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.

We are currently performing gene silencing experiments in human monocyte-derived DCs (MDDCs) with the dual aim of generating relevant knowledge on (i) the transcriptional regulation of DC differentiation, and (ii) the safe and rationale in vitro manipulation of gene expression in DCs.

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

We performed co-cultures of IFN-DCs with zoledronate-stimulated gamma delta T lymphocytes. Gamma delta T-cell activation, as demonstrated by their up-modulation of activation marker expression levels (e.g. CD25 and CD69), was observed. The studies demonstrated for the first time the existence of a bidirectional activating interaction between DCs and gamma delta T lymphocytes activated by aminobiphosphonates.

We identified a novel DC subset called IKDC producing IFNg and expressing TRAIL which can recognize and kill transformed tumor cells in vitro and in vivo. Cultures were generated of tumor cells with IKDC producing IFNg and expressing TRAIL which can recognize and kill transformed tumor cells in vitro and in vivo. IKDC generate long contacts and synapses with tumor cells and ultimately kill them within 4 hours.

These DC were generated by culturing enriched monocytes for 5 days.

These DC were used in a maturation step for subsequent production of a DC vaccine.

These cells acquire a mature phenotype along with an enhanced secretion of several cytokines/chemokines. Moreover, these DCs are very potent in inducing naive CD4(+) T cells to differentiate into interferon-gamma (IFN-gamma)-secreting type 1 T helper (Th1) cells.

These cells do not acquire a mature phenotype and do not lead to an enhanced secretion of several cytokines/chemokines.

These DC were used to elicit CTL responses in a mouse model.

These cells are incubated in Fc-block (1:50 in PBS + 2 % FCS) 30 min on ice, to be stained for FACS.

Indium labelled E/L-S DC (without antigen loading) is administered i.v. to a patient to study the reaction with regard to DC migration.

We are defining the differential roles of intestinal lymph DC subsets in activation of naïve and memory T cells. Two of the three DC subsets are highly potent activators of naïve CD4+ T cells, and do not appear to be constitutively suppressed in any way.

My laboratory continued to analyse the ability of invariant NKT (iNKT) cells to assist priming of antigen specific T and B cell responses. We have demonstrated that activation of human DC by Toll like receptor ligands (TLR-L) modulates the lipid biosynthetic pathway, resulting in enhanced recognition of CD1d-associated lipids by iNKT cells; we have clarified the mechanisms by which CD1d-restricted lymphocytes translate T cell receptor (TCR) recognition of lipids with similar group heads into distinct biological responses; and we demonstrated that pathogen-derive...