Highly efficient expression of transgenic proteins by naked DNA-transfected dendritic cells through terminal differentiation.
Dendritic cells (DCs) play a key role in the induction and control of immunity. Genetic engineering of DCs is a promising approach for the development of a broad range of immunomodulatory strategies, for purposes ranging from genetic immunization to tolerance induction. The development of DC-based immunotherapies is limited by the inability to efficiently transfect DCs using naked DNA. Here we demonstrate that after plasmid DNA delivery, the transgene expression level controlled by the human immediate-early cytomegalovirus promoter (hIE-CMVp) is higher in mature DCs than in immature DCs and is further increased after terminal differentiation of DCs by agonist anti-CD40 monoclonal antibody (mAb) or after DC interaction with CD4(+) T cells. CD40 signaling of DCs resulted in nuclear translocation of the transcription factors nuclear factor-kappaB (NF-kappaB), activator of protein-1 (AP-1), and cyclic adenosine monophosphate (cAMP)-responsive element, necessary for the activation of hIE-CMVp. Transgene expression by DCs diminished after the inhibition of these transcription factors or the blockade of adhesion molecules involved in the DC-T-cell synapse. Importantly, CD40 signaling of DCs results in the highly efficient expression and presentation of transgenic antigens and the induction of "in vivo" cytotoxic T-cell (CTL) responses specific for transgenic antigen peptides, demonstrating the functional potential of genetically engineered DCs.
Larregina
Adriana T
AT
W1051 Biomedical Science Tower, 200 Lothrop St, Pittsburgh, PA 15213, USA. adrianal@pitt.edu
Morelli
Adrian E
AE
Tkacheva
Olga
O
Erdos
Geza
G
Donahue
Cara
C
Watkins
Simon C
SC
Thomson
Angus W
AW
Falo
Louis D
LD
D000818:Animals; D017951:Antigen Presentation; D000941:Antigens; D019013:CD40 Antigens / Q000378:metabolism; D002454:Cell Differentiation; D003587:Cytomegalovirus / Q000235:genetics; D004274:DNA, Recombinant / Q000235:genetics*; D003713:Dendritic Cells / Q000166:cytology* / Q000276:immunology / Q000378:metabolism*; D015870:Gene Expression; D006801:Humans; D007167:Immunotherapy / Q000379:methods; D051379:Mice; D008810:Mice, Inbred C57BL; D008822:Mice, Transgenic; D010047:Ovalbumin / Q000276:immunology; D011401:Promoter Regions, Genetic; D011994:Recombinant Proteins / Q000235:genetics*; D015398:Signal Transduction; D013602:T-Lymphocytes, Cytotoxic / Q000276:immunology
D000941:Antigens; D019013:CD40 Antigens; D004274:DNA, Recombinant; D011994:Recombinant Proteins; D010047:Ovalbumin