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.

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.

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.

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.

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

It was found that lymph nodes that drain sites of mature DC or adjuvant inoculation recruited L-selectin- CCR7- effector cells.

MAGE-A3 specific CD4+ T cells were found also in a high percentage of melanoma patients but CD4+ T cells showed an unpolarized or Th2 skewed phenotype. We have optimized the MAGE-A3 peptides to load onto HLA-DR*1101 tetramers.

Analysing antigen loading strategies for DC, we found that the priming capacity of MelanA/HLA-A2-specific autologous CD8+ T cells by lipofected DC was higher compared to electroprated DC.

It was found that lymph nodes that drain sites of mature DC or adjuvant inoculation recruited memory CD8+ T cells.

Stimulation of Mycobacterium tuberculosis (MTb)-antigen specific T cells ex-vivo in the presence of anti-IL-10 antibodies results in their increased proliferation and IFN- production.

Injection of lentiviral vectors activated OVA-specific CD4+ T cells and this CD4 help was shown to be necessary for an adequate primary and memory CTL response.

Perforine 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.

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 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.

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.

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.

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.

It was found that these cells do not migrate to lymph nodes in the steady state.