CD4+CD25+Foxp3+IFNg+CD178+ Human Induced Treg (Itreg) Contribute to Suppression of Alloresponses by Apoptosis of Responder Cells

Abstract

Recently, a new subset of induced immunoregulatory T cells (iTreg) that produced IFNg was reported. In vivo, iTreg with the phenotype CD4+CD25+Foxp3+IFNg+ were shown to be associated with good long-term graft outcome in renal transplant recipients. In vitro, CD4+CD25+Foxp3+IFNg+ were shown to inhibit allogeneic T cell responses antigen-unspecifically as well as antigen-specifically. Inhibition was characterized by decreased activation of responder T cells. Others reported that iTreg initiate immunosuppression by apoptosis induction in effector cells. In the present study, we investigated whether apoptosis and Fas/FasL-dependent pathways contribute to the inhibition of T cell activation mediated by IFNg-producing CD4+CD25+Foxp3+ iTreg. Early apoptosis and necrosis rates as well as co-expression of immunostimulatory and immunosuppressive proteins in/on CD4+CD25+Foxp3+, CD4+IFNg+Foxp3+ and CD4+CD25+IFNg+ PBL were analyzed using cells from healthy controls and four-color flow cytometry, PMA/Ionomycin-stimulated PBL, and MLC. 16h PMA/Ionomycin stimulation induced iTreg subsets with the phenotype CD4+CD25+Foxp3+, CD4+IFNg+Foxp3+, and CD4+CD25+IFNg+ co-expressing CD178, CD95, CD152, CD279, Granzyme A, Granzyme B, Perforin, IL-10, and TGFβ1 (all p< 0.05). Using, in addition, five-color fluorescence flow cytometry, approximately 1% of stimulated CD4+ PBL were CD25+Foxp3+IFNg+CD178+ (unstimulated 0%). 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+IFNg+CD178+ iTreg. Apoptosis was inhibited dose-dependently in the presence of increasing concentrations of monoclonal antibody against CD178, CD95, or IFNg (all p< 0.05). CD4+CD25+IFNg+ 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 with CD4+CD25+IFNg+ and CD4+CD25+CD178+ PBL separated from primary MLC and added to secondary MLC with autologous or third-party cells as stimulator. In summary, polyclonal or allogeneic stimulation of PBL induces an iTreg subpopulation with the phenotype CD4+CD25+Foxp3+IFNg+CD178+. These iTreg, when isolated from primary cell cultures, enable immediate apoptosis in stimulated responder and effector cells. We speculate that CD4+CD25+Foxp3+IFNg+ iTreg detected in the blood of renal transplant recipients with good long-term graft outcome induce immunosuppression in part by Fas/FasL-dependent mechanisms. Because CD4+CD25+Foxp3+IFNg+ iTreg express IFNg receptors and are induced by IFNg, they represent the first line of iTreg during an initiating immune response. IFNg-activated effector cells with strongly up-regulated CD95 expression might be the perfect target for this early apoptosis-inducing iTreg subset.