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

Abstract

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.

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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.

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Acknowledgements

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.

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Authors and Affiliations

Authors

Contributions

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.

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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)

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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). https://doi.org/10.1038/nbt.2593

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