Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Technical Report
  • Published:

A reductionist cell-free major histocompatibility complex class II antigen processing system identifies immunodominant epitopes

Nature Medicine volume 16, pages 1333–1340 (2010)Cite this article

Subjects

Abstract

Immunodominance is defined as restricted responsiveness of T cells to a few selected epitopes from complex antigens. Strategies currently used for elucidating CD4+ T cell epitopes are inadequate. To understand the mechanism of epitope selection for helper T cells, we established a cell-free antigen processing system composed of defined proteins: human leukocyte antigen-DR1 (HLA-DR1), HLA-DM and cathepsins. Our reductionist system successfully identified the physiologically selected immunodominant epitopes of two model antigens: hemagglutinin-1 (HA1) from influenza virus (A/Texas/1/77) and type II collagen (CII). When applied for identification of new epitopes from a recombinant liver-stage antigen of malaria falciparum (LSA-NRC) or HA1 from H5N1 influenza virus ('avian flu'), the system selected single epitopes from each protein that were confirmed to be immunodominant by their capacity to activate CD4+ T cells from H5N1-immunized HLA-DR1–transgenic mice and LSA-NRC–vaccinated HLA-DR1–positive human volunteers. Thus, we provide a new tool for the identification of physiologically relevant helper T cell epitopes from antigens.

Access through your institution
Buy or subscribe

This is a preview of subscription content, access via your institution

Access options

Access through your institution

Buy this article

  • Purchase on SpringerLink
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Sensitivity to cathepsins and identification of rHA1-derived peptides by mass spectrometry.
Figure 2: Identification of type II collagen-derived peptides eluted from DR1.
Figure 3: Identification of DR1 restricted epitope of H5N1 rHA1 by the cell-free antigen processing system.
Figure 4: Biological validation of the immunodominant epitope of H5N1 rHA1 identified by the reductionist antigen processing system.
Figure 5: Identification of the DR1-restricted epitope of LSA-NRC and biological validation.

Similar content being viewed by others

Accession codes

Accessions

GenBank/EMBL/DDBJ

References

  1. Sercarz, E.E. et al. Dominance and crypticity of T cell antigenic determinants. Annu. Rev. Immunol. 11, 729–766 (1993).

    Article  CAS  Google Scholar 

  2. Sercarz, E.E. Immune focusing vs diversification and their connection to immune regulation. Immunol. Rev. 164, 5–10 (1998).

    Article  CAS  Google Scholar 

  3. Lamb, J.R., Eckels, D.D., Lake, P., Woody, J.N. & Green, N. Human T-cell clones recognize chemically synthesized peptides of influenza haemagglutinin. Nature 300, 66–69 (1982).

    Article  CAS  Google Scholar 

  4. Rosloniec, E.F. et al. An HLA-DR1 transgene confers susceptibility to collagen-induced arthritis elicited with human type II collagen. J. Exp. Med. 185, 1113–1122 (1997).

    Article  CAS  Google Scholar 

  5. Jensen, P.E. Recent advances in antigen processing and presentation. Nat. Immunol. 8, 1041–1048 (2007).

    Article  CAS  Google Scholar 

  6. Amigorena, S., Drake, J.R., Webster, P. & Mellman, I. Transient accumulation of new class II MHC molecules in a novel endocytic compartment in B lymphocytes. Nature 369, 113–120 (1994).

    Article  CAS  Google Scholar 

  7. Kropshofer, H. et al. Editing of the HLA-DR-peptide repertoire by HLA-DM. EMBO J. 15, 6144–6154 (1996).

    Article  CAS  Google Scholar 

  8. Chou, C.L. & Sadegh-Nasseri, S. HLA-DM recognizes the flexible conformation of major histocompatibility complex class II. J. Exp. Med. 192, 1697–1706 (2000).

    Article  CAS  Google Scholar 

  9. Riberdy, J.M., Newcomb, J.R., Surman, M.J., Barbosa, J.A. & Cresswell, P. HLA-DR molecules from an antigen-processing mutant cell line are associated with invariant chain peptides. Nature 360, 474–477 (1992).

    Article  CAS  Google Scholar 

  10. Sloan, V.S. et al. Mediation by HLA-DM of dissociation of peptides from HLA-DR. Nature 375, 802–806 (1995).

    Article  CAS  Google Scholar 

  11. Sadegh-Nasseri, S., Chen, M., Narayan, K. & Bouvier, M. The convergent roles of tapasin and HLA-DM in antigen presentation. Trends Immunol. 29, 141–147 (2008).

    Article  CAS  Google Scholar 

  12. Plüger, E.B. et al. Specific role for cathepsin S in the generation of antigenic peptides in vivo. Eur. J. Immunol. 32, 467–476 (2002).

    Article  Google Scholar 

  13. Riese, R.J. et al. Essential role for cathepsin S in MHC class II–associated invariant chain processing and peptide loading. Immunity 4, 357–366 (1996).

    Article  CAS  Google Scholar 

  14. Chapman, H.A. Endosomal proteases in antigen presentation. Curr. Opin. Immunol. 18, 78–84 (2006).

    Article  CAS  Google Scholar 

  15. Pisoni, R.L., Acker, T.L., Lisowski, K.M., Lemons, R.M. & Thoene, J.G. A cysteine-specific lysosomal transport system provides a major route for the delivery of thiol to human fibroblast lysosomes: possible role in supporting lysosomal proteolysis. J. Cell Biol. 110, 327–335 (1990).

    Article  CAS  Google Scholar 

  16. Maric, M. et al. Defective antigen processing in GILT-free mice. Science 294, 1361–1365 (2001).

    Article  CAS  Google Scholar 

  17. Boja, E.S. & Fales, H.M. Overalkylation of a protein digest with iodoacetamide. Anal. Chem. 73, 3576–3582 (2001).

    Article  CAS  Google Scholar 

  18. Schey, K.L. & Finley, E.L. Identification of peptide oxidation by tandem mass spectrometry. Acc. Chem. Res. 33, 299–306 (2000).

    Article  CAS  Google Scholar 

  19. Narayan, K. et al. Staphylococcal enterotoxin A induces small clusters of HLA-DR1 on B cells. PLoS ONE 4, e6188 (2009).

    Article  Google Scholar 

  20. Van den Steen, P.E. et al. Cleavage of denatured natural collagen type II by neutrophil gelatinase B reveals enzyme specificity, post-translational modifications in the substrate and the formation of remnant epitopes in rheumatoid arthritis. FASEB J. 16, 379–389 (2002).

    Article  CAS  Google Scholar 

  21. Van den Steen, P.E. et al. Generation of glycosylated remnant epitopes from human collagen type II by gelatinase B. Biochemistry 43, 10809–10816 (2004).

    Article  CAS  Google Scholar 

  22. Stern, L.J. & Wiley, D.C. The human class II MHC protein HLA-DR1 assembles as empty αβ heterodimers in the absence of antigenic peptide. Cell 68, 465–477 (1992).

    Article  CAS  Google Scholar 

  23. Krzych, U. et al. T lymphocytes from volunteers immunized with irradiated Plasmodium falciparum sporozoites recognize liver and blood stage malaria antigens. J. Immunol. 155, 4072–4077 (1995).

    CAS  PubMed  Google Scholar 

  24. Fidock, D.A. et al. Plasmodium falciparum liver stage antigen-1 is well conserved and contains potent B and T cell determinants. J. Immunol. 153, 190–204 (1994).

    CAS  PubMed  Google Scholar 

  25. Hillier, C.J. et al. Process development and analysis of liver-stage antigen 1, a preerythrocyte-stage protein-based vaccine for Plasmodium falciparum. Infect. Immun. 73, 2109–2115 (2005).

    Article  CAS  Google Scholar 

  26. Cummings, J.F. et al. Recombinant liver stage antigen-1 (LSA-1) formulated with AS01 or AS02 is safe, elicits high titer antibody and induces IFN-γ/IL-2 CD4+ T cells but does not protect against experimental Plasmodium falciparum infection. Vaccine 28, 5135–5144 (2010).

    Article  CAS  Google Scholar 

  27. Deussing, J. et al. Cathepsins B and D are dispensable for major histocompatibility complex class II–mediated antigen presentation. Proc. Natl. Acad. Sci. USA 95, 4516–4521 (1998).

    Article  CAS  Google Scholar 

  28. Parker, C.E., Papac, D.I., Trojak, S.K. & Tomer, K.B. Epitope mapping by mass spectrometry: determination of an epitope on HIV-1 IIIB p26 recognized by a monoclonal antibody. J. Immunol. 157, 198–206 (1996).

    CAS  PubMed  Google Scholar 

  29. Chicz, R.M. et al. Predominant naturally processed peptides bound to HLA-DR1 are derived from MHC-related molecules and are heterogeneous in size. Nature 358, 764–768 (1992).

    Article  CAS  Google Scholar 

  30. Hunt, D.F. et al. Characterization of peptides bound to the class I MHC molecule HLA-A2.1 by mass spectrometry. Science 255, 1261–1263 (1992).

    Article  CAS  Google Scholar 

  31. Strug, I. et al. Vaccinia peptides eluted from HLA-DR1 isolated from virus-infected cells are recognized by CD4+ T cells from a vaccinated donor. J. Proteome Res. 7, 2703–2711 (2008).

    Article  CAS  Google Scholar 

  32. Ireland, J., Herzog, J. & Unanue, E.R. Cutting edge: unique T cells that recognize citrullinated peptides are a feature of protein immunization. J. Immunol. 177, 1421–1425 (2006).

    Article  CAS  Google Scholar 

  33. Beltrami, A. et al. Citrullination-dependent differential presentation of a self-peptide by HLA-B27 subtypes. J. Biol. Chem. 283, 27189–27199 (2008).

    Article  CAS  Google Scholar 

  34. Gebe, J.A. & Kwok, W.W. Tracking antigen specific CD4+ T-cells with soluble MHC molecules. Methods Mol. Med. 136, 39–50 (2007).

    Article  CAS  Google Scholar 

  35. Narayan, K. et al. HLA-DM targets the hydrogen bond between the histidine at position β81 and peptide to dissociate HLA-DR–peptide complexes. Nat. Immunol. 8, 92–100 (2007).

    Article  CAS  Google Scholar 

  36. Suri, A. et al. In APCs, the autologous peptides selected by the diabetogenic I-Ag7 molecule are unique and determined by the amino acid changes in the P9 pocket. J. Immunol. 168, 1235–1243 (2002).

    Article  CAS  Google Scholar 

  37. Natarajan, S.K., Stern, L.J. & Sadegh-Nasseri, S. Sodium dodecyl sulfate stability of HLA-DR1 complexes correlates with burial of hydrophobic residues in pocket 1. J. Immunol. 162, 3463–3470 (1999).

    CAS  PubMed  Google Scholar 

Download references

Acknowledgements

We wish to thank S. Landry, A. Hamad, J. Pomerantz and K. Narayan for reading the manuscript and insightful discussions, S. Khoruzhenko for protein production, S. Kalb-Ramirez and D. Wang for the initial mass spectrometry, F. Korangy and D. Pardoll (Johns Hopkins University) for Escherichia coli transformed with an expression vector encoding influenza HA, D. Zaller (Merck) for the original DR1-transgenic mice, L. Rein for testing the human samples and the US National Institutes of Health Tetramer Facility for providing DR1 and CLIP monomers. This work was supported by R01 grants AI063764 and GM053549, a grant from Johns Hopkins Malaria Research Institute to S.S.-N., a US National Science Foundation predoctoral award to I.Z.H. and a Johns Hopkins University Rheumatology T32 Fellowship to A.K.

Author information

Author notes

  1. Isamu Z Hartman

    Present address: Present address: Department of Molecular Genetics, University of Texas Southwestern Medical Center, Dallas, Texas, USA.,

  2. Isamu Z Hartman and AeRyon Kim: These authors contributed equally to this work.

Authors and Affiliations

  1. Graduate Program in Immunology, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA

    Isamu Z Hartman, AeRyon Kim & Scheherazade Sadegh-Nasseri

  2. Department of Pharmacology, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA

    Robert J Cotter & Wendell Griffith

  3. Department of Pathology, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA

    Kimberly Walter, Sarat K Dalai & Scheherazade Sadegh-Nasseri

  4. Department of Biological Chemistry, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA

    Tatiana Boronina & Robert N Cole

  5. Division of Malaria Vaccine Development, Walter Reed Army Institute of Research, Silver Spring, Maryland, USA

    David E Lanar, Robert Schwenk & Urszula Krzych

  6. Mass Spectrometry and Proteomics Facility, Institute for Basic Biomedical Sciences, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA

    Robert N Cole

Authors
  1. Isamu Z Hartman
    View author publications

    Search author on:PubMed Google Scholar

  2. AeRyon Kim
    View author publications

    Search author on:PubMed Google Scholar

  3. Robert J Cotter
    View author publications

    Search author on:PubMed Google Scholar

  4. Kimberly Walter
    View author publications

    Search author on:PubMed Google Scholar

  5. Sarat K Dalai
    View author publications

    Search author on:PubMed Google Scholar

  6. Tatiana Boronina
    View author publications

    Search author on:PubMed Google Scholar

  7. Wendell Griffith
    View author publications

    Search author on:PubMed Google Scholar

  8. David E Lanar
    View author publications

    Search author on:PubMed Google Scholar

  9. Robert Schwenk
    View author publications

    Search author on:PubMed Google Scholar

  10. Urszula Krzych
    View author publications

    Search author on:PubMed Google Scholar

  11. Robert N Cole
    View author publications

    Search author on:PubMed Google Scholar

  12. Scheherazade Sadegh-Nasseri
    View author publications

    Search author on:PubMed Google Scholar

Contributions

I.Z.H. and A.K. designed and executed experiments, analyzed data and wrote the paper; R.J.C., W.G., T.B. and R.N.C. provided mass spectrometry data and analyses; K.W. cloned DM; S.K.D. contributed to in vivo testing; R.S., D.E.L. and U.K. did LSA-1 experiments in humans; and S.S.-N. designed experiments, supervised the project, obtained funding and wrote the paper.

Corresponding author

Correspondence to Scheherazade Sadegh-Nasseri.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–14 and Supplementary Tables 1–4 (PDF 773 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Hartman, I., Kim, A., Cotter, R. et al. A reductionist cell-free major histocompatibility complex class II antigen processing system identifies immunodominant epitopes. Nat Med 16, 1333–1340 (2010). https://doi.org/10.1038/nm.2248

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue date:

  • DOI: https://doi.org/10.1038/nm.2248

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing