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J Immunol Methods. 2011 Jun 30;369(1-2):33-41. doi: 10.1016/j.jim.2011.04.003. Epub 2011 Apr 8.

Isolation of viable antigen-specific CD8+ T cells based on membrane-bound tumor necrosis factor (TNF)-α expression.

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1
Department of Microbiology, University of Pennsylvania, Philadelphia, PA 19104, USA.

Abstract

Current technology to isolate viable cytokine-producing antigen-specific primary human T cells is limited to bi-specific antibody capture systems, which suffer from limited sensitivity and high background. Here, we describe a novel procedure for isolating antigen-specific human T cells based on their ability to produce tumor necrosis factor (TNF)-α. Unlike many cytokines, TNF-α is initially produced in a biologically active membrane-bound form that is subsequently cleaved by TNF-α converting enzyme (TACE) to release the soluble form of TNF-α. By preventing this cleavage event, we show that TNF-α can be 'trapped' on the surface of the T cells from which it originates and directly labeled for viable isolation of these antigen-specific T cells. Together with other existing sorting procedures to isolate activated T cells, this new technique should permit the direct isolation of multi-functional T lymphocytes for further protein and gene expression analyses, as well as a detailed functional assessment of the potential role that TNF-α producing T cells play in the adaptive immune system.

PMID:
21501617
PMCID:
PMC3116017
DOI:
10.1016/j.jim.2011.04.003
[Indexed for MEDLINE]
Free PMC Article
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