Engineered CAR T cells from GBM patients

CAR T cells were engineered starting from GBM patients’ PBMCs (as approved by the Institutional Review Board) which were separated by a density gradient (Lymphoprep, Alere Technologies AS, Oslo, Norway; #1114545) and then plated in RPMI 1640 (Gibco; #52400-017) with 1% FBS, 1% glutamine and 1% penicillin-streptomycin. Non-adherent cells were pre-stimulated for 48 h in 7 µg/mL Phytohemagglutinin (PHA-M, Sigma-Aldrich; #L8902) at a concentration of 1 × 10⁶ cells/mL in a culture medium made by RPMI 1640 supplemented with 10% heat-inactivated defined FBS (HyClone Laboratories, Utah, USA; #SH30070.03), 500 UI/mL recombinant human Interleukin-2 (rhIL-2, Proleukin, Clinigen Healthcare Ltd, Staffordshire DE14 2WW, UK; #801313AY). Isolated T lymphocytes were transduced by a retroviral vector bearing second-generation anti-GD2 CAR and GFP control vector^{11 (link),12 (link)}.