Recombinant human interleukin il 2

To isolate human T cells, peripheral blood mononuclear cells were extracted from whole blood of healthy donors by Ficoll density gradient centrifugation. T cells were enriched with Dynabeads^® Human T-Activator CD3/CD28 (Thermo Fisher Scientific, USA) and followed by stimulation for 24–36 h in the X-Vivo medium (Lonza, CH) supplemented with 50 IU/ml recombinant human interleukin (IL)-2 (PeproTech, USA) and then transduced with the lentiviral particles at multiplicity of infection (MOI) = 40. Mouse T cells isolated from spleen and lymph nodes of C57BL/6 mice by the Pan T Cell Isolation Kit II (Miltenyi Biotec) were activated with Dynabeads^® Mouse T-Activator CD3/CD28 (Thermo Fisher Scientific) and recombinant murine IL-2 (ProSpec) for 48 h, and then infected with retroviral particles at MOI = 10.

Recombinant human interleukin (IL)-2, IL-21 (PeproTech, Rocky Hill, NJ, USA), and IL-18 (MBL International, Woburn, MA, USA) were used to stimulate NK cells in WB samples. Fluorescein isothiocyanate (FITC)-conjugated anti-human CD3 monoclonal antibody (mAb) and phycoerythrin (PE)-cyanine (Cy)5-conjugated anti-human CD56 mAb were used to evaluate NK cell purity; PE-conjugated anti-human CD107a mAbs were used as markers of degranulation. All fluorescent mAbs were obtained from BD Biosciences (San Jose, CA, USA).

Recombinant human interleukin (IL)-2 and IL-15 (PeproTech, Rocky Hill, NJ, USA) were used to expand NK cells. Phycoerythrin (PE)-conjugated anti-human HLA-E (clone 3D12) (BioLegend, San Diego, CA, USA) was used to measure HLA-E expression on generically modified K562-HLA-E during culture. Allophycocyanin (APC)-Cy7-conjugated anti-human CD3, PE-Cy7-conjugated anti-human CD56, pacific blue-conjugated anti-human CD16, fluorescein isothiocyanate (FITC)-conjugated anti-human CD57, pacific blue-conjugated anti-human NKp46, PE-conjugated anti-human NKp30, peridinin chlorophyll protein complex (PerCP)-conjugated anti-human NKG2D (eBioscience, San Diego, CA, USA), FITC-conjugated anti-human CD69, APC-conjugated anti-human DNAM-1 (BD Biosciences, San Jose, CA, USA), APC-conjugated anti-human NKG2A, PE-conjugated anti-human NKG2C, PerCP-conjugated anti-human KIR2DL1 (R & D Systems, Minneapolis, MN, USA), FITC-conjugated anti-human KIR2DL2/L3, and BV421-conjugated anti-human KIR3DL1 (BioLegend) were used to evaluate purity and surface expression of NK cell receptors. The PE-conjugated anti-human CD107a mAb was used as a surrogate marker for quantifying degranulation. BV421-conjugated anti-human interferon (IFN)-γ and FITC-conjugated anti-FcεRIγ subunit (FcɛRγ) (Millipore, CA, USA) were used for intracellular staining.

The HTLV-I-transformed T cell lines C5/MJ, HUT-102, MT-2, MT-4, and SLB-1; ATL-derived (HTLV-I-positive) cell lines KOB, MT-1, ST1, and TL-OmI; and T-ALL (HTLV-I-negative) cell lines CCRF-CEM and MOLT-4 were grown in Roswell Park Memorial Institute (RPMI)-1640 medium supplemented with 10% heat-inactivated fetal bovine serum (FBS), penicillin (100 units/mL), and streptomycin (100 µm/mL). Recombinant human interleukin (IL)-2 (0.5 nM) (PeproTech, NJ, USA) was added to the culture of KOB and ST1. The Burkitt lymphoma cell lines BJAB and Raji were grown in RPMI-1640 medium supplemented with 20% FBS, penicillin, and streptomycin. The colon adenocarcinoma cell line SW480 was grown in Dulbecco’s modified Eagle’s medium (D-MEM) supplemented with 10% FBS, penicillin, and streptomycin.

Human hepatocarcinoma cell lines that lacked TM4SF5 expression (SNU449 and empty-vector control SNU449C_p cells) or that expressed wild-type (WT) TM4SF5 [(exogenous expression) SNU449T₇, (endogenous expression) HepG2, Huh7, and HepG2 cells] were used for in vitro experiments and were previously described [17 (link)]. Cells were purchased from either the Korean Cell Bank (Seoul, Korea) or American Type Culture Collection (ATCC, Manassas, VA, USA). Hepatocytes were cultured in Roswell Park Memorial Institute (RPMI) 1640 Medium or Dulbecco’s Modified Eagle Medium (DMEM) (Cytiva, Marlborough, MA, USA) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin (GenDEPOT Inc., Barker, TX, USA) at 37 °C in 5% CO₂. Cells were stably infected with empty pLJM1-EGFP (Addgene) or TM4SF5-encoding pLJM1-EGFP lentiviral vectors. Human NK92 cells were cultured in α-Minimum Essential Medium (MEM) (Invitrogen, Grand Island, NY, USA) containing 200 U/mL recombinant human interleukin (IL)-2 (PeproTech, Rocky Hill, NJ, USA), 12.5% FBS, and 12.5% fetal horse serum (GenDEPOT Inc.). Stable cells were cultured in RPMI 1640 (WelGene) containing 10% FBS, geneticin (G418; 250 μg/mL), and antibiotics (Invitrogen, Grand Island, NY, USA). Cells were passaged every 3–4 days at ratios specified by the suppliers. Cells were monitored for mycoplasma contamination.