Requirement for cell-to-cell contact for suppressor activity of GP-iTreg. Transwell experiments were performed in 24-well plates. A total of 106 CFSE-labeled GPIIb/IIIa-reactive CD4+ T cells were stimulated with 105 GPIIb/IIIa-pulsed DCs in the lower chamber. A total of 106 GPIIb/IIIa-specific iTreg activated with 105 GPIIb/IIIa-pulsed DCs were either added directly to the lower chamber or placed in the upper chamber. After 3 days of culture, CFSE-labeled GPIIb/IIIa-reactive T cells in the lower chamber were harvested. The fluorescence intensity of GPIIb/IIIa-reactive T cells was determined by flow cytometry. Proliferation was measured as the percentage of proliferating GP-reactive T cells. Results are representative of 16 independent experiments (A) and shown as mean plus or minus SD with error bars representing SD (B).
