Avaliação de dois diferentes protocolos de maturação de células dendríticas no perfil de ativação e migração celular

Abstract

INTRODUCTION: With an indispensable role, dendritic cells, important antigen presenters with phenotypic and functional heterogeneity, link innate and adaptive immune responses. They are responsible for regulating and modulating effector responses against microorganisms and tumor cells, and also maintain immunological tolerance under homeostatic conditions. Their plasticity works according to stimuli and microenvironment in which they are inserted. Immunotherapy using these cells is under constant study to obtain sufficient activation of a cellular immune response. Several protocols and patents have been and are still being tested for differentiation and maturation of an appropriate profile of dendritic cells for effective immunotherapy. OBJECTIVE: To test the activation and migration profile presented by the culture of dendritic cells submitted to maturation by different stimulus protocols (TNF-α and TNF α IL-12 and CCL5) in an in vitro assay. METHODOLOGY: Peripheral blood from healthy volunteers (n=10) was used for the isolation of monocytes and lymphocytes. Adherent cells were differentiated into dendritic cells that were subjected to maturation by 2 different protocols (protocol 1 with TNF-α stimulation; protocol 2 regarding stimulation with TNF-α, IL-12 and CCL5) and then co-culture with cells non-adherent. To characterize the phenotypic profile of total cells (adherent and non-adherent), the mean fluorescence intensity of surface markers (CD209, CD11c, HLA-DR, CD123, CD209, CD80, CD86 CD83, CD3, CD8, CD4) was analyzed, CD25, FOX-P3, NKG2D and CD54) and cytokines (granzymes, perforins, IL-10, IFN-y, TNF-α, IL-12, TGF-β, IL-4 and IL-17) by cytometry of flow. The migration of mature dendritic cells was seen on staining and counting of cells in polystyrene membrane cultured with a chemotactic gradient (MCP-1 and CCL5). RESULTS AND DISCUSSION: The results show significant increases in the expression of adhesion/co-stimulatory molecules CD209, CD11c, HLA-DR, CD123, CD80, CD86 and CD83 and cytokines (IFN-γ, TNF-α, IL-12, IL-10 and TGF -β) of dendritic cells from protocol 2, compared to protocol 1. Thus, we see the activation of important subtypes of T cells (Th1 and Th17, with reduced Treg) and Natural Killer (NK) that produce IFN-γ, with decreased expression of IL-10 in the same protocol. CONCLUSION: Concluding our findings, we demonstrate that the stimulation protocol with TNF-α, IL-12 and CCL5 was able to generate a profile of mature dendritic cells and effective for activation of balanced subtypes of T lymphocytes and NK cells, important in the antitumor immune response.