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.

Data on the role of STAT3 signalling in directing DC-mediated responses toward immunogenicity or tolerance will be generated. To this purpose, we will identify (i) genes and (ii) micro-RNA selectively controlled by STAT3 in human MDDCs treated with proinflammatory or tolerogenic stimuli (including TLR ligands, cytokines and vitamin D3). This will shed light on the role of STAT3 signalling.

Pathways-based analysis will be done in collaboration with P15 (Cavalieri) to identify the intracellular target of Japanin and data will become available from this study.

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.

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

Concerning the pathway based analysis and database we have started the curation of the TLR4-CD14 pathway in DCs and we have described the events occurring at the DC membrane level. We have also started global gene expression analyses to better difine the role of NFAT in DCs.

During 2008, we extended all the data regarding TLR3 signaling to TLR8 signaling. Cytokines assays and immunoblotting studies suggest that Src kinases play a crucial role in the control of both MyD88- and TRIF-dependent pathways. We also extended microarray analysis on human MoDC stimulated with R848 (TLR8 agonist), pretreated or not with PP2. In summary, the new generated data on TLR8 triggered gene expression are similar to previous data obtained with TLR3 stimulation, and confirmed that src kinases inhibition is associated to inhibition of key genes in the infl...

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 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...

The importance of alveolar macrophages and more recently DC in the immune response to Mycobacterium tuberculosis (MTb) has been well documented, but the relative contribution of plasmacytoid DC during bacterial infections is largely unknown. Thus, data was obtained on the contribution of pDC in acute MTb infection. We examined whether plasmacytoid pDC are infected and/or activated by MTb, and whether they play a role in regulating bacterial clearance during acute MTb infection and if so how. We have compared these DC with myeloid DC and macrophages with respect t...

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 ...

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 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.

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 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...

Data was obtained on RNA transfected DC which proved superior in priming naïve T cells in vitro compared to DC loaded with native peptides.

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 ...