title: An efficient method for cloning human autoantigen-specific T cells.
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.
authors.affiliation: Autoimmunity and Transplantation Division, The Walter and Eliza Hall Institute of Medical Research, 1G Royal Parade, Parkville, Victoria 3050, Australia. mannering@wehi.edu.au
authors.forename: Stuart I
authors.initials: SI
authors.lastname: Mannering
authors.forename: James A
authors.initials: JA
authors.lastname: Dromey
authors.forename: Jessica S
authors.initials: JS
authors.lastname: Morris
authors.forename: Daniel J
authors.initials: DJ
authors.lastname: Thearle
authors.forename: Kent P
authors.initials: KP
authors.lastname: Jensen
authors.forename: Leonard C
authors.initials: LC
authors.lastname: Harrison
mesh_terms: D001324:Autoantigens / Q000276:immunology*; D015496:CD4-Positive T-Lymphocytes / Q000166:cytology* / Q000276:immunology; D018929:Cell Culture Techniques / Q000379:methods*; D002999:Clone Cells / Q000276:immunology*; D016207:Cytokines / Q000378:metabolism; D003922:Diabetes Mellitus, Type 1 / Q000276:immunology; D005434:Flow Cytometry; D005968:Glutamate Decarboxylase / Q000276:immunology; D006801:Humans; D016133:Polymerase Chain Reaction; D011384:Proinsulin / Q000276:immunology
chemical_list: D001324:Autoantigens; D016207:Cytokines; D011384:Proinsulin; D005968:Glutamate Decarboxylase