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 233

expressing the large T-antigen. Subsequent stimulation of the cells with the drugs
ionomycin (a calcium ionophore) and PMA induces polyclonal T-cell activation
by causing direct and rapid increase in intracellular [Ca 2+ ] [25]. Although the
composite NFAT promoter resulted in stronger induction of luciferase reporter
expression following T-cell activation, the full-length IL-2 promoter had much
lower basal levels of luciferase reporter expression in the absence of any stimula-
tion (Figure 1). These results indicate that unlike the synthetic NFAT promoter,
the IL-2 promoter in the plasmid background was strictly controled as there was
no considerable constitutive reporter expression in un-stimulated cells. Further-
more, costimulation with ionomycin and PMA resulted in a significant increase
in IL-2 promoter-driven luciferase expression compared to unstimulated cells.
It was shown that reporter gene expression driven by the full-length IL-2
promoter in transfected human peripheral blood T lymphocytes and the Jurkat
T- cell line paralleled that of the endogenous IL-2 gene following T-cell activa-
tion [26]. Their results also indicated that the NFAT sites were less implicated in
transcriptional regulation in normal T cells than they were in the tumor T-cell
line. A later study showed that activation of NFAT element is insufficient for
activation of the IL-2 promoter, and that the NFAT and IL-2 promoters differ
in their requirements for co-stimulation [27]. Furthermore, Hooijberg E et al.,
used a self-inactivating retroviral vector in which GFP reporter expression was
controlled by multiple (3 or 6) repeats of NFAT elements followed by a minimal
IL-2 promoter to visualize and isolate antigen-stimulated Jurkat cells, primary
T-cell blasts and antigen-specific T-cell clones [28]. Although the design resulted
in activation-induced GFP expression, the authors reported that a percentage of
the gene-modified T-cells expressed GFP constitutively independently of acti-
vation. In addition, not all T-cells in an antigen-specific clonal population had
upregulated GFP expression following activation. The sum of these studies and
our results suggest that the full-length IL-2 promoter may serve as a more specific
and stringent activation-dependent promoter in T-cells.
Subsequently, we examined the potential of the full-length IL-2 promoter
within the context of a retroviral vector to drive stable and activation-induced
reporter expression in transduced Jurkat T-cells. The cDNA for human IL-2
promoter was incorporated into a self-inactivating retroviral vector replacing
the enhancer and promoter machinery in the 3'LTR of MoMLV. The resulting
SINIL-2pr construct incorporated EGFP fused to HSVTK as a reporter/suicide
gene (Figure 2). VSV-G pseudotyped retroparticles were generated and were used
to transduce the Jurkat T-cell line. Flow cytometry analysis showed that expres-
sion of the EGFP reporter was markedly enhanced post co-stimulation of the
gene-modified Jurkat T-cells with ionomycin and PMA (Figure 3). Furthermore,
activation-induced EGFP expression from the SINIL-2 design was specific to