Lentiviral transduction of human hematopoietic cells by HIV-1- and SIV-based vectors containing a bicistronic cassette driven by various internal promoters.

BACKGROUND: Lentiviral gene transfer into hematopoietic cells has been mostly optimized with vectors carrying a single reporter gene. For many clinical applications, lentiviral vectors should contain more than one gene because transduced cells should be enriched by a selectable marker or killed for safety reasons after use. Thus, we compared various vectors containing a bicistronic cassette driven by different ubiquitous promoters for their ability to transduce human T-lymphocytes, CD34+-cells, and dendritic cells (DCs) derived from CD34+-cells or monocytes. METHODS: We designed HIV or SIV constructs containing a bicistronic cassette composed of two reporter genes (thy1/GFP) linked by an internal ribosome entry site sequence and driven by the cytomegalovirus (CMV) or elongation factor 1alpha (EF1alpha) promoters. The woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) was or not inserted within the constructs, the Vpx accessory protein was or not used for SIV vectors. Target cells were infected at the same multiplicity of infection, transduction efficiency was analyzed both by flow cytometry and vector integration. RESULTS: For T-cells, HIV-based vectors/WPRE+ in which the thy1/GFP cassette was driven by the EF1alpha promoter were more efficient than SIV-based vectors. For CD34+-cells and CD34+-derived DCs, better thy1/GFP expression was achieved when the CMV promoter drove the cassette inserted into HIV-based vectors/WPRE+. Conversely, for monocyte-derived DCs, the cassette yielded better thy1/GFP expression when inserted into SIV-based vectors/WPRE+ and driven by the CMV or EF1alpha promoters, the use of Vpx significantly improving the expression levels. CONCLUSIONS: Our results provide guidelines for improving the transduction of T-cells, CD34+-cells or DCs with lentiviral bicistronic vectors designed for clinical applications.

CD4+CD25+ regulatory T-cell deficiency in patients with hepatitis C-mixed cryoglobulinemia vasculitis.

Patients who are chronically infected with hepatitis C virus (HCV) often develop mixed cryoglobulinemia (MC), a B-cell proliferative disorder with polyclonal activation and autoantibody production. We investigated if MC is associated with a deficit of CD4(+)CD25(+) immunoregulatory T (Treg) cells, which have been shown to control autoimmunity. Because Treg cells express higher amounts of CD25 than activated CD4(+) T cells, we analyzed blood CD4(+)CD25(high) Treg cells in 69 untreated patients chronically infected with HCV. Treg cell frequency in patients without MC (8.8% +/- 2.3%) or with asymptomatic MC (7.4% +/- 2.1%) was comparable to that of healthy controls (7.9% +/- 1.3%). In contrast, it was significantly reduced in symptomatic MC patients (2.6% +/- 1.2%, P <.001) even when compared to a panel of untreated HCV(-) patients with different inflammatory disorders (6.2% +/- 0.8%, P <.0001). In symptomatic MC patients, the purified remaining CD4(+)CD25(+) T cells retained suppressive activity in vitro. These results, together with experimental data showing that depletion of Treg cells induces autoimmunity, suggest a major role of Treg cell deficiency in HCV-MC vasculitis and this is the first report of a quantitative Treg cell deficiency in virus-associated autoimmunity.