An optimal anticancer vaccine probably requires the cooperation of both CD4(+) Th cells and CD8(+) CTLs. A promising tool in cancer immunotherapy is, therefore, the genetic modification of dendritic cells (DCs) by introducing the coding region of a tumor Ag, of which the antigenic peptides will be presented in both HLA class I and class II molecules. This can be achieved by linking the tumor Ag to the HLA class II-targeting sequence of an endosomal or lysosomal protein. In this study we compared the efficiency of the targeting signals of invariant chain, lysosome...

In this study, we compared dendritic cells (DCs) differentiated from positively selected monocytes (CD14-DCs) to DCs differentiated from adherence-selected monocytes (adh-DCs) with emphasis on lentiviral transduction. Using a second-generation, triple-helix containing, self-inactivating lentiviral vector at a multiplicity of infection (MOI) of 15, we observed enhanced transduction of CD14-DCs (72.8 +/- 5.3%, mean fluorescence intensity [MFI] = 166 +/- 76) compared to adh-DCs (32.3 +/- 13.1%, MFI = 119 +/- 76, n = 5). More importantly, the efficiency to transduce ...

The use of tumor antigen-loaded dendritic cells (DC) is one of the most promising approaches to inducing a tumor-specific immune response. We compared electroporation of mRNA to lentiviral transduction for the delivery of tumor antigens to human monocyte-derived and murine bone marrow-derived DC. Both lentiviral transduction and mRNA electroporation induced eGFP expression in on average 81% of human DC. For murine DC, eGFP mRNA electroporation (62%) proved to be more efficient than lentiviral transduction (47%). When we used tNGFR as a transgene we observed lenti...

Until now, studies utilizing mRNA electroporation as a tool for the delivery of tumor antigens to human monocyte-derived dendritic cells (DC) have focused on DC electroporated in an immature state. Immature DC are considered to be specialized in antigen capture and processing, whereas mature DC present antigen and have an increased T-cell stimulatory capacity. Therefore, the consensus has been to electroporate DC before maturation. We show that the transfection efficiency of DC electroporated either before or after maturation was similarly high. Both immature and...

Dendritic cells (DC) are professional antigen-presenting cells that are used in vaccine approaches to cancer. Classically, mature monocyte-derived DC are generated in vitro in the presence of interleukin (IL)-4, granulocyte macrophage-colony stimulating factor, and inflammatory cytokines (G4-DC). Recently, it has been described that DC can also be generated in the presence of IL-3 and interferon (IFN)-beta and that these DC are efficiently matured using polyriboinosinic polyribocytidylic acid (I3-DC). In this study, a series of in vitro experiments was performed ...

Human CD83 is a marker molecule for mature dendritic cells (DC) and is also expressed on activated B and T cells. Although CD83 has been implicated in immune responses, its function on DC and T cells remains unclear. In this study, we wanted to assess the role of CD83 expressed on DC and T cells in the immune response. Down-regulation of CD83 expression on human DC through RNA interference (RNAi) results in a less potent induction of allogeneic T cell proliferation, reduced IFN-gamma secretion by established T cells and decreased capacity in the priming of functi...

Dendritic cells (DCs) are an attractive tool for immunomodulation, targeting mature DCs (mDCs) for immunization or immature/semimature DCs (iDCs) for tolerization. Therefore, introducing antigens into DCs has become a prime topic in various immunological disciplines. Numerous studies have shown that lentiviruses are an efficient vehicle for this purpose. This study evaluates the effects of lentiviral transduction on iDC activation. Immature DCs are efficiently transduced with increasing doses of lentivirus without affecting cell viability. Transduction at low mul...

CD4(+)CD25(+) regulatory T cells (Treg) have been described as an important hurdle for immunotherapy. Engagement of glucocorticoid-induced TNF receptor-related protein (GITR) has emerged recently as an important mechanism to control the suppression of CD4(+)CD25(+) Treg. Furthermore, it has been documented extensively that GITR ligation is costimulatory for naive and activated T cells in the murine setting. However, little is known about the role of the human GITR ligand (huGITRL). We wanted to explore whether huGITRL could enhance antigen-specific T cell priming...

INTRODUCTION: In vivo bioluminescence imaging (BLI) is a promising technique for non-invasive tumour imaging. D: -luciferin can be administrated intraperitonealy or intravenously. This will influence its availability and, therefore, the bioluminescent signal. The aim of this study is to compare the repeatability of BLI measurement after IV versus IP administration of D: -luciferin and assess the correlation between photon emission and histological cell count both in vitro and in vivo. MATERIALS AND METHODS: Fluc-positive R1M cells were subcutaneously inoculated i...

A20 is a zinc finger protein with ubiquitin-modifying activity. A20 has been described as negatively regulating signaling induced by the TNF receptor and TLR family in a number of cell types, including mouse bone marrow-derived dendritic cells (DCs). However, the expression and effect of A20 in activated human monocyte-derived DCs have not been previously evaluated. We report that DCs activated with the TLR3 ligand poly(I:C) up-regulate A20. Down-regulating A20 demonstrated its role in the functional activation of DCs. A20 down-regulated DCs showed higher activat...

Lentiviral based constructs represent a recent development in the generation of transgenic animals. The ease of use, and the fact that the same backbone vectors can be used to down-modulate endogenous gene expression and to produce transgenic animals overexpressing a gene of interest, have fuelled growing interest in this technology. In this study, we have used a lentiviral delivery system to generate transgenic mice expressing altered levels (up or downregulated) of a gene of interest. Although this lentiviral-based approach led to high levels of transgenesis an...

Dendritic cells (DCs) are pivotal regulators of immune reactivity and immune tolerance. The observation that DCs can recruit naive T cells has invigorated cancer immunology and led to the proposal of DCs as the basis for vaccines designed for the treatment of cancer. Designing effective strategies to load DCs with antigens is a challenging field of research. The successful realization of gene transfer to DCs will be highly dependent on the employed vector system. Here, we review various viral and non-viral gene transfer systems, and discuss their distinct charact...

Lentiviral vectors have emerged as promising tools for both gene therapy and immunotherapy purposes. They exhibit several advantages over other viral systems in that they are less immunogenic and are capable of transducing a wide range of different cell types, including dendritic cells (DC). DC transduced ex vivo with a whole range of different (tumor) antigens were capable of inducing strong antigen-specific T-cell responses, both in vitro and in vivo. Recently, the administration of lentiviral vectors in vivo has gained substantial interest as an alternative me...

The discovery of tumor-associated antigens, which are either selectively or preferentially expressed by tumors, together with an improved insight in dendritic cell biology illustrating their key function in the immune system, have provided a rationale to initiate dendritic cell-based cancer immunotherapy trials. Nevertheless, dendritic cell vaccination is in an early stage, as methods for preparing tumor antigen presenting dendritic cells and improving their immunostimulatory function are continuously being optimized. In addition, recent improvements in immunomon...

Tumour immunotherapy has become a treatment modality for cancer, harnessing the immune system to recognize and eradicate tumour cells specifically. It is based on the expression of tumour associated antigens (TAA) by the tumour cells and aims at the induction of TAA-specific effector T cell responses, whilst overruling various mechanisms that can hamper the anti-tumour immune response, e.g. regulatory T cells (Treg). (Re-) activation of effector T cells requires the completion of a carefully orchestrated series of specific steps. Particularly important is the pro...

Recently, new methods have been introduced describing assessment of antigen-specific CD4+ T-cell immunity according to the induction of CD154 (CD40L) on CD4+ T cells during short-term activation. In our study, we have evaluated the influence of different stimulation conditions on the flow cytometric analysis of CD154 expression after antigenic in vitro activation. We used different cell preparation methods, antigen sources, and time periods of in vitro stimulation and analyzed their impact on intra and extracellular detection of antigen-induced CD154 expression o...

In this study, we investigated the development of T cell responses in mice after administration of a mannosylated ovalbumin peptide (M-OVA(323-339)). Immunization with M-OVA(323-339) in complete adjuvant resulted in enhanced antigen presentation in draining lymph nodes. Monitoring the fate of CFSE-labeled ovalbumin peptide-specific TCR transgenic CD4(+) T cells revealed that immunization with M-OVA(323-339) induced normal clonal expansion, recirculation and CD62L expression of antigen-specific T cells in vivo. However, these T cells developed only poor effector f...