Optimization Of Procedures In Order To Get Mature Dendritic Cells For Vaccine Use

Abstract

Dentritic cells (DCs) are professional antigen-presenting cells (APC), and they play a major role in the generation and regulation on immunity against tumour cells. The exquisite capacity of dendritic cells (DCs) to stimulate primary T-cell responses provides the basis to use DCs as an adjuvant for immuno-therapy against cancer. Malignant melanoma has been shown to be susceptible to T cell-mediated immunity and, therefore, is a candidate for immuno-therapy. Several in vitro and in vivo studies show the ability of vaccination with DCs to elicit tumour-specific T-cell immunity. Vaccination with peptide- or lysate-pulsed DC shows the clinical efficiency in the induction of a curative tumour-specific therapy in metastatic melanoma and other malignancies. Studies in our laboratory demostrated that DCs obtained by PBMCs (Peripheral Blood Mononuclear Cells) of sane donor genereted in vitro in GM-CSF and IL-4, loaded with melanoma antigens (tumour protein lysate) and maturated in 24 hours with autologous cocktail of cytokines (MCM=Monocyte Conditioned Medium), present mature phenotype to explore the possibility of DC-based immune-therapy in patients affected by malignant melanoma. Autologous cytokines (MCM) are obtained by stimulated PBMCs of same sane donor, whereas tumour protein lysate is obtained by patients affected by metastatic malignant melanoma. In our study, we observed that, before pulsing, immature DCs showed a highter capacity of phagocytosis (test with FITC-dextran) that progressively reduced after induction of maturation. Besides, the incubation of immature DCs with autologous cytokines (MCM) resulted in mature DC expressing the full array of co-stimulatory molecules (CD80, CD86, CD83), cytokines and chemokines making them the most effective antigen-presenting cells in vitro. We observed that the use of frozen PBMCs produces the same results of fresh cells, except for a small loss of cells, after thawing. Our results coincide with data present in literature. In conclusion, DCs constitute a promising tool for the development of therapy against cancer. Supported by Ministry Of Health, RF00-352.