Detection of GAD65-specific T-cells by major histocompatibility complex class II tetramers in type 1 diabetic patients and at-risk subjects.

Soluble HLA-DR401 or -DR404 tetramers containing a peptide corresponding to an immunodominant epitope from human GAD65 were used to analyze peripheral blood T-cells of newly diagnosed type 1 diabetic patients and at-risk subjects. Peripheral blood mononuclear cells were expanded on antigen-presenting cells presenting GAD65 peptide and subsequently activated with specific plate-bound class II-peptide monomers. T-cell activation defined in flow cytometry by CD4(high) and/or CD25 markers were observed in all type 1 diabetic patients and some at-risk subjects, but not in normal control subjects. The activated T-cells stained positive with tetramers containing the GAD65 epitope 555-567. Tetramer-positive cells were CD4(high) T-cells with high avidity for an immunodominant GAD65 T-cell epitope. Phenotyping of T-cells utilizing HLA class II tetramers provides a new tool to characterize the autoimmune response in type 1 diabetes.