The transfer of T cell receptor (TCR) genes into patient T cells is a promising approach for the treatment of both viral infections and cancer. Although efficient methods exist to identify antibodies for the treatment of these diseases, comparable strategies to identify TCRs have been lacking. We have developed a high-throughput DNA-based strategy to identify TCR sequences by the capture and sequencing of genomic DNA fragments encoding the TCR genes. We establish the value of this approach by assembling a large library of cancer germline tumor antigen–reactive TCRs. Furthermore, by exploiting the quantitative nature of TCR gene capture, we show the feasibility of identifying antigen-specific TCRs in oligoclonal T cell populations from either human material or TCR-humanized mice. Finally, we demonstrate the ability to identify tumor-reactive TCRs within intratumoral T cell subsets without knowledge of antigen specificities, which may be the first step toward the development of autologous TCR gene therapy to target patient-specific neoantigens in human cancer.

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We are grateful to A. Pfauth, F. van Diepen and B. Hooibrink for assistance with flow cytometry and to M. Nagasawa, T. Heidebrecht, E. Siteur-van Rijnstra, K. Weijer, M. Böhne, M. van der Maas, W. van de Kasteele and T. de Jong for technical assistance. The Bloemenhove Clinic (Heemstede, The Netherlands) provided fetal tissues. We thank A. Kaiser, S. Naik and J. Rohr for critical discussions. C.L. is a fellow in the PhD Fellowship Program of Boehringer Ingelheim Fonds – Foundation for Basic Research in Biomedicine. M.A.T., I.Z.M., D.A.B. and D.M.C. are supported by the Molecular and Cell Biology program of the Russian Academy of Sciences RFBR 12-04-33139 and 12-04-00229-a. G.M.B. is the recipient of a Leukaemia and Lymphoma Research Bennett Senior Non-clinical Fellowship (12004). T.B. is supported by the Deutsche Forschungsgemeinschaft through Sonderforschungsbereich TR36. This work was supported by grants from the Dutch Cancer Society (NKI 2009-4282 to T.N.M.S., J.B.A.G.H. and G.M.B. and NKI 2006-3530 to T.N.M.S. and H.S.) and the Danish Council for Strategic Research (09-065152 to S.R.H. and T.N.M.S.).

Author information

Author notes

    • Nicolas Legrand
    •  & Gavin M Bendle

    Present addresses: AXENIS, Institut Pasteur, Paris, France (N.L.) and School of Cancer Sciences, University of Birmingham, Birmingham, UK (G.M.B.).

    • Bianca Heemskerk
    •  & Pia Kvistborg

    These authors contributed equally to this work.

    • Gavin M Bendle
    •  & Ton N M Schumacher

    These authors jointly directed this work.


  1. Division of Immunology, The Netherlands Cancer Institute, Amsterdam, The Netherlands.

    • Carsten Linnemann
    • , Bianca Heemskerk
    • , Pia Kvistborg
    • , Kaspar Bresser
    • , Remko Schotte
    • , Samira Michels
    • , Raquel Gomez-Eerland
    • , Chengyi Jenny Shu
    • , Christian U Blank
    • , John B A G Haanen
    • , Gavin M Bendle
    •  & Ton N M Schumacher
  2. Central Genomics Facility, The Netherlands Cancer Institute, Amsterdam, The Netherlands.

    • Roelof J C Kluin
    • , Marja Nieuwland
    • , Arno Velds
    •  & Ron M Kerkhoven
  3. Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Moscow, Russia.

    • Dmitriy A Bolotin
    • , Ilgar Z Mamedov
    • , Maria A Turchaninova
    •  & Dmitriy M Chudakov
  4. Max Delbrück Center for Molecular Medicine, Berlin, Germany.

    • Xiaojing Chen
    •  & Thomas Blankenstein
  5. Institute of Immunology, Charité Campus Benjamin Franklin, Berlin, Germany.

    • Xiaojing Chen
    •  & Thomas Blankenstein
  6. Department of Cell Biology and Histology, Academic Medical Center of the University of Amsterdam, Center for Immunology Amsterdam, Amsterdam, The Netherlands.

    • Remko Schotte
    • , Nicolas Legrand
    •  & Hergen Spits
  7. Laboratory of Experimental Hematology, Department of Hematology, Leiden University Medical Center, Leiden, The Netherlands.

    • Lorenz Jahn
    • , Pleun Hombrink
    •  & Mirjam H M Heemskerk
  8. AIMM Therapeutics, Amsterdam, The Netherlands.

    • Nicolas Legrand
    •  & Hergen Spits
  9. Tytgat Institute for Liver and Intestinal Research, Academic Medical Center of the University of Amsterdam, Amsterdam, The Netherlands.

    • Hergen Spits
  10. Center for Cancer Immune Therapy, Herlev Hospital, Copenhagen, Denmark.

    • Sine Reker Hadrup


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C.L. designed, performed, analyzed and interpreted all experiments and wrote the paper. B.H. designed, performed, analyzed and interpreted experiments for the analysis of bulk antigen-specific T cell responses and T cell subsets in TILs and helped in designing, performing, analyzing and interpreting CG-TCR validation experiments. P.K. identified CG antigen–specific T cell responses and helped in designing, performing, analyzing and interpreting CG-TCR validation experiments. R.J.C.K. and A.V. performed bioinformatic analysis to identify chromosomal rearrangements. X.C. designed, performed, analyzed and interpreted experiments in AaBbDII mice. R.S., N.L. and R.G.-E. designed, performed, analyzed and interpreted experiments with HIS mice. L.J. and P.H. designed, performed, analyzed and interpreted experiments to raise allogeneic TCRs against CD79b. C.J.S. identified CG antigen–specific T cell responses in TILs, contributed to RNA-bait library design and performed initial validations. K.B. and S.M. assisted in TCR validation experiments. M.N. and R.M.K. performed TCR gene capture and Illumina sequencing and analyzed and interpreted data. C.U.B. and J.B.A.G.H. provided patient material. M.A.T., I.Z.M., D.A.B. and D.M.C. developed the CDR3 identification algorithm, performed bioinformatic analysis to identify CDR3s and analyzed and interpreted data. H.S. designed and interpreted experiments in HIS mice. S.R.H. identified CG antigen–specific T cell responses and provided patient material. M.H.M.H. designed and interpreted experiments to raise allogeneic TCRs against CD79b. T.B. designed and interpreted experiments in AaBbDII mice. G.M.B. co-supervised the project, designed, performed, analyzed and interpreted all experiments and wrote the paper. T.N.M.S. developed the concept of TCR gene capture, supervised the project, designed and interpreted all experiments and wrote the paper.

Competing interests

The authors declare no competing financial interests.

Corresponding authors

Correspondence to Gavin M Bendle or Ton N M Schumacher.

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