PROPOSAL SUMMARY Chimeric Antigen Receptor (CAR) T cells are genetically engineered to target and eliminate tumor cells by expressing a synthetic receptor that directs the cytotoxic function of T cells against tumor antigens 1,2. In the context of some hematologic malignancies, this living cellular therapy has proved highly potent and effective at eliminating cancer cells 3–5. However, in the context of solid tumors such as melanoma which is composed of a complex immune-tumor microenvironment, CAR T cells become dysfunctional and fail to show the same clinical promise 6,7. Elucidating the mechanisms that mediate this dysfunction and establishing a therapeutic platform to increase CAR T efficacy in solid tumors could provide a potent immunotherapy to treat wider breadth of cancers. Myeloid cells comprise a large portion of infiltrating immune cells and have the behavioral plasticity to be either suppressive-tumor promoting, or inflammatory-tumor eliminating 8–11. Antibody based immunotherapies that aim to harness the potential anti-tumor properties of myeloid cells have been developed but have limited application as monotherapies and demonstrate greater efficacy in combination with T cell initiating immunotherapies 12–15. Together, these data suggest the opportunity to combine CAR T cells with myeloid modulating immunotherapeutic to evaluate a novel combination platform to treat solid tumors. To evaluate potential synergies of CAR T and myeloid immunotherapies we propose a systems level longitudinal analysis with Mass Cytometry by Time Of Flight (CyTOF) and local and systmic cytokine profiling to evaluate immunological phenotype and function across the immune system. This approach will allow observations of emergent properties and immune trajectory analysis in settings of combination therapy. We hypothesize that CAR T cells become exhausted and dysfunctional in solid tumors because they encounter an immunosuppressive and inhibitory tumor microenvironment dictated by tumor-resident myeloid cells. Therefore, we predict that in combination with myeloid activating immunotherapies, CAR T cells will have decreased phenotypic markers of exhaustion, greater penetrance into the TME, and increased potency, resulting in progressive TME remodeling and tumor rejection. To interrogate the role of different myeloid populations, we will evaluate a dendritic cell modulating agent (aCD40), and a macrophage modulating therapy (aCD47) in combination with CAR T therapy.This proposal will elucidate mechanism of CAR T cell dysfunction in a solid tumor mouse model of melanoma and investigate a potential therapeutic platform to expand potency and efficacy of engineered cells for solid tumors.