An efficient method for cloning human autoantigen-specific T cells

J Immunol Methods. 2005 Mar;298(1-2):83-92. doi: 10.1016/j.jim.2005.01.001.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Autoantigens / immunology*
  • CD4-Positive T-Lymphocytes / cytology*
  • CD4-Positive T-Lymphocytes / immunology
  • Cell Culture Techniques / methods*
  • Clone Cells / immunology*
  • Cytokines / metabolism
  • Diabetes Mellitus, Type 1 / immunology
  • Flow Cytometry
  • Glutamate Decarboxylase / immunology
  • Humans
  • Polymerase Chain Reaction
  • Proinsulin / immunology

Substances

  • Autoantigens
  • Cytokines
  • Proinsulin
  • Glutamate Decarboxylase