Massively parallel interrogation and mining of natively paired human TCRαβ repertoires


T cells engineered to express antigen-specific T cell receptors (TCRs) are potent therapies for viral infections and cancer. However, efficient identification of clinical candidate TCRs is complicated by the size and complexity of T cell repertoires and the challenges of working with primary T cells. Here we present a high-throughput method to identify TCRs with high functional avidity from diverse human T cell repertoires. The approach used massively parallel microfluidics to generate libraries of natively paired, full-length TCRαβ clones, from millions of primary T cells, which were then expressed in Jurkat cells. The TCRαβ–Jurkat libraries enabled repeated screening and panning for antigen-reactive TCRs using peptide major histocompatibility complex binding and cellular activation. We captured more than 2.9 million natively paired TCRαβ clonotypes from six healthy human donors and identified rare (<0.001% frequency) viral-antigen-reactive TCRs. We also mined a tumor-infiltrating lymphocyte sample from a patient with melanoma and identified several tumor-specific TCRs, which, after expression in primary T cells, led to tumor cell killing.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.

Fig. 1: Overview of the massively parallel TCRαβ repertoire mining technology.
Fig. 2: Bioinformatic analysis of six paired TCRαβ repertoires.
Fig. 3: Identification of virus-specific TCRαβ clones by library panning and cellular activation screens.
Fig. 4: Experimental validation of viral-specific TCRαβ clones.
Fig. 5: Identification and experimental validation of antitumor antigen TCRαβ clones from TILs.

Data availability

Presort TCRα–TCRβ repertoire fastq sequence files are deposited at the Sequence Read Archive (, under BioProject ID PRJNA541985. All other data are available from the corresponding author upon reasonable request.


  1. 1.

    Yee, C. Adoptive T cell therapy: points to consider. Curr. Opin. Immunol. 51, 197–203 (2018).

    CAS  PubMed  Google Scholar 

  2. 2.

    Barrett, A. J., Prockop, S. & Bollard, C. M. Virus-specific T cells: broadening applicability. Biol. Blood Marrow Transplant. 24, 13–18 (2018).

    CAS  PubMed  Google Scholar 

  3. 3.

    Romano, M., Fanelli, G., Albany, C. J., Giganti, G. & Lombardi, G. Past, present, and future of regulatory T cell therapy in transplantation and autoimmunity. Front. Immunol. 10, 43 (2019).

    CAS  PubMed  PubMed Central  Google Scholar 

  4. 4.

    Chodon, T. et al. Adoptive transfer of MART-1 T-cell receptor transgenic lymphocytes and dendritic cell vaccination in patients with metastatic melanoma. Clin. Cancer Res. 20, 2457–2465 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  5. 5.

    Rapoport, A. P. et al. NY-ESO-1-specific TCR-engineered T cells mediate sustained antigen-specific antitumor effects in myeloma. Nat. Med. 21, 914–921 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  6. 6.

    Tendeiro Rego, R., Morris, E. C. & Lowdell, M. W. T-cell receptor gene-modified cells: past promises, present methodologies and future challenges. Cytotherapy 21, 341–357 (2019).

    CAS  PubMed  Google Scholar 

  7. 7.

    Zhang, J. & Wang, L. The emerging world of TCR-T cell trials against cancer: a systematic review. Technol. Cancer Res. Treat. 18, 1533033819831068 (2019).

    CAS  PubMed  PubMed Central  Google Scholar 

  8. 8.

    Sadelain, M., Rivière, I. & Riddell, S. Therapeutic T cell engineering. Nature 545, 423–431 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  9. 9.

    Dawson, N. A. J. & Levings, M. K. Antigen-specific regulatory T cells: are police CARs the answer? Transl. Res. 187, 53–58 (2017).

    CAS  PubMed  Google Scholar 

  10. 10.

    Linnemann, C. et al. High-throughput identification of antigen-specific TCRs by TCR gene capture. Nat. Med. 19, 1534–1541 (2013).

    CAS  PubMed  Google Scholar 

  11. 11.

    Scheper, W. et al. Low and variable tumor reactivity of the intratumoral TCR repertoire in human cancers. Nat. Med. 25, 89–94 (2019).

    CAS  PubMed  Google Scholar 

  12. 12.

    Yossef, R. et al. Enhanced detection of neoantigen-reactive T cells targeting unique and shared oncogenes for personalized cancer immunotherapy. JCI Insight 3, 122467 (2018).

    PubMed  Google Scholar 

  13. 13.

    Hu, Z. et al. A cloning and expression system to probe T-cell receptor specificity and assess functional avidity to neoantigens. Blood 132, 1911–1921 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  14. 14.

    Li, Y. et al. Directed evolution of human T-cell receptors with picomolar affinities by phage display. Nat. Biotechnol. 23, 349–354 (2005).

    CAS  PubMed  Google Scholar 

  15. 15.

    Wagner, E. K. et al. Human cytomegalovirus-specific T cell receptor engineered for high affinity and soluble expression using mammalian cell display. J. Biol. Chem. 294, 5790–5804 (2019).

  16. 16.

    Border, E. C., Sanderson, J. P., Weissensteiner, T., Gerry, A. B. & Pumphrey, N. J. Affinity-enhanced T-cell receptors for adoptive T-cell therapy targeting MAGE-A10: strategy for selection of an optimal candidate. Oncoimmunology 8, e1532759 (2019).

    PubMed  Google Scholar 

  17. 17.

    Linette, G. P. et al. Cardiovascular toxicity and titin cross-reactivity of affinity-enhanced T cells in myeloma and melanoma. Blood 122, 863–871 (2015).

    Google Scholar 

  18. 18.

    Guo, X.-Z. J. et al. Rapid cloning, expression, and functional characterization of paired αβ and γδ T-cell receptor chains from single-cell analysis. Mol. Ther. Methods Clin. Dev. 3, 15054 (2016).

    PubMed  PubMed Central  Google Scholar 

  19. 19.

    Azizi, E. et al. Single-cell map of diverse immune phenotypes in the breast tumor microenvironment. Cell 174, 1293–1308 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  20. 20.

    Adler, A. S. et al. Rare, high-affinity anti-pathogen antibodies from human repertoires, discovered using microfluidics and molecular genomics. mAbs 9, 1282–1296 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  21. 21.

    Wang, B. et al. Functional interrogation and mining of natively paired human VH:VL antibody repertoires. Nat. Biotechnol. 36, 152–155 (2018).

    PubMed  PubMed Central  Google Scholar 

  22. 22.

    Kieke, M. C. et al. Selection of functional T cell receptor mutants from a yeast surface-display library. Proc. Natl Acad. Sci. USA 96, 5651–5656 (1999).

    CAS  PubMed  Google Scholar 

  23. 23.

    Smith, S. N., Harris, D. T. & Kranz, D. M. in Yeast Surface Display 1319, 95–141 (Springer, 2015).

  24. 24.

    Kuhns, M. S., Davis, M. M. & Garcia, K. C. Deconstructing the form and function of the TCR/CD3 complex. Immunity 24, 133–139 (2006).

    CAS  PubMed  Google Scholar 

  25. 25.

    Tsuji, T. et al. Rapid construction of antitumor T-cell receptor vectors from frozen tumors for engineered T-cell therapy. Cancer Immunol. Res. 6, 594–604 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  26. 26.

    Shugay, M. et al. Towards error-free profiling of immune repertoires. Nat. Methods 11, 653–655 (2014).

    CAS  PubMed  Google Scholar 

  27. 27.

    Heather, J. M., Ismail, M., Oakes, T. & Chain, B. High-throughput sequencing of the T-cell receptor repertoire: pitfalls and opportunities. Brief. Bioinformatics 19, 554–565 (2018).

    CAS  PubMed  Google Scholar 

  28. 28.

    Exley, M., Porcelli, S., Furman, M., Garcia, J. & Balk, S. CD161 (NKR-P1A) costimulation of CD1d-dependent activation of human T cells expressing invariant Vα24JαQ T cell receptor α chains. J. Exp. Med. 188, 867–876 (1998).

    CAS  PubMed  PubMed Central  Google Scholar 

  29. 29.

    Kjer-Nielsen, L. et al. MR1 presents microbial vitamin B metabolites to MAIT cells. Nature 491, 717–723 (2012).

    CAS  PubMed  Google Scholar 

  30. 30.

    Gold, M. C. et al. MR1-restricted MAIT cells display ligand discrimination and pathogen selectivity through distinct T cell receptor usage. J. Exp. Med. 211, 1601–1610 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  31. 31.

    Dellabona, P., Padovan, E., Casorati, G., Brockhaus, M. & Lanzavecchia, A. An invariant Vα24–JαQ/Vβ11 T cell receptor is expressed in all individuals by clonally expanded CD4-8 T cells. J. Exp. Med. 180, 1171–1176 (1994).

    CAS  PubMed  Google Scholar 

  32. 32.

    Porcelli, S., Yockey, C. E., Brenner, M. B. & Balk, S. P. Analysis of T cell antigen receptor (TCR) expression by human peripheral blood CD4-8− α/β T cells demonstrates preferential use of several Vβ genes and an invariant TCRα chain. J. Exp. Med. 178, 1–16 (1993).

    CAS  PubMed  Google Scholar 

  33. 33.

    Held, K., Beltrán, E., Moser, M., Hohlfeld, R. & Dornmair, K. T-cell receptor repertoire of human peripheral CD161hiTRAV1-2+ MAIT cells revealed by next generation sequencing and single cell analysis. Hum. Immunol. 76, 607–614 (2015).

    CAS  PubMed  Google Scholar 

  34. 34.

    Dash, P. et al. Quantifiable predictive features define epitope-specific T cell receptor repertoires. Nature 547, 89–93 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  35. 35.

    Glanville, J. et al. Identifying specificity groups in the T cell receptor repertoire. Nature 547, 94–98 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  36. 36.

    Sanjana, N. E., Shalem, O. & Zhang, F. Improved vectors and genome-wide libraries for CRISPR screening. Nat. Methods 11, 783–784 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  37. 37.

    Cole, D. K. et al. Germ line-governed recognition of a cancer epitope by an immunodominant human T-cell receptor. J. Biol. Chem. 284, 27281–27289 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  38. 38.

    Trautmann, L. et al. Selection of T cell clones expressing high-affinity public TCRs within human cytomegalovirus-specific CD8 T cell responses. J. Immunol. 175, 6123–6132 (2005).

    CAS  PubMed  Google Scholar 

  39. 39.

    Day, E. K. et al. Rapid CD8+ T cell repertoire focusing and selection of high-affinity clones into memory following primary infection with a persistent human virus: human cytomegalovirus. J. Immunol. 179, 3203–3213 (2007).

    CAS  PubMed  Google Scholar 

  40. 40.

    Yang, X. et al. Structural basis for clonal diversity of the public T cell response to a dominant human cytomegalovirus epitope. J. Biol. Chem. 290, 29106–29119 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  41. 41.

    Sibener, L. V. et al. Isolation of a structural mechanism for uncoupling T cell receptor signaling from peptide–MHC binding. Cell 174, 672–687.e27 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  42. 42.

    Birnbaum, M. E. et al. Deconstructing the peptide–MHC specificity of T cell recognition. Cell 157, 1073–1087 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  43. 43.

    Rosenberg, S. A. et al. Durable complete responses in heavily pretreated patients with metastatic melanoma using T-cell transfer immunotherapy. Clin. Cancer Res. 17, 4550–4557 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  44. 44.

    Shugay, M. et al. VDJdb: a curated database of T-cell receptor sequences with known antigen specificity. Nucleic Acids Res. 46, D419–D427 (2018).

    CAS  PubMed  Google Scholar 

  45. 45.

    Yost, K. E. et al. Clonal replacement of tumor-specific T cells following PD-1 blockade. Nat. Med. 25, 1251–1259 (2019).

    CAS  PubMed  PubMed Central  Google Scholar 

  46. 46.

    Gerdemann, U. et al. Rapidly generated multivirus-specific cytotoxic T lymphocytes for the prophylaxis and treatment of viral infections. Mol. Ther. 20, 1622–1632 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  47. 47.

    Barrett, A. J. & Bollard, C. M. The coming of age of adoptive T-cell therapy for viral infection after stem cell transplantation. Ann. Transl. Med. 3, 62 (2015).

    PubMed  PubMed Central  Google Scholar 

  48. 48.

    O’Leary, M. C. et al. FDA approval summary: tisagenlecleucel for treatment of patients with relapsed or refractory B-cell precursor acute lymphoblastic leukemia. Clin. Cancer Res. 25, 1142–1146 (2019).

    PubMed  Google Scholar 

  49. 49.

    Bouchkouj, N. et al. FDA approval summary: axicabtagene ciloleucel for relapsed or refractory large B-cell lymphoma. Clin. Cancer Res. 25, 1702–1708 (2019).

    PubMed  Google Scholar 

  50. 50.

    Edgar, R. C. & Flyvbjerg, H. Error filtering, pair assembly and error correction for next-generation sequencing reads. Bioinformatics 31, 3476–3482 (2015).

    CAS  PubMed  Google Scholar 

  51. 51.

    Lefranc, M.-P. et al. IMGT, the international ImMunoGeneTics information system. Nucleic Acids Res. 37, D1006–D1012 (2009).

    CAS  PubMed  Google Scholar 

  52. 52.

    Edgar, R. C. Search and clustering orders of magnitude faster than BLAST. Bioinformatics 26, 2460–2461 (2010).

    CAS  Google Scholar 

  53. 53.

    Exley, M., Garcia, J., Balk, S. P. & Porcelli, S. Requirements for CD1d recognition by human invariant Vα24+ CD4-CD8 T cells. J. Exp. Med. 186, 109–120 (1997).

    CAS  PubMed  PubMed Central  Google Scholar 

  54. 54.

    Han, M. et al. Invariant or highly conserved TCRα are expressed on double-negative (CD3+CD4CD8) and CD8+ T cells. J Immunol 163, 301–311 (1999).

    CAS  PubMed  Google Scholar 

  55. 55.

    Démoulins, T., Gachelin, G., Bequet, D. & Dormont, D. A biased Vα24+ T-cell repertoire leads to circulating NKT-cell defects in a multiple sclerosis patient at the onset of his disease. Immunol. Lett. 90, 223–228 (2003).

    PubMed  Google Scholar 

  56. 56.

    Greenaway, H. Y. et al. NKT and MAIT invariant TCRα sequences can be produced efficiently by VJ gene recombination. Immunobiology 218, 213–224 (2013).

    CAS  PubMed  Google Scholar 

  57. 57.

    Van Rhijn, I. et al. A conserved human T cell population targets mycobacterial antigens presented by CD1β. Nat. Immunol. 14, 706–713 (2013).

    PubMed  PubMed Central  Google Scholar 

  58. 58.

    Lepore, M. et al. Parallel T-cell cloning and deep sequencing of human MAIT cells reveal stable oligoclonal TCRβ repertoire. Nat. Commun. 5, 3866 (2014).

    PubMed  Google Scholar 

  59. 59.

    Funston, G. M., Kallioinen, S. E., de Felipe, P., Ryan, M. D. & Iggo, R. D. Expression of heterologous genes in oncolytic adenoviruses using picornaviral 2A sequences that trigger ribosome skipping. J. Gen. Virol. 89, 389–396 (2008).

    CAS  PubMed  Google Scholar 

  60. 60.

    Lyons, G. E. et al. Influence of human CD8 on antigen recognition by T-cell receptor-transduced cells. Cancer Res. 66, 11455–11461 (2006).

    CAS  PubMed  Google Scholar 

  61. 61.

    Thakral, D., Dobbins, J., Devine, L. & Kavathas, P. B. Differential expression of the human CD8β splice variants and regulation of the M-2 isoform by ubiquitination. J. Immunol. 180, 7431–7442 (2008).

    CAS  PubMed  Google Scholar 

  62. 62.

    Zufferey, R. et al. Self-inactivating lentivirus vector for safe and efficient in vivo gene delivery. J. Virol. 72, 9873–9880 (1998).

    CAS  PubMed  PubMed Central  Google Scholar 

  63. 63.

    Han, A., Glanville, J., Hansmann, L. & Davis, M. M. Linking T-cell receptor sequence to functional phenotype at the single-cell level. Nat. Biotechnol. 32, 684–692 (2014).

  64. 64.

    Chheda, Z. S. et al. Novel and shared neoantigen derived from histone 3 variant H3.3K27M mutation for glioma T cell therapy. J. Exp. Med. 215, 141–157 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  65. 65.

    Goff, S. L. et al. Enhanced receptor expression and in vitro effector function of a murine–human hybrid MART-1-reactive T cell receptor following a rapid expansion. Cancer Immunol. Immunother. 59, 1551–1560 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

Download references


This work was partially funded by National Institute of Allergy and Infectious Diseases grant R43AI120313-01 to D.S.J. and National Cancer Institute grant R43CA232942 to M.J.S. N.O. is supported with funding from the Medical Research Council, Engineering and Physical Sciences Research Council Centre for Doctoral Training (EP/L014904/1). R. Mizrahi, M. Adams, R. Leong and J. Leong (GigaGen) assisted with the development of Gibson assembly protocols. E. Stone and S. Keating (GigaGen) provided useful discussions about general T cell immunology. R. Guest (Immetacyte) provided assistance with isolation and expansion of TILs.

Author information




Conceptualization: M.J.S., J.S.B., A.S.A., E.H.M., R.E.H., M.C. and D.S.J.; methodology: M.J.S., E.K.W., J.M.H., N.O., J.S.B., A.S.A., M.A.A., E.H.M. and D.S.J.; software: R.C.E. and Y.W.L.; validation: M.J.S., A.L.N. and E.K.W.; investigation: M.J.S., A.L.N., N.O., J.S.B. and E.K.W.; data curation: M.J.S., E.K.W., J.M.H., Y.W.L., A.S.A. and D.S.J.; writing—original draft preparation: M.J.S. and D.S.J.; writing—review and editing: M.J.S., E.K.W., N.O., J.S.B., J.M.H., A.S.A., Y.W.L., E.H.M., M.C. and D.S.J.; visualization: M.J.S., E.K.W., J.M.H., Y.W.L., A.S.A. and D.S.J.; supervision: M.J.S., J.S.B., A.S.A. and D.S.J.; project administration: M.J.S. and D.S.J.; funding acquisition: M.J.S. and D.S.J.

Corresponding author

Correspondence to David S. Johnson.

Ethics declarations

Competing interests

M.J.S., A.L.N., E.K.W., A.S.A., M.A.A., Y.W.L., R.C.E. and D.S.J. are salaried employees of GigaGen, which is an affiliate of GigaMune. GigaMune pays cash to GigaGen for research services. M.J.S., A.L.N., E.K.W., A.S.A., M.A.A., R.C.E., Y.W.L., E.H.M., M.C. and D.S.J. are holders of equity shares in GigaMune. J.M.H. and M.C. hold research positions at the Massachusetts General Hospital. Massachusetts General Hospital has entered into a research collaboration with GigaMune. M.C. is currently an employee of AstraZeneca. M.C. owns equity in Revitope Oncology and Gritstone Oncology. M.C. received consultant fees from Merck Laboratories. J.S.B. and R.E.H. are salaried employees of Immetacyte. R.E.H. is a holder of equity shares in Immetacyte. The viral TCRs and TCR repertoire mining methods are described in US Patent and Trademark Office (USPTO) provisional patent application 62/821808, assigned to GigaMune (M.J.S., A.L.N., E.K.W., Y.W.L., A.S.A., M.A.A. and D.S.J.). The PMEL TCRs are described in USPTO provisional patent application 62/842691, assigned to GigaMune (M.J.S., A.S.A., M.A.A. and D.S.J.). Methods for generating TCR libraries are described in patents WO2012083225A2, US20160362470A1, US20170247684A1 and US20170247683A1, assigned to GigaGen or GigaMune (M.J.S., A.S.A., E.H.M. and D.S.J.).

Additional information

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary information

Supplementary Information

Supplementary Figs. 1–41.

Reporting Summary

Supplementary Table 1

Summary of HLA types, CMV and EBV status, number of input cells, number of sequencing reads and number of clonotypes for each of the seven libraries.

Supplementary Table 2

Summary of sequencing data for primary T cells, linked TCR libraries, cloned TCR libraries and Jurkat TCR libraries for each of the seven libraries generated in the study.

Supplementary Table 3

Summary of the MHC dextramers used in the Jurkat panning experiments.

Supplementary Table 4

CDR3, V-gene and J-gene sequences for each TCR identified in the Jurkat panning experiments, plus their frequencies in the initial primary T cell sequencing data.

Supplementary Table 5

CDR3, V-gene and J-gene sequences for each TCR identified in the Jurkat panning experiments, plus their frequencies in the initial primary T cell sequencing data and at each step of the Jurkat panning experiments. Also included are MHC dextramer-binding and Jurkat activation validation data for monoclonal TCR constructs.

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Spindler, M.J., Nelson, A.L., Wagner, E.K. et al. Massively parallel interrogation and mining of natively paired human TCRαβ repertoires. Nat Biotechnol 38, 609–619 (2020).

Download citation