Project Summary The B cell compartment is comprised of many individual cells, each of which may express a distinct B cell receptor (BCR). Upon antigen engagement, B cells proliferate resulting in clonal expansion, and may further diversify their BCR genes through somatic hypermutation (SHM) and class switch recombination. These two processes allow for affinity maturation toward the insulting antigen and the distribution of Ab reactivity across isotypes which interface with other components of the immune system defining an Abs effector function. Clonal expansion and diversification result in the presence of expanded groups of B cells with similar BCRs (clonal lineages). The overarching goal of this program is to investigate the evolution and durability of immune responses to allo, self, and foreign antigens. Although sequencing of BCR genes allows the visualization of cell expansions and diversification that occur during the evolution of B cell responses, sequence information alone is insufficient to evaluate changes in antigen binding that occur as immune responses evolve. The Antigen Receptor Sequencing, Cloning, Expression, and Analysis Core (Core B) will facilitate the objectives of this program by allowing the analysis of these two distinct but related features of humeral immunity. We will support the aims of the projects through the following activities: i) Generating antigen receptor sequence information. Projects require repertoire level sequence information form bulk and sorted lymphocytes, BCR for Projects 1 and 3, TCR for Project 2. Core B will centralize the processing and quality control involved in generating next-generation of antigen receptor libraries. We will coordinate with the Projects and with Core D, the Data and Informatics Service Core, for analysis. ii) Generating Abs by cloning and expressing the BCR of single-sorted antigen-reactive B cells as recombinant Ab. Both Projects 1 and 3 of this proposal are evaluating the reactivity of antigen-reactive B cells to a variety of specificities, allo- and auto-HLA, and viral pathogen derived antigens. Using a high- throughput Ab cloning and expression system, Core B will produce recombinant Abs from the cloned BCR of single-sorted antigen reactive B cells across reactive with these antigens. iii) Evaluating the specificity, relative affinity, and cross reactivity of Abs. Because understanding the effects of SHM, the relation of antigen reactivity within and across expanded lineages, the relative affinity of Abs to their antigens and related structures requires physical measurements of binding, Core B will evaluate Abs reactivity profiles using multiplexed cytometric arrays. iv) Core B will generate Ab and Fab expression vectors to support the bio-physical measurements of Ab binding and structural studies performed by Core C. Collectively, these activities will promote synergy between the Projects and the Cores of this proposal and accelerate the research by providing access to spec...