Data has been obtained from a Balb-neuT mice model to study new strategies for advanced breast cancer. A colony has been established of transgenic Balb-neuT mice (founders provided by Forni, Torino IT) that over-express the rat HER-2/neu oncogene under the MMTV promoter. These mice develop mammary carcinomas with later lung and liver metastases; the course of disease and the type of tumours that develop closely recapitulate many features of human breast cancer. A collaborative study has commenced between Austyn and Cerundolo to use this model to study new strategi...

Knowledge and gene expression data were gained on differentiation patterns of T cells.

We have obtained valuable data on chemically well-defined TLR ligand conjugates and their effectiveness to induce a strong immune response via the cross-presentation pathway. We have studied long peptides which were covalently conjugated to either the TLR2 ligand or TLR9 ligand, Pam3CysSK4 or CpG, respectively have been used to study the uptake, antigen presentation, and induction of specific T-cells. A pronounced enhancement in antigen presentation by DC in vitro was facilitated by the conjugated peptides compared to peptide alone, or peptide mixed with the fre...

In 2007 we established routine monitoring of tumor-specific T-cell subsets by multicolor flow cytometry and obtained immunological data from this. For this purpose we used multimers kindly provided by Pierre Coulie (Brussels) together with surface markers (such as CD8, CD4, CD3, CD45RA, CCR7, CD25, CD27, CD137, CD127) in different 8-color panels on the BD FACSCanto II. Additionally we used functional markers such as CD107a and intracellular cytokine staining for the further analysis of individual T-cell populations and T-cell clones. Monitoring of the ongoing vacc...

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 ...

Data were obtained on responses to viral infections in TLR9 -/- mice, C57Bl/6 mice and IFNalpha receptor -/- mice. Toll receptor 9 (TLR9) is an important pattern recognition receptor. It is stimulated by foreign DNA or CpG motivs. We found that TLR9 -/- mice were very susceptible to infection with the mouse pox virus Ectromelia. In contrast to DC from C57Bl/6 mice, DC from TLR9 -/- produced little if any interferon alpha. However, MVA-BN induced IFNalpha in presence or absence of TLR9 -/-. Moreover, coinfection with MVA-BN and Ectromilia protected TLR9 -/- mice ...

Our lab gathered data on the responses to EBV and HIV in hu-SCID mice.

Our lab has obtained data on the mechanism of how IL-10 regulates the immune response to MTb infection in mice. Control and clearance of intracellular pathogens such as MTb is dependent on the production of TNF and the induction of the T-helper 1 (Th1) cytokine IFN-gamma by IL-12. Conversely, IL-10 is a suppressive cytokine essential for dampening the immune response to a number of intracellular pathogens to limit host immune pathology. IL-10 has been shown to exert its suppressive effect by directly acting on the antigen presenting cell (APC), therefore functi...

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 lab has gathered data in establishing a per-clinical model of breast cancer.

We obtained data on immune tolerance in mice breast-fed by antigen-exposed lactating mothers. We have previously generated a monoclonal antibody, 2C44, that reacted to a peptide derived from the Leishmania LACK protein bound to I-Ad MHC class II molecules. We have successfully used the 2C44 mAb to visualize LACK-presenting DCs in mice infected with the L. major. In 2007, we have attempted to identify the APCs that are responsible for the development of immune tolerance in mice that have been breast-fed by antigen-exposed lactating mothers. Recent experiments perf...

Our laboratory obtained data from studies of DC phagosomes and cross-presentation using K/O and mutant mouse strains. Cross presentation is the process by which Dendritic cells (DC) phagocytose pathogens or dying cell fragments, and present proteolytic peptides derived from these antigens in association with MHC class I molecules. The reasons why DCs are the only antigen presenting cells that efficiently cross present antigens are not well understood. We have demonstrated that the NADPH oxidase NOX2 is recruited to DC early phagosomes mediating a sustained produc...

We obtained data from an evaluation whether antigens could be rerouted from the endosomal into the cytosolic compartment by combining antigen with cell penetrating peptides to further enhance cross-presentation. We made significant progress using biodegradable polymers to enhance cross-presentation of loaded antigens, and the best results were obtained by maturing DCs with a combination of the TLR ligands R848 and polyI:C. Furthermore, we have shown that DC electroporated with melanoma antigen RNA express the proteins ex vivo and in vivo.

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

We investigated the effects of tumour hypoxia on DC differentiation and maturation and gathered data in mice. This is a an important topic as sites of inflammation are often characterized by both low oxygen tension and an extensive infiltration of inflammatory leukocytes, including DC, which need to move against oxygen gradients. We showed that oxygen availability is necessary to promote full monocyte-derived DC differentiation and maturation. Low oxygen tension (hypoxia) inhibits expression of several differentiation and maturation markers (CD1a, CD40, CD80, CD8...

My laboratory obtained data from a continued analysis of the ability of invariant NKT (iNKT) cells to assist priming of antigen specific T and B cell responses. We have carried out three complementary lines of research: 1) We have demonstrated that activation of human DC by Toll like receptor ligands (TLR-L) modulates the lipid biosynthetic pathway, resulting in enhanced recognition of CD1d-associated lipids by iNKT cells. 2) We have clarified the mechanisms by which CD1d-restricted lymphocytes translate T cell receptor (TCR) recognition of lipids with similar g...

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). ...

Data have been obtained defining the differential roles of intestinal lymph DC subsets in activation of naïve and memory T cells in rats. We have shown that two of the three DC subsets are highly potent activators of naïve CD4+ T cells, and do not appear to be constitutively suppressed in any way. We are currently examining the phenotype of the activated cells and determining if Tregs are induced. We have initiated (in collaboration with Pfizer) a study of nano-particles as potential deliverers of intestinal vaccines.

We have obtained data on the immuno-physiology of rat and mouse intestinal dendritic cells. We have used a known adjuvant, E. coli heat-labile toxin (Etx) and a potential adjuvant R-848, a small TLR7/8 ligand. We have shown that R-848 causes massive changes in DC migration and leads to their activation in lymph nodes but not lymph. These changes are due to TNF-alpha (migration) and Type 1 interferons (activation), but are not accompanied by obvious oral adjuvant effects. In contrast Etx causes only minor changes in migration and activation but is a potent oral adju...