Interleukin il 2

The SNK-6 and NK-92 cells were routinely kept in Sun Yat-sen University Cancer Center (SYSUCC) and were incubated in a humidified incubator with 5 % CO₂ at 37 °C. The SNK-6 cell line was cultured in RPMI-1640 (Gibco, USA) medium containing 2 mmol/l glutamine, 100 U/ml penicillin, and 100 μg/ml streptomycin, supplemented with 1000 U/ml interleukin (IL)-2 (Sigma-Aldrich, USA) and 10 % human AB serum (Gemini Bioproducts, Woodland, CA, USA). The NK-92 cells were maintained in α-MEM (Life Technologies, Karlsruhe, Germany) containing 20 % FBS (Gibco, USA), 2 mM l-glutamate, 100 mg/ml penicillin, and 100 mg/ml streptomycin (Life Technologies) and supplemented with 10 ng/ml IL-2 (Sigma-Aldrich, USA).

The co-culture of the MSCs and T cells was carried out using 24-well plates (contact culture) and the Transwell system (isolated culture) in which the T cells and MSCs were physically separated by a membrane permeable for soluble factors. The inner hanging cell culture insets with 0.4-μm pore size membrane of a 24-well plate were purchased from Millipore (Billerica, MA, USA). The BM-, AT-, WJ- and PL-MSCs were seeded at 10⁵ cells/well in regular 24-well plates and 24-well Transwell plates containing α-MEM, 10% FBS and 100 U/ml penicillin/streptomycin. After 24 h, 10 μg/ml mitomycin C (MMC; Sigma) were added to inhibit MSC proliferation, and the cells were incubated for 2 h at 37°C followed by 5 extensive washes with medium. A total of 10⁵ T cells/well was added and stimulated with 10 g/ml phytohemagglutinin (PHA; Sigma) and 10 ng/ml interleukin (IL)-2 (Sigma). IL-2/PHA-activated T cells were cultured in the presence or absence of MSCs. The cultures were plated in triplicate and incubated for 5 days before the addition of 5-bromo-20-deoxyuridine (BrdU). After 18 h, proliferation was assessed using the BrdU-Assay kit (Roche Applied Science, Penzberg, Germany) according to the manufacturer’s instructions.