The dead cells were stained (Mini67-1KT or Mini26-1KT (PKH67/PKH26 green/red fluorescent cell linker mini kit for general cell membrane labelling) from Sigma) for a process of dead cell uptake by DC.

The D2SC1/Flt3-DC were stained (Mini67-1KT or Mini26-1KT (PKH67/PKH26 green/red fluorescent cell linker mini kit for general cell membrane labelling) from Sigma) for a process of dead cell uptake.

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

MoDC were stimulated with yeast.

From the CD14- PBMCs, untouched naive CD4+ T cells were isolated with a combination of magnetic sorting.

The T blasts are used to immunize MAGE-3 positive patients.

The 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, are used for vaccination.

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

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

The autologous T lymphocytes were stimulated with (frozen) MoDC at a ratio of 1 DC per 10 T cells.

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

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.

Monocytes were cultured in serum free medium in the presence of IL-4 and GM-CSF, and concentrated cytokines were added.

We have used “conventional” techniques to monitor the vaccine induced specific anti-tumor T cell responses (thymidine-incorporation, ELISA, LDA and ELISPOT assays). We have set the staining conditions for the HLA-DR*1101 tetramers loaded with tetanus toxoid and MAGE-3 peptides corresponding promiscuous CD4+ T cell epitopes. Antigen-specific CD4+ T cells are visualized after in vitro short-term expansion; we are currently optimizing the conditions for the ex-vivo staining.

We have demonstrated that the target of FcgammaRs with the antigen induces the internalization of the immune complex, leading to the maturation of CD11c+CD11b+ conventional DCs. These DCs become thus efficient to present antigenic peptides to helper CD4 T cells and cytotoxic CD8 T cells via cross-presentation pathway.

We used specific inhibitors to study the role of src-family tyrosine kinases in the maturation of human monocyte derived dendritic cells upon stimulation with several toll like receptor (TLR) agonists. The effect of these kinase inhibitors on the capacity of DC to be activated by a TLR2 (PAM3CSK4), TLR3 (Poly I:C), TLR5 (Flagellin), and TLR8 (Poly U) agonist was evaluated.

We studied the correlation between CD8 multiple cell surface markers and functional profiles studied at single cell level. For that purpose, we subdivided peripheral CD8 T cells into eleven different cell subtypes based on the association of multiple cell surface markers. In each subtype, we isolated single-cells. In each single cell, we quantified the expression of multiple genes. Moreover, we isolated and studied cells from different normal donors.

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.

We investigated the interactions between human monocyte derived DCs (MDDC), generated in the presence of GM-CSF and IL-4 (IL-4 DC), and antigen-stimulated circulating gamma delta T lymphocytes, bearing the Vgamma2 TCR. The studies demonstrated for the first time the existence of a bidirectional activating interaction between DCs and gamma delta T lymphocytes activated by aminobiphosphonates. These data suggest a potential adjuvant role of this early cross-talk in the therapeutic activity of aminobiphosphonate drug that are currently used as anti-tumor drugs.