IFN gamma-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(+)IFN gamma(+) 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(+)IFN gamma(+) PBL separated from PMA/Ionomycin-stimulated PBL of healthy controls inhibited secondary cell cultures of autologous PBL Furthermore, CD4(+)CD25(+)IFN gamma(+) 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 IFN gamma(+) 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 IFN gamma(+) 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 IFN gamma(+) iTreg in specific pretransplant MLC. Quantity of induced IFN gamma(+) iTreg depends on the strength of the alloresponse and both parameters are inversely associated with long-term graft outcome. (C) 2012 Elsevier B.V. All rights reserved.