These DC were co-electroporated with constitutively active TLRs and TAA encoding mRNA. They were used to compare different maturation methods.

We found that LPS-stimulated mDCs are able to induce up-regulation of co-stimulatory molecules on co-cultured pDCs. Likewise, CpG-stimulated pDCs activate co-cultured mDCs. The cross talk between these two populations of DC is very efficient since it can be observed at mDC/pDC ratio ranging from 1/10 to 10/1.

The cDNA’s encoding these early expressed HIV antigens have been human codon optimized and modified with lysosomal targeting sequences.

The DC were matured with inflammatory cytokines followed by electroporation with TAA encoding mRNA. They were used to compare different maturation methods.

These cells were stained by Mini67-1KT or Mini26-1KT (PKH67/PKH26 green/red fluorescent cell linker mini kit for general cell membrane labelling, from Sigma) in order to analyse their uptake of dead cells.

These DC still have functional receptors and are exposed to the receptor antagonist laminarin in order to block them.

The cultured cells were subsequently pulsed with tumor antigen derived peptides (one per culture) at a concentration of 5 µg/mL in approximately 5 mL and at a cell density of 20 x 10e6/mL for 1 h.

We have electroporated these DC with melanoma antigen RNA for a cross-presentation study and showed that they express the proteins ex vivo and in vivo.

An HSV vector expressing full length tyrosinase, gp100 and MART-1 was used to transduce DC from melanoma patients which were then to be used for vaccination.

Using pharmacological inhibitors, or macrophages and DC from mice carrying a null mutation in MAP kinase signalling molecule(s), we have evidence for a role of certain MAP kinases in the regulation of IL-10 and IL-12, IFN-gamma production.

We used dendritic cells loaded with anti-DEC205-antigen constructs, consisting of a single-chain variable fragment (scFv) directed against DEC-205, genetically linked to different parts of the MAGE-A3 model antigen (i.e. the KKl and EVDPIGHLY peptides), in a study to compare antigen-loading strategies.

DC pulsed with tumor antigens were evaluated for their immunotherapy potential in EG7-OVA and P815 tumor models. Depletion of natural regulatory T cells in tumor bearing mice resulted in rejection of P815 cells, but not EG7-OVA, suggesting that regulatory T cells have a stronger impact on immune responses against weakly immunogenic or auto-antigen (P1A).

Attenuated Expression of A20 Markedly Increases the Efficacy of Double Stranded RNA-Activated Dendritic Cells as an Anti-Cancer Vaccine A20 is a zinc finger protein with ubiquitine-modifying activity. It has been described that A20 negatively regulates signaling induced by the tumor necrosis factor receptor and toll like receptor (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 monocyte-derived DCs have not been previously evaluated. We report that DC...

The receptors of these DC were blocked by addition of laminarin, mannan and chitin.

We also show that DCs derived from ashen mice, which are defective for the small GTPase Rab27a, fail to cross present antigens efficiently, due to increased phagosome acidification and antigen degradation. This defect in Rab27a-deficient DCs results from the impaired recruitment to phagosomes of the NOX2 membrane components. Therefore phagosomal alkalinization by NOX2 is controlled by Rab27a, and is required for efficient cross presentation in DCs.

DCs pre-loaded in vitro with ICs are at least 1000-fold more potent than ICs injected directly into mice or DCs loaded with the same amount of non-complexed protein. FcgammaRs on DCs were required for efficient priming of Ag-specific CD8+ cells in vivo and induction of tumor protection.

The DC were matured with TLR ligand R848 and polyI:C. Using these DC, we obtained best results in our evaluation whether antigens could be rerouted from the endosomal into the cytosolic compartment by combining antigen with cell penetrating peptides to further enhance cross-presentation.

DCs were pulsed with allogenic melanoma peptides (SK-Mel 24, HLA-A1 and HLA-A2 positive) plus KLH as immunological tracer and subsequently used to produce a vaccine for melanoma.

These DC were electroporated, co-cultured with purified CD8 T cells using MACS cell separation to asses the in vitro CD8 T cell stimulatory capacity of DC.

These dendritic cells were obtained from PBMC from which monocytes had been isolated that differentiated into DC after cell culture.