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 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 ...

Our lab has obtained data on indirect activation of DC by immunomodulatory mediators. TLRs signal directly for DC activation but also promote production of immunomodulatory mediators by many other cell types. These mediators could be sufficient to activate DC indirectly, thereby potentially allowing responses to organisms with which DC do not come into direct contact. We tested this hypothesis and, surprisingly, found that indirect signals alone led only to partial DC activation and resulted in clonal expansion of T cells lacking typical Th1 or Th2 function. Th...

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).

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 was obtained on the properties of the CD16, CD1c and BDCA-3 DC populations and their production of cytokines in response to different Toll-like receptor (TLR). We studied two major blood populations of DCs, myeloid DCs (mDCs) and plasmacytoid DCs (pDCs). Among mDCs, three populations could be further identified by specific markers: CD16, CD1c and BDCA-3. The properties of these DC populations and their production of cytokines in response to different Toll-like receptor (TLR) agonists were evaluated. The TLR agonists used were: PAM3CSK4, Poly I:C, LPS, Flagel...

We obtained data using specific inhibitors on the role of src-family tyrosine kinases in the maturation of human monocytes 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. Src kinases were found to be required for the initiation of some maturation characteristics but not of others, since their inhibition was able to dissociate cytokines and chemokines ...

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...

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 Fabs that bind strongly to L-SIGN, but to a lesser degree or not at all to dendritic cell-specific ICAM-grabbing nonintegrin (DC-SIGN). The C-type lectin L-SIGN is expressed on liver and lymph node endothelial cells,where it serves as a receptor for a variety of carbohydrate ligands, including ICAM-3, Ebola, and HIV. To consider targeting liver/lymph node-specific ICAM-3-grabbing nonintegrin (L-SIGN) for therapeutic purposes in autoimmunity and infectious disease, we isolated and characterized Fabs that bind strongly to L-SIGN, but to a lesse...

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 have data available on a novel M-CSF dependent DC developmental pathway that is independent of Flt3L. These DC have unique characteristics and precursor origin. The cells can be found in vivo in Flt3L gene deleted (-/-) mice injected with recombinant M-CSF.

We obtained transcriptional and pathway analysis data of human monocyte-derived DCs stimulated with yeast, spheroplasts, pseudohyphae, spores, purified RNA and not purified RNA and LPS. We propose the role of cell wall in the mechanisms of DC-Pathogen interaction as an experiment that could serve as reference in bioinformatics approaches for the reconstruction of DC activation signalling pathways. We have stimulated human Monocyte derived Dendritic Cells (hMoDC) with yeast, spheroplasts, pseudohyphae, spores, purified RNA and not purified RNA, LPS. We have measur...

Data is available from a re-analysis of existing microarray data; we started experimental work to assess the reliability and completeness of the DC_Eu.Gene pathway set using stimulation of DCs with S.cerevisiae cells, pseudohyphae, spores in comparison with the results obtained with LPS, R848, LPS and R848, Zymosan and Curdlan.

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.

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 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...

Data is available on the interaction between microorganism-associated molecular patterns and individual toll-like receptors on dendritic cells that turn on signal transduction pathways leading to the activation of different transcription factors. Microbial invasions are perceived by pattern recognition receptor (PRR)-expressing cells of the innate immune system. PRRs bind a number of microbial products collectively referred to as microorganism-associated molecular patterns (MAMPs). Toll-like receptors (TLRs) are the best-characterized PRRs. The interaction of MAM...

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 ...

We investigated and obtained data on the interactions between human monocyte derived DCs (MDDC), generated in the presence of GM-CSF and IL-4 (IL-4 DC), and antigen-stimulated circulating gamma delta T lymphocytes, bearing the Vgamma2 TCR. Co-cultures of IFN-DCs with zoledronate-stimulated gamma delta T lymphocytes were also performed. gamma delta T-cell activation, as demonstrated by their up-modulation of activation marker expression levels (e.g. CD25 and CD69), was observed. These studies demonstrated for the first time the existence of a bidirectional activatin...