Our lab obtained data on the ability of NK cell IFN-gamma production to predict long term survival in advanced GISTs treated with IM. Background: Mutant isoforms of the KIT or PDGF receptors expressed by gastrointestinal stromal tumors (GISTs) are considered the therapeutic targets for imatinib mesylate (IM) (Gleevec®, Novartis), a specific inhibitor of these tyrosine kinase receptors. Predictive factors for the response to IM pertain to the intrinsic features of GIST tumor cells but not to the host. Besides its direct antiproliferative activity on tumor cells, I...

A phase I/II study of immunization of HIV infected individuals stable under HAART has been initiated which will provide us with immunological data. These patients are being vaccinated with cytokine cocktail matured DCs electroporated with mRNA encoding Tat, Rev and Nef. The cDNA’s encoding these early expressed HIV antigens have been human codon optimized and modified with lysosomal targeting sequences. Four vaccines separated by 4 weeks. Two weeks after the 4th vaccine, an analytical HAART interruption was started. So far, 17 patients have been included in t...

During the previous period we obtained data from our continued pilot clinical trials of DC-based immunotherapy of melanoma patients. After an initial cohort of patients treated with monocyte-derived DCs matured with inflammatory cytokines and subsequently electroporated with mRNA encoding 6 tumor-associated antigens, we persued this trial in a next cohort where DC-based vaccines were combined with low dose IFN-alfa (3 x 10e6 U per wk). In a significantly higher number of patients we observed vaccine-induced depigmentation and vitiligo. Further improvement and ca...

Summary of the completed RCC clinical study and the data obtained: RCC subjects were deficient in T cell IFN-gamma and IL-2 production pre-vaccination. The IL-2 defect was corrected in 7/12 patients post-vaccination. Vaccination resulted in an increase in tumor antigen-specific T cells in 8/12 patients. 7/12 subjects had a response to more than one antigen post-vaccination. Median overall survival was ~25 months compared to ~11 months for historical controls (confirmed by matched pair analysis).

We have further data available on a clinical trial on vaccination of B-CLL patients with autologous, apoptotic, leukemic cells (Apo-DC). Cohort 1 with the vaccine alone and cohort 2 (5 patients) combining the vaccine together with GM-CSF as an adjuvant has finished accrual and all patients in these two cohorts have been monitored for 52 weeks. The clinical and immune monitoring data is being currently analyzed. The third cohort consisting of the vaccine and GM-CSF administered after a single dose of cyclophosphamide (300 mg/ sq. meter) is currently being accrued...

Our group has generated pathway data from human DC treated with pathogen derivatives.

Data has been obtained on the activation of human DC by Toll like receptor ligands (TLR-L) that modulates the lipid biosynthetic pathway, resulting in enhanced recognition of CD1d-associated lipids by iNKT cells. DC derived soluble factors further increase CD1d-restricted iNKT cell activation, as defined by IFN-alpha secretion. Finally, using soluble tetrameric iNKT TCR as a staining reagent we demonstrate specific up-regulation of CD1d-bound ligands upon TLR-mediated APC maturation. The ability of innate stimuli to modulate the lipid profile of DC resulting in iNK...

We have obtained data from a study where we addressed a model antigen to exosomes secreted by tumor cells in vivo, to allow capture of antigen-bearing exosomes by DCs and cross-presentation of the antigen. For this purpose, the model antigen OVA was fused to the C1C2 exosome-binding domain of MFG-E8/lactadherin. Tumors secreting the exosome-bound antigen grow slower than tumors secreting soluble OVA, because they induce activation of OVA-specific CD8+ T cells into killer cells. In addition, inducing in vivo secretion of the C1C2-coupled antigen by DNA vaccination i...

We have obtained data from an analysis of antigen loading of DCs via targeting DC-SIGN, which resulted in enhanced class II-mediated presentation. Constructs for fusion proteins of the DC specific receptor DC-SIGN fused to GFP have been generated.

Our group has obtained imaging data on the role of DALIS and related aggregates in antigen presentation and their interaction with the regulation of translation in response to pathogens. We have implemented in collaboration with lead coordinator Carl Figdor, the organization of the imaging core-facility of the DC-THERA NoE. Experimentally, we have been applying confocal microscopy to the study of DALIS and related aggregates in order to understand their role in antigen presentation and their interaction with the regulation of translation in response to pathogens....

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...

Data has been obtained in a study of antigen recognition of blood-derived T cells. In this study, an IFNgamma ELISPOT was done with PBMC loaded with peptide pools, each consisting of 10 15-mers, which overlap with 11 aminoacids.

Data have been obtained on the antigen recognition of DTH infiltrating T cells. To obtain the data, activation of CD4 and CD8 T cells upon recognition of EBV-B cells expressing vaccinal antigens was evaluated by CD137 upregulation, CD107a and CD40L expression using flow cytometry. Luminex technology was used to quantify the TNFalpha and IFNgamma secretion.

Our lab obtained data on the activation of immature monocyte-derived dendritic cells after transduction with high doses of lentiviral vectors. Dendritic cells (DCs) are an attractive tool for immunomodulation, targeting mature DCs (mDCs) for immunization or immature/semimature DCs (iDCs) for tolerization. Therefore, introducing antigens into DCs has become a prime topic in various immunological disciplines. Numerous studies have shown that lentiviruses are an efficient vehicle for this purpose. This study evaluates the effects of lentiviral transduction on iDC ...

We have obtained data from in vitro studies to demonstrate that DC can be activated via the dectin-1/Syk pathway to become APC capable of priming CD8+ T cells. In addition, we have shown that the same DC can interact with Tregs and convert them into cells that co-express ROR?t and Foxp3 and produce IL-17. We have shown that this pathway can serve as an alternative to TLR signalling for allowing DC to become effector cells capable of priming CD4+ T cells. We have used agonists of the dectin-1/Syk pathway as adjuvants in vivo to induce DC activation and CD4+ T cell ...

At the end of 2004 BruCells has initiated two identical clinical studies entitled: “A Phase IB/II Study of Immunotherapy with Autologous Dendritic Cells Pulsed with Multiple HLA-A2 and MAGE-3.DP4 Peptides in Metastatic Cutaneous Melanoma Patients”. Patients received sequential immunizations with autologous dendritic cells pulsed with 8 HLA-restricted HLA-A2 peptides and a MAGE-3.DP4 peptide. They were randomized to receive immunizations either with I3 DC (DC generated with interferon-beta and interleukin-3) or with G4 DC (DC generated with GM-CSF and IL-4). At ...

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...

Our lab gathered data on DC activation by pathogen-derived stimuli. We focused on the study of the response of MDDCs to the combined stimulation with TLR ligands and we observed that simultaneous activation of TLR4 and TLR8 signaling cascades results in a marked inhibition of the secretion of the proinflammatory chemokine CCL2 with respect to stimulation through a single TLR. This inhibition is specific for both CCL2 and TLR agonist combination and could represent a novel regulatory mechanism evolved to maintain immunological balance (Del Cornò et al.). Future st...