Self-Antigen Localisation and B Cell Self-Tolerance

B cells are a crucial component of the adaptive immune system that allow for the specific recognition of, and subsequent production of antibodies against, a wide spectrum of foreign antigens. The required diversity of the B cell repertoire is achieved firstly during early B cell development through V(D)J recombination of immunoglobulin variable region genes and, after antigen-dependent differentiation into germinal centre (GC) B cells, via mutation of variable region genes by somatic hypermutation (SHM). While these two mechanisms make it possible to generate a large and diverse B cell repertoire to fight infections, their largely random nature of also results in the production of B cells that target self-antigens. Under normal circumstances there are multiple complementary mechanisms that regulate the development and survival of such B cells during development and during immune responses to prevent the production of potentially pathogenic autoantibodies. Previous work by others and our group have demonstrated that self-reactive B cells directed against membrane self-antigens generated during B cell development or during the immune response through SHM are efficiently deleted from the repertoire. However, in many autoimmune conditions, pathogenic autoantibodies are generated against intracellular, organelle-associated antigens. This process is presumably blocked under normal circumstances, but it remains unknown how during B cell development and the immune response tolerance against intracellular self-antigens is maintained. To investigate these questions, a variety of transgenic mouse models were developed expressing the HEL4X model self-antigen within various intracellular organelles, including the endoplasmic reticulum (ER), cis-Golgi (CG) and trans-Golgi (TG), in addition to versions expressed on the cell surface plasma membrane (m) or a secreted, soluble form (SC). To explore central and peripheral tolerance, HEL4X self-antigen transgenic mice were crossed to various BCR transgenic lines the produced B cells with varying affinities for the self-antigen. Developing B cells with high or intermediate affinity for membrane bound self-antigen were efficiently deleted early in their development. In contrast, developing B cells with high affinity for self-antigens targeted to the ER, CG or TG are not impacted by (are ignorant of) their own intracellularly expressed self-antigen and not significantly impacted by the small but detectable amounts of self-antigen that was located extracellularly in each case. Notably, secreted self-antigen provided positive selection signals apparently through potentiating tonic BCR signalling. In addition, TG as well as ER localised self-antigen was found to promote B1 B cell production and natural secretion of anti-HEL4X IgM autoantibodies. Within the GC, and in contrast to the primary repertoire, B cells that became reactive to HEL4X following SHM were eliminated regardless of the intracellular or extracellular targeting of the self-antigen. This occurred independent of self-antigen expression by GC B cells themselves and was mediated via externalised self-antigen in the case of HEL4X targeted to the ER, CG, or TG. Nevertheless, suppression of early production of anti-HEL4X autoantibodies only occurred when HEL4X was expressed on the plasma membrane, indicating that the pace with which self-reactive GC B cells were removed was slower in response to intracellular and secreted self-antigens. Overall, the work described in this thesis demonstrates that the threshold for removing self-reactive B cells in the GC is considerably higher than in the primary repertoire, potentially due them being poised for differentiation into autoantibody-producing plasma cells.