Project Summary Immune checkpoint blockade (ICB) has produced extraordinary clinical responses in more than 25 tumor types. However, only a small number of patients benefit from this therapy owing to the immunosuppressive tu- mor microenvironment (TME). As one of the major components of the TME, tumor-associated macrophages (TAMs) usually possess profound inhibitory activity against tumor-killing T cells and facilitate tumor escape from immunotherapy. Clinical findings have shown that the presence of suppressive pro-tumorigenic TAMs correlates with reduced survival in bladder cancer patients treated with immunotherapy. Due to the plasticity of macrophages, excitement has been growing to reshape the pro-tumorigenic TAMs toward the anti-tumorigenic phenotype to stimulate the immunity against cancer. Emerging evidence reveals that this process of macro- phage polarization is inextricably affected by metabolites in the TME, such as free fatty acid (FFA). Lack of data regarding the role of FFA signals in macrophages prevents us from designing an elegant approach to re- polarizing TAM to foster a better anti-tumor T cell response. We propose to dissect the molecular basis of the FFA-mediated signal pathway in TAMs differentiation and its role in resistance to PD-1 blockade, which can be leveraged to restore sensitivity to ICB therapy. Our analysis of published datasets revealed that TAMs from human bladder tumors uniquely express elevated levels of a fatty acid receptor, G Protein-Coupled Receptor 84 (GPR84). These GPR84 expressing cells exhibited enriched hallmarks of anti-tumorigenic function com- pared to their counterpart. Further analysis reveals that expression of GPR84 significantly correlates with longer survival in bladder cancer patients. Our work shows that genetic ablation of GPR84 leads to enhanced production of inhibitory molecules including Arginase 1 via activation of CCAAT/enhancer-binding protein beta (C/EBPβ). By contrast, GPR84 activation by its agonist 6-OAU can reprogram pro-tumorigenic macrophages to produce anti-tumorigenic signature molecules, such as tumor necrosis factor-α (TNFα). Mechanistically, we found that GPR84 potentiates the activity of Nuclear factor kappa B (NF-κB) to enhance TNFα production. Central hypothesis: GPR84 serves as a metabolic signaling checkpoint for determining the function of macro- phage by restricting the immunosuppressive while promoting the immune-stimulating phenotype. Treatment with GPR84 agonists significantly retards tumor growth and increases the anti-tumor efficacy of anti-PD-1 mAb therapy in a MB49 bladder cancer model. Aim 1: Determine whether the lack of GPR84 promotes the polariza- tion of immunosuppressive TAMs. Aim 2: Dissect the molecular and epigenetic mechanisms by which GPR84 signaling promotes an immune-stimulating phenotype in macrophages. Aim 3: Determine whether targeting GPR84-mediated macrophage repolarization enhances the anti-tumor efficacy of ICB. Results will inform the ...