Primary human T cell isolation protocol

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)].