HIV Protocols pp 359-374 | Cite as

Quantifying HIV-1-Specific CD8+ T-Cell Responses Using ELISPOT and Cytokine Flow Cytometry

  • Barbara L. Shacklett
  • J. William Critchfield
  • Donna Lemongello
Part of the Methods In Molecular Biology™ book series (MIMB, volume 485)


Since the initial description and characterization of the agent that causes AIDS, human immunodeficiency virus (HIV-1), numerous research groups have characterized immune responses to this virus. Much effort has been directed towards identifying potential correlates of protection that may be useful for the development of vaccines and immunotherapies. In addition, several investigations have focused on comparing patients with rapid vs. slow disease progression profiles in an attempt to identify the characteristics of a “successful” immune response. Although many gaps remain in our understanding of the host–pathogen relationship, great progress has been made during the past 20 years in elucidating the adaptive, cell-mediated response to HIV-1. These investigations have benefited in recent years from the development of new approaches to the analysis of antigen-specific CD8+ T-cell function, notably the ELISPOT assay and cytokine flow cytometry. This chapter provides simple protocols for these two methods.

Key words

Cytokine ELISPOT flow cytometry T-cell 



This work is supported by NIH/NIAID AI057020 and by the California Universitywide AIDS Research Program (UARP), grant CH05-D-606. The authors thank their colleagues in the Department of Medical Microbiology and Immunology, UC Davis, for assistance with protocol optimization. We also thank Drs. Douglas Nixon (University of California, San Francisco) and Michael Betts (University of Pennsylvania) for sharing protocols and technical information.


  1. 1.
    Altman, J. D., Moss, P. A., Goulder, P. J., et al. (1996) Phenotypic analysis of antigen-specific T lymphocytes. Science 274, 94–96.CrossRefPubMedGoogle Scholar
  2. 2.
    Ogg, G. S., Jin, X., Bonhoeffer, S., et al. (1998) Quantitation of HIV-1-specific cytotoxic T lymphocytes and plasma load of viral RNA. Science 279, 2103–2106.CrossRefPubMedGoogle Scholar
  3. 3.
    Coligan, J., Kruisbeek, A., Margulies, D., et al. (1999), Current Protocols in Immunology, Wiley Interscience, New York.Google Scholar
  4. 4.
    Nixon, D. F., Townsend, A. R., Elvin, J. G., et al. (1988) HIV-1 gag-specific cytotoxic T lymphocytes defined with recombinant vaccinia virus and synthetic peptides. Nature 336, 484–487.CrossRefPubMedGoogle Scholar
  5. 5.
    Doherty, P. C. (1998) The new numbers game for virus-specific CD8+ T cells. Science 280, 227.CrossRefPubMedGoogle Scholar
  6. 6.
    Tan, L. C., Gudgeon, N., Annels, N. E., et al. (1999) A re-evaluation of the frequency of CD8+ T cells specific for EBV in healthy virus carriers. J Immunol 162, 1827–1835.PubMedGoogle Scholar
  7. 7.
    Lalvani, A., Brookes, R., Hambleton, S., et al. (1997) Rapid effector function in CD8+ memory T cells. J Exp Med 186, 859–865.CrossRefPubMedGoogle Scholar
  8. 8.
    Russell, N. D., Hudgens, M. G., Ha, R., et al. (2003) Moving to human immunodeficiency virus type 1 vaccine efficacy trials: defining T cell responses as potential correlates of immunity. J Infect Dis 187, 226–242.CrossRefPubMedGoogle Scholar
  9. 9.
    Anthony, D. D., Lehmann, P. V. (2003) T-cell epitope mapping using the ELISPOT approach. Methods 29, 260–269.CrossRefPubMedGoogle Scholar
  10. 10.
    Addo, M. M., Yu, X. G., Rathod, A., et al. (2003) Comprehensive epitope analysis of human immunodeficiency virus type 1 (HIV-1)-specific T-cell responses directed against the entire expressed HIV-1 genome demonstrate broadly directed responses, but no correlation to viral load. J Virol 77, 2081–2092.CrossRefPubMedGoogle Scholar
  11. 11.
    Perfetto, S. P., Chattopadhyay, P. K., Roederer, M. (2004) Seventeen-colour flow cytometry: unravelling the immune system. Nat Rev Immunol 4, 648–655.CrossRefPubMedGoogle Scholar
  12. 12.
    Betts, M. R., Nason, M. C., West, S. M., et al. (2006) HIV nonprogressors preferentially maintain highly functional HIV-specific CD8+ T cells. Blood 107, 4781–4789.CrossRefPubMedGoogle Scholar
  13. 13.
    Picker, L. J., Singh, M. K., Zdraveski, Z., et al. (1995) Direct demonstration of cytokine synthesis heterogeneity among human memory/effector T cells by flow cytometry. Blood 86, 1408–1419.PubMedGoogle Scholar
  14. 14.
    Komanduri, K. V., Viswanathan, M. N., Wieder, E. D., et al. (1998) Restoration of cytomegalovirus-specific CD4+ T-lymphocyte responses after ganciclovir and highly active antiretroviral therapy in individuals infected with HIV-1. Nat Med 4, 953–956.CrossRefPubMedGoogle Scholar
  15. 15.
    Betts, M. R., Koup, R. A. (2004) Detection of T-Cell Degranulation: CD107a and b, in: Cytometry, New Developments, Vol. 75, pp. 497–512 (Darzynkiewicz, Z., Roederer, M. and Tanke, H. J., Eds.) Elsevier Press, San Diego, CA.CrossRefGoogle Scholar
  16. 16.
    Kern, F., Surel, I. P., Brock, C., et al. (1998) T-cell epitope mapping by flow cytometry. Nat Med 4, 975–978.CrossRefPubMedGoogle Scholar
  17. 17.
    Betts, M. R., Ambrozak, D. R., Douek, D. C., et al. (2001) Analysis of Total Human Immunodeficiency Virus (HIV)-Specific CD4(+) and CD8(+) T-Cell Responses: Relationship to Viral Load in Untreated HIV Infection. J Virol 75, 11983–11991.CrossRefPubMedGoogle Scholar
  18. 18.
    Cherepnev, G., Volk, H. D., Kern, F. (2004) Use of Peptides and Peptide Libraries as T-Cell Stimulants in Flow Cytometric Studies, in (Darzynkiewicz, Z., Roederer, M. and Tanke, H. J., Eds.), Methods in Cell Biology, Vol. 75, pp. 453–479 Elsevier, San Diego.Google Scholar
  19. 19.
    Herr, W., Protzer, U., Lohse, A. W., et al. (1998) Quantification of CD8+ T lymphocytes responsive to human immunodeficiency virus (HIV) peptide antigens in HIV-infected patients and seronegative persons at high risk for recent HIV exposure. J Infect Dis 178, 260–265.CrossRefPubMedGoogle Scholar
  20. 20.
    Janetzki, S., Schaed, S., Blachere, N. E., et al. (2004) Evaluation of Elispot assays: influence of method and operator on variability of results. J Immunol Methods 291, 175–183.CrossRefPubMedGoogle Scholar
  21. 21.
    Larsson, M., Jin, X., Ramratnam, B., et al. (1999) A recombinant vaccinia virus based ELISPOT assay detects high frequencies of Pol-specific CD8 T cells in HIV-1-positive individuals. AIDS 13, 767–777.CrossRefPubMedGoogle Scholar
  22. 22.
    Currier, J. R., Kuta, E. G., Turk, E., et al. (2002) A panel of MHC class I restricted viral peptides for use as a quality control for vaccine trial ELISPOT assays. J Immunol Methods 260, 157–172.CrossRefPubMedGoogle Scholar
  23. 23.
    Harari, A., Rizzardi, G. P., Ellefsen, K., et al. (2002) Analysis of HIV-1- and CMV-specific memory CD4 T-cell responses during primary and chronic infection. Blood 100, 1381–1387.CrossRefPubMedGoogle Scholar
  24. 24.
    Sandberg, J. K., Fast, N. M., Nixon, D. F. (2001) Functional heterogeneity of cytokines and cytolytic effector molecules in human cd8(+) t lymphocytes. J Immunol 167, 181–187.PubMedGoogle Scholar
  25. 25.
    Betts, M. R., Brenchley, J. M., Price, D. A., et al. (2003) Sensitive and viable identification of antigen-specific CD8+ T cells by a flow cytometric assay for degranulation. J Immunol Methods 281, 65–78.CrossRefPubMedGoogle Scholar
  26. 26.
    Roederer, M., De Rosa, S., Gerstein, R., et al. (1997) 8 color, 10-parameter flow cytometry to elucidate complex leukocyte heterogeneity. Cytometry 29, 328–339.CrossRefPubMedGoogle Scholar
  27. 27.
    Mwau, M., McMichael, A. J., Hanke, T. (2002) Design and validation of an enzyme-linked immunospot assay for use in clinical trials of candidate HIV vaccines. AIDS Res Hum Retroviruses 18, 611–618.CrossRefPubMedGoogle Scholar
  28. 28.
    Betts, M. R., Price, D. A., Brenchley, J. M., et al. (2004) The functional profile of primary human antiviral CD8+ T cell effector activity is dictated by cognate peptide concentration. J Immunol 172, 6407–6417.PubMedGoogle Scholar
  29. 29.
    CDC (1987) Recommendations for Prevention of HIV Transmission in Health-Care Settings. MMWR 36, 3S–18S.Google Scholar
  30. 30.
    Hudgens, M. G., Self, S. G., Chiu, Y. L., et al. (2004) Statistical considerations for the design and analysis of the ELISpot assay in HIV-1 vaccine trials. J Immunol Methods 288, 19–34.CrossRefPubMedGoogle Scholar
  31. 31.
    Jennes, W., Kestens, L., Nixon, D., et al. (2002) Enhanced ELISPOT detection of antigen-specific T cell responses from cryopreserved specimens with addition of both IL-7 and IL-15-the Amplispot assay. J Immunol Methods 270, 99–108.PubMedGoogle Scholar
  32. 32.
    Ott, P. A., Berner, B. R., Herzog, B. A., et al. (2004) CD28 costimulation enhances the sensitivity of the ELISPOT assay for detection of antigen-specific memory effector CD4 and CD8 cell populations in human diseases. J Immunol Methods 285, 223–235.CrossRefPubMedGoogle Scholar
  33. 33.
    Altfeld, M. A., Trocha, A., Eldridge, R. L., et al. (2000) Identification of dominant optimal HLA-B60- and HLA-B61-restricted cytotoxic T-lymphocyte (CTL) epitopes: rapid characterization of CTL responses by enzyme-linked immunospot assay. J Virol 74, 8541–8549.CrossRefPubMedGoogle Scholar
  34. 34.
    Larsson, M., Wilkens, D. T., Fonteneau, J. F., et al. (2002) Amplification of low-frequency antiviral CD8+ T-cell responses using autologous dendritic cells. AIDS 16, 171–180.CrossRefPubMedGoogle Scholar
  35. 35.
    Lamoreaux, L., Roederer, M., Koup, R. (2006) Intracellular cytokine optimization and standard operating procedure. Nature Protocols 1, 1507–1516.CrossRefPubMedGoogle Scholar
  36. 36.
    Maecker, H. T., Rinfret, A., D’Souza, P., Darden, J., Roig, E., Landry, C. et al. (2005) Standardization of cytokine flow cytometry assays. BMC Immunol 6, 13.CrossRefPubMedGoogle Scholar
  37. 37.
    Perfetto, S. P., Chattopadhyay, P. K., Lamoreaux, L., et al. (2006) Amine reactive dyes: an effective tool to discriminate live and dead cells in polychromatic flow cytometry. J Immunol Methods 313, 199–208.CrossRefPubMedGoogle Scholar

Copyright information

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

Authors and Affiliations

  • Barbara L. Shacklett
    • 1
  • J. William Critchfield
    • 1
  • Donna Lemongello
    • 1
  1. 1.Department of Medical Microbiology and Immunology and Division of Infectious Diseases Department of Internal MedicineSchool of Medicine, University of CaliforniaDavisUSA

Personalised recommendations