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.

The dead cells were stained and used in the uptake of dead cells by DC.

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

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 marginal zone dendritic cells, interacting with the cholera toxin (CT) adjuvant, lead to effective priming of an immune response in vivo. For the first time, we have followed adjuvant targeting of MZ DC in vivo.

After 6 hour of stimulation, DCs were collected, washed 3 times with PBS, treated with zymolyase, washed twice and cells, lysated with a hypotonic solution (KCl 0.05%), were plated on YPD. Survival of yeast cells, spores or hyphae after uptake was reported as percentage of colony forming units after 3 days relative to the total number of cells growing in the absence of DCs exposure. To assess the importance of ROS production in DC killing ability, DPI (10 microM) was added 30 minutes before stimulation and survival of microorganisms was assessed using the same...

These DCs have been generated from monocytes after elutriation and have not been pulsed with irradiated tumor cells (apoptotic bodies).

The tumor antigen derived peptide pulsed monocyte-derived DC were diluted in medium containing 20 ng/mL TNF-?, plus IL-4 and GM-CSF.

The crude population of DC is divided into the three known splenic subsets (CD8+, CD4+, DN DC) using Fluorescence activated cell sorting (BD FACS Aria). This procedure allowed to obtain highly pure fractions of each subset. However, because DC are a very rare population, this method is very material- and cost-intensive. Furthermore, it is not surprising that the obtained cell numbers of each subset after isolation are below 107. However, these cell numbers should be sufficient for proteomic investigations. The proteomes of all three subsets using mass spectrometry is in progress.

Our current efforts in a study of functional homogeinity involve a transition to 8 colours surface staining, and the evaluation of antigen-specific cell sets identified with MHC tetramers. For the later process, we initiated data collection of EBV, CMV and HIV specific T cell sets.

These T cells are specific for the 0T-1-OVA antigen. They were used to study the expression of twenty different genes either mediating effector functions, or coding for different receptors involved in T cell differentiation and memory generation.

These T cells are specific for the P14-GP33 antigen. They were used to study the expression of twenty different genes either mediating effector functions, or coding for different receptors involved in T cell differentiation and memory generation.