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High-throughput determination of the antigen specificities of T cell receptors in single cells

Nature Biotechnology volume 36, pages 11561159 (2018) | Download Citation

Abstract

We present tetramer-associated T-cell receptor sequencing (TetTCR-seq) to link T cell receptor (TCR) sequences to their cognate antigens in single cells at high throughput. Binding is determined using a library of DNA-barcoded antigen tetramers that is rapidly generated by in vitro transcription and translation. We applied TetTCR-seq to identify patterns in TCR cross-reactivity with cancer neoantigens and to rapidly isolate neoantigen-specific TCRs with no cross-reactivity to the wild-type antigen.

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Acknowledgements

We thank B. Wendel for discussions and for producing recombinant HLA-A2; M.M. Davis and H. Huang at Stanford University for discussion of the lentiviral transduction protocol and providing a template TCR construct and HLA-A2 construct; W. Uckert at the Max Delbruck Center for Molecular Medicine for sharing the Jurkat 76 cell line; A. Brock at University of Texas Austin for sharing the HEK 293T cell line; J. Lou at the Institute of Biophysics, Chinese Academy of Sciences, for helping with HCV APL prediction; P. Parker, K. Patel and H. Pan for assistance with initial prototyping; and the NIH tetramer center for additional pMHC tetramer reagents. We also thank anonymous blood donors and staff members at We Are Blood for sample collection. This work was supported by NIH grants R00AG040149 (N.J.), S10OD020072 (N.J.) and R33CA225539 (N.J.), by NSF CAREER Award 1653866 (N.J.), by Welch Foundation grant F1785 (N.J.), by the Robert J. Kleberg, Jr. and Helen C. Kleberg Foundation (N.J.) and by National Natural Science Foundation of China major international (regional) joint research project 81220108006 (W.J.) and NSFC-NHMRC joint research grant 81561128016 (W.J.). N.J. is a Cancer Prevention and Research Institute of Texas (CPRIT) Scholar and a Damon Runyon-Rachleff Innovator. S.-Q.Z. is a recipient of Thrust 2000 – Archie W. Straiton Endowed Graduate Fellowship in Engineering No. 1. A.A.S. is a recipient of the Cockrell School of Engineering fellowship and the Thrust 2000 – Mario E. Ramirez Endowed Graduate Fellowship in Engineering.

Author information

Author notes

    • Shu-Qi Zhang
    • , Ke-Yue Ma
    • , Alexandra A Schonnesen
    •  & Mingliang Zhang

    These authors contributed equally to this work.

Affiliations

  1. McKetta Department of Chemical Engineering, University of Texas at Austin, Austin, Texas, USA.

    • Shu-Qi Zhang
  2. Institute for Cellular and Molecular Biology, University of Texas at Austin, Austin, Texas, USA.

    • Ke-Yue Ma
    •  & Ning Jiang
  3. Department of Biomedical Engineering, University of Texas at Austin, Austin, Texas, USA.

    • Alexandra A Schonnesen
    • , Chenfeng He
    • , Eric Sun
    • , Chad M Williams
    •  & Ning Jiang
  4. Department of Endocrinology and Metabolism, Shanghai Jiao Tong University Affiliated Sixth People's Hospital, Shanghai, China.

    • Mingliang Zhang
    •  & Weiping Jia
  5. Shanghai Key Laboratory of Diabetes Mellitus, Shanghai Clinical Center of Diabetes, Shanghai, China.

    • Mingliang Zhang
    •  & Weiping Jia
  6. LIVESTRONG Cancer Institutes, Dell Medical School, University of Texas at Austin, Austin, Texas, USA.

    • Ning Jiang

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Contributions

S.-Q.Z. conceived and developed the technology platform. S.-Q.Z and N.J. conceived and designed the study. S.-Q.Z. and K.-Y.M. designed, performed and analyzed data for the majority of experiments; A.A.S. and M.Z. performed TCR cloning, transduction and pMHC tetramer staining studies; C.H. wrote the script for converting sequencing data into TCR sequences, DNA-BC and MIDs, and predicted HCV APLs; C.M.W. and E.S. performed in vitro cell culture and functionality experiments; W.J. cosupervised study and codesigned some experiments; N.J. supervised the study; S.-Q.Z. and N.J. wrote the manuscript with feedback from all authors.

Competing interests

N.J. is a scientific advisor for ImmuDX LLC and Immune Arch Inc. A provisional patent application has been filed by the University of Texas at Austin for the method described here.

Corresponding authors

Correspondence to Weiping Jia or Ning Jiang.

Supplementary information

PDF files

  1. 1.

    Supplementary Text and Figures

    Supplementary Note and Supplementary Figures 1–20

  2. 2.

    Life Sciences Reporting Summary

Excel files

  1. 1.

    Supplementary Table 1

    Peptide library name, sequence, and fluorescent encoding for each of the six main experiments

  2. 2.

    Supplementary Table 2

    TetTCR-seq summary for experiment 1

  3. 3.

    Supplementary Table 3

    TetTCR-Seq summary for experiment 2

  4. 4.

    Supplementary Table 4

    Description of neoantigen and wild-type peptides used for experiments 3 and 4

  5. 5.

    Supplementary Table 5

    TetTCR-seq summary for experiment 3

  6. 6.

    Supplementary Table 6

    TetTCR-seq summary for experiment 4

  7. 7.

    Supplementary Table 7

    Description of neoantigen and wild-type peptides used for experiments 5 and 6

  8. 8.

    Supplementary Table 8

    TetTCR-seq summary for experiment 5

  9. 9.

    Supplementary Table 9

    TetTCR-seq summary for experiment 6

  10. 10.

    Supplementary Table 10

    Primer and DNA template sequences for generating DNA-BC pMHC tetramers and amplification of the DNA-BC

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DOI

https://doi.org/10.1038/nbt.4282