Clone hi30

Whole blood samples were collected from healthy donors (IRB No. SMC 2018-12-021). Peripheral blood mononuclear cells (PBMCs) were isolated from the whole blood by Ficoll density gradient centrifugation and were screened to determine the HLA-type. The PBMCs were then cultured using an NK cell expansion medium system (KBM NK Kit) (Kohjin Bio, Sakado, Japan) for a total of 10–14 days according to the manufacturer’s protocol. Ultimately, highly pure NK cells were isolated using CD56 MicroBeads (Miltenyi Biotec Inc., Auburn, CA, USA) with a VarioMACS system (Miltenyi Biotec Inc.). Cultured NK cells were harvested and analyzed by flow cytometry to determine their purity, and CD3⁻CD56⁺ NK cells were determined to be 90–95% pure.
The following monoclonal antibodies were used to stain cultured NK cells: anti-CD45-BUV395 (1:200, Clone HI30, BD Biosciences), anti-CD3-APC-Cy7 (1:100, Clone SP34–2, BD Biosciences), anti-CD56-PE (1:100, Clone CMSSB, Invitrogen), anti-CD16-BV421 (1:100, Clone 3G8, BD Biosciences), anti-CD19-PE-CF594 (1:200, Clone HIB19, BD Biosciences), anti-CD14-PE-CF594 (1:200, Clone MQP9, BD Biosciences), anti-NKG2D-APC (1:100, Clone 1D11, Invitrogen), anti-NKp46-BV510 (1:100, Clone 9E2, Biolegend), anti-CD94-PerCP-Cy5.5 (1:100, Clone DX22, Biolegend), and anti-CD69-PE-Cy7 (1:100, Clone FN50, Invitrogen).

To expand activated T cells, PBMCs were added to flasks pre-coated with anti-CD3 (1:500, Clone OKT-3, Invitrogen) and anti-CD28 (1:500, Clone CD28.2, Invitrogen) monoclonal antibodies and then incubated in serum-free KBM551 medium (Kohjin Bio) containing 200 IU/ml human recombinant interleukin-2 (rhIL-2) (R&D Systems) at 37 °C in a 5% CO₂ atmosphere. Four days later, the cells were transferred into a culture bag (Kohjin Bio) and were cultured for a total of 10–14 days. Cultured T cells were harvested and analyzed by flow cytometry. The following monoclonal antibodies were used to stain cultured T cells: anti-CD45-BUV395 (1:200, Clone HI30, BD Biosciences), anti-CD3-PE-Cy7 (1:100, Clone SP34-2, BD Biosciences), anti-CD56-PE (1:100, Clone CMSSB, Invitrogen), anti-CD19-PE-CF594 (1:200, Clone HIB19, BD Biosciences), anti-CD14-PE-CF594 (1:200, Clone MQP9, BD Biosciences), anti-CD4-BV605 (1:100, Clone RPA-T4, BD Biosciences), anti-CD8-APC-R700 (1:100, Clone SK1, BD Biosciences), anti-CCR7-PerCP-Cy5.5 (1:100, Clone G043H7, Biolegend), and anti-CD45RA-APC-H7 (1:100, Clone HI100, BD Biosciences).