Analysis of LC3-Associated Phagocytosis and Antigen Presentation

  • Laure-Anne Ligeon
  • Susana Romao
  • Christian Münz
Part of the Methods in Molecular Biology book series (MIMB, volume 1519)


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.

Key words

Autophagy Confocal microscopy LC3-associated phagosome MHC class II MHC class II antigen presentation Phagosome maturation Phagosome assay 



Research in our laboratory is supported by grants from Cancer Research Switzerland (KFS-3234-08-2013), Worldwide Cancer Research (14-1033), KFSPMS and KFSPHHLD of the University of Zurich, the Sobek Foundation, the Swiss Vaccine Research Institute and the Swiss National Science Foundation (310030_162560 and CRSII3_160708).


  1. 1.
    Mizushima N, Yoshimori T, Ohsumi Y (2011) The role of Atg proteins in autophagosome formation. Annu Rev Cell Dev Biol 27:107–132CrossRefPubMedGoogle Scholar
  2. 2.
    Paludan C, Schmid D, Landthaler M et al (2005) Endogenous MHC class II processing of a viral nuclear antigen after autophagy. Science 307:593–596CrossRefPubMedGoogle Scholar
  3. 3.
    Schmid D, Pypaert M, Münz C (2007) Antigen-loading compartments for major histocompatibility complex class II molecules continuously receive input from autophagosomes. Immunity 26:79–92CrossRefPubMedGoogle Scholar
  4. 4.
    Romao S, Gasser N, Becker AC et al (2013) Autophagy proteins stabilize pathogen-containing phagosomes for prolonged MHC IIantigen processing. J Cell Biol 203:757–766CrossRefPubMedPubMedCentralGoogle Scholar
  5. 5.
    Sanjuan MA, Dillon CP, Tait SWG et al (2007) Toll-like receptor signalling in macrophages links the autophagy pathway to phagocytosis. Nature 450:1253–1257CrossRefPubMedGoogle Scholar
  6. 6.
    Ma J, Becker C, Lowell CA et al (2012) Dectin-1-triggered recruitment of light chain 3 protein to phagosomes facilitates major histocompatibility complex class II presentation of fungal-derived antigens. J Biol Chem 287:34149–34156CrossRefPubMedPubMedCentralGoogle Scholar
  7. 7.
    Delamarre L, Couture R, Mellman I et al (2006) Enhancing immunogenicity by limiting susceptibility to lysosomal proteolysis. J Exp Med 203:2049–2055CrossRefPubMedPubMedCentralGoogle Scholar
  8. 8.
    Savina A, Jancic C, Hugues S et al (2006) NOX2 controls phagosomal pH to regulate antigen processing during crosspresentation by dendritic cells. Cell 126:205–218CrossRefPubMedGoogle Scholar
  9. 9.
    Martinez J, Malireddi RKS, Lu Q et al (2015) Molecular characterization of LC3-associated phagocytosis reveals distinct roles for Rubicon, NOX2 and autophagy proteins. Nat Cell Biol 17:893–906CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2017

Authors and Affiliations

  • Laure-Anne Ligeon
    • 1
  • Susana Romao
    • 1
  • Christian Münz
    • 1
  1. 1.Viral Immunobiology, Institute of Experimental ImmunologyUniversity of ZürichZürichSwitzerland

Personalised recommendations