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Generation of peptide–MHC class I complexes through UV-mediated ligand exchange

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Abstract

Major histocompatibility complex (MHC) class I molecules present peptide ligands on the cell surface for recognition by appropriate cytotoxic T cells. MHC-bound peptides are critical for the stability of the MHC complex, and standard strategies for the production of recombinant MHC complexes are based on in vitro refolding reactions with specific peptides. This strategy is not amenable to high-throughput production of vast collections of MHC molecules. We have developed conditional MHC ligands that form stable complexes with MHC molecules but can be cleaved upon UV irradiation. The resulting empty, peptide-receptive MHC molecules can be charged with epitopes of choice under native conditions. Here we describe in-depth procedures for the high-throughput production of peptide-MHC (pMHC) complexes by MHC exchange, the analysis of peptide exchange efficiency by ELISA and the parallel production of MHC tetramers for T-cell detection. The production of the conditional pMHC complex by an in vitro refolding reaction can be achieved within 2 weeks, and the actual high-throughput MHC peptide exchange and subsequent MHC tetramer formation require less than a day.

*Note: In the version of this article originally published online, the Reagent Setup listing for wash buffer should have read: “20 mM Tris pH 8, 100 mM NaCl.” This error has been corrected in the HTML and PDF versions of the article.

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Figure 1: UV-mediated peptide exchange.
Figure 2: Conditional peptide ligand.
Figure 3: Synthesis of the photolabile amino acid residue.
Figure 4: HLA A2.1–selective, UV-sensitive conditional MHC ligand containing a (2-nitro)phenylglycine residue.
Figure 5: Flow scheme of the various procedures involved in MHC peptide exchange.
Figure 6: Flow scheme of the assembly of biotinylated MHC class I monomers.
Figure 7: MHC class I tetramer formation.
Figure 8: ELISA setup and results.
Figure 9: Typical HPLC profile of an MHC PE-streptavidin titration.
Figure 10: Flow cytometry.

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Change history

  • 19 October 2006

    In the version of this article originally published online, the Reagent Setup listing for wash buffer should have read: “20 mM Tris pH 8, 100 mM NaCl.” This error has been corrected in the HTML and PDF versions of the article.

References

  1. Fremont, D.H., Matsumura, M., Stura, E.A., Peterson, P.A. & Wilson, I.A. Crystal structures of two viral peptides in complex with murine MHC class I H-2Kb. Science 257, 919–927 (1992).

    Article  CAS  Google Scholar 

  2. Silver, M.L., Guo, H.C., Strominger, J.L. & Wiley, D.C. Atomic structure of a human MHC molecule presenting an influenza virus peptide. Nature 360, 367–369 (1992).

    Article  CAS  Google Scholar 

  3. Bouvier, M. & Wiley, D.C. Importance of peptide amino and carboxyl termini to the stability of MHC class I molecules. Science 265, 398–402 (1994).

    Article  CAS  Google Scholar 

  4. Schumacher, T.N. et al. Peptide selection by MHC class I molecules. Nature 350, 703–706 (1991).

    Article  CAS  Google Scholar 

  5. Bakker, A.H. & Schumacher, T.N.M. MHC multimer technology: current status and future prospects. Curr. Opin. Immunol. 17, 428–433 (2005).

    Article  CAS  Google Scholar 

  6. Ljunggren, H.G. et al. Empty MHC class I molecules come out in the cold. Nature 346, 476–480 (1990).

    Article  CAS  Google Scholar 

  7. Schumacher, T.N. et al. Direct binding of peptide to empty MHC class I molecules on intact cells and in vitro. Cell 62, 563–567 (1990).

    Article  CAS  Google Scholar 

  8. Garboczi, D.N., Hung, D.T. & Wiley, D.C. HLA-A2-peptide complexes: refolding and crystallization of molecules expressed in Escherichia coli and complexed with single antigenic peptides. Proc. Natl. Acad. Sci. USA 89, 3429–3433 (1992).

    Article  CAS  Google Scholar 

  9. Buchli, R. et al. Real-time measurement of in vitro peptide binding to soluble HLA-A*0201 by fluorescence polarization. Biochemistry 43, 14852–14863 (2004).

    Article  CAS  Google Scholar 

  10. Greten, T.F. et al. Direct visualization of antigen-specific T cells: HTLV-1 Tax11–19- specific CD8(+) T cells are activated in peripheral blood and accumulate in cerebrospinal fluid from HAM/TSP patients. Proc. Natl. Acad. Sci. USA 95, 7568–7573 (1998).

    Article  CAS  Google Scholar 

  11. Toebes, M. et al. Design and use of conditional MHC class I ligands. Nat. Med. 12, 246–251 (2006).

    Article  CAS  Google Scholar 

  12. Bouvier, M. & Wiley, D.C. Structural characterization of a soluble and partially folded class I major histocompatibility heavy chain/beta 2m heterodimer. Nat. Struct. Biol. 5, 377–384 (1998).

    Article  CAS  Google Scholar 

  13. Fahnestock, M.L., Tamir, I., Narhi, L. & Bjorkman, P.J. Thermal stability comparison of purified empty and peptide-filled forms of a class I MHC molecule. Science 258, 1658–1662 (1992).

    Article  CAS  Google Scholar 

  14. Bosques, C.J. & Imperiali, B. Photolytic control of peptide self-assembly. J. Am. Chem. Soc. 125, 7530–7531 (2003).

    Article  CAS  Google Scholar 

  15. Checovich, W.J., Bolger, R.E. & Burke, T. Fluorescence polarization – A new tool for cell and molecular biology. Nature 375, 254–256 (1995).

    Article  CAS  Google Scholar 

  16. Cobbold, M. et al. Adoptive transfer of cytomegalovirus-specific CTL to stem cell transplant patients after selection by HLA-peptide tetramers. J. Exp. Med. 202, 379–386 (2005).

    Article  CAS  Google Scholar 

  17. Dudley, E.M. et al. Adoptive cell transfer therapy following non-myeloablative but lymphodepleting chemotherapy for the treatment of patients with refractory metastatic melanoma. J. Clin. Oncol. 23, 2346–2357 (2005).

    Article  CAS  Google Scholar 

  18. Soen, Y., Chen, D.S., Kraft, D.L., Davis, M.M. & Brown, P.O. Detection and characterization of cellular immune responses using peptide-MHC microarrays. PLoS Biol. 1, e65 (2003).

    Article  Google Scholar 

  19. Chen, D.S. et al. Marked differences in human melanoma antigen-specific T cell responsiveness after vaccination using a functional microarray. PLoS Med. 2, e265 (2005).

    Article  Google Scholar 

  20. Stone, J.D., Demkowicz, W.E. & Stern, L.J. HLA-restricted epitope identification and detection of functional T cell responses by using MHC-peptide and costimulatory microarrays. Proc. Natl. Acad. Sci. USA 102, 3744–3791 (2005).

    Article  CAS  Google Scholar 

  21. Hlavac, F., Connan, F., Hoebeke, J., Guillet, J.G. & Choppin, J. Direct detection of peptide-dependent HLA variability by surface plasmon resonance. Mol. Immunol. 33, 573–582 (1996).

    Article  CAS  Google Scholar 

  22. Sette, A. et al. Peptide binding to the most frequent HLA-A class I alleles measured by quantitative molecular binding assays. Mol. Immunol. 31, 813–822 (1994).

    Article  CAS  Google Scholar 

  23. Altman, J.D. et al. Phenotypic analysis of antigen-specific T lymphocytes. Science 274, 94–96 (1996).

    Article  CAS  Google Scholar 

  24. Guillaume, P. et al. Soluble major histocompatibility complex-peptide octamers with impaired CD8 binding selectively induce Fas-dependent apoptosis. J. Biol. Chem. 278, 4500–4509 (2003).

    Article  CAS  Google Scholar 

  25. Wellings, D.A. & Atherton, E. Standard Fmoc protocols. Methods Enzymol. 289, 44–67 (1997).

    Article  CAS  Google Scholar 

  26. Ghrayeb, J. et al. Secretion cloning vectors in Escherichia coli. EMBO J. 3, 2437–2442 (1984).

    Article  CAS  Google Scholar 

  27. Blanchet, J.S. et al. A new generation of Melan-A/MART-1 peptides that fulfill both increased immunogenicity and high resistance to biodegradation: implication for molecular anti-melanoma immunotherapy. J. Immunol. 167, 5852–5861 (2001).

    Article  CAS  Google Scholar 

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Correspondence to Ton N M Schumacher or Huib Ovaa.

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The technology described in this manuscript is the subject of a patent application. Based on Netherlands Cancer Institute policy on management of intellectual property, M.T., H.O and T.N.M.S. would be entitled to a portion of received royalty income in case of future licensing.

Supplementary information

Supplementary Fig. 1

Synthetic scheme for the synthesis of N-Fluorenylmethyloxycarbonyl-(2-nitro)phenylglycine. (PDF 72 kb)

Supplementary Fig. 2

Standard titration curve. (PDF 54 kb)

Supplementary Methods (DOC 51 kb)

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Rodenko, B., Toebes, M., Hadrup, S. et al. Generation of peptide–MHC class I complexes through UV-mediated ligand exchange. Nat Protoc 1, 1120–1132 (2006). https://doi.org/10.1038/nprot.2006.121

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