Abstract Immunotherapy has made an enormous impact in the treatment of multiple types of cancer, however most patients fail to benefit and methods to monitor response are lacking. To bridge this gap, we have developed non-invasive positron emission tomography (PET) tracers to interrogate immune activity in the tumor microenvironment. Immune cell release of interferon-γ (IFN-γ) is a hallmark of CD8+ cytotoxic T cell (CTL) and Th1-mediated immune activity, both of which contribute to anti-tumor immunity. Our results thus far show that antibody-based immunoPET tracers targeting IFN-γ can detect anti-tumor immunity after administration of immunotherapy, which correlates to treatment outcomes in preclinical tumor models. The current proposal will advance these studies by examining the potential of IFN-γ PET as a pre-treatment predictor of immunotherapy response. We will utilize novel Collaborative Cross recombinant inbred mouse models developed during the course of this project, which exhibit a wide range of response rates to immune checkpoint inhibitors (ICI) due to select variants in their genetic background. These models mimic the diversity of the human population, yet limit tumor heterogeneity as a variable by utilizing genetically identical tumor lines, allowing us to focus on the role of host immunity. We find pre-existing intratumoral IFN-γ expression correlates to ICI outcomes in these models, and we will now test whether IFN-γ PET imaging prior to therapy is predictive of ICI response. Positive results will support the use of IFN-γ PET as part of the formulation of patient treatment strategy. In a necessary step for clinical translation, we will also prepare to submit an investigational new drug (IND) application. Dosimetry, stability studies, and toxicology will be performed. Collectively, the proposed studies will support the clinical use of IFN-γ PET to address critical unmet needs, including predictive biomarker discovery and treatment monitoring technology for cancer immunotherapy. While this proposal focuses on oncology, immune monitoring technologies may also have additional application in multiple inflammatory and autoimmune conditions.