Osteosarcoma (OS) is the most common primary bone malignancy, predominantly affecting children and adolescents. About 80% of OS patients will develop metastasis within 10 years, and approximately 40% of these patients will succumb to their disease despite intense, debilitating treatments. The unrewarding clinical outcomes have landed this disease among the “most wanted” for development of new and effective therapies. Immunotherapy is dramatically changing how we treat patients with cancer. These novel antibody-based therapies do not target cancer cells directly; instead, they block immune checkpoint molecules such as CTLA-4 and PD-1, which inhibit host antitumor T-cell responses. Tumors use a variety of mechanisms to evade, deceive and suppress the host immune system. MicroRNAs (miRNAs), which repress gene expression through translational repression or posttranscriptional degradation, are secreted from cells in the tumor microenvironment (TME) in extracellular vesicles (EVs) and can perform their regulatory function in recipient cells such as T cells. We have uncovered novel, tumor-intrinsic factors that mold the tumor-immune environment. In particular, a family of conserved small RNAs (microRNAs) secreted in tumor-derived extracellular vesicles lead to destabilization of the CD28 co-receptor when T cells take them up. T cells become activated after encountering tumor antigens, primarily in lymph nodes, in a CD28-dependent process that results in proliferation and differentiation. Activated T cells must then traffic to and infiltrate into the tumor in order to directly attack the tumor cells, a process dependent on continued CD28 expression and signaling. Furthermore, release from PD-1 “exhaustion” requires reactivation of the CD28 pathway. Our central hypothesis is that immune suppressive microRNA containing EVs, secreted by osteosarcoma cells, suppress host T-cell activation and function, resulting in a deficient immune response that allows for increased tumor progression and growth. Here, we will test this hypothesis through the following specific aims: Aim 1 is designed to establish the mechanisms of CD28 silencing by OS-derived EVs in humans, mice, and dogs, and Aim 2 will define the relationship between CD28 silencing and resistance to immune checkpoint blockade in pre-clinical mouse models of OS. Impact: Our proposed mechanistic characterization of antitumor immune regulation and preclinical evaluation will form the foundation for developing novel immunotherapy strategies in the treatment of OS.