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Linear Fidelity in Quantification of Anti-Viral CD8(+) T Cells
Enumeration of anti-viral CD8(+) T cells to make comparisons between mice, viruses and vaccines is a frequently used approach, but controversy persists as to the most appropriate methods. Use of peptide-MHC tetramers (or variants) and intracellular staining for cytokines, in particular IFNγ, after a...
Autores principales: | , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Public Library of Science
2012
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3379996/ https://www.ncbi.nlm.nih.gov/pubmed/22745779 http://dx.doi.org/10.1371/journal.pone.0039533 |
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author | Flesch, Inge E. A. Hollett, Natasha A. Wong, Yik Chun Tscharke, David C. |
author_facet | Flesch, Inge E. A. Hollett, Natasha A. Wong, Yik Chun Tscharke, David C. |
author_sort | Flesch, Inge E. A. |
collection | PubMed |
description | Enumeration of anti-viral CD8(+) T cells to make comparisons between mice, viruses and vaccines is a frequently used approach, but controversy persists as to the most appropriate methods. Use of peptide-MHC tetramers (or variants) and intracellular staining for cytokines, in particular IFNγ, after a short ex vivo stimulation are now common, as are a variety of cytotoxicity assays, but few direct comparisons have been made. It has been argued that use of tetramers leads to the counting of non-functional T cells and that measurement of single cytokines will fail to identify cells with alternative functions. Further, the linear range of these methods has not been tested and this is required to give confidence that relative quantifications can be compared across samples. Here we show for two acute virus infections and CD8(+) T cells activated in vitro that DimerX (a tetramer variant) and intracellular staining for IFNγ, alone or in combination with CD107 to detect degranulation, gave comparable results at the peak of the response. Importantly, these methods were highly linear over nearly two orders of magnitude. In contrast, in vitro and in vivo assays for cytotoxicity were not linear, suffering from high background killing, plateaus in maximal killing and substantial underestimation of differences in magnitude of responses. |
format | Online Article Text |
id | pubmed-3379996 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2012 |
publisher | Public Library of Science |
record_format | MEDLINE/PubMed |
spelling | pubmed-33799962012-06-28 Linear Fidelity in Quantification of Anti-Viral CD8(+) T Cells Flesch, Inge E. A. Hollett, Natasha A. Wong, Yik Chun Tscharke, David C. PLoS One Research Article Enumeration of anti-viral CD8(+) T cells to make comparisons between mice, viruses and vaccines is a frequently used approach, but controversy persists as to the most appropriate methods. Use of peptide-MHC tetramers (or variants) and intracellular staining for cytokines, in particular IFNγ, after a short ex vivo stimulation are now common, as are a variety of cytotoxicity assays, but few direct comparisons have been made. It has been argued that use of tetramers leads to the counting of non-functional T cells and that measurement of single cytokines will fail to identify cells with alternative functions. Further, the linear range of these methods has not been tested and this is required to give confidence that relative quantifications can be compared across samples. Here we show for two acute virus infections and CD8(+) T cells activated in vitro that DimerX (a tetramer variant) and intracellular staining for IFNγ, alone or in combination with CD107 to detect degranulation, gave comparable results at the peak of the response. Importantly, these methods were highly linear over nearly two orders of magnitude. In contrast, in vitro and in vivo assays for cytotoxicity were not linear, suffering from high background killing, plateaus in maximal killing and substantial underestimation of differences in magnitude of responses. Public Library of Science 2012-06-20 /pmc/articles/PMC3379996/ /pubmed/22745779 http://dx.doi.org/10.1371/journal.pone.0039533 Text en Flesch et al. http://creativecommons.org/licenses/by/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited. |
spellingShingle | Research Article Flesch, Inge E. A. Hollett, Natasha A. Wong, Yik Chun Tscharke, David C. Linear Fidelity in Quantification of Anti-Viral CD8(+) T Cells |
title | Linear Fidelity in Quantification of Anti-Viral CD8(+) T Cells |
title_full | Linear Fidelity in Quantification of Anti-Viral CD8(+) T Cells |
title_fullStr | Linear Fidelity in Quantification of Anti-Viral CD8(+) T Cells |
title_full_unstemmed | Linear Fidelity in Quantification of Anti-Viral CD8(+) T Cells |
title_short | Linear Fidelity in Quantification of Anti-Viral CD8(+) T Cells |
title_sort | linear fidelity in quantification of anti-viral cd8(+) t cells |
topic | Research Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3379996/ https://www.ncbi.nlm.nih.gov/pubmed/22745779 http://dx.doi.org/10.1371/journal.pone.0039533 |
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