Cytotoxic effects of canine nk cells on canine mammary tumor cells

Background & Aim Natural killer (NK) cells play a crucial role in immune responses against various pathogens. NK cells are innate lymphocytes that rapidly provide defenses against tumors and viral infections allowing pathogen elimination or limiting viral spread. Their fast responses mainly rely on the expression of multiple germ-line encoded activating receptors among which natural cytotoxic receptors (NCRs) and NKG2D play the most relevant role in the recognition and killing of infected cells. NK cells have been characterized in a variety of mammals, but canine NK cells remain incompletely characterized. Mammary tumors account for ∼30% of all tumors in the female canine and approximately 50% of the tumors are malignant. Canine mammary gland tumor cell lines are excellent experimental models for identification of cancer-associated genes and understanding cancer biology and progression. In this study, we isolated canine peripheral blood-derived NK cells and defined their immunophenotype and cytotoxic effects on canine mammary tumor cells. In this study, we compared characteristics of canine NK cells against two canine mammary carcinoma cell lines, REM134 and CF41.Mg. Methods, Results & Conclusion Canine NK cells are proliferated and activated in CD5 negative population under specific culture condition. We peripheral blood mononuclear cells (PBMCs) were isolated from female beagle dogs (n=3). Subsequently, CD5 negative cells were isolated by immunomagnetic separation from PBMCs. For activation and expansion of NK cells, CD5 negative cells and co-cultured K562 cells in the presence of IL-2, IL-15, and IL-21 for 21 days. We found that expression markers of activated NK cells such as NKp30, NKp44, NKp46, NKG2D, CD244, perforin, granzyme B, and tumor necrosis factor alpha were highly upregulated. In addition, we found upregulated production of interferon gamma of activated NK cells against target cells such as REM134 and CF41.Mg. Also, we observed that cytotoxicity of activated NK cells against target cells more sensitively reacted to CF41.Mg than REM134. Taken together, we recommend the development of an experimental application of CF41Mg, which has not been reported in canine mammary carcinoma research.