PROJECT SUMMARY/ABSTRACT Immune checkpoint blockade has revolutionized the therapeutic landscape of patients with metastatic cancer. However, despite remarkable outcomes in some patients, only a minority achieves complete and durable clinical response. To overcome this, combination of radiotherapy and immune checkpoint blockade has gained popularity to turn up the heat on cold tumors. While tumor irradiation can elicit an immunogenic cell death triggering tumor antigen presentation and T-cell priming, immune checkpoint blockade can enhance the expansion of tumor-reactive T cells which culminates with regression of distant non-irradiated metastases, referred as “abscopal effect”. The synergy of radiotherapy and current immune checkpoint blockade has shown to improve oncological outcomes in some patients but rates of abscopal effect remain scarce. Our long-term goals are to develop new effective therapeutic combinations to boost abscopal response and improve clinical outcome of patients with advanced cancers. To that end, there is a critical need to shed the light on the underlying mechanisms of poor response to radiotherapy. Our group has generated compelling functional evidence implicating the immune checkpoint molecule B7-H3 as a key mediator of tumor immune evasion that may act both locally by impairing T cell-mediated cytotoxicity and systemically through release of immunomodulatory tumor-derived extracellular vesicles (tdEVs). The overall objectives in this application are to gain a better understanding of the role of B7-H3 In local and systemic antitumor immunity and evaluate the therapeutic potential of targeting B7-H3 function with immune checkpoint inhibitors. In line with this, our central hypothesis is that tumor B7-H3 acts as a “don’t touch me” signal which inhibits the cytotoxic function of CD8 T cells. We will test this hypothesis through two aims. In Aim 1, we will dissect the mechanisms of B7-H3-induced CD8 T cell dysfunction. To that end, we will use proximity labeling technologies and time-lapse imaging of T-cell mediated cytotoxicity. We will also employ immunocompetent mouse models humanized for B7-H3 to evaluate the safety and therapeutic efficacy of a newly developed B7-H3 inhibitor. In Aim 2, we will elucidate the mechanisms of systemic immunosuppression mediated by B7-H3+ tdEVs in response to radiotherapy using genetically engineered mouse tumor cells and in vivo proximity-dependent biotinylation. We have assembled a multi- disciplinary team with complementary expertise in extracellular vesicle biology, radiation oncology, tumor immunology and proteomics to validate B7-H3 as a therapeutic target to boost abscopal response in patients with advanced cancers treated with radiotherapy.