人B细胞交叉提呈肿瘤来源自噬小体(DRibbles)抗原诱导T细胞应答及其抗肿瘤作用的研究

The previous studies have demonstrated that tumor derived-autophagosomes (DRibbles) could induce DCs activation and corss-present DRibbles antigens to induce specific T cell response and anti-tumor response through CLEC9A/CLEC9AL. Our recent studies show that DRibbles could induce mouse B cell activation and present DRibbles-containing antigens to induce specific T cell responses and anti-tumor response through HMGB1/TLR2. However, it is currently unknown whether DRibbles could activate human B cells and induce antigen-specific T cell responses and anti-tumor response. In this research, we will discuss: ①whether DRibbles could activate human B cells and the invloved molecular mechanisms, including B cells recognition and internalization of DRibbles and the signal pathway of B cells activation. ②the T cell responses and molecular mechanisms induced by human B cells loaded with DRibbles with the model antigen CMV pp65. ③the anti-tumor efficacy of the human B cells loaded with DRibbles with the humanized immune reconstituted HepG2 HCC murine model. The research will supply experimental clue for the clinical application of human B/DRibbles vaccine.

已有研究证实：肿瘤细胞来源自噬小体(DRibble)富含肿瘤抗原并通过其内源性分子佐剂(CLEC9A/CLEC9AL)有效活化DCs、诱导具有抗瘤效果的T细胞应答；我们近期发现：DRibbles能通过HMGB1/TLR2激活小鼠B细胞成为APC、有效诱导特异性T细胞应答及抗瘤作用，但DRibbles对人B细胞的作用及机制有待研究。本项目拟：①探讨DRibbles活化人B细胞及其分子机制（人B细胞识别、内吞DRibbles及活化的分子机制）；②以CMV pp65为模式抗原，探讨人B细胞交叉递呈pp65+DRibbles抗原诱导特异性T细胞应答及其分子机制（处理、递呈DRibbles抗原的途径？B细胞刺激剂的协同作用？）；③采用人免疫重建荷人肝癌裸鼠模型，探讨人B细胞/DRibbles疫苗的抗瘤作用及机制（抗瘤效果？CD4Th细胞参与？），为人B细胞/DRibbles疫苗的临床应用提供实验依据

1、.研究发现：不同肿瘤来源的DRibbles均能有效刺激人PBMC中B细胞的增值，且与肿瘤细胞裂解液中包含的可溶性蛋白质抗原相比，DRibbles作为一种颗粒性抗原载体更能有效地诱导人PBMC及B细胞的增殖；.2、.研究发现：DRibbles刺激能同时上调人初始B细胞和记忆B细胞表面MHC分子、活化标志CD25及共刺激分子CD86、CD80等的表达；并且，DRibbles刺激人B细胞分泌细胞因子IL-1α、IL-6、IL-10、TNF-α和趋化因子MCP1、MIP1α、IP-10、IL-8及造血生长因子G-CSF、GM-CSF的水平均显著高于细胞裂解液；.3、.研究发现：流式细胞仪和激光共聚焦仪观察显示人B细胞体外能直接摄取DRibbles，并且DRibbles在B细胞内与溶酶体共定位；.4、.研究证实：人B细胞能有效加工递呈DRibbles抗原并诱导特异性CD8+T和CD4+T细胞分泌IFN-γ；.5、.研究证实：在人免疫重建荷人肝癌模型中，B/DRibbles 疫苗能有效抑制HepG2 皮下移植瘤生长。