PROJECT SUMMARY As one of two lineages of extant jawless vertebrates, lampreys represent a pivotal branch of vertebrate phylogeny; they offer a critical source of information for understanding the evolutionary origins of our adaptive immune system. While immunoglobulin domain-based receptors mediate adaptive immunity in all jawed vertebrate groups, the variable lymphocyte receptor (VLR) system in lampreys and hagfishes is based on receptors constructed of leucine-rich repeats (LRR). The VLRs are assembled by a gene conversion-like process that sequentially copies sequences donated by fragmentary LRR cassettes into an incomplete germline VLR gene. Although structurally unrelated to immunoglobulin and T-cell receptors, the function of three types of VLRs are nonetheless rooted in B- (VLRB) and T-like lymphocytes (VLRA and VLRC), indicating that the basic genetic programs for these cell types are a fundamental feature of adaptive immunity in all vertebrates. The proposed studies focus on the diversity, functions and translational application of the lamprey VLRB antibodies, which are expressed in a cell-surface form on B-like lymphocytes and as secreted polymeric antibody by mature plasma cells. Our first aim is to characterize the development of the VLRB system beginning with early feeding larvae and thereafter in multiple tissues of three-to-four-year larvae and adults. CRISPR/Cas9 strategies will be used to perturb the function of candidate B-like cell regulators and signal mediators in embryos and larvae. We will characterize the developing VLRB repertoire by analyzing high fidelity long-read sequence data for each lymphopoietic tissue and developmental stage. We will also analyze scRNA-seq data from early larva and ammocoete tissues to identify and characterize subsets of B-like cells and their stages in differentiation. In aim two we will characterize the composite VLRB receptor. VLRB is expressed as a GPI-linked protein on the surface of lamprey B-like cells, therefore implying that antigen-induced signaling requires associated transmembrane domain proteins with cytoplasmic signaling capacity. We will conduct pull down assays on extracts from lamprey VLRB lymphocytes using VLRB specific monoclonal antibodies and use mass spectrometry to identify coprecipitated proteins. This analysis will be guided and supplemented by a set of candidate signal mediators that we have identified from VLRB cell transcriptomes and scRNA-seq datasets generated in these studies. In aim three we will produce and characterize monoclonal lamprey VLRB antibodies for basic and translational applications. The analysis is focused on VLRB antibodies against highly lethal brain (glioblastoma and astrocytoma) and ovarian tumors.