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.

One volume fresh medium containing 2x concentrated cytokines was added to the cell culture of monocytes in serum free medium in the presence of 100 ng/mL GM-CSF (Leukine, Berlex) and 20 ng/mL IL-4 (Cell Genix).

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).

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).

The leukapheresis product is taken from a HLA-A2 positive healthy donor and is subsequently separated into monocytes and lymphocytes.

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 have completed and published our work on biodegradable poly (D, L-lactide-co-glycolide) (PLGS) micropsheres (MS) coencapsulating ligands for endosomally expressed TLRs plus exogeneous Antigen (Ag) to deliver its cargo to endosomes of murine Dendritic cells (DCs), thus initiating TLR mediated DC maturation as well as processing of MHC class I and class II restricted epitopes.

The organization of bmDC, B lymphocytes and plasma cells in the BM of the long bones was studied by immunochistochemistry. Using CX3CR1GFP mice, in which bmDC are green fluorescent (GFP), the bmDC localization was analyzed and it was found that the cells were organized in unique clusters, mainly located in the endosteum of the BM. These DC co-localize with B cells and these clusters are occupying architecturally definable niches and are reminiscent to the niches that were found in the cranial BM parenchyma. By using Fibronectin antibody the stroma of the BM co...

Src kinases were found to be required for the initiation of some maturation characteristics of human monocytes derived dendritic cells upon stimulation with several toll like receptor (TLR) agonists but not of others, since their inhibition was able to dissociate cytokines and chemokines production from the induction of surface maturation markers.

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.

These K/O mice were studied with regard to responses to viral infections. We used the mouse pox virus Ectromelia and MVA-BN. Coinfection with MVA-BN and Ectromilia protected the IFNalpha receptor -/- mice. The data show that MVA-BN can induce potent innate immune responses able to control viral replication to allow the specific immune response to eliminate Ectromelia in immune deficient mice.

Hu-SCID mice were used to study responses to EBV and HIV.

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.