Low-avidity T cells are not stained but are activated by GAD-pulsed APCs and soluble GAD TMr. (A) ³H-TdR incorporation was measured on GAD TMr^high and GAD TMr^low T cells cultured for 60 hours on B cells pulsed with either control peptide (□) or with increasing concentrations of GAD_555-567 peptide (▦, ▪). As positive control, anti-CD3 and anti-CD28 mAbs (5 μg/mL each) were added to unpulsed B cells (▧). (B) High-avidity (GAD TMr^high) and low-avidity (GAD TMr^low) T cells were stimulated with 10 μg/mL GAD (black line) or control (dashed line) TMr for 3 hours at 37°C and IFN-γ secretion measured with a capture assay. Percents of GAD TMr-stimulated IFN-γ⁺ cells are indicated. (C) T cells were stimulated as described with GAD (▪) or control (□) TMrs and stained with a fluorochrome-conjugated anti-CD69 mAb, and the percent increase in CD69⁺ cells was calculated. The mean ± SE of 2 independent experiments is shown.