Abstract
Although each T lymphocyte expresses a T-cell receptor (TCR) that recognizes cognate antigen and controls T-cell activation, different T cells bearing the same TCR can be functionally distinct. Each TCR is a heterodimer, and both α- and β-chains contribute to determining TCR antigen specificity. Here we present a methodology enabling integration of information about TCR specificity with information about T cell function. This method involves sequencing of TCRα and TCRβ genes, and amplifying functional genes characteristic of different T cell subsets, in single T cells. Because this approach retains information about individual TCRα-TCRβ pairs, TCRs of interest can be expressed and used in functional studies, for antigen discovery, or in therapeutic applications. We apply this approach to study the clonal ancestry and differentiation of T lymphocytes infiltrating a human colorectal carcinoma.
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Change history
14 January 2015
In the version of this article initially published, the concentration of the V-region primers in the Online Methods section was given as 0.6 μM. The correct concentration is 0.06 μM. The error has been corrected in the HTML and PDF versions of the article.
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Acknowledgements
We thank members of the Davis laboratory and the Y.-H. Chien laboratory for helpful discussions. We thank E. Newell for critical reading of the manuscript and helpful suggestions. We thank C. Bolen for assistance with data analysis. We thank X. Ji for deep sequencing. Tissue was obtained through the Stanford University Tissue Bank. The Stanford Shared FACS Facility provided use of equipment and the Stanford Functional Genomics Facility provided deep sequencing services. A.H. is supported by a grant from the Simons Foundation. L.H. is supported by a fellowship from the German Research Foundation (D.F.G.). M.M.D. is funded by the US National Institutes of Health and is an investigator of the Howard Hughes Medical Institute.
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Contributions
A.H. conceived the project and experiments, designed methodology and primers, performed experiments, analyzed data, assisted in the optimization of the software pipeline, generated figures and wrote the manuscript. J.G. designed the software, analyzed data and generated figures. L.H. designed and performed experiments, analyzed data and generated figures. M.M.D. conceived the project and experiments and wrote the manuscript.
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Supplementary information
Supplementary Text and Figures
Supplementary Figures 1–8, Supplementary Tables 1, 3, 5, 7 and Supplementary Note (PDF 22832 kb)
Supplementary Table 2
Penotyping primers for first two PCR reactions (XLSX 57 kb)
Supplementary Table 4
TCR sequences from the TCR validation panel (XLSX 62 kb)
Supplementary Table 6
Reads counts per well of each phenotyping parameter illustrated in Fig. 2. (XLSX 119 kb)
Supplementary Table 8
Reads counts per well of each phenotyping parameter illustrated in Fig. 2. (XLSX 108 kb)
Supplementary Table 9
Paired TCR alpha/beta sequences for 309 CD4+ T cells from adjacent colon for which a TCR beta chain was obtained. (XLSX 71 kb)
Supplementary Table 10
Reads counts per well of each tumor CD4+ T cell analyzed. (XLSX 106 kb)
Supplementary Table 11
Reads counts per well of each adjacent colon CD4+ T cell analyzed. (XLSX 89 kb)
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Han, A., Glanville, J., Hansmann, L. et al. Linking T-cell receptor sequence to functional phenotype at the single-cell level. Nat Biotechnol 32, 684–692 (2014). https://doi.org/10.1038/nbt.2938
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DOI: https://doi.org/10.1038/nbt.2938
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