Skip to main content

Advertisement

SpringerLink
Log in

Measurement of cytotoxic T lymphocyte activity of human cytomegalovirus seropositive individuals by a highly sensitive coupled luminescent method

  • Original Investigation
  • Published:
Medical Microbiology and Immunology Aims and scope Submit manuscript

Abstract

A coupled luminescent method (CLM) based on glyceraldehyde-3-phosphate dehydrogenase released from injured target cells was used to evaluate the cytotoxicity of antigen-specific HLA class I-restricted CTLs. In contrast to established methods, CLM does not require the pretreatment of target cells with radioactive or toxic labeling substances. CTLs from healthy HLA-A2 positive donors were stimulated by autologous dendritic cells (DCs) pulsed with HLA-A2 restricted HCMV-pp65 nonamer peptides. HLA-A2 positive T2 cells or autologous monocytes pulsed with HCMV-pp65 nonamer peptide served as target cells. Lysis was detected only in HCMV-pp65-pulsed target cells incubated with CTLs from seropositive donors stimulated by HCMV-pp65-pulsed DCs. After 3 days, stimulation 38% of T2 cells and 17% of monocytes were lysed at an effector to target ratio of 8:1. In conclusion, CLM represents a highly sensitive, fast, material-saving and non-toxic/non-radioactive method for the measurement of antigen-specific CTL cytotoxic activity.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3

Similar content being viewed by others

References

  1. Brunner KT, Mauel J, Cerottini JC, Chapuis B (1968) 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

    CAS  PubMed  Google Scholar 

  2. von Zons P, Crowley-Nowick P, Friberg D, Bell M, Koldovsky U, Whiteside TL (1997) Comparison of europium and chromium release assays: cytotoxicity in healthy individuals and patients with cervical carcinoma. Clin Diagn Lab Immunol 4:202–207

    Google Scholar 

  3. Granberg C, Blomberg K, Hemmila I, Lovgren T (1988) Determination of cytotoxic T lymphocyte activity by time-resolved fluorometry using europium-labelled concanavalin A-stimulated cells as targets. J Immunol Methods 114:191–195

    Article  CAS  PubMed  Google Scholar 

  4. Blomberg K, Granberg C, Hemmila I, Lovgren T (1986) Europium-labelled target cells in an assay of natural killer cell activity. I. A novel non-radioactive method based on time-resolved fluorescence. J Immunol Methods 86:225–229

    Article  CAS  PubMed  Google Scholar 

  5. Blomberg K, Granberg C, Hemmila I, Lovgren T (1986) Europium-labelled target cells in an assay of natural killer cell activity. II. A novel non-radioactive method based on time-resolved fluorescence. significance and specificity of the method. J Immunol Methods 92:117–123

    Article  CAS  PubMed  Google Scholar 

  6. Zimmermann SY, Esser R, Rohrbach E, Klingebiel T, Koehl U (2005) A novel four-colour flow cytometric assay to determine natural killer cell or T-cell-mediated cellular cytotoxicity against leukaemic cells in peripheral or bone marrow specimens containing greater than 20% of normal cells. J Immunol Methods 296:63–76

    Article  CAS  PubMed  Google Scholar 

  7. Ogbomo H, Hahn A, Geiler J, Michaelis M, Doerr HW, Cinatl J Jr (2006) NK sensitivity of neuroblastoma cells determined by a highly sensitive coupled luminescent method. Biochem Biophys Res Commun 339:375–379

    Article  CAS  PubMed  Google Scholar 

  8. Ogbomo H, Michaelis M, Kreuter J, Doerr HW, Cinatl J Jr (2007) Histone deacetylase inhibitors suppress natural killer cell cytolytic activity. FEBS Lett 581:1317–1322

    Article  CAS  PubMed  Google Scholar 

  9. Asanuma H, Sharp M, Maecker HT, Maino VC, Arvin AM (2000) Frequencies of memory T cells specific for varicella-zoster virus, herpes simplex virus, and cytomegalovirus by intracellular detection of cytokine expression. J Infect Dis 181:859–866

    Article  CAS  PubMed  Google Scholar 

  10. Khan N, Hislop A, Gudgeon N, Cobbold M, Khanna R, Nayak L, Rickinson AB, Moss PA (2004) Herpesvirus-specific CD8 T cell immunity in old age: cytomegalovirus impairs the response to a coresident EBV infection. J Immunol 173:7481–7489

    CAS  PubMed  Google Scholar 

  11. Provenzano M, Mocellin S, Bettinotti M, Preuss J, Monsurro V, Marincola FM, Stroncek D (2002) Identification of immune dominant cytomegalovirus epitopes using quantitative real-time polymerase chain reactions to measure interferon-gamma production by peptide-stimulated peripheral blood mononuclear cells. J Immunother 25:342–351

    Article  CAS  PubMed  Google Scholar 

  12. Wills MR, Carmichael AJ, Mynard K, Jin X, Weekes MP, Plachter B, Sissons JG (1996) The human cytotoxic T-lymphocyte (CTL) response to cytomegalovirus is dominated by structural protein pp65: frequency, specificity, and T-cell receptor usage of pp 65-specific CTL. J Virol 70:7569–7579

    CAS  PubMed  Google Scholar 

  13. McLaughlin-Taylor E, Pande H, Forman SJ, Tanamachi B, Li CR, Zaia JA, Greenberg PD, Riddell SR (1994) Identification of the major late human cytomegalovirus matrix protein pp65 as a target antigen for CD8+ virus-specific cytotoxic T lymphocytes. J Med Virol 43:103–110

    Article  CAS  PubMed  Google Scholar 

  14. Kern F, Bunde T, Faulhaber N, Kiecker F, Khatamzas E, Rudawski IM, Pruss A, Gratama JW, Volkmer-Engert R, Ewert R, Reinke P, Volk HD, Picker LJ (2002) Cytomegalovirus (CMV) phosphoprotein 65 makes a large contribution to shaping the T cell repertoire in CMV-exposed individuals. J Infect Dis 185:1709–1716

    Article  CAS  PubMed  Google Scholar 

  15. Papanicolaou GA, Latouche JB, Tan C, Dupont J, Stiles J, Pamer EG, Sadelain M (2003) Rapid expansion of cytomegalovirus-specific cytotoxic T lymphocytes by artificial antigen-presenting cells expressing a single HLA allele. Blood 102:2498–2505

    Article  CAS  PubMed  Google Scholar 

  16. Boppana SB, Britt WJ (1996) Recognition of human cytomegalovirus gene products by HCMV-specific cytotoxic T cells. Virology 222:293–296

    Article  CAS  PubMed  Google Scholar 

  17. Riddell SR, Rabin M, Geballe AP, Britt WJ, Greenberg PD (1991) Class I MHC-restricted cytotoxic T lymphocyte recognition of cells infected with human cytomegalovirus does not require endogenous viral gene expression. J Immunol 146:2795–2804

    CAS  PubMed  Google Scholar 

  18. Gilbert MJ, Riddell SR, Li CR, Greenberg PD (1993) Selective interference with class I major histocompatibility complex presentation of the major immediate-early protein following infection with human cytomegalovirus. J Virol 67:3461–3469

    CAS  PubMed  Google Scholar 

  19. Riddell SR, Greenberg PD (1994) Therapeutic reconstitution of human viral immunity by adoptive transfer of cytotoxic T lymphocyte clones. Curr Top Microbiol Immunol 189:9–34

    CAS  PubMed  Google Scholar 

  20. Diamond DJ, York J, Sun JY, Wright CL, Forman SJ (1997) Development of a candidate HLA A*0201 restricted peptide-based vaccine against human cytomegalovirus infection. Blood 90:1751–1767

    CAS  PubMed  Google Scholar 

  21. Falk K, Rotzschke O, Deres K, Metzger J, Jung G, Rammensee HG (1991) Identification of naturally processed viral nonapeptides allows their quantification in infected cells and suggests an allele-specific T cell epitope forecast. J Exp Med 174:425–434

    Article  CAS  PubMed  Google Scholar 

  22. Falk K, Rotzschke O, Stevanovic S, Jung G, Rammensee HG (1991) Allele-specific motifs revealed by sequencing of self-peptides eluted from MHC molecules. Nature 351:290–296

    Article  CAS  PubMed  Google Scholar 

  23. Rammensee HG, Falk K, Rotzschke O (1993) Peptides naturally presented by MHC class I molecules. Annu Rev Immunol 11:213–244

    Article  CAS  PubMed  Google Scholar 

  24. Hunt DF, Henderson RA, Shabanowitz J, Sakaguchi K, Michel H, Sevilir N, Cox AL, Appella E, Engelhard VH (1992) Characterization of peptides bound to the class I MHC molecule HLA-A2.1 by mass spectrometry. Science 255:1261–1263

    Article  CAS  PubMed  Google Scholar 

  25. Crumpacker DB, Alexander J, Cresswell P, Engelhard VH (1992) Role of endogenous peptides in murine allogenic cytotoxic T cell responses assessed using transfectants of the antigen-processing mutant 174xCEM.T2. J Immunol 148:3004–3011

    CAS  PubMed  Google Scholar 

  26. Maecker HT, Ghanekar SA, Suni MA, He XS, Picker LJ, Maino VC (2001) Factors affecting the efficiency of CD8+ T cell cross-priming with exogenous antigens. J Immunol 166:7268–7275

    CAS  PubMed  Google Scholar 

  27. Ghanekar SA, Nomura LE, Suni MA, Picker LJ, Maecker HT, Maino VC (2001) Gamma interferon expression in CD8(+) T cells is a marker for circulating cytotoxic T lymphocytes that recognize an HLA A2-restricted epitope of human cytomegalovirus phosphoprotein pp65. Clin Diagn Lab Immunol 8:628–631

    CAS  PubMed  Google Scholar 

  28. Engstrand M, Lidehall AK, Totterman TH, Herrman B, Eriksson BM, Korsgren O (2003) Cellular responses to cytomegalovirus in immunosuppressed patients: circulating CD8+ T cells recognizing CMVpp65 are present but display functional impairment. Clin Exp Immunol 132:96–104

    Article  CAS  PubMed  Google Scholar 

  29. Waller EC, Day E, Sissons JG, Wills MR (2008) Dynamics of T cell memory in human cytomegalovirus infection. Med Microbiol Immunol 197:83–96

    Article  PubMed  Google Scholar 

  30. Martin H, Mandron M, Davrinche C (2008) Interplay between human cytomegalovirus and dendritic cells in T cell activation. Med Microbiol Immunol 197:179–184

    Article  PubMed  Google Scholar 

  31. Besold K, Plachter B (2008) Recombinant viruses as tools to study human cytomegalovirus immune modulation. Med Microbiol Immunol 197:215–222

    Article  PubMed  Google Scholar 

  32. Banchereau J, Steinman RM (1998) Dendritic cells and the control of immunity. Nature 392:245–252

    Article  CAS  PubMed  Google Scholar 

  33. Dhodapkar MV, Steinman RM, Sapp M, Desai H, Fossella C, Krasovsky J, Donahoe SM, Dunbar PR, Cerundolo V, Nixon DF, Bhardwaj N (1999) Rapid generation of broad T-cell immunity in humans after a single injection of mature dendritic cells. J Clin Invest 104:173–180

    Article  CAS  PubMed  Google Scholar 

  34. Corey MJ, Kinders RJ, Brown LG, Vessella RL (1997) A very sensitive coupled luminescent assay for cytotoxicity and complement-mediated lysis. J Immunol Methods 207:43–51

    Article  CAS  PubMed  Google Scholar 

  35. Jenne L, Arrighi JF, Jonuleit H, Saurat JH, Hauser C (2000) Dendritic cells containing apoptotic melanoma cells prime human CD8+ T cells for efficient tumor cell lysis. Cancer Res 60:4446–4452

    CAS  PubMed  Google Scholar 

  36. Michaelis M, Doerr HW, Cinatl J Jr (2009) Oncomodulation by human cytomegalovirus: evidence becomes stronger. Med Microbiol Immunol 198:79–81

    Article  PubMed  Google Scholar 

  37. Martin H, Mandron M, Davrinche C (2008) Interplay between human cytomegalovirus and dendritic cells in T cell activation. Med Microbiol Immunol 197:179–184

    Article  PubMed  Google Scholar 

  38. Waller EC, Day E, Sissons JG, Wills MR (2008) Dynamics of T cell memory in human cytomegalovirus infection. Med Microbiol Immunol 197:83–96

    Article  PubMed  Google Scholar 

  39. Su BY, Su CY, Yu SF, Chen CJ (2007) Incidental discovery of high systemic lupus erythematosus disease activity associated with cytomegalovirus viral activity. Med Microbiol Immunol 196:165–170

    Article  PubMed  Google Scholar 

Download references

Acknowledgments

The authors gratefully acknowledge the support by the organization “Hilfe für krebskranke Kinder, Frankfurt/Main e.V.”, by the foundation “Frankfurter Stiftung für krebskranke Kinder”, and by the European Commission-funded Co-operative Research and Specific Targeted Research Projects; COOP-CT-2004, Contract Nr. 512864 and LSHB-CT-2004, Contract Nr. 512054, respectively.

Author information

Authors and Affiliations

  1. Institut für Medizinische Virologie, Zentrum der Hygiene, Klinikum der Johann Wolfgang Goethe-Universität, Paul-Ehrlich-Str. 40, 60596, Frankfurt am Main, Germany

    Henry Ogbomo, Janina Geiler, Anke Leutz, Kristina von Kietzell, Martin Michaelis, Hans Wilhelm Doerr & Jindrich Cinatl Jr.

Authors
  1. Henry Ogbomo
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Janina Geiler
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Anke Leutz
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Kristina von Kietzell
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Martin Michaelis
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Hans Wilhelm Doerr
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Jindrich Cinatl Jr.
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Jindrich Cinatl Jr..

Rights and permissions

Reprints and permissions

About this article

Cite this article

Ogbomo, H., Geiler, J., Leutz, A. et al. Measurement of cytotoxic T lymphocyte activity of human cytomegalovirus seropositive individuals by a highly sensitive coupled luminescent method. Med Microbiol Immunol 198, 257–262 (2009). https://doi.org/10.1007/s00430-009-0126-5

Download citation

  • Received:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00430-009-0126-5

Keywords

Search

Navigation