Human il 15

Canine NK cells were isolated using previously described methods [19 (link)]. Peripheral blood mononuclear cells (PBMCs) were isolated from a beagle dog (Genia Inc., Korea). Subsequently, CD5 negative (CD5^lo) cells were isolated by immunomagnetic separation and cultured in a cell culture flask at 37°C in a 5% CO₂ incubator supplemented with 500 U/mL human IL-2, 10 ng/mL human IL-15, and 5 ng/mL canine IL-21 (all from R&D System, Minneapolis, USA). After 21 days, cell surface markers were analyzed via FACS flow cytometry of activated CD5^lo cells. The activated canine NK cell markers were identified by labeling, CD5^lo cells (1 × 10⁵) with antibodies against surface markers including mouse anti-dog CD3-FITC (clone CA17.2A12, Bio-Rad, Hercules, USA), rat anti-dog CD4-FITC (clone YKIX302.9, Bio-Rad), rat anti-dog CD5-PE (clone YKIX322.3, Bio-Rad), mouse anti-dog CD21-APC (clone CA2.1D6, Bio-Rad), rat anti-dog CD45-APC (clone YKIX716.13, Bio-Rad), and rat anti-dog major histocompatibility complex (MHC)-II-FITC (clone YKIX334.2, eBioscience, San Diego, USA) for 1 hr. The labeled cells were washed twice with phosphate buffered saline and analyzed using a FACSCalibur™ flow cytometer (Becton Dickinson, USA) with Cell Quest Pro software (Becton Dickinson) for data analysis.

Umbilical cord blood (UCB) samples were provided by Chungnam National University Hospital (Daejeon, Republic of Korea). Samples of human CB were obtained from umbilical veins of normal and full-term infants after written informed consent by their mothers, and the protocol was approved by the guidance of the Korea Research Institute of Bioscience and Biotechnology (KRIBB) Institutional Review Board (KRIBB-IRB-20051216-05). In vitro NK cell differentiation from CD34⁺Lin⁻ was performed as previously described [31] (link). Briefly, the isolated CD34⁺Lin⁻ were maintained in MyeloCult H5100 supplemented with stem cell factor (SCF, 30 ng/ml) and flt-3 ligand (FL, 50 ng/ml) for 14 days at 37°C, 5% CO2. The culture medium was refreshed every 3 days. Then, the medium was changed to differentiation medium containing human IL-15 (30 ng/ml, R&D Systems) and cultured for another 14 days. Every 3–4 days, half of the medium was discarded and replenished by fresh medium containing freshly added cytokines. MyeloCult H5100 (Stem Cell Technologies) supplemented with 10⁻⁶ M freshly dissolved hydrocortisone (HC, Sigma) and 50 µg/ml gentamicin was used as culture medium.