ABSTRACT
Objective:To investigate the effect of tripartite motif-containing 23 (Trim23) on the differentiation and maturation of dendritic cells and the possible mechanism.Methods:Mouse bone marrow-derived dendritic cells (BMDCs) were prepared from bone marrow cells of C57BL/6 mice with the presence of Flt3L. Real-time quantitative PCR and Western blot were used to detect the expression of Trim23 in BMDCs after LPS stimulation. An overexpression vector for full-length Trim23 (Trim23 OE) was constructed and transfected into BMDCs, and the pcDNA3.1 empty vector was used as control. Flow cytometry was used to detect the expression of CD80, CD86, CD40 and MHCⅡ on the surface of vector-transfected BMDCs after LPS stimulation and ELISA was used to detect the secretion of IL-12p40, TNF-α, IL-6 and IL-10 by these cells. CD8 + and CD4 + T cells were isolated from spleen and lymph nodes of OT-Ⅰ and OT-Ⅱ mice by magnetic beads and co-cultured with LPS-treated BMDCs in the presence of ovalbumin (OVA). Flow cytometry was used to detect the proliferation and differentiation of CD8 + and CD4 + T cells. Western blot was performed to analyze the phosphorylation of p38, ERK1/2 and AKT in BMDCs. Two overexpression vectors for Trim23 mutants lacking RING or ARF domain (Trim23 ΔRING and Trim23 ΔARF) were constructed and transfected into BMDCs. Then flow cytometry and ELISA were used to detect the expression of surface molecules and cytokines. Results:The expression of Trim23 in BMDCs was significantly down-regulated after LPS stimulation. The expression of MHCⅡ, CD86 and CD80 and the secretion of TNF-α and IL-6 decreased significantly in BMDCs overexpressing Trim23. Furthermore, overexpression of Trim23 inhibited the ability of BMDCs to induce the proliferation and differentiation of CD4 + T cells and the proliferation of CD8 + T cells. Western blot showed that the phosphorylation of p38 and ERK1/2 decreased significantly in Trim23-overexpressing BMDCs. Compared with wildtype Trim23, overexpression of Trim23 ΔRING had no significant influence on the expression of surface molecules (MHCⅡ and CD86) and the secretion of cytokines (TNF-α and IL-6) in BMDCs stimulated by LPS. Conclusions:Trim23 overexpression inhibited the maturation and immune activation of BMDCs via MAPK signal pathway and its RING domain. This study provided reference for targeting Trim23 to improve the immune response of dendritic cell-based tumor vaccines.
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.
ABSTRACT
Background: CD40 is a type α-membrane protein of the tumor necrosis factor receptor super-family, and CD40-induced responses may mediate growth and angiogenesis in carcinoma cells.Objectives: Define the effect of CD40 ligation on AGS gastric cancer cell line and the role of vascular endothelial growth factor/vascular endothelial growth factor receptor (VEGF/VEGFR) signals in this process. Methods: We treated AGS cells with 1 μg/mL soluble CD40 ligand (sCD40L) with or without pre-incubation of either anti-VEGF mAb (MAB293) or VEGFR tyrosine kinase inhibitor (SU5416). We determined the growth effects by cell counts or [3H]-thymidine incorporation assay and VEGF levels in cell-free supernatant using enzymelinked immunosorbent assays. Results: The engagement of CD40-induced AGS cells proliferation accompanied by a significant increase autocrine VEGF through PI3K activation (p