Skip to main content

Thank you for visiting 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.

Combinatorial tetramer staining and mass cytometry analysis facilitate T-cell epitope mapping and characterization


It is currently not possible to predict which epitopes will be recognized by T cells in different individuals. This is a barrier to the thorough analysis and understanding of T-cell responses after vaccination or infection. Here, by combining mass cytometry with combinatorial peptide–MHC tetramer staining, we have developed a method allowing the rapid and simultaneous identification and characterization of T cells specific for many epitopes. We use this to screen up to 109 different peptide–MHC tetramers in a single human blood sample, while still retaining at least 23 labels to analyze other markers of T-cell phenotype and function. Among 77 candidate rotavirus epitopes, we identified six T-cell epitopes restricted to human leukocyte antigen (HLA)-A*0201 in the blood of healthy individuals. T cells specific for epitopes in the rotavirus VP3 protein displayed a distinct phenotype and were present at high frequencies in intestinal epithelium. This approach should be useful for the comprehensive analysis of T-cell responses to infectious diseases or vaccines.

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

Relevant articles

Open Access articles citing this article.

Access options

Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.

Figure 1: T-cell epitope discovery strategy.
Figure 2: Auto-gating strategy and validation with scrambled three-metal coding.
Figure 3: Analysis of the phenotype of antigen-specific T cells in the blood of 17 normal donors.
Figure 4: Phenotypic comparison of IEL and blood-derived rotavirus-specific CD8+ T cells.


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

    Article  CAS  Google Scholar 

  2. Davis, M.M., Altman, J.D. & Newell, E.W. Interrogating the repertoire: broadening the scope of peptide-MHC multimer analysis. Nat. Rev. Immunol. 11, 551–558 (2011).

    Article  CAS  Google Scholar 

  3. Day, C.L. et al. Ex vivo analysis of human memory CD4 T cells specific for hepatitis C virus using MHC class II tetramers. J. Clin. Invest. 112, 831–842 (2003).

    Article  CAS  Google Scholar 

  4. Moon, J.J. et al. Naive CD4(+) T cell frequency varies for different epitopes and predicts repertoire diversity and response magnitude. Immunity 27, 203–213 (2007).

    Article  CAS  Google Scholar 

  5. Ornatsky, O., Baranov, V.I., Bandura, D.R., Tanner, S.D. & Dick, J. Multiple cellular antigen detection by ICP-MS. J. Immunol. Methods 308, 68–76 (2006).

    Article  CAS  Google Scholar 

  6. Bendall, S.C. et al. Single-cell mass cytometry of differential immune and drug responses across a human hematopoietic continuum. Science 332, 687–696 (2011).

    Article  CAS  Google Scholar 

  7. Newell, E.W., Sigal, N., Bendall, S.C., Nolan, G.P. & Davis, M.M. Cytometry by time-of-flight shows combinatorial cytokine expression and virus-specific cell niches within a continuum of CD8(+) T cell phenotypes. Immunity 142–152 (2012).

  8. Chang, C.X. et al. Sources of diversity in T cell epitope discovery. Front. Biosci. 16, 3014–3035 (2011).

    Article  CAS  Google Scholar 

  9. Lundegaard, C., Lund, O. & Nielsen, M. Prediction of epitopes using neural network based methods. J. Immunol. Methods 374, 26–34 (2011).

    Article  CAS  Google Scholar 

  10. Babji, S. & Kang, G. Rotavirus vaccination in developing countries. Curr. Opin. Virol. (2012).

  11. Wei, J. et al. A naturally processed epitope on rotavirus VP7 glycoprotein recognized by HLA-A2.1-restricted cytotoxic CD8+ T cells. Viral Immunol. 22, 189–194 (2009).

    Article  CAS  Google Scholar 

  12. Wei, J. et al. Identification of an HLA-A*0201-restricted cytotoxic T-lymphocyte epitope in rotavirus VP6 protein. J. Gen. Virol. 87, 3393–3396 (2006).

    Article  CAS  Google Scholar 

  13. Ward, R. Mechanisms of protection against rotavirus infection and disease. Pediatr. Infect. Dis. J. 28, S57–S59 (2009).

    Article  Google Scholar 

  14. Newell, E.W., Klein, L.O., Yu, W. & Davis, M.M. Simultaneous detection of many T-cell specificities using combinatorial tetramer staining. Nat. Methods 6, 497–499 (2009).

    Article  CAS  Google Scholar 

  15. Hadrup, S.R. et al. Parallel detection of antigen-specific T-cell responses by multidimensional encoding of MHC multimers. Nat. Methods 6, 520–526 (2009).

    Article  CAS  Google Scholar 

  16. Lundegaard, C. et al. NetMHC-3.0: accurate web accessible predictions of human, mouse and monkey MHC class I affinities for peptides of length 8–11. Nucleic Acids Res. 36, W509–W512 (2008).

    Article  CAS  Google Scholar 

  17. Rippinger, C.M., Patton, J.T. & McDonald, S.M. Complete genome sequence analysis of candidate human rotavirus vaccine strains RV3 and 116E. Virology 405, 201–213 (2010).

    Article  CAS  Google Scholar 

  18. Cole, D.K. et al. Germ line-governed recognition of a cancer epitope by an immunodominant human T-cell receptor. J. Biol. Chem. 284, 27281–27289 (2009).

    Article  CAS  Google Scholar 

  19. Vita, R. et al. The immune epitope database 2.0. Nucleic Acids Res. 38, D854–D862 (2010).

    Article  CAS  Google Scholar 

  20. Sallusto, F., Geginat, J. & Lanzavecchia, A. Central memory and effector memory T cell subsets: function, generation, and maintenance. Annu. Rev. Immunol. 22, 745–763 (2004).

    Article  CAS  Google Scholar 

  21. Gorfu, G., Rivera-Nieves, J. & Ley, K. Role of beta7 integrins in intestinal lymphocyte homing and retention. Curr. Mol. Med. 9, 836–850 (2009).

    Article  CAS  Google Scholar 

  22. Ravkov, E.V., Myrick, C.M. & Altman, J.D. Immediate early effector functions of virus-specific CD8+CCR7+ memory cells in humans defined by HLA and CC chemokine ligand 19 tetramers. J. Immunol. 170, 2461–2468 (2003).

    Article  CAS  Google Scholar 

  23. Pittet, M.J. et al. High frequencies of naive Melan-A/MART-1-specific CD8(+) T cells in a large proportion of human histocompatibility leukocyte antigen (HLA)-A2 individuals. J. Exp. Med. 190, 705–716 (1999).

    Article  CAS  Google Scholar 

  24. DeNucci, C.C., Pagan, A.J., Mitchell, J.S. & Shimizu, Y. Control of alpha4beta7 integrin expression and CD4 T cell homing by the beta1 integrin subunit. J. Immunol. 184, 2458–2467 (2010).

    Article  CAS  Google Scholar 

  25. Feng, N. et al. Inhibition of rotavirus replication by a non-neutralizing, rotavirus VP6-specific IgA mAb. J. Clin. Invest. 109, 1203–1213 (2002).

    Article  CAS  Google Scholar 

  26. Matthijnssens, J. et al. Recommendations for the classification of group A rotaviruses using all 11 genomic RNA segments. Arch. Virol. 153, 1621–1629 (2008).

    Article  CAS  Google Scholar 

  27. Fiocchi, C. & Youngman, K.R. Isolation of human intestinal mucosal mononuclear cells. Curr. Prot. Immunol. Chapter 7, Supplement 19, 7.30 (2001).

    Google Scholar 

  28. Ramachandiran, V. et al. A robust method for production of MHC tetramers with small molecule fluorophores. J. Immunol. Methods 319, 13–20 (2007).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  30. Bakker, A.H. et al. 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. USA 105, 3825–3830 (2008).

    Article  CAS  Google Scholar 

  31. Lissina, A. et al. Protein kinase inhibitors substantially improve the physical detection of T-cells with peptide-MHC tetramers. J. Immunol. Methods 340, 11–24 (2009).

    Article  CAS  Google Scholar 

  32. Fienberg, H.G., Simonds, E.F., Fantl, W.J., Nolan, G.P. & Bodenmiller, B. A platinum-based covalent viability reagent for single-cell mass cytometry. Cytometry A 81, 467–475 (2012).

    Article  Google Scholar 

  33. Bodenmiller, B. et al. Multiplexed mass cytometry profiling of cellular states perturbed by small-molecule regulators. Nat. Biotechnol. 30, 858–867 (2012).

    Article  CAS  Google Scholar 

  34. Parks, D.R., Roederer, M. & Moore, W.A. A new “Logicle” display method avoids deceptive effects of logarithmic scaling for low signals and compensated data. Cytometry A 69, 541–551 (2006).

    Article  Google Scholar 

Download references


We thank members of the Davis, Holden Maecker and Garry Nolan labs for sharing advice and experience concerning mass cytometry and antibody clone usage, especially M. Leipold for help with the mass cytometry instrument, and X. He, F. Wen, W. O'Gorman, A. Han, S. Bendall, O. Goldberger and Y.-H. Chien for helpful discussions. This work was supported by the Bill and Melinda Gates Foundation Grand Challenges Exploration phase I and II grants, National Institutes of Health grants U19-AI057229 and U19-AI090019, and The Howard Hughes Medical Institute. E.W.N. was supported by The American Cancer Society's Steven Stanley and Edward Albert Bielfelt Post-Doctoral Fellowship and by funding through the Singapore Immunology Network.

Author information

Authors and Affiliations



E.W.N. conceived and designed the experiments, wrote the manuscript, made and validated CyTOF reagents, wrote analysis scripts, helped with tetramer staining experiments, helped adapt IEL cell preparations for mass cytometry, adapted the epitope prediction algorithm and performed all data analysis. N.S. helped conceive and design the experiments, made and validated CyTOF reagents, made all multiplex tetramer-staining cocktails and performed all tetramer-staining experiments. N.N. optimized the IEL processing procedure for use of IEL samples with mass cytometry and processed all IEL samples for the experiments. B.A.K. adapted the epitope prediction algorithm and performed all epitope predictions. H.B.G. helped conceive and design the experiments. M.M.D. helped conceive and design the experiments, and wrote the manuscript. All authors made corrections and provided critical feedback on the manuscript.

Corresponding authors

Correspondence to Evan W Newell or Mark M Davis.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–7, Supplementary Tables 1–2 and Supplementary Matlab Scripts (PDF 6093 kb)

Supplementary Movie

3D gating example of rotavirus specific cells in one donor (MOV 20002 kb)

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Newell, E., Sigal, N., Nair, N. et al. Combinatorial tetramer staining and mass cytometry analysis facilitate T-cell epitope mapping and characterization. Nat Biotechnol 31, 623–629 (2013).

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI:

This article is cited by


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