PROJECT SUMMARY Cancer is the second-leading cause of death in the United States, causing nearly 600,000 deaths each year. A critical aspect of cancer progression is evasion of the immune system. The B7 and CD28 families of ligands and receptors mediate essential costimulatory or coinhibitory pathways; pathways that promote or suppress the immune system, respectively. B7x (B7-H4, B7S1 or VTCN1) is a newly discovered member of the B7-family that inhibits T-cell proliferation and effector functions. It is frequently overexpressed in a wide variety of human cancers, and is generally correlated with advanced disease status and poorer clinical outcomes. Previously, we demonstrated in a mouse model that B7x promotes tumor metastasis and promotes immunosuppressive cell populations such as myeloid-derived suppressor cells (MDSCs). Further, we also showed that blockade of B7x reduces tumor metastasis, alters the tumor-infiltrating immune population to favor anti-tumor effector cells, and confers long-term immunity against tumor rechallenge. Thus, we propose two aims: (1) Elucidate the regulation and function of B7x and its role in tumor progression (2) Develop and characterize anti-B7x therapy and its effect on tumor-infiltrating immune cells. For our first aim, we will explore tumor-associated hypoxia as a mechanism for B7x expression. Further, we will characterize the effects B7x has on the survival and generation of MDSCs. Lastly, we will investigate the role of B7x in tumor development and progression in vivo by generating a mouse spontaneous cancer model. In our second aim, we will develop and characterize B7x-targeted therapy. First, we have generated several clones of human B7x-specific monoclonal antibodies, which we will test for efficacy with our experimental metastasis model in vivo. Next, in context of the long-term anti-tumor immunity conferred by anti-B7x therapy, we will investigate how B7x blockade alters memory T-cell populations. Lastly, we will test B7x blockade in combination with established checkpoint inhibitors and determine if combination therapy has synergistic effects. With these studies, we seek to gain a better understanding of the functional contribution of B7x to tumor progression, and establish it as a prime target for immunotherapy.