title: Retrovirus-mediated gene transfer into T cells: 95% transduction efficiency without further in vitro selection.
abstract: This study was designed to retrovirally transduce T cells by a protocol that would be simple, short, cost effective, applicable for clinical use, and efficient enough to avoid further selection of transduced T cells. Because retrovirally mediated infection is depending on the cell cycle, we first optimized the conditions for activating T cells in the presence of immobilized CD3 monoclonal antibodies and recombinant interleukin 2. Cell cycle analysis indicated that CD8+ and total T cells reach a maximum of cycling within 4 days whereas CD4+ T cells attain their maximum of cycling only by day 6. Taking into account these data, CD4+, CD8+, and total T cells were preactivated for 5 and 3 days, respectively, and then infected for 24 hr with supernatant containing retrovirus pseudotyped with gibbon-ape leukemia virus envelope, using a cell centrifugation protocol. Results show that approximately 95% of CD4+, CD8+, and total T cells can be transduced, this transduction efficiency being significantly higher than that obtained with amphotropic retrovirus vectors. Furthermore, under permanent growth stimulation, transduced T cells can be expanded approximately 1,000-fold in 4 weeks of culture with maintenance of transgene expression. However, Immunoscope analysis revealed alterations of T cell repertoire diversity after 2-3 weeks in culture that was not due to retroviral transduction per se. Overall, these data provide evidence that T cells can be transduced at levels that may alleviate the need for both further selection of transduced cells and in vitro expansion, thereby preserving the repertoire diversity of the transduced T cells to be reinfused.
authors.affiliation: Biologie et Thérapeutique des Pathologies Immunitaires, ESA CNRS 7087 CERVI, CHU Pitié Salpétrière, Paris, France.
authors.forename: M
authors.initials: M
authors.lastname: Movassagh
authors.forename: O
authors.initials: O
authors.lastname: Boyer
authors.forename: M C
authors.initials: MC
authors.lastname: Burland
authors.forename: V
authors.initials: V
authors.lastname: Leclercq
authors.forename: D
authors.initials: D
authors.lastname: Klatzmann
authors.forename: F M
authors.initials: FM
authors.lastname: Lemoine
mesh_terms: D000911:Antibodies, Monoclonal / Q000378:metabolism; D017252:CD3 Complex / Q000276:immunology; D015496:CD4-Positive T-Lymphocytes / Q000378:metabolism; D018414:CD8-Positive T-Lymphocytes / Q000378:metabolism; D002453:Cell Cycle / Q000235:genetics; D002498:Centrifugation; D005697:Galactosides / Q000378:metabolism; D018014:Gene Transfer Techniques; D006801:Humans; D007211:Indoles / Q000378:metabolism; D007376:Interleukin-2 / Q000378:metabolism; D007700:Kinetics; D017989:Leukemia Virus, Gibbon Ape / Q000235:genetics; D008213:Lymphocyte Activation; D011994:Recombinant Proteins / Q000378:metabolism; D012190:Retroviridae / Q000235:genetics*; D013601:T-Lymphocytes / Q000378:metabolism*; D013997:Time Factors; D014161:Transduction, Genetic; D014407:Tumor Cells, Cultured; D001616:beta-Galactosidase / Q000378:metabolism
chemical_list: D000911:Antibodies, Monoclonal; D017252:CD3 Complex; D005697:Galactosides; D007211:Indoles; D007376:Interleukin-2; D011994:Recombinant Proteins; D001616:beta-Galactosidase; C044888:5-bromo-4-chloro-3-indolyl beta-galactoside