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 免疫反应是一个尚未得到很好研究的核心挑战，这主要是由于缺乏有效的免疫。 用于研究蜂窝通信的组织模型。自2013年以来最新发现的贩卖小泡 诺贝尔医学奖照亮了理解远距离、非接触细胞的新途径 调节免疫中的通讯。然而，对这种多样化和纳米尺寸的囊泡的研究，即 外体是极具挑战性的，因为在区分动态和异质性方面存在巨大的困难。 体内系统中存在的囊泡种群。我们的研究工作通过以下方式解决了关键技术差距 开发一系列工具包，包括高效、高通量的外切体微流控方法 分离、分型、分子工程和转基因，以及纳米递送。最近，我们观察到， 分泌性外切体的分子包装随细胞培养环境的变化而变化 以及周围的社区。2D细胞培养系统的体外研究不能 解释体内外切体免疫调节机制。我们假设一个3D仿生淋巴结 组织系统可以作为体内类组织模型，有效地研究在体外切体分子 刺激下的包装和分泌动力学，用于将胞外体货物与细胞水平互连 回应。这项为期五年的研究将聚焦于准确阐明免疫调节的三个关键挑战 在分子水平上通过外体途径：1)开发3D、可编程的仿生淋巴组织 具有明确的免疫适应性以模拟体内免疫组织微环境的铸造厂；2) 建立单细胞单外切体研究方法，用于单细胞单外切体的高灵敏度检测和分型 对理解免疫异质性具有动态和统计学意义的外体种群；3) 发现可以选择性地包装免疫刺激microRNA的基序，以及MHC结合肽 转化为外体进行靶向免疫调节，可以建立货物内化的互联 细胞水平的反应。长期目标是增进我们对免疫调节的理解。 异质性，并最终能够以分子的精度精确地编程免疫反应。

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.