Induced Treg with the phenotype CD4(+)CD25(+)Foxp3(+)IFN gamma(+) were shown to be associated with good long-term graft outcome in renal transplant recipients and inhibition of allogeneic T-cell responses in vitro. In the present study, we investigated whether apoptosis and Fas/FasL-dependent pathways contribute to the inhibition of T-cell activation. Early apoptosis and necrosis rates as well as co-expression of immunostimulatory and immunosuppressive proteins in/on CD4(+)CD25(+)Foxp3(+), CD4(+)IFN gamma(+)Foxp3(+) and CD4(+)CD25(+)IFN gamma(+) PBL were analyzed using cells from healthy controls and four-color flow cytometry, PMA/Ionomycin-stimulated PBL, and MLC. Sixteen hours PMA/Ionomycin stimulation induced iTreg subsets with the phenotype CD4(+)CD25(+)Foxp3(+), CD4(+)IFN gamma(+)Foxp3(+) and CD4(+)CD25(+)IFN gamma(+) co-expressing CD95, CD152, CD178, CD279, Granzyme A, Granzyme B, Perforin, IL-10, and TGF beta(1). CD178(+) iTreg increased within 3 h after PMA/Ionomycin stimulation in parallel to early apoptotic Annexin(+)/PI- PBL, suggesting CD178-mediated apoptosis of responder cells by CD4(+)CD25(+)Foxp3(+)IFN gamma(+)CD178(+) iTreg. CD4(+)CD25(+)IFN gamma(+) and CD4(+)CD25(+)CD178(+) PBL separated from primary cell cultures and added to autologous PMA/Ionomycin stimulated secondary cell cultures induced apoptosis immediately. Early apoptosis was not antigen-specific as shown in secondary MLC with separated CD4(+)CD25(+)IFN gamma(+) and CD4(+)CD25(+)CD178(+) PBL and third-party cells as stimulator. CD4(+)CD25(+)Foxp3(+)IFN gamma(+)CD178(+) iTreg differentiate after cell stimulation and induce antigen-unspecific apoptosis of activated CD95(+) responder/effector cells in vitro that might contribute to iTreg-mediated inhibition of T-cell activation. (c) 2012 American Society for Histocompatibility and Immunogenetics. Published by Elsevier Inc. All rights reserved.