Development of a technique for specific labelling phagosome-derived membranous structures in dendritic cells

项目总结/摘要 树突状细胞（DC）是在建立适应性免疫中发挥关键作用的免疫细胞 通过一个叫做交叉呈现的过程。交叉展示的一个主要途径是从 颗粒状病原体被DC吞噬到称为吞噬体的细胞内空泡中。的 吞噬体是组装主要组织相容性复合物1类（MHC-I）和抗原性复合物1类（MHC-II）的位点。 肽复合物，并启动MHC-I：肽复合物转运至 细胞表面。MHC-I：肽复合物如何从吞噬体转运到细胞 表面不太清楚，但可能是通过吞噬体脱落膜结构。 然而，现有的活细胞成像技术不能提供这一过程的全面图片。 这项研究的长期目标是开发一种新的技术， 标记来自DC中含有病原体的吞噬体的所有膜结构， 交叉展示这项拟议中的技术使用高度工程化的微粒， 颗粒病原体，同时携带可释放的膜染料。最具创新性的特点是， 所提出的技术是使用一种特殊的材料，首先将染料保持在微粒中， 微粒被吞噬，然后在吞噬后释放染料。核心假设 这项研究的一个重要方面是，通过将膜染料单独递送到具有微粒的吞噬体中， 使染料可从微粒释放，从微粒衍生的膜结构 吞噬体可以被染料染色。这个假设是在我们自己的基础上提出的 初步结果。本申请的目的，这是实现长期目标的下一步- 术语目标是确定微粒是否可以用于特异性标记吞噬体来源的 模型DC中的膜结构。具体目标是（1）建立一个用于制造 （2）确定微粒是否可以被模型DC吞噬， 载有微粒的吞噬体是否可以散布被染料染色的膜结构。 该项目的成功将证明该技术的可行性，并为该技术的进一步推广奠定基础 发展这项技术的完全开发版本有望大大提高我们的 理解交叉呈现。

PROJECT SUMMARY/ABSTRACT Dendritic cells (DCs) are immune cells that play a critical role in establishing adaptive immunity through a process termed cross-presentation. A major route of cross-presentation starts with the phagocytosis of a particulate pathogen by a DC into an intracellular vacuole called phagosome. The phagosome is a site for assembling major histocompatibility complex class 1 (MHC-I) and antigenic peptides into MHC-I:peptide complexes and initiating the transport of the MHC-I:peptide complexes to the cell surface. How the MHC-I:peptide complexes are transported from the phagosome to the cell surface is not well understood but probably through shedding membranous structures by the phagosome. However, existing live-cell imaging techniques cannot provide a comprehensive picture of this process. The long-term goal of this proposed research is to develop a new technique capable of specifically labelling all membranous structures derived from a pathogen-containing phagosome in a DC undergoing cross-presentation. This proposed technique uses highly engineered microparticles crafted to mimic particulate pathogens while carrying a releasable membrane dye. The most innovative feature of this proposed technique is the use of a special material to first hold the dye in the microparticle before the microparticle is phagocytosed and then release the dye after the phagocytosis. The central hypothesis of this study is that by delivering a membrane dye solely into a phagosome with a microparticle and rendering the dye releasable from the microparticle, the membranous structures derived from the phagosome can be stained by the dye. This hypothesis has been formulated on the basis of our own preliminary results. The objective of this application, which is the next step toward attainment of the long- term goal, is to determine whether the microparticles can be used to specifically label phagosome-derived membranous structures in model DCs. The specific aims are to (1) establish a protocol for fabricating the microparticles, and (2) determine whether the microparticles can be phagocytosed by model DCs and whether the microparticle-laden phagosome can disseminate membranous structures stained by the dye. Success of this project will prove the feasibility of this technique and lay a foundation for its further development. The fully developed version of this technique promises to significantly advance our understanding of cross-presentation.