Recombinant hil 2

NK-92 cells and OVCAR-3 cells (ovarian cancer cell line) were purchased from Shanghai Baili Biotechnology Co., Ltd (Shanghai, China). NK-92 cells were cultured in α-MEM (Gibco, Gaithersburg, MD, USA) with 10% fetal bovine serum (HyClone Laboratories, Logan, UT, USA), 100 U/ml penicillin/streptomycin (Gibco), and 100 U/ml recombinant hIL-2 (R&D Systems, Minneapolis, MN, USA). OVCAR-3 cells were cultured in RPMI-1640 Medium (Gibco) with 20% fetal bovine serum and 100 U/ml penicillin/streptomycin. Cells were subcultured every 2 days.

RBC depleted naïve KaLwRij splenocytes were enriched using a CD8a+ T‐cell isolation kit (Miltenyi Biotec), as per manufacturer's instructions. Murine T cells were then cocultured with gamma‐irradiated (30 Gy) 5TGM1 cells at 10:1 effector:target ratio in T‐cell medium (10% FCS RPMI‐1640 medium, 0.05 mM β‐mercaptoethanol, 1× non‐essential amino acids and 2 ng/mL recombinant hIL‐2 (R&D Systems), stimulated with or without hMPO (2 μg/mL). After 72 h, T cells were harvested and seeded at 3 × 10⁴ cells/well with 1 × 10⁴ irradiated 5TGM1 cells in 1% FCS T‐cell medium in a 96‐well plate and cultured for 24 h. Cytotoxicity was determined using a lactate dehydrogenase (LDH) assay kit (Promega Corporation) as per manufacturer's instructions.
For human T‐cell cytotoxicity assays, Vγ9Vδ2 T cells were cultured with or without hMPO (2 μg/mL) for 2 h in 10% heat‐inactivated FCS DMEM. T‐cells were then seeded at 1 × 10⁵/well in a 96‐well plate with 1 × 10⁴ RPMI‐8226 cells and cultured for 4 h. RPMI‐8226 LDH release was determined as outlined above.

Donor human PBMCs were isolated from healthy volunteers (Scripps Research Institute blood donor service) using Ficoll-Paque (GE Healthcare Life Sciences) and preactivated overnight with recombinant hIL-2 (R&D Systems). The CD33⁺ SIRPα⁺ MOLM-13 (DSMZ, Braunschweig, Germany) tumor cell line (Target) was prelabeled with CellTrace Violet (Thermo Fisher Scientific) and preincubated with test antibodies at dose concentrations up to 200 nmol/L before coculturing with PBMCs at a final effector-to-target ratio of 80:1 for 3 hours at 37°C. Propidium iodide (PI) was added to the coculture, the target cytotoxicity was determined using a Mirroball cytometer, and data were analyzed using Cellista V4.33 (SPT Labtech) as CellTrace Violet/PI-positive versus total target cells.