Proliferation assays were performed in triplicate in 96-well round-bottom plates. T cell clones were stimulated with irrelevant or specific peptides in the presence of irradiated nontransfected or DQ-transfected HEK293 cells as APCs. After 72-hour stimulation, cultures were pulsed with 1 μCi of [3H] thymidine and harvested 18 hours later. SI was calculated by dividing the net counts of specific peptides (counts per minute of DQ8cis- or DQ8trans-stimulated cells subtracted from counts per minute of nontransfected cells) by the counts per minute of irrelevant peptide. For cytokine enzyme-linked immunosorbent assay (ELISA), IFN-γ (clone MD-1), IL-4 (clone 8D4–8), and IL-10 (clone JES3-19F1) capturing antibodies (BioLegend) were coated onto 96-well round-bottom plates. Supernatants (50 μl) from cultures of T cell clones were collected after 48 hours of stimulation and added to each well. After overnight incubation, bound cytokines were detected by biotinylated anti–IFN-γ (clone 4 s.B3), anti–IL-4 (clone MP4-25D2), and anti–IL-10 (clone JES3-12G8) antibodies (BioLegend) and quantified using a Victor2 D time-resolved fluorometer (PerkinElmer). Unless otherwise stated, peptide concentrations were everywhere 2.5 μM for the wild-type insulin peptide and 500 nM for all other peptides. For half-maximal effective concentration (EC50) determination in Fig. 4B, secreted IFN-γ was measured by ELISA after co-incubation of T cell clones with serial-diluted mutated DQ8cis/trans proteins for 48 hours. For EC50 determination in fig. S4A, various concentrations of each biotinylated peptide were incubated in wells coated with DQ8 and DQ8trans proteins. After washing, the remaining biotinylated peptides were labeled using europium-conjugated streptavidin (PerkinElmer) and quantified using the Victor2 D time-resolved fluorometer (PerkinElmer). EC50 values were calculated via a nonlinear regression model in Prism software. All experiments except EC50 determination in fig. S4A were performed in the presence of anti-CD28 antibodies (1 μg/ml).