Project Summary/Abstract Affinity maturation increases the binding strength of the B cell receptor for its target antigen, and is a fundamental mechanism affecting vaccine efficacy, autoimmune diseases, and the development of immunotherapies. Historically, this process was believed to occur only in endothermic vertebrates (i.e. birds and mammals), taking place in specialized structures called germinal centers, which form in the B cell follicles of the spleen and lymph nodes during an immune response. Here B cells mutate their immunoglobulin genes to produce a greater diversity of receptors. The B cells then migrate within the germinal center to interact with T follicular helper cells and follicular dendritic cells, which present intact antigen for Darwinian selection of B cell clones. More recently, and despite an absence of germinal centers and true follicular dendritic cells, it has been definitively shown that cold-blooded vertebrates are capable of some level of affinity maturing their antibody responses. This finding leads to the hypothesis that a primordial B cell selection structure, or germinal center precursor, preceded the complex stringent selection structures present in mammals. This project will investigate B cell selection and affinity maturation in the nurse shark, a model organism that belongs to the oldest extant taxonomic group (Chondrichthyes) to possess B cell receptors, T cell receptors, and major histocompatibility complex. It has previously been demonstrated that nurse sharks are capable of both affinity maturation and immunological memory. Additionally, the nurse shark has an immunoglobulin class consisting of only heavy chain that does not associate with light chain, IgNAR, well suited for B cell receptor repertoire analysis. These characteristics make the nurse shark an ideal model to study the evolution of B cell selection and affinity maturation. The evolution of B cell selection will be addressed through three avenues of investigation: first, the sites of B cell selection will be identified in the nurse shark spleen, then assessed for their cellular architecture, B cell repertoire, and cytokine transcriptome through immunofluorescent staining, laser capture microdissection, and high throughput sequencing. Second, the nurse shark primary and affinity matured B cell repertoires will be analyzed by sequencing and isolation of antigen-specific IgNAR clones via phage display; these will be sequenced to evaluate clone diversity, then expressed as recombinant proteins to assess their binding affinity. Finally, the nurse shark memory response will be characterized to determine the diversity and affinity of clones selected for long-term immune protection and identify the signaling cytokines that maintain long-lived B cells in sharks. The results of these aims will provide a greater understanding of the evolution of B cell selection, somatic hypermutation, and affinity maturation.