Our principle objective is to understand the cellular and molecular mechanisms leading to the development of a mucosal immune response. Such a response acts to prevent, limit, contain, and resolve bacterial and viral infections that commence via the respiratory or gastrointestinal epithelium. Since the IgA isotype of antibody is primarily responsible for humoral immunity expressed at mucosal surfaces we are particularly concerned with the aspects of B cell development leading to antibody isotype restriction and commitment to expression of the IgA isotype. Our research program has four main aims: 1) to discern those characteristics of vaccines and adjuvants that make them effective at stimulating and priming for a secretory antibody response by the mucosal route; 2) to determine the cellular and molecular requirements for stimulating IgA memory cells to proliferate and generate clones making IgA antibodies; 3) to assess whether germinal centers, especially those in Peyer's patches (PP) in the walls of the gut, are sites of isotype switching, variable region gene mutations, and diversification of B cells to pre-memory or preplasma cells; and 4) to determine whether mucosal follicles in respiratory and gastrointestinal tract function tract function similarly as sites of IgA memory cell development and whether 'cross-priming' can occur when antigen is applied to either mucosal surface. To address these goals we will prepare hapten-conjugates of cholera toxin, the most efficacious non-replicating antigen known, to stimulate a mucosal IgA response via the gut route. These conjugates as well as Mycoplasma pulmonis, reovirus type 1, and Morganella morganii (a gram-negative enteric bacteria) will be administered by the mucosal route to prime mucosal lymphoid tissue for specific IgA responses. Clonal murine B cell cultures will be used to establish growth, differentiation, and regulatory stimuli for IgA memory cells by adding subsets of T-helper cells, dendritic cells, cytokines, Ig-binding factors, and putative regulatory T cells. Germinal center (GC) and non-GC PP cells will be resolved based on MAbs and lectins that define surface phenotypes and their functional potential will be tested in vitro and in vivo. Molecular genetic analyses will be used to probe for isotype switching, point mutation, and shifts in mRNA for secreted versus mucosal stimulation. Both allotype congenic immunocompetent and severe combine immunodeficient mice will be used to assess functional potential and protective role of subsets of GC and non-GC PP cells.
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