Canine NK cell atlas: Genomic profiling of blood and tissue-resident NK cells including genomic biomarkers from first-in-dog immunotherapy trial

Natural killer (NK) cells are key effectors in anti-tumor responses with great potential to extend the promise of immunotherapy. However, additional work is needed to understand NK immunoregulation across tissues and activation states in both dogs and people. Here, we used bulk RNA sequencing (RNAseq) with CIBERSORTx deconvolution combined with single cell (SC) RNAseq to interrogate the NK gene expression and signaling pathways across canine tissues, including spleen, liver, sarcomas, lung, placenta, ovary, and uterus. Additionally, we compared peripheral NK gene expression signatures among dogs receiving three novel NK-targeting canine immunotherapies, including autologous NK transfer, allogeneic NK transfer, and molecularly targeted radiotherapy with IL-2 immunocytokine. Overall, canine spleen had the largest absolute number of NK cells, followed by placenta, and sarcoma had the lowest. Unique signatures were observed across organs consistent with conventional, activated, and stem-like NK cells in the spleen, lung, and placenta, respectively. Importantly, integrated analysis of NK trials showed distinct signatures with greater DGEs related to activation and recruitment post treatment in responders compared to non-responders. This comprehensive genomic analysis provides insight into the canine NK profiles across tissues and in response to immunotherapy, increasing our understanding of canine NK cells and advancing mechanistic investigations into novel therapeutic approaches.