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An efficient method for cloning human autoantigen-specific T cells.

Stuart I Mannering·James A Dromey·Jessica S Morris·Daniel J Thearle·Kent P Jensen·Leonard C Harrison
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Abstract

T-cell clones are valuable tools for investigating T-cell specificity in infectious, autoimmune and malignant diseases. T cells specific for clinically-relevant autoantigens are difficult to clone using traditional methods. Here we describe an efficient method for cloning human autoantigen-specific CD4+ T cells pre-labelled with CFSE. Proliferating, antigen-responsive CD4+ cells were identified flow cytometrically by their reduction in CFSE staining and single cells were sorted into separate wells. The conditions (cytokines, mitogens and tissue culture plates) for raising T-cell clones were optimised. Media supplemented with IL-2+IL-4 supported growth of the largest number of antigen-specific clones. Three mitogens, PHA, anti-CD3 and anti-CD3+anti-CD28, each stimulated the growth of similar numbers of antigen-specific clones. Cloning efficiency was similar in flat- and round-bottom plates. Based on these findings, IL-2+IL-4, anti-CD3 and round-bottom plates were used to clone FACS-sorted autoantigen-specific CFSE-labelled CD4+ T cells. Sixty proinsulin- and 47 glutamic acid decarboxylase-specific clones were obtained from six and two donors, respectively. In conclusion, the CFSE-based method is ideal for cloning rare, autoantigen-specific, human CD4+ T cells.

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AutoantigensCD4-Positive T-LymphocytesCell Culture TechniquesClone CellsCytokinesDiabetes Mellitus, Type 1Flow CytometryGlutamate DecarboxylaseHumansPolymerase Chain ReactionProinsulinJournal ArticleResearch Support, Non-U.S. Gov't

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Stuart I Mannering, James A Dromey, Jessica S Morris, Daniel J Thearle, Kent P Jensen, Leonard C Harrison. 2005. An efficient method for cloning human autoantigen-specific T cells.. https://doi.org/10.1016/j.jim.2005.01.001

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