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.

Blood samples and tumour biopsies will be taken from these newly diagnosed glioblastoma patients in order to carry out a preclinical study in which circulating tumour-specific CD8+ T-cells will be detected in their blood. In order to obtain the blood samples and tumour biopsies, a file has been submitted to the Ethics Committee of the Erasme Hospital and has been recently approved.

Monocytes were transferred into culture bags by sterile welding and cultured for 5 days in serum free medium (CellGro, Cell Genix) in the presence of 100 ng/mL GM-CSF (Leukine, Berlex) and 20 ng/mL IL-4 (Cell Genix).

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

Melanoma patients were vaccinated with 4 peptides (MAGE-A3, gp100, NA17 and tyrosinase) presented by HLA-A2 and monitored. We measured responses to these peptides injected with Montanide, but did not know whether the Montanide adjuvant was important for these T cell responses. We therefore injected the same peptides without adjuvant, and no response was observed. We conclude that Montanide had a clear adjuvant effect for the CD8 T cell responses measured against these four peptides.

Differentiation of monocytes into dendritic cells is promoted, along with recombinant IL-4, by addition of granulocyte-macrophage colony-stimulating factor (GMCSF 1000U/ml, Chemicon).

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.

We have worked to strengthen vaccine developments with an improved humanized mouse model. We focus on recombinant vaccines against HIV driven by a powerful poxvirus vaccine system termed MVA BN. To improve our understanding MVA BN has been analyzed thoroughly on the nucleotide level, safety in cell cultures, immune deficient mice as well as in healthy, HIV–infected and other immune deficient humans.

The patients are immunized with autologous DC loaded with KLH, as immunological tracer, and an allogeneic peptidome (i.e. natural tumour peptides, NTPs), obtained from melanoma cell line SK-Mel24.

These patients undergo leukapheresis and later intradermal administration of the produced vaccine (V administrations of autologous dendritic cells generated from adherent peripheral blood monocytes and subject to quality controls - with eventual additional ones depending on clinical outcome).

An HLA-A2 positive healthy donor underwent leukapheresis with the aim to generate monocyte derived DC under GMP conditions.

The patients are immunized with autologous DC loaded with KLH, as immunological tracer, and an allogeneic peptidome (i.e. natural tumour peptides, NTPs), obtained from melanoma cell line SK-Mel24.

These patients undergo leukapheresis and later intradermal administration of the produced vaccine (V administrations of autologous dendritic cells generated from adherent peripheral blood monocytes and subject to quality controls - with eventual additional ones depending on clinical outcome).

HLA-DP4/MAGE3 molecules have been prepared to stain ex vivo PBL from immunized melanoma patients for a study of anti-tumor responses in melanoma patients that were vaccinated with autologous DCs loaded with a combination of tumor Ag peptides.

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.

Patients with resected HPV16-positive cervical cancer were vaccinated with an overlapping set of long peptides comprising the sequences of the HPV16 E6 and E7 oncoproteins emulsified in Montanide ISA-51.