B细胞交叉提呈肿瘤细胞来源自噬小体(DRibble)抗原及其诱导抗肿瘤T细胞应答的机制研究

Our previous studies have demonstrated that autophagosome-enriched vaccine named DRibble (DRiPs-containing blebs), derived from tumor cells after inhibition of protein degradation, sequester both long-lived and short-lived proteins, as well as damage-associated molecular pattern (DAMP) molecules, and are efficient antigen carriers for crosspriming of tumor-reactive CD8+ T cells by DCs. However, DCs are present in very low number in peripheral blood and it is very difficult to expand DCs ex vivo from non-stem cell sources, without significant in vivo expansion with cytokines, clinical grade DCs are very difficult to produce in large quantity. One of the principal obstacles to the clinical use of DCs is the potential need for continuous administration of large quantities of DCs. Therefore, investigating the ability of other more abundant APC types, such as activated B cells as a promising alternative to DCs, to induce a CD8+ T cell response might help to design better vaccination strategy. Our recent studies showed that DRibbles efficiently induced B cells activation, antibody production, cytokine secretion and antigen cross-presentation mainly depending on their protein component via TLR2/MyD88 dependent manner. Moreover, we found that DRibbles-activated B cells could present antigens in the DRibbles to directly activate effector T cells and induce na?ve CD8+ T cell response. However, the involved mechanisms have not been studied so far. In this study, we will investigate that ① the molecular mechanism of B cells activation induced by Dribbles, involved B cell recognition and internalization of DRibbles and signal transduction pathway of B cells activation. ② the molecular mechanism of B cell processing and cross-presentation of DRibbles and response of na?ve CD8 T cells induced by DRibble-activated B cells. ③ the anti-tumor efficacy induced by B cells loaded with DRibbles. Therefore, investigating the ability of activated B cells, as a promising alternative to DCs, to induce a CD8+ T cell response might improve the understanding the role of B cells in tumor immunity and help to design better vaccination strategy and optimize DRibbles based vaccines for cancer immunotherapy.

申请者前期研究证实：肿瘤细胞来源自噬小体(DRibble)富含肿瘤抗原并含多种具内源性佐剂作用的DAMP分子、能活化DCs诱导具有抗瘤效果的T细胞应答，但DCs存在数量少、扩增制备难等制约因素；近期实验发现：DRibble能刺激B细胞活化并诱导初始T细胞应答，但其中分子机制以及B细胞/DRibble疫苗的抗瘤作用有待深入研究。 本项目拟研究：①DRibble活化B细胞的分子机制(B细胞识别、内吞DRibble；活化信号途径的分子机制)；②B细胞交叉提呈DRibble抗原及诱导初始T细胞应答的分子机制(处理、提呈DRibble抗原的途径？共刺激分子/细胞因子/Th细胞辅助?)；③B细胞/DRibble疫苗体内诱导抗肿瘤免疫应答的机制(最佳免疫方案？诱导记忆性T细胞？抗肿瘤效果？)。 探讨上述问题将进一步理解DRibble与B细胞抗原提呈功能的关系，为DRibble疫苗临床应用提供的新思路。

1.研究发现：DRibbles活化B细胞依赖TLR2-MyD88分子，以及经典NF-κB信号通路和非经典NF-kB活化途径。DRibbles所含内源性危险信号分子HMGB1与B细胞的活化有密切关系。Caveolae(细胞质膜微囊)介导的细胞内吞作用(endocytosis)与DRibbles诱导B细胞活化有密切关系。.2.研究证实：B细胞能有效交叉递呈DRibbles抗原并刺激效应CD4+T和CD8+T细胞的再活化及分泌IFN-γ，该作用依赖B细胞的TLR2/MyD88信号通路。DRibbles所含内源性危险信号分子HMGB1与B细胞交叉递呈DRibbles刺激效应CD4+T和CD8+T细胞的再活化有密切关系。Caveolae(细胞质膜微囊)介导的细胞内吞(endocytosis)与B细胞交叉递呈DRibbles抗原并刺激效应CD4+T和CD8+T细胞再活化有密切关系。.3.研究证实：B/DRibbles加强免疫能增强DRibbles疫苗诱导的免疫应答及其抗肿瘤作用；联合应用CpG和抗CD40抗体预处理B细胞能有效加强B/DRibbles疫苗诱导的免疫应答及其抗肿瘤作用。.4.研究发现：B/DRibbles首次免疫能诱导抗原特异性初始T细胞免疫应答及抗肿瘤作用；联合CpG和抗CD40抗体能加强B/DRibbles疫苗的抗肿瘤作用。.5.研究初步发现：人源性DRibbles抗原体外能诱导人记忆性B细胞（CD19+CD27+）（图 19A）和初始B细胞（CD19+CD27-）的活化。.6.研究初步发现：Dribbles通过TLR4-MyD88信号途径活化巨噬细胞并上调CD40L，并通过CD40/CD40L增强Dribbles诱导的B细胞活化水平及其诱导的T细胞应答。

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