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Abstract
Background:
Despite of aggressive treatments, the long-term survival rate is about 30% in stage 4 neuroblastoma (NBL). Recently, dendritic cell (DC)-based immunotherapy is emerging as a promising tool in cancer treatment. But it is very difficult to get sufficient amount of autologous tumor as the source of tumor antigen in DC-based immunotherapy. The purpose of this study was to test whether DCs loaded with allogeneic NBL tumor antigens can prime effective antitumor immune responses.
Methods:
Peripheral blood mononuclear cells (PBMCs) were differentiated into immature DCs in the presence of GM-CSF and IL-4. As the source of tumor antigens, human neuroblastoma cell lines, SK-N-MC, SK-N-SH, and IMR-32, were used after induction of apoptosis by UV irradiation. The immature DCs were loaded with apoptotic tumor cells, and then cultured with PBMCs for priming the tumor antigen-specific T lymphocytes. The tumor-specific cytotoxicity of T lymphocytes against NBL cells was analysed after coculture.
Results:
Incubation of DCs with apoptotic tumor cells effectively loaded DCs with tumor antigens and induced maturation of DCs. The tumor antigen-challenged T lymphocytes effectively killed the NBL cells, which were used as tumor antigens. Furthermore, the T lymphocytes showed a broad ranged cytotoxicity to all of the NBL cell lines, which were not challenged as tumor antigens.
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Fig. 1
Scheme of the experimental design for stimulation of T lymphocytes by tumor antigen-loaded dendritic cells.
Fig. 2
Photomicrograph of peripheral blood monocyte- derived immature (left, ×400) and mature human dendritic cells (right, ×1,000), Papanicolaou staining.
Fig. 3
Phenotypic analysis of peripheral blood monocyte-derived dendritic cells (PB-DC) by two color flow cytometry analysis using MoAb combinations of CH6, a new monoclonal antibody against human dendritic cells, and other markers of human dendritic cells.
Fig. 4
UV-induced apoptosis of tumor cells (SK-N-MC). Apoptosis of the tumor cells was induced by UV irradiation for 2 hours at room temperature. Apoptosis and cell death were analysed by staining the cells with annexin V.
Fig. 5
Immunophenotypic changes of peripheral blood monocyte-derived dendrtitic cells before and after tumor antigen loading with apoptotic tumor cells (SK-N-SH).
Fig. 6
Tumor-specific cytotoxicity by tumor antigen- challenged T lymphocytes. T lymphocytes were stimulated by tumor (SK-N-MC) antigen-loaded dendritic cells, and analysed for the tumor-specific cytotoxicity against three NBL cell lines; SK-N-MC (●—●), SK-N-SH (■—■), and IMR-32 (▲—▲), and two control cell lines; SK-BR-3 (△—△) and K-562 (○—○) at various effecto r：target (E：T) ratios.
Fig. 7
Tumor-directed cytotoxicity of T lymphocytes against various tumor cell lines at various effector：target (E：T) ratios. T lymphocytes from different donors were stimulated by DCs loaded with apoptotic SK-N-MC cells, and tumor-specific cytotoxic effector function was measured.
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