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High Efficiency Ex Vivo Cloning of Antigen-Specific Human Effector T Cells
While cloned T cells are valuable tools for the exploration of immune responses against viruses and tumours, current cloning methods do not allow inferences to be made about the function and phenotype of a clone's in vivo precursor, nor can precise cloning efficiencies be calculated. Additional...
Autores principales: | , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Public Library of Science
2014
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4219695/ https://www.ncbi.nlm.nih.gov/pubmed/25368986 http://dx.doi.org/10.1371/journal.pone.0110741 |
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author | Neller, Michelle A. Lai, Michael H.-L. Lanagan, Catherine M. O′Connor, Linda E. Pritchard, Antonia L. Martinez, Nathan R. Schmidt, Christopher W. |
author_facet | Neller, Michelle A. Lai, Michael H.-L. Lanagan, Catherine M. O′Connor, Linda E. Pritchard, Antonia L. Martinez, Nathan R. Schmidt, Christopher W. |
author_sort | Neller, Michelle A. |
collection | PubMed |
description | While cloned T cells are valuable tools for the exploration of immune responses against viruses and tumours, current cloning methods do not allow inferences to be made about the function and phenotype of a clone's in vivo precursor, nor can precise cloning efficiencies be calculated. Additionally, there is currently no general method for cloning antigen-specific effector T cells directly from peripheral blood mononuclear cells, without the need for prior expansion in vitro. Here we describe an efficient method for cloning effector T cells ex vivo. Functional T cells are detected using optimised interferon gamma capture following stimulation with viral or tumour cell-derived antigen. In combination with multiple phenotypic markers, single effector T cells are sorted using a flow cytometer directly into multi-well plates, and cloned using standard, non antigen-specific expansion methods. We provide examples of this novel technology to generate antigen-reactive clones from healthy donors using Epstein-Barr virus and cytomegalovirus as representative viral antigen sources, and from two melanoma patients using autologous melanoma cells. Cloning efficiency, clonality, and retention/loss of function are described. Ex vivo effector cell cloning provides a rapid and effective method of deriving antigen-specific T cells clones with traceable in vivo precursor function and phenotype. |
format | Online Article Text |
id | pubmed-4219695 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2014 |
publisher | Public Library of Science |
record_format | MEDLINE/PubMed |
spelling | pubmed-42196952014-11-12 High Efficiency Ex Vivo Cloning of Antigen-Specific Human Effector T Cells Neller, Michelle A. Lai, Michael H.-L. Lanagan, Catherine M. O′Connor, Linda E. Pritchard, Antonia L. Martinez, Nathan R. Schmidt, Christopher W. PLoS One Research Article While cloned T cells are valuable tools for the exploration of immune responses against viruses and tumours, current cloning methods do not allow inferences to be made about the function and phenotype of a clone's in vivo precursor, nor can precise cloning efficiencies be calculated. Additionally, there is currently no general method for cloning antigen-specific effector T cells directly from peripheral blood mononuclear cells, without the need for prior expansion in vitro. Here we describe an efficient method for cloning effector T cells ex vivo. Functional T cells are detected using optimised interferon gamma capture following stimulation with viral or tumour cell-derived antigen. In combination with multiple phenotypic markers, single effector T cells are sorted using a flow cytometer directly into multi-well plates, and cloned using standard, non antigen-specific expansion methods. We provide examples of this novel technology to generate antigen-reactive clones from healthy donors using Epstein-Barr virus and cytomegalovirus as representative viral antigen sources, and from two melanoma patients using autologous melanoma cells. Cloning efficiency, clonality, and retention/loss of function are described. Ex vivo effector cell cloning provides a rapid and effective method of deriving antigen-specific T cells clones with traceable in vivo precursor function and phenotype. Public Library of Science 2014-11-04 /pmc/articles/PMC4219695/ /pubmed/25368986 http://dx.doi.org/10.1371/journal.pone.0110741 Text en © 2014 Neller 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 Neller, Michelle A. Lai, Michael H.-L. Lanagan, Catherine M. O′Connor, Linda E. Pritchard, Antonia L. Martinez, Nathan R. Schmidt, Christopher W. High Efficiency Ex Vivo Cloning of Antigen-Specific Human Effector T Cells |
title | High Efficiency Ex Vivo Cloning of Antigen-Specific Human Effector T Cells |
title_full | High Efficiency Ex Vivo Cloning of Antigen-Specific Human Effector T Cells |
title_fullStr | High Efficiency Ex Vivo Cloning of Antigen-Specific Human Effector T Cells |
title_full_unstemmed | High Efficiency Ex Vivo Cloning of Antigen-Specific Human Effector T Cells |
title_short | High Efficiency Ex Vivo Cloning of Antigen-Specific Human Effector T Cells |
title_sort | high efficiency ex vivo cloning of antigen-specific human effector t cells |
topic | Research Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4219695/ https://www.ncbi.nlm.nih.gov/pubmed/25368986 http://dx.doi.org/10.1371/journal.pone.0110741 |
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