title: CD4+ CD25+ Foxp3+ IFNγ+ CD178+ human induced Treg (iTreg) contribute to suppression of alloresponses by apoptosis of responder cells.
abstract: 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 TGFβ(1). CD178(+) iTreg increased within 3h 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.
authors.affiliation: Department of Transplantation-Immunology, Institute of Immunology, University of Heidelberg, Im Neuenheimer Feld 305, 69120 Heidelberg, Germany. Volker.Daniel@med.uni-heidelberg.de
authors.forename: Volker
authors.initials: V
authors.lastname: Daniel
authors.forename: Mahmoud
authors.initials: M
authors.lastname: Sadeghi
authors.forename: Haihao
authors.initials: H
authors.lastname: Wang
authors.forename: Gerhard
authors.initials: G
authors.lastname: Opelz
mesh_terms: D000911:Antibodies, Monoclonal / Q000276:immunology / Q000494:pharmacology; D015703:Antigens, CD / Q000378:metabolism; D017209:Apoptosis / Q000187:drug effects / Q000276:immunology*; D015704:CD4 Antigens / Q000378:metabolism; D002462:Cell Membrane / Q000378:metabolism; D002478:Cells, Cultured; D053222:Fas Ligand Protein / Q000276:immunology / Q000378:metabolism; D051858:Forkhead Transcription Factors / Q000378:metabolism; D053804:Granzymes / Q000378:metabolism; D006801:Humans; D007371:Interferon-gamma / Q000276:immunology / Q000378:metabolism; D053645:Interleukin-2 Receptor alpha Subunit / Q000378:metabolism; D042541:Intracellular Space / Q000378:metabolism; D007959:Lymphocyte Culture Test, Mixed; D054353:Perforin / Q000378:metabolism; D017471:Receptors, Interferon / Q000378:metabolism; D050378:T-Lymphocytes, Regulatory / Q000187:drug effects / Q000276:immunology* / Q000378:metabolism*; D019014:fas Receptor / Q000276:immunology / Q000378:metabolism; D000096743:Interferon gamma Receptor
chemical_list: D000911:Antibodies, Monoclonal; D015703:Antigens, CD; D015704:CD4 Antigens; D053222:Fas Ligand Protein; D051858:Forkhead Transcription Factors; D053804:Granzymes; D007371:Interferon-gamma; D053645:Interleukin-2 Receptor alpha Subunit; D054353:Perforin; D017471:Receptors, Interferon; D019014:fas Receptor; D000096743:Interferon gamma Receptor; C418974:FOXP3 protein, human