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Multiple myeloma (MM) cells express certain tumour-associated antigens (TAAs) that could serve as targets for active-specific immunotherapy. The aim of the present study was to test the MM/dendritic cell (DC) fusion as a vaccination strategy. We fused MM cells with DC to generate fusion cells (FCs) and tested their antigen presenting cell (APC) function in mixed lymphocyte reactions and cytotoxicity assays. First, the HS Sultan and SK0-007 HAT sensitive human MM cell lines and DCs generated from peripheral blood of normal donors were fused in the presence of 50% polyethylene glycol to form FCs. Next, tumour cells freshly isolated from patients were similarly fused with autologous DCs to generate FCs. The FCs demonstrated a biphenotypic profile, confirmed both by flow-cytometry and dual immunofluorescence microscopy. These FCs induced MM-specific cytotoxicity. FCs, but not MM cells or DCs alone, were potent stimulators of autologous patient T cells. More importantly, FC-primed autologous peripheral blood mononuclear cells demonstrated major histocompatibility complex-restricted MM-specific cytolysis. These studies therefore demonstrated that MM/DC FC can trigger an autologous immune response to MM cells and formed the framework for a clinical trial currently underway. [ABSTRACT FROM AUTHOR]

Please cite this paper as: Enhanced T-cell activation by immature dendritic cells loaded with HSP70-expressing heat-killed melanoma cells. Experimental Dermatology 2010; 19: 108–116. Vaccination protocols that utilize dendritic cells (DCs) to elicit therapeutic immunity against tumors are the subject of intense research. Given that the capacity of DCs to cross-present antigens is physiologically low, there is considerable interest to develop strategies that enhance that pathway. In order to best exploit the enhanced cross-presentation of antigens bound to heat shock protein 70 (HSP70), we analysed melanoma cell preparations for their HSP70 expression. Western blotting revealed strong upregulation of HSP70 after heat-killing in contrast to UV-B irradiation. When the uptake of heat-killed necrotic cells by DCs at various levels of maturation was assessed, 61 ± 7% of immature DCs (iDCs) internalized fluorescence-labelled necrotic material. Apoptotic material from UV-B-irradiated cells was internalized by only 48 ± 5% of iDCs. Maturation-inducing cytokines did not affect the uptake when added simultaneously with the tumor cell preparations. Loading DCs with heat-necrotic or apoptotic melanoma cells slightly reduced CD83 expression while leaving CD208 (DC-LAMP) expression unchanged. As determined by IFN-γ-detecting enzyme-linked-immunospot assays, iDCs loaded with heat-killed melanoma cells activated autologous T cells most effectively when used without any further maturation, whereas DCs loaded with apoptotic material required maturation. In conclusion, HSP70-expressing melanoma cells could be generated by heat-killing. Loading iDCs with heat-killed melanoma cells resulted in a superior priming of autologous T cells in vitro. [ABSTRACT FROM AUTHOR]