Recently, a new subset of induced regulatory T-cells (iTreg) was detected. IFNg-producing CD3+CD4+CD25+Foxp3+ PBL were shown to have an immunoregulatory role in long-term renal transplant recipients. This iTreg subset is more frequently detectable in the blood of patients with good than in the blood of patients with impaired long-term kidney graft function. In-vitro, separated CD3+CD4+CD25+Foxp3+IFNg+ PBL from primary MLCs of healthy controls suppressed secondary MLCs antigen-unspecifically. In the present study we tried to find indications of antigen-specific MLC suppression mediated by separated CD4+CD25+IFNg+ PBL. PBL of a healthy control (HC1) were stimulated in 6-day-MLC with irradiated PBL of 2 HLA class-I/II incompatible healthy controls (HC2, HC3). CD4+CD25+IFNg+ PBL were separated from primary MLC and added to secondary 6-day-MLC. Secondary MLC HC1 versus HC2 showed 9.3% blasts, secondary MLC HC1 versus HC2 co-cultured with separated CD4+CD25+IFNg+ PBL from primary MLC HC1 versus HC3 exhibited 5.0% blasts, whereas secondary MLC HC1 versus HC2 cocultured with separated CD4+CD25+IFNg+ PBL from primary MLC HC1 versus HC2 exhibited only 1.7% blasts. Similar findings were observed for secondary MLC HC1 versus HC3 co-cultured with corresponding CD4+CD25+IFNg+ PBL from primary MLC: 7.0% blast formation in primary MLC, 2.1% blasts in third-party, and 2.0% blasts in autologous secondary MLC. These data suggest that primary MLC-derived CD4+CD25+IFNg+ PBL inhibit the allogeneic immune response in secondary MLC unspecifically. However, the strongest suppression was observed in antigen-specific MLC/iTreg combinations, suggesting antigen-specific suppression by a subset of separated CD4+CD25+IFNg+ PBL. Next, we studied whether patients with good long-term graft outcome (n=3; current serum creatinine 0.9-1.3 mg/dl) might show a higher frequency of iTreg in MLC than patients with intermediate (n=3, current serum creatinine 1.8-2.7 mg/dl) or poor long-term graft function (n=2, current serum creatinine 11 and 14 mg/dl). All patients were studied >15 years posttransplant (range: 18 - 29 years). Recipients shared a maximum of 3 HLA class I/II mismatches with the original graft donor. PBL obtained prior to transplantation and stored in liquid nitrogen were used. Recipient PBL were stimulated with either donor or pooled third-party PBL for 6 days. Then, CD4+CD25+ PBL co-expressing Foxp3+IFN-g+, GARP+Foxp3+, or GARP+IFNg+ were determined using four-color fluorescence flow-cytometry. Cell proliferation was measured using CFSE-staining of responder cells. Patients with impaired long-term graft outcome showed a stronger anti-donor response than patients with good long-term graft outcome. They exhibited stronger cell proliferation in recipient/donor MLC and increased iTreg induction in recipient/donor as well as recipient/third-party MLC. Thus, strong iTreg and blast induction in pretransplant MLCs with donor cells was associated with impaired long-term graft outcome. We conclude that CD4+CD25+Foxp3+IFNg+ PBL represent a heterogenous population containing iTreg that suppress allogeneic T-cell responses and may be involved in inhibition of the specific posttransplant alloresponse. As published, CD4+CD25+Foxp3+IFNg+ iTreg express IFNg receptors, are induced by IFNg and represent the first line of iTreg during an initiating immune response. Therefore, increased MLC responses are associated with increased induction of IFNg-sensitive CD4+CD25+Foxp3+IFNg+ iTreg in-vitro, thus explaining our finding that high pretransplant iTreg frequencies in-vitro are associated with poor long-term graft outcome.