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.

We evaluated the capacity of autologous DC and HEK293 cells transfected with relevant HLA alleles to function as T cell targets in Elispot assays upon transfection with tumor antigen encoding plasmids. Due to strong background from the autologous DC we decided that HEK293 transfected with one HLA-allele at a time plus simultaneously transfected with up to 5 tumor antiges would be optimal to screen for antigen specificity in the patients T cells. However, during a second T cell culture to expand large numbers of T cells for this screening procedure a drastic ex...

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

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.

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.

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.

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.

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

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.

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

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

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 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.

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.

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

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

The dead cells were stained and used in the uptake of dead cells by DC.

The DC were matured with TLR ligand R848 and polyI:C. Using these DC, we obtained best results in our evaluation whether antigens could be rerouted from the endosomal into the cytosolic compartment by combining antigen with cell penetrating peptides to further enhance cross-presentation.