PurposeThe presence and functional competence of intratumoral CD8+T cells is often a barometer for successful immunotherapeutic responses in cancer. Despite this understanding and the extensive number of clinical-stage immunotherapies focused on potentiation (co-stimulation) or rescue (checkpoint blockade) of CD8+T cell antitumor activity, dynamic biomarker strategies are often lacking. To help fill this gap, immuno-PET nuclear imaging has emerged as a powerful tool forin vivomolecular imaging of antibody targeting. Here, we took advantage of immuno-PET imaging using89Zr-IAB42M1-14, anti-mouse CD8 minibody, to characterize CD8+T-cell tumor infiltration dynamics following ICOS (inducible T-cell co-stimulator) agonist antibody treatment alone and in combination with PD-1 blocking antibody in a model of mammary carcinoma.Procedures.Female BALB/c mice with established EMT6 tumors received 10 µg, IP of either IgG control antibodies, ICOS agonist monotherapy, or ICOS/PD-1 combination therapy on days 0, 3, 5, 7, 9, 10, or 14. Imaging was performed at 24 and 48 h post IV dose of89Zr IAB42M1-14. In addition to89Zr-IAB42M1-14 uptake in tumor and tumor-draining lymph node (TDLN), 3D radiomic features were extracted from PET/CT images to identify treatment effects. Imaging mass cytometry (IMC) and immunohistochemistry (IHC) was performed at end of study.Results89Zr-IAB42M1-14 uptake in the tumor was observed by day 11 and was preceded by an increase in the TDLN as early as day 4. The spatial distribution of89Zr-IAB42M1-14 was more uniform in the drug treatedvs.control tumors, which had spatially distinct tracer uptake in the periphery relative to the core of the tumor. IMC analysis showed an increased percentage of cytotoxic T cells in the ICOS monotherapy and ICOS/PD-1 combination group compared to IgG controls. Additionally, temporal radiomics analysis demonstrated early predictiveness of imaging features.ConclusionTo our knowledge, this is the first detailed description of the use of a novel immune-PET imaging technique to assess the kinetics of CD8+T-cell infiltration into tumor and lymphoid tissues following ICOS agonist and PD-1 blocking antibody therapy. By demonstrating the capacity for increased spatial and temporal resolution of CD8+T-cell infiltration across tumors and lymphoid tissues, these observations underscore the widespread potential clinical utility of non-invasive PET imaging for T-cell-based immunotherapy in cancer.Supplementary InformationThe online version contains supplementary material available at 10.1007/s11307-022-01781-7.