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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.

ZnT8-Specific CD4+ T Cells Display Distinct Cytokine Expression Profiles between Type 1 Diabetes Patients and Healthy Adults