Skip to main content

Unique Strengths of ELISPOT for T Cell Diagnostics

Part of the Methods in Molecular Biology book series (MIMB,volume 792)

Abstract

The T cell system plays an essential role in infections, allergic reactions, tumor and transplant rejection, as well as autoimmune diseases. It does so by the selective engagement of different antigen-specific effector cell lineages that differentially secrete cytokines and other effector molecules. These T cell subsets may or may not have cytolytic activity, can preferentially migrate to different tissues, and display variable capabilities to expand clonally. The quest of T cell immune diagnostics is to understand which specific effector function and T cell lineage is associated with a given clinical outcome, be it positive or adverse. No single assay can measure all of the relevant parameters. In this chapter, we review the unique contributions that ELISPOT assays can make toward understanding T cell-mediated immunity. ELISPOT assays have an unsurpassed sensitivity in detecting low frequency antigen-specific T cells that secrete effector molecules, including granzyme and perforin. They provide robust, highly reproducible data – even by first time users. Because ELISPOT assays require roughly tenfold less cell material than flow cytometry, ELISPOT is ideally suited for all measurements requiring parallel testing under multiple conditions. These include defining (a) T cell reactivity to individual peptides of extensive libraries, thereby establishing the fine–specificity of the response, and determinant mapping; (b) reactivity to different concentrations of the antigen in serial dilutions to measure the avidity of the T cell response; or (c) different secretory products released by T cells which define their respective effector lineage/functions. Further, because T cells survive ELISPOT assays unaffected, they can be retested for the acquisition of additional information in follow-up assays. These strengths of ELISPOT assays the weaknesses of flow cytometry-based measurements. Thus, the two assays systems compliment each other in the quest to understand T cell-mediated immunity in vivo.

Key words

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

Buying options

Protocol
USD   49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD   89.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD   119.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD   169.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Learn about institutional subscriptions

Springer Nature is developing a new tool to find and evaluate Protocols. Learn more

References

  1. Sekaly, R.P. (2008) The failed HIV Merck vaccine study: a step back or a launching point for future vaccine development? J. Exp. Med 205, 7–12.

    Google Scholar 

  2. Tigno, J.T., Lehmann, P.V., and Tary-Lehmann, M. (2009) Dissociated induction of cytotoxicity and DTH by CFA and CpG. J Immunother 32, 389–394.

    Google Scholar 

  3. Streeck, H., Frahm, N., and Walker, B.D. (2009) The role of IFN-gamma Elispot assay in HIV vaccine research. Nat Protoc 4, 461–469.

    Google Scholar 

  4. Rininsland, F. Helms, T., Asaad, R.J., Boehm, B.O., and Tary-Lehmann, M. (2000) Granzyme B ELISPOT assay for ex vivo measurements of T cell immunity. J Immunol Methods 240, 143–155.

    Google Scholar 

  5. Kleen, T. O., Asaad, R.J., Landry, S.J., Boehm, B.O., and Tary-Lehmann, M. (2005) Tc1 effector diversity shows dissociated expression of granzyme B and interferon-gamma in HIV infection. AIDS 18, 383–392.

    Google Scholar 

  6. Kuerten, S., Kleen, T., Assad, R.J., Lehmann, P.V., and Tary-Lehmann, M. (2007) Dissociated production of perforin, granzyme B and IFN-γ by HIV-specific CD8+ cells in HIV infection. AIDS Research and Human Retroviruses 24, 62–71.

    Google Scholar 

  7. S., Asaad, R.J., Schoenberger, S.P., Lehmann, P.V., and Tary-Lehmann, M. (2008) The TRAIL of helpless CD8+ T cells in HIV infection. AIDS Res Hum Retroviruses 24, 1–9.

    Google Scholar 

  8. Zhu, J., Yamane, and H., Paul, W.E. (2010) Differentiation of effector T cell populations. Annu. Rev. Immunol 28, 445–489.

    Google Scholar 

  9. McElrath, M.J., De Rosa, S.C., Moodie, Z., Dubey, S., Kierstead. L,, Janes, H., et al. (2008) HIV-1 vaccine-induced immunity in the test-of-concept Step Study: a case-cohort analysis. Lancet 372, 1894–905.

    Google Scholar 

  10. Schlingmann, T.R., Shive, C.L., Targoni, O.S., Tary-Lehmann, M., Lehmann, P.V. (2009) Increased per cell IFN-gamma productivity indicates recent in vivo activation of T cells. Cellular Immunology 258,131–137.

    Google Scholar 

  11. Helms, T., Boehm, B.O., Assad, R.J, Trezza, R.T., Lehmann, P.V., and Tary-Lehmann, M. (2000) Direct visualization of cytokine-­producing, recall antigen-specific CD4 memory T cells in healthy individuals and HIV patients. J Immunol 164, 3723–3732.

    Google Scholar 

  12. Hesse, M.D., Karulin, A.Y., Boehm, B.O., Lehmann, P.V., and Tary-Lehmann, M. (2001) A T cell clone’s avidity is a function of its activation state, J Immunol 167, 1353–1361.

    Google Scholar 

  13. Schmittel, A., Keilholz, U., and Scheibenbogen, C. (1997) Evaluation of the interferon-gamma ELISPOT-assay for quantification of peptide specific T lymphocytes from peripheral blood. J Immunol Methods. 210, 67–74.

    Google Scholar 

  14. Zhang, W., Caspell, R., Karulin, A.Y., Ahmad, M., Haicheur, N., Abdelsalam, A., et al. (2009) ELISPOT assays provide reproducible results among different laboratories for T-cell immune monitoring--even in hands of ELISPOT-inexperienced investigators, J Immunotoxicol 6 , 227–234.

    Google Scholar 

  15. Kuerten, S., Rottlaender, A., Rodi, M., Velasco, V.B. Jr, Schroeter, M., Kaiser, C., et al. (2010) The clinical course of EAE is reflected by the dynamics of the neuroantigen-specific T cell compartment in the blood. Clin Immunol. 137:422–432.

    Google Scholar 

  16. Kreher, C. R., Dittrich, M. T., Guerkov, R., Boehm, B. O., and Tary-Lehmann, M. (2003) CD4+ and CD8+ cells in cryopreserved human PBMC maintain full functionality in cytokine ELISPOT assays, J Immunol Methods 278, 79–93.

    Google Scholar 

  17. Ott, P.A., Herzog, B.A., Quast, S., Hofstetter, H.H., Boehm, B.O., Tary-Lehmann, M., et al. (2005) Islet-cell antigen-reactive T cells show different expansion rates and Th1/Th2 differentiation in type 1 diabetic patients and healthy controls. Clin Immunol 115, 102–114.

    Google Scholar 

  18. Anthony, D. D., and Lehmann, P.V. (2003) T-cell epitope mapping using the ELISPOT approach. Methods 29, 260–271.

    Google Scholar 

  19. Yip, H. C., Karulin, A.Y., Tary-Lehmann, M., Hesse, M.D., Radeke, H., Heeger, P.S., et al. (1999) Adjuvant-guided type-1 and type-2 immunity: infectious/noninfectious dichotomy defines the class of response. J Immunol 162, 39423949.

    Google Scholar 

  20. Targoni, O. S., and Lehmann, P.V. (1998) Endogenous myelin basic protein inactivates the high avidity T cell repertoire. J Exp Med 187, 2055–2063.

    Google Scholar 

  21. Janetzki. S., Britten, C.M., Kalos, M., Levitsky, H.I., Maecker, H.T., Melief, C.J., et al. (2009) “MIATA”-minimal information about T cell assays. Immunity 31, 527–528.

    Google Scholar 

  22. Karulin, A. Y., Hesse, M.D., Tary-Lehmann, M., and Lehmann, P.V. (2000) Single-cytokine-producing CD4 memory cells predominate in type 1 and type 2 immunity. J Immunol 164, 1862–1872.

    Google Scholar 

  23. Quast, S., Zhang, W., Shive, C., Kovalovski, D., Ott, P.A., Herzog, B.A, et al. (2005) IL-2 absorption affects IFN-gamma and IL-5, but not IL-4 producing memory T cells in double color cytokine ELISPOT assays. Cell Immunol 237, 28–36.

    Google Scholar 

  24. Forsthuber, T., Yip, H. C., and Lehmann, P. V. (1996) Induction of TH1 and TH2 immunity in neonatal mice. Science 271, 1728–1730.

    Google Scholar 

  25. Ott, P. A., Tary-Lehmann, M., and Lehmann, P. V. (2007) The secretory IFN-gamma response of single CD4 memory cells after activation on different antigen presenting cell types. Clin Immunol 124, 267–276.

    Google Scholar 

  26. Hofstetter H.H., Karulin A., Forsthuber, T.G., Ott, P.A., Tary-Lehmann, M., and Lehmann P.V. (2005) The cytokine signature of MOG-specific CD4 cells in the EAE of C57BL/6 mice. J Neuroimmunol 170, 105114.

    Google Scholar 

Download references

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Paul V. Lehmann .

Editor information

Editors and Affiliations

Rights and permissions

Reprints and permissions

Copyright information

© 2012 Springer Science+Business Media, LLC

About this protocol

Cite this protocol

Lehmann, P.V., Zhang, W. (2012). Unique Strengths of ELISPOT for T Cell Diagnostics. In: Kalyuzhny, A. (eds) Handbook of ELISPOT. Methods in Molecular Biology, vol 792. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-61779-325-7_1

Download citation

  • DOI: https://doi.org/10.1007/978-1-61779-325-7_1

  • Published:

  • Publisher Name: Humana Press, Totowa, NJ

  • Print ISBN: 978-1-61779-324-0

  • Online ISBN: 978-1-61779-325-7

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics