Journal
CLINICAL IMMUNOLOGY
Volume 157, Issue 2, Pages 216-225Publisher
ACADEMIC PRESS INC ELSEVIER SCIENCE
DOI: 10.1016/j.clim.2015.02.012
Keywords
ELISpot; CD8(+) T cells; Autoantigen; MHC class I; Diabetes
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Funding
- German Federal Ministry of Education and Research (BMBF) [82DZD00101]
- DFG Research Center and Cluster of Excellence-Center for Regenerative Therapies Dresden [FZ 111]
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Quantification of antigen-specific CD8(+) T cells is important for monitoring infection, vaccination, and response to therapy in cancer and immune-mediated diseases. Cytokine enzyme-linked-immunospot (ELISpot) assays are often used for this purpose. We found that substantial spot formation in IFN gamma ELISpot assays occurred independently of CD8(+) T cells even when classical MHC class I restricted peptides are used for stimulation. Using fractionated cells and intracellular cytokine staining, the non-CD8(+) T cell IFN-gamma production was attributed to the CD4(+) T cell fraction. We therefore refined a cell line-based ELISpot assay combining HLA-A*0201 expressing K562 cells for antigen presentation with purified CD8(+) T cells and demonstrated that it specifically detected CD8(+) T cell responses with detection limits comparable to traditional ELISpot assays and dextramer-based quantification. The assay was further adapted to whole antigen responses with antigen (pre-proinsulin)-expressing HLA-A*0201K562 cells. Thus, we revealed and corrected a weak spot of the CD8(+) ELISpot assay. (C) 2015 Elsevier Inc. All rights reserved.
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