We have generated data on the kinetic of the transcriptional profiling of D1 cells treated with either DMSO or LPS.

We have data on the kinetic of the transcriptional profiling of human MoDC treated with IL2 in vitro.

Our laboratory has obtained data on the kinetic of the transcriptional profiling of purified mouse DC treated with IL10.

A large reference ‘golden experiment’ will be carried out in which the transcriptome of DC vaccines of 50-100 patients will be compared and data transcriptomic data will become available.

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.

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...

Data has been obtained on expression and regulations of Notch ligands on DC subsets.

We obtained data from studies of CD8 T cell properties in man comparing HIV, CMV and EBV-specific cells. It is usually assumed that effector gene expression in CD8 T cells may be similar in all immune responses, CD8 T cells in vivo differentiating into cytotoxic T cells that also produce g-inf. In contrast we published in 2007 that CD8 follow several differentiation waves and that effector gene expression is heterogeneous, different effector genes having different kinetics of expression-down regulation and associating randomly, suggesting that different immune re...

In our lab, we have gene expression data in T cells available in responses to LCMV-GP33 protein and listeria-Ova. We studied TcR-Tg clones specific of each antigen (P14-GP33 specific, 0T-1-OVA specific), as well as endogenous polyclonal T cells, specific for the same antigens, identified by MHC pentamers loaded with specific peptides. In each individual T cell, we studied the expression of twenty different genes either mediating effector functions, or coding for different receptors involved in T cell differentiation and memory generation. We were surprised to obs...

Global gene expression data have been obtained using Affymetrix mouse chips by designing dendritic cell-based bioassays. To identify possible differentially expressed genes by DC after activation with stimuli typically involved in inducing Th1 or Th2 type responses we have performed comparative kinetic global gene expression analyses of DC activated with different TLR agonists typically associated with Th1 (LPS, CpG, Poly I:C) or Th2 responses (Pam3Cys). For these analyses, we have used the Affymetrix GeneChip® technology and, in particular, mouse chips showing pr...

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.

We can provide data for two experiments composed of 14 microarrays each. To obtain the data, DCs were stimulated with different form of fungi. We perform RNA preparation, labelling, hybridization, and scanning according to the Affymetrix reference protocols and BioPolo (Prof. Ricciardi-Castagnoli and Dott. Francesca Zolezzi) and preliminary data normalization according to AMDA (BioPolo).

We can provide Illumina microarray data for four experiments composed of 4 microarrays each on DCs stimulated with pathogenic and non pathogenic fungi. RNA preparation, labeling, hybridization on a HT12 array (Illumina), and scanning were performed according to Illumina instructions by Genomics Lab, Wellcome Trust Centre for Human Genetics (Univeristy of Oxford).

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

Our lab obtained data on functional homogeneity from a gene expression analysis at single-cell level for the expression of 8 effector genes from normal donors. Until 2006 we subdivided CD8 PBL from normal donors in 14 different cell sets based in the expression levels and the co-expression of 5 different cell surface markers and studied functional homogeneity by screening cells from each subset at single-cell level for the expression of 8 effector genes (Blood, 2007). Although these studies allowed us isolate some homogeneous cell types, we yet found heterogenei...

Collaborative work with P34 (Sica) has been started to study the effects of Japanin on DC exposed to hypoxia (below) and to perform transcriptional profiling of the treated cells. This work, which could not have been undertaken at this stage without support from DC-THERA, has now led to a joint publication in 2008 (Mancino et al.). Transcriptional data are available.

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...

Agilent and Affymetrix data were obtained from an expression analysis of DC treated +/- poly I:C +/- kinase inhibitors. We have previously shown that activation of Src family kinases is absolutely required for cytokine production in DC stimulated by agonists of several Toll like receptors. Inhibition of these kinases by the specific inhibitors PP1/2 uncoupled cytokine production from the up-regulation of co-stimulatory molecules in DC. We investigated gene expression profiles in DC stimulated with PolyI:C, pretreated or not with PP2, microarray analysis was perfo...

Affymetrix data will be gathered through an integrated Functional Genomics approach to study DC maturation with an unprecedented detail. D1 cells will be stimulated with LPS and harvested at different time-points for GeneChip analysis and for tandem mass spectrometry-based analysis of the integral plasma membrane proteome. A protocol for obtaining highly pure integral membrane proteins has already been set up in the last two months in the lab of Paola Castagnoli in collaboration with the lab of Juri Rappsilber at IFOM. A pilot project has already been performed o...