We attempted to improve the adoptive transfer protocol for immunotherapy by stimulating T cells with monocyte derived DC pulsed with tumor lysate, instead of simply adding tumor lysate into PBMC cultures. T cells raised exhibited some tumor reactivity and outgrowth of a distinct T cell population could be observed.

All lymphocytes (6,4 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.

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 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.

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.

B cell proliferation (polyclonal, oligoclonal, monoclonal) was observed in human Hemato-Lymphoid System Rag2-/-gc-/- mice after being infected with EBV and mounting an immune response.

Cytotoxic T cells proliferation was observed in human Hemato-Lymphoid System Rag2-/-gc-/- mice after being infected with EBV and mounting an immune response.

These T cells stem from an elutriation process from a metastatic lesion of stage III/IV melanoma patients. Subsequently, these T cells will be expanded by co-culture with tumor lysate loaded DC.

These T cells were generated from the Leukapheresis product, after elutration, of a metastatic lesion of stage III/IV melanoma patients, expanded by co-culture with tumor lysate loaded DC.

These Treg were induced after treatment of primed mice with intact anti-CTLA-4 antibodies. These regulatory T cells inhibit Th1 responses, in vitro and in vivo, and repress experimental intestinal inflammation, by a mechanism involving IL-10 and IDO.

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 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.

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.

iNKT cells were stimulated in vivo with the synthetic CD1d ligand alpha-GalCer. This significantly enhanced immune responses to protein and peptide based vaccines, due to rapid iNKT dependent DC maturation.

These T cells were obtained from isolation during elutriation and were used for subsequent adoptive transfer.

These T cells were isolated during elutriation of a leukapheresis product (patients with stage III or stage IV melanoma).

These primed T cells have been obtained from untouched naive CD4+ T cells co-cultured with fungi -pulsed DCs.

Purified CD8 T cells using MACS cell separation were co-cultured with electroporated DC and subequently became evaluated on their cytokine secretion, cytotoxicity and % of antigen specific T cells.

These T cells are to be purified using MACS cell separation to contain only CD8+ T cells. The latter are then co-cultured with electroporated DC, stimulated and become evaluated on their cytokine secretion, cytotoxicity and % of antigen specific T cells.