3D biomimetic lymph node engineered extracellular vesicles for understanding the heterogeneity of adaptive immunity

摘要 了解免疫异质性如何利用细胞多样性来实现免疫系统的适应性和灵活性。 免疫应答是一个尚未得到充分研究的核心挑战，主要是由于缺乏有效的免疫力 研究细胞通讯的组织模型。自2013年以来最近发现的贩运囊泡 诺贝尔医学奖照亮了理解远距离，非接触细胞的新途径 通信调节免疫力。然而，对这种多样的纳米大小的囊泡的研究，即 外泌体，是极具挑战性的，由于巨大的困难，区分动态和异质性， 在体内系统中存在的囊泡群体。我们的研究工作通过以下方式解决关键技术差距： 开发了一系列工具集，包括高效、高通量的外泌体微流控方法 分离、分型、分子工程和转染以及纳米递送。最近，我们观察到， 分泌的外泌体的分子包装在细胞培养环境改变时是高度可变的 以及周围的社区。使用2D细胞培养系统的体外研究无法 解释体内外泌体免疫调节机制。我们假设一个3D仿生淋巴结 组织系统可作为有效研究体内外泌体分子的类体内组织模型 刺激后的包装和分泌动力学，用于将外泌体货物与细胞水平相互连接 应答这项为期五年的研究将重点关注精确阐明免疫调节的三个关键挑战 通过外泌体途径在分子水平上：1）开发3D可编程仿生淋巴结组织 具有明确的免疫适应性的铸造，用于模拟体内免疫组织微环境; 2） 开发单细胞单外泌体研究方法，用于高灵敏度检测和单细胞外泌体亚型分型。 对于理解免疫异质性具有动态和统计学意义的外泌体群体; 3） 发现可以选择性包装免疫刺激microRNA以及MHC结合肽的基序 用于靶向免疫调节，这可以建立货物内化的互连 细胞水平的反应。长期目标是促进我们对免疫调节的理解 异质性，并最终能够在分子精度上精确地编程免疫应答。

ABSTRACT Understanding how immune-heterogeneity leverages cellular diversity to achieve the adaptivity and flexibility in immune-responses is a core challenge that has not been well studied, largely due to the lack of a valid immunity tissue model for investigation of cellular communications. Recent discovery of trafficking vesicles since 2013 Nobel Prize of Medicine shined the light on the new avenue for understanding long-distance, non-contact cellular communications in modulating immunity. However, the study of such diverse and nano-sized vesicles, namely exosomes, is extremely challenging, due to immense difficulties in differentiating dynamic and heterogeneous vesicle populations presented in the in vivo system. Our research work addresses key technology gaps by developing a series of tool sets, including high-efficient and high-throughput microfluidic approach for exosome isolation, subtyping, molecular engineering and transfection, and nano-delivery. Recently, we observed that molecular packaging of secreted exosomes is highly variable upon the change of cellular culture environment as well as surrounding community. The in vitro investigations with 2D cell culture systems are incapable of interpreting in vivo exosome immunity modulation mechanism. We hypothesize that a 3D biomimetic lymph node tissue system could serve as the in vivo -like tissue model for effectively studying in vivo exosome molecular packaging and secretion dynamics upon stimulations, for interconnecting exosomal cargoes with cellular level responses. This five-year study will focus on three key challenges for precisely elucidating immune-modulation at the molecular level via the exosome route: 1) Develop a 3D, programmable biomimetic lymph node tissue foundry with well-defined immunological adaptations for mimicking in vivo immune tissue microenvironment; 2) Develop a single cell single exosome study approach for highly sensitive detection and subtyping of single exosome populations with dynamic and statistical significance for understanding immunity heterogeneity; 3) Discover motifs that can selectively pack immuno-stimulating microRNAs, as well as the MHC-binding peptides into exosomes for targeted immunity modulation, which can establish the interconnection of cargo internalization with cellular level responses. The long-term goal is to advance our understanding in immunity regulation heterogeneity and eventually be able to precisely programme immune responses at the molecular precision.