, Volume 8, Issue 6, pp 563–571 | Cite as

Measurement of CTL-induced cytotoxicity: The caspase 3 assay



Cytotoxic T lymphocytes (CTL) are critical effector cells of the immune system. Measurement of target cell damage has historically been an important measure of CTL function. CTL kill their target cells predominantly by inducing programmed cell death, or apoptosis. The gold standard for CTL-mediated cytotoxicity has been the 51Cr release assay. However, measurement of target cell lysis by 51Cr release does not provide mechanistic information on the fate of target cells, especially at the single cell level. Given the recent advances in our understanding of programmed cell death, newer assays are required which evaluate the status of the apoptotic pathways in target cells. We have developed a flow cytometry-based assay for CTL-mediated cytotoxicity based on specific binding of antibody to activated caspase 3 in target cells. Our assay is convenient and more sensitive than the 51Cr release assay. The use of this assay should allow mechanistic studies of the intracellular events resulting from CTL attack.

apoptosis caspase cytotoxic T lymphocyte 51Cr release flow cytometry 


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. 1.
    Bashford CL, Menestrina G, Henkart PA, Pasternak CA. Cell damage by cytolysin. Spontaneous recovery and reversible inhibition by divalent cations. J Immunol 1988; 141: 3965–3974.Google Scholar
  2. 2.
    Hameed A, Olsen KJ, Lee M-K, Lichtenheld MG, Podack ER. Cytolysis by Capermeable transmembrane channels. Pore formation causes extensive DNA degradation and cell lysis. J Exp Med 1989; 169: 765–777.Google Scholar
  3. 3.
    Podack ER, Dennert G. Assembly of two types of tubules with putative cytolytic function by cloned natural killer cells. Nature 1983; 302: 442–445.Google Scholar
  4. 4.
    Henkart PA, Millard PJ, Reynolds CW, Henkart MP. Cytolytic activity of purified cytoplasmic granules from cytotoxic rat large granular lymphocyte tumors. J Exp Med 1984; 160: 75–93.Google Scholar
  5. 5.
    Millard PJ, Henkart MP, Reynolds CW, Henkart PA. Purification and properties of cytoplasmic granules from cytotoxic rat LGL tumors. J Immunol 1984; 132: 3197–3204.Google Scholar
  6. 6.
    Berke G. The CTL's kiss of death. Cell 1995; 81: 9–12.Google Scholar
  7. 7.
    Henkart PA, Williams MS, Zacharchuk CM, Sarin A. Do CTL kill target cells by inducing apoptosis? Semin Immunol 1997; 9: 135–144.Google Scholar
  8. 8.
    Russell JH, Ley TJ. Lymphocyte-mediated cytotoxicity. Annu Rev Immunol 2002; 20: 323–370.Google Scholar
  9. 9.
    Ashkenazi A, Dixit V. Apoptosis control by death and decoy receptors. Curr Opin Cell Biol 1999; 11: 255–260.Google Scholar
  10. 10.
    Ashkenazi A, Dixit VM. Death receptors: Signaling and modulation. Science 1998; 281: 1305–1308.Google Scholar
  11. 11.
    Trapani JA, Smyth MJ. Functional significance of the perforin/ granzyme cell death pathway. Nat Rev Immunol 2002; 2: 735–747.Google Scholar
  12. 12.
    Shi L, Mai S, Israels S, et al. Granzyme B (GraB) autonomously crosses the cell membrane and perforin initiates apoptosis and GraB nuclear localization. J Exp Med 1997; 185: 855–866.Google Scholar
  13. 13.
    Kam C-M, Hudig D, Powers JC. Granzymes (lymphocyte serine proteases): Characterization with natural and synthetic substrates and inhibitors. Biochem Biophys Acta 2000; 1477: 307–323.Google Scholar
  14. 14.
    Edwards KM, Davis JE, Browne KA, Sutton VR, Trapani JA. Anti-viral strategies of cytotoxic T lymphocytes are manifested through a variety of granule-bound pathways of apoptosis induction. Immunol and Cell Biol 1999; 77.Google Scholar
  15. 15.
    Trapani JA. Dual mechanisms of apoptosis induction by cytotoxic lymphocytes. Int Rev Cytol 1998; 182: 111–192.Google Scholar
  16. 16.
    Heusel JW, Wesselschmidt RL, Shresta S, Russell JH, Ley TJ. Cytotoxic lymphocytes require granzyme B for the rapid induction of DNA fragmentation and apoptosis in allogeneic target cells. Cell 1994; 76: 977–987.Google Scholar
  17. 17.
    Shi L, Kam CM, Powers JC, Aebersold R, Greenberg AH. Purification of three cytotoxic lymphocyte granule serine proteases that induce apoptosis through distinct substrate and target cell interactions. J Exp Med 1992; 176: 1521–1529.Google Scholar
  18. 18.
    Trapani JA, Jans P, Smyth MJ, et al. Perforin-dependent nuclear entry of granzyme B precedes apoptosis, and is not a consequence of nuclear membrane dysfunction. Cell Death Different 1998; 5: 488–496.Google Scholar
  19. 19.
    Beresford PJ, Xia Z, Greenberg AH, Lieberman J. Granzyme A loading induces rapid cytolysis and a novel form of DNA damage independently of caspase activation. Immunity 1999; 10: 585–594.Google Scholar
  20. 20.
    Shresta S, Graubert TA, Thomas DA, Raptis SZ, Ley TJ. Granzyme A initiates an alternative pathway for granule-mediated apoptosis. Immunity 1999; 10: 595–405.Google Scholar
  21. 21.
    Sutton VR, Vaux DL, Trapani JA. Bcl-2 prevents apoptosis induced by perforin and granzyme B, but not that mediated by whole cytotoxic lymphocytes. J Immunol 1997; 158: 5783–5790.Google Scholar
  22. 22.
    Green DR, Reed JC. Mitochondria and apoptosis. Science 1998; 281: 1309–1312.Google Scholar
  23. 23.
    Barry M, Heibein JA, Pinkoski MJ, et al. Granyzme b short-circuits the need for caspase 8 activity during granule-mediated cytotoxic T-lymphocyte killing by directly cleaving bid. Mol Cell Biol 2000; 20: 3781–3794.Google Scholar
  24. 24.
    Heibein JA, Goping IS, Barry M, et al. Granzyme B-mediated cytochrome c release is regulated by the Bcl-2 family members Bid and Bax. J Exp Med 2000; 192: 1391–1401.Google Scholar
  25. 25.
    Sutton VR, Davis JE, Cancilla M, et al. Initiation of apoptosis by granzyme B requires direct cleavage of bid, but not direct granzyme B-mediated caspase activation. J Exp Med 2000; 192: 1403–1413.Google Scholar
  26. 26.
    Sutton VR, Wowk ME, Cancilla M, Trapani JA. Caspase activation by granzyme B is indirect, and caspase autoprocessing requires the release of proapoptotic mitochondrial factors. Immunity 2003; 18: 319–329.Google Scholar
  27. 27.
    Li P, Nijihawan D, Budihardjo I, et al. Cytochrome c and dATP-dependent formation of Apaf-1/caspase-9 complex initiates an apoptotic protease cascade. Cell 1997; 91: 479–489.Google Scholar
  28. 28.
    Goping IS, Barry M, Liston P, et al. Granzyme B-induced apoptosis requires both direct caspase activation and relief of caspase inhibition. Immunity 2003; 18: 355–365.Google Scholar
  29. 29.
    Duband-Goulet I, Courvalin JC, Buendia B. LBR, a chromatin and lamin binding protein from the inner nuclear membrane, is proteolyzed at late stages of apoptosis. J Cell Sci 1998; 111: 1441–1451.Google Scholar
  30. 30.
    Sakahira H, Enari M, Nagata S. Cleavage of CAD inhibitor in CAD activation and DNA degradation during apoptosis. Nature 1998; 391: 96–99.Google Scholar
  31. 31.
    Enari M, Sakahira H, Yokoyama H, et al. A caspase-activated DNase that degrades DNA during apoptosis, and its inhibitor ICAD. Nature 1998; 391: 43–50.Google Scholar
  32. 32.
    Liu X, Zou H, Slaughter C, Wang X. DFF, a heterodimeric protein that functions downstream of caspase-3 to trigger DNA fragmentation during apoptosis. Cell 1997; 89: 175–184.Google Scholar
  33. 33.
    Brunner KT, Mauel J, Cerottini J-C, Chapuis B. Quantitative assay of the lytic action of immune lymphoid cells of 51Cr labeled allogenic target cells in vitro: Inhibition by isoantibody and by drugs. Immunology 1968; 14: 181–196.Google Scholar
  34. 34.
    Lichtenfels R, Biddison WE, Schulz H, Vogt AB, Martin R. CARE-LASS (calcein-release-assay), an improved fluorescence-based test system to measure cytotoxic T lymphocyte activity. J Immunol Methods 1994; 172: 227–239.Google Scholar
  35. 35.
    Wierda WG, Mehr DS, Kim YB. Comparison of fluorochrome-labeled and 51Cr-labeled targets for natural killer cytotoxicity assay. J Immunol Methods 1989; 122: 15–24.Google Scholar
  36. 36.
    Korzeniewski C, Callewaert DM. An enzyme-release assay for natural cytotoxicity. J Immunol Methods 1983; 64: 313–320.Google Scholar
  37. 37.
    Szekeres J, Pacsa AS, Pejtsik B. Measurement of lymphocyte cytotoxicity by assessing endogenous alkaline phosphatase activity of the target cells. J Immunol Methods 1981; 40: 151–154.Google Scholar
  38. 38.
    Schafer H, Schafer A, Kiderlen AF, Masihi KN, Burger R. A highly sensitive cytotoxicity assay based on the release of reporter enzymes, from stably transfected cell lines. J Immunol Methods 1997; 204: 89–98.Google Scholar
  39. 39.
    Bachy M, Bonnin-Rivalland A, Tilliet V, Trannoy E. Beta galactosidase release as an alternative to chromium release in cytotoxic T-cell assays. J Immunol Methods 1999; 230: 37–46.Google Scholar
  40. 40.
    Sheehy ME, McDermott AB, Furlan SN, Klenerman P, Nixon DF. A novel technique for the fluorometric assessment of T lymphocyte antigen specific lysis. J Immunol Methods 2001; 249: 99–110.Google Scholar
  41. 41.
    Lecoeur H, Fevrier M, Garcia S, Riviere Y, Gougeon ML. A novel flow cytometric assay for quantitation and multiparametric characterization of cell-mediated cytotoxicity. J Immunol Methods 2001; 253: 177–187.Google Scholar
  42. 42.
    Sanderson CJ, Taylor GA. The kinetics of Cr release from target cells in cell mediated cytotoxicity and the relationship to the kinetics of killing. Cell Tissue Kinet 1975; 8: 23–32.Google Scholar
  43. 43.
    Shiver JW, Henkart PA. A noncytotoxic mast cell tumor line exhibits potent IgE-dependent cytotoxicity after transfection with the cytolysin/perforin gene. Cell 1991; 64: 1175–1181.Google Scholar
  44. 44.
    Shiver JW, Su L, Henkart PA. Cytotoxicity with target DNA breakdown by rat basophilic leukemia cells expressing both cytolysin and granzyme A. Cell 1992; 71: 315–322.Google Scholar
  45. 45.
    Davis JE, Sutton VR, Smyth MJ, Trapani JA. Dependence of granzyme B-mediated cell death on a pathway regulated by Bcl-2 or its viral homolog, BHRF1. Cell Death Differ 2000; 7: 973–983.Google Scholar
  46. 46.
    Jagarlamoody SM, Aust JC, Tew RH, McKhann CF. In vitro detection of cytotoxic cellular immunity against tumor-specific antigens by a radioisotopic technique. Proc Natl Acad Sci USA 1971; 68: 1346–1350.Google Scholar
  47. 47.
    Cohen AM, Burdick JF, Ketcham AS. Cell-mediated cytotoxicity: An assay using 125 I-iododeoxyuridine-labeled target cells. J Immunol 1971; 107: 895–898.Google Scholar
  48. 48.
    Le Mevel BP, Oldham RK, Wells SA, Herberman RB. An evaluation of 125I-iododeoxyuridine as a cellular label for in vitro assays: Kinetics of incorporation and toxicity. J Natl Cancer Inst 1973; 51: 1551–1558.Google Scholar
  49. 49.
    Oldham RK, Siwarski D, McCoy JL, Plata EJ, Herberman RB. Evaluation of a cell-mediated cytotoxicity assay utilizing 125 iododeoxyuridine-labeled tissue-culture target cells. Natl Cancer Inst Monogr 1973; 37: 49–58.Google Scholar
  50. 50.
    Munger WE, Berrebi GA, Henkart PA. Possible involvement of CTL granule proteases in target cell DNA breakdown. Immunol Rev 1988; 103: 99–109.Google Scholar
  51. 51.
    Matzinger P. The JAM test. A simple assay for DNA fragmentation and cell death. J Immunol Methods 1991; 145: 185–192.Google Scholar
  52. 52.
    Shi Y. Mechanisms of caspase activation and inhibition during apoptosis. Mol Cell 2002; 9: 459–470.Google Scholar
  53. 53.
    Walker PR, Saas P, Dietrich PY. Role of Fas ligand (CD95L) in immune escape: The tumor cell strikes back. J Immunol 1997; 158: 4521–4524.Google Scholar
  54. 54.
    Dong H, Strome SE, Salomao DR, et al. Tumor-associated B7-H1 promotes T-cell apoptosis: A potential mechanism of immune evasion. Nat Med 2002; 8: 793–800.Google Scholar
  55. 55.
    Trambas CM, Griffiths GM. Delivering the kiss of death. Nature Immunology 2003; 4: 399–403.Google Scholar
  56. 56.
    Liu L, Chahroudi A, Silvestri G, et al. Visualization and quantification of T cell-mediated cytotoxicity using cell-permeable fluorogenic caspase substrates. Nature Med 2002; 8: 185–189.Google Scholar
  57. 57.
    Brickner AG, Warren EH, Caldwell JA, et al. The immunogenicity of a new human minor histocompatibility antigen results from differential antigen processing. J Exp Med 2001; 193: 195–206.Google Scholar
  58. 58.
    Kataoka T, Shinohara N, Takayama H, et al. Concanamycin A, a powerful tool for characterization and estimation of contribution of perforin-and Fas-based lytic pathways in cell-mediated cytotoxicity. J Immunol 1996; 156: 3678–3686.Google Scholar
  59. 59.
    Goyal L. Cell death inhibition: Keeping caspases in check. Cell 2001; 104: 805–808.Google Scholar
  60. 60.
    Cheng EH, Wei MC, Weiler S, et al. BCL-2, BCL-X(L) sequester BH3 domain-only molecules preventing BAX-and BAK-mediated mitochondrial apoptosis. Mol Cell 2001; 8: 705–711.Google Scholar
  61. 61.
    Waterhouse NJ, Trapani JA. CTL: Caspases Terminate Life, but that's not the whole story. Tissue Antigens 2002; 59: 175–183.Google Scholar
  62. 62.
    Bernardi P, Petronilli V, Di Lisa F, Forte M. A mitochondrial perspective on cell death. Trends Biochem Sci 2001; 26: 112–117.Google Scholar

Copyright information

© Kluwer Academic Publishers 2003

Authors and Affiliations

  1. 1.Program in Infectious DiseasesFred Hutchinson Cancer Research CenterUSA
  2. 2.Department of Laboratory MedicineUniversity of WashingtonSeattleUSA

Personalised recommendations