To understand whether these regulators of gene expression could somehow contribute to the fine tuning of CCL2 expression in MD-DCs, a quantitative microarray approach that detects 723 human mature miR (miRBase 10.1, Dec 2007), was used to measure miR expression profile in these cells, either untreated or stimulated with LPS, R848, or their combination for 8 h. The analysis of the microarray data generated from 4 independent experiments is in progress.

Concerning the pathway based analysis and database we have started the curation of the TLR4-CD14 pathway in DCs and we have described the events occurring at the DC membrane level. We have also started global gene expression analyses to better difine the role of NFAT in DCs.

During 2008, we extended all the data regarding TLR3 signaling to TLR8 signaling. Cytokines assays and immunoblotting studies suggest that Src kinases play a crucial role in the control of both MyD88- and TRIF-dependent pathways. We also extended microarray analysis on human MoDC stimulated with R848 (TLR8 agonist), pretreated or not with PP2. In summary, the new generated data on TLR8 triggered gene expression are similar to previous data obtained with TLR3 stimulation, and confirmed that src kinases inhibition is associated to inhibition of key genes in the infl...

Work has continued to characterise the function of a novel tick-derived DC modulator that perturbs various signalling pathways of human DC but not others; transcriptional profiling and pathways-based analysis will be undertaken in the next phase and data will become available.

We have used fluorescence microscopy to analyse the endocytic pathway in DC and to track C-type lectins that are internalised from the cell surface. From these analyses, there is data available. As part of this work, we have used microscopy to study the effect of Dectin-1 uptake on its ability to signal for downstream responses. Finally, we have shown that fluorescently-labelled antibodies against a novel C-type lectin, DNGR-1, can be used to mark DC upon injection into mice and might therefore be useful for in vivo imaging studies. Microscopy will continue to b...

Data is available from an evaluation of different stimulation conditions for the cytometric assessment of antigen-specific Th-cells based on CD154 expression induced during short-term in vitro activation, a method established by us during previous DC-THERA activities. This is important and essential for implementation of the technology into clinical immune monitoring in e.g. vaccination trials. Moreover, the affect of stimulation length was determined. Differences with respect to the induced frequencies of antigen-specific, activated CD154+ Th-cells were minor. I...

There is data available on translation during DC/T cell interaction in presence or absence of cognate antigenic peptides. Efforts on the definition of a novel way of monitoring DC and T cell activation as well as studying viral infections were finalized. The puromycin-based technology (SUnSET, surface sensing of translation) was applied to monitor translation by FACs during DC/T cell interaction in presence or absence of cognate antigenic peptides. It was demonstrated that translation activation is an excellent read-out for early antigen specific T cell activati...

Data was obtained on the regulatory T cells (Tregs) and their role in effective anti-tumor immune responses in FoxP3-DTR DEREG mice. Although multiple mechanisms of Treg action have been proposed, the actual mechanism involved in the suppression of anti tumor T cell responses are still unclear. Treg inactivation by anti-CD25 antibodies or by depletion in FoxP3-DTR DEREG mice resulted in effective rejection of the tumors upon adoptive transfer of antigen-specific CD8+ T cells. Treg inactivation or depletion induces a marked arrest in the migration of anti-tumor C...

Proteomic data has been obtained using used yeast as a model organism to optimize technological approaches for the quantitative assessment of molecular components in yeast proteomics. We compared protein levels of essentially all endogenous proteins in haploid yeast cells to their diploid counterparts. Our analysis spanned more than four orders of magnitude in protein abundance with no discrimination against membrane or low level regulatory proteins (deGodoy et al.).

Data were obtained from profiling of human monocyte-derived DC exposed to different maturation cocktails and various activators or inhibitors.

Transcriptional data is available on macrophage knockout BALB/c and C57BL/6 mice using different stimuli at different time points in the presence or absence of a kinase inhibitors.

Pathway data is available form a comparative pathways-based analysis of proteomic data performed from same sources.

Data will become available on the safety, antitumoral immune response and DC migration in the organism in patients with advance renal cell carcinoma and melanoma from this trial using cellular immunotherapy with dendritic cells loaded with tumor antigens. This study is not yet open for participant recruitment. ClinicalTrials.gov Identifier: NCT00610389 Purpose Background: cellular immunotherapy with dendritic cells (DC) loaded with tumor antigens has shown clinical activity, although in a small number of patients. Therefore, it is mandatory to improve the resul...

Data was obtained on the efficacy in a phase I/II study investigating multiple injections of CLL-DCV01 in patients with previously treated B-cell chronic lymphocytic leukemia. This study is fully enrolled (n=9). Efficacy data will be assessed via tumor burden data collected over the next two months. Absolute lymphocyte counts and immunomonitoring data will be compiled by the end of Q1’08.

Data was generated on DC activation by different forms of the non-pathogenic yeast Saccharomyces cerevisiae. A special focus was laid on the response of MDDCs to the non pathogenic fungi.

We further our understanding of different DC populations and their soluble products in adults but in particular in neonatal mice. We have concentrated on the evaluation of the interplay between the hematopoetic factor Flt3L and IFNalpha,beta and have data available from our studies. We found that the cooperation between IFNalpha,beta and Flt3L (FL) plays an important role in the defense against Herpes simplex virus type 1 (HSV-1) in neonates. Treatment of neonatal mice with recombinant IFNalpha has a short-term, FL-independent and a long-term, FL-dependent protect...

DC-THERA applicant : Anita Sapoznikov Institution: The Weizmann Institute of Science, Israel Supervisor: Dr. Steffen Jung Data has been obtained on the cellular organization of the marrow of long bones. The bone marrow (BM) is a primary lymphoid organ, where diverse hematopoietic lineages arise from common multi-potential progenitors [1]. However, aside from this established role in hematopoiesis, the BM also serves as secondary lymphoid organ in the generation of adaptive T and B cell immune responses [2,3]. It has been shown that the BM is also populated by m...

We have obtained data on the immunogenicity induced by chemotherapeutics.

We have developed structural, kinetic and functional data to study the binding of ligands to CD1 molecules and activation of CD1d restricted iNKT cells. The results of these studies have led to: i) The development of protocols to refold in vitro CD1 molecules, which were used for the generation of CD1 tetramers. Ability to generate CD1d tetramers has provided us with the opportunity of comparing a broad panel of CD1d binding compounds for their ability to stimulate iNKT cells. ii) Identification of a novel population of NKT cells, which does not express the ca...

Data has been generated in HHD2 transgenic mice on dendritic cell derived-exosomes (DEX), nanomeric vesicles harboring MHC/peptide complexes capable of promoting primary T cell responses and tumor rejection in the presence of adjuvants. We showed that, in the absence of adjuvants, DEX mediate potent antigen dependent-antitumor effects against preestablished tumors in mice pretreated with immunopotentiating dosing of cyclophosphamide (CTX). CTX could i) abolish the suppressive function of CD4+CD25+Foxp3+ regulatory T cells (Treg), ii) markedly enhance the mag...