Analysis of LC3-Associated Phagocytosis and Antigen Presentation.
The noncanonical macroautophagy pathway, LC3-associated phagocytosis (LAP) has recently emerged as an important catabolic process involved during exogenous antigen processing. It has been described that in human macrophages and dendritic cells the direct recruitment of LC3 to the phagosomal membrane is associated with its maturation impairment, allowing the stabilization of the cargo to prolong antigen presentation on major histocompatibility complex (MHC) class II molecules.In this chapter, we describe methods to monitor, manipulate, and understand the role of LAP during MHC class II presentation. We show how to enhance LAP formation resulting in antigen presentation by using zymosan or beads coated with Candida albicans extract. Then, we describe how to determine the localization of Rab7 or Lamp2 on LC3-phagosomes by confocal microscopy, a useful technique to follow phagosome maturation. Finally, we propose an assay to understand how MHC class II antigen presentation can be modulated by the LAP pathway.