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...

Work has continued to characterise the function of a novel tick-derived DC modulator that perturbs various signalling pathways of human DC but not others; transcriptional profiling and pathways-based analysis will be undertaken in the next phase and data will become available.

We are currently performing gene silencing experiments in human monocyte-derived DCs (MDDCs) with the dual aim of generating relevant knowledge and data on (i) the transcriptional regulation of DC differentiation, and (ii) the safe and rationale in vitro manipulation of gene expression in DCs. One of the two different approaches that have been set up in parallel is: Lentiviral transduction of shRNAs: to this aim, shRNA specific for IRF4 and STAT3 are in the process to be cloned into the pLVTHM lentiviral vector (obtained from the repository Addgene). This v...

We are currently performing gene silencing experiments in human monocyte-derived DCs (MDDCs) with the dual aim of generating relevant knowledge and data on (i) the transcriptional regulation of DC differentiation, and (ii) the safe and rationale in vitro manipulation of gene expression in DCs. One of the two different approaches that have been set up in parallel is: Transient transfection of synthetic siRNA: to this aim, commercially available siRNA Smart Pool from Dharmacon targeted against the STAT3 transcription factor have been successfully transfected i...

Data has been obtained on the homing capacity of DC transfected with RNA encoding E/L-Selectin. We have efficiently transfected DC with RNA encoding a functional protein (E/L-Selectin) which allows entry of DC into LN from HEV. Mouse E/L-S transfected DC, when given i.v., homed to LN, whereas non transfected DC home only to the spleen. 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 th...

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...

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 have obtained data on the STAT-1 pathway in response of DC to Th1-inducing stimuli. We have identified some genes differentially expressed in the different conditions. In particular, the most relevant gene upregulated in presence of stimuli typically inducing Th1 responses (LPS, CpG, Poly I:C) but not in presence of the Th2 stimulus, Pam3Cys, was Stat-1. We then evaluated whether STAT-1 was phosphorylated in DC following activation with TLR-dependent Th1 and Th2 stimuli and we found that only in presence of TLR stimuli able to induce Th1 responses STAT-1 was act...

We have obtained data from our studies demonstrating that DC and other cell types can be activated in vitro by transfection with single stranded viral or synthetic RNA containing 5’ phosphates. This form of activation is mediated by the helicase RIG-I and leads to production of high levels of type I IFNs. Notably, this observation may explain the ability of mRNA transfection to activate DC for immunisation protocols: such RNA, even if synthesised in the presence of cap analog, always contains a certain percentage of uncapped molecules bearing 5’ tri-phosphates.

Data was obtained using CpG-DNA as TLR9 ligand, to establish an in vitro system to mature myeloid DCs and plasmacytoid (p) DCs and on the efficacy of CpG-DNA-Ag complexes for cross-presentation of Ag by DCs was analyzed in vivo. Mature DCs upregulated CD80, CD86, MHC class II and produced cell type specific cytokines. CD8+ myeloid DCs “cross-presented” exogeneous Ags (Ovalbumin) while pDCs produce type 1 Interferons. The efficacy of CpG-DNA-Ag complexed for cross-presentation of Ag by DCs was analyzed in vivo. Primary and secondary CTL response against the C8 ...

We obtained data on priming of an immune response through a series of critical interactions between the cholera toxin (CT) adjuvant and the dendritic cells (DC) of the splenic marginal zone (MZ). The in vivo mechanisms of action of most vaccine adjuvants are poorly understood. Splenic marginal zone dendritic cells mediate the cholera toxin adjuvant effect. We demonstrated in mice that a series of critical interactions between the cholera toxin adjuvant and the dendritic cells of the splenic marginal zone lead to effective priming of an immune response. Thus, for ...

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...

Our lab generated integrated microarray data on cellular pathways and gene ontology annotation. This has been done in the context of the program DC_Eu.Gene freely available for the DC-THERA users, using ENSEMBL as a conversion table.

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...

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...

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...

Based on the data we recently reported concerning the role of GM-CSF in regulating human DC differentiation (Conti et al., EJI 2008), we will perform a detailed analysis of mRNA and microRNA expression in immature and TLR ligand-stimulated GM-DCs in order to identify specific mechanisms that control the generation/activation of these cells. Data obtained in this study will become available. Moreover, the role of microRNAs in controlling the switch of GM-CSF-exposed monocyte precursors toward DCs or macrophages will also be investigated.

Our lab generated data on the mechanisms for DC-induced Th1 development and the molecular pathways for inducing Th1 cells producing IFN-gamma solely, or Th1 cells producing IFN-gamma and IL-10. We have reported that plasmacytoid precursor dendritic cells (pDC) activated with the TLR-9 ligand CpG induce strong development of Th1 responses (Boonstra et al, 2003). In contrast, bone marrow-derived myeloid DC (BM-myeloid DC) and splenic CD8alpha+ and CD8alpha- DC, despite expression of TLR9 mRNA, were poor at directing Th1 responses when activated with CpG. We have ...

We will start a collaboration with G. Schuler (P06) to examine the modulation of dendritic cell function by Denileukin Difitox (ONTAK) and will provide pathway analysis data.

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.