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A new cloning and expression system yields and validates TCRs from blood lymphocytes of patients with cancer within 10 days

Nature Medicine volume 19, pages 1542–1546 (2013)Cite this article

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Abstract

Antigen-specific T cell therapy, or T cell receptor (TCR) gene therapy, is a promising immunotherapy for infectious diseases and cancers. However, a suitable rapid and direct screening system for antigen-specific TCRs is not available. Here, we report an efficient cloning and functional evaluation system to determine the antigen specificity of TCR cDNAs derived from single antigen-specific human T cells within 10 d. Using this system, we cloned and analyzed 380 Epstein-Barr virus–specific TCRs from ten healthy donors with latent Epstein-Barr virus infection and assessed the activity of cytotoxic T lymphocytes (CTLs) carrying these TCRs against antigenic peptide–bearing target cells. We also used this system to clone tumor antigen–specific TCRs from peptide-vaccinated patients with cancer. We obtained 210 tumor-associated antigen–specific TCRs and demonstrated the cytotoxic activity of CTLs carrying these TCRs against peptide-bearing cells. This system may provide a fast and powerful approach for TCR gene therapy for infectious diseases and cancers.

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Figure 1: Schematic of the hTEC10 system.
Figure 2: Analysis of EBV-specific human TCRαβ pairs obtained by hTEC10.
Figure 3: hTEC10 analysis of peptide vaccine–specific TCRs from peptide-vaccinated patients with HCC.
Figure 4: Repertoire analysis of cytokine-secreting CD8+ T cells by stimulation with a specific peptide.

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References

  1. Morgan, R.A. et al. Cancer regression in patients after transfer of genetically engineered lymphocytes. Science 314, 126–129 (2006).

    Article  CAS  Google Scholar 

  2. Johnson, L.A. et al. Gene therapy with human and mouse T-cell receptors mediates cancer regression and targets normal tissues expressing cognate antigen. Blood 114, 535–546 (2009).

    Article  CAS  Google Scholar 

  3. Linnemann, C., Schumacher, T.N. & Bendle, G.M. T-cell receptor gene therapy: critical parameters for clinical success. J. Invest. Dermatol. 131, 1806–1816 (2011).

    Article  CAS  Google Scholar 

  4. Uttenthal, B.J., Chua, I., Morris, E.C. & Stauss, H.J. Challenges in T cell receptor gene therapy. J. Gene Med. 14, 386–399 (2012).

    Article  CAS  Google Scholar 

  5. Ozawa, T., Tajiri, K., Kishi, H. & Muraguchi, A. Comprehensive analysis of the functional TCR repertoire at the single-cell level. Biochem. Biophys. Res. Commun. 367, 820–825 (2008).

    Article  CAS  Google Scholar 

  6. Dash, P. et al. Paired analysis of TCRα and TCRβ chains at the single-cell level in mice. J. Clin. Invest. 121, 288–295 (2011).

    Article  CAS  Google Scholar 

  7. Kuzushima, K. et al. Tetramer-assisted identification and characterization of epitopes recognized by HLA A*2402-restricted Epstein-Barr virus-specific CD8+ T cells. Blood 101, 1460–1468 (2003).

    Article  CAS  Google Scholar 

  8. Miyahara, Y. et al. Determination of cellularly processed HLA-A2402–restricted novel CTL epitopes derived from two cancer germ line genes, MAGE-A4 and SAGE. Clin. Cancer Res. 11, 5581–5589 (2005).

    Article  CAS  Google Scholar 

  9. Okamoto, S. et al. Improved expression and reactivity of transduced tumor-specific TCRs in human lymphocytes by specific silencing of endogenous TCR. Cancer Res. 69, 9003–9011 (2009).

    Article  CAS  Google Scholar 

  10. Zhou, J., Dudley, M.E., Rosenberg, S.A. & Robbins, P.F. Selective growth, in vitro and in vivo, of individual T cell clones from tumor-infiltrating lymphocytes obtained from patients with melanoma. J. Immunol. 173, 7622–7629 (2004).

    Article  CAS  Google Scholar 

  11. Mizukoshi, E., Nakamoto, Y., Tsuji, H., Yamashita, T. & Kaneko, S. Identification of α-fetoprotein–derived peptides recognized by cytotoxic T lymphocytes in HLA-A24+ patients with hepatocellular carcinoma. Int. J. Cancer 118, 1194–1204 (2006).

    Article  CAS  Google Scholar 

  12. Lim, A. et al. Frequent contribution of T cell clonotypes with public TCR features to the chronic response against a dominant EBV-derived epitope: application to direct detection of their molecular imprint on the human peripheral T cell repertoire. J. Immunol. 165, 2001–2011 (2000).

    Article  CAS  Google Scholar 

  13. Argaet, V.P. et al. Dominant selection of an invariant T cell antigen receptor in response to persistent infection by Epstein-Barr virus. J. Exp. Med. 180, 2335–2340 (1994).

    Article  CAS  Google Scholar 

  14. Parkhurst, M.R. et al. T cells targeting carcinoembryonic antigen can mediate regression of metastatic colorectal cancer but induce severe transient colitis. Mol. Ther. 19, 620–626 (2011).

    Article  CAS  Google Scholar 

  15. Butterfield, L.H. et al. A phase I/II trial testing immunization of hepatocellular carcinoma patients with dendritic cells pulsed with four α-fetoprotein peptides. Clin. Cancer Res. 12, 2817–2825 (2006).

    Article  CAS  Google Scholar 

  16. Mizukoshi, E. et al. Comparative analysis of various tumor-associated antigen-specific T-cell responses in patients with hepatocellular carcinoma. Hepatology 53, 1206–1216 (2011).

    Article  CAS  Google Scholar 

  17. Ueno, T., Tomiyama, H., Fujiwara, M., Oka, S. & Takiguchi, M. Functionally impaired HIV-specific CD8 T cells show high affinity TCR-ligand interactions. J. Immunol. 173, 5451–5457 (2004).

    Article  CAS  Google Scholar 

  18. Morita, S., Kojima, T. & Kitamura, T. Plat-E: an efficient and stable system for transient packaging of retroviruses. Gene Ther. 7, 1063–1066 (2000).

    Article  CAS  Google Scholar 

  19. Kinsella, T.M. & Nolan, G.P. Episomal vectors rapidly and stably produce high-titer recombinant retrovirus. Hum. Gene Ther. 7, 1405–1413 (1996).

    Article  CAS  Google Scholar 

  20. Mizukoshi, E. et al. Cytotoxic T cell responses to human telomerase reverse transcriptase in patients with hepatocellular carcinoma. Hepatology 43, 1284–1294 (2006).

    Article  CAS  Google Scholar 

  21. Giudicelli, V., Chaume, D. & Lefranc, M.P. IMGT/V-QUEST, an integrated software program for immunoglobulin and T cell receptor V-J and V-D-J rearrangement analysis. Nucleic Acids Res. 32, W435–W440 (2004).

    Article  CAS  Google Scholar 

  22. Onishi, M. et al. Applications of retrovirus-mediated expression cloning. Exp. Hematol. 24, 324–329 (1996).

    CAS  PubMed  Google Scholar 

  23. Ryan, M.D., King, A.M. & Thomas, G.P. Cleavage of foot-and-mouth disease virus polyprotein is mediated by residues located within a 19 amino acid sequence. J. Gen. Virol. 72, 2727–2732 (1991).

    Article  CAS  Google Scholar 

  24. Leisegang, M. et al. Enhanced functionality of T cell receptor-redirected T cells is defined by the transgene cassette. J. Mol. Med. (Berl) 86, 573–583 (2008).

    Article  CAS  Google Scholar 

  25. Goldstein, A. Biostatistics: An Introductory Text (Macmillan, New York, 1964).

  26. Jin, A. et al. A rapid and efficient single-cell manipulation method for screening antigen-specific antibody-secreting cells from human peripheral blood. Nat. Med. 15, 1088–1092 (2009).

    Article  CAS  Google Scholar 

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Acknowledgements

We thank S. Hirota, A. Takishita, K. Shitaoka and M. Horii for technical assistance and K. Hata for secretarial work. This research was supported by grants from the Hokuriku Innovation Cluster for Health Science and a Grant-in-Aid from the Ministry of Education, Culture, Sports, Science and Technology in Japan (11F01415 and 24650630). Retroviral pMX vector, pMX-IRES-EGFP vector and PLAT-E cell line were kindly provided by T. Kitamura (University of Tokyo), human CD8–expressing TG40 cell line by T. Ueno and C. Motozono (Kumamoto University) with permission from T. Saito (Riken), T2-A24 cell line by K. Kuzushima (Aichi Cancer Center Research Institute), Phoenix-A cell line by G. Nolan (Stanford University) and C1R-A24 cell line from M. Takiguchi (Kumamoto University).

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  1. Eiji Kobayashi and Eishiro Mizukoshi: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Immunology, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, Toyama, Japan

    Eiji Kobayashi, Hiroyuki Kishi, Tatsuhiko Ozawa, Hiroshi Hamana, Terumi Nagai, Aishun Jin & Atsushi Muraguchi

  2. Department of Gastroenterology, Graduate School of Medicine, Kanazawa University, Kanazawa, Ishikawa, Japan

    Eishiro Mizukoshi, Hidetoshi Nakagawa & Shuichi Kaneko

  3. College of Basic Medical Science, Harbin Medical University, Harbin, China

    Aishun Jin

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  1. Eiji Kobayashi
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Contributions

E.K., E.M., H.H., T.N. and A.J. performed and analyzed the experiments. E.K., E.M., H.K., S.K., H.N. and A.M. designed the experiments. E.M. and T.O. contributed reagents. E.K., H.K. and A.M. wrote the manuscript, and E.K., E.M., H.K., S.K. and A.M. edited the manuscript.

Corresponding author

Correspondence to Hiroyuki Kishi.

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The authors declare no competing financial interests.

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Supplementary Figures 1–14 and Supplementary Tables 1–5 (PDF 1698 kb)

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Kobayashi, E., Mizukoshi, E., Kishi, H. et al. A new cloning and expression system yields and validates TCRs from blood lymphocytes of patients with cancer within 10 days. Nat Med 19, 1542–1546 (2013). https://doi.org/10.1038/nm.3358

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