Abstract.Cancer is associated with a reduction in immature and mature circulating dendritic cells (DCs), and with an impaired migratory capacity, compared with healthy donors. Therefore, modern approaches to the in vitro generation of DCs loaded with tumor antigens and their use for inducing antitumor immune responses in vivo are being investigated. The purpose of the present study was to investigate the phenotypic and functional characteristics of peripheral blood DC subsets in patients with non-small cell lung cancer (NSCLC), and the development of an antitumor cytotoxic response by mononuclear cells (MNCs) from patients using in vitro generated antigen-primed DCs. Heparinized peripheral venous blood samples were obtained from 10 healthy donors and 20 patients with a histologically verified diagnosis of NSCLC. The ability of antigen-activated DCs to stimulate the activity of MNCs against autologous tumor cells was evaluated using a cytotoxic test. Peripheral blood DC subsets from patients with NSCLC were identified to be decreased and to exhibit an impaired ability to mature, compared with healthy donors. Furthermore, DCs generated from MNCs from patients with NSCLC were able to stimulate a specific cytotoxic response when loaded with autologous tumor lysates or RNA and matured, in vitro. A perforin and granzyme B-dependent mode of cytotoxicity was primarily induced. The ability of DCs loaded with tumor antigens to increase the cytotoxic activity of MNCs against NSCLC cells in vitro indicates the effective induction and co-stimulation of T lymphocytes by the generated DCs.

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BackgroundSince dendritic cells (DC) are involved in the development of autoimmune inflammation, DC are considered to be target cells for specific therapy of rheumatoid arthritis (RA)[1]. The development of treatment strategies requires comprehensive research into the qualitative characteristics of DC subtypes both ex vivo from RA patients and in vitro, to determine the possibility of inducing functionally mature DC in RA.ObjectivesTo study different subpopulations of DC from peripheral blood and to induce simular DC in cultureMethodsTo generate different subsets of DC we used rhIL-3 (BioVision, USA), R848 (Resiquimod, BioVision, USA), LPS (25 ng/ml, E. coli O114:B4; Sigma, USA), rhGM-CSF and rhIL-4 (PeproTech, USA). Phenotypic characterization of immature mDC(Lin-HLADR+CD123-CD11c+) and pDC(Lin-HLA-DR+CD123+CD11c-) in the was estimated by the positive expression of CD80, CD83, CCR7, IL-10, IL-12, IFN-α. Analysis of the DC target populations contents was performed using flow cytometer “BD FACSVerse”.ResultsDC from RA patients were characterized by low expression levels of CD80 and CD83 on both populations cells and high expression of CCR7 only on pDC. An increase in mDC and pDC producing IL-12 and IFN-α and a decrease in mDC and pDC producing IL-4 and IL-10 were shown in RA. Immature DC (iDC), similar to mDC in terms of their phenotypic characteristics, and mature mDC were generated and expression of surface markers was analyzed. DC induced according to this protocol expressed CD80, CD83 and CCR7 at similar levels for all groups at each stage of maturation analysis. Healthy donors (HD) DC had increased expression of CD80 in response to TNF-α stimulation, while mDC from RA patients had significantly increased expression of both CD80 and CD83. The expression levels of CCR7 on immature mDC was very low for both HD and RA patients, and increased upon addition of TNF-α. Analysis of DC cytokine-producing activity demonstrated that induced mature DC produced cytokines upon addition of specific stimulators, but cells from RA patients showed no significant differences in IL-10, IL-12, and IFN-α production compared with HD. iDC with plasmacytoid phenotypic characteristics and mature pDC were generated and the expression of surface markers was analyzed. Using IL-3, R848, and LPS as stimulating factors significantly increased the expression levels of CD80 and CD83 after maturation in RA and HD groups. Additionally, the DC maturation process was accompanied by a simultaneous increase in CCR7 expression when pDC from RA patients or HD were stimulated by LPS. The expression levels of CD80, CD83 and CCR7 did not differ significantly between RA patients and HD at all stages of pDC maturation. We observed no differences in terms of IL-10 and IL-12 production by mature pDC between the RA and HD groups. However, pDC from RA patients demonstrated significantly higher production of IFN-α compared with pDC from HD.ConclusionsIn summary, the current study indicated DC might be target cells for the therapy of rheumatoid arthritis. Additio...

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Currently, one of the most promising approaches for the treatment of oncological patients is the selective activation of T-cell antitumor immunity using immune cells. The generation of functionally active DCs in vitro constitutes a promising approach in the development of DC-based anticancer vaccines to mobilize patient defense systems, because their activation by tumor-specific antigens to induce cytotoxic responses, and their increased efficiency of antigen presentation to induce cytotoxic T lymphocytes (CTLs) via costimulatory molecules and cytokines can be controlled. The purpose of this study was to investigate the