Expansion and Fibronectin-Enhanced Retroviral Transduction of Primary Human T Lymphocytes for Adoptive Immunotherapy

Human lymphocytes remain one of the most promising target cells for gene therapy. Gene modified lymphocytes have been successfully used to treat ADA-deficient patients and to control GvHD after allogeneic bone marrow transplantation. Since activation and proliferation of T cells is 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 phytohemagglutinin (PHA) and/or the anti-CD3 antibody OKT3 in the presence of different concentrations of recombinant interleukin-2 (11-2). Using OKT3 (10 or 30 ng/ml) and 11-2 (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 centrifugational 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 (20.0±7.5%) than centrifugational transduction in uncoated culture flasks (13.6±5.1%)(p=0.041). To both rapidly characterize transduced cells and to isolate these from residual nontransduced but biologically equivalent cells, an amphotropic Mo-MuLV based retroviral vector containing the intracytoplasmatically-truncated human low-affinity nerve growth factor receptor (△LNGFR) 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 supernatantbased retroviral gene transfer into primary human T lymphocytes can be enhanced by extracellular matrix proteins such as fibronectin approaching levels of transduction that can otherwise only be achieved by cocultivation of virus-producing cells with target cells. 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.