 5 days was able to specifically reduce the number of MOG-reactive B lymphocytes to approximately 50% in comparison to control animals. As the transgenic IgHMOG mice are not the model of choice because they are characterized by high titers of MOG-reactive antibodies which are able to intercept applied immunotoxin, the targeting and depletion of MOG-reactive B lymphocytes turned out to be too challenging in this model. The use of a MOG-induced EAE model in DBA/1 mice for in vivo evaluation of the MOG-ETA' autoimmunotoxin better represents the nature of the inflammatory reaction to be found in multiple sclerosis lesions. The application of the MOG-ETA' autoimmunotoxin had a positive therapeutic effect on EAE-induced animals as the disease development was almost completely prevented in the treated group. The control group was injected with an immunotoxin that was comprised of a non-relevant binding ligand but the same toxic moiety as in the autoimmunotoxin MOG-ETA'. As these animals revealed a severe clinical course of 10 days after EAE induction, the specific in vivo mode of action of the MOG-ETA' autoimmunotoxin was clearly demonstrated. In conclusion, the present work provides the proof-of-principle for a novel approach that may have a strong positive impact on antigen-specific therapies in autoimmune disorders.