Introduction: High serum levels of soluble CD30 (sCD30) are considered predictive of poor outcome in kidney, islet and lung transplantation. Our preliminary data show that patients with a high response of IFN-γ after allogeneic stimulation in vitro exhibit also a higher release of sCD30. However, the mechanisms by which sCD30 release is regulated and how this molecule affects allograft survival are poorly understood. We investigated the effect on sCD30 release of neutralizing antibodies against IFN-γ and other cytokines as well as the involvement of metalloproteinases ADAM10 and ADAM17/TACE in the ectodomain shedding of CD30 during the alloimmune response. Methods: Purified naive CD4+CD45RA+ T cells from healthy controls (n=8) were stimulated with HLA-DR mismatched stimulator cells in mixed lymphocyte culture (MLC) and incubated in the presence or absence of neutralizing anti-IFN-γ, anti-IL-2, anti-TGF-β, anti-IL-10 and anti-CD30L antibodies or their respective isotype controls (0.5-32 μg/ml). At 144h of culture, supernatants were harvested, and the released sCD30 and the soluble fraction of ADAM10 and ADAM17/TACE were quantified by ELISA. T cell memory phenotypes defined by expression of CD45RO and CD27 and ADAM10 and TACE expression were analyzed by flow cytometry. Results: Compared to autologous stimulation, allogeneic stimulation resulted in two-fold increased sCD30 release into the supernatant. Anti-IFN-γ (p=0.0004) and anti-IL-2 (p=0.0013) abrogated the release of sCD30 in a dose-dependent manner (p< 0.0001), whereas anti-TGF-β, anti-IL-10 and anti-CD30L had no effect when compared to their respective isotype controls. Measurement of ADAM10 and ADAM17/TACE in supernatants from MLC showed differential regulation of these enzymes, whereby allogeneic stimulation resulted in an increased secretion of ADAM10 (p=0.047) and a reduced secretion of ADAM17/TACE (p=0.008). The levels of these enzymes did not change in the presence of neutralizing antibodies, thus indicating that the blocking effect of anti-IFN-γ and anti-IL-2 cannot be attributed to a direct effect of metalloproteinases. Flow cytometry analysis showed that the blocking effect on sCD30 release exerted by anti-IFN-γ and anti-IL-2 was either due to a failure to differentiate from naive to central memory cells, or failure to preserve a central memory phenotype in the responder cells. ADAM10 and ADAM17/TACE were detected on a very low percentage (1-3%) of MLC responder cells which suggests either a high secretion rate into the supernatant or internalization of the proteins. Conclusion: Allostimulation of CD4+ T lymphocytes results, in an IFN-γ and IL-2-dependent manner, in an increased release of sCD30 by central memory (CD27+CD45RO+) T cells. Soluble fractions of metalloproteinases ADAM10 and ADAM17/TACE, which are responsible for CD30 shedding, are differentially secreted into the supernatant but are poorly expressed on MLC responder T cells. These results support clinical findings showing an association of high pretransplant serum sCD30 levels and a high pretransplant IFN-γ T cell in vitro response with poor kidney allograft prognosis.