Article | Published:

Immunosequencing identifies signatures of cytomegalovirus exposure history and HLA-mediated effects on the T cell repertoire

Nature Genetics volume 49, pages 659665 (2017) | Download Citation


An individual's T cell repertoire dynamically encodes their pathogen exposure history. To determine whether pathogen exposure signatures can be identified by documenting public T cell receptors (TCRs), we profiled the T cell repertoire of 666 subjects with known cytomegalovirus (CMV) serostatus by immunosequencing. We developed a statistical classification framework that could diagnose CMV status from the resulting catalog of TCRβ sequences with high specificity and sensitivity in both the original cohort and a validation cohort of 120 different subjects. We also confirmed that three of the identified CMV-associated TCRβ molecules bind CMV in vitro, and, moreover, we used this approach to accurately predict the HLA-A and HLA-B alleles of most subjects in the first cohort. As all memory T cell responses are encoded in the common format of somatic TCR recombination, our approach could potentially be generalized to a wide variety of disease states, as well as other immunological phenotypes, as a highly parallelizable diagnostic strategy.

Access optionsAccess options

Rent or Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.


  1. 1.

    , , , & Most α/β T cell receptor diversity is due to terminal deoxynucleotidyl transferase. J. Exp. Med. 194, 1385–1390 (2001).

  2. 2.

    & T-cell antigen receptor genes and T-cell recognition. Nature 334, 395–402 (1988).

  3. 3.

    et al. A direct estimate of the human αβ T cell receptor diversity. Science 286, 958–961 (1999).

  4. 4.

    , , , & High frequency of herpesvirus-specific clonotypes in the human T cell repertoire can remain stable over decades with minimal turnover. J. Virol. 87, 697–700 (2013).

  5. 5.

    et al. Comprehensive assessment of T-cell receptor β-chain diversity in αβ T cells. Blood 114, 4099–4107 (2009).

  6. 6.

    et al. Overlap and effective size of the human CD8+ T cell receptor repertoire. Sci. Transl. Med. 2, 47ra64 (2010).

  7. 7.

    , , & The molecular basis for public T-cell responses? Nat. Rev. Immunol. 8, 231–238 (2008).

  8. 8.

    , , & Determinants of public T cell responses. Cell Res. 22, 33–42 (2012).

  9. 9.

    & Immunophenotyping of normal lymphocytes. Clin. Lab. Haematol. 16, 21–32 (1994).

  10. 10.

    et al. Lymphocyte subset reference ranges in adult Caucasians. Clin. Immunol. Immunopathol. 60, 190–208 (1991).

  11. 11.

    & Controlling cytomegalovirus: helping the immune system take the lead. Viruses 6, 2242–2258 (2014).

  12. 12.

    & Human cytomegalovirus: clinical aspects, immune regulation, and emerging treatments. Lancet Infect. Dis. 4, 725–738 (2004).

  13. 13.

    On the interpretation of χ2 from contingency tables, and the calculation of P. J. R. Stat. Soc. 85, 87–94 (1922).

  14. 14.

    et al. TCR-β repertoire analysis of antigen-specific single T cells using a high-density microcavity array. Biotechnol. Bioeng. 106, 311–318 (2010).

  15. 15.

    et al. Clonotype analysis of cytomegalovirus-specific cytotoxic T lymphocytes. J. Am. Soc. Nephrol. 20, 344–352 (2009).

  16. 16.

    et al. Predictable αβ T-cell receptor selection toward an HLA-B*3501-restricted human cytomegalovirus epitope. J. Virol. 81, 7269–7273 (2007).

  17. 17.

    et al. The impact of a large and frequent deletion in the human TCR β locus on antiviral immunity. J. Immunol. 188, 2742–2748 (2012).

  18. 18.

    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).

  19. 19.

    et al. TCR repertoire analysis by next generation sequencing allows complex differential diagnosis of T cell–related pathology. Am. J. Transplant. 13, 2842–2854 (2013).

  20. 20.

    et al. Cytomegalovirus-specific CD8+ T cells targeting different peptide/HLA combinations demonstrate varying T-cell receptor diversity. Immunology 135, 27–39 (2012).

  21. 21.

    et al. Characterization of antigen-specific repertoire diversity following in vitro restimulation by a recombinant adenovirus expressing human cytomegalovirus pp65. Eur. J. Immunol. 33, 760–768 (2003).

  22. 22.

    et al. CMV-specific T cells generated from naïve T cells recognize atypical epitopes and may be protective in vivo. Sci. Transl. Med. 7, 285ra63 (2015).

  23. 23.

    et al. Efficiency of T-cell receptor expression in dual-specific T cells is controlled by the intrinsic qualities of the TCR chains within the TCR–CD3 complex. Blood 109, 235–243 (2007).

  24. 24.

    et al. Clonotype selection and composition of human CD8 T cells specific for persistent herpes viruses varies with differentiation but is stable over time. J. Immunol. 183, 319–331 (2009).

  25. 25.

    et al. Clonotype and repertoire changes drive the functional improvement of HIV-specific CD8 T cell populations under conditions of limited antigenic stimulation. J. Immunol. 188, 1156–1167 (2012).

  26. 26.

    , , & Comparative analysis of CD8+ T cell responses against human cytomegalovirus proteins pp65 and immediate early 1 shows similarities in precursor frequency, oligoclonality, and phenotype. J. Infect. Dis. 185, 1025–1034 (2002).

  27. 27.

    et al. Cytomegalovirus seropositivity drives the CD8 T cell repertoire toward greater clonality in healthy elderly individuals. J. Immunol. 169, 1984–1992 (2002).

  28. 28.

    et al. Deep sequencing of antiviral T-cell responses to HCMV and EBV in humans reveals a stable repertoire that is maintained for many years. PLoS Pathog. 8, e1002889 (2012).

  29. 29.

    et al. Combining next-generation sequencing and immune assays: a novel method for identification of antigen-specific T cells. PLoS One 8, e74231 (2013).

  30. 30.

    et al. In vitro expansion of antigen-specific CD8+ T cells distorts the T-cell repertoire. J. Immunol. Methods 405, 199–203 (2014).

  31. 31.

    et al. A single TCRα-chain with dominant peptide recognition in the allorestricted HER2/neu-specific T cell repertoire. J. Immunol. 184, 1617–1629 (2010).

  32. 32.

    et al. Large TCR diversity of virus-specific CD8 T cells provides the mechanistic basis for massive TCR renewal after antigen exposure. J. Immunol. 186, 7039–7049 (2011).

  33. 33.

    et al. Single-cell T-cell receptor-β analysis of HLA-A*2402-restricted CMV-pp65-specific cytotoxic T-cells in allogeneic hematopoietic SCT. Bone Marrow Transplant. 49, 87–94 (2014).

  34. 34.

    et al. Recognition of distinct cross-reactive virus-specific CD8+ T cells reveals a unique TCR signature in a clinical setting. J. Immunol. 192, 5039–5049 (2014).

  35. 35.

    et al. Characterization of human cytomegalovirus peptide–specific CD8+ T-cell repertoire diversity following in vitro restimulation by antigen-pulsed dendritic cells. Blood 99, 213–223 (2002).

  36. 36.

    et al. Avidity for antigen shapes clonal dominance in CD8+ T cell populations specific for persistent DNA viruses. J. Exp. Med. 202, 1349–1361 (2005).

  37. 37.

    et al. Generation of cytomegalovirus-specific human T-lymphocyte clones by using autologous B-lymphoblastoid cells with stable expression of pp65 or IE1 proteins: a tool to study the fine specificity of the antiviral response. J. Virol. 74, 3948–3952 (2000).

  38. 38.

    et al. The transfer of adaptive immunity to CMV during hematopoietic stem cell transplantation is dependent on the specificity and phenotype of CMV-specific T cells in the donor. Blood 114, 5071–5080 (2009).

  39. 39.

    , , & CMV-specific TCR–transgenic T cells for immunotherapy. J. Immunol. 183, 6819–6830 (2009).

  40. 40.

    et al. Cytomegalovirus-specific regulatory and effector T cells share TCR clonality—possible relation to repetitive CMV infections. Am. J. Transplant. 12, 669–681 (2012).

  41. 41.

    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).

  42. 42.

    et al. Persistent survival of prevalent clonotypes within an immunodominant HIV gag-specific CD8+ T cell response. J. Immunol. 186, 359–371 (2011).

  43. 43.

    et al. TCR β-chain sharing in human CD8+ T cell responses to cytomegalovirus and EBV. J. Immunol. 181, 7853–7862 (2008).

  44. 44.

    , , , & T cell receptor αβ diversity inversely correlates with pathogen-specific antibody levels in human cytomegalovirus infection. Sci. Transl. Med. 4, 128ra42 (2012).

  45. 45.

    , , , & The memory cytotoxic T-lymphocyte (CTL) response to human cytomegalovirus infection contains individual peptide-specific CTL clones that have undergone extensive expansion in vivo. J. Virol. 73, 2099–2108 (1999).

  46. 46.

    , , & Long-term stable expanded human CD4+ T cell clones specific for human cytomegalovirus are distributed in both CD45RAhigh and CD45ROhigh populations. J. Immunol. 173, 5843–5851 (2004).

  47. 47.

    et al. Impact of clonal competition for peptide–MHC complexes on the CD8+ T-cell repertoire selection in a persistent viral infection. Blood 111, 4283–4292 (2008).

  48. 48.

    et al. Multiplex identification of antigen-specific T cell receptors using a combination of immune assays and immune receptor sequencing. PLoS One 10, e0141561 (2015).

  49. 49.

    & Selecting and maintaining a diverse T-cell repertoire. Nature 402, 255–262 (1999).

  50. 50.

    , , & Positive and negative selection of the T cell repertoire: what thymocytes see (and don't see). Nat. Rev. Immunol. 14, 377–391 (2014).

  51. 51.

    et al. Impact of TCR reactivity and HLA phenotype on naive CD8 T cell frequency in humans. J. Immunol. 184, 6731–6738 (2010).

  52. 52.

    et al. Role of the MHC restriction during maturation of antigen-specific human T cells in the thymus. Eur. J. Immunol. 46, 560–569 (2016).

  53. 53.

    et al. A structural voyage toward an understanding of the MHC-I-restricted immune response: lessons learned and much to be learned. Immunol. Rev. 250, 61–81 (2012).

  54. 54.

    A very high level of crossreactivity is an essential feature of the T-cell receptor. Immunol. Today 19, 395–404 (1998).

  55. 55.

    et al. A single autoimmune T cell receptor recognizes more than a million different peptides. J. Biol. Chem. 287, 1168–1177 (2012).

  56. 56.

    et al. Allo-HLA reactivity of virus-specific memory T cells is common. Blood 115, 3146–3157 (2010).

  57. 57.

    , , & An alloresponse in humans is dominated by cytotoxic T lymphocytes (CTL) cross-reactive with a single Epstein–Barr virus CTL epitope: implications for graft-versus-host disease. J. Exp. Med. 179, 1155–1161 (1994).

  58. 58.

    , , , & Cross-recognition of HLA DR4 alloantigen by virus-specific CD8+ T cells: a new paradigm for self-/nonself-recognition. Blood 114, 2244–2253 (2009).

  59. 59.

    , , & IMGT/JunctionAnalysis: the first tool for the analysis of the immunoglobulin and T cell receptor complex V–J and V–D–J JUNCTIONs. Bioinformatics 20 (Suppl. 1), i379–i385 (2004).

  60. 60.

    et al. Using synthetic templates to design an unbiased multiplex PCR assay. Nat. Commun. 4, 2680 (2013).

  61. 61.

    & Statistical significance for genomewide studies. Proc. Natl. Acad. Sci. USA 100, 9440–9445 (2003).

  62. 62.

    et al. Dynamics of the cytotoxic T cell response to a model of acute viral infection. J. Virol. 89, 4517–4526 (2015).

Download references


The authors would like to thank M. Chung and other technical staff in the Adaptive Biotechnologies immunosequencing laboratory for their work on this project, S. House for helping compile the list of CMV-reactive TCRβ sequences from the literature, and C. Linkem and K. Boland for assistance with sample tagging for the immuneACCESS project. This work was funded in part by an award from the W.M. Keck Foundation Medical Research Program to H.S.R. and C.S.C.

Author information

Author notes

    • Ryan O Emerson
    •  & William S DeWitt

    These authors contributed equally to this work.

    • Mark Rieder
    •  & Harlan S Robins

    These authors jointly directed this work.


  1. Adaptive Biotechnologies, Seattle, Washington, USA.

    • Ryan O Emerson
    • , William S DeWitt
    • , Marissa Vignali
    • , Joyce K Hu
    • , Edward J Osborne
    • , Cindy Desmarais
    • , Mark Klinger
    • , Mark Rieder
    •  & Harlan S Robins
  2. Computational Biology Program, Fred Hutchinson Cancer Research Center, Seattle, Washington, USA.

    • William S DeWitt
    •  & Harlan S Robins
  3. Fred Hutchinson Cancer Research Center, Seattle, Washington, USA.

    • Jenna Gravley
    • , Christopher S Carlson
    •  & John A Hansen


  1. Search for Ryan O Emerson in:

  2. Search for William S DeWitt in:

  3. Search for Marissa Vignali in:

  4. Search for Jenna Gravley in:

  5. Search for Joyce K Hu in:

  6. Search for Edward J Osborne in:

  7. Search for Cindy Desmarais in:

  8. Search for Mark Klinger in:

  9. Search for Christopher S Carlson in:

  10. Search for John A Hansen in:

  11. Search for Mark Rieder in:

  12. Search for Harlan S Robins in:


J.G. and J.A.H. obtained the DNA samples and determined the CMV status and HLA type of the subjects. R.O.E., C.S.C., M.R., and H.S.R. conceived and designed the experiments. M.R. generated the sequence data. R.O.E., W.S.D., M.V., and C.D. analyzed the results. R.O.E. and W.S.D. performed the statistical analyses. M.V. and C.D. performed the literature searches of CMV-specific TCRs. J.K.H., E.J.O., and M.K. performed and analyzed in vitro confirmation experiments. R.O.E., W.S.D., M.V., M.K., and H.S.R. wrote the manuscript.

Competing interests

H.S.R. has employment, equity ownership, patents, and royalties with Adaptive Biotechnologies, and C.S.C. has consultancy, equity ownership, patents, and royalties with Adaptive Biotechnologies; R.O.E., W.S.D., M.V., C.D., J.K.H., E.J.O., M.K., and M.R. have employment and equity ownership with Adaptive Biotechnologies.

Corresponding author

Correspondence to Ryan O Emerson.

Supplementary information

PDF files

  1. 1.

    Supplementary Text and Figures

    Supplementary Figures 1–4

Excel files

  1. 1.

    Supplementary Table 1

    Detailed demographic and phenotypic information for subjects in Cohorts 1 and 2.

  2. 2.

    Supplementary Table 2

    List of the 164 CMV-associated TCRβs.

  3. 3.

    Supplementary Table 3

    List of 1054 previously published CMV-reactive TCRβs.

  4. 4.

    Supplementary Table 4

    Overlap between CMV-associated TCRβs and previously published, TCR-reactive TCRβs

  5. 5.

    Supplementary Table 5

    List of antigens used in the MIRA experiment.

  6. 6.

    Supplementary Table 6

    Result of the MIRA experiment.

About this article

Publication history





Further reading