CD4+CD25+Foxp3+IFNγ+ Treg are immunosuppressive in vitro and increase with intensity of the alloresponse in pretransplant MLC

IFNγ-producing CD3+CD4+CD25+Foxp3+ induced Treg are more frequently detectable in patients with good than in patients with impaired long-term kidney graft function.We investigated the in-vitro function of separated CD3+CD4+CD25+Foxp3+IFNγ+ PBL that were induced by phorbol-12-myristate-13-acetate(PMA)/Ionomycin or alloantigenic stimulation. Additionally, we studied iTreg induction and cell proliferation in MLC with pretransplant obtained PBL.CD4+CD25+IFNγ+ PBL separated from PMA/Ionomycin-stimulated PBL of healthy controls inhibited secondary cell cultures of autologous PBL. Furthermore, CD4+CD25+IFNγ+ PBL separated from primary MLC and added to secondary MLC suppressed allogeneic T-cell activation in secondary MLC unspecifically, irrespective of the stimulator cell. However, the strongest suppression was observed in specific MLC. Patients with poor long-term graft outcome were able to form IFNγ+ iTreg in pretransplant MLC. Eight patients with a serum creatinine level ranging from 0.9 to 14 mg/dl 18–29 years posttransplant were studied. In MLC with pretransplant obtained recipient and donor cells, strong IFNγ+ iTreg (p = 0.007) and strong blast induction (p = 0.047) were associated with impaired long-term graft outcome. Long-term graft outcome was not associated with cell proliferation and iTreg induction in unspecific MLC with third-party cells as stimulator.The data indicate that patients with impaired long-term graft outcome are able to form high numbers of IFNγ+ iTreg in specific pretransplant MLC. Quantity of induced IFNγ+ iTreg depends on the strength of the alloresponse and both parameters are inversely associated with long-term graft outcome.