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...

We have obtained data from a study where we addressed a model antigen to exosomes secreted by tumor cells in vivo, to allow capture of antigen-bearing exosomes by DCs and cross-presentation of the antigen. For this purpose, the model antigen OVA was fused to the C1C2 exosome-binding domain of MFG-E8/lactadherin. Tumors secreting the exosome-bound antigen grow slower than tumors secreting soluble OVA, because they induce activation of OVA-specific CD8+ T cells into killer cells. In addition, inducing in vivo secretion of the C1C2-coupled antigen by DNA vaccination i...

Our lab has generated data on receptors on dendritic cells necessary for capture of exosomes. In 2007, we have finalized analysis of the receptors on DCs responsible for capture of exosomes secreted by mature DCs. We have shown that expression of LFA-1, but not of Mac-1, integrin, on DCs is required for capture of ICAM-1-bearing exosomes. Furthermore, the CD8+ subpopulation of DCs in lymph nodes expresses LFA-1 at higher levels than the CD8- subpopulation, and is responsible for in vivo capture of injected exosomes. We have thus proposed a new role for LFA-1 on DCs...

Our laboratory has obtained data on the role of DC-derived cytokines in NK cell production of IFN-gamma.

Our lab has obtained data on the role of IL-10 and IDO in relation to Th1 and inflammatory responses in a colitis model tested on mice. IDO appears critical for the regulation of TNBS colitis upon CTLA-4 engagement, as the beneficial effect of anti-CTLA-4 treatment was lost in IDO-deficient mice. By contrast, the absence of IDO did not alter the course of inflammation in mice injected with TNBS only, an unexpected finding which suggests that IDO-dependent counter-regulation requires other factors/cells in addition to IDO production and T cell activation by TNBS. Ou...

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...

Studies have continued and data obtained on the effects of biolistic (Gene Gun) delivery of vectors engineered to express distinct chemokines at the local skin site of mice. In particular, attention has been focussed on the recruitment of distinct subsets of DC to skin. This work, which is being performed in collaboration with PowderMed, a former SME now owned by Pfizer, may lead to the development of more targeted strategies to induce distinct T cell responses using this technology. A publication has resulted from this work.

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 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 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 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...

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...

We have obtained data by fluorescence microscopy analysing DC distribution in spleen and how this is affected during inflammation driven by stromal cells or hematopoietic cells. One of our images of DC was selected as the cover for the June 2007 issue of Nature Immunology.

Flow cytometric analysis data were obtained of translation activation in DC-activated T cells.

We have obtained imaging data by confocal microscopy of translation activation in DC-activated T cells and have pursued our efforts on the definition of a novel way of monitoring DC and T cell activation as well as studying viral infections. We also have now applied the puromycin-based technology developed in our laboratory (SUnSET, surface sensing of translation) to monitor translation by FACs in individual or cell populations. We have demonstrated that translation activation is an excellent read-out for early antigen specific T cell activation by DCs. We hope tha...

Multi-photon imaging data on DC-CD8+ T cell interactions in intact lymph nodes (LNs) have been obtained. The initiation of cytotoxic immune responses requires the direct interaction between naïve CD8+ T lymphocytes and dendritic cells. Multi-photon imaging showed that during priming, naïve T cells and dendritic cells (DCs) establish sequentially brief (i.e. minutes) and long (hours) antigen-specific contacts. We observed that ICAM-1 expression by mature DCs is critical for long-lasting contacts with CD8+ T cells, but dispensable for short-lived antigen-specific i...

We have been following up on the effort to generate proteomics map data of lysosomes purified from non-activated and activated mouse DCs. The decision to focus on the most important organelle for DC activity has been driven from the need to describe in greater detail the proteins involved in the DC response both qualitatively and quantitatively. The partner has been trying to quantify the relative amount of 400 proteins. They have also indexed better this list and are currently comparing it to a list of proteins present in macrophages lysosomes (in collaboration ...

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

We have established an in-depth proteomic map of murine dendritic cell subsets. We have been working in the attempt to overcome the limits in sample availability, exploiting the recent significant improvements of proteomic technology. The results allowed to lower the required sample amount significantly, an essential feature to analyze limited cell populations like dendritic cells. The group completed the measurements of murine dendritic cell subsets and established an in-depth proteomic map of them. These preliminary results show more than 4500 identified proteins...

We obtained data on the protein changes that occur at the cell surface of a maturing DC, induced by a prototypical maturation stimulus such as LPS. Results obtained from the LC-MS/MS analysis of the integral plasma membrane proteome of D1 cells stimulated for 24 hours with LPS have been compared to the results obtained from immature D1 cells. As expected, DC up-regulated the cell surface expression of all proteins known to be part of the so-called “immunological synapse”, namely MHC class II (region E and K), CD40, CD80, CD86 and ICAM. Interestingly, LPS stimul...