We are planning clinical trials of melanoma patients treated with DC and tumour lysate plus adoptive transfer of T cells which will result in corresponding data.

Understanding the distribution, function, and lineage relationship of CD8+ T-cell subpopulations is of fundamental value for the monitoring of the immune system in several experimental and clinical situations. However, the available data concerning the description of effector and memory CD8+ subsets in humans remain rather fragmentary because different studies favored the usage of distinct and restricted sets of cell surface markers and functional parameters. We obtained coexpression data of 18 genes simultaneously in individual cells from CD8+ T cell subsets (as...

Gene expression data were generated on DC activated with different TLR agonists typically associated with the respective response.

Pathway-level data have been obtained from a pilot experiment on yeast cells treated with peptides with potential antifungal action. We have compared results from both genomics and proteomics experiments and search for similarities at the level of pathways and have used Eu.Gene to compare the two different data sources demonstrating the feasibility of using this program to this task. Overall correlation between changes in RNA and protein expression was poor but increased when looking at components of immunologically-relevant pathways and Gene ontology terms. The...

We have obtained data on the induction of CTL responses in vivo by immunization of in vitro matured DC versus CTL responses induced by sustained delivery of a maturation signal from a comparison in a murine model. PolyI:C12U (Ampligen) did not activate the murine bone-marrow derived DC. Similar experiments are being performed using a stabilised form of polyI:C.

We have data available from a comparison of the use of fresh peripheral blood mononuclear cells (PBMC) versus fresh heparinised whole blood. Only when peptide pools as an antigen source are used PBMC exhibited a slightly higher stimulation capacity compared to whole blood. However, slightly higher background was observed when using PBMC to stimulate with antigen. Lowest background was observed when either peptides or protein were used for stimulation enabling assessment also of rare antigen-specific Th-cells below frequencies of 0.05% of peripheral CD4+ Th-cell...

The group has data available on a newly identified DC subset called IKDC producing IFNg and expressing TRAIL which can recognize and kill transformed tumor cells in vitro and in vivo. They performed a video-confocal microscopy overnight on cultures of tumor cells with IKDC. We visualized that IKDC generate long contacts and synapses with tumor cells and ultimately kill them within 4 hours.

We have obtained imaging data by confocal microscopy of translation activation in DC-activated T cells and have pursued our efforts on the definition of a novel way of monitoring DC and T cell activation as well as studying viral infections. We also have now applied the puromycin-based technology developed in our laboratory (SUnSET, surface sensing of translation) to monitor translation by FACs in individual or cell populations. We have demonstrated that translation activation is an excellent read-out for early antigen specific T cell activation by DCs. We hope tha...

We obtained data on the control of innate immunity by the mammalian target of Rapamycin (m-TOR) by enhancing TLR driven production of proinflammatory cytokines and type 1 interferons and by repressing posttranslational processing, via the inflammasome, of IL-1beta. We will further analyse the impact of the m-TOR inhibitor Rapamycin on innate immunity.

Data has been obtained on the effect of exposure of DCs or gamma-delta T lymphocytes to HIV-1 and on the miR profile expressed in HIV-exposed DCs. Based on our previous findings demonstrating a bidirectional activating interaction between immature MDDCs and gamma-delta T lymphocytes (Conti et al., JI. 2005), as well as on the demonstration that HIV-1 exposed MDDCs exhibit an impaired functional maturation (Fantuzzi et al., J Virol 2004), we are currently investigating whether exposure of DCs or gamma-delta T lymphocytes to HIV-1 can directly modulate their functi...

We have obtained data on a possible cooperation between myeloid and plasmacytoid DCs in response to different microbial stimuli and a characterization of the mechanisms of this cooperation. For this, we have studied co-culture of mDCs and pDCs in response to CpGs, LPS and bacterial particles. Taking advantage on the fact that human myeloid and plasmacitoid DC show a selective response to LPS and CpG respectively, we have previously identified a cross-talk between two type of cells. Indeed it was observed that LPS-stimulated mDCs are able to induce up-regulation o...

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 will be obtained from a study that we plan on DC receptor characteristics and signalling, focussing on antigen receptors.

Affymetrix and Illumina microarray data is available on 3X106 DCs that were either not stimulated or stimulated with different stimuli. Cells were harvested after 4 hours of stimulation. RNA extraction was done with TRIzol reagent (Invitrogen). microarray hybridization were performed on the basis of technology used. Affymetrix array data files (CEL files) were pre-processed and normalized using the Robust Multichip Average procedure (RMA). Annotations were updated following a procedure devised by Dai and co-workers (Dai et al., 2005). Computation was performed wit...

Our laboratory has data on autologous dendritic cells loaded with apoptotic leukemic cells from patients with B-CLL used as vaccine in patients. In preclinical experiments DC-loaded with apoptotic leukemic cells were shown to induce a strong preferentially Th1 T-cell response. Apoptotic tumor cells were more effective than lysate, RNA and cell hybrids. A clinical trial has been initiated where autologous dendritic cells are loaded with apoptotic leukemic cells from patients with B-CLL. So far “vaccines” have been produced for six patients. Four patients are...

Data has been obtained from the application of a DC-based assay aimed at identifying new adjuvant molecules potentially capable to induce Th1 responses, determining the effects of new chemical compounds on dendritic cell capacity to produce type I IFNs. The selected molecules could be potential new vaccine adjuvants.

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...

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...

We have data on a novel innate signalling pathway in DC. Using curdlan as a specific agonist of dectin-1, we have shown that this C-type lectin couple to Syk kinase leads to activation of ERK, JNK and p38 MAPKs, as well as NF-kappaB. However, signalling terminates rapidly once the receptor is internalised, thereby explaining previous observations that large, non-phagocytisable Dectin-1 ligands are much more potent agonists than smaller ones. This is the first example of a bona fide signalling pathway from a cell surface innate receptor other than a TLR. We will ...

We have obtained data using electron microscopy to document the production and secretion of tubulovesicular structures by cells overexpressing VSV-G glycoprotein. These tubulovesicular structures contaminate recombinant viral preparations and play an important role in determining their innate activating potential. The protocols used in those studies have been published and expression vectors for GFP-RIG-I have been made available (outside of the DC-THERA Directory).