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 ...

Our lab obtained data on maturation of DC by TLR agonists.

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.

Data were obtained on the effect of enhanced CD83 expression on dendritic cells and T cells and its correlation with effective immune responses. Human CD83 is a marker molecule for mature dendritic cells (DC) and is also expressed on activated B and T cells. Although CD83 has been implicated in immune responses, its function on DC and T cells remains unclear. In this study, we wanted to assess the role of CD83 expressed on DC and T cells in the immune response. Down-regulation of CD83 expression on human DC through RNA interference (RNAi) results in a less potent...

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.

Our laboratory obtained data on the induction of immune responses after targeting antigens to DC via TLR agonists.

We have obtained data from an analysis of the uptake, conservation and cross-presentation of the model antigen OVA after Fc receptor-mediated uptake by DC and from an analysis of the immunological consequences of the antigen depot. We found that MHC class I presentation is relatively short-lived in contrast to MHC class II. However, CD8 cross-priming capacity of OVA-loaded DC was functionally retained for many days, while peptide-pulsed DC had lost their priming capacity after 24 hours. Strikingly, OVA protein antigen was conserved intracellularly in DC for many ...

Our group has MRI data available on the migration of DCs in vivo in melanoma patients. From isolated lymphnodes obtained after surgery we could identify single DC after staining with Prussian blue for iron. We are in the process of analysing the T cell rosettes (activation stage) that surround these DC to get insight in the functional activity. It was also demonstrated that DCs transfected with RNA produce derived proteins in vivo in the lymph nodes. Also efforts were spent to get further information on the dynamics of immature and mature DCs. We discovered (van...

We have data available on the induction of effective therapeutic antitumor immunity by direct in vivo administration of ovalbumin (OVA) encoding lentiviral vectors. Ex vivo lentivirally transduced dendritic cells (DC) have been described to induce CD8+ and CD4+ T-cell responses against various tumor-associated antigens (TAAs) in vitro and in vivo. We report here that direct administration of ovalbumin (OVA) encoding lentiviral vectors caused in vivo transduction of cells that were found in draining lymph nodes (LNs) and induced potent anti-OVA cytotoxic T cells ...

Our lab obtained data on the induction of antigen-specific CD8+ cytotoxic T cells by dendritic cells co-electroporated with a dsRNA analogue and tumor antigen mRNA. The maturation state of dendritic cells (DCs) is an important determinant for the initiation and regulation of adaptive immune responses. In this study, we wanted to assess whether functional activation of human monocyte-derived DCs can be achieved by electroporation of an activation signal in the form of double-stranded (ds) RNA and whether simultaneous electroporation of the dsRNA with tumor antigen...

Our lab conducted an immunomonitoring study, such as on effector memory T cells, which led to corresponding immunological data.

Using the MLPC/tetramer method described last year, we obtained data by pursuing the immunological monitoring of melanoma patients vaccinated with 4 peptides (MAGE-A3, gp100, NA17 and tyrosinase) presented by HLA-A2. We measured responses to these peptides injected with Montanide, but did not know whether the Montanide adjuvant was important for these T cell responses. Indeed, melanoma patients may mount spontaneous responses to these antigenic peptides. We therefore injected the same peptides without adjuvant, and no response was observed. We conclude that Montan...

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...

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...

Global gene expression data have been obtained using Affymetrix mouse chips by designing dendritic cell-based bioassays. To identify possible differentially expressed genes by DC after activation with stimuli typically involved in inducing Th1 or Th2 type responses we have performed comparative kinetic global gene expression analyses of DC activated with different TLR agonists typically associated with Th1 (LPS, CpG, Poly I:C) or Th2 responses (Pam3Cys). For these analyses, we have used the Affymetrix GeneChip® technology and, in particular, mouse chips showing pr...

We are collecting immunological data from a melanoma patient, aiming to identify the antigen(s) recognized by T cells from this patient that experienced an outstanding response to immunotherapy. This patient received several adoptive T cell transfers and regressed from stage IV melanoma to a disease free state and has remained tumor free for several years. In a collaboration with Dr. O. Winqvist, Karolinska Institute, we attempted to improve the adoptive transfer protocol by stimulating T cells with monocyte derived DC pulsed with tumor lysate, instead of simply a...

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...

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 expression and regulations of Notch ligands on DC subsets.