Measuring oxidation within LC3-associated phagosomes that optimizes MHC class II restricted antigen presentation

Abstract

LC3-associated phagocytosis (LAP) uses components of the molecular machinery of macroautophagy and is involved in the presentation of extracellular antigens by Major Histocompatibility Complex (MHC) class II molecules. It is initiated by receptor-mediated phagocytosis and results in the formation of LAPosomes: single-membrane vesicles that are decorated with the macroautophagy protein LC3B. LAPosomes have been described to prolong antigen presentation in macrophages but the molecular mechanism of this process is just beginning to be understood. Known key regulators of LAPosome formation are Reactive Oxygen Species (ROS), which can modulate the pH and the oxidative state within LAPosomes. Here, we present two complementary methods to monitor oxidation in LAPosomes and to study its function in MHC class II restricted antigen presentation, both in primary human macrophages: (I) Coating the LAP-trigger zymosan with OxyBURST allows semi-quantitative assessment of oxidation levels within LAPosomes by confocal microscopy. (II) The co-culture of macrophages with CD4⁺T cells to assess the effects of LAP on Candida albicans antigen presentation by measuring IL-17A and IFN-γ secretion.

Keywords: Autophagy; CD4(+) T cells; LAP; NADPH oxidase 2 (NOX2); Reactive oxygen species (ROS).