Canine il 21

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.

Peripheral blood mononuclear cells were isolated by discontinuous density gradient centrifugation with Hypaque-Ficoll gradients (Histopaque 1.119, Sigma-Aldrich, St. Louis, MO, USA; Lymphoprep^TM 1.077, Axis-Shield PoC AS, Oslo, Norway). Heparinized whole blood was diluted 1:2 with phosphate-buffered saline (PBS), carefully layered onto the discontinuous density gradients, and centrifuged at 400 × g for 25 min. PBMCs were then collected and washed twice with PBS. Canine PBMCs (3.5 × 10⁶) were incubated in a 24-well tissue culture plate with 100-Gy-irradiated-K562 cells (0.5 × 10⁶) in the presence of 100 IU/ml human interleukin (IL)-2 (PeproTec, Rocky Hill, NJ, USA), 10 IU/ml canine IL-15, and 5 ng/ml canine IL-21 (R&D Systems, Minneapolis, MN, USA) in RPMI-1640 and 10% fetal bovine serum (FBS, Gibco, Carlsbad, CA, USA) for 21 days (24 (link)). 100-Gy gamma irradiation is a sufficient dose to induce complete K562 cell death regardless of cytokine stimulation (16 (link), 17 (link), 24 (link), 27 (link)). Fresh medium with IL-2 and rcIL-15 was provided every other day.