These DCs have been generated from monocytes after elutriation and have not been pulsed with irradiated tumor cells (apoptotic bodies).

The tumor antigen derived peptide pulsed monocyte-derived DC were diluted in medium containing 20 ng/mL TNF-?, plus IL-4 and GM-CSF.

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.

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.

These T cells are specific for the P14-GP33 antigen. They were used to study the expression of twenty different genes either mediating effector functions, or coding for different receptors involved in T cell differentiation and memory generation.

These T cells are specific for the 0T-1-OVA antigen. They were used to study the expression of twenty different genes either mediating effector functions, or coding for different receptors involved in T cell differentiation and memory generation.

These monocytes were extracted from a Leukapheresis product from cancer patients using elutriation.

These T cells were obtained from isolation during elutriation and were used for subsequent adoptive transfer.

1,25(OH)2D3 was added to the freshly isolated monocytes; this prevented the generation of IFN-DCs and directed already differentiated IFN-DCs toward a more immature stage.

All lymphocytes (6,4 x 10e9) were frozen in 90% A-plasma and 10% GMP-grade DMSO.

The mature peptide pulsed DC were frozen at 5 x 10e6 cells per vial in freezing medium for subsequent stimulation of autologous T cells.

The majority of monocytes (1 x 10e9) were frozen in 90% A-plasma and 10% GMP-grade DMSO.

Tests were performed on frozen peripheral blood leukocytes (PBL) obtained from leukapheresis or buffy-coat collections performed before and after the 6 vaccinations in Cycle 1 and after the 3 vaccinations of Cycle 2. The fresh PBL samples were collected from UTCM (Hôpital Erasme, ULB) and were transferred to Ludwig Institute where the freezing of the samples and analyses were performed.

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.

Untouched naive CD4+ T cells are co-coltured with fungi -pulsed DCs with the aim to prime naive T cells.

These dendritic cells were generated with GM-CSF and IL-4. They were used in a clinical trial in melanoma patients who were randomized to receive immunizations either with these G4 DC or with I3 DC (DC generated with interferon-beta and interleukin-3).

These T lymphocytes are exposed to HIV-1 to examine if this can directly modulate their functions or interfere with their cross-talk. Preliminary results indicated that, although virus exposure of gamma-delta T cells does not significantly affect their properties, HIV-exposed DCs exhibit a reduced capacity to deliver activation and proliferative signals to gamma-delta T lymphocytes. Moreover, a dysregulated pattern of cytokines and chemokines produced by both cell populations is observed in the presence of the virus.

These DC had been generated from monocytes after elutriation of leukapheresis product (patients with stage III or stage IV melanoma), were not pulsed and were frozen.

These DCs were generated from adherent peripheral blood monocytes and will subsequently be used as a vaccine for HLA-A1 and/or HLA-A2 positive patients with stage III/IV melanoma.

These stained were stained for a process of dead cell uptake by DC.