beta-galactosidase transduced T lymphocytes: a comparison between stimulation by either PHA and IL-2 or a mixed lymphocyte reaction.

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.