The VH repertoire and clonal diversification of B cells in myositis and vasculitis.
PhD thesis, University of Glasgow.
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Autoimmune inflammatory reactions occur in a number of disorders to a variety of self antigens but the precise cellular and molecular mechanisms resulting in pathology are largely unresolved. In some instances T cell mediated reactions are thought to be the main contributors to disease mechanisms but it is becoming increasingly evident that B cells, and their cognate antibodies, play a significant and contributory role in disease pathogenesis. Therefore establishing the occurrence of highly-specific B cell antigen-driven adaptive immune responses within autoimmune disorders would provide a valuable understanding into the roles of B cells within these disorders.
The work of this study sought to determine the incidence of these B cell antigen-driven adaptive immune responses in the target tissues of the autoimmune disorders myositis and vasculitis, two autoimmune disorders characterised by a wide range of autoantibodies which have been implicated in the pathological mechanisms of these diseases. This study aimed to test the hypothesis that infiltrating B cells within the target tissue of myositis and vasculitis patients were being stimulated by antigen present within the tissue resulting in clonal diversification and affinity maturation mechanisms which would contribute to the pathological mechanisms of these disorders.
Initially the cellular phenotypes and organisations of the inflammatory infiltrating cell population were characterised by immunohistochemical techniques, with particular interest on the infiltrating B cell and plasma cell populations. No typical ectopic germinal centre structures were observed in the target tissues from either disorder; cellular aggregations varied from loose aggregations to dense cellular follicles. Both B cell and plasma cell phenotypes were included in these infiltrating populations in correlation with varying numbers of cells positive for helper, cytotoxic and regulatory T cell, follicular dendritic cell, macrophage and proliferating cell markers. Double fluorescent labelling of B cells with the Ki67 proliferation marker indicated the possible expansion and clonal diversification of these cells within the target tissues. To address the main objective of this study and the possible antigen-driven mechanisms and active participation of these infiltrating B cells within the target tissue of these disorders, Ig gene repertoires, mutational characteristics, clonal diversification and affinity maturation were examined from infiltrating cells microdissected from areas of aggregation within the target tissues of both disorders. From the muscle infiltrating cells, Ig gene selections which were both patient and disease specific, mutational characteristics and oligoclonal expansion of B cells were observed establishing the involvement of muscle infiltrating B cells in the antigen-driven responses within inflamed muscle. Alternatively in the inflamed skin of vasculitis patients very few Ig gene rearrangements could be identified and no clonally related sequences or oligoclonal expansion of B cells were observed despite mutational characteristics indicating that antigen-driven diversification had occurred within these cells. Collectively the results indicate a role for B cells in the antigen-driven responses towards autoantigens within both disorders, either within an active antigen-driven response within the target tissue or at alternative sites resulting in cell migration into the target tissues.
In the final part of this study recombinant antigens were used to identify antigen-specific B and plasma cells within the inflammatory infiltrating cell population in some cases of myositis. Pilot experiments were conducted to establish the sequence characteristics of Ig genes from these antigen-specific cells.
Results of this study demonstrate the influence of B cell antigen-driven responses, either directly or indirectly, within the target tissues of myositis and vasculitis patients respectively, although the exact mechanisms leading to autoimmune reactions, and additional roles of B cells within these reactions, remain unresolved. The work presented from this study provides a foundation for further work to fully ascertain the role of B cells and antibodies within the target tissues of autoimmune disorders and in characterising the disease-associated antibodies, identifying stimulating antigens as well as assessing the effects of somatic hypermutation on the affinity and specificity of autoantigen specific antibodies. Increased understanding of B cells in these disorders will ultimately assist in the diagnosis and management of these diseases.
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