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...

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 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 of the capacity of splenic DC, pulsed in vitro with protein antigens or peptides, to induce immunity after transfer into syngeneic animals. To improve existing strategies, the amplitude and polarisation of T cell responses was monitored in the presence or absence of regulatory T cells. Natural regulatory T cells (CD4+ CD25+) were depleted by injection of anti-CD25 mAbs, whereas regulatory T cells were induced by injection of (presumably agonistic) anti-CTLA-4 mAbs. Our observations demonstrate that natural and CTLA-4 induced regulatory T cells a...

The use of gene guns in ballistically delivering DNA vaccine coated gold micro-particles to skin can potentially damage targeted cells, including Langerhans cells, therefore influencing transfection efficiencies. We have assessed and obtained data on cell death in the viable epidermis by non-invasive near infrared two-photon microscopy following micro-particle bombardment of murine skin. We showed that the ballistic delivery of micro-particles to the viable epidermis can result in localised cell death. Furthermore, experimental results show the degree of cell deat...

Data have been obtained on Mart-1 specific T cell priming and/or NK cell triggering in HHD2 mice. We have used DC exosomes along with various TLR ligands or cytokines such as IL-2 or alpha IFN to promote Mart-1 specific T cell priming and/or NK cell triggering in HHD2 mice. We demonstrated that only TLR3 and 9 ligation is able to induce CTL priming in vivo. However, exosomes in the absence of adjuvants can induce NK cell activation. DC exosome-mediated NK cell triggering depends upon NKG2D ligands presented on the exosome surface

We obtained data from a study of the correlation between CD8 multiple cell surface markers and functional profiles studied at single cell level. For that purpose, we subdivided peripheral CD8 T cells into eleven different cell subtypes based on the association of multiple cell surface markers. In each subtype, we isolated single-cells. In each single cell, we quantified the expression of multiple genes. Moreover, we isolated and studied cells from different normal donors. These results were published in Blood. We showed that - this strategy allowed the identifi...

Data was obtained on CD4+ T cell responses against pathogens or tumors in humans. Our goal was to generate peptide-MHC class II multimers to follow antigen-specific CD4+ T cells in immunized individuals. The study was a collaboration with Monica Moro in P. Dellabona’s lab. to study CD4+ T cell responses against pathogens or tumors in humans. Soluble recombinant HLADR1101 MHC class II molecules receptive for loading with either pathogen or tumor-derived peptides were constructed. Although all these molecules could stain CD4+ T cells that had been stimulated for f...

Our lab has immunological data available on human Hemato-Lymphoid System Rag2-/-gc-/- mice infected with EBV. Both Epstein Bar Virus (EBV) and Human Immunodeficiency Virus (HIV) are human specific lymphotropic viruses. Human Hemato-Lymphoid System Rag2-/-gc-/- mice were infected with EBV and mount an immune response (i.e. cytotoxic T cell proliferation, some control of EBV driven B cell proliferation, perforine and granzyme expressing T cell infiltration in B cell infected areas in lymphoid organs in situ), however, specific T cells could not be detected directl...

Data is available on human Hemato-Lymphoid System Rag2gc-/- mice infected with both CXCR4 as well as CCR5 tropic HIV-1 strains. HIV causes a disseminated infection and spreads in all newly generated lymphoid tissues, thus closely resembling HIV infection in humans. However, as in EBV infected animals, adaptive T and B cell responses were not consistently detected. Thus, although these “humanized” mice represent a major step forward in generating an in vivo preclinical testing system, they still harbor limitations as inconsistent/weak adaptive immune respons...

We have data available on some functional immune responses in a small animal model to study the development and function of the human hematopoietic and immune system as well as human specific infections in vivo. We established “human hemato-lymphoid-system mice” by transplanting human CD34+ cord blood cells into irradiated newborn Rag2-/-gc-/- mice, leading to de novo development of human B, T, and dendritic cells; formation of structured primary and secondary lymphoid organs; and production of some limited functional immune responses.

We have data available on the role of chemokine receptors in regulating migration of mature DC and subsets of effector and memory T cells in secondary lymphoid organs. CCR7 is a chemokine receptor that is expressed by mature DCs and central memory T cells (TCM) and regulate migration of these cells in secondary lymphoid organs in the steady state. T lymphocytes lacking the lymph node homing receptors L-selectin and CCR7 do not migrate to lymph nodes in the steady state. In contrast it was found that lymph nodes that drain sites of mature DC or adjuvant inoculatio...

Our lab obtained data on the kinetics of effector gene expression and association in TCR-Tg CD8 cells responding to the male antigen and to Listeria-OVA with regard to « in vivo » responses to Ags. Surprisingly, instead of a single program of differentiation leading to the coordinated expression of all effector genes; we found that individual effector genes had different kinetics of expression and associated randomly generating several new effector cell types. These results are published in J. Exp. Med. Since these results were obtained in TCR-Tg mice, and thus ...

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...

We obtained data from an in vitro system to study ex-vivo responses able to detect bona fide in vivo primed CD4+ T cells. We focused on the monitoring of tumour antigen specific CD4+ T cell responses. We select the following tumour associated antigens: i) the E6 and E7 proteins of human papilloma viruses, ii) the carcinoembryonic antigen (CEA) and iii) the tumour specific antigen MAGE-A3. We investigated spontaneous CD4+ T cell responses to these antigens in normal donors and in patients with high-grade cervical lesions, pancreas adenocarcinoma and advanced ...

Data have been obtained on the use of nano-particles for intestinal vaccine delivery from our continued investigation of the use of nano-particles as oral vaccine vehicles (Cerovic). We have identified an important role for complement C1q in the uptake of scrapie agent by dendritic cells (Flores-Langarica).We have shown that a protein from Schistosomes has dramatic effects on intestinal physiology, including dendritic cells (Nassar). We have shown that steady-state intestinal denritic cells are not constitutively biased towards stimluating Th2 responses (Milling). ...