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.
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References
Chapman, H. A. (2006) Endosomal proteases in antigen presentation. Curr. Opin. Immunol. 18, 78–84.
Bryant, P., and Ploegh H. (2004) Class II MHC peptide loading by the professionals. Curr. Opin. Immunol. 16, 96–102.
Bevan, M. J. (2004) Helping the CD8+ T-cell response. Nat. Rev. Immunol. 4, 595–602.
Schmid, D., Dengjel, J., Schoor, O., Stevanovic, S., and Münz. C. (2006) Autophagy in innate and adaptive immunity against intracellular pathogens. J. Mol. Med. 84, 194–202.
Münz, C. (2006) Autophagy and antigen presentation. Cell Microbiol. 8, 891–898.
Brazil, M. I., Weiss, S., and Stockinger, B. (1997) Excessive degradation of intracellular protein in macrophages prevents presentation in the context of major histocompatibility complex class II molecules. Eur. J. Immunol. 27, 1506–1514.
Dorfel, D., Appel, S., Grunebach, F., Weck, M. M., Muller, M. R., Heine, A., and Brossart, P. (2005) Processing and presentation of HLA class I and II epitopes by dendritic cells after transfection with in vitro transcribed MUC1 RNA. Blood 105, 3199–3205.
Nimmerjahn, F., Milosevic, S., Behrends, U., Jaffee, E. M., Pardoll, D. M., Bornkamm, G. W., and Mautner, J. (2003) Major histocompatibility complex class II-restricted presentation of a cytosolic antigen by autophagy. Eur. J. Immunol. 33,1250–1259.
Paludan, C., Schmid, D., Landthaler, M., Vockerodt, M., Kube, D., Tuschl, T., and Münz, C. (2005) Endogenous MHC class II processing of a viral nuclear antigen after autophagy. Science 307, 593–596.
Schmid, D., Pypaert, M., and Münz, C. (2007) MHC class II antigen loading compartments continuously receive input from autophagosomes. Immunity 26,79–92.
Kabeya, Y., Mizushima, N., Ueno, T., Yamamoto, A., Kirisako, T., Noda, T., Kominami, E., Ohsumi, Y., and Yoshimori, T. (2000) LC3, a mammalian homologue of yeast Apg8p, is localized in autophagosome membranes after processing. Embo J. 19, 5720–5728.
Mizushima, N., Yamamoto, A., Matsui, M., Yoshimori, T., and Ohsumi, Y. (2004) In vivo analysis of autophagy in response to nutrient starvation using transgenic mice expressing a fluorescent autophagosome marker. Mol. Biol. Cell 15,1101–1111.
Dengjel, J., Schoor, O., Fischer, R., Reich, M., Kraus, M., Muller, M., Kreymborg, K., Altenberend, F., Brandenburg, J., Kalbacher, H., Brock, R. R., Driessen, C., Rammensee, H. G., and Stevanovic, S. (2005) Autophagy promotes MHC class II presentation of peptides from intracellular source proteins. Proc. Natl. Acad. Sci. USA 102, 7922–7927.
Martin, K., and Helenius, A. (1991) Transport of incoming influenza virus nucleocapsids into the nucleus. J. Virol. 65, 232–244.
Fonteneau, J. F., Larsson, M., Somersan, S., Sanders, C., Münz, C., Kwok, W. W., Bhardwaj, N., and Jotereau, F. (2001) Generation of high quantities of viral and tumor-specific human CD4+ and CD8+ T-cell clones using peptide pulsed mature dendritic cells. J. Immunol. Methods 258, 111–126.
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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Schmid, D., Münz, C. (2008). Localization and MHC Class II Presentation of Antigens Targeted for Macroautophagy. In: Deretic, V. (eds) Autophagosome and Phagosome. Methods in Molecular Biology™, vol 445. Humana Press. https://doi.org/10.1007/978-1-59745-157-4_14
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DOI: https://doi.org/10.1007/978-1-59745-157-4_14
Publisher Name: Humana Press
Print ISBN: 978-1-58829-853-9
Online ISBN: 978-1-59745-157-4
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