title: A new way to generate cytolytic tumor-specific T cells: electroporation of RNA coding for a T cell receptor into T lymphocytes.
abstract: Effective T cell receptor (TCR) transfer until now required stable retroviral transduction. However, retroviral transduction poses the threat of irreversible genetic manipulation of autologous cells. We, therefore, used optimized RNA transfection for transient manipulation. The transfection efficiency, using EGFP RNA, was >90%. The electroporation of primary T cells, isolated from blood, with TCR-coding RNA resulted in functional cytotoxic T lymphocytes (CTLs) (>60% killing at an effector to target ratio of 20:1) with the same HLA-A2/gp100-specificity as the parental CTL clone. The TCR-transfected T cells specifically recognized peptide-pulsed T2 cells, or dendritic cells electroporated with gp100-coding RNA, in an IFNgamma-secretion assay and retained this ability, even after cryopreservation, over 3 days. Most importantly, we show here for the first time that the electroporated T cells also displayed cytotoxicity, and specifically lysed peptide-loaded T2 cells and HLA-A2+/gp100+ melanoma cells over a period of at least 72 h. Peptide-titration studies showed that the lytic efficiency of the RNA-transfected T cells was similar to that of retrovirally transduced T cells, and approximated that of the parental CTL clone. Functional TCR transfer by RNA electroporation is now possible without the disadvantages of retroviral transduction, and forms a new strategy for the immunotherapy of cancer.
authors.affiliation: Department of Dermatology, University Hospital of Erlangen, Erlangen, Germany. niels.schaft@derma.imed.uni-erlangen.de
authors.forename: Niels
authors.initials: N
authors.lastname: Schaft
authors.forename: Jan
authors.initials: J
authors.lastname: Dörrie
authors.forename: Ina
authors.initials: I
authors.lastname: Müller
authors.forename: Verena
authors.initials: V
authors.lastname: Beck
authors.forename: Stefanie
authors.initials: S
authors.lastname: Baumann
authors.forename: Tanja
authors.initials: T
authors.lastname: Schunder
authors.forename: Eckhart
authors.initials: E
authors.lastname: Kämpgen
authors.forename: Gerold
authors.initials: G
authors.lastname: Schuler
mesh_terms: D017209:Apoptosis; D001483:Base Sequence; D045744:Cell Line, Tumor; D018274:Electroporation / Q000379:methods*; D005434:Flow Cytometry; D006801:Humans; D007963:Leukocytes, Mononuclear / Q000276:immunology; D008545:Melanoma / Q000276:immunology*; D008969:Molecular Sequence Data; D012313:RNA / Q000096:biosynthesis* / Q000235:genetics; D011948:Receptors, Antigen, T-Cell / Q000235:genetics*; D016176:T-Lymphocyte Subsets / Q000276:immunology*; D013602:T-Lymphocytes, Cytotoxic / Q000276:immunology*
chemical_list: D011948:Receptors, Antigen, T-Cell; D012313:RNA