title: Expansion and fibronectin-enhanced retroviral transduction of primary human T lymphocytes for adoptive immunotherapy.
abstract: Human lymphocytes remain among the most promising target cells for gene therapy. Gene-modified lymphocytes have been used successfully to treat adenosine deaminase (ADA)-deficient patients and to control GvHD after allogeneic BMT. Because activation and proliferation of T cells are necessary for efficient retrovirus-mediated gene transfer and subsequent selection of transduced cells, mononuclear cells (MNC) from steady-state and G-CSF-stimulated peripheral blood were activated by short exposure to the mitogen PHA, the anti-CD3 antibody OKT3, or both in the presence of different concentrations of recombinant IL-2. Using OKT3 (10 or 30 ng/ml) and IL-2 (100 U/ml), T cells expanded efficiently during a 14-day culture period. Cell expansion was similar under serum-free conditions. The immunophenotypic profile over time showed a marked increase in CD8+ cells, leading to a reversed CD4/CD8 ratio of 1:2 and a slight increase in CD56+ cells. Supernatant-based centrifugal transduction of primary human T lymphocytes was compared with supernatant transduction on the extracellular matrix protein fibronectin. Transduction with cell-free retrovirus-containing supernatant in tissue culture flasks coated with human plasma fibronectin led to significantly higher transduction efficiencies (20% +/- 7.5%) than centrifugal transduction in uncoated culture flasks (13.6% +/- 5.1%)(p = 0.041). To both rapidly characterize transduced cells and isolate these from residual nontransduced but biologically equivalent cells, an amphotropic Moloney murine leukemia virus (MoMuLV)-based retroviral vector containing the intracytoplasmically truncated human low-affinity nerve growth factor receptor (deltaLNGFR) cDNA as a marker gene was used. FACS sorting of T cells after transduction resulted in >90% LNGFR+ cells and was much faster than enrichment of transduced cells through growth in G418-selection medium. These results show that supernatant-based retroviral gene transfer into primary human T lymphocytes can be enhanced by fibronectin. Ectopic expression of a cell surface protein can be used to rapidly and conveniently quantitate transduction efficiency through FACS analysis and to efficiently enrich transduced cells through FACS sorting.
authors.affiliation: Department of Hematology and Oncology, University Hospital of the Albert-Ludwigs-Universität Freiburg, Germany.
authors.forename: M
authors.initials: M
authors.lastname: Stockschläder
authors.forename: M
authors.initials: M
authors.lastname: Haiss
authors.forename: S
authors.initials: S
authors.lastname: Exner
authors.forename: O
authors.initials: O
authors.lastname: Schmah
authors.forename: H
authors.initials: H
authors.lastname: Veelken
authors.forename: M
authors.initials: M
authors.lastname: Follo
authors.forename: R
authors.initials: R
authors.lastname: Rüger
authors.forename: J
authors.initials: J
authors.lastname: Finke
mesh_terms: D018014:Gene Transfer Techniques; D005822:Genetic Vectors; D016179:Granulocyte Colony-Stimulating Factor / Q000276:immunology; D006801:Humans; D016130:Immunophenotyping; D016219:Immunotherapy, Adoptive; D008213:Lymphocyte Activation; D016853:Muromonab-CD3 / Q000276:immunology; D012190:Retroviridae; D013601:T-Lymphocytes / Q000276:immunology*
chemical_list: D016853:Muromonab-CD3; D016179:Granulocyte Colony-Stimulating Factor