A panel of 17 GAD65250–266-, 13 InsB11–24-, and 16 IA-2957–972–derived peptides with a single alanine substitution was synthesized to determine the antigenic motif. Glycine or phenylalanine substitution was used if the original residue was an alanine. GAD65250–266, InsB11–24, and IA-2957–972 T cell clones obtained from T1D patients were stimulated with 500 nM (for GAD65250–266 and IA-2957–972) or 2.5 μM (for InsB11–24) of wild-type or modified peptides in the presence of irradiated DQ8cis- or DQ8trans-expressing HEK293 cells as APCs. For GAD65250–266 and IA-2957–972, cultures were pulsed after 72 hours of stimulation with 1 μCi of [3H] thymidine and harvested 18 hours later. For InsB11–24, culture supernatants were collected after 48 hours of stimulation and assayed for IFN-γ secretion by cytokine ELISA. The % activity was calculated by dividing the SI of mutated peptides by the SI of the wild-type peptide. All stimulation experiments were performed in the presence of anti-CD28 antibodies (1 μg/ml).

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