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...

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...

We have explored fundamental aspects of the anti-tumor responses. We obtained data examining the cell populations which infiltrate progressive versus regressing P815 mastocytoma in order to identify cells which display immunosuppressive properties. We found changes in regulatory T cells populations as well as in the “myeloid suppressor cells”. Our preliminary data suggest that two populations of regulatory T cells coexist (natural and induced) in progressing tumors, and that a population of Gr1+ cells may affect tumor resistance in vivo.

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...

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...

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...

Data was obtained on the properties of the CD16, CD1c and BDCA-3 DC populations and their production of cytokines in response to different Toll-like receptor (TLR). We studied two major blood populations of DCs, myeloid DCs (mDCs) and plasmacytoid DCs (pDCs). Among mDCs, three populations could be further identified by specific markers: CD16, CD1c and BDCA-3. The properties of these DC populations and their production of cytokines in response to different Toll-like receptor (TLR) agonists were evaluated. The TLR agonists used were: PAM3CSK4, Poly I:C, LPS, Flagel...

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...

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 obtained data from our activity to establish the identification of versatile central memory CD8+ T-cells capable of high IL-2 secretion according to new markers. Versatile human CD8+ T-cells represent up to 30% of the CD8+ central memory pool in healthy adults and are characterised by unique functional capabilities (ability to secrete IL-2 after restimulation in concordance with low effector cytokine (e.g. IFN) and lack of cytotoxic mediator (e.g. perforin) expression (Frentsch et al., unpublished)). They might present an attractive candidate memory/effector C...

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...

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 immunogenicity induced by chemotherapeutics.

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

We obtained data on the immunotherapy potential of DC, pulsed with tumor antigens, through its evaluation in EG7-OVA and P815 tumor models. Depletion of natural regulatory T cells in tumor bearing mice resulted in rejection of P815 cells, but not EG7-OVA, suggesting that regulatory T cells have a stronger impact on immune responses against weakly immunogenic or auto-antigen (P1A). Treg are detected in growing P815 tumor and there is some evidence that 2 regulatory T cells develop sequentially. T cell activation was monitored by tetramer staining and in vivo CTL as...

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.

Data will be available from in-vitro pre-clinical tests on the pharmacodynamic activity of the fusion vaccines. This test will verify the capacity of the fusion vaccine to present certain tumour-antigen derived peptides corresponding to antigens present in the fused cancer cells. BruCells has met with the Dutch MEB for a Scientific Advice Meeting on January 10, 2008 in The Hague. The Dutch authorities consider that the proposed in-vitro pre-clinical tests are adequate to check the pharmacodynamic activity of the fusion vaccines and that no animal model tests would be necessary.

This test will verify whether the fusion vaccine possesses the properties of both parental cell types for inducing primary CD8+ T-cell responses (data will become available). Detection of circulating tumour-specific CD8+ T-cells. BruCells has met with the Dutch MEB for a Scientific Advice Meeting on January 10, 2008 in The Hague. The Dutch authorities consider that the proposed in-vitro pre-clinical tests are adequate to check the pharmacodynamic activity of the fusion vaccines and that no animal model tests would be necessary.

This test, described for glioblastoma, performed with PBMC from newly diagnosed glioblastoma patients will verify the ability of tumour-specific T-cells to recognize the fusion vaccine. Data will be made available. BruCells has met with the Dutch MEB for a Scientific Advice Meeting on January 10, 2008 in The Hague. The Dutch authorities consider that the proposed in-vitro pre-clinical tests are adequate to check the pharmacodynamic activity of the fusion vaccines and that no animal model tests would be necessary.

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 ...