T cell clones were generated by staining cultured T cells with tetramers, sorting gated tetramer-positive CD4+ cells using FACSAria (at single-cell purity, applying a singlet gate to the lymphocyte population), and expanding in a 96-well plate in the presence of 1.0 × 105 irradiated PBMCs and phytohemagglutinin (2 μg/ml; Remel Inc., Lenexa, KS). After expansion of each T cell clone into a single well of a 48-well plate, clones were stained again with tetramer and also stimulated in parallel with the corresponding peptides (500 nM), adding HLA-DQ–matched irradiated PBMCs as APCs and measuring thymidine uptake to verify epitope specificity.

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