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.

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

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).

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.

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.

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

Human DC exposed to TLR ligands and activated iNKT cells in vitro. They had enhanced expression of maturation markers, suggesting that a cooperative action of TLR ligands and iNKT cells on DC function is a generalizable phenomenon across species. These studies highlight the potential for manipulating the interactions between TLR ligands and iNKT cell activation in the design of effective vaccine adjuvants.

We have efficiently transfected DC with RNA encoding a functional protein (E/L-Selectin) which allows entry of DC into LN from HEV. The novel adherence capacity of human E/L-S transfected DC was demonstrated via sticking to sialyl-LewisX coated slides using a parallel plate flow microscope. RNA transfected human DC could be frozen and thawed without loosing their functionality.

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

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.

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

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.

These DC were exposed to different maturation cocktails and various activators or inhibitors and profiled.

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

We wanted to assess whether functional activation of human monocyte-derived DCs can be achieved by electroporation of an activation signal in the form of double-stranded (ds) RNA and whether simultaneous electroporation of the dsRNA with tumor antigen encoding mRNA can lead to the induction of a cytotoxic T-lymphocyte (CTL) response.