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.

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