Direct Comparison of Canine and Immune Responses Using Transcriptomic and Functional Analyses

The canine spontaneous cancer model is increasingly utilized to evaluate new combined cancer immunotherapy approaches. While the major leukocyte subsets and phenotypes are closely related in dogs and humans, the functionality of T cells and antigen presenting cells in the two species has not been previously compared in detail. Such information would be important in interpreting immune response data and evaluating the potential toxicities of new cancer immunotherapies in dogs. To address this question, we used in vitro assays to compare the transcriptomic, cytokine, and proliferative responses of activated canine and human T cells, and also compared responses in activated macrophages. Transcriptomic analysis following T cell activation revealed shared expression of 515 significantly upregulated genes and 360 significantly downregulated immune genes. Pathway analysis identified 33 immune pathways shared between canine and human activated T cells, along with 34 immune pathways that were unique to each species. Activated human T cells exhibited a marked Th1 bias, whereas canine T cells were transcriptionally less active overall. Despite similar proliferative responses to activation, canine T cells produced significantly less IFN-g than human T cells. Moreover, canine macrophages were significantly more responsive to activation by IFN-g than human macrophages, as reflected by co-stimulatory molecule expression and TNF-a production. Thus, these studies revealed overall broad similarity in responses to immune activation between dogs and humans, but also uncovered important key quantitative and qualitative differences, particularly with respect to T cell responses, that should be considered in designing and evaluating cancer immunotherapy studies in dogs. Following Ficoll separation, PBMC for both species were washed with PBS, counted and added to 24-well plates in triplicate cultures, at a cell density of 2 X 10^6 cells per mL in complete culture medium. For T cell activation, phytohemagglutinin (PHA) (Sigma-Aldrich, St. Louis, MO) was added to cultures at a concentration of 5 ug/ml for 72 hours (cytokine and proliferation measurement) or for 24h (RNA sequencing).