Project Summary Dogs are large, outbred animals that develop cancer spontaneously in the presence of an intact immune system and in the setting of shared environmental exposures with humans. Humans and dogs also share a paired evolutionary history which has led to greater similarities between canine and human genomes and microbiomes than between mouse and human. Together, these traits make dogs an advantageous translational model to study cancer immunology and cancer immunotherapy. Dog clinical trials allow for the study of complex immune interactions during therapy while also addressing long-term efficacy and toxicity of cancer immunotherapies. However, immune dissection requires the development of robust and validated assays and reagents, and high impact studies using dog immunotherapy are premature until a deeper understanding of dog immunology and immune biomarkers is realized, especially for natural killer (NK) cells. This proposal will build on our exciting preliminary data characterizing dog NK cells, including first-in-dog clinical trials of adoptive transfer of dog NK cells and novel cytokine delivery with inhaled IL-15. In collaboration with the School of Medicine, the School of Veterinary Medicine, and the Data Intensive Biology Lab which performs cross-species genomic, transcriptomic, and metagenomic sequence analyses, we will examine dog NK differential gene expression across 1) multiple stimulatory and steady-state conditions; 2) from dog blood and tumor bio-specimens; and 3) from responding and non-responding dogs treated with inhaled IL-15 on an investigational dog clinical trial. We will utilize multiple applications of high-throughput sequencing technologies (RNASeq and single cell RNA sequencing) to establish more robust and validated gene signatures of resting and activated dog NK cells from both ex vivo and in vivo conditions with further analysis of dog sarcoma-infiltrating NK cells. We will then compare these gene signatures to data from high throughput sequencing of murine and human NK cells to bridge species differences and facilitate clinical translation. Ultimately, knowledge of the role of canine NK cells in immune defense and cancer therapy is limited, and it remains unclear whether this is due to biological differences across species or limited reagents for investigation. Results from this proposal will extend the important link that canine studies provide between murine pre-clinical studies and human clinical trials, for cancer immunotherapy research in general but especially for NK studies where high impact clinical translation has remained elusive.