PROJECT SUMMARY The proposed studies address unresolved principal features of the adaptive immune system we have identified in lampreys and hagfish. Instead of the immunoglobulin (Ig) domain-based system of all jawed vertebrates, adaptive immunity in the jawless vertebrates is mediated by highly diverse sets of leucine-rich repeat (LRR) proteins, named Variable LRR Receptors (VLRs). We have shown that three types of VLR in lampreys (VLRA, VLRB and VLRC), while structurally unrelated to Ig and T cell receptors (TCRs), are nonetheless expressed by cells with functional characteristics and gene expression profiles similar to those of mammalian B cells (VLRB), αβT cells (VLRA), and γδT cells (VLRC). Here we will characterize the diversity of the agnathan T-like cells and the structures of their VLR signaling complexes. Along with VLRA and VLRC cells, we will characterize two novel VLRs that we recently identified, VLRD and VLRE, that are distantly related to VLRA and VLRC. We will investigate the receptor diversity of VLRD and VLRE and the characteristics of subpopulations of lymphocytes that express them. We have found orthologs of VLRD and VLRE in all six currently available lamprey genomes. We will extend our phylogenetic survey of the presence and genomic organization of these new receptors to more divergent lampreys and hagfishes. Like VLRA and VLRC, VLRD and VLRE are transmembrane proteins with short cytoplasmic tails that lack recognizable signaling motifs. This implies the presence of VLR-associated signaling molecules, as in jawed vertebrate TCR signaling. We will investigate the nature of signaling partners in the composite VLRA/C/D/E receptors using pulldown/mass spectrometry strategies with our monoclonal antibodies. We have identified transcripts for several candidate transmembrane proteins with ITAM motifs that are expressed by VLRA+ and VLRC+ lymphocytes. We have also identified a sea lamprey ZAP70-like kinase gene with preferential expression in the VLRA and VLRC T-like cells. We will use the sea lamprey ZAP70 tandem SH2 domains as bait to identify potential interacting ITAM proteins from lysates of activated lamprey lymphocytes. We will use single-cell RNA sequencing (scRNA-seq) to delineate the subsets of T-like cells from lamprey larval tissues. We will further characterize the early development of the T-like cells using CRISPR/Cas9 to mutate candidate regulators of VLRA/C/D/E assembly and signaling, followed by assessments of VLR expression, repertoire diversity, and lymphocyte development. We will also characterize the emergence of T- like lymphocytes in development with scRNA-seq analysis of cells isolated from early feeding larvae throughout the onset of VLR assembly. These investigations will elucidate T-like cell function in a divergent branch of vertebrate phylogeny. They will highlight common mechanisms of adaptive immunity across vertebrates, ancient features that are retained in the agnathan lineage from a common vertebra...