Stable Aβ-specific Treg generation by in vivo and in vitro Aβ immune stimulation. (A) Experimental scheme for the in vivo depletion of Tregs and in vitro Aβ antigen-specific Treg production and culture. (B) To compare suppressive capacities, PBS+, Aβ+, and KLH+ Tregs were cocultured with e670-labeled Teffs at ratios of 4:1, 2:1 and 1:1 (Teff:Treg). After 3 days, the percent suppression was analyzed. (C) Intracellular cytokine and transcription factor ecpressed by PBS+ and Aβ+ Tregs were analyzed by flow cytometry. Tregs were stimulated 12 h with PMA and inomycin in the present of brefeldin A. (D) Representative immunoblot and (E) quantification for 42 different cytokines and chemokines extracellularly secreted from PBS+ and Aβ+ Tregs after stimulation PMA and inomycin. (F) Tregs were labeled with anti- CD62L, CD69, CD127, PD-1, GITR, CTLA4, IL-10, Ki-67, and Helios antibodies and analyzed by flow cytometry. Data are presented as the mean ± SEM. n = 3-5; *P < 0.05, **P < 0.01.