Lentivirus-Mediated Gene Transfer and Expression in Established Human Tumor Antigen-Specific Cytotoxic T Cells and Primary Unstimulated T Cells

Abstract

In this report, we evaluated the efficiency of stable gene transfer into established CD8(+) human tumor antigen-specific cytotoxic T cell (CTL) lines and peripheral blood lymphocytes (PBL) by oncoretroviral and lentiviral vectors. In the oncoretroviral vector, the green fluorescent protein (GFP) reporter gene was regulated by the murine stem cell virus (MSCV) promoter. In three human immunodeficiency virus type 1 (HIV-1)-based lentiviral vectors, the GFP transgene was regulated by either a chimeric MSCV/HIV-1 promoter, or cellular promoters from human housekeeping genes PGK and EF1 alpha. We found that several lines of proliferating tumor-specific CTL were poorly (=2%) transduced by the oncoretroviral vector that transduced Jurkat T cell line efficiently (=80%). In contrast, three lentiviral vectors transduced 38-63% of these proliferating CTL. More interestingly, all lentiviral vectors packaged without the HIV-1 accessory proteins transduced human bulk PBL and purified CD4(+) and CD8(+) lymphocyte subsets without prior stimulation. Detailed analysis indicated that the lentiviral vectors containing the EF1 alpha or PGK ubiquitous promoter can transduce unstimulated PBL and achieve low-level transgene expression in the absence of any T-cell activation. However, T-cell activation subsequent to the transduction of unstimulated PBL is required for high-level transgene expression. Transduced PBL expressing transgene delivered by the lentiviral vectors still preserved resting and naïve cell phenotypes. Taken together, prior T cell stimulation and HIV-1 accessory proteins are dispensable for lentivirus-mediated gene transfer into resting naïve and memory T lymphocytes. These results will have significant implications for the study of T-cell biology and for the improvement of clinical gene therapies of acquired immune deficiency syndrome (AIDS) and cancer.

MicroRNA-146a and MicroRNA-146b Regulate Human Dendritic Cell Apoptosis and Cytokine Production by Targeting TRAF6 and IRAK1 Proteins

“…The lentiviral constructs were verified by sequencing analysis, and expression of TRAF6 and IRAK1 was confirmed in 293T cells (American Type Culture Collection) by Western blotting. Lentiviral production in 293T cells was performed as described previously (26). Viral supernatants of 293T cells were concentrated by ultracentrifugation and the viral titer was determined, which ranged from 2.1 to 9.6 ϫ 10 7 transducing units/ml.…”

Promoting human embryonic stem cell renewal or differentiation by modulating Wnt signal and culture conditions

“…The retroviral vectors (LTR-neo-CMV promoter-Wnt1 or Wnt3a gene with the HA tag) were produced in 293T cells by transient transfection as previously described [24]. Stably transduced cells were selected by G418 (500 mg/ml).…”

Stable gene transfer and expression in human primary T cells by the Sleeping Beauty transposon system

“…13 Recently, attention has been focused on HIV-1-derived lentiviruses, which have been shown to transduce a variety of slowly or nondividing cells, including unstimulated T cells. [14][15][16][17] However, the procedure required to produce high-titer lentiviral vectors remains complex and costly. Furthermore, both oncoretroviral and lentiviral vector integration show a preference for transcriptionally active genes, including proto-oncogenes and signaling genes.…”