Generation of CD2⁺CD8⁺NK Cells from c-kit⁺Bone Marrow Cells in Porcine

Abstract

Natural killer (NK) cells provide one of the initial barriers of cellular host defense against pathogens, in particular intracellular pathogens. Because bone marrow-derived hematopoietic stem cells (HSCs), lymphoid protenitors, can give rise to NK cells, NK ontogeny has been considered to be exclusively lymphoid. Here, we show that porcine c-kit + bone marrow cells (c-kit + BM cells) develop into NK cells in vitro in the presence of various cytokines [interleukin (IL)-2, IL-7, IL-15, IL-21, stem cell factor (SCF), and fms-like tyrosine kinase-3 ligand (FLT3L)]. Adding hydrocortisone (HDC) and stromal cells greatly increases the frequency of c-kit + BM cells that give rise to CD2 + CD8 + NK cells. Also, intracellular levels of perforin, granzyme B, and NKG2D were determined by RT-PCR and western blotting analysis. It was found that of perforin, granzyme B, and NKG2D levels significantly were increased in cytokine-stimulated c-kit + BM cells than those of controls. And, we compared the ability of the cytotoxicity of CD2 + CD8 + NK cells differentiated by cytokines from c-kit + BM cells against K562 target cells for 28 days. Cytokines-induced NK cells as effector cells were incubated with K562 cells as target in a ratio of 100:1 for 4 h once a week. In results, CD2 + CD8 + NK cells induced by cytokines and stromal cells showed a significantly increased cytotoxicity 21 days later. Whereas, our results indicated that c-kit + BM cells not pretreated with cytokines have lower levels of cytotoxicity. Taken together, this study suggests that cytokines-induced NK cells from porcine c-kit + BM cells may be used as adoptive transfer therapy if the known obstacles to xenografting (e.g. immune and non-immune problems) were overcome in the future.