Isolation and Transduction of Genetically Modified T Cells

Human Peripheral Blood Mononuclear Cells (PBMCs) were isolated from buffy coats of healthy donors (German Red Cross, Dresden, Germany) using density centrifugation with Pancoll separating solution (1,077g/ml) (PanBiotech, Aidenbach, Germany). Thereafter, primary T cells were isolated using pan T cell isolation kit according to the manufacturer’s instructions (Miltenyi Biotec, Bergisch Gladbach, Germany). The detailed transduction of the genetically modified RevCAR and Dual-RevCAR T cells were described in details previously (24 (link)). During transduction, T cells were cultured with IL-15, IL-7 and IL-2 (Miltenyi Biotec). However, 20-24 h before the assays, RevCAR T cells were cultured in RPMI complete medium lacking these cytokines. The research conducted with human T cells was approved by the local ethics committee of the Medical Faculty Carl Gustav Carus, Technische Universität Dresden, Germany (EK138042014).