Differences between mouse and human γδ T cells have hindered a better understanding of the role of these innate lymphocytes in human cancer, due in part to the lack of relevant transgenic models. Key advances in the previous funding cycle of this grant underscored the crucial role of γδ T cells in human ovarian cancer and how butyrophilins govern their phenotypes. Building upon our new preliminary results, our central hypothesis is that that BTNL9, in a lipid-dependent manner, activates crucial subsets of anti-tumor δ1 T cells in an “innate-like”, CDR3-independent fashion, through binding to specific variable γ regions. Furthermore, novel allogeneic cellular therapies using exhaustion-resilient δ1 T cells as a CAR T cell platform, in a BTNL9-dependent manner, will be more effective at in halting the progression of epithelial human cancers, compared to conventional αβ CAR T cells, particularly in tumors with low antigenic expression. These hypotheses will be tested in these Aims: In Specific Aim 1, we will elucidate the detailed mechanism by which BTNL9 activates various subsets of γδ1 T cells in a lipid-dependent manner. Based on this knowledge, we will also define the crucial role ofBTNL9- dependent Vδ1 TILs in anti-tumor immunity in human cancer, in conjunction with other γδ T cell subsets. In Specific Aim 2, we will characterize the heterogeneity and trajectory of evolution of human cancer- infiltrating γδ1 T cells. These studies will define the anti-tumor activity of different γδ T cell subsets and the molecular drivers of the evolution of protective phenotypes in vivo in human cancer. In Specific Aim 3, we will evaluate new allogeneic cell therapies that exploit the resilience of Vδ1 T cell effector activity in human epithelial cancer. These experiments will provide a rationale for ensuing CAR T cell therapies using Vδ1 T cells and γδ TCR signals as a potentially superior platform for controlling the progression of CAR T cell-refractory malignancies, as compared to autologous conventional αβ T cells. These studies will not only address the knowledge gap concerning the interaction between butyrophilins and γδ T cells in human cancer, but also establish a mechanistic rationale for novel cellular therapies that can effectively combat aggressive and common solid human cancers, paving the way for "off the shelf" CAR T cell therapies.