A low antigen dose selectively promotes expansion of high-avidity autoreactive T cells with distinct phenotypic characteristics: A study of human autoreactive CD4+T cells specific for GAD65

Abstract

T cells specific for pancreatic islet proteins can be detected in type 1 diabetes patients and at-risk individuals, suggesting a failure of the central tolerance and negative selection. We addressed the question, how antigen dose shapes the diversity of CD4+ autoreactive T cells specific for glutamate decarboxylase 65 (GAD65) in a healthy HLA-DR*0404+ individual, with a persistent GAD65-specific T-cell response. CD4+T cells from this subject were stimulated with decreasing concentrations of the GAD65 555-567 (557I) peptide, and T-cell clones were derived from the tetramer-binding cell population. Functional and structural avidity, TcR-Vβ usage, and cytokine profiles were investigated at a clonal level. T-cell clones established with a low antigen dose (0.1 and 1 μg/ml) displayed higher avidity in contrast to the clones established with the highest antigen dose (10 μg/ml; Mann–Whitney U test, p = 0.003 and 0.006, respectively). The T-cell clones stimulated with the lowest peptide dose also had a higher tetramer-binding affinity than clones stimulated with the highest dose (p = 0.026). The majority (60.0%) of the high-avidity clones expressed TcR-Vβ5.1 chain whereas only one (12.5%) low-avidity clone did. All clones displayed Th0/Th2 cytokine profiles, but intermediate and high-avidity clones produced more IL-10 than low-avidity clones (p = 0.032). The results demonstrate an important role of the antigen dose in the determination of characteristics of the responding T-cell repertoire. High IL-13 and IL-10 production by GAD65-reactive T cells suggests a more anti-inflammatory profile of this healthy individual underlying protection from T1D.

Keywords::

• Antigen dose
• autoreactive T cells
• cytokines
• TcR repertoire
• T1D

Acknowledgments

We thank the blood donors, Dr Xiaoping Wu for expert assistance in single-cell sorting, and Heikki Hölttä for skillful assistance in cytokine detection.

Declaration of interest: This study was supported by grants from American Association of Diabetes, Boehringer Ingelheim Fonds, Finnish Cultural Foundation, Juvenile Diabetes Research Foundation, K. Albin Johanssons Stiftelse, Research and Science Foundation of Farmos, Swedish Cultural Foundation in Finland, Sigrid Jusélius Foundation, and Victoriastiftelsen. The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.