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

项目摘要/摘要 树突状细胞(DC)是在建立获得性免疫中起关键作用的免疫细胞 通过一种称为交叉演示的过程。交叉演示的主要途径是从 DC将颗粒病原体吞噬到称为吞噬小体的细胞内空泡中。这个 吞噬小体是组装主要组织相容性复合体1类(MHC-I)和抗原的部位 多肽进入MHC-I：多肽复合体和启动MHC-I：多肽复合体的转运 细胞表面。MHC-I：多肽复合体是如何从吞噬小体转运到细胞的 表面并不是很清楚，但可能是吞噬小体脱落了膜结构。 然而，现有的活细胞成像技术不能提供这一过程的全面图像。 这项拟议研究的长期目标是开发一种新的技术，能够 标记来自DC中含有病原体的吞噬小体的所有膜结构 交叉演示。这项拟议的技术使用了高度工程化的微粒来模拟 携带可释放膜染料的微粒病原体。它最具创新性的特点是 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，然后再将染料 微粒被吞噬，然后在吞噬后释放染料。中心假说 这项研究的意义在于，通过将膜染料单独输送到带有微粒的吞噬小体中，并 使染料可从微粒中释放，膜结构源自 吞噬小体可以被染料染色。这一假设是在我们自己的基础上提出的 初步结果。这项申请的目标是朝着实现长期目标的下一步- 术语的目标是确定微粒是否可以被用来专门标记吞噬小体衍生的 模型树突状细胞的膜性结构。具体目标是(1)建立一种用于制造 微粒，以及(2)确定微粒是否能被模型DC吞噬和 携带微粒的吞噬小体是否能扩散染有染料的膜结构。 该项目的成功将证明该技术的可行性，并为其进一步发展奠定基础。 发展。这项技术的完全开发版本有望显著推进我们的 理解交叉陈述。

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.