An efficient method for cloning human autoantigen-specific T cells
- Author(s)
- Mannering, SI; Dromey, JA; Morris, JS; Thearle, DJ; Jensen, KP; Harrison, LC;
- Details
- Publication Year 2005-03,Volume 298,Issue #1-2,Page 83-92
- Journal Title
- JOURNAL OF IMMUNOLOGICAL METHODS
- Publication Type
- Journal Article
- 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. (c) 2005 Elsevier B.V. All rights reserved.
- Publisher
- ELSEVIER SCIENCE BV
- Keywords
- PROINSULIN EPITOPES; CLONES; IDENTIFICATION; POPULATIONS; ANTIGENS/; PEPTIDE; CD4(+); CHAIN
- Publisher's Version
- https://doi.org/10.1016/j.jim.2005.01.001
- Terms of Use/Rights Notice
- Refer to copyright notice on published article.
Creation Date: 2005-03-01 12:00:00