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.

Granzyme expressing T cells were observed in human Hemato-Lymphoid System Rag2-/-gc-/- mice after being infected with EBV and mounting an immune response. They infiltrated in B cell infected areas in lymphoid organs in situ.

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...

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.

HPV-18 E6 specific CD4+ T cells were found in a high percentage of patients with cervical lesions and we showed that the quality of the response (i.e. the level of IFN-? produced) could predict the clinical outcome after surgery.

Human DC exposed to TLR ligands and activated iNKT cells in vitro. They had enhanced expression of maturation markers, suggesting that a cooperative action of TLR ligands and iNKT cells on DC function is a generalizable phenomenon across species. These studies highlight the potential for manipulating the interactions between TLR ligands and iNKT cell activation in the design of effective vaccine adjuvants.

We have efficiently transfected DC with RNA encoding a functional protein (E/L-Selectin) which allows entry of DC into LN from HEV. The novel adherence capacity of human E/L-S transfected DC was demonstrated via sticking to sialyl-LewisX coated slides using a parallel plate flow microscope. RNA transfected human DC could be frozen and thawed without loosing their functionality.