Vagaries of the ELISpot assay : specific detection of antigen responsive cells requires purified CD8(+) T cells and MHC class I expressing antigen presenting cell lines
Copyright © 2015 Elsevier Inc. All rights reserved.
| Veröffentlicht in: | Clinical immunology (Orlando, Fla.). - 1999. - 157(2015), 2 vom: 15. Apr., Seite 216-25 |
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| 1. Verfasser: | |
| Weitere Verfasser: | , , , , , , , |
| Format: | Online-Aufsatz |
| Sprache: | English |
| Veröffentlicht: |
2015
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| Zugriff auf das übergeordnete Werk: | Clinical immunology (Orlando, Fla.) |
| Schlagworte: | Journal Article Research Support, Non-U.S. Gov't Autoantigen CD8(+) T cells Diabetes ELISpot MHC class I HLA-A*02:01 antigen HLA-A2 Antigen Histocompatibility Antigens Class I mehr... |
| Zusammenfassung: | Copyright © 2015 Elsevier Inc. All rights reserved. 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 IFNγ 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 IFNγ 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 |
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| Beschreibung: | Date Completed 29.06.2015 Date Revised 27.04.2015 published: Print-Electronic Citation Status MEDLINE |
| ISSN: | 1521-7035 |
| DOI: | 10.1016/j.clim.2015.02.012 |