Adenoviral transduction of human 'clinical grade' immature dendritic cells enhances costimulatory molecule expression and T-cell stimulatory capacity.
The therapeutic use of dendritic cells (DC) in antigen-specific anti-tumor vaccines, requires sufficient numbers of functional DC, the preparation of which should comply with the code of Good Manufacturing Practice. In addition, the expression of tumor specific antigen should be possible in these DC. As a preclinical step, the method reported here was developed in healthy volunteers. Monocytes (Mo) were isolated by leukapheresis from 12 donors, purified by elutriation and then cultured for 6 days in sealed bags in AIM-V serum free medium with granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-13 (IL-13). Between 6x10(8) and 1x10(9) immature DC (iDC) could be differentiated from one leukapheresis. Cells displayed a characteristic iDC phenotype (CD1a(+), CD14(-), CD80(+), CD86(+), HLA DR(+), CD83(-)), and had potent allogeneic and antigen dependent autologous T cell-stimulatory capacity. Moreover, iDC could be further differentiated into mature DC by CD40 ligation as assessed by CD83 expression and the upregulation of HLA-DR and costimulatory molecules. After infection with a recombinant adenovirus encoding for beta-galactosidase (betaGal), 50% to 80% of iDC expressed betaGal without toxicity. Adenovirus infection increased the expression of both costimulatory molecules and CD83, and also increased allogeneic stimulatory capacity. Thus, the method developed here allows us to use large numbers of functional iDC as will be required for therapeutic uses in man. These DC can express a transgenic protein.
Service d'immunologie biologique, Assistance Publique-Hôpitaux de Paris, Hôpital Henri Mondor, Créteil, France. helene.rouard@hmn.ap-hop-paris.fr
H
H
Rouard
A
A
Léon
B
B
Klonjkowski
J
J
Marquet
L
L
Tennezé
A
A
Plonquet
S G
SG
Agrawal
J P
JP
Abastado
M
M
Eloit
J P
JP
Farcet
M H
MH
Delfau-Larue
D000256:Adenoviridae; D017951:Antigen Presentation; D015703:Antigens, CD; D019496:Cancer Vaccines; D002454:Cell Differentiation; D002469:Cell Separation / Q000379:methods; D002478:Cells, Cultured; D046508:Culture Techniques / Q000379:methods*; D003713:Dendritic Cells / Q000166:cytology* / Q000276:immunology*; D018014:Gene Transfer Techniques; D005822:Genetic Vectors; D006684:HLA-DR Antigens / Q000096:biosynthesis; D006801:Humans; D007136:Immunoglobulins / Q000096:biosynthesis; D007937:Leukapheresis; D007959:Lymphocyte Culture Test, Mixed; D008562:Membrane Glycoproteins / Q000096:biosynthesis; D011994:Recombinant Proteins / Q000276:immunology; D013601:T-Lymphocytes / Q000276:immunology; D019076:Transgenes; D001616:beta-Galactosidase / Q000235:genetics / Q000276:immunology; D000097802:CD83 Antigen
D015703:Antigens, CD; D019496:Cancer Vaccines; D006684:HLA-DR Antigens; D007136:Immunoglobulins; D008562:Membrane Glycoproteins; D011994:Recombinant Proteins; D001616:beta-Galactosidase; D000097802:CD83 Antigen