We obtained data assessing the entire CD4+ Th-cells specific for defined antigens also for murine cells (based on the strategies developed in work performed in 2005 (Frentsch et al., Nature Medicine 2005)). For intracellular assessment of CD154 induced in the course of short-term activation target cells are stimulated with antigen for 6h in the presence of the secretion inhibitor BrefA (for 4h). For extracellular analysis of live antigen-specific Th-cells cells are stimulated in the presence of an anti-CD40 blocking antibody and biotinylated anti-CD154 antibody. W...

Our group has obtained proteomic data on the three known splenic subsets (CD8+, CD4+, DN DC). Despite the significant advances in mass spectrometry, which has enabled much of proteomics, due to various analytical challenges so far no eukaryotic total cell proteome has been sequenced completely. An alternative approach is to purify single organelles or DC components in order to generate the proteomic data. An additional problem is to obtain highly pure fractions of DC from crude population of untouched splenic cells with a purity of 70-80%. This includes several...

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 ...

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 from a phase 2 study testing PME-CD40L DC in RCC patients with clear cell histology. The Phase 2 trial has the same general design, dosing regimen, endpoints, and immunomonitoring plan as the completed RCC trial. However, the Phase 2 study tests the improved RCC product (i.e., PME-CD40L DC) and is restricted to RCC patients with clear cell histology. As predicted from the in vitro data, the improved PME-CD40L DC product does indeed lead to restoration of both IL-2 and IFN-? responses. Also consistent with our in vitro observation is the...

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.

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...

Data has been obtained on MVA BN (a powerful poxvirus vaccine system) from a thorough analysis on the nucleotide level on safety in cell cultures in immune deficient mice as well as in healthy, HIV–infected and other immune deficient humans. We have furthered our interaction with Dr.Markus Manz, (Partner group 27) IBR, Bellinzona, Switzerland to strengthen vaccine developments with an improved humanized mouse model. We focus on recombinant vaccines against HIV driven by a powerful poxvirus vaccine system termed MVA BN. To improve our understanding MVA BN has b...

Data was obtained on the regulatory T cells (Tregs) and their role in effective anti-tumor immune responses in FoxP3-DTR DEREG mice. Although multiple mechanisms of Treg action have been proposed, the actual mechanism involved in the suppression of anti tumor T cell responses are still unclear. Treg inactivation by anti-CD25 antibodies or by depletion in FoxP3-DTR DEREG mice resulted in effective rejection of the tumors upon adoptive transfer of antigen-specific CD8+ T cells. Treg inactivation or depletion induces a marked arrest in the migration of anti-tumor C...

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...

Our lab has obtained data on the mechanism of how IL-10 regulates the immune response to MTb infection in mice. Control and clearance of intracellular pathogens such as MTb is dependent on the production of TNF and the induction of the T-helper 1 (Th1) cytokine IFN-gamma by IL-12. Conversely, IL-10 is a suppressive cytokine essential for dampening the immune response to a number of intracellular pathogens to limit host immune pathology. IL-10 has been shown to exert its suppressive effect by directly acting on the antigen presenting cell (APC), therefore functi...

Data has been obtained from our work aiming at defining the role of CCR6 in directing migration of TH17 cells at sites of inflammation or infection. We found that CCR6–deficient (CCR6-KO) mice were resistant to induction of EAE but became susceptible when transferred with small number of CCR6-sufficient T cells. CCR6 was found to be required on a first wave of TH-17 cells that entered the CNS through the choroid plexus epithelial cells, which constitutively expressed CCL20 in both mice and humans. CCR6+ T cells triggered entry of a second wave of T cells that mi...

We have microarray data on macrophages and DC from different genetic backgrounds of mice, stimulated with CpG, in the presence or absence of a MEK inhibitor, and further data on the role of certain MAP kinases in the regulation of IL-10 and IL-12, IFN-gamma production. Using pharmacological inhibitors, or macrophages and DC from mice carrying a null mutation in MAP kinase signalling molecule(s), we have evidence for a role of certain MAP kinases in the regulation of IL-10 and IL-12, IFN-gamma production. The work on the role of the MAP kinases, TPL-2 and ERK in u...

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

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...

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...

Our lab has obtained data on the role of p50 NF-kB in differentiation, survival and APC function in mice. Tumor growth is supported by tumor stroma, which is made by matrix and infiltrating cells, such as tumor associated macrophages (TAM) and tumor associated dendritic cells (TADC). We have recently reported that TAM display massive nuclear localization of the p50 NF-kB inhibitory homodimer, which correlates with impaired inflammatory functions (Saccani et al., 2006).The functional significance of this observation was demonstrated in p50 NF-kB deficient mice, whi...

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.

We are continuing our studies of the roles of DC in scrapie uptake and dissemination and have obtained data on intestinal DC subsets all of which can acquire scrapie ME7 form the intestinal lumen and transport it via lymph. In current studies we are examining the effects of indomethacin-induced small intestinal inflammation on both intestinal DC biology and on scrapie uptake, transport and delivery. We are characterising (in collaboration with Sebastian Amigorena) rat lymph exosomes and will determine if they transport intestinally-delivered scrapie ME7. We are ...

We obtained data on the influence of bLf, whose immunomodulant properties are now recognized, on MDDC differentiation/activation. We found that bLF interferes with the activation of MD-DC induced by different TLR agonists, but not by T cell mediated signals. Although bLF did not significantly influence monocyte differentiation in DCs, we found that it markedly reduced the up-modulation of CD83, co-stimulatory and MHC molecules (i.e. CD80, CD86, MHC class I and II), and cytokine secretion (IL-12, TNF-? and IL-23) induced by LPS or polyI-C. Consistently with an impa...