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 230

cyclosporin-A-sensitive Activation-induced eGFP expression
in Jurkat Cells Transduced With SINIL-2pr Retroparticles
In order to examine whether the SINIL-2pr configuration leads to activation-
induced expression in Jurkat T-cells, we compared the level of EGFP reporter
expression in Jurkat T-cells transduced with SINIL-2pr retroparticles at a multi-
plicity of infection (MOI) of 5, (three consecutive transduction rounds) follow-
ing drug stimulation relative to that of untreated cells (Figure 3). Mean EGFP
expression (MnX) was quantified at ~48 hr post -activation using flow cytom-
etry. Figure 3C shows the flow cytometry profile of one representative experiment
whereby a mixed population of SINIL-2pr-transduced Jurkat T-cells had detect-
able EGFP fluorescence (MnX = 3.22) relative to control non gene-modified cells
(MnX = 1.33, Figure 3A). Since the SINIL-2pr design is expected to display a
“self-inactivation” phenotype with no expression driven from the incorporated
IL-2 promoter in the absence of any T-cell activation, we speculated that this
low level of EGFP expression resulted from the basal level of activation in Jurkat
T-cells since these cells are dividing continuously in culture. Co-stimulation of
the mixed SINIL-2pr-transduced population with 1 µ M ionomycin and 10 ng/
ml PMA for ~6 hr resulted in enhanced EGFP reporter expression (MnX = 8.37,
Figure 3D) as compared to stimulated null cells (MnX = 1.62, Figure 3B). Results
obtained from three independent experiments (Figure 3E) showed an average 2.0
± 0.1 - fold increase (mean ± SEM) in relative mean EGFP expression in SINIL-
2pr - transduced Jurkat cells following ionomycin and PMA treatment which
was significantly higher than that in untreated cells (P = 0.011). This activation-
induced expression was abrogated when the cells were pretreated for 30 minutes
with 300 nM CsA, a potent inhibitor of calcineurin and therefore of the signal
transduction cascade that leads to IL-2 promoter transcriptional activation, indi-
cating that the enhanced expression of EGFP was under the regulatory control of
the IL-2 promoter.
cyclosporin A-sensitive Activation-induced eGFP expression
in Jurkat P116 Cells Transduced with SINIL-2pr Retroparticles
To determine if the low levels of EGFP reporter expression obtained in Jurkat cells
transduced with SINIL-2pr prior to drug stimulation were indeed a consequence
of basal state of activation in this tumor T-cell line, we tested the SINIL-2pr de-
sign in Jurkat P116 T-cells that are deficient in ZAP-70, a kinase implicated in
early steps in T-cell activation. Though expandable in culture, Jurkat P116 cells
exhibit a longer doubling time compared to that of the parent cell line reflecting
a lower basal activation state (data not shown). Figure 4 shows the flow cytometry
profile of one representative experiment whereby a mixed polyclonal population