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Published on: October 29, 2015
Isolation and characterization of canine natural killer cells
Helen T Michael1, Daisuke Ito, Valarie McCullar
1Department of Veterinary Clinical Sciences, College of Veterinary Medicine, University of Minnesota, 1352 Boyd Avenue, St. Paul, MN 55108, United States. helen.michael@nih.gov
Insights
Canine natural killer (NK) cells, crucial for innate immunity, were successfully isolated and expanded from peripheral blood. These expanded canine NK cells exhibit cytotoxic activity, advancing their characterization in veterinary immunology.
Area of Science:
- Immunology
- Veterinary Science
- Cell Biology
Background:
- Natural killer (NK) cells are vital lymphocytes mediating innate immunity by killing target cells without prior sensitization.
- While human and murine NK cell functions are well-understood, canine NK cells remain incompletely characterized.
- Defining canine NK cell immunophenotype and function is crucial for advancing veterinary immunology and comparative studies.
Purpose of the Study:
- To isolate and culture canine peripheral blood NK cells.
- To define the immunophenotype of canine NK cells.
- To assess the cytotoxic capabilities of expanded canine NK cells.
Main Methods:
- Peripheral blood mononuclear cells (PBMCs) were isolated from healthy dogs.
- T cells were depleted using immunomagnetic separation.
- Residual non-T, non-B lymphocytes were cultured with IL-2, IL-15, or feeder cells for expansion and characterization.
Main Results:
- Non-T, non-B lymphocytes survived and expanded in culture, with IL-2 essential for survival and IL-15 for expansion.
- Culture with mouse embryonic liver feeder cells and IL-2 also supported survival and expansion.
- Expanded cells uniformly expressed CD45 and MHC I, demonstrating significant cytotoxic activity against target cells.
- MHC II and CD11/18 expression was observed in subsets of these canine NK cells.
Conclusions:
- Canine NK cells can be successfully expanded from peripheral blood mononuclear cells.
- Culture conditions involving T-cell depletion and supplementation with cytokines (IL-2, IL-15) or feeder cells are effective for canine NK cell expansion.
- Expanded canine NK cells exhibit characteristics consistent with NK cells from other species, including cytotoxic activity.
Abstract:
NK cells are non-T, non-B lymphocytes that kill target cells without previous activation. The immunophenotype and function of these cells in humans and mice are well defined, but canine NK cells remain incompletely characterized. Our objectives were to isolate and culture canine peripheral blood NK cells, and to define their immunophenotype and killing capability. PBMC were obtained from healthy dogs and T cells were depleted by immunomagnetic separation. The residual cells were cultured in media supplemented with IL-2, IL-15 or both, or with mouse embryonic liver (EL) feeder cells. Non-T, non-B lymphocytes survived and expanded in these cultures. IL-2 was necessary and sufficient for survival; the addition of IL-15 was necessary for expansion, but IL-15 alone did not support survival. Culture with EL cells and IL-2 also fostered survival and expansion. The non-T, non-B lymphocytes uniformly expressed CD45, MHC I, and showed significant cytotoxic activity against CTAC targets. Expression of MHC II, CD11/18 was restricted to subsets of these cells. The data show that cells meeting the criteria for NK cells in other species, i.e., non-T, non-B lymphocytes with cytotoxic activity, can be expanded from canine PBMC by T-cell depletion and culture with cytokines or feeder cells.
