Abstract Immune cells protect us from disease by detecting and responding to foreign molecules. Understanding the molecular basis of this response is critical if we are to generate new therapies for treatment or prevention of diseases involving immune cells. The objective of our research is to characterize a newly discovered antigen detecting protein (butyrophilin 3, BTN3) which influences the immune response mediated by gamma delta T cells. Gamma delta T cells are cytotoxic T cells that respond quickly to foreign threats and serve a variety of roles, including direct lysis of infected or malignant cells, and as such, their activation holds great promise for therapeutic manipulation. In contrast to T cells that express the more prevalent alpha beta T cell receptor and respond to peptide antigens, T cells that express the Vgamma9Vdelta2 T cell receptor respond to small phosphorous-containing compounds known as phosphoantigens. Butyrophilin 3A1 (BTN3A1) is the receptor for phosphoantigens and mediates their activation of T cells through unclear mechanisms. We developed a library of novel synthetic phosphoantigens, as well as a library of butyrophilin constructs and point mutations, both of which are valuable tools for understanding the underlying biology of butyrophilins. Here, we propose aims that test the underlying hypothesis that ligand binding to the intracellular domain of BTN3A1 produces conformational and organizational changes that are required for interaction with counter receptors on T cells. Understanding how BTN3A1 and the related 3A2, 3A3, and 2A1 isoforms function at the molecular level is important because it 1) will help optimize past and present clinical trials that have examined phosphoantigens and phosphoantigen- expanded cells as immunotherapies, and 2) will identify new molecular targets or strategies within this complex for therapeutic manipulation. Our studies in Aim 1 will show a structural basis for how phosphoantigens affect the full length endogenous BTN3A1 using multiple biophysical and molecular biological approaches. In Aim 2, we will investigate the function of BTN3A1 in phosphoantigen-induced Vgamma9Vdelta2 T cell lysis of phosphoantigen containing cells and associated cytokine production. Together, this will allow us to build a structure-function model of BTN3 with regards to how its domain organization, oligomerization status, protein- protein interactions, and relationship to BTN2A1 influence its function. This will largely be done in the context of biological membranes through use of a novel in vitro membrane nanodisc/cryo-EM model system. Our ultimate goal is to present a clear structural model that demonstrates how phosphoantigen-induced conformational and/or compositional changes in the BTN3 complex promote effector functions of T cells. This will enable clinical development of therapies that modulate butyrophilin function to overcome immune checkpoints. These findings will come at a point when the biological...