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ELISPOT and functional T cell analyses using HLA mono-specific target cells

Horlock, Claire, Stott, Bryony, Dyson, Julian, Ogg, Graham, McPherson, Tess, Jones, Louise, Sewell, Andrew K., Wooldridge, Linda, Cole, David, Stebbing, Justin and Savage, Philip 2009. ELISPOT and functional T cell analyses using HLA mono-specific target cells. Journal of Immunological Methods 350 (1-2) , pp. 150-160. 10.1016/j.jim.2009.08.011

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Abstract

Simple T cell assays specific for any chosen HLA class I or class II/peptide combination, are of enormous value in cancer immunotherapy, clinical trials, vaccine and infectious disease research. The reliable measurement of T cell activity can be difficult due to the presence of other alleles on target cells, particularly for the non-HLA-A2 alleles, and the varying baseline characteristics of the different APCs employed. In the absence of pulsing with HLA-A2 restricted peptides, T2 cells are functionally HLA class I and II negative. By coating these cells with recombinant HLA peptide complexes, HLA mono-specific cells are produced that present only a defined single epitope, and generate minimal background immune activation. In ELISPOT, intracellular cytokine staining (ICS) and killing assays using T cells specific for HLA-A2/peptide complexes, the HLA mono-specific cells gave comparable results, to those using standard peptide pulsed HLA-A2 positive T2 cells without significant background. Successful T cell assays for non-HLA-A2 T cells were also performed, with PBMCs recognizing HLA-A24 and HLA-DR15/peptide complexes. The data, obtained with ELISPOT, ICS and FACS-based killing assays, all demonstrate high specificity of T cell activity and low levels of background activity. HLA mono-specific cells are simple to prepare, and can be used with any stable recombinant HLA allele/peptide combination; providing a useful system for improved T cell functional analyses across all HLA allotypes. This represents a significant advance in the generation of reliable functional T cell data.

Item Type: Article
Date Type: Publication
Status: Published
Schools: Medicine
Systems Immunity Research Institute (SIURI)
Subjects: Q Science > QR Microbiology > QR180 Immunology
Uncontrolled Keywords: ELISPOT, cytotoxicity, T cell, assays, in vitro, immunotherapy
Publisher: Elsevier
ISSN: 0022-1759
Last Modified: 04 Jun 2017 03:45
URI: http://orca.cf.ac.uk/id/eprint/26016

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