In vitro effects of β-glucan combined with agonistic anti-CD40 monoclonal antibody on immune functions of dendritic cells / 中华微生物学和免疫学杂志

ABSTRACT

Objective:To investigate the effects of β-glucan combined with agonistic anti-CD40 monoclonal antibody (5C11) on the immune functions of dendritic cells (DCs) and the possible molecular mechanism.Methods:Mononuclear cells were separated from fresh concentrated white cells (granulocytes) of healthy subjects using Ficoll density gradient centrifugation, and induced by GM-CSF and IL-4 to differentiate into immature DCs. Following various stimulation (5C11 alone, β-glucan alone, 5C11 combined with β-glucan), flow cytometry was used to detect the expression of CD40, CD80, CD83, CD86 and MHC-Ⅱ molecule HLA-DR on the surface of DCs. ELISA was used to detect the secretion of cytokines including IL-12, IL-6, TNF-α and IL-10. Western blot was used to detect the phosphorylation of proteins related to MAPK signaling pathway.Results:Flow cytometry suggested that β-glucan significantly induced the expression of co-stimulatory molecule CD40 on the surface of DCs ( P<0.05). After the DCs were co-stimulated with β-glucan and 5C11, CD80, CD83 and CD86 expression were further significantly increased, and a strong synergistic effect on CD83 expression, a key marker of DC maturation, was observed ( P<0.01). ELISA showed that β-glucan combined with 5C11 could significantly promote the secretion of cytokines such as IL-12, IL-6 and TNF-α by DCs, and have a synergistic effect on the secretion of IL-12, a critical cytokine in regulating DC functions ( P<0.01). Western blot indicated that the phosphorylation of p38 MAPK and p44/42 MAPK in DCs was increased significantly after combined treatment, and the phosphorylation started earlier and lasted longer compared to that in DCs stimulated with 5C11 or β-glucan alone ( P<0.01). Conclusions:This study suggested that β-glucan combined with 5C11 had a synergistic effect on promoting the maturation and improving the immune functions of DCs, providing a new strategy for the preparation of anti-tumor DC vaccines.