A self-inactivating retrovector incorporating the IL-2 promoter for activation-induced transgene expression in genetically engineered T-cells.
BACKGROUND
T-cell activation leads to signaling pathways that ultimately result in induction of gene transcription from the interleukin-2 (IL-2) promoter. We hypothesized that the IL-2 promoter or its synthetic derivatives can lead to T-cell specific, activation-induced transgene expression. Our objective was to develop a retroviral vector for stable and activation-induced transgene expression in T-lymphocytes.


RESULTS
First, we compared the transcriptional potency of the full-length IL-2 promoter with that of a synthetic promoter composed of 3 repeats of the Nuclear Factor of Activated T-Cells (NFAT) element following activation of transfected Jurkat T-cells expressing the large SV40 T antigen (Jurkat TAg). Although the NFAT3 promoter resulted in a stronger induction of luciferase reporter expression post stimulation, the basal levels of the IL-2 promoter-driven reporter expression were much lower indicating that the IL-2 promoter can serve as a more stringent activation-dependent promoter in T-cells. Based on this data, we generated a self-inactivating retroviral vector with the full-length human IL-2 promoter, namely SINIL-2pr that incorporated the enhanced green fluorescent protein (EGFP) fused to herpes simplex virus thymidine kinase as a reporter/suicide "bifunctional" gene. Subsequently, Vesicular Stomatitis Virus-G Protein pseudotyped retroparticles were generated for SINIL-2pr and used to transduce the Jurkat T-cell line and the ZAP-70-deficient P116 cell line. Flow cytometry analysis showed that EGFP expression was markedly enhanced post co-stimulation of the gene-modified cells with 1 muM ionomycin and 10 ng/ml phorbol 12-myristate 13-acetate (PMA). This activation-induced expression was abrogated when the cells were pretreated with 300 nM cyclosporin A.


CONCLUSIONS
These results demonstrate that the SINIL-2pr retrovector leads to activation-inducible transgene expression in Jurkat T-cell lines. We propose that this design can be potentially exploited in several cellular immunotherapy applications.
Jaalouk
Diana E
DE
Lady Davis Institute for Medical Research, McGill University, Montreal, Quebec, Canada. djaalouk@mdanderson.org
Lejeune
Laurence
L
Couture
Clément
C
Galipeau
Jacques
J
D016572:Cyclosporine / Q000494:pharmacology; D000229:Dependovirus / Q000235:genetics; D005818:Genetic Engineering; D006801:Humans; D007376:Interleukin-2 / Q000235:genetics*; D019169:Jurkat Cells; D008156:Luciferases / Q000235:genetics; D008213:Lymphocyte Activation; D050778:NFATC Transcription Factors / Q000235:genetics*; D011401:Promoter Regions, Genetic; D012190:Retroviridae / Q000235:genetics; D013601:T-Lymphocytes / Q000378:metabolism*; D019076:Transgenes
D007376:Interleukin-2; D050778:NFATC Transcription Factors; C494010:NFATC4 protein, human; D016572:Cyclosporine; D008156:Luciferases