SpringerProtocols: Abstract: Use of Interferon-γ ELISPOT in Monitoring Immune Responses in Humans

Use of Interferon-γ ELISPOT in Monitoring Immune Responses in Humans

By: Mark Matijevic², Robert G. Urban²

Abstract

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The interferon (IFN)-γ enzyme-linked immunospot (ELISPOT) assay has become a useful tool for immunologists seeking to quantify immune responses on a per-cell basis. The assay is sensitive and allows for the enumeration of low-frequency T-cells. Many have applied this assay to clinical trials as a way to measure biological activity in a patient cohort. It is critical that each laboratory attempting to use the assay in their facility perform rigorous development and qualification work to establish an assay that suits their particular needs. This chapter serves as a demonstration of two practical and slightly different approaches to using the ELISPOT assay to monitor immune activity in the human periphery: (1) assays using whole samples of peripheral blood mononuclear cells with and without the use of additional antigen presenting cells and (2) assays using enriched T-cell populations. Detailed protocols and procedures will be covered, as well as a demonstration of results obtained from three separate applications.

Affiliation(s): (2) MGI Pharma Biologics, Lexington, MA

Book Title: Handbook of ELISPOT: Methods and Protocols

Series: Methods in Molecular Biology | Volume: 302 | Pub. Date: Mar-15-2005 | Page Range: 237-251 | DOI: 10.1385/1-59259-903-6:237

Subject: Immunology

Key Words: ELISPOT - PBMCs - clinical trial - immune response - IFN-γ

References

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1.	Pittet M. J., Zippelius A., Speiser D. E., Assenmacher M., Guillaume P., Valmori D., Lienard D., Lejeune F., Cerottini J.-C., and Romero P. (2001) Ex vivo IFN-γ secretion by circulating CD8 T-lymphocytes: implications of a novel approach for T-cell monitoring in infectious and malignant diseases. J. Immunol. 166, 7634–7640.

2.	Schmittel A., Keilholz U., Bauer S., Kuhne U., Stevanovich S., Thiel E., et al. (2001) Application of the IFN-γ ELISPOT assay to quantify T-cell responses against proteins. J. Immunol. Methods. 247, 17–24.

3.	Schmittel A., Keilholz U., and Scheibenbogen C. (1997) Evaluation of the IFN-γ ELISPOT-assay for quantification of peptide specific T-lymphocytes from peripheral blood. J. Immunol. Methods. 210, 167–174.

4.	Di Fabio S., Mbawuike I. N., Kiyono H., Fujihashi K., Couch R. B., and McGhee J. R. (1994) Quantitation of human influenza virus-specific cytotoxic T-lymphocytes: correlation of cytotoxicity and increased numbers of IFN-γ producing CD8₊ T-cells. Int. Immunol. 6, 11–19.

5.	McCutcheon M., Wehner N., Wensky A., Kushner M., Doan S., Hsiao L., et al. (1997) A sensitive ELISPOT assay to detect low frequency human T-lymphocytes. J. Immunol. Methods. 210, 149–166.

6.	Larsson M., Jin X., Ramratnam B., Ogg G. S., Engelmayer J., Demoitie M. A., 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.

7.	Kumar A., Weiss W., Tine J. A., Hoffman S. L., and Rogers W. O. (2001) ELISPOT assay for detection of peptide specific IFN-γ secreting cells in rhesus macaques. J. Immunol. Methods. 247, 49–60.

8.	Corne P., Huguet M. F., Briant L., Segondy M., Reynes J., and Vendrell J. P. (1999) Detection and enumeration of HIV-1 producing cells by ELISPOT (enzyme-linked immunospot) assay. J. Acquir. Immune Defic. Syndr. Hum. Retrovirol. 20, 442–447

9.	Griffioen M., Borghi M., Schrier P. I., and Osanto S. (2001) Detection and quantification of CD8₊ T-cells specific for HLA-A0201-binding melanoma and viral peptides by the IFN-γ ELISPOT assay. Int. J. Cancer.* 93, 549–555.

10.	Clay T. M., Hobeika A. C., Mosca P. J., Lyerly H. K., and Morse M. A. (2001) Assays for monitoring cellular immune responses to active immunotherapy of cancer. Clin. Cancer Res. 7, 1127–1135.

11.	Klencke B., Matijevic M., Urban R. G., Lathey J. L., Hedley M. L., Berry M., et al. (2002) Encapsulated plasmid DNA treatment for human papillomavirus 16-asso-ciated anal dysplasia: a phase I study of ZYC101. Clin. Cancer Res. 8, 1028–1037.

12.	Pass H. A., Schwarz S. L., Wunderlich J. R., and Rosenberg S. A. (1998) Immunization of patients with melanoma peptide vaccines: immunologic assessment using the ELISPOT assay. Cancer J. Sci. Am. 4, 316–323.

13.	Lewis J. J., Janetzki S., Schaed S., Panageas K. S., Wang S., Williams L., et al. (2000) Evaluation of CD8(₊) T-cell frequencies by the Elispot assay in healthy individuals and in patients with metastatic melanoma immunized with tyrosinaseide. Int. J. Cancer. 87, 391–398.

14.	Mwau M., McMichael A. J., and Hanke T. (2002) Design and validation of an enzyme-linked immunospot assay for use in clinical trials of candidate HIV vaccines. AIDS Res. Human Retroviruses. 18, 611–618.

15.	Smith J. G., Liu X., Kaufhold R. M., Clair J., and Caulfield M. J. (2001) Development and validation of a gamma interferon ELISPOT assay for quantification of cellular immune responses to varicella-zoster virus. Clin. Diagn. Lab. Immunol. 8, 871–879.

16.	Lathey J. L., Sathiyaseelan J., Matijevic M., and Hedley M. L. (2003) Validation of pretrial ELISPOT measurements for predicting assay performance during a clinical trial. BioProcess Int. September, 34–41.

17.	Flynn K., Beltz G., Altman J., Ahmed R., Woodland D., and Doherty P. (1998) Virus-specific CD8₊ T-cells in primary and secondary influenza pneumonia. Immunity. 8, 683–691.

18.	Anderson K., Cresswell P., Gammon M., Hermes J., Williamson A., and Zweerink H. (1991) Endogenously synthesized peptide with an endoplasmic reticulum signal sequence sensitizes antigen processing mutant cells to class I-restricted, cell-mediated lysis. J. Exp. Med. 174, 489–492.

19.	Currier J. R., Kuta E. G., Turk E., Earhart L. B., Loomis-Price L., Janetzki S., 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.

20.	Britten C. M., Meyer R. G., Kreer T., Drexler I., Wolfel T., and Herr W. (2002) The use of HLA-A0201-transfected K562 as standard antigen-presenting cells for CD8₊ T-lymphocytes in IFN-γ ELISPOT assays. J. Immunol. Methods.* 259, 95–110.

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