Ht1080 cells

Lentiviral vector supernatants were produced by transfection of 10 µg lentiviral vector, 12 µg gag-pol, 6 µg rev, and 2 µg VSVg packaging plasmids into 293T cells (ATCC, LGC Standards, Wesel, Germany) in 10 cm plates. All plasmids were mixed and transfected using calcium-phosphate in the presence of 25 µM chloroquine (Sigma-Aldrich, Seelze, Germany, #C6628). Media exchange was performed 6 h and viral supernatants were harvested 30 and 54 h after transfection. Pooled viral supernatants were filtered through 0.22-μm filters (Millex-GP, Millipore, #SLGP033RS) and 100-fold concentrated using an ultracentrifugation step for 2 h at 25,000 rpm and 4 °C. The infectious titer was determined by applying serial dilutions of viral supernatants onto 10⁵ HT1080 cells (DSMZ, Braunschweig, Germany) in the presence of 4 µg/ml protamine sulfate (Sigma-Aldrich, #P3369).

Human embryonic kidney (HEK293T) cells and human hepatoma (HepG2) cells were purchased from ATCC (Manassas,VA), and human fibrosarcoma HT1080 cells were obtained from DSMZ (Braunschweig, Germany). All cells were grown in Dulbecco’s Modified Eagle’s Medium supplemented with penicillin (100 U/mL), streptomycin (100 μg/mL), 2 mM glutamine (all from Gibco/BRL, Karlsruhe, Germany), and 10% heat-inactivated fetal bovine serum (PAN, Aidenbach, Germany) at 37°C in a humidified atmosphere with 5% CO₂.
Primary human T lymphocytes from peripheral blood (PB) samples from healthy adult volunteers who provided written informed consent that is documented in the department of Otorhinolaryngology & head/neck surgery (ENT). This study including the documentation was approved by the ethics committee of the Heinrich-Heine-University of Düsseldorf (ethics No. 4687). After Ficoll-Hypaque density gradient centrifugation, outgrowth of >95% T cells was achieved by incubation of the mononuclear cells on immobilized CD3 and CD28 antibodies (BD Bioscience, San Jose, CA) in combination with IL-2 (100 IU/ml, Chiron, Marburg, Germany) in complete Iscove’s Modified Dulbecco’s Medium (Sigma-Aldrich, Deisenhofen, Germany) as described previously [28 (link), 35 (link), 36 (link)].