Highly efficient expression of transgenic proteins by naked DNA-transfected dendritic cells through terminal differentiation

Abstract

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 ( IntroductionDendritic cells (DCs) have a key role in initiating and controlling immune responses. In addition to being the most potent antigenpresenting cells (APCs), DCs determine the nature and magnitude of immune responses and provide a link between innate and acquired immunity. 1,2 Genetic engineering of DCs offers potential for the development of immune-regulatory strategies for purposes ranging from immunization to tolerance induction. The capacity of DCs engineered to express transgenic antigens, cytokines, or T-cell costimulatory molecules to induce or bias T helpers 1 and 2 (T H 1/T H 2)-skewed immune responses, or to promote tolerance, is the subject of current investigation. [3][4][5] DNA-based immunization has potential advantages over proteinbased vaccines. [5][6][7][8] These include the simultaneous delivery of transgenic antigens and immunoregulatory genes to DCs. Despite advances in the understanding of DC biology, the development of genetic immunization strategies using DCs transfected with plasmid DNA has been limited by low transfection efficiencies. 9 Currently, the most efficient method for DC transduction is infection by recombinant adenovirus (rAd) at a relatively high multiplicity of infection (MOI). However, the applicability of viral vectors is limited by their potential to interfere with DC function and by the coadministration of viral antigens that elicit strong Band T-cell responses in the host that may limit the readministration of viral vectors. [10][11][12] Indirect evidence suggests that the signaling pathways involved in maturation/terminal differentiation of DCs may enhance the level of transgene expression driven by the human immediate-early cytomegalovirus promoter (hIE-CMVp). We and others have shown that rAd induces the maturation of mouse bone marrow (BM)-derived DCs (BMDCs) mainly by nuclear translocation of nuclear factor-B (NF-B). 11 NF-B is one of the factors necessary to initiate mRNA transcription by hIE-CMVp, and nuclear translocation of NF-B occurs immediately after CMV infection. 13,14 Furthermore, low doses of rAds, engineered to be internalized by cell surface CD40, resulted in enhanced hIE-CMVpdriven transgene expression by DCs. 15 CD40 ligand (L) (CD154) is expressed transiently on the surfaces of activated T cells and mediates the terminal differentiation of DCs. This phe...

Immunization with Lentiviral Vector-Transduced Dendritic Cells Induces Strong and Long-Lasting T Cell Responses and Therapeutic Immunity

“…We have recently shown that nonviral transfection of DCs can be achieved using naked DNA with very high transfection efficiencies (46). Although the use of plasmid DNA to transfect DCs results in nonintegrating transfection, we demonstrated more sustained expression of H-2K b -SIINFEKL complexes on the surface of transfected DCs compared with DCs pulsed with SIINFEKL.…”

“…This may be a critical issue for immunotherapy, particularly in the setting of tumor-bearing hosts or victims of viral infections such as HIV, in whom immunizations will need to overcome pre-existing immunosuppressive mechanisms. We recently demonstrated, by direct comparison, that DCs expressing transgenic Ag after nonviral transfection induced more potent T cell immunity than peptide/ protein-pulsed DCs (46). As yet there are no reports comparing the immunogenicity of lvv-transduced DCs with that of peptide/protein-pulsed DCs.…”

“…Genes encoding enhanced GFP (EGFP) or truncated cytoplasmic chicken OVA (first 138 aa were deleted) were cloned into the vector downstream of the CMV promoter using BamHI and XhoI sites. Our previous studies demonstrated that this cytosolically expressed transgenic Ag can be efficiently presented (46). To enhance nuclear import, a DNA fragment containing a central polypurine tract sequence and the central termination sequence, which together form a triple-stranded DNA flap (TRIP), was inserted in the unique site of ClaI in front of CMV promoter as previously described (48).…”

Ex vivo generation of genetically modified dendritic cells for immunotherapy: implications of lymphocyte contamination

“…In general, DCs generated from adherent celldepleted PB monocytes are used for the development of gene-modified DC vaccines that express known tumor antigens, using either non-viral or viral vectors. 7,9,10,[31][32][33] We, and others, have shown the potential of HIV-1-based lentiviral vectors in delivering transgene to human monocyte-derived DCs and the induction of T-cell responses. 25,[34][35][36] The initial aim of the current study was to develop and optimize a clinically feasible method to generate gene-modified DC vaccines using an SIN lentiviral vector for cancer immunotherapy.…”

“…The enhanced transgene expression detected in DCs following stimulation with TNF-a Immune responses to gene-modified dendritic cells N Chinnasamy et al and sCD40L correlated with their terminal differentiation as assessed by further upregulation of costimulatory molecules (CD80, CD86, CD40, and CD54) and MHC molecules, and also by expression of maturation marker CD83 (data not shown) as suggested previously. 31 Similarly, increased expression of eGFP in transduced lymphocytes contaminants after exposure to TNF-a and sCD40L might be due to changes in their activation status, probably by receiving costimulatory and immunostimulatory factors from mature DCs due to the direct effect of TNF-a and sCD40L.…”