Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • On the Market
  • Published:

Visualization and quantification of T cell–mediated cytotoxicity using cell-permeable fluorogenic caspase substrates

Nature Medicine volume 8, pages 185–189 (2002)Cite this article

Abstract

We have developed a non-radioactive flow-cytometry assay to monitor and quantify the target-cell killing activities mediated by cytotoxic T lymphocytes (CTLs). This flow-cytometry CTL (FCC) assay is predicated on measurement of CTL-induced caspase activation in target cells through detection of the specific cleavage of fluorogenic caspase substrates. Here we show that this assay reliably detects antigen-specific CTL killing of target cells, and demonstrate that it provides a more sensitive, more informative and safer alternative to the standard 51Cr-release assay most often used to quantify CTL responses. The FCC assay can be used to study CTL-mediated killing of primary host target cells of different cell lineages, and enables the study of antigen-specific cellular immune responses in real time at the single-cell level. As such, the FCC assay can provide a valuable tool for studies of infectious disease pathogenesis and development of new vaccines and immunotherapies.

This is a preview of subscription content, access via your institution

Relevant articles

Open Access articles citing this article.

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: FCC assay detected strong NP396–404-specific CTL response.
Figure 2: Comparison of CTL activities specific for a panel of LCMV epitopes measured by FCC and 51Cr-release assays.
Figure 3: LCMV-specific CTL killing of primary target cells detected by FCC assay.
Figure 4: The CTL-mediated killing of target cells was directly visualized using the microscopic FCC assay.

References

  1. Doherty, P.C. & Christensen, J.P. Accessing complexity: the dynamics of virus-specific T-cell responses. Annu. Rev. Immunol. 18, 561–592 (2000).

    Article  CAS  Google Scholar 

  2. Brunner, K.T., Mauel, J., Cerottini, J.C. & Chapuis, B. Quantitative assay of the lytic action of immune lymphoid cells on 51-Cr-labelled allogeneic target cells in vitro; inhibition by isoantibody and by drugs. Immunology 14, 181–196 (1968).

    CAS  PubMed  PubMed Central  Google Scholar 

  3. Nociari, M.M., Shalev, A., Benias, P. & Russo, C. A novel one-step, highly sensitive fluorometric assay to evaluate cell-mediated cytotoxicity. J. Immunol. Meth. 213, 157–167 (1998).

    Article  CAS  Google Scholar 

  4. Altman, J.D. et al. Phenotypic analysis of antigen-specific T lymphocytes. Science 274, 94–96 (1996); erratum: 280, 1821 (1998).

    Article  CAS  Google Scholar 

  5. Butz, E.A. & Bevan, M.J. Massive expansion of antigen-specific CD8+ T cells during an acute virus infection. Immunity 8, 167–175 (1998).

    Article  CAS  Google Scholar 

  6. Maino, V.C. & Picker, L.J. Identification of functional subsets by flow cytometry: intracellular detection of cytokine expression. Cytometry 34, 207–215 (1998).

    Article  CAS  Google Scholar 

  7. Appay, V. et al. HIV-specific CD8(+) T cells produce antiviral cytokines but are impaired in cytolytic function. J. Exp. Med. 192, 63–75 (2000).

    Article  CAS  Google Scholar 

  8. Lee, P.P. et al. Characterization of circulating T cells specific for tumor-associated antigens in melanoma patients. Nature Med. 5, 677–685 (1999).

    Article  CAS  Google Scholar 

  9. Zajac, A.J. et al. Viral immune evasion due to persistence of activated T cells without effector function. J. Exp. Med. 188, 2205–2213 (1998).

    Article  CAS  Google Scholar 

  10. Sheehy, M.E., McDermott, A.B., Furlan, S.N., Klenerman, P. & Nixon, D.F. A novel technique for the fluorometric assessment of T lymphocyte antigen specific lysis. J. Immunol. Meth. 249, 99–110 (2001); erratum: 252, 219–220 (2001).

    Article  CAS  Google Scholar 

  11. Lecoeur, H., Fevrier, M., Garcia, S., Riviere, Y. & Gougeon, M.L. A novel flow cytometric assay for quantitation and multiparametric characterization of cell-mediated cytotoxicity. J. Immunol. Meth. 253, 177–187 (2001).

    Article  CAS  Google Scholar 

  12. Atkinson, E.A. et al. Cytotoxic T lymphocyte-assisted suicide. Caspase 3 activation is primarily the result of the direct action of granzyme B. J. Biol. Chem. 273, 21261–21266 (1998).

    Article  CAS  Google Scholar 

  13. Packard, B.Z., Toptygin, D.D., Komoriya, A. & Brand, L. Profluorescent protease substrates: intramolecular dimers described by the exciton model. Proc. Natl. Acad. Sci. USA 93, 11640–11645 (1996).

    Article  CAS  Google Scholar 

  14. Komoriya, A., Packard, B.Z., Brown, M.J., Wu, M.L. & Henkart, P.A. Assessment of caspase activities in intact apoptotic thymocytes using cell-permeable fluorogenic caspase substrates. J. Exp. Med. 191, 1819–1828 (2000).

    Article  CAS  Google Scholar 

  15. Murali-Krishna, K. et al. Counting antigen-specific CD8 T cells: A reevaluation of bystander activation during viral infection. Immunity 8, 177–187 (1998).

    Article  CAS  Google Scholar 

  16. Thornberry, N.A. et al. A combinatorial approach defines specificities of members of the caspase family and granzyme B. Functional relationships established for key mediators of apoptosis. J. Biol. Chem. 272, 17907–17911 (1997).

    Article  CAS  Google Scholar 

  17. Ploegh, H.L. Viral strategies of immune evasion. Science 280, 248–253 (1998).

    Article  CAS  Google Scholar 

  18. Collins, K.L., Chen, B.K., Kalams, S.A., Walker, B.D. & Baltimore, D. HIV-1 Nef protein protects infected primary cells against killing by cytotoxic T lymphocytes. Nature 391, 397–401 (1998).

    Article  CAS  Google Scholar 

  19. Ruppert, J. et al. Prominent role of secondary anchor residues in peptide binding to HLA-A2.1 molecules. Cell 74, 929–937 (1993).

    Article  CAS  Google Scholar 

  20. Liu, L., Usherwood, E.J., Blackman, M.A. & Woodland, D.L. T-cell vaccination alters the course of murine herpesvirus 68 infection and the establishment of viral latency in mice. J. Virol. 73, 9849–9857 (1999).

    CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

We thank R. Ahmed for provision of reagents and helpful advice; P. Henkart, R. Mittler and S. Staprans for critical review of the manuscript; and T. Cinotte for technical assistance. This study was supported by NIH grant P01AI46007 and R2AI49089. M.B.F. is an Elizabeth Glaser Scientist of the Pediatric AIDS Foundation.

Author information

Authors and Affiliations

  1. Emory Vaccine Center and Emory University School of Medicine, Atlanta, Georgia, USA

    Luzheng Liu, Ann Chahroudi, Guido Silvestri, Mary E. Wernett, William J. Kaiser, Jeffrey T. Safrit, John D. Altman & Mark B. Feinberg

  2. OncoImmunin, Gaithersburg, Maryland, USA

    Akira Komoriya & Beverly Z. Packard

Authors
  1. Luzheng Liu
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Ann Chahroudi
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Guido Silvestri
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Mary E. Wernett
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. William J. Kaiser
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Jeffrey T. Safrit
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Akira Komoriya
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. John D. Altman
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Beverly Z. Packard
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Mark B. Feinberg
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Mark B. Feinberg.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Liu, L., Chahroudi, A., Silvestri, G. et al. Visualization and quantification of T cell–mediated cytotoxicity using cell-permeable fluorogenic caspase substrates. Nat Med 8, 185–189 (2002). https://doi.org/10.1038/nm0202-185

Download citation

  • Issue Date:

  • DOI: https://doi.org/10.1038/nm0202-185

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing