Project Summary/Abstract Triple-negative breast cancer (TNBC) is highly aggressive and uniformly lethal in the metastatic setting despite recent approvals of immuno-therapeutics for this disease. Outcomes could potentially be improved by closing gaps in our understanding of immunological control of TNBC. A subset of human TNBCs (6-20%) strongly overexpress the immune receptor VISTA (V-domain immunoglobulin suppressor of T cell activation), which bears close homology to the drug target PD-L1. Clinical trials in humans are now evaluating the safety and efficacy of targeting VISTA’s extracellular domain for cancer. However, we lack a mechanistic understanding of how VISTA signals to the interior of the cell. Preliminary data show that VISTA+ TNBCs have immunologically “cold” tumor microenvironments and decreased proliferative index. These effects are controlled by both the extracellular and intracellular domains of VISTA. By purifying proteins bound to the intracellular domain, we have established that VISTA can recruit and sequester cytoplasmic clathrin-adaptor proteins, causing defects in plasma membrane receptor endocytosis and trafficking. Consequently, important immunological and cancer growth receptors like TLR4 and EGFR do not function effectively when VISTA is expressed. This leads to the hypothesis that VISTA’s intracellular domain is a cell-intrinsic repressor of membrane receptor trafficking and that targeting both VISTA’s extracellular and intracellular domains is critical to improve VISTA therapeutics. This hypothesis will be tested in three specific aims: (1) To mechanistically dissect how VISTA blocks receptor trafficking, we will characterize how proteins bind to VISTA’s intracellular domain; (2) Define the mechanistic roles of VISTA’s intracellular domain binding partners in receptor trafficking and activity; and (3) Evaluate therapeutic strategies for VISTA+ triple-negative breast cancers. These aims will be accomplished through biochemical analysis of purified proteins and cell lines with engineered mutations to disrupt clathrin adaptor binding to VISTA’s intracellular domain. Analysis will include the effects of VISTA in both tumor cells that overexpress VISTA to model human TNBCs and immune cells that naturally express VISTA for normal physiology. Combination regimens to explore the effects of blocking both extracellular and intracellular functions of VISTA will be tested in immunocompetent mouse models of triple-negative breast cancer. This work should address the need to develop novel immunomodulatory agents to improve outcomes for TNBC. Although VISTA-targeted therapeutics are currently in clinical development, all current approaches exclusively target VISTA’s extracellular domain. Our studies have discovered that VISTA’s intracellular domain is a critical determinant of receptor function that we propose to extensively characterize to catalyze improved VISTA-targeted therapies.