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 reason, the production of transgene-expressing DC derived from CD34 + cells transduced either shortly after ex vivo purification or during their differentiation into DC were evaluated.


METHODS
CD34+ cells were transduced with lentivectors encoding for GFP before or after 21 days of culture with FLT3-ligand, thrombopoietin and stem cell 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 allostimulatory capacity was evaluated on GFP+ and GFP- sorted cells.


RESULTS
Immature (G4-induced) DC obtained from amplified CD34 + cells were transducible by lentiviral vectors while mature (GT4-induced) DC were rather refractory. Moreover, since differentiated DC did not proliferate, large quantities of vectors were required to generate transgene-expressing cells with this protocol. In contrast, greater numbers of both immature and mature GFP- expressing DC were obtained with CD34+ cells exposed to lentivector shortly after purification. By the time of DC induction, GFP+ cells had increased by approximately 170-fold. After DC induction with G4, 32% of CD1a+, HLA-DR+, or CD40+ cells expressed GFP. CD1a+E-cadherin+ GFP+ Langerhans-like DC were also obtained. Incubation with TNF induced mature CD83+GFP+ DC that displayed a higher allostimulatory capacity than cells induced with G4 alone.


CONCLUSIONS
The transduction of a small number of CD34+ cells with minimal doses of lentivector may allow for the production of a large number of DC expressing selected antigens useful for immunotherapy.
Salmon
P
P
Department of Genetics and Microbiology, Faculty of Medicine, Geneva University Hospital, Switzerland.
Arrighi
J F
JF
Piguet
V
V
Chapuis
B
B
Zubler
R H
RH
Trono
D
D
Kindler
V
V
D015703:Antigens, CD; D018949:Antigens, CD1 / Q000235:genetics; D018952:Antigens, CD34 / Q000032:analysis; D002454:Cell Differentiation / Q000187:drug effects; D002455:Cell Division / Q000187:drug effects; D003713:Dendritic Cells / Q000502:physiology*; D005312:Fetal Blood / Q000187:drug effects / Q000502:physiology; D005434:Flow Cytometry; D005822:Genetic Vectors; D016178:Granulocyte-Macrophage Colony-Stimulating Factor / Q000494:pharmacology; D049452:Green Fluorescent Proteins; D006684:HLA-DR Antigens / Q000235:genetics; D019650:Hematopoietic Stem Cell Mobilization; D006412:Hematopoietic Stem Cells / Q000166:cytology / Q000187:drug effects / Q000502:physiology*; D006801:Humans; D007136:Immunoglobulins / Q000235:genetics; D007231:Infant, Newborn; D015847:Interleukin-4 / Q000494:pharmacology; D007700:Kinetics; D016086:Lentivirus / Q000235:genetics*; D008164:Luminescent Proteins / Q000032:analysis / Q000235:genetics; D008562:Membrane Glycoproteins / Q000235:genetics; D008565:Membrane Proteins / Q000494:pharmacology; D011994:Recombinant Proteins / Q000494:pharmacology; D019089:Stem Cell Factor / Q000494:pharmacology; D013926:Thrombopoietin / Q000494:pharmacology; D013997:Time Factors; D014161:Transduction, Genetic; D014162:Transfection; D000097802:CD83 Antigen
D015703:Antigens, CD; D018949:Antigens, CD1; D018952:Antigens, CD34; D016178:Granulocyte-Macrophage Colony-Stimulating Factor; D049452:Green Fluorescent Proteins; D006684:HLA-DR Antigens; D007136:Immunoglobulins; D015847:Interleukin-4; D008164:Luminescent Proteins; D008562:Membrane Glycoproteins; D008565:Membrane Proteins; D011994:Recombinant Proteins; D019089:Stem Cell Factor; D013926:Thrombopoietin; D000097802:CD83 Antigen; C084881:flt3 ligand protein