A novel approach to genetic engineering of T-cell subsets by hematopoietic stem cell infection with a bicistronic lentivirus.

Lentiviral modification of hematopoietic stem cells (HSCs) paved the way for in vivo experimentation and therapeutic approaches in patients with genetic disease. A disadvantage of this method is the use of a ubiquitous promoter leads not only to genetic modification of the leukocyte subset of interest e.g. T-cells, but also all other subsequent leukocyte progeny of the parent HSCs. To overcome this limitation we tested a bicistronic lentivirus, enabling subset specific modifications. Designed novel lentiviral constructs harbor a global promoter (mPGK) regulating mCherry for HSCs selection and a T-cell specific promoter upstream of eGFP. Two T-cell specific promoters were assessed: the distal Lck-(dLck) and the CD3δ-promoter. Transduced HSCs were FACS sorted by mCherry expression and transferred into sublethally irradiated C57/BL6 mice. Successful transplantation and T-cell specific expression of eGFP was monitored by peripheral blood assessment. Furthermore, recruitment response of lentiviral engineered leukocytes to the site of inflammation was tested in a peritonitis model without functional impairment. Our constructed lentivirus enables fast generation of subset specific leukocyte transgenesis as shown in T-cells in vivo and opens new opportunities to modify other HSCs derived subsets in the future.

Dose/time-dependent modulation of the endothelial function through induction agents: non-depleting versus depleting agents.

BACKGROUND: Polyclonal anti-thymocyte globulins (ATGs) and anti-CD25 antibodies are agents used for induction of immunosuppression in solid-organ transplantation. We aimed to investigate the effect of different regimens of these immunosuppressive induction agents on transendothelial migration of peripheral blood mononuclear cells (PBMC) and evaluated the endothelial apoptosis after treatment. METHODS: Human microvascular endothelial cells were either activated with tumor necrosis factor-α/interferon-γ or not and further treated with 25 or 125 µg/mL ATG (Thymoglobulin, Sanofi-Aventis, Germany) for 2 hours or 24 hours, or with 5 µg/mL Basiliximab (Simulect, Novartis, Germany) for 2 hours or 24 hours. PBMC were either activated with phytohaemagglutinin (PHA) or not and further treated with 25 or 125 µg/mL ATG or with 5 µg/mL Basiliximab for 2 h and then used for transendothelial migration assays. Apoptosis of endothelial cells was detected by means of Annexin-V staining after 2-hour incubation with either 25 or 125 µg/mL ATG or 5 µg/mL Basiliximab. RESULTS: Prophylactic 24-hour administration of ATG to naive endothelial cells without PBMC treatment reduced transendothelial migration. Prophylactic 24-hour administration of ATG and Basiliximab to naive endothelial cells after PBMC treatment with the same agents reduced the transendothelial migration after 24 hours. In both cases, no effect could be observed after 2-hour treatment. Basiliximab but not ATG showed a reduction of transmigration after 2-hour treatment of PBMCs without naive EC treatment. Apoptosis of endothelial cells after treatment increased in both cases, being in case of ATG dose-dependent, increasing from 1.2% after either 25 µg/mL ATG to 8.7% after 125 µg/mL ATG. CONCLUSIONS: Immunosuppressive induction agents modulate the endothelial activity in a dose- and time-dependent manner. Our results suggest that administration of induction agents over longer time periods could provide a potential benefit regarding endothelial immunomodulation. Increased doses may, however, show a deleterious effect on endothelial survival.