We have developed a method to generate alloreactive regulatory T cells in vitro in the presence of interferon (IFN)-gamma and donor antigen presenting cells (APCs). We hypothesized that these IFN-gamma-conditioned T cells (Tcon) would reduce transplantation-associated arteriosclerosis. Tcon were generated from mouse (CBA.Ca, H-2(k)) CD4(+) T cells cultured in the presence of IFN-gamma for 14 days. These cultures were pulsed with bone marrow-derived B6 (H-2(b)) APC. 1 x 10(5) CD25(-)CD4(+) effector T cells from naive H-2(k) mice were then cotransferred with 4 x 10(5) Tcon into CBA-rag(-/-) mice. One day later, these mice received a fully allogenic B6 CD31(-/-) abdominal aorta transplant. Transfer of CD25(-)CD4(+) effectors resulted in 29.7 +/- 14.5% luminal occlusion of allogeneic aortic grafts after 30 days. Cotransfer of Tcon reduced this occlusion to 11.7 +/- 13.1%; P < 0.05. In addition, the CD31(-) donor endothelium was fully repopulated by CD31(+) recipient endothelial cells in the absence of Tcon, but not in the presence of Tcon. In some experiments, we cotransplanted B6 skin with aortic grafts to ensure enhanced reactivation of the regulatory cells, which led to an additional reduction in vasculopathy (1.9 +/- 3.0% luminal occlusion). In the presence of Tcon, CD4(+) T cell infiltration into grafts was markedly reduced by a regulatory mechanism that included reduced priming and proliferation of CD25(-)CD4(+) effectors. These data illustrate the potential of ex vivo generated regulatory T cells for the inhibition of transplant-associated vasculopathy.