We have data available on dendritic cell development from hematopoietic stem and progenitor cells in steady-state and in inflammatory conditions, and, in particular, gene-expression data of Lin–c-KitintFlt3+M-CSFR+ cells in mouse bone marrow. Lymphoid tissue plasmacytoid and conventional dendritic cells (DCs) are continuously regenerated from hematopoietic stem cells. The cytokine dependence and biology of plasmacytoid and conventional DCs suggest that regeneration might proceed through common DC-restricted developmental intermediates. By selecting for cytokin...

In our lab, we have gene expression data in T cells available in responses to LCMV-GP33 protein and listeria-Ova. We studied TcR-Tg clones specific of each antigen (P14-GP33 specific, 0T-1-OVA specific), as well as endogenous polyclonal T cells, specific for the same antigens, identified by MHC pentamers loaded with specific peptides. In each individual T cell, we studied the expression of twenty different genes either mediating effector functions, or coding for different receptors involved in T cell differentiation and memory generation. We were surprised to obs...

We have data available on the inadequate nature of the antitumor CTL response, generated in a syngeneic mouse tumor model expressing an Ag derived from the early region 1A of human adenovirus type 5. We demonstrated using this syngeneic mouse tumor model that the inadequate nature of the antitumor CTL response is not due to direct Ag presentation by the tumor cells, but results from presentation of tumor-derived Ag by nonactivated CD11c(+) APC. Although this event results in division of naive CTL in tumor draining lymph nodes, it does not establish a productive i...

We have obtained data addressing the effectiveness of TLR ligand-long peptide conjugates in vivo by either direct vaccination or ex vivo DC loading prior to vaccination. Both approaches show similar effectiveness in CTL priming suggesting that TLRL-peptide conjugates efficiently target DC in vivo.

Data has been obtained on DCs loaded with Ag-IgG immune complexes (ICs) as a means of prophylactic immunization in mice. Therapeutic vaccinations strongly delay tumor growth or even prevent tumors from growing out. By depleting CD4+ and CD8+ cell populations prior to tumor challenge, CD8+ cells were identified as the main effector cells involved. Importantly, DCs pre-loaded in vitro with ICs are at least 1000-fold more potent than ICs injected directly into mice or DCs loaded with the same amount of non-complexed protein. FcgammaRs on DCs were required for effici...

Protein expression data is available for our pre-clinical work aiming at developing DC based vaccines expressing the tumor antigens PSA or Her2/neu. Monocyte rich fractions were produced by elutriation under GMP conditions and cultured for 5 days with GM-CSF and IL-4. Conditions for transfection with electroporation (Amaxa) or with Adenovirus constructs have been established. Protein expression (Her2/neu, PSA) following transfection with a panel of different full length or truncated Her2/neu or PSA constructs was assessed using Facs analysis (Her2/neu) or assessme...

Data characterizing the antigen-presenting DC in immunized mice is available. Our goal was to generate a probe that could allow the visualization of specific activated DCs following either the immunization of mice with an antigen in adjuvant or an infection with leishmania major parasites. We thus tried to prepare a mAb reacting to the immunodominant LACK156-173 peptide of leishmania bound to I-Ad MHC class II molecules. To this aim, we injected I-Ad/LACK recombinant dimers to TCR transgenic mice which exhibited an increased frequency of LACK-specific T cells. ...

Although many bacteria and protozoa can invade dendritic cells (DC), it is not clear whether infected DC process pathogen-derived antigen and stimulate antigen-specific CD4+ T cells in vivo. To address this issue and obtain data on this question, we have infected susceptible BALB/c (H2-d) mice with a recombinant Leishmania major parasite expressing a fluorescent tracer. We have directly visualized antigen-presenting cells using a mAb reacting to an antigenic peptide derived from the parasite LACK antigen bound to I-Ad MHC class II molecule. I-Ad/LACK complexes wer...

Data was obtained on the infiltration and destruction of solid tumors by cytotoxic T lymphocytes (CTLs). Although the immune system evolved to fight infections, it may also attack and destroy solid tumors. In most cases, tumor rejection is initiated by CD8+ CTLs, which infiltrate solid tumors, recognize tumor antigens and kill tumor cells. We used a combination of two-photon intravital microscopy and immunofluorescence on ordered sequential sections to analyze the infiltration and destruction of solid tumors by CTLs. We show that in the periphery of a thymoma gr...

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...

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 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.

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 ...

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 ...

Our lab generated data on alternatives to the ‘classical’ maturation method (i.e. a cocktail of inflammatory cytokines) for moDC (e.g. ‘clinical grade reagents such as Ampligen TM and Hiltonol TM). Moreover the possibility to co-electroporate moDC with constitutively active TLRs and TAA encoding mRNA was persued. The in vitro charging of monocyte derived DC (moDC) with antigens has been optimised. In contrast to previous studies, the electroporation of TAA encoding mRNA after in vitro maturation of the moDC resulted in a better electroporation efficiency an...

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...

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.

We obtained data on priming of an immune response through a series of critical interactions between the cholera toxin (CT) adjuvant and the dendritic cells (DC) of the splenic marginal zone (MZ). The in vivo mechanisms of action of most vaccine adjuvants are poorly understood. Splenic marginal zone dendritic cells mediate the cholera toxin adjuvant effect. We demonstrated in mice that a series of critical interactions between the cholera toxin adjuvant and the dendritic cells of the splenic marginal zone lead to effective priming of an immune response. Thus, for ...

Among the battery of cell surface receptors expressed by DCs, the receptor for the Fc portion of IgG (FcgammaRs) recognizes the antibodies coupled to their specific antigens, called immune complexes (IC). We have data available from our demonstration that the target of FcgammaRs with the antigen induces the internalization of the IC, leading to the maturation of CD11c+CD11b+ conventional DCs. These DCs become thus efficient to present antigenic peptides to helper CD4 T cells and cytotoxic CD8 T cells via cross-presentation pathway. This capability might then enhan...

Data was obtained on RNA transfected DC which proved superior in priming naïve T cells in vitro compared to DC loaded with native peptides.