Biology Reference
In-Depth Information
induction of the IL-2 promoter as it was sensitive to pre-treatment of the cells
with cyclosporin A (Figure 3E), an immunosuppressant drug that blocks IL-2
promoter transcriptional activation by interfering with the enzymatic activity of
the phosphatase calcineurin [29].
However, although there was no significant reporter expression from the IL-2
promoter in the plasmid backbone, the SINIL-2 pr design did result in measur-
able EGFP expression in unstimulated transduced Jurkat cells. We speculated that
this basal level of EGFP expression reflects a basal status of activation in the Jurkat
tumor T-cell line since the cells are constantly dividing in culture that is not the
case for unactivated primary T-cells that are quiescent in the absence of stimula-
tion. To investigate this we transduced the Jurkat P116 T-cells. Due to their de-
fectiveness in ZAP-70, a kinase involved in early steps of TCR signaling, the cells
divide at a much slower rate compared to the parent cell line reflecting a lower
basal status of activation. However, since T-cell activation by co-stimulation with
ionomycin and PMA bypasses these early steps, Jurkat P116 cells can be activated
with these drugs. Interestingly, the Jurkat P116 cells transduced with SINIL-2
pr had lower basal levels of EGFP expression compared to gene-modified Jurkat
cells (Figure 4). Furthermore, as speculated, the mutant cells resulted in a stronger
induction of reporter expression following drug stimulation whereby the segrega-
tion between the GFP +ve and the GFP -ve cells was more evident.
Zhang P-X and Fuleihan RL used a recombinant adeno-associated virus
(AAV) vector incorporating the IL-2 promoter to drive activation-dependent lu-
ciferase reporter expression in the Jurkat T-cell line [30]. However, there was low
efficiency of gene transfer with very few clonal AAV integration events. Despite
in vitro selection in G418, some of the transferred material was lost or silenced
over time. Furthermore, to induce luciferase reporter expression following T-cell
activation, the cells had to be subjected to additional stimulation such as heat
shock and irradiation due to the nature of the vector used. Hence, taking all this
into consideration, the AAV system would be greatly limited if applied to primary
T-cells.
Since cytotoxic CD8+ T-cells do not significantly induce IL-2 promoter as
compared to CD4+ T-cells, we propose that the SINIL-2pr system would be espe-
cially useful in applications such as negative selection of alloreactive CD4+ T-cells
when the disparity between donor and recipient is at the MHC Class II locus
and for preferential enrichment of antigen-specific CD4+ T-cells which have been
shown to confer an advantage in several immunotherapy applications. In one
study, unselected donor T-lymphocytes with a predominance of CD4+ popula-
tion resulted in a stronger expansion of virus-specific T-cells [31]. Similarly, an-
other study showed that there was a strikingly higher proportion of CD4+ EBV-
Search WWH ::
Custom Search