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.

  • Article
  • Published:

Yeast display of MHC-II enables rapid identification of peptide ligands from protein antigens (RIPPA)

Cellular & Molecular Immunology volume 18, pages 1847–1860 (2021)Cite this article

Subjects

Abstract

CD4+ T cells orchestrate adaptive immune responses via binding of antigens to their receptors through specific peptide/MHC-II complexes. To study these responses, it is essential to identify protein-derived MHC-II peptide ligands that constitute epitopes for T cell recognition. However, generating cells expressing single MHC-II alleles and isolating these proteins for use in peptide elution or binding studies is time consuming. Here, we express human MHC alleles (HLA-DR4 and HLA-DQ6) as native, noncovalent αβ dimers on yeast cells for direct flow cytometry-based screening of peptide ligands from selected antigens. We demonstrate rapid, accurate identification of DQ6 ligands from pre-pro-hypocretin, a narcolepsy-related immunogenic target. We also identify 20 DR4-binding SARS-CoV-2 spike peptides homologous to SARS-CoV-1 epitopes, and one spike peptide overlapping with the reported SARS-CoV-2 epitope recognized by CD4+ T cells from unexposed individuals carrying DR4 subtypes. Our method is optimized for immediate application upon the emergence of novel pathogens.

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

Fig. 1: The leucine zipper motif enhanced proper folding and surface expression of functional DR4 in yeast.
Fig. 2: Proper folding and surface expression of peptide-linked or “empty” DQ6 with a LZ domain in yeast.
Fig. 3: Binding of peptides to “empty” MHC-II displayed on yeast.
Fig. 4: RIPPA identified all DQ6 binders from HCRT.
Fig. 5: DR4-binding peptides derived from the SARS-CoV-2 S protein identified by yeast display vs computational prediction.
Fig. 6: RIPPA performs better than selected computational prediction algorithms.
Fig. 7: DR4 binders derived from the SARS-CoV-2 (COVID) S protein.

Similar content being viewed by others

References

  1. Rossjohn J, Gras S, Miles JJ, Turner SJ, Godfrey DI, McCluskey J. T cell antigen receptor recognition of antigen-presenting molecules. Annu Rev Immunol. 2015;33:169–200.

    Article  CAS  PubMed  Google Scholar 

  2. La Gruta NL, Gras S, Daley SR, Thomas PG, Rossjohn J. Understanding the drivers of MHC restriction of T cell receptors. Nat Rev Immunol. 2018;18:467–78.

    Article  PubMed  CAS  Google Scholar 

  3. Latorre D, Kallweit U, Armentani E, Foglierini M, Mele F, Cassotta A, et al. T cells in patients with narcolepsy target self-antigens of hypocretin neurons. Nature. 2018;562:63–8.

    Article  CAS  PubMed  Google Scholar 

  4. Luo G, Ambati A, Lin L, Bonvalet M, Partinen M, Ji X, et al. Autoimmunity to hypocretin and molecular mimicry to flu in type 1 narcolepsy. Proc Natl Acad Sci USA. 2018;115:E12323–E12332.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Jiang W, Birtley JR, Hung SC, Wang W, Chiou SH, Macaubas C, et al. In vivo clonal expansion and phenotypes of hypocretin-specific CD4(+) T cells in narcolepsy patients and controls. Nat Commun. 2019;10:5247.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Mateus J, Grifoni A, Tarke A, Sidney J, Ramirez SI, Dan JM, et al. Selective and cross-reactive SARS-CoV-2 T cell epitopes in unexposed humans. Science. 2020;370:89–94.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Grifoni A, Weiskopf D, Ramirez SI, Mateus J, Dan JM, Moderbacher CR, et al. Targets of T cell responses to SARS-CoV-2 coronavirus in humans with COVID-19 disease and unexposed individuals. Cell. 2020;181:1489–501 e1415.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Le Bert N, Tan AT, Kunasegaran K, Tham C, Hafezi M, Chia A, et al. SARS-CoV-2-specific T cell immunity in cases of COVID-19 and SARS, and uninfected controls. Nature. 2020;584:457–62.

    Article  PubMed  CAS  Google Scholar 

  9. Braun, J. et al. SARS-CoV-2-reactive T cells in healthy donors and patients with COVID-19. Nature 2020;587:270–4.

    Article  CAS  PubMed  Google Scholar 

  10. Han A, Glanville J, Hansmann L, Davis MM. Linking T-cell receptor sequence to functional phenotype at the single-cell level. Nat Biotechnol. 2014;32:684–92.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Huang H, Wang C, Rubelt F, Scriba TJ, Davis MM. Analyzing the Mycobacterium tuberculosis immune response by T-cell receptor clustering with GLIPH2 and genome-wide antigen screening. Nat Biotechnol. 2020;38:1194–202.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Newell EW, Davis MM. Beyond model antigens: high-dimensional methods for the analysis of antigen-specific T cells. Nat Biotechnol. 2014;32:149–57.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Yang J, James E, Roti M, Huston L, Gebe JA, Kwok WW. Searching immunodominant epitopes prior to epidemic: HLA class II-restricted SARS-CoV spike protein epitopes in unexposed individuals. Int Immunol. 2009;21:63–71.

    Article  CAS  PubMed  Google Scholar 

  14. Saligrama N, Zhao F, Sikora MJ, Serratelli WS, Fernandes RA, Louis DM, et al. Opposing T cell responses in experimental autoimmune encephalomyelitis. Nature. 2019;572:481–7.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Sibener LV, Fernandes RA, Kolawole EM, Carbone CB, Liu F, McAffee D, et al. Isolation of a structural mechanism for uncoupling T cell receptor signaling from peptide-MHC binding. Cell. 2018;174:672–87 e627.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Rappazzo CG, Huisman BD, Birnbaum ME. Repertoire-scale determination of class II MHC peptide binding via yeast display improves antigen prediction. Nat Commun. 2020;11:4414.

    Article  PubMed  PubMed Central  Google Scholar 

  17. Chen B, Khodadoust MS, Olsson N, Wagar LE, Fast E, Liu CL, et al. Predicting HLA class II antigen presentation through integrated deep learning. Nat Biotechnol. 2019;37:1332–43.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Nielsen M, Andreatta M, Peters B, Buus S. Immunoinformatics: predicting peptide–MHC binding. Annu Rev Biomed Data Sci. 2020;3:191–215.

    Article  Google Scholar 

  19. Reynisson B, Barra C, Kaabinejadian S, Hildebrand WH, Peters B, Nielsen M. Improved prediction of MHC II antigen presentation through integration and motif deconvolution of mass spectrometry MHC eluted ligand data. J Proteome Res. 2020;19:2304–15.

    Article  CAS  PubMed  Google Scholar 

  20. Abelin JG, Harjanto D, Malloy M, Suri P, Colson T, Goulding SP, et al. Defining HLA-II ligand processing and binding rules with mass spectrometry enhances cancer epitope prediction. Immunity. 2019;51:766–79 e717.

    Article  CAS  PubMed  Google Scholar 

  21. Nanaware PP, Jurewicz MM, Leszyk J, Shaffer SA, Stern LJ. HLA-DO modulates the diversity of the MHC-II self-peptidome. Mol Cell Proteomics. 2019;18:490–503.

    Article  CAS  PubMed  Google Scholar 

  22. Khodadoust MS, Olsson N, Wagar LE, Haabeth OA, Chen B, Swaminathan K, et al. Antigen presentation profiling reveals recognition of lymphoma immunoglobulin neoantigens. Nature. 2017;543:723–7.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Purcell AW, Ramarathinam SH, Ternette N. Mass spectrometry-based identification of MHC-bound peptides for immunopeptidomics. Nat Protoc. 2019;14:1687–707.

    Article  CAS  PubMed  Google Scholar 

  24. Jiang W, Strohman MJ, Somasundaram S, Ayyangar S, Hou T, Wang N, et al. pH-susceptibility of HLA-DO tunes DO/DM ratios to regulate HLA-DM catalytic activity. Sci Rep. 2015;5:17333.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Sidney J, Steen A, Moore C, Ngo S, Chung J, Peters B, et al. Divergent motifs but overlapping binding repertoires of six HLA-DQ molecules frequently expressed in the worldwide human population. J Immunol. 2010;185:4189–98.

    Article  CAS  PubMed  Google Scholar 

  26. Osterbye T, Nielsen M, Dudek NL, Ramarathinam SH, Purcell AW, Schafer-Nielsen C, et al. HLA class II specificity assessed by high-density peptide microarray interactions. J Immunol. 2020;205:290–9.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Justesen S, Harndahl M, Lamberth K, Nielsen LL, Buus S. Functional recombinant MHC class II molecules and high-throughput peptide-binding assays. Immunome Res. 2009;5:2.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  28. Hung SC, Hou T, Jiang W, Wang N, Qiao SW, Chow IT, et al. Epitope selection for HLA-DQ2 presentation: implications for celiac disease and viral defense. J Immunol. 2019;202:2558–69.

    Article  CAS  PubMed  Google Scholar 

  29. Boder ET, Wittrup KD. Yeast surface display for screening combinatorial polypeptide libraries. Nat Biotechnol. 1997;15:553–7.B

    Article  CAS  PubMed  Google Scholar 

  30. Boder ET, Jiang W. Engineering antibodies for cancer therapy. Annu Rev Chem Biomol. Eng. 2011;2:53–75.

    Article  CAS  PubMed  Google Scholar 

  31. Busch R, Pashine A, Garcia KC, Mellins ED. Stabilization of soluble, low-affinity HLA-DM/HLA-DR1 complexes by leucine zippers. J Immunol Methods. 2002;263:111–21.

    Article  CAS  PubMed  Google Scholar 

  32. Serra P, Garabatos N, Singha S, Fandos C, Garnica J, Solé P, et al. Increased yields and biological potency of knob-into-hole-based soluble MHC class II molecules. Nat Commun. 2019;10:4917.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  33. Jiang W, Boder ET. High-throughput engineering and analysis of peptide binding to class II MHC. Proc Natl Acad Sci USA. 2010;107:13258–63.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Boder ET, Bill JR, Nields AW, Marrack PC, Kappler JW. Yeast surface display of a noncovalent MHC class II heterodimer complexed with antigenic peptide. Biotechnol Bioeng. 2005;92:485–91.

    Article  CAS  PubMed  Google Scholar 

  35. Adler LN, Jiang W, Bhamidipati K, Millican M, Macaubas C, Hung SC, et al. The other function: class II-restricted antigen presentation by B cells. Front Immunol. 2017;8:319.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  36. Mellins ED, Stern LJ. HLA-DM and HLA-DO, key regulators of MHC-II processing and presentation. Curr Opin Immunol. 2014;26:115–22.

    Article  CAS  PubMed  Google Scholar 

  37. Rinderknecht CH, Belmares MP, Catanzarite TL, Bankovich AJ, Holmes TH, Garcia KC, et al. Posttranslational regulation of I-Ed by affinity for CLIP. J Immunol. 2007;179:5907–15.

    Article  CAS  PubMed  Google Scholar 

  38. Wen F, Esteban O, Zhao HM. Rapid identification of CD4+T-cell epitopes using yeast displaying pathogen-derived peptide library. J Immunol Methods. 2008;336:37–44.

    Article  CAS  PubMed  Google Scholar 

  39. Wen F, Sethi DK, Wucherpfennig KW, Zhao H. Cell surface display of functional human MHC class II proteins: yeast display versus insect cell display. Protein Eng Des Sel. 2011;24:701–9.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Birnbaum ME, Mendoza JL, Sethi DK, Dong S, Glanville J, Dobbins J, et al. Deconstructing the peptide-MHC specificity of T cell recognition. Cell. 2014;157:1073–87.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Esteban O, Zhao H. Directed evolution of soluble single-chain human class II MHC molecules. J Mol Biol. 2004;340:81–95.

    Article  CAS  PubMed  Google Scholar 

  42. Han F, Lin L, Warby SC, Faraco J, Li J, Dong SX, et al. Narcolepsy onset is seasonal and increased following the 2009 H1N1 pandemic in China. Ann Neurol. 2011;70:410–7.

    Article  PubMed  Google Scholar 

  43. Partinen M, Saarenpää-Heikkilä O, Ilveskoski I, Hublin C, Linna M, Olsén P, et al. Increased incidence and clinical picture of childhood narcolepsy following the 2009 H1N1 pandemic vaccination campaign in Finland. PLoS ONE. 2012;7:e33723.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  44. Schinkelshoek MS, Fronczek R, Kooy-Winkelaar E, Petersen J, Reid HH, van der Heide A, et al. H1N1 hemagglutinin-specific HLA-DQ6-restricted CD4+ T cells can be readily detected in narcolepsy type 1 patients and healthy controls. J Neuroimmunol. 2019;332:167–75.

    Article  CAS  PubMed  Google Scholar 

  45. Siebold C, Hansen BE, Wyer JR, Harlos K, Esnouf RE, Svejgaard A, et al. Crystal structure of HLA-DQ0602 that protects against type 1 diabetes and confers strong susceptibility to narcolepsy. Proc Natl Acad Sci USA. 2004;101:1999–2004.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  46. Wrapp D, Wang N, Corbett KS, Goldsmith JA, Hsieh CL, Abiona O, et al. Cryo-EM structure of the 2019-nCoV spike in the prefusion conformation. Science. 2020;367:1260–3.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  47. Cai Y, Zhang J, Xiao T, Peng H, Sterling SM, Walsh RM JR, et al. Distinct conformational states of SARS-CoV-2 spike protein. Science. 2020;369:1586–92.

    Article  CAS  PubMed  Google Scholar 

  48. Fast, E, Chen, B. Potential T-cell and B-cell epitopes of 2019-nCoV. bioRxiv: 10.1101/2020.02.19.955484 [Preprint]. (2020) [cited 20 Feb 2020]. Available from https://doi.org/10.1101/2020.02.19.955484.

  49. Grifoni A, Sidney J, Zhang Y, Scheuermann RH, Peters B, Sette A. A sequence homology and bioinformatic approach can predict candidate targets for immune responses to SARS-CoV-2. Cell Host Microbe. 2020;27:671–.e672.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  50. Starwalt SE, Masteller EL, Bluestone JA, Kranz DM. Directed evolution of a single-chain class II MHC product by yeast display. Protein Eng. 2003;16:147–56.

    Article  CAS  PubMed  Google Scholar 

  51. Feldhaus MJ, Siegel RW, Opresko LK, Coleman JR, Feldhaus JM, Yeung YA, et al. Flow-cytometric isolation of human antibodies from a nonimmune Saccharomyces cerevisiae surface display library. Nat Biotechnol. 2003;21:163–70.

    Article  CAS  PubMed  Google Scholar 

  52. Korber B, Fischer WM, Gnanakaran S, Yoon H, Theiler J, Abfalterer W, et al. Tracking changes in SARS-CoV-2 spike: evidence that D614G increases infectivity of the COVID-19 virus. Cell. 2020;182:812–.e819.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  53. Pal D. Spike protein fusion loop controls SARS-CoV-2 fusogenicity and infectivity. J Struct Biol. 2021;213:107713.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  54. Lokman SM, Rasheduzzaman M, Salauddin A, Barua R, Tanzina AY, Rumi MH, et al. Exploring the genomic and proteomic variations of SARS-CoV-2 spike glycoprotein: a computational biology approach. Infect. Genet. Evol. 2020;84:104389.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

We thank Dr. Eric T. Boder in the Department of Chemical and Biomolecular Engineering, University of Tennessee, Knoxville, for providing necessary plasmids; Cynthia Jia for assistance with the preparation of reagents; and Dr. Hong Jin, Dr. Robbert Van Der Most, and Dr. Sofia Buonocore for critical review of the manuscript. This work was funded by the Lucile Packard Foundation for Children’s Health and a research grant from GlaxoSmithKline. R.L. was funded by the China Scholarship Council Funding (CSC) during his academic research at the Stanford University School of Medicine.

Author information

Author notes

  1. These authors contributed equally: Rongzeng Liu, Wei Jiang

Authors and Affiliations

  1. Department of Pediatrics–Human Gene Therapy, Stanford University School of Medicine, Stanford, CA, USA

    Rongzeng Liu, Wei Jiang & Elizabeth D. Mellins

  2. Stanford Immunology, Stanford University School of Medicine, Stanford, CA, USA

    Rongzeng Liu, Wei Jiang & Elizabeth D. Mellins

  3. Department of Immunology, Henan University of Science and Technology School of Medicine, Luoyang, China

    Rongzeng Liu

Authors
  1. Rongzeng Liu
    View author publications

    Search author on:PubMed Google Scholar

  2. Wei Jiang
    View author publications

    Search author on:PubMed Google Scholar

  3. Elizabeth D. Mellins
    View author publications

    Search author on:PubMed Google Scholar

Contributions

R.L., W.J., and E.D.M. conceived the project, designed the experiments, analyzed the results, and wrote the manuscript. R.L. performed plasmid construction, yeast transformation, flow cytometry, and peptide binding on yeast with assistance from W.J. All authors agreed with the submission.

Corresponding authors

Correspondence to Wei Jiang or Elizabeth D. Mellins.

Ethics declarations

Competing interests

The authors declare no competing interests.

Supplementary information

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Liu, R., Jiang, W. & Mellins, E.D. Yeast display of MHC-II enables rapid identification of peptide ligands from protein antigens (RIPPA). Cell Mol Immunol 18, 1847–1860 (2021). https://doi.org/10.1038/s41423-021-00717-5

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue date:

  • DOI: https://doi.org/10.1038/s41423-021-00717-5

Keywords

This article is cited by

Search

Advanced search

Quick links