Analysis of Frequency and Phenotype of Antigen-Specific T Cells

  • Angus Stock
  • Vincenzo Cerundolo
Part of the Methods in Molecular Biology™ book series (MIMB, volume 514)


Over the last decade, our understanding of the cellular immune system has been greatly advanced through the development of methods to identify antigen-specific T cells directly ex vivo. The major reagents and techniques used for this purpose are (i) tetramerised MHC:peptide complexes (tetramers) which bind to specific T-cell receptors (TCR) and (ii) assays that detect T cells which synthesise cytokines in response to cognate stimulation (intracellular cytokine staining (ICS)). Here, we provide a detailed description of the procedure for generating and using class I MHC:peptide tetramers to label peptide-specific T cells and for carrying out ICS to measure antigen-specific T lymphocytes.

Key words

Tetramers antigen-specific T cells MHC class I intracellular cytokine staining CTL 


  1. 1.
    Jung T, Schauer U, Heusser C, Neumann C, Rieger, C. Detection of intracellular cytokines by flow cytometry. J Immunol Methods 1993;159:197–207CrossRefPubMedGoogle Scholar
  2. 2.
    Vikingsson A, Pederson K, Muller D. Enumeration of IFN-gamma producing lymphocytes by flow cytometry and correlation with quantitative measurement of IFN-gamma. J Immunol Methods 1994;173:219–28.CrossRefPubMedGoogle Scholar
  3. 3.
    Murali-Krishna K, Altman JD, Suresh M, et al. Counting antigen-specific CD8 T cells: a re-evaluation of bystander activation during viral infection. Immunity 1998;8:177–87.CrossRefPubMedGoogle Scholar
  4. 4.
    Flynn KJ, Belz GT, Altman JD, Ahmed R, Woodland DL, Doherty PC. Virus-specific CD8+ T cells in primary and secondary influenza pneumonia. Immunity 1998;8:683–91.CrossRefPubMedGoogle Scholar
  5. 5.
    Altman JD, Moss PA, Goulder PJ, et al. Phenotypic analysis of antigen-specific T lymphocytes. Science 1996;274:94–6.CrossRefPubMedGoogle Scholar
  6. 6.
    Klenerman P, Cerundolo V, Dunbar PR. Tracking T cells with tetramers: new tales from new tools. Nat Rev Immunol 2002;2:263–72.CrossRefPubMedGoogle Scholar
  7. 7.
    Garboczi DN, Hung DT, Wiley DC. HLA-A2-peptide complexes: refolding and crystallization of molecules expressed in Escherichia coli and complexed with single antigenic peptides. Proc Natl Acad Sci U S A 1992;89:3429–33.Google Scholar
  8. 8.
    Stock AT, Jones CM, Heath WR, Carbone FR. CTL response compensation for the loss of an immunodominant class I-restricted HSV-1 determinant. Immunol Cell Biol 2006;84:543–50.CrossRefPubMedGoogle Scholar
  9. 9.
    Tscharke DC, Woo WP, Sakala IG, et al. Poxvirus CD8+ T-cell determinants and cross-reactivity in BALB/c mice. J Virol 2006;80:6318–23.CrossRefPubMedGoogle Scholar
  10. 10.
    Hammarlund E, Lewis MW, Hansen SG, et al. Duration of antiviral immunity after smallpox vaccination. Nat Med 2003;9:1131–7.CrossRefPubMedGoogle Scholar
  11. 11.
    Whelan JA, Dunbar PR, Price DA, et al. Specificity of CTL interactions with peptide-MHC class I tetrameric complexes is temperature dependent. J Immunol 1999;163:4342–8.PubMedGoogle Scholar
  12. 12.
    Schwarz K, van Den Broek M, Kostka S, et al. Overexpression of the proteasome subunits LMP2, LMP7, and MECL-1, but not PA28 alpha/beta, enhances the presentation of an immunodominant lymphocytic choriomeningitis virus T cell epitope. J Immunol 2000;165:768–78.PubMedGoogle Scholar
  13. 13.
    Koelle DM, Chen HB, Gavin MA, Wald A, Kwok WW, Corey L. CD8 CTL from genital herpes simplex lesions: recognition of viral tegument and immediate early proteins and lysis of infected cutaneous cells. J Immunol 2001;166:4049–58.PubMedGoogle Scholar

Copyright information

© Humana Press, a part of Springer Science+Business Media, LLC 2009

Authors and Affiliations

  • Angus Stock
    • 1
  • Vincenzo Cerundolo
    • 1
  1. 1.Nuffield Department of Clinical MedicineWeatherall Institute of Molecular MedicineUK

Personalised recommendations