Generation of CD2＋CD8＋ NK cells from c-kit＋ BM cells 169 cillin (100 U/ml)/streptomycin (100 U/ml). At the start of cultures, IL-2 (30 ng/ml) , IL-7 (10 ng/ml), IL-15 (10 ng/ml), IL-21 (30 ng/ml), SCF (30 ng/ml), and FLT3L (50 ng/ml) (R&D Systems, Minneapolis, MN, USA) were added. Cul- tures were refed weekly by 50% volume change of media, supplemented with the above-listed cytokines (except IL-3), with HDC. For 28 days culture, once a week cytokines-acti- vated cells were harvested, counted, and analyzed. FACS analysis Phenotypes of the cytokines-induced NK cells were char- acterized by multi-color flow cytometry analysis (FACS Calibur with two lasers, Becton Dickinson) using combina- tions of the porcine antibodies to facilitate two color analy- sis. Cells were stained unfixed in 96 well round bottom mi- crotitre plates (Sterilin, Cambridge, UK). In each case, cells were incubated on ice with the CD2-fluorescein isothiocy- anate (FITC) and CD8-phycoerythrin (PE) mAbs for 20 min, washed and further incubated on ice for another 20 min with fluorescent dye conjugated secondary antibody (either isotype specific FITC, Alexa 488, Phycoerythrin, Alexa 633 or Streptavidin-PERCP-Southern Biotechnology Association, Molecular Probes, BD Biosciences). The cells were diluted and washed with FACS diluent; PBS pH 7.4 plus 0.6% bo- vine serum albumin, 0.1% sodium azide and 2% horse serum. After the external staining the cells were given one final wash and fixed by the addition of 50 ml of FACS-fix (450 ml sterile distilled water plus 60 ml 10× calcium/mag- nesium free PBS pH 7.4 [Sigma] and 15 ml formaldehyde solution [38% (w/w); Analar]) diluted 1 in 4 with FACS di- luent and placed on ice for 1 h. The cells were then washed with FACS diluent and 50 ml of permeabilization solution [FACS diluent plus 0.05% Tween 20 (Sigma)] added to every well. The plates were then sealed and carefully floated on a 37oC water bath for 15 min. The seal was removed and the cells washed with FACS diluent to remove the detergent. Reverse transcription and real-time polymerase chain reaction Total RNA from cytokines-activated cells (5×106 cells/ml) was prepared by adding TRIzol Reagent (Invitrogen, Cali- fornia, USA) according to the manufacturer’s protocol [26]. The RNA solution was stored at －70oC until used. Semi- quantitative reverse transcriptase reactions were conducted using MuLV reverse transcriptase. One milligram of RNA was incubated with oligo-dT15 for 5 min at 70oC and then mixed with a 5× first-strand buffer, 10 mM dNTPs, and 0.1 M DTT. The reaction mixture was further incubated for 5 min at 37oC and for 60 min after the addition of MuLV reverse transcriptase (2 U). Reactions were terminated after 10 min at 70oC, and total RNA was depleted by adding RNase H. The polymerase chain reaction (PCR) was con- ducted with the incubation mixture (2 ml cDNA, 4 mM 5’ and 3’ primers, a 10× buffer [10 mM Tris-HCl, pH 8.3, 50 mM KCl, 0.1% Triton X-100], 250 μM of dNTP, 25 mM of MgCl2, and 1 unit of Taq polymerase [Promega, Wisconsin, USA]). The following incubation conditions were used: a 30 sec denaturation time at 94oC, an annealing time of 30 sec at 55oC, an extension time of 45 sec at 72oC, and a final extension of 5 min at 72oC. For RT-PCR analysis, one mi- crogram of RNA was submitted to reverse transcription with the Molony murine leukemia virus reverse transcrip- tase (Invitrogen, California, USA). Two microliters of cDNA obtained from each sample were submitted to a qPCR assay using the SYBR green Master mix method (Life technology, Applied Biosystem, California, USA) in the ABO sequence detection system, The primers (Bioneer, Daejeon, Korea) used in this experiment are indicated as following [perforin F 5’ - CTG GCT CAC ACG AGG TGA TG - 3’; perforin R 5’ - TCA GGT GAG GCA AGC ACT TG - 3’], [granzyme B F 5’ - AAC ACC CAG CTG TGT GTA GG - 3’; granzyme B R 5’ - TGA GAC TTT GGT GCA GGC TC - 3’] and [NKG2D F 5’ -TCC TCC CAG CAT GGA GAT AAG GGA- 3’; NKG2D R 5’ - GTT AGG TGA GAG GAT GGA ACC ATC TTC - 3’], designed from published porcine sequences [27]. Each PCR product signal was normalized relative to that of G3PDH obtained from the same RNA. Preparation of protein lysate and immunoblot analysis Lysates of cytokines-stimulated c-kit＋ BM cells were sepa- rated as previously described [28]. Briefly, cytokines-stimu- lated CD2＋CD8＋ NK cells (5×106 cells/ml) washed 3 times in cold phosphate-buffered saline with 1 mM sodium ortho- vanadate and were lysed in lysis buffer (20 mM Tris-HCl, pH 7.4, 2 mM EDTA, 2 mM ethyleneglycotetraacetic acid, 50 mM β-glycerophosphate, 1 mM sodium orthovanadate, 1 mM dithiothreitol, 1% Triton X-100, 10% glycerol, 10 μg/ ml aprotinin, 10 μg/ml pepstatin, 1 mM benzimide, and 2 mM PMSF) for 30 min with rotation at 4oC. The lysates were clarified by centrifugation at 16,000 g for 10 min at 4 o C and stored at －20oC until needed. Then, whole cell lysates were analyzed by immunoblot- ting methods as reported previously [29]. Briefly, proteins were separated on 10% sodium dodecyl sulfate-polyacry- lamide gels and were transferred by electroblotting to a poly- vinylidenedifluoride membrane. Membranes were blocked for 60 min in Tris-buffered saline containing 3% bovine se- rum albumin, 20 mM NaF, 2 mM EDTA, and 0.2% Tween 20 at room temperature. The membrane was incubated for 60 min with specific primary antibody [1：1,000 for perfor- in (Mouse IgG2b; clone No: F-1), granzyme B (Mouse IgG1; clone No: 2C5) and NKG2D (Mouse IgG1; clone No: 1D11) and 1：2,000 for β-actin (Mouse IgG1; clone No: C-4)] at 4oC, washed 3 times with the same buffer, and incubated for an additional 60 min with horseradish peroxidase con- jugated secondary antibody. The total levels of perforin, granzyme B, NKG2D, and β-actin were visualized by chem- iluminescence using Supersignal West Dura Extended Duration Substrate (Pierce, Rockford, IL, USA), and the in- tensity of the immunoreactive bands was quantified using a densitometer (Sanyo, Tokyo, Japan). Experiments were performed independently five times. Cytotoxic assay of cytokines-induced porcine CD2＋CD8＋ NK cells The cytotoxicity of procine NK cells was quantified using a 51Cr-release assay according to the procedure described previously in [30]. Briefly, in the NK cytotoxicity assay, the cytokines-induced CD2＋CD8＋ NK cells were used as the effector cells. The CD2＋CD8＋ NK cells were incubated in 100-mm culture dish containing 7 ml of DMEM with 20% porcine serum. And, the erythroleukemic tumor cell line; K562 cells obtained from ATCC (American Type Culture Collection, Rockville, MD, USA) were used as targets, This line was maintained in a stationary suspension culture in complete medium: RPMI 1640 supplemented with 10%