INVOLVEMENT OF PERFORIN/GRANZYME B-DEPENDENT SIGNALING PATHWAY IN CYTOTOXIC ACTIVITY OF DENDRITIC CELLS TOWARDS HUMAN GLIOBLASTOMA CELLS

Granule-mediated cytotoxicity of effector cells is a universal mechanism of tumor growth inhibition and induction of tumor cell death. The aim of present study was to evaluate expression of lytic molecules in DCs generated in presence of IFNα (IFN-DCs), and to analyze the role of granule-mediated mechanism for IFN-DC cytotoxic activity against tumor cell lines. IFN-DCs were generated by culturing of plastic-adherent peripheral blood mononuclear cells in presence of GM-CSF and IFNα for 4 d followed by LPS addition for 24 h. The tumor cell lines were obtained from malignant tissues from patients with glioblastoma multiforme. Maturation of IFN-DCs in presence of LPS was accompanied by accumulation of intracellular perforin and granzyme B molecules. Perforin expression showed a direct correlation with intracellular lysosome-associated membrane protein-1 (LAMP-1/CD107a) expression in LPS-stimulated IFN-DCs. However, CD107a expression did not increase under LPS stimulation. At the same time, LPS caused upregulated degranulation in IFN-DCs, as shown by an increase of surface CD107a expression on IFN-DCs. LPS activation of DCs generated from the same donors in the presence of GM-CSF and IL-4 (IL-4-DCs) did not influence perforin and granzyme B expression in IL-4-DCs which was significantly lower than in IFN-DCs. Intracellular pool of CD107a molecules was increased in response to LPS stimulation of IL-4-DCs, but surface CD107a expression did not change on IL-4-DCs. Studies of cytotoxic activity of LPS-stimulated IFN-DCs revealed that concanamicyn A (CMA), an inhibitor of vacuolar H+-ATPase and of perforin/granzyme B-mediated signaling pathway, caused reduced cytotoxicity of donor DCs towards glioblastoma cell lines. Involvement of perforin/granzyme B-signaling pathway into the DCs cytotoxicity was confirmed with glioblastoma cell lines, since blockage of this mechanism with vacuolar H+ ATPase blocker (CMA) caused inhibition of the IFN-DC cytotoxicity. Differently reduced DC cytotoxic activity by CMA may suggest that the glioblastoma cell lysis can be mediated via perforin/granzyme B-independent mechanisms.