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.
Viral Immunobiology, Institute of Experimental Immunology, University of Zürich, Winterthurerstrasse 190, Zürich, 8057, Switzerland.
Laure-Anne
LA
Ligeon
Viral Immunobiology, Institute of Experimental Immunology, University of Zürich, Winterthurerstrasse 190, Zürich, 8057, Switzerland.
Susana
S
Romao
Viral Immunobiology, Institute of Experimental Immunology, University of Zürich, Winterthurerstrasse 190, Zürich, 8057, Switzerland. muenzc@immunology.uzh.ch.
Christian
C
Münz
D017951:Antigen Presentation; D001343:Autophagy; D000071187:Autophagy-Related Protein 5 / Q000378:metabolism; D002478:Cells, Cultured; D003713:Dendritic Cells / Q000166:cytology / Q000378:metabolism; D020868:Gene Silencing; D000949:Histocompatibility Antigens Class II / Q000378:metabolism; D006801:Humans; D016086:Lentivirus / Q000378:metabolism; D018613:Microscopy, Confocal / Q000379:methods*; D008869:Microtubule-Associated Proteins / Q000378:metabolism; D010587:Phagocytosis; D010588:Phagosomes / Q000378:metabolism; D014161:Transduction, Genetic
D000071187:Autophagy-Related Protein 5; D000949:Histocompatibility Antigens Class II; C477012:MAP1LC3A protein, human; D008869:Microtubule-Associated Proteins
Autophagy; Confocal microscopy; LC3-associated phagosome; MHC class II; MHC class II antigen presentation; Phagosome assay; Phagosome maturation