Abstract
Advances in immunology and immune therapies require knowledge of antigenic peptide sequences that are presented on MHC class II and class I molecules of antigen presenting cells. The most specialized antigen presenting cells are dendritic cells (DCs). In the past, the small number of DCs that could be isolated from mouse spleen prevented direct analysis of the MHC II peptide repertoire presented by DCs. Here we describe a protocol that integrates immunological methods (in vivo enrichment of mouse spleen DCs by Flt3L treatment and immunoprecipitation of MHC II-peptide complexes), mass spectrometry analysis and peptide synthesis (LC-MS/MS and quantitation analysis for non tryptic peptides) to identify and quantitate the endogenous peptides that are bound to MHC II molecules on DCs. The described method produces quantitative data that are reproducible and reliable enough to cover a wide range of peptide copy numbers. We propose the application of this method in future studies to quantitatively investigate the MHC II repertoire on DCs presented during viral infections or different immunizations in vaccine development research.
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References
Banchereau J, Steinman RM (1998) Dendritic cells and the control of immunity. Nature 392:245–252
Mellman I, Steinman RM (2001) Dendritic cells: specialized and regulated antigen processing machines. Cell 106:255–258
Bozzacco L, Yu H, Zebroski HA et al (2011) Mass spectrometry analysis and quantitation of peptides presented on the MHC II molecules of mouse spleen dendritic cells. J Proteome Res 10:5016–5030
Bozzacco L, Trumpfheller C, Huang Y et al (2010) HIV gag protein is efficiently cross-presented when targeted with an antibody towards the DEC-205 receptor in Flt3 ligand-mobilized murine DC. Eur J Immunol 40:36–46
Maraskovsky E, Brasel K, Teepe M et al (1996) Dramatic increase in the numbers of functionally mature dendritic cells in Flt3 ligand-treated mice: multiple dendritic cell subpopulations identified. J Exp Med 184:1953–1962
O'Keeffe M, Hochrein H, Vremec D et al (2002) Effects of administration of progenipoietin 1, Flt-3 ligand, granulocyte colony-stimulating factor, and pegylated granulocyte–macrophage colony-stimulating factor on dendritic cell subsets in mice. Blood 99:2122–2130
Curti A, Fogli M, Ratta M et al (2001) Stem cell factor and FLT3-Ligand are strictly required to sustain the long-term expansion of primitive CD34(+)DR(−) dendritic cell precursors. J Immunol 166:848–854
D'Amico A, Wu L (2003) The early progenitors of mouse dendritic cells and plasmacytoid predendritic cells are within the bone marrow hemopoietic precursors expressing Flt3. J Exp Med 198:293–303
Bozzacco L, Yu H, Dengjel J et al (2012) Strategy for identifying dendritic cell-processed CD4+ T cell epitopes from HIV gag p24 peptides. PloS one (in press)
Gerber SA, Rush J, Stemman O et al (2003) Absolute quantification of proteins and phosphoproteins from cell lysates by tandem MS. Proc Natl Acad Sci U S A 100:6940–6945
Dranoff G, Jaffee E, Lazenby A et al (1993) Vaccination with irradiated tumor cells engineered to secrete murine granulocyte–macrophage colony-stimulating factor stimulates potent, specific, and long-lasting anti-tumor immunity. Proc Natl Acad Sci U S A 90:3539–3543
Metlay JP, Witmer-Pack MD, Agger R et al (1990) The distinct leukocyte integrins of mouse spleen dendritic cells as identified with new hamster monoclonal antibodies. J Exp Med 171:1753–1771
Kang Y-S, Yamazaki S, Iyoda T et al (2003) SIGN-R1, a novel C-type lectin expressed by marginal zone macrophages in spleen, mediates uptake of the polysaccharide dextran. Int Immunol 15:177–186
Pulendran B, Lingappa J, Kennedy MK et al (1997) Developmental pathways of dendritic cells in vivo: distinct function, phenotype, and localization of dendritic cell subsets in FLT3 ligand-treated mice. J Immunol 159:2222–2231
Shelver WL (2005) Immunochemical protocols. In: Burns R (ed) Methods in molecular biology. Humana Press, Totowa, NJ
Hames BD, Rickwood D (1998) Gel electrophoresis of proteins: a practical approach. Oxford University Press, Oxford
Bolt MW, Mahoney PA (1997) High-efficiency blotting of proteins of diverse sizes following sodium dodecyl sulfate-polyacrylamide, gel elecrophoresis. Anal Biochem 247:185–192
Acknowledgments
We thank Dr. Svetlana Mojsov, the Rockefeller University, New York and Dr. Jörn Dengjel, University of Freiburg, Freiburg, for critical discussion and analytical tools. We are grateful to Marguerite Nulty for help with the references. This work was supported by grants from National Institute of Allergy and Infectious Diseases (NIAID), AI13013, 081677, and AI051573 to Ralph M. Steinman.
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Bozzacco, L., Yu, H. (2013). Identification and Quantitation of MHC Class II-Bound Peptides from Mouse Spleen Dendritic Cells by Immunoprecipitation and Mass Spectrometry Analysis. In: Fulton, K., Twine, S. (eds) Immunoproteomics. Methods in Molecular Biology, vol 1061. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-589-7_14
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DOI: https://doi.org/10.1007/978-1-62703-589-7_14
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Publisher Name: Humana Press, Totowa, NJ
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