Localization and MHC Class II Presentation of Antigens Targeted for Macroautophagy

  • Dorothee Schmid
  • Christian Münz
Protocol
Part of the Methods in Molecular Biology™ book series (MIMB, volume 445)

summary

Intracellular antigens can be presented on major histocompatibility complex (MHC) class II molecules after degradation via macroautophagy. To enhance MHC class II presentation of potential vaccine antigens, we have developed a method to target antigens for autophagic degradation via fusion to the Atg8/LC3 protein: Atg8/LC3 is specifically incorporated into autophagosomes via coupling to phosphatidylethanolamine, and subsequently degraded in MHC class II loading compartments (MIICs). Antigens fused to the N-terminus of Atg8/LC3 follow the same pathway and get preferentially presented on MHC class II molecules. The localization of Atg8/LC3 fusion antigens in MIICs can be visualized by confocal microscopy, and MHC class II presentation can be quantified in a presentation assay with antigen-specific CD4+ T-cell clones. These assays are good measures of autophagosome formation and lysosomal degradation of macroautophagy cargo and therefore are useful for studying regulation of the autophagic pathway under various experimental conditions and physiological perturbations.

Key Words

Atg8/LC3 influenza matrix protein 1 MHC class II MHC class II loading compartment (MIIC) confocal microscopy CD4+ T-cell clones IFN-\(\gamma\) ELISA 
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Notes

Acknowledgments

We thank Rajiv Khanna and Irene Joab for the gift of cell lines and Ari Helenius for the gift of the MP1-specific antiserum. This work was supported by the Arnold and Mabel Beckman Foundation, the Alexandrine and Alexander Sinsheimer Foundation, the Burroughs Wellcome Fund, the National Cancer Institute (R01CA108609 and R01CA101741) (to C.M.), and in part by a General Clinical Research Center grant (M01-RR00102) from the National Center for Research Resources at the National Institutes of Health (to the Rockefeller University Hospital). D.S. is a recipient of a predoctoral fellowship from the Schering Foundation.

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Copyright information

© Humana Press, a part of Springer Science+Business Media, LLC 2008

Authors and Affiliations

  • Dorothee Schmid
    • 1
  • Christian Münz
    • 1
  1. 1.Laboratory of Viral Immunobiology and Christopher H. Browne Center for Immunology and Immune DiseasesThe Rockefeller UniversityNY

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