The cultured cells were subsequently pulsed with tumor antigen derived peptides (one per culture) at a concentration of 5 µg/mL in approximately 5 mL and at a cell density of 20 x 10e6/mL for 1 h.

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.

DC pulsed with tumor antigens were evaluated for their immunotherapy potential in EG7-OVA and P815 tumor models. Depletion of natural regulatory T cells in tumor bearing mice resulted in rejection of P815 cells, but not EG7-OVA, suggesting that regulatory T cells have a stronger impact on immune responses against weakly immunogenic or auto-antigen (P1A).

Cytotoxic T cells proliferation was observed in human Hemato-Lymphoid System Rag2-/-gc-/- mice after being infected with EBV and mounting an immune response.

We found that LPS-stimulated mDCs are able to induce up-regulation of co-stimulatory molecules on co-cultured pDCs. Likewise, CpG-stimulated pDCs activate co-cultured mDCs. The cross talk between these two populations of DC is very efficient since it can be observed at mDC/pDC ratio ranging from 1/10 to 10/1.

In the process of total RNA extraction from yeast cells, the supernatants were precipitated in RNase-free centrifuge tube by adding 1/10 volume 3 M NaAcetate (pH 5.3) and 2.5 volumes of cold ETOH 100%.

These DC were co-electroporated with constitutively active TLRs and TAA encoding mRNA. They were used to compare different maturation methods.

Long peptides which were covalently conjugated to either the TLR2 ligand or TLR9 ligand, Pam3CysSK4 or CpG have been used to study the uptake, antigen presentation, and induction of specific T-cells.

Co-cultures of mDCs and pDCs in response to CpGs, LPS and bacterial particles were studied to identify a possible cooperation between myeloid and plasmacytoid DCs in response to different microbial stimuli, and to characterize the mechanisms of this cooperation.

Our current efforts in a study of functional homogeinity involve a transition to 8 colours surface staining, and the evaluation of antigen-specific cell sets identified with MHC tetramers. For the later process, we initiated data collection of EBV, CMV and HIV specific T cell sets.

CLL-DCV01 is used for multiple injections in patients with previously treated B-cell Chronic Lymphocytic Leukemia.

Down-regulation of CD83 expression on human DC through RNA interference (RNAi) results in a less potent induction of allogeneic T cell proliferation, reduced IFN-gamma secretion by established T cells and decreased capacity in the priming of functional tumor antigen-specific CD8+ T lymphocytes. In addition, CD83 mRNA-electroporated DC are stronger T cell stimulators. However, CD83 overexpression on Melan-A/MART-1-specific tumor-infiltrating lymphocytes (TIL) circumvents the need for CD83 expression on DC. Co-culture of immature DC with TIL or K562 cells overex...

The crude population of DC was divided into the three known splenic subsets (CD8+, CD4+, DN DC) using Fluorescence activated cell sorting (BD FACS Aria). This procedure allowed to obtain highly pure fractions of each subset. However, because DC are a very rare population, this method is very material- and cost-intensive. Furthermore, it is not surprising that the obtained cell numbers of each subset after isolation are below 107. However, these cell numbers should be sufficient for proteomic investigations. The proteomes of all three subsets using mass spectrometry is in progress.

The crude population of DC is divided into the three known splenic subsets (CD8+, CD4+, DN DC) using Fluorescence activated cell sorting (BD FACS Aria). This procedure allowed to obtain highly pure fractions of each subset. However, because DC are a very rare population, this method is very material- and cost-intensive. Furthermore, it is not surprising that the obtained cell numbers of each subset after isolation are below 107. However, these cell numbers should be sufficient for proteomic investigations. The proteomes of all three subsets using mass spectrometry is in progress.

The cell populations which infiltrate progressive versus regressing P815 mastocytoma were examined in order to identify cells which display immunosuppressive properties. We found changes in regulatory T cells populations as well as in the “myeloid suppressor cells”.

CEA specific CD4+ T cells were found both in normal donors and in patients with high-grade cervical lesions, pancreas adenocarcinoma and advanced melanoma but CD4+ T cells from the patients compared to normal donors showed impaired IFN-gamma production.

4 ml of chloroform (Fischer BP1145-1) were added to buffered and incubated S. cerevisia cells in a process of total RNA extractions.

We observed that ICAM-1 expression by mature DCs is critical for long-lasting contacts with CD8+ T cells, but dispensable for short-lived antigen-specific interactions. Serial brief T cell-dendritic cell contacts induced early CD8+ T cell activation, proliferation and effector CTL differentiation in the first few days after immunization.

Manufacturing of cGMP RNA to be used as an API (active pharmaceutical ingredient) in the planned Tri-Mix DC RNA Trial will be performed after obtaining the manufacturing license for RNA loaded DCs.

Chitin (500 micrograms/ml) was added to DCs in order to block their beta-glucan receptors.