PBMCs were prepared from the blood of T1D participants or healthy participants by Ficoll underlay. CD4+ T cells were isolated using the Miltenyi CD4+ T cell isolation kit, plated in 48-well plates with adherent antigen-presenting cells (APCs), and stimulated with the selected islet-peptide set or a pool of five consecutive peptides of 17 amino acids in length with an 11-residue overlap spanning the H1HA and H1NP protein sequences. The cells were cultured for 2 weeks in the presence of T cell medium and IL-2 (Roche; added every 24 to 48 hours starting on day 7) at 37°C and stained with PE tetramers. Subsequently, cells were stained with CD4 PerCP (BD Biosciences), CD3 FITC (fluorescein isothiocyanate), and CD25 APC monoclonal antibodies (eBioscience) and analyzed by flow cytometry. Cells from pools that gave positive staining were analyzed again with the corresponding individual peptide tetramers to identify each antigenic peptide. Our criterion for positivity was distinct staining that labeled a compact tetramer-positive CD4+ population and was more than threefold above background (cells from an unstimulated well) in the same experiment.

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