Analysis of potential Tre-related cytopathic effects.

<p>Exponentially growing Jurkat T cells were transduced with LV-cCtr, or LV-cTre encoding constitutively expressed Tre-recombinase, or mock transduced. After enriching GFP⁺ cells by FACS (LV-cCtr or LV-cTre), cells were cultured for up to 15 weeks. Every week cells were harvested and analyzed. (A) Western blot analysis of the indicated proteins using rabbit polyclonal anti-Tre serum and mouse anti-α-Tubulin antibodies. Protein signals were visualized using an Odyssey Infrared Imaging System (LI-COR). (B) Metabolic activity measured by MTT assay; (C) Apoptosis assessed by Annexin V assay at week 15 of constitutive Tre expression; (D) Cell cycle progression monitored by DNA staining at week 15 of constitutive Tre expression. (E) Functionality of human primary transduced CD4⁺ T cells tested by flow cytometric analysis of CD154 and IFNγ expression after PMA/ionomycin stimulation. (F) Secretion pattern of Th1-, Th2- and Th17-specific cytokines in primary transduced CD4⁺ T cells after PMA/ionomycin stimulation as determined by multiplex ELISA. The pattern matches that of non-transduced cells (mock). (G) IFNγ- and IL4-specific Elispot analysis of human primary transduced CD4⁺ T cells after PMA/ionomycin stimulation. (H) CD34⁺ HSC were transduced with LV-Tre (Tat inducible promoter configuration) and 100 cells were seeded into cytokine-containing methyl-cellulose. Culture dishes were incubated for 14 days at adequate conditions, before counting colonies. (I) HSC differentiation assay as in panel F using an LV constitutively expressing Tre-recombinase (LV-cTre).