Phenotypes of DCs and induced GP-specific CD4⁺CD25⁺Treg. (A) Freshly isolated monocytes cultured for 1 hour (day 0), immature DCs (day 5), and mature DCs (day 7) were analyzed by flow cytometry to determine the levels of expression of CD83, CD86, and HLA-DR. To detect the tolerogenic properties of GP-iTreg-modulated DCs, GPIIb/IIIa-prepulsed immature DCs were cocultured with GPIIb/IIIa-specific iTreg for 2 additional days (day 7). Then DCs were negatively selected by depleting iTreg with CD4 microbeads and analyzed by flow cytometry. Results were representative of 16 independent experiments. (B) Phenotypes of the induced GP-specific CD4⁺CD25⁺ Treg from ITP patients. CD4⁺CD25⁻CD45RA⁺ T cells isolated from the PBMCs of ITP patients were stimulated with mDCs prepulsed with trypsin-digested GPIIb/IIIa or GPIb/IX in the presence or absence of TGF-β1 (5 ng/mL). Seven days later, the CD4⁺CD25⁺ cells were purified by positive selection and analyzed for the expression of Foxp3, CTLA-4, GITR, and CD25.