Skip to main content

Multiplex MHC Class I Tetramer Combined with Intranuclear Staining by Mass Cytometry

Part of the Methods in Molecular Biology book series (MIMB,volume 1989)

Abstract

Antigen-specific CD8+ T cells play a crucial role in the host protective immune response against viruses, tumors, and other diseases. Major histocompatibility complex (MHC) class I tetramers allow for a direct detection of such antigen-specific CD8+ T cells. Using mass cytometry together with multiplex MHC class I tetramer staining, we are able to screen more than 1000 different antigen candidates simultaneously across tissues in health and disease, while retaining the possibility to deliver an in-depth characterization of antigen-specific CD8+ T cells and associated phenotypes. Here we describe the method for a MHC class I tetramer multiplexing approach together with intracellular antibody staining for a parallel phenotypic cell characterization using mass cytometry in human specimens.

Key words

This is a preview of subscription content, log in via an institution.

Buying options

Protocol
USD   49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD   189.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Hardcover Book
USD   249.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Learn about institutional subscriptions

Springer Nature is developing a new tool to find and evaluate Protocols. Learn more

References

  1. Wong P, Pamer EG (2003) CD8 T cell responses to infectious pathogens. Annu Rev Immunol 21:29–70. https://doi.org/10.1146/annurev.immunol.21.120601.141114

    Article  CAS  PubMed  Google Scholar 

  2. Zhang N, Bevan MJ (2011) CD8(+) T cells: foot soldiers of the immune system. Immunity 35(2):161–168. https://doi.org/10.1016/j.immuni.2011.07.010

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Wherry EJ, Kurachi M (2015) Molecular and cellular insights into T cell exhaustion. Nat Rev Immunol 15(8):486–499. https://doi.org/10.1038/nri3862

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Wherry EJ (2011) T cell exhaustion. Nat Immunol 12(6):492–499

    Article  CAS  PubMed  Google Scholar 

  5. Newell EW, Becht E (2018) High-dimensional profiling of tumor-specific immune responses: asking T cells about what they “see” in cancer. Cancer Immunol Res 6(1):2–9. https://doi.org/10.1158/2326-6066.CIR-17-0519

    Article  CAS  PubMed  Google Scholar 

  6. Whiteside TL, Zhao Y, Tsukishiro T et al (2003) Enzyme-linked Immunospot, cytokine flow cytometry, and tetramers in the detection of T-cell responses to a dendritic cell-based multipeptide vaccine in patients with melanoma. Clin Cancer Res 9(2):641–649

    CAS  PubMed  Google Scholar 

  7. Altman JD, Moss PA, Goulder PJ et al (1996) Phenotypic analysis of antigen-specific T lymphocytes. Science 274(5284):94–96

    Article  CAS  PubMed  Google Scholar 

  8. Altman JD, Davis MM (2003) MHC-peptide tetramers to visualize antigen-specific T cells. Curr Protoc Immunol Chapter 17:Unit 17.3. https://doi.org/10.1002/0471142735.im1703s53

    Article  PubMed  Google Scholar 

  9. Simoni Y, Fehlings M, Kloverpris HN et al (2018) Human innate lymphoid cell subsets possess tissue-type based heterogeneity in phenotype and frequency. Immunity 48(5):1060. https://doi.org/10.1016/j.immuni.2018.04.028

    Article  CAS  PubMed  Google Scholar 

  10. Simoni Y, Fehlings M, Kloverpris HN et al (2017) Human innate lymphoid cell subsets possess tissue-type based heterogeneity in phenotype and frequency. Immunity 46(1):148–161. https://doi.org/10.1016/j.immuni.2016.11.005

    Article  CAS  PubMed  Google Scholar 

  11. Simoni Y, Newell EW (2017) Toward meaningful definitions of innate-lymphoid-cell subsets. Immunity 46(5):760–761. https://doi.org/10.1016/j.immuni.2017.04.026

    Article  CAS  PubMed  Google Scholar 

  12. Simoni Y, Newell EW (2018) Dissecting human ILC heterogeneity: more than just three subsets. Immunology 153(3):297–303. https://doi.org/10.1111/imm.12862

    Article  CAS  PubMed  Google Scholar 

  13. Wong MT, Chen J, Narayanan S et al (2015) Mapping the diversity of follicular helper T cells in human blood and tonsils using high-dimensional mass cytometry analysis. Cell Rep 11(11):1822–1833. https://doi.org/10.1016/j.celrep.2015.05.022

    Article  CAS  PubMed  Google Scholar 

  14. Wong MT, Ong DE, Lim FS et al (2016) A high-dimensional atlas of human T cell diversity reveals tissue-specific trafficking and cytokine signatures. Immunity 45(2):442–456. https://doi.org/10.1016/j.immuni.2016.07.007

    Article  CAS  PubMed  Google Scholar 

  15. Newell EW, Klein LO, Yu W et al (2009) Simultaneous detection of many T-cell specificities using combinatorial tetramer staining. Nat Methods 6(7):497–499. https://doi.org/10.1038/nmeth.1344

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Newell EW, Sigal N, Bendall SC et al (2012) Cytometry by time-of-flight shows combinatorial cytokine expression and virus-specific cell niches within a continuum of CD8+ T cell phenotypes. Immunity 36(1):142–152. https://doi.org/10.1016/j.immuni.2012.01.002

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Newell EW, Sigal N, Nair N et al (2013) Combinatorial tetramer staining and mass cytometry analysis facilitate T-cell epitope mapping and characterization. Nat Biotechnol 31(7):623–629. https://doi.org/10.1038/nbt.2593

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Fehlings M, Chakarov S, Simoni Y et al (2018) Multiplex peptide-MHC tetramer staining using mass cytometry for deep analysis of the influenza-specific T-cell response in mice. J Immunol Methods 453:30–36. https://doi.org/10.1016/j.jim.2017.09.010

    Article  CAS  PubMed  Google Scholar 

  19. Fehlings M, Simoni Y, Penny HL et al (2017) Checkpoint blockade immunotherapy reshapes the high-dimensional phenotypic heterogeneity of murine intratumoural neoantigen-specific CD8(+) T cells. Nat Commun 8(1):562. https://doi.org/10.1038/s41467-017-00627-z

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Simoni Y, Becht E, Fehlings M et al (2018) Bystander CD8(+) T cells are abundant and phenotypically distinct in human tumour infiltrates. Nature 557(7706):575–579. https://doi.org/10.1038/s41586-018-0130-2

    Article  CAS  PubMed  Google Scholar 

  21. Rodenko B, Toebes M, Hadrup SR et al (2006) Generation of peptide-MHC class I complexes through UV-mediated ligand exchange. Nat Protoc 1(3):1120–1132. https://doi.org/10.1038/nprot.2006.121

    Article  CAS  PubMed  Google Scholar 

  22. Bakker AH, Hoppes R, Linnemann C et al (2008) Conditional MHC class I ligands and peptide exchange technology for the human MHC gene products HLA-A1, -A3, -A11, and -B7. Proc Natl Acad Sci U S A 105(10):3825–3830. https://doi.org/10.1073/pnas.0709717105

    Article  PubMed  PubMed Central  Google Scholar 

  23. Toebes M, Coccoris M, Bins A et al (2006) Design and use of conditional MHC class I ligands. Nat Med 12(2):246–251. https://doi.org/10.1038/nm1360

    Article  CAS  PubMed  Google Scholar 

  24. Leong ML, Newell EW (2015) Multiplexed peptide-MHC tetramer staining with mass cytometry. Methods Mol Biol 1346:115–131. https://doi.org/10.1007/978-1-4939-2987-0_9

    Article  CAS  PubMed  Google Scholar 

  25. Finck R, Simonds EF, Jager A et al (2013) Normalization of mass cytometry data with bead standards. Cytometry A 83(5):483–494. https://doi.org/10.1002/cyto.a.22271

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Cheng Y, Wong MT, van der Maaten L et al (2016) Categorical analysis of human T cell heterogeneity with one-dimensional soli-expression by nonlinear stochastic embedding. J Immunol 196(2):924–932. https://doi.org/10.4049/jimmunol.1501928

    Article  CAS  PubMed  Google Scholar 

  27. Becht E, Dutertre C-A, Kwok IWH, et al (2018) Evaluation of UMAP as an alternative to t-SNE for single-cell data. bioRxiv. https://doi.org/10.1101/298430

  28. Qiu P, Simonds EF, Bendall SC et al (2011) Extracting a cellular hierarchy from high-dimensional cytometry data with SPADE. Nat Biotechnol 29(10):886–891. https://doi.org/10.1038/nbt.1991

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Amir el AD, Davis KL, Tadmor MD et al (2013) viSNE enables visualization of high dimensional single-cell data and reveals phenotypic heterogeneity of leukemia. Nat Biotechnol 31(6):545–552. https://doi.org/10.1038/nbt.2594

    Article  CAS  Google Scholar 

  30. van Unen V, Hollt T, Pezzotti N et al (2017) Visual analysis of mass cytometry data by hierarchical stochastic neighbour embedding reveals rare cell types. Nat Commun 8(1):1740. https://doi.org/10.1038/s41467-017-01689-9

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgments

The authors thank all members of Evan Newell lab.

Competing interests: E.W.N. is a board director and shareholder of immunoSCAPE Pte.Ltd. M.F. is Director, Scientific Affairs and shareholder of immunoSCAPE Pte. Ltd. All other authors declare no competing financial interests.

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Evan W. Newell .

Editor information

Editors and Affiliations

Rights and permissions

Reprints and permissions

Copyright information

© 2019 Springer Science+Business Media, LLC, part of Springer Nature

About this protocol

Check for updates. Verify currency and authenticity via CrossMark

Cite this protocol

Simoni, Y., Fehlings, M., Newell, E.W. (2019). Multiplex MHC Class I Tetramer Combined with Intranuclear Staining by Mass Cytometry. In: McGuire, H., Ashhurst, T. (eds) Mass Cytometry. Methods in Molecular Biology, vol 1989. Humana, New York, NY. https://doi.org/10.1007/978-1-4939-9454-0_11

Download citation

  • DOI: https://doi.org/10.1007/978-1-4939-9454-0_11

  • Published:

  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-4939-9453-3

  • Online ISBN: 978-1-4939-9454-0

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics