ZnT8-Specific CD4+ T cells display distinct cytokine expression profiles between type 1 diabetes patients and healthy adults

PLoS One. 2013;8(2):e55595. doi: 10.1371/journal.pone.0055595. Epub 2013 Feb 4.

Abstract

Determination of antigen-specific T cell repertoires in human blood has been a challenge. Here, we show a novel integrated approach that permits determination of multiple parameters of antigen-specific T cell repertoires. The approach consists of two assays: the Direct assay and the Cytokine-driven assay. Briefly, human PBMCs are first stimulated with overlapping peptides encoding a given antigen for 48 hours to measure cytokine secretion (Direct assay). Peptide-reactive T cells are further expanded by IL-2 for 5 days; and after overnight starvation, expanded cells are stimulated with the same peptides from the initial culture to analyze cytokine secretion (Cytokine-driven assay). We first applied this integrated approach to determine the type of islet-antigen-specific T cells in healthy adults. Out of ten donors, the Direct assay identified GAD65-specific CD4(+) T cells in three adults and zinc transporter 8 (ZnT8)-specific CD4(+) T cells in five adults. The intracytoplasmic cytokine staining assay showed that these islet-antigen-specific CD4(+) T cells belonged to the CD45RO(+) memory compartment. The Cytokine-driven assay further revealed that islet-antigen-specific CD4(+) T cells in healthy adults were capable of secreting various types of cytokines including type 1 and type 2 cytokines as well as IL-10. We next applied our integrated assay to determine whether the type of ZnT8-specific CD4(+) T cells is different between Type 1 diabetes patients and age/gender/HLA-matched healthy adults. We found that ZnT8-specific CD4(+) T cells were skewed towards Th1 cells in T1D patients, while Th2 and IL-10-producing cells were prevalent in healthy adults. In conclusion, the Direct assay and the Cytokine-driven assay complement each other, and the combination of the two assays provides information of antigen-specific T cell repertoires on the breadth, type, and avidity. This strategy is applicable to determine the differences in the quality of antigen-specific T cells between health and disease.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adult
  • Antigens, Differentiation, T-Lymphocyte / genetics*
  • Antigens, Differentiation, T-Lymphocyte / immunology
  • Case-Control Studies
  • Cation Transport Proteins / genetics*
  • Cation Transport Proteins / immunology
  • Cell Proliferation / drug effects
  • Cytokines / biosynthesis*
  • Cytokines / immunology
  • Diabetes Mellitus, Type 1 / genetics*
  • Diabetes Mellitus, Type 1 / immunology
  • Diabetes Mellitus, Type 1 / pathology
  • Female
  • Gene Expression / drug effects
  • Gene Expression / immunology*
  • Glutamate Decarboxylase / genetics
  • Glutamate Decarboxylase / immunology
  • Humans
  • Immunoassay
  • Immunologic Memory / genetics
  • Immunologic Memory / immunology
  • Immunophenotyping
  • Interleukin-2 / pharmacology
  • Lymphocyte Activation / drug effects
  • Male
  • Middle Aged
  • Peptides / immunology
  • Peptides / pharmacology
  • Th1 Cells / drug effects
  • Th1 Cells / immunology
  • Th1 Cells / pathology
  • Th1-Th2 Balance / drug effects
  • Th2 Cells / drug effects
  • Th2 Cells / immunology
  • Th2 Cells / pathology
  • Zinc Transporter 8

Substances

  • Antigens, Differentiation, T-Lymphocyte
  • Cation Transport Proteins
  • Cytokines
  • Interleukin-2
  • Peptides
  • SLC30A8 protein, human
  • Zinc Transporter 8
  • Glutamate Decarboxylase
  • glutamate decarboxylase 2

Grant support

This study was supported by funding from Juvenile Diabetes Research Foundation (grant number: 17-2010-406; http://www.jdrf.org) and Baylor Health Care System. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.