Detection of antigen specificity of the tolerance induced by GP-iTreg. (A) A total of 2 × 105 GPIIb/IIIa-specific iTreg from 16 ITP patients were cocultured with CFSE-labeled GPIIb/IIIa-reactive T cells (2 × 105) and stimulated with 2 × 104 autologous imDCs (irradiated at 30 Gy) loaded with (i) GPIIb/IIIa, or (ii) both GPIIb/IIIa and GPIb/IX. Meanwhile, GPIIb/IIIa-specific iTreg (2 × 105) were cocultured with CFSE-labeled GPIb/IX-reactive T cells (2 × 105) and stimulated with 2 × 104 DCs loaded (iii) with GPIb/IX, (iv) concurrently with GPIIb/IIIa and GPIb/IX, or (v) separately with GPIIb/IIIa and GPIb/IX. For control purposes, CFSE-labeled (vi) GPIIb/IIIa- or (vii) GPIb/IX-reactive T cells (2 × 105) were cultured with autologous DCs (2 × 104) loaded with the respective antigen. After 3 days of culture, proliferation was measured as the percentage of proliferating GP-reactive T cells and shown as mean plus or minus SD with error bars representing SD. (B) A representative flow cytometric diagram was shown.
