We used specific inhibitors to study the role of src-family tyrosine kinases in the maturation of human monocyte derived dendritic cells upon stimulation with several toll like receptor (TLR) agonists. The effect of these kinase inhibitors on the capacity of DC to be activated by a TLR2 (PAM3CSK4), TLR3 (Poly I:C), TLR5 (Flagellin), and TLR8 (Poly U) agonist was evaluated.

We have stimulated human Monocyte derived Dendritic Cells (hMoDC) with yeast, spheroplasts, pseudohyphae, spores, purified RNA and not purified RNA, LPS.

Commercially available siRNA Smart Pool from Dharmacon targeted against the STAT3 transcription factor have been successfully transfected in these human MDDCs.

On these cells, transcriptional profiling was performed and a comparative pathway-based analysis was done.

We studied the response of human MoDC to yeast, spheroplasts, pseudohyphae and spores. Analysis of IL-6, IL-8, TNF?, IL-1?, IFN?, IL-10, IL-12p70, IL-12p40 secretion was performed with a multiplex assay.

These stained were stained for a process of dead cell uptake by DC.

These DC had been generated from monocytes after elutriation of leukapheresis product (patients with stage III or stage IV melanoma), were not pulsed and were frozen.

The mature peptide pulsed DC were frozen at 5 x 10e6 cells per vial in freezing medium for subsequent stimulation of autologous T cells.

These DCs were generated from adherent peripheral blood monocytes and will subsequently be used as a vaccine for HLA-A1 and/or HLA-A2 positive patients with stage III/IV melanoma.

Untouched naive CD4+ T cells are co-coltured with fungi -pulsed DCs with the aim to prime naive T cells.

These dendritic cells were generated with GM-CSF and IL-4. They were used in a clinical trial in melanoma patients who were randomized to receive immunizations either with these G4 DC or with I3 DC (DC generated with interferon-beta and interleukin-3).

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.

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

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

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.

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.

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

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.

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.

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.