Development of DC-based vaccine therapy by utilizing exosomes collected from healthy volunteer-derived DCs

本研究的目的是开发一种制备树突状细胞(DC)的方法，这种树突状细胞具有在个体之间具有相同的抗原递呈能力，作为癌症患者的治疗工具。利用健康志愿者外周血单个核细胞(PBMC)来源的树突状细胞(DC)分泌的外切体作为研究对象是本研究的独特之处。研究期间获得的数据如下：1)DC来源的外切体的定性和定量评价方法：我们发展了一种结合超速离心法、蔗糖密度法和BDLA-DR结合珠的新方法来提纯和定性定量评价外切体。结果表明，在健康志愿者DC来源的外体上，关键抗原提呈相关分子，包括MHC-II类分子、CD80、CD86和ICAM-1均有高度且均一的表达。另一方面，这些分子在患者来源的DC的外切体上的表达很弱。2)DC来源的Exosome对DC分化和功能的影响：2-1：健康志愿者DC来源的Exosome对CD4T细胞存活的延长。2-2：健康志愿者来源的DC的Exosome促进患者来源DC的分化。2-3提高健康志愿者DC来源的Exosome的NK细胞活性。2-4：从肿瘤细胞冲击的DC收集的Exosome诱导细胞毒性T细胞(CTL)。5)：从恶性积液中收集的外切体可延长调节性T细胞的存活时间。3)其他：从CE4基因转染树突状细胞分泌的外切体可能表达CEA。

Aim of this study is to develop a method for preparing dendritic cells (DCs), which have homogeneous ability of antigen presentation between individuals, as therapeutic tool for cancer patients. It is a unique point that exosomes secreted by DCs generated from healthy volunteers' peripheral blood mononuclear cells (PBMCs) are utilized for this ourpose.Data obtained during the research period are as follows:1) Methodology for qualitative and quantitative evaluation of DC-derived exosomes:We developed a new methodology for purification and qualitative and quantitative evaluation of exosomes by combination of ultracentrifugation, sucrose density, and BDLA-DR-bound beads. As a result, it was shown tat critical antigen presentation-related molecules, including MHC class II, CD80, CD86, and ICAM-1, are expressed highly and homogeneously on exosomes derived from healthy volunteer's DCs. On the other hand, expressions of these molecules were weak on exosomes derived from patient-derived DCs.In conclusion, it was strongly indicated that expression of HLA-DR on purified exosomes is a marker for qualitative and quantitative evaluation of DC-derived exosomes.2)Effects of DC"derived exosomes on differentiation and function of DCs:2-1: Survival prolongation of CD4+ T cells by exosomes collected from healty volunteer-derived DCs.2-2: Improved differentiation of patient-derived DCs by exosomes collected from healty volunteerderived DCs.2-3 increased NK cell activity by exosomes collected from healty volunteerderived DCs.2-4: Induction of cytotoxic T cells (CTLs) by exosomes collected from tumor cell-pulsed DCs.2-5: Survival prolongation of regulatory T cells by exosomes collectede from malignant effusions.3)Others:An interesting possibility that exosomes secreted from CE4-transfected DCs express CEA was indicated.