Transduction of CD34+cells with lentiviral vectors enables the production of large quantities of transgene-expressing immature and mature dendritic cells

Background Genetically engineered dendritic cells (DC) presenting specific antigens to T cells may be of great interest for immunotherapy. For this re ason, the production of transgene-expressing DC derived from CD34+ cells tr ansduced either shortly after ex vivo purification or during their differen tiation into DC were evaluated. Methods CD34+ cells were transduced with lentivectors encoding for GFP befo re or after 21 days of culture with FLT3-ligand, thrombopoietin and stem ce ll factor and induction into DC with GM-CSF+IL-4 (G4) or G4+TNF (GT4). GFP and DC-specific marker expression was assessed by flow cytometry, and allos timulatory capacity was evaluated on GFP+ and GFP- sorted cells. Results Immature (G4-induced) DC obtained from amplified CD34+ cells were t ransducible by lentiviral vectors while mature (GT4-induced) DC were rather refractory. Moreover, since differentiated DC did not proliferate, large q uantities of vectors were required to generate transgene-expressing cells w ith this protocol. In contrast, greater numbers of both immature and mature GFP-expressing DC were obtained with CD34+ cells exposed to lentivector sh ortly after purification. By the time of DC induction, GFP+ cells had incre ased by approximately 170-fold. After DC induction with G4, 32% of CD1a+, H LA-DR+, or CD40+ cells expressed GFP. CD1a+E-cadherin+ GFP+ Langerhans-like DC were also obtained. Incubation with TNF induced mature CD83+ GFP+ DC th at displayed a higher allostimulatory capacity than cells induced with G4 a lone. Conclusion The transduction of a small number of CD34+ cells with minimal d oses of lentivector may allow for the production of a large number of DC ex pressing selected antigens useful for immunotherapy. Copyright (C) 2001 Joh n Wiley & Sons, Ltd.