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...
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| Autores principales: | , , , , , , |
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| Formato: | article |
| Lenguaje: | EN |
| Publicado: |
Public Library of Science (PLoS)
2014
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| Materias: | |
| Acceso en línea: | https://doaj.org/article/d11adaf970244ecea3c97633192bcdc9 |
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| Sumario: | 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. |
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