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.