Somatic Diversification of Immunoglobulin Genes in Galt

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DESCRIPTION (provided by applicant): The intestinal commensal microbiota is required for development of lymphoid tissues and mucosal immunity in vertebrates. However, little is known of the mechanism by which these processes occur. Previous work in rabbits has demonstrated that the intestinal commensal microbiota is also required for somatic hypermutation (SHM) and gene conversion (GC) of Ig genes during development of the preimmune antibody repertoire, and for B cell selection, both of which occur in gut-associated lymphoid tissues (GALT). By introducing select bacterial strains into sterile GALT, we found that some but not all commensal organisms have the capacity to induce GALT development and SHM and GC of Ig genes. We identified two commensal microorganisms, B. fragilis and B. subtilis, that together, induced these processes; however, introduced singly, neither organism induced these processes. The goal of this study is to determine the mechanism by which these bacteria induce GALT development and SHM and GC of Ig genes, and why two bacteria are required. The first specific aim is to determine how bacteria enter the host, and if the difference in follicle-inducing bacteria and non-follicle-inducing bacteria resides in how they are taken up across the epithelium of GALT. Experiments to test this include introducing bacteria into germfree appendix and then performing in situ hybridization and immunofluorescence to determine in which cells the bacteria are localized. The next specific aim, by using soluble receptors expressed in recombinant adenovirus, will investigate whether dendritic cells (DC) contribute to GALT development and Ig gene diversification by secretion of the B cell growth factor, BAFF, or by inhibiting the transepithelial migration of DC in vivo. In Aim 3, the requirement for T cell help will be investigated by inhibiting the activation of T cells with soluble CTLA4 and B-T cell interactions with soluble CD40, both produced in recombinant adenovirus. In Aim 4, experiments are designed to search for a B cell superantigen that induces GALT development and somatic diversification of the primary antibody repertoire. These experiments are important because the lumen of the intestine contains 10X more bacterial cells than all other cells of the human body combined, and yet we know little about how these organisms contribute to human health and diseases such as allergy and inflammatory bowel disease.