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 235

specific T-cells transduced after early sensitization with EBV-presenting B-cells
[19]. In adoptive immunotherapy of cancer, CD4+ T-cells were also shown to
play an essential role by providing early stimuli for proliferation of tumor-reactive
T-cells [32]. In addition, co-expression of the EGFP reporter and the HSVTK
suicide gene as a fusion protein in the SINIL-2pr design allows not only for selec-
tion of the engineered T-cells but also conditional destruction following GCV
treatment. The utility of the EGFP-HSVTK fusion as a “bifunctional” reporter/
suicide transgene and its potential use in primary human T-cells was already dem-
onstrated in previously published work by our group [33].
conclusion
We conclude that the SINIL-2 pr design can serve as platform for stable, specific,
and activation-induced expression in T-cell lines. Further experiments need to be
done to investigate the function of this system in primary T-cells and to exploit
its use in several T-cell immunotherapy applications such as negative selection of
alloreactive T-cells and their elimination from the donor T-cell population prior
to infusion or for ex vivo sorting of antigen-specific T-cells and their subsequent
in vivo tracking.
Methods
cell Lines and Plasmids
The IL-2 promoter-luciferase plasmid (IL-2Luc) and the NFAT3 promoter-lu-
ciferase plasmid (NFATLuc) were previously described [34]. The Jurkat human
acute lymphoblastic leukemia T-cell line was obtained from the American Type
Culture Collection (ATCC, Rockville, MD). Jurkat cells expressing the SV40
large T antigen (Jurkat TAg) were previously described [34]. ZAP-70-deficient
Jurkat P116 cells were generously provided by Dr. R. T. Abraham (Department
of Immunology, Mayo Clinic, Minnesota). All T-cell lines were maintained in
RPMI media (Gibco-BRL, Gaithesburg, MD) supplemented with 10% heat-in-
activated FBS (Gibco-BRL) and 1% penicillin-streptomycin. pJ6 Ω Bleo plasmid
and 293GPG retroviral packaging cell line were generous gifts from Dr. Richard.
C. Mulligan (Children's Hospital, Boston, MA). 293GPG cells were maintained
in media composed of DMEM (Gibco-BRL), 10% heat-inactivated FBS supple-
mented with 0.3 mg/ml G418 (Mediatech, Herndon, VA), 2 µ g/ml puromycin
(Sigma, Oakville, ONT), and 1 µ g/ml tetracycline (Fisher Scientific, Nepean,
ONT).