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beta-galactosidase transduced T lymphocytes: a comparison between stimulation by either PHA and IL-2 or a mixed
M Di Ianni1, C Casciari, R Ciurnelli
1Department of Clinical Medicine, Pathology and Pharmacology, Perugia University, Italy.
Background:
Retroviral-mediated gene transfer stably introduces exogenous genes into normal and neoplastic cells of the hematopoietic system.
Methods:
We used two retroviral vectors [the first, FLac, expresses a chimeric protein (Sh-ble::LacZ) between the product of the phleomycin resistance gene (Sh-ble) and the bacterial beta-galactosidase encoded by the LacZ gene; the second, NuNL vector, contains a fusion sequence (LacZ::Neo) that expresses the LacZ and the neomycin resistance genes] to transduce T lymphocytes derived from the peripheral blood of healthy human donors. Two lymphocyte activation procedures were employed: a) phytohemagglutinin/interleukin-2 (PHA/IL-2) polyclonal stimulation; b) allogeneic stimulation in a mixed irradiated or non irradiated lymphocyte reaction, both supplemented with IL-2 (MLR/IL-2). Infection was achieved by co-cultivating activated T cells with the producing amphotropic cell line pretreated with mitomycin C for 96 hours. Infection and transduction efficiency were assayed by LacZ gene expression, which is detected as indigo blue staining with the chromogenic substrate 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside (X..Gal).
Results:
The highest percentage of transduced T cells was reached on the 3rd PHA/IL-2 and on 9th MLR/IL-2 activation days. In these conditions with FLac vector we obtained up to 80% X-Gal+ cells after PHA/IL-2 activation and 66% and 44%, respectively, with non irradiated and irradiated MLR/IL-2, respectively. Up to 40% X-Gal+ cells were obtained with NuNL vector after PHA/IL-2 stimulation, 40% with irradiated and 48% with non irradiated MLR/IL-2 activated cells. In term of transduction efficiency, large variability was observed among patients. There were no immunophenotypical differences between FLac or NuNL vector-transduced cells activated by either of the two techniques and the control cells.
Conclusions:
Our results indicate that: a) the use of FLac or NuNL vector retroviral-mediated gene transfer into T-lymphocytes derived from peripheral blood and stimulated by either PHA/IL-2 or a MLR produces a high percentage of transduced T cells; b) MLR is a good system for generating a transduced alloreactive lymphocyte population. The combination of high transduction efficiency and the capacity to obtain alloreactive transduced lymphocytes should open up the possibility of generating new in vitro and in vivo studies with selectable genes for in vivo therapeutic use.