Generation of CD2＋CD8＋ NK cells from c-kit＋ BM cells 171 Fig. 3. The expression of perforin, granzyme B, and NKG2D mRNA in length of time. Normal control (N/C) is the cytokines-not treated cells. Before 7 days the treatment of cytokines, perforin and granzyme B mRNAs were not expressed and but 14 days later, these cells expressed perforin and granzyme B mRNA (A, B, *p＜0.01). Whereas, NKG2D mRNA was expressed 21 days later (C, *p＜0.01). Interestingly, perforin, granzyme B, and NKG2D mRNAs were not expressed within a week (A ∼C, p＞0.05). All expression is presented as the fold relative to the N/C. Results are representative of five independent experiments. cells when stromal cells was not used as a feeder cells (data not shown), suggesting stromal cells is necessary for the development of NK cells in porcine. Analysis of perforin, granzyme B, and NKG2D mRNA expression by RT-PCR The cytokines-induced CD2＋CD8＋ NK cells were exami- ned for the perforin, granzyme B, and NKG2D gene expres- sion by RT-PCR. The c-kit＋ BM cells not pretreated with cytokines were also examined for the same genes. Fig. 3 shows that the cytokines-stimulated c-kit＋ BM cells have a stronger perforin, granzyme B, and NKG2D mRNA ex- pression than the c-kit＋ BM cells not pretreated with cyto- kines in length of time. As shown Fig. 3, perforin, granzyme B, and NKG2D mRNA level were increased for 7th, 14th, 21th, and 28th days compare to day 0 (just before the treat- ment of cytokines). The perforin and granzyme B mRNA levels have more than about 2-fold 14 days later (Fig. 3A and 3B, *p＜0.01). NKG2D mRNA expression was in- creased about 1.5-fold 21 days later compare to day 0 (Fig. 3C, *p＜0.01). M easurement of perforin, granzyme B, and NKG2D protein levels Because cytokines may induce NK cell cytotoxicity against cancer cells through a secretion of perforin [32], granzyme B [33], and NKG2D [34] in human or porcine, we examined whether the treatment of cytokines increased perforin, granzyme B, and NKG2D activity using western blotting and fluorescence image. Cytokines-stimulated c-kit＋ BM cells were examined for the perforin, granzyme B, and NKG2D protein levels. The c-kit＋ BM cells not pretreated with cytokines were also examined for these protein levels in length of time. Fig. 4 shows that cytokines-stimulated c-kit＋ BM cells have a stronger perforin, granzyme B, and NKG2D band than the c-kit＋ BM cells not pretreated with cytokines in length of time for 28 days. Fig. 4 shows that the cytokines-stimulated c-kit＋ BM cells have increased perforin, granzyme B, and FasL levels than the c-kit＋ BM cells not pretreated with cytokines in length of time. As shown Fig. 4A and 4B, perforin and granzyme B levels were significantly increased 14 days later treatment of cytokines (*p＜0.01). Whereas, NKG2D protein was significantly in- creased 21 days later treatment of cytokines (Fig. 4C; *p＜ 0.01). Cytotoxic activities of cytokines-stimulated NK cells The in vitro cytotoxic property of effector cells (cytokines- induced CD2＋CD8＋ NK cells) against target cells (K562 cells) was analyzed. The ratios of effector cells and target cells were 100：1. Cytokines-induced CD2＋CD8＋ NK cells evoked a cytotoxic activity against target cells two weeks later. Cytotoxic activity induced by cytokines was seen in each time-point after cytokines treatment (Fig. 5); 1.28± 0.87% (without cytokines) and 1.53±0.52% (with cytokines) at day 0, 1.32±0.76% (without cytokines) and 2.84±0.98% (with cytokines) at 7 day, 1.07±0.98% (without cytokines) and 7.65±2.27% (with cytokines) at day 14, 1.52±0.85% (without cytokines) and 22.76±4.75% (with cytokines) at day 21, and 1.98±0.91% (without cytokines) and 19.53± 4.98% (with cytokines) at day 28 (Fig. 5; *p＜0.01). A sig- nificant increase in cytotoxic activity in response to treat- ment of cytokines was observed 14 days later treatment of cytokines. The early (before 14 days treatment of cyto- kines) cytotoxic changes were not significantly difference (p＞0.05). These results show that cytokines-induced CD2＋ CD8＋ NK cells could be more cytotoxic activities against target cells than the c-kit＋ BM cells not pretreated with cytokines.