In vivo detection of antigen-specific CD8+ T cells by immuno-positron emission tomography


The immune system’s ability to recognize peptides on major histocompatibility molecules contributes to the eradication of cancers and pathogens. Tracking these responses in vivo could help evaluate the efficacy of immune interventions and improve mechanistic understanding of immune responses. For this purpose, we employ synTacs, which are dimeric major histocompatibility molecule scaffolds of defined composition. SynTacs, when labeled with positron-emitting isotopes, can noninvasively image antigen-specific CD8+ T cells in vivo. Using radiolabeled synTacs loaded with the appropriate peptides, we imaged human papillomavirus-specific CD8+ T cells by positron emission tomography in mice bearing human papillomavirus-positive tumors, as well as influenza A virus–specific CD8+ T cells in the lungs of influenza A virus–infected mice. It is thus possible to visualize antigen-specific CD8+ T-cell populations in vivo, which may serve prognostic and diagnostic roles.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.

Fig. 1: SynTac design, validation and labeling strategy.
Fig. 2: Analysis of synTac specificity in vitro.
Fig. 3: Detection of bulk CD8+ T cells.
Fig. 4: PET–CT imaging with the HPV E7 synTac.
Fig. 5: PET–CT imaging with the IAV NP synTac.
Fig. 6: SynTac PET–CT imaging following different radiolabeling strategies.

Data availability

The original PET–CT DICOM files that support the findings of this study are available from the corresponding author upon reasonable request. They are not available from public repositories due to their large size (~1 GB per mouse). Source data are provided with this paper.


  1. 1.

    Rashidian, M. et al. Predicting the response to CTLA-4 blockade by longitudinal noninvasive monitoring of CD8+ T cells. J. Exp. Med. 214, 2243–2255 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  2. 2.

    Wu, A. M. Antibodies and antimatter: the resurgence of immuno-PET. J. Nucl. Med. 50, 2–5 (2009).

    CAS  PubMed  Google Scholar 

  3. 3.

    Tavare, R. et al. An effective immuno-PET imaging method to monitor CD8-dependent responses to immunotherapy. Cancer Res. 76, 73–82 (2016).

    CAS  PubMed  Google Scholar 

  4. 4.

    Dey, S. et al. Tracking antigen-specific T cells: technological advancement and limitations. Biotechnol. Adv. 37, 145–153 (2019).

    CAS  PubMed  Google Scholar 

  5. 5.

    Alam, I. S. et al. Imaging activated T cells predicts response to cancer vaccines. J. Clin. Invest. 128, 2569–2580 (2018).

    PubMed  PubMed Central  Google Scholar 

  6. 6.

    Larimer, B. M. et al. Granzyme B PET imaging as a predictive biomarker of immunotherapy response. Cancer Res. 77, 2318–2327 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  7. 7.

    Gibson, H. M. et al. IFN-γ PET imaging as a predictive tool for monitoring response to tumor immunotherapy. Cancer Res. 78, 5706–5717 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  8. 8.

    Marciscano, A. E. & Thorek, D. L. J. Role of noninvasive molecular imaging in determining response. Adv. Radiat. Oncol. 3, 534–547 (2018).

    PubMed  PubMed Central  Google Scholar 

  9. 9.

    Vyas, J. M., Van der Veen, A. G. & Ploegh, H. L. The known unknowns of antigen processing and presentation. Nat. Rev. Immunol. 8, 607–618 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  10. 10.

    Skeate, J. G., Woodham, A. W., Einstein, M. H., Da Silva, D. M. & Kast, W. M. Current therapeutic vaccination and immunotherapy strategies for HPV-related diseases. Hum. Vaccin. Immunother. 12, 1418–1429 (2016).

    PubMed  PubMed Central  Google Scholar 

  11. 11.

    Sebzda, E. et al. Selection of the T cell repertoire. Annu. Rev. Immunol. 17, 829–874 (1999).

    CAS  PubMed  Google Scholar 

  12. 12.

    Davis, M. M. et al. Ligand recognition by αβ T cell receptors. Annu. Rev. Immunol. 16, 523–544 (1998).

    CAS  PubMed  Google Scholar 

  13. 13.

    Huppa, J. B. & Davis, M. M. T-cell-antigen recognition and the immunological synapse. Nat. Rev. Immunol. 3, 973–983 (2003).

    CAS  PubMed  Google Scholar 

  14. 14.

    Doherty, P. C. The tetramer transformation. J. Immunol. 187, 5–6 (2011).

    CAS  PubMed  Google Scholar 

  15. 15.

    Altman, J. D. et al. Phenotypic analysis of antigen-specific T lymphocytes. Science 274, 94–96 (1996).

    CAS  PubMed  Google Scholar 

  16. 16.

    Duncan, A. R. & Winter, G. The binding site for C1q on IgG. Nature 332, 738–740 (1988).

    CAS  PubMed  Google Scholar 

  17. 17.

    Wines, B. D., Powell, M. S., Parren, P. W., Barnes, N. & Hogarth, P. M. The IgG Fc contains distinct Fc receptor (FcR) binding sites: the leukocyte receptors Fc γ RI and Fc γ RIIa bind to a region in the Fc distinct from that recognized by neonatal FcR and protein A. J. Immunol. 164, 5313–5318 (2000).

    CAS  PubMed  Google Scholar 

  18. 18.

    Feltkamp, M. C. et al. Vaccination with cytotoxic T lymphocyte epitope-containing peptide protects against a tumor induced by human papillomavirus type 16-transformed cells. Eur. J. Immunol. 23, 2242–2249 (1993).

    CAS  PubMed  Google Scholar 

  19. 19.

    Rotzschke, O. et al. Isolation and analysis of naturally processed viral peptides as recognized by cytotoxic T cells. Nature 348, 252–254 (1990).

    CAS  PubMed  Google Scholar 

  20. 20.

    Gallimore, A. et al. A protective cytotoxic T cell response to a subdominant epitope is influenced by the stability of the MHC class I/peptide complex and the overall spectrum of viral peptides generated within infected cells. Eur. J. Immunol. 28, 3301–3311 (1998).

    CAS  PubMed  Google Scholar 

  21. 21.

    Rashidian, M. et al. Noninvasive imaging of immune responses. Proc. Natl Acad. Sci. USA 112, 6146–6151 (2015).

    CAS  PubMed  Google Scholar 

  22. 22.

    Woodham, A. W. et al. Nanobody-antigen conjugates elicit HPV-specific antitumor immune responses. Cancer Immunol. Res. 6, 870–880 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  23. 23.

    Rashidian, M. et al. The use of (18)F-2-fluorodeoxyglucose (FDG) to label antibody fragments for immuno-PET of pancreatic cancer. ACS Cent. Sci. 1, 142–147 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  24. 24.

    Walboomers, J. M. et al. Human papillomavirus is a necessary cause of invasive cervical cancer worldwide. J. Pathol. 189, 12–19 (1999).

    CAS  PubMed  Google Scholar 

  25. 25.

    Stanley, M. A., Pett, M. R. & Coleman, N. HPV: from infection to cancer. Biochem. Soc. Trans. 35, 1456–1460 (2007).

    CAS  PubMed  Google Scholar 

  26. 26.

    Feltkamp, M. C. et al. Cytotoxic T lymphocytes raised against a subdominant epitope offered as a synthetic peptide eradicate human papillomavirus type 16-induced tumors. Eur. J. Immunol. 25, 2638–2642 (1995).

    CAS  PubMed  Google Scholar 

  27. 27.

    Kanodia, S. et al. Expression of LIGHT/TNFSF14 combined with vaccination against human papillomavirus Type 16 E7 induces significant tumor regression. Cancer Res. 70, 3955–3964 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  28. 28.

    Thomas, P. G., Keating, R., Hulse-Post, D. J. & Doherty, P. C. Cell-mediated protection in influenza infection. Emerg. Infect. Dis. 12, 48–54 (2006).

    CAS  PubMed  PubMed Central  Google Scholar 

  29. 29.

    Wu, A. M. Engineered antibodies for molecular imaging of cancer. Methods 65, 139–147 (2014).

    CAS  PubMed  Google Scholar 

  30. 30.

    Sheeley, D. M., Merrill, B. M. & Taylor, L. C. Characterization of monoclonal antibody glycosylation: comparison of expression systems and identification of terminal α-linked galactose. Anal. Biochem. 247, 102–110 (1997).

    CAS  PubMed  Google Scholar 

  31. 31.

    Roggenbuck, D., Mytilinaiou, M. G., Lapin, S. V., Reinhold, D. & Conrad, K. Asialoglycoprotein receptor (ASGPR): a peculiar target of liver-specific autoimmunity. Auto. Immun. Highlights 3, 119–125 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  32. 32.

    Pyzik, M. et al. Hepatic FcRn regulates albumin homeostasis and susceptibility to liver injury. Proc. Natl Acad. Sci. USA 114, E2862–E2871 (2017).

    CAS  PubMed  Google Scholar 

  33. 33.

    Sockolosky, J. T. & Szoka, F. C. The neonatal Fc receptor, FcRn, as a target for drug delivery and therapy. Adv. Drug Deliv. Rev. 91, 109–124 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  34. 34.

    Weissleder, R., Schwaiger, M. C., Gambhir, S. S. & Hricak, H. Imaging approaches to optimize molecular therapies. Sci. Transl. Med. 8, 355ps316 (2016).

    Google Scholar 

  35. 35.

    Mall, S. et al. Immuno-PET imaging of engineered human T cells in tumors. Cancer Res. 76, 4113–4123 (2016).

    CAS  PubMed  Google Scholar 

  36. 36.

    Seo, J. W. et al. CD8(+) T-cell density imaging with (64)Cu-labeled Cys-diabody informs immunotherapy protocols. Clin. Cancer Res. 24, 4976–4987 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  37. 37.

    Quayle, S. N. et al. CUE-101, a novel HPV16 E7-pHLA-IL-2-Fc fusion protein, enhances tumor antigen-specific T cell activation for the treatment of HPV16-driven malignancies. Clin. Cancer Res. 26, 1953–1964 (2020).

    PubMed  Google Scholar 

  38. 38.

    Guimaraes, C. P. et al. Site-specific C-terminal and internal loop labeling of proteins using sortase-mediated reactions. Nat. Protoc. 8, 1787–1799 (2013).

    PubMed  PubMed Central  Google Scholar 

  39. 39.

    Steurer, W. et al. Ex vivo coating of islet cell allografts with murine CTLA4/Fc promotes graft tolerance. J. Immunol. 155, 1165–1174 (1995).

    CAS  PubMed  Google Scholar 

  40. 40.

    Yan, L., Woodham, A. W., Da Silva, D. M. & Kast, W. M. Functional analysis of HPV-like particle-activated Langerhans cells in vitro. Methods Mol. Biol. 1249, 333–350 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  41. 41.

    Carvalho, L. H., Hafalla, J. C. & Zavala, F. ELISPOT assay to measure antigen-specific murine CD8(+) T cell responses. J. Immunol. Methods 252, 207–218 (2001).

    CAS  PubMed  Google Scholar 

  42. 42.

    Schmidt, F. I. et al. Phenotypic lentivirus screens to identify functional single domain antibodies. Nat. Microbiol. 1, 16080 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

Download references


A.W.W. is supported by the Arnold O. Beckman Postdoctoral Fellowship. R.W.C. is supported in part by funding from the Cancer Research Institute Irvington Postdoctoral Fellowship. M.R. was supported by the National Institutes of Health (grant 1K22CA226040-01). H.L.P. is supported by the Lustgarten Foundation (award ID 388167). The laboratory of H.L.P. receives financial support in the form of a sponsored research agreement from VIR in the general area of immunity to flu virus. We acknowledge Cue Biopharma for partial support of this work. The synTac technology was developed with support provided by the National Institutes of Health (U01GM094665, U54GM094662, R01AI145024 and R01CA198095 to S.C.A.). We acknowledge the Wollowick Family Foundation Chair in Multiple Sclerosis and Immunology (to S.C.A.) and Janet & Martin Spatz and the Helen & Irving Spatz Foundation. Additional support provided by the Albert Einstein Macromolecular Therapeutics Development Facility, the Einstein-Rockefeller-CUNY Center for AIDS Research (P30AI124414) and the Albert Einstein Cancer Center (P30CA013330).

Author information




A.W.W., S.H.Z., E.L.Z., S.C.A. and H.L.P. conceived of and designed experiments. S.H.Z., R.J.C., R.D.S., S.J.G. and S.C.A. designed and assembled the plasmids encoding the heavy and light chains of the synTacs. A.W.W., E.L.Z. and M.R. performed preliminary HPV, IAV and PET studies, respectively. A.W.W., S.H.Z., E.L.Z., S.C.K., R.W.C., M.R., S.J.G., J.L.D., M.M., P.K.D. and A.B.P. performed experiments. A.W.W., S.H.Z., E.L.Z., S.C.A. and H.L.P. analyzed and interpreted data. A.W.W., S.H.Z., S.C.A. and H.L.P. drafted the manuscript. All authors reviewed and edited the manuscript.

Corresponding authors

Correspondence to Steven C. Almo or Hidde L. Ploegh.

Ethics declarations

Competing interests

The synTac technology was developed in the laboratory of S.C.A. and was licensed to Cue Biopharma, Inc., in which he holds equity, receives royalties and serves as chair of its scientific advisory board. S.J.G. and R.D.S. receive royalties from Cue Biopharma, Inc. The laboratory of H.P. receives financial support in the form of a sponsored research agreement from VIR Biotechnology in the general area of immunity to flu virus, but H.L.P. has no equity stake in VIR.

Additional information

Peer review information Nina Vogt was the primary editor on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.

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

Supplementary information

Supplementary Information

Supplementary Figs. 1–14, Tables 1–6 and Source Data for Supplementary Figs. 1 and 3.

Reporting Summary

Source data

Source Data Fig. 1

Unmodified Coomassie-stained gel; unprocessed western blot.

Source Data Fig. 6

Unmodified Coomassie-stained gels.

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Woodham, A.W., Zeigler, S.H., Zeyang, E.L. et al. In vivo detection of antigen-specific CD8+ T cells by immuno-positron emission tomography. Nat Methods (2020).

Download citation


Sign up for the Nature Briefing newsletter for a daily update on COVID-19 science.
Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing