172 KH Lim, et al Fig. 5. Cytotoxic activity of CD2＋CD8＋ NK cells compared with normal control. Cytokines-activated porcine CD2＋CD8＋ NK cells were incubated with K562 cells as target in a ratio of 100：1 for 4 h. Cytotoxicity was measured by 51Cr release assay for 18 h. Mean cytotoxicity percent of K562 cells are shown (sample number=5, *p＜0.01). DISCUSSION NK cells are the lymphocytes that mature in the bone marrow, derived from HSC [35]. These NK cells mediate critical functions in innate immunity by directly destroying infected or transformed cells [15]. The factors regulating NK differentiation are poorly understood. However, it is certain that it involves cell to cell interactions that render NK responsive to soluble factors from the microenviron- ment, and several factors such as IL-15 are reported to be involved in differentiation of NK cells. Although the intact BM microenvironment is essential for NK differentiation, the stromal cells requirements might be replaced, at least partially, by early-acting cytokines including SCF, FLT3L, and IL-7 [16,17]. These cytokines were reported to induce Fig. 4. The analysis of perforin, granzyme B, and NKG2D protein in length of time. The expressions of perforin, granxyme B, and NKG2D proteins were determined by Western blotting. Densitometric analysis of each protein is shown. Within a week after the treatment of cytokines, perforin and granzyme B proteins were not expressed but two weeks later, these cells expressed perforin and granzyme B mRNAs (A, B, *p＜ 0.01). Whereas, NKG2D mRNA was significantly expressed 21 days later treatment of cytokines (C, *p＜0.01). All expression is presented as the fold relative to the N/C. Results are representative of five independent experiments. CD122 responsiveness through their receptors in NK cells [15]. Interestingly, despite that IL-15 has been known to be essential for NK generation [36,37], transition from HSC to premature NK does not require IL-15 [38]. Thus the ge- neration of premature NK is regarded as IL-15-independent mechanism. Once committed to the NK lineage, premature NK cells acquire the phenotypic and functional character- istics of mature NK cells by IL-15 stimulation through their receptors. In human, IL-2 can also promote the acquisition of CD19 and KIRs on premature NK or immature NK cells [39], although IL-2 deficient mice exhibit normal population of NK cells, suggesting IL-2 is not necessary for the devel- opment of NK cells in mice. Also, in previous reports, it is observed that combination of stromal cells and HDCs may greatly increase the in vitro NK-cell differentiation from CD34＋CD38－/＋ cells in the presence of appropriate cytokines [10,35,40]. Although much is known about the function of NK cells in humans and mice, NK cells activity in swine remains preliminary, and their role in animal viral diseases still is obscure. The recent progress in these ani- mal species has been reviewed by Boysen and Storset [41] and Gerner et al. [42]. In porcine, NK cells may account for a total of 5 to 10% of circulating lymphocytes and cur- rently are identified as belonging to a subset of cells that coexpress CD2 and CD8 molecules [32]. Although mRNAs related to many cytotoxic molecules have been detected, un- til now no studies have been conducted to define their role in the generic function of porcine NK cells. But it is known that porcine NK cells can secret perforin, granzyme B and NKG2D, and kill in vitro targets [31-33]. Their function can be modulated by direct stimulation with cytokines such as interleukin-2 (IL-2), IL-12, IL-15, IL-18 [43,44]. Against this background, we selected 6 cytokines (IL-2, IL-7, IL-15, IL-21, SCF, and FLT3L), HDCs, and stromal cells as candi- dates for factors that induce differentiation of porcine HSCs, based on our hypothesis that such factors also play pivotal roles in the differentiation from porcine HSCs to