PROJECT SUMMARY/ABSTRACT A diverse accumulation of infiltrating immune cells is a prominent feature of nearly all cancers1. By directing the effector functions of cancer-killing T cells, the immune system has the ability to specifically target and mediate clearance of large established tumors2–5. However, T cell effector function is typically constrained within tumors, allowing disease progression. Recent insights into how tumors limit T cell function have translated into new classes of cancer treatments such as immune checkpoint blockade (αCTLA-4 and PD-1) and the adoptive transfer of large numbers of autologous tumor specific T cells6,7. While immune based therapies have transformed the practice of oncology over a short period of time, such treatments only work in a small percentage of patients, as cancers deploy multiple and non-redundant means to evade the immune system. As our knowledge of the biologic drivers of tumor induced T cell dysfunction remains limited, the bounds of cancer immunotherapy remain undefined. A high abundance of cell death is found in many tumors and is associated with poor prognosis. Cell death results in the release of intracellular potassium (K+), thereby increasing the extracellular potassium ([K+]e) the tumor microenvironment. We first recognized that this that high [K+]e suppresses T cell antitumor function and cytokine production, limiting antitumor immunity (Eil R et al, Nature 2016 & Science 2019). Mechanistically, K+ mediated suppression of T cell signaling required the phosphatase activity of PP2A. Genetically reprogramming of T cell K+ transport translated into improved functionality in vitro and in vivo. While these findings point to an unrecognized ionic checkpoint resulting from cancer cell death byproducts, others have proposed cell death and tissue damage to increase immune activity due to the release of damage associated molecular patterns (DAMPs) and inflammasome activation in myeloid cells. However, inflammasome signaling is also suppressed by high [K+]e. Thus,our central hypothesis is thatintracellular K+ is a central regulator of immunobiology that can be leveraged to augment T cell antitumor function. Intrahepatic Cholangiocarcinoma (ICC) is the second most common cause of primary liver cancer and is fatal in nearly all cases unless surgically resected owing to resistance to cytotoxic and targeted therapies19,20. ICC is characterized by a high degree of genomic intratumoral heterogeneity and necrosis as well as a recently appreciated responsiveness to T cell-based immunotherapy, providing an ideal context in which to pursue the research proposed herein. If successful, the research proposed in this submission will, 1) define the degree of immune suppression enforced by cell death byproducts in the context of human intrahepatic cholangiocarcinoma; 2) test novel cancer treatments based on augmenting T cell ion transport 3) clarify previously unrecognized aspects of cellular biology controlled by intr...