Abstract
The use of the IFN-γ ELISPOT assay to evaluate cellular immune responses has gained increasing popularity, especially as a surrogate measure for CTL responses. We developed and validated some modifications of the IFN-γ ELISPOT assay to optimize immunological monitoring of various cancer vaccine trials. Taking into consideration that the main mechanism of cell-mediated cytotoxicity is the release of cytolytic granules that contain, among others, cytolytic protein Granzyme B (GrB), we developed the GrB ELISPOT assay. Extensive studies demonstrated that the GrB ELISPOT assay is specific, accurately measures the rapid release of GrB, is more sensitive than the 51Cr-release assay, and that it may be successfully applied to measuring CTL precursory frequency in PBMC from cancer patients. Assuming that immunological assays that demonstrate recognition of native tumor cells (tumor-specific) may be more clinically relevant than assays that demonstrate recognition of tumor protein or peptide (antigen-specific), we developed and validated the Autologous Tumor IFN-γ ELISPOT assay using PBMC from idiotype vaccinated lymphoma patients as effectors and autologous B cell lymphoma tumor cells as targets. The precursor frequency of tumor-reactive T cells was significantly higher in the postvaccine PBMC, compared with prevaccine samples in all patients tested. Furthermore, the specificity of these T cells was established by the lack of reactivity against autologous normal B cells. These results demonstrate the feasibility of evaluating tumor-specific T cell responses when autologous, primary tumor cells are available as targets. Modifications of ELISPOT assay described in this chapter allow more comprehensive assessment of low frequency tumor-specific CTL and their specific effector functions and can provide valuable insight with regards to immune responses in cancer vaccine trials.
Key words: granzyme B, ELISPOT
References
- 1.Rosenberg S. Progress in human tumor immunology and immunotherapy. Nature. 2001;411:380–4. doi: 10.1038/35077246. [DOI] [PubMed] [Google Scholar]
- 2.Finn O., Lotze M. A decade in the life of tumor immunology. Clin Cancer Res. 2001;7:759s–760s. [PubMed] [Google Scholar]
- 3.Kwak L. Translational development of active immunotherapy for hematologic malignancies. Semin Oncol. 2003;30(3 Suppl 8):17–22. doi: 10.1016/s0093-7754(03)00232-x. [DOI] [PubMed] [Google Scholar]
- 4.Keilholz U., Weber J., Finke J., et al. Immunologic monitoring of cancer vaccine therapy: results of a workshop sponsored by the Society for Biological Therapy. J Immunother. 2002;25:97–138. doi: 10.1097/00002371-200203000-00001. [DOI] [PubMed] [Google Scholar]
- 5.Regner M., Lobigs M., Blanden R., Mullbacher A. Effector cytolotic function but not IFN-gamma production in cytotoxic T cells triggered by virus-infected target cells in vitro. Scand J Immunol. 2001;54:366–74. doi: 10.1046/j.1365-3083.2001.00955.x. [DOI] [PubMed] [Google Scholar]
- 6.Trinchieri G., Perussia B. Human natural killer cells: biologic and pathologic aspects. Lab Invest. 1984;50:489–513. [PubMed] [Google Scholar]
- 7.Smyth M., Trapani J. Granzymes: exogenous proteinases that induce target cell apoptosis. Immunol Today. 1995;16:202–6. doi: 10.1016/0167-5699(95)80122-7. [DOI] [PubMed] [Google Scholar]
- 8.Podack E. Execution and suicide: cytotoxic lymphocytes enforce Draconian laws through separate molecular pathways. Curr Opin Immunol. 1995;7:11–6. doi: 10.1016/0952-7915(95)80023-9. [DOI] [PubMed] [Google Scholar]
- 9.Berke G. The CTL’s kiss of death. Cell. 1995;81:9–12. doi: 10.1016/0092-8674(95)90365-8. [DOI] [PubMed] [Google Scholar]
- 10.Liu C., Persechini P., Young J. Perforin and lymphocyte-mediated cytolysis. Immunol Rev. 1995;146:145–75. doi: 10.1111/j.1600-065x.1995.tb00688.x. [DOI] [PubMed] [Google Scholar]
- 11.Jenne D., Tschopp J. Granzymes, a family of serine proteases released from granules of cytolytic T lymphocytes upon T cell receptor stimulation. Immunol Rev. 1988;103:53–71. doi: 10.1111/j.1600-065x.1988.tb00749.x. [DOI] [PubMed] [Google Scholar]
- 12.Rininsland F., Helms T., Asaad R., Boehm B., Tary-Lehmann M., Granzyme B. ELISPOT assay for ex vivo measurements of T cell immunity. J Immunol Methods. 2000;240:143–55. doi: 10.1016/S0022-1759(00)00191-5. [DOI] [PubMed] [Google Scholar]
- 13.Shafer-Weaver K., Sayers T., Strobl S., et al. The Granzyme B ELISPOT assay: an alternative to the 51Cr-release assay for monitoring cell-mediated cytotoxicity. J Transl Med. 2003;1:14. doi: 10.1186/1479-5876-1-14. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 14.Whiteside T. Immunologic monitoring of clinical trials in patients with cancer: technology versus common sense. Immunol Invest. 2000;29:149–62. doi: 10.3109/08820130009062299. [DOI] [PubMed] [Google Scholar]
- 15.Schmittel A., Keilholz U., Thiel E., Scheibenbogen C. Quantification of tumor-specific T lymphocytes with the ELISPOT assay. J Immunother. 2000;23:289–95. doi: 10.1097/00002371-200005000-00001. [DOI] [PubMed] [Google Scholar]
- 16.Asai T., Storkus W., Mueller-Berghaus J., et al. In vitro generated cytolytic T lymphocytes reactive against head and neck cancer recognize multiple epitopes presented by HLA-A2, including peptides derived from the p53 and MDM-2 proteins. Cancer Immun. 2002;2:3. [PubMed] [Google Scholar]
- 17.Malyguine A., Strobl S., Shafer-Weaver K., et al. A Modified Human ELISPOT Assay to Detect Specific Responses to Primary Tumor Cell Targets. J Transl Med. 2004;2:9. doi: 10.1186/1479-5876-2-9. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 18.Bendandi M., Gocke C., Kobrin C., et al. Complete molecular remissions induced by patient-specific vaccination plus granulocyte-monocyte colony-stimulating factor against lymphoma. Nat Med. 1999;5:1171–1177. doi: 10.1038/13928. [DOI] [PubMed] [Google Scholar]
- 19.Derby E., Reddy V., Nelson E., et al. Correlation of human CD56+ cell cytotoxicity and IFN-gamma production. Cytokine. 2001;13:85–90. doi: 10.1006/cyto.2000.0804. [DOI] [PubMed] [Google Scholar]
- 20.Russell J., Ley T. Lymphocyte-mediated cytotoxicity. Annu Rev Immunol. 2002;20:323–70. doi: 10.1146/annurev.immunol.20.100201.131730. [DOI] [PubMed] [Google Scholar]
- 21.Griffiths G. Protein sorting and secretion during CTL killing. Semin Immunol. 1997;9:109–15. doi: 10.1006/smim.1997.0059. [DOI] [PubMed] [Google Scholar]
- 22.Henkart P. CTL effector functions. Semin Immunol. 1997;9:85–6. doi: 10.1006/smim.1997.0064. [DOI] [PubMed] [Google Scholar]
- 23.Smyth M., Street S., Trapani J. Cutting Edge: Granzymes A and B are not essential for perforin-mediated tumor rejection. J Immunol. 2003;171:515–518. doi: 10.4049/jimmunol.171.2.515. [DOI] [PubMed] [Google Scholar]
- 24.Davis J., Smyth M., Trapani J. Granzyme A and B-deficient killer lymphocytes are defective in eliciting DNA fragmentation but retain potent in vivo anti-tumor capacity. Eur J Immunol. 2001;31:39–47. doi: 10.1002/1521-4141(200101)31:1<39::AID-IMMU39>3.0.CO;2-1. [DOI] [PubMed] [Google Scholar]
- 25.Sayers T., Wiltrout T., Sowder R., Munger W., Smyth M., Henderson L. Purification of a factor from the granules of a rat natural killer cell line (RNK) that reduces tumor cell growth and changes tumor morphology. Molecular identity with a granule serine protease (RNKP-1) J Immunol. 1992;148:292–300. [PubMed] [Google Scholar]
- 26.Smyth M., Kelly J., Sutton V., et al. Unlocking the secrets of cytotoxic granule proteins. J Leukoc Biol. 2001;70:18–29. [PubMed] [Google Scholar]
- 27.Kwak L., Pennington R., Boni L., Ochoa A., Robb R., Popescu M. Cutting Edge: Liposomal formulation of a self lymphoma antigen induces potent protective anti-tumor immunity. J Immunol. 1998;160:3637–3461. [PubMed] [Google Scholar]
- 28.Schultze J., Cardoso A., Freeman G., et al. Follicular lymphomas can be induced to present alloantigen efficiently: a conceptual model to improve their tumor immunogenicity. Proc Natl Acad Sci USA. 1995;92:8200–8204. doi: 10.1073/pnas.92.18.8200. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 29.Nuchtern J., Biddison W., Klausner R. Class II MHC molecules can use the endogenous pathway of antigen presentation. Nature. 1990;343:74–6. doi: 10.1038/343074a0. [DOI] [PubMed] [Google Scholar]