B cell Receptor repertoire, cloning and expression Core

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Core C: B cell Receptor repertoire, cloning and expression Core Project Summary B cells express a widely diverse repertoire of B cell antigen receptors (BCRs) that can be further diversified by isotype-switching of heavy chain constant regions and by somatic hypermutation (SHM) in the antigen-binding domains of both heavy and light chains. B cells responding to protein antigens, such as viral antigens or transplant antigens, are often expanded in the germinal center, which stringently selects for high affinity memory B cells and antibody-secreting cells (ASCs). In contrast, B cells responding to carbohydrate antigens, such bacterial capsule components or blood group antigens, rarely enter the germinal center, but are nonetheless highly-selected based on reactivity and cross-reactivity to various antigens. The processes of clonal expansion and selection leads to the formation of B cell lineages that are related by BCR sequence and antigen-specificity. Importantly, the characteristics of the BCR (V gene use, SHM, isotype, affinity, cross-reactivity) often determine the functions of individual B cells, the tissues in which they reside, and the effector activities of the antibodies they produce. However, we have a limited understanding of antigen-specific B cells in the non-lymphoid tissues of humans. Therefore, the projects of this U19 will characterize B cells specific for different types of antigens (glycans in Project 1, viral antigens in Project 2 and xenoantigens in Project 3), obtained from lymphoid, mucosal and adipose tissues of normal human donors. Importantly, each project will determine the biochemical and molecular characteristics of antibodies made by individual B cells, determine the clonal relationships between antigen-specific B cells in different locations and use cytometric bead arrays to characterize the specificities, cross-reactivities and affinities of antibodies made by individual B cells. In order to achieve these goals and accelerate the research activities of each project, Core C will sort individual B cells from selected populations into 384-well plates, amplify VH and VL gene segments, clone the amplicons into IgG expression vectors, transfect the vectors into eukaryotic cells and produce recombinant monoclonal antibodies. Core C will also perform BCR heavy chain repertoire sequencing on sorted memory B cells and ASCs from the same tissues/donors used by the projects to sort antigen-specific B cells. Finally, Core C will develop cytometric bead arrays to enable each project to assay the reactivity profiles of serum samples, culture supernatants and monoclonal antibodies. Together, these activities will promote synergy and cooperation between projects and will accelerate the pace of research by centralizing procedures that require specialized expertise and instrumentation and by standardizing reagent development and production for each of the individual projects.