We have data available from an analysis of the proteome of the phagocytic/endocytic compartments that are formed upon cell entry of antigens via specific pathways. The ultimate goal is to get a better insight into the protein complexes that are involved in antigen processing, and learn whether the different entry routes for antigens into DCs might have distinct functional characteristics.

We obtained data on MHC II transport during human monocyte-derived DC maturation. We have published that the E3-ubiquitin ligase MARCH I is responsible for peptide-loaded MHC II ubiquitination and their subsequent internalization in immature DC.s The ligase MARCH I has been shown to be down-regulated upon activation. (De gassart et al, PNAS 2008). We will also investigate further the role of the ubiquitin ligases of the MARCH family in antigen processing, in particular we will focus on cross-presentation and CD1a molecule transport.

We obtained data on the control of innate immunity by the mammalian target of Rapamycin (m-TOR) by enhancing TLR driven production of proinflammatory cytokines and type 1 interferons and by repressing posttranslational processing, via the inflammasome, of IL-1beta. We will further analyse the impact of the m-TOR inhibitor Rapamycin on innate immunity.

We obtained data on antigen delivery by a vector with ovalbumin and CMVpp65 in mice or human, respectively. We used a MHC II presentation pathway targeting vector originally published by Wang et al. 1999. Proof of principle studies for antigen delivery have been performed with ovalbumin (murine studies) and CMVpp65 (human studies).

We will explore the immunogenicity of necrotic cells as a tool to dissect the molecular determinants involved in “danger” recognition and will obtain data on this.

We have obtained data on CD8T lymphocyte populations in humans from our effort to identify and characterize these populations. We compared the distribution of phenotypically distinct cell sets using seven color staining in the blood, lymph nodes and spleen. Due to our location in a pediatric hospital, we have access to these organs removed from patients that undergo surgery due to clinical situations that do not affect the immune system (most frequently malformations). We found that the addition of other markers allow to subdivide N, CM, TEM and TEMRA populations ...

We have obtained data based on the characterization of a novel C-type lectin expressed primarily in mouse CD8?+ DC and their putative human equivalents. This lectin, named DNGR-1, can serve as a receptor for antigen targetting to DC. Notably, tumour antigens coupled to anti-DNGR-1 antibodies and given together with a suitable adjuvant elicit potent CTL responses that can promote tumour regression. We will continue to explore the use of DNGR-1 as a tool for targeting antigens to DC in vivo. In particular, we will examine if such targeting in the absence of adjuva...

Proteomic map data will be generated for phagosomes formed after phagocytosis through different phagocytic receptors (FcRs, DEC-205, mannose receptor). We will do this by applying a unique methodology dedicated to the identification of phagosome proteins in dendritic cells, under various experimental conditions, that we developed at the Curie Institute. This methodology incorporates a unique sample preparation procedure based on 96 well plate complex sample trypsinization, a nano-LC-based peptide separation coupled to MSMS analysis sequencing. This procedure led us...

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...

Data obtained from a proteomic analysis of human exosomes performed by Sebastian AMIGORENA from the DC-THERA consortium allowed us to suspect a number of proteins as a potential candidate target for our hybridoma 13. We have immunized mice with human DC derived-exosomes and generated hybridomas that produce antibodies recognizing at least the human exosomes. Out of 20 hybridomas, only one appears to recognize specifically DC exosomes and not tumor exosomes nor autologous monocytes or DC or PBL. The Ab produced by this N°13 hybridoma is binding to a protein of abo...

Central to all T cell responses is the interaction between the T cell receptor and peptide antigen presented by the MHC molecules. This interaction selects the dominant T cell clones with the most favourable (not necessarily highest) affinity and determines the extent of cross-reaction with epitope variants. I am currently collaborating with the structural biology group of Professor Yvonne Jones in the Nuffield Department of Medicine (NDM) at the Wellcome Trust Centre for Human Genetics. This collaboration has led to structural data on the CD1b in complex with s...

We obtained microarray and RT-PCR data on the priming of CD8+ T cells in mice and their regulation of proliferation and development into effector and/or memory cells. The positive influence of a reactive lymph node on the priming of CD8+ T-cells was already analyzed in 2007. We set up experiments to determine if this effect would benefit a response against a second infection. We infected mice with CD8+ T-cells primed with or without the influence of a reactive lymph node with a flu-virus encoding the specific antigen. After several days we determined the viral lo...

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...

Proteomic data was obtained from a proteomic analysis of DC-derived organelles such as the exosomes. Following the extensive proteomic analysis of immature and mature DC-derived exosomes, ICAM-1 was identified as the major protein on mature DC-derived exosomes, required for their functional activity. In order to functionally validate the proteomic data, the antigen-presenting function has been investigated. Exosomes bearing functional MHC-peptide complexes have been shown to be presented at the surface of recipient DCs as intact "antigen-presenting microdomains", w...

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...

Our lab has proteomic data available on the interaction between RIG-I and NS1 in influenza A. We have used fluorescence microscopy to analyse the interaction between RIG-I, a viral sensing protein, and NS1, an inhibitor of interferon production that is encoded by influenza A virus. This approach enabled us to determine that the two proteins form a complex in influenza infected cells.

Proteomic data has been obtained using used yeast as a model organism to optimize technological approaches for the quantitative assessment of molecular components in yeast proteomics. We compared protein levels of essentially all endogenous proteins in haploid yeast cells to their diploid counterparts. Our analysis spanned more than four orders of magnitude in protein abundance with no discrimination against membrane or low level regulatory proteins (deGodoy et al.).

A comprehensive protein inventory of clinical grade immature and cytokine cocktail matured (Il-6, IL-1 beta, TNF alpha, PGE2; 48 hours) monocyte derived human dendritic cells (DC) from a healthy donor has been established by using high accuracy, high sensitivity protein identification technology. We have identified 2794 proteins in DCs by liquid chromatography tandem mass spectrometry. Prior to MS analysis, DC were lysed and divided into a soluble fraction containing cytosolic proteins and into an insoluble fraction enriched for membrane containing proteins. High...

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...