title: Recombinant adenoviral vector-lipofectAMINE complex for gene transduction into human T lymphocytes.
abstract: We have evaluated, as a vector for gene transfer into human T lymphocytes, a recombinant adenovirus (rAd-MFG-AP) carrying a modified, membrane-exposed, alkaline phosphatase (AP) as reporter gene. CD3+ cells were selected from the buffy coat of healthy donors by the immunomagnetic technique. The positive cell population, comprising 96+/-2% CD3+ cells, was cultured with clinical-grade cytokine(s) for 3-7 days prior to rAd-MFG-AP transduction and the transgene expression was evaluated 48 hr later by indirect immunofluorescence flow cytometry assay with an anti-alkaline phosphatase antibody. The best efficiency of transduction was achieved on incubation of CD3+ cells with IL-2 plus either IL-12 (AP+ cells, 12+/-3%) or IL-7 (AP+ cells, 11+/-3%). To increase further the efficiency of transduction, we have combined LipofectAMINE and rAd-MFG-AP with the aim to enhance the uptake of viral particles into the target cells. The percentage of CD3+ cells transduced by rAd-MFG-AP-LipofectAMINE complex was 24+/-4% (range, 20-35%) after incubation with IL-2 plus IL-7 and 22+/-4% (range, 18-32%) after incubation with II-2 plus IL-12. Forty-eight hours after the incubation with rAd-MFG-AP, the transduced T lymphocytes were subjected to fluorescence-activated cell sorting and fractionated into AP+ and AP- cell subpopulations. The AP+ cell fraction, comprising 96.8% of AP+ cells, was evaluated by FACScan analysis for T lymphocyte surface antigens. The immunophenotyping of the transduced T lymphocytes has shown that there was not a particular subtype of T lymphocytes more susceptible to rAd-MFG-AP transduction. In addition, the transgene expression did not modify T lymphocyte functions, as demonstrated by results obtained by cytotoxicity assay before and after rAd-MFG-AP-LipofectAMINE complex transduction. In conclusion, human T lymphocytes can be efficiently transduced, under clinically applicable conditions, by adenovirus-LipofectAMINE complex after 7 days of culture with IL-2 and IL-12 or IL-7.
authors.affiliation: Division of Medical Oncology, Istituto Nazionale Tumori, Milan, Italy. dinicola@istitutotumori.mi.it
authors.forename: M
authors.initials: M
authors.lastname: Di Nicola
authors.forename: M
authors.initials: M
authors.lastname: Milanesi
authors.forename: M
authors.initials: M
authors.lastname: Magni
authors.forename: M
authors.initials: M
authors.lastname: Bregni
authors.forename: C
authors.initials: C
authors.lastname: Carlo-Stella
authors.forename: P
authors.initials: P
authors.lastname: Longoni
authors.forename: R
authors.initials: R
authors.lastname: Tomanin
authors.forename: F
authors.initials: F
authors.lastname: Ravagnani
authors.forename: M
authors.initials: M
authors.lastname: Scarpa
authors.forename: C
authors.initials: C
authors.lastname: Jordan
authors.forename: A M
authors.initials: AM
authors.lastname: Gianni
mesh_terms: D000256:Adenoviridae / Q000235:genetics*; D000469:Alkaline Phosphatase / Q000235:genetics; D017252:CD3 Complex / Q000378:metabolism; D002411:Cation Exchange Resins / Q000378:metabolism*; D003602:Cytotoxicity, Immunologic; D005434:Flow Cytometry; D018014:Gene Transfer Techniques; D005822:Genetic Vectors; D006801:Humans; D007376:Interleukin-2 / Q000276:immunology; D015851:Interleukin-7 / Q000276:immunology; D050356:Lipid Metabolism; D008055:Lipids; D008213:Lymphocyte Activation; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D013601:T-Lymphocytes / Q000378:metabolism / Q000502:physiology; D014161:Transduction, Genetic
chemical_list: D017252:CD3 Complex; D002411:Cation Exchange Resins; D007376:Interleukin-2; D015851:Interleukin-7; D008055:Lipids; C086724:Lipofectamine; D011994:Recombinant Proteins; D000469:Alkaline Phosphatase