Our lab has obtained data on the molecular signatures for alternatively activated DC. This type of activation characterizes the state of DC in certain pathophysiological conditions, such as during the resolution of inflammation as well as in chronic inflammatory conditions and in tumors. AA-DC were obtained by culturing immature DC in the presence of a maturative agonists (such as, LPS, CD40L or TNF) and calcitriol, IL-10 or prostaglandin E2. AA-DC showed a decrease in the production of IL-12 but retained most of the ability to secrete other cytokines, such as TN...

We have further data available on the expression and function of FcgammaR in mice. We had previously shown that conventional DCs express the inhibitory type II FcgammaR and the activatory type I and type III FcgammaRs. Therefore, we next investigated the expression and function of these murine FcgammaR in CD11c+CD11b-B220+ plasmacytoid DCs (pDCs). pDCs express mostly FcgammaRIIB while the expression of FcgammaRI and FcgammaRIII is only detected by RT-PCR at low but significant level. Moreover, the ITAM-containing intracellular chain associated to FcgammaRI and Fcg...

Among the battery of cell surface receptors expressed by DCs, the receptor for the Fc portion of IgG (FcgammaRs) recognizes the antibodies coupled to their specific antigens, called immune complexes (IC). We have data available from our demonstration that the target of FcgammaRs with the antigen induces the internalization of the IC, leading to the maturation of CD11c+CD11b+ conventional DCs. These DCs become thus efficient to present antigenic peptides to helper CD4 T cells and cytotoxic CD8 T cells via cross-presentation pathway. This capability might then enhan...

We have obtained data on the ability of mMature DCs to efficiently present antigens associated with MHC molecules to T lymphocytes. Dendritic cells (DCs) are major actors of innate and adaptive immune responses. Immature DCs express a great diversity of receptors at their cell surface, which provide them the ability to uptake antigen and induce an intracellular signal. DCs become mature after encountering soluble mediators or conserved pathogen associated molecular patterns (PAMPs) or after interacting with other cells. Mature DCs acquire the ability to efficient...

Data was obtained of the capacity of splenic DC, pulsed in vitro with protein antigens or peptides, to induce immunity after transfer into syngeneic animals. To improve existing strategies, the amplitude and polarisation of T cell responses was monitored in the presence or absence of regulatory T cells. Natural regulatory T cells (CD4+ CD25+) were depleted by injection of anti-CD25 mAbs, whereas regulatory T cells were induced by injection of (presumably agonistic) anti-CTLA-4 mAbs. Our observations demonstrate that natural and CTLA-4 induced regulatory T cells a...

The use of gene guns in ballistically delivering DNA vaccine coated gold micro-particles to skin can potentially damage targeted cells, including Langerhans cells, therefore influencing transfection efficiencies. We have assessed and obtained data on cell death in the viable epidermis by non-invasive near infrared two-photon microscopy following micro-particle bombardment of murine skin. We showed that the ballistic delivery of micro-particles to the viable epidermis can result in localised cell death. Furthermore, experimental results show the degree of cell deat...

We have compiled a comprehensive protein profile of DCs and obtained data on changes in protein level and post-translational modifications involved in the regulation of specific signalling pathways or cell processes. First, we have set up a system for SILAC labelling of bone marrow derived murine dendritic cells (BM-DC). The principal method for generating BM-DC with GM-CSF was adapted from Lutz et al. (J Immunol Methods 1999, 223: 77-92). After some modifications of the protocol the FACS analysis of DC, which were generated in conditions described by Lutz et al....

Data have been obtained on Mart-1 specific T cell priming and/or NK cell triggering in HHD2 mice. We have used DC exosomes along with various TLR ligands or cytokines such as IL-2 or alpha IFN to promote Mart-1 specific T cell priming and/or NK cell triggering in HHD2 mice. We demonstrated that only TLR3 and 9 ligation is able to induce CTL priming in vivo. However, exosomes in the absence of adjuvants can induce NK cell activation. DC exosome-mediated NK cell triggering depends upon NKG2D ligands presented on the exosome surface

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 lab has obtained data on the dependence of IL-10 as well as IL-12 production on signalling adaptor molecules. Dendritic cells (DC) arise in the bone marrow and circulate through blood or tissue; upon pathogen challenge DC subsequently migrate to lymphoid organs to initiate immune responses (Banchereau et al., 1998). DC act as sentinels for pathogens forming the interface between the innate and adaptive immune response. In addition they are flexible in driving both Th1 and Th2 responses (Boonstra, Rajsbaum et al., 2003). Mouse DC can be derived from bone marro...

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...

Our lab obtained first data from a new clinical trial of a DC vaccine administered into irradiated tumours in RCC. Three patients were enrolled and DC vaccine was produced. However only one patient received the vaccine due to cancer progression in the other two patients. The DC were labelled with 111In. The following patients will receive DC labelled with Endorem and the vaccine will be imaged with MRI. The patient treated is in complete remission 6 months after treatment.