New therapeutic vaccines by using small membrane vesicle secreted by dendritic cells: New development for chronic virus infections

在这项研究中，我们研究了树突状细胞(DC)来源的外周小体(Ex)是否可以在表达HLA-A0201的转基因小鼠(HHD)中成为抗病毒免疫的候选细胞。用表达丙型肝炎病毒NS3-5A的重组腺病毒(Rec-Ad)感染DC的培养上清液，并加入或不加DC成熟试剂如脂多糖、IL-12、CpG、佛波醇酯、离子霉素、CD40L或ATP。经斑点印迹分析，Ex含有CD63、CD81、CD44、I-Ab、HLAA2、HSP和RAEδ。用这些Exs诱导细胞毒性T淋巴细胞(CTL)或自然杀伤细胞反应。ELISPOT法检测的干扰素γ合成显示分泌细胞数量增加。根据EX刺激后脾细胞RAEδ和HSP浓度的升高以及脾细胞NKG2D阳性细胞的出现率，我们推测，在Ex刺激后，NK细胞数量的增加部分地诱导了干扰素γ的合成和细胞溶解功能。在Ex对CTL反应的影响中，CD8+T细胞而不是CD4+T细胞可能是重要的。在测试的TLRs中，TLR-2除了作为众所周知的LFA分子外，还作为Ex的受体发挥了重要作用。

In this study, we investigated whether exosome (Ex) derived from dendritic cells (DC) can be attractive candidate for antiviral immunity in the transgenic mice (HHD) expressing HLA-A0201. We isolated Ex from culture supernatant of DC that were infected with recombinant Adenovirus (Rec Ad) expressing NS3-5A of Hepatitis C virus (HCV) with or without maturation reagents of DC such as LPS, IL-12, CpG, phorbor ester, ionomycin, CD40L or ATP. By estimating dot blot assay, Ex contained CD63, CD81, CD44, I-Ab, HLA-A2, HSP, and Raeδ. Responses of cytotoxic T lymphocytes (CTL) or natural killer cells were induced by these Exs. IFN γsyntheses estimated by ELISPOT assay showed increased numbers of secreting cells. From the findings of increased concentration of Raeδand HSP in Exs and the frequency of NKG2D-positive cells in the splenic cells after the Ex stimulation, we hypothesized that NK cell increases in number induce IFN γ synthesis and cytolytic function in part just after the Ex stimulation. CD8+ T cells, but not CD4+ T cells may be important for the effects of Ex on CTL responses. Among the TLRs tested, TLR-2 suggested an important role as receptor of Ex in addition to the well known LFA molecules.