Department of Transplantation-Immunology, Institute of Immunology, University of Heidelberg, Im Neuenheimer Feld 305, 69120 Heidelberg, Germany.
Hum Immunol. 2013 Feb;74(2):151-62. doi: 10.1016/j.humimm.2012.09.010. Epub 2012 Sep 24.
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.
具有表型 CD4(+)CD25(+)Foxp3(+)IFNγ(+)的诱导性调节性 T 细胞（Treg）与肾移植受者的长期移植物预后良好相关，并在体外抑制同种异体 T 细胞反应。在本研究中，我们研究了细胞凋亡和 Fas/FasL 依赖性途径是否有助于抑制 T 细胞激活。使用来自健康对照者和四色流式细胞术、PMA/离子霉素刺激的 PBL 和 MLC 的 PBL 的早期凋亡和坏死率以及共表达的免疫刺激和免疫抑制蛋白，分析了 CD4(+)CD25(+)Foxp3(+)、CD4(+)IFNγ(+)Foxp3(+)和 CD4(+)CD25(+)IFNγ(+)PBL 中的 CD4(+)CD25(+)Foxp3(+)、CD4(+)IFNγ(+)Foxp3(+)和 CD4(+)CD25(+)IFNγ(+)PBL 的早期凋亡和坏死率以及共表达的免疫刺激和免疫抑制蛋白。PMA/离子霉素刺激 16 小时诱导具有表型 CD4(+)CD25(+)Foxp3(+)、CD4(+)IFNγ(+)Foxp3(+)和 CD4(+)CD25(+)IFNγ(+)的 iTreg 亚群共表达 CD95、CD152、CD178、CD279、Granzyme A、Granzyme B、穿孔素、IL-10 和 TGFβ(1)。PMA/离子霉素刺激后 3 小时内，CD178(+)iTreg 增加，与早期凋亡的 Annexin(+) / PI(-) PBL 平行，表明 CD178 通过 CD4(+)CD25(+)Foxp3(+)IFNγ(+)CD178(+)iTreg 介导应答细胞的凋亡。从原代细胞培养物中分离的 CD4(+)CD25(+)IFNγ(+)和 CD4(+)CD25(+)CD178(+)PBL 并添加到自体 PMA/离子霉素刺激的次级细胞培养物中，可立即诱导细胞凋亡。如次级 MLC 中分离的 CD4(+)CD25(+)IFNγ(+)和 CD4(+)CD25(+)CD178(+)PBL 与第三方细胞作为刺激物所示，早期凋亡不是抗原特异性的。CD4(+)CD25(+)Foxp3(+)IFNγ(+)CD178(+)iTreg 在细胞刺激后分化，并诱导体外激活的 CD95(+)应答/效应细胞的抗原非特异性凋亡，这可能有助于 iTreg 介导的 T 细胞激活抑制。
