CD4+CD25+Foxp3+IFNγ+CD178+ human induced Treg (iTreg) contribute to suppression of alloresponses by apoptosis of responder cells

Induced Treg with the phenotype CD4+CD25+Foxp3+IFNγ+ 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+IFNγ+Foxp3+ and CD4+CD25+IFNγ+ 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+IFNγ+Foxp3+ and CD4+CD25+IFNγ+ co-expressing CD95, CD152, CD178, CD279, Granzyme A, Granzyme B, Perforin, IL-10, and TGFβ1. 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+IFNγ+CD178+ iTreg. CD4+CD25+IFNγ+ 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+IFNγ+ and CD4+CD25+CD178+ PBL and third-party cells as stimulator.CD4+CD25+Foxp3+IFNγ+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.