title: Isolation of viable antigen-specific CD8+ T cells based on membrane-bound tumor necrosis factor (TNF)-α expression.
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.
authors.affiliation: Department of Microbiology, University of Pennsylvania, Philadelphia, PA 19104, USA.
authors.forename: Danielle
authors.initials: D
authors.lastname: Haney
authors.forename: Máire F
authors.initials: MF
authors.lastname: Quigley
authors.forename: Tedi E
authors.initials: TE
authors.lastname: Asher
authors.forename: David R
authors.initials: DR
authors.lastname: Ambrozak
authors.forename: Emma
authors.initials: E
authors.lastname: Gostick
authors.forename: David A
authors.initials: DA
authors.lastname: Price
authors.forename: Daniel C
authors.initials: DC
authors.lastname: Douek
authors.forename: Michael R
authors.initials: MR
authors.lastname: Betts
mesh_terms: D018414:CD8-Positive T-Lymphocytes / Q000276:immunology* / Q000378:metabolism; D002462:Cell Membrane; D002469:Cell Separation / Q000379:methods*; D002478:Cells, Cultured; D015395:Histocompatibility Antigens Class I / Q000276:immunology; D006801:Humans; D007694:Killer Cells, Natural / Q000276:immunology; D014409:Tumor Necrosis Factor-alpha / Q000276:immunology* / Q000378:metabolism
chemical_list: D015395:Histocompatibility Antigens Class I; D014409:Tumor Necrosis Factor-alpha