γδ T cells constitute an important component of the immune response against infectious agents and cancerous transformations, yet the biochemical mechanisms by which they detect antigen through their somatically recombined T cell receptor (TCR) remain unclear. Unlike αβTCRs, which are restricted to recognizing antigens in the context of Major Histocompatibility Complex (MHC) molecules, γδTCRs can recognize a diversity of ligands ranging from self MHC to intact, unprocessed, viral glycoproteins. Our recent work has established CD1 molecules as ligands for a subpopulation of human Vδ1 γδ T cells, producing robust functional, biochemical and structural evidence. We seek to extend our studies to the human gut, where γδ T cells, and in particular, Vδ1+ T cells, predominate. Our preliminary data suggests that CD1 recognition is robust and present in all individuals examined, and that there exist important functional differences between CD1 reactive γδ T cells in tumors versus healthy adjoining tissue. Thus, the long-term goal of this proposal is to fully characterize this CD1 reactive population in tumors versus healthy tissue, examining their functional effector phenotypes, TCR repertoire and immunomodulatory signals, in addition to the TCR, that shape the recruitment, activation and potential expansion of these cells in the context of a highly relevant human disease, colorectal cancer. Our first aim, “Characterization of CD1-specific γδ T cells in normal and diseased tissue.”, seeks to use classical cellular expansions complemented by direct ex vivo functional and transcript analysis to profile CD1 reactive and non- reactive T cell populations derived from tumor and adjoining healthy tissue. These data will provide insight into the signals that regulate γδ T cells within the tumor microenvironment compared to healthy tissue. Our second aim, “Elucidation of the molecular mechanisms by which γδ TCRs bind to CD1/lipid complexes.”, will focus on characterizing the interaction between the γδ TCRs expressed by these cells and CD1/lipid antigen. We will use protein biochemistry, biophysics and x-ray crystallography to elucide the molecular mechanisms by which the γδ TCR recognizes CD1/lipid. Our effort will significantly expand our understanding of the specific signals that regulate γδ T cell activity in human health and disease. Our third aim, “Determine the presence and role of ligand, co-stimulatory and/or co-receptor molecules in CD1 specific γδ T cell activation and phenotype in the colon” will characterize the ligand and immunomodulatory signals that may regulate the activity of CD1 reactive γδ T cells in the context of human colorectal cancer. We will combine RNAseq and differentiation assays using cord blood derived, naïve Vδ1 cells to test the relevance of candidate signals. This will be complemented by in vitro derived native Vδ1 T cells through the OP9/DL1 system. γδ T cells can be either pro-inflammatory or regulatory, therefore we seek to unders...