A Self-Inactivating Retrovector Incorporating the IL-2 Promoter for Activation- Induced Transgene Expression in Genetically Engineered T-Cells - Genetic Engineering: Recent Developments in Applications - page 227

Activation of T-cells, physiologically by triggering of the T-cell receptor (TCR)
complex or pharmacologically by using reagents such as ionomycin and PMA
results in the production of IL-2 which plays a key role in T-cell proliferation
and differentiation. Therefore, IL-2 gene transcription by induction of the IL-2
promoter is specific to activated T-cells [21]. Plasmid vectors in which reporter
gene expression is under the control of the full-length IL-2 promoter or synthetic
promoters composed of either single or multiple binding motifs within the IL-2
promoter have been used in many investigative studies of T-cell activation [22-
24]. In these studies, reporter gene expression was induced following activation
of the transfected T-cell lines. Hence, we propose that incorporation of the IL-2
promoter or its synthetic derivatives into a transcriptionaly silent retroviral vector
would lead to stable and activation-induced transgene expression in T-cells. Our
objective is to develop a retroviral vector for stable activation-induced transgene
expression in T-lymphocytes.
In this study, we first compared the transcriptional potency and stringency of
the full-length IL-2 promoter with that of a synthetic promoter composed of 3
NFAT elements following stimulation of T-cells. Transient transfections of Jurkat
Tag cells with plasmids harboring either one of the two promoters upstream of a
luciferase reporter showed that although the NFAT3 synthetic promoter resulted
in a stronger induction of reporter expression following T-cell activation, the IL-2
promoter had a more stringent activation-dependent expression. Consequently,
we tested if a self-inactivating retroviral vector incorporating the full-length IL-2
promoter namely SINIL-2 pr would serve as an integrated platform for stable and
specific activation-induced transgene expression in T-cell lines.
results
Activation-induced Luciferase reporter expression in Jurkat
tag cells
We compared the transcriptional capacity of the full-length IL-2 promoter with
that of a synthetic promoter composed of 3 NFAT elements in transient transfec-
tion assays using plasmids harboring either one of the two promoters upstream
of a luciferase reporter to determine which would allow for more stringent T-cell
activation-dependent reporter expression. Jurkat T-cells expressing the large T Ag
were transfected with IL-2 promoter-luciferase plasmid (Figure 1A) or NFAT3
promoter-luciferase plasmid (Figure 1B). Co-stimulation of transfected cells
with 1 µ M ionomycin and 10 ng/ml PMA for ~6 hr resulted in 6.4 ± 0.4 -
fold increase in IL-2 promoter-driven luciferase reporter expression (P = 0.002)
and 14.6 ± 1.3 - fold increase in NFAT3 promoter-driven luciferase expression