Abstract Recent emerging tumor-targeted immunotherapy strategies are met with positive and durable outcomes in the clinic. Yet, at least half of cancer patients remain non-responsive despite attempts to engineer their own immune system to attack and destroy the tumor. There is an urgent need for imaging biomarkers to universally guide immunotherapy, regardless of tumor characteristics or treatment modality. The ability to non-invasively and quantitatively image T cell infiltration, anti-tumor activity and expression of tumor suppressors within the tumor through positron emission tomography (PET) will expeditiously identify targets of secondary intervention to improve outcomes. In our proposed study, we will explore 18F-labeled diabody targeting interferon- (IFN-) to interrogate the immune surveillance-signaling axis. We hypothesize that imaging of this immune effector molecule, a hallmark of CD8+ cytotoxic T cell (CTL) and Th1-mediated response, will better predict therapeutic outcomes over total CD8+ or CD3+ T cell imaging alone. We will further monitor the relationship of IFN- with the checkpoint molecules PD-1 and its ligand PD-L1 through PET in an induced anergic microenvironment. The established and well-characterized immune competent neu (the rat homolog of HER2) transgenic and neu+ TUBO xenograft mice will be utilized for active and passive treatment using a HER2/neu DNA vaccine and an anti-neu monoclonal antibody, respectively. Our second aim seeks to examine the potential of our PET radiotracers in neu+ tumor-bearing diversity outbred mice, which recapitulate genetic heterogeneity in humans. Genetic analysis will be conducted to identify loci associated with T cell infiltration, IFN- expression and PD- 1/PD-L1 upregulation. The goal is to identify genetic markers to predict patient susceptibility to immunotherapy and identify potential novel targets for intervention or imaging. Finally, as a progression toward clinical application, we will evaluate the human analog of the IFN- PET tracer against tumor tissue sections via a retrospective histopathological review. A comparative analysis of ex vivo binding of the imaging probe against histological scores will be conducted. Positive findings will warrant clinical translation for informed application of therapeutic strategies. Taken together, the proposed study can potentially address the critical need to develop image-guided tools to monitor immune-facilitated treatment as prompted by landmark cancer immunotherapy breakthroughs.