Our lab has data available on the effect of endogenous IL-10 production by DC subsets resulting in low levels of IL-12p70 production in mice. We have shown that plasmacytoid pDC respond to both CpG and the TLR-7 ligand R848 to produce very high levels of IL-12p70 (in the ng/ml range per 5 x 105 cells/ml) and IFN-gamma. However, BM-myeloid and splenic CD8alpha+ DC produced at least fivefold less IL-12p70, and CD8alpha- DC produced undetectable levels of IL-12p70 upon stimulation with CpG. We show that induction of endogenous IL-10 production by these latter DC ...

Our lab generated data on alternatives to the ‘classical’ maturation method (i.e. a cocktail of inflammatory cytokines) for moDC (e.g. ‘clinical grade reagents such as Ampligen TM and Hiltonol TM). Moreover the possibility to co-electroporate moDC with constitutively active TLRs and TAA encoding mRNA was persued. The in vitro charging of monocyte derived DC (moDC) with antigens has been optimised. In contrast to previous studies, the electroporation of TAA encoding mRNA after in vitro maturation of the moDC resulted in a better electroporation efficiency an...

We annotated DC specific pathways and integrated them in the pathway tool. Initially we have annotated DC specific pathways using information publically available on the web. A preliminary curated non exhaustive pathway list can be found in the DC-THERA version of the program DC_Eu.Gene.

We developed additional antigen-loading strategies for DC and obtained data from first experiments on which constructs lead to antigen presentation and which do not. On the one hand we compared lipofection of tumor-antigen-encoding RNA to RNA electroporation into DC, and found that electroporation results in antigen expression in all cells, while lipofection results in higher antigen expression only in a portion of the cells (other cells are negative). Interestingly, the priming capacity of MelanA/HLA-A2-specific autologous CD8+ T cells by lipofected DC was high...

We have developed structural, kinetic and functional data to study the binding of ligands to CD1 molecules and activation of CD1d restricted iNKT cells. The results of these studies have led to: i) The development of protocols to refold in vitro CD1 molecules, which were used for the generation of CD1 tetramers. Ability to generate CD1d tetramers has provided us with the opportunity of comparing a broad panel of CD1d binding compounds for their ability to stimulate iNKT cells. ii) Identification of a novel population of NKT cells, which does not express the ca...

Our group has generated pathway data from human DC treated with pathogen derivatives.

We obtained data on the biological activity of an LPS-like molecule extracted from the freshwater cyanobacterium Oscillatoria Planktothrix FP1 that we named CyP. We found that CyP acts as a potent and selective antagonist of bacterial LPS. CyP did not induce any detectable response in human DCs and competitively inhibited LPS binding to the extracellular domain of TLR4. Addition of CyP together with LPS completely inhibited both MyD88- and TRIF-dependent pathways and suppressed the whole LPS-induced gene transcription program. CyP was effective in protecting mice ...

Data has been obtained on the activation of human DC by Toll like receptor ligands (TLR-L) that modulates the lipid biosynthetic pathway, resulting in enhanced recognition of CD1d-associated lipids by iNKT cells. DC derived soluble factors further increase CD1d-restricted iNKT cell activation, as defined by IFN-alpha secretion. Finally, using soluble tetrameric iNKT TCR as a staining reagent we demonstrate specific up-regulation of CD1d-bound ligands upon TLR-mediated APC maturation. The ability of innate stimuli to modulate the lipid profile of DC resulting in iNK...

Pathway-level data have been obtained from a pilot experiment on yeast cells treated with peptides with potential antifungal action. We have compared results from both genomics and proteomics experiments and search for similarities at the level of pathways and have used Eu.Gene to compare the two different data sources demonstrating the feasibility of using this program to this task. Overall correlation between changes in RNA and protein expression was poor but increased when looking at components of immunologically-relevant pathways and Gene ontology terms. The...

We have obtained data on the induction of CTL responses in vivo by immunization of in vitro matured DC versus CTL responses induced by sustained delivery of a maturation signal from a comparison in a murine model. PolyI:C12U (Ampligen) did not activate the murine bone-marrow derived DC. Similar experiments are being performed using a stabilised form of polyI:C.

We have obtained data on a possible cooperation between myeloid and plasmacytoid DCs in response to different microbial stimuli and a characterization of the mechanisms of this cooperation. For this, we have studied co-culture of mDCs and pDCs in response to CpGs, LPS and bacterial particles. Taking advantage on the fact that human myeloid and plasmacitoid DC show a selective response to LPS and CpG respectively, we have previously identified a cross-talk between two type of cells. Indeed it was observed that LPS-stimulated mDCs are able to induce up-regulation o...

Data will be obtained from a study that we plan on DC receptor characteristics and signalling, focussing on antigen receptors.

Data has been obtained from the application of a DC-based assay aimed at identifying new adjuvant molecules potentially capable to induce Th1 responses, determining the effects of new chemical compounds on dendritic cell capacity to produce type I IFNs. The selected molecules could be potential new vaccine adjuvants.

We have data on a novel innate signalling pathway in DC. Using curdlan as a specific agonist of dectin-1, we have shown that this C-type lectin couple to Syk kinase leads to activation of ERK, JNK and p38 MAPKs, as well as NF-kappaB. However, signalling terminates rapidly once the receptor is internalised, thereby explaining previous observations that large, non-phagocytisable Dectin-1 ligands are much more potent agonists than smaller ones. This is the first example of a bona fide signalling pathway from a cell surface innate receptor other than a TLR. We will ...

We have obtained data using electron microscopy to document the production and secretion of tubulovesicular structures by cells overexpressing VSV-G glycoprotein. These tubulovesicular structures contaminate recombinant viral preparations and play an important role in determining their innate activating potential. The protocols used in those studies have been published and expression vectors for GFP-RIG-I have been made available (outside of the DC-THERA Directory).

Our group obtained data on molecular signaling triggered by LPS in dendritic cells and the role of CD14 in the regulation of the DC life cycle through NFAT activation. Toll-like receptors (TLRs) are the best-characterized pattern recognition receptors (PRRs). Individual TLRs recruit diverse combinations of adapter proteins, and thereby trigger signal transduction pathways leading to the activation of various transcription factors, including nuclear factor (NF)-kB, activation protein (AP)-1, and interferon regulatory factors (IRFs). Interleukin-2 (IL-2) is one of th...

Our data demonstrate that treatment of primed mice with intact anti-CTLA-4 antibodies induces the development of regulatory T cells expressing high levels of ICOS and producing IL-10. These regulatory T cells inhibit Th1 responses, in vitro and in vivo, and repress experimental intestinal inflammation, by a mechanism involving IL-10 and IDO. These ICOS+ regulatory T cells appear distinct from the naturally occurring regulatory T cells described above, suggesting that two populations can inhibit DC-induced Th1 responses in vivo. Experiments are under way to identi...

Data was obtained on the DC-activating, TLR-4-mediated stimulus, LPS, on rat intestinal and hepatic DC in vivo. DC present peripheral Ags to T cells in lymph nodes, but also influence their differentiation (tolerance/immunity, Th1/Th2). To investigate how peripheral conditions affect DC properties and might subsequently regulate T cell differentiation, we examined the effects of a potent DC-activating, TLR-4-mediated stimulus, LPS, on rat intestinal and hepatic DC in vivo. Our results suggest that any explanation of switching between tolerance and immunity as wel...

We obtained data on the Pharmacological compound 1alpha,25-Dihydroxyvitamin D3 [1,25(OH)2D3], the biologically active metabolite of Vitamin D3, and its analogues, and its effect on the DC differentiation/activation pathway induced by type I IFN. 1,25(OH)2D3 prevented the generation of IFN-DCs when added to freshly isolated monocytes, and is capable to redirect already differentiated IFN-DCs toward a more immature stage. Interestingly the suppressive effect of 1,25(OH)2D3 was associated with a potent impairment of DC migration in response to inflammatory and lymph ...

Data has been obtained on the role of Src kinases in DCs during synergic engagement of TLR8 with either TLR4 or TLR3. Using specific inhibitors we have previously found that src-family tyrosine kinases are required for cytokines and chemokines production during maturation of human monocyte-derived dendritic cells (DCs) upon stimulation with several toll like receptor (TLR) agonists. We have now evaluated the role of Src kinases in DCs during synergic engagement of TLR8 with either TLR4 or TLR3. We tested if src-family tyrosine kinases inhibitors had any effect on...