Aims: Natural CD4(+)CD25(+) regulatory cells (nTregs) have been implicated in maintaining peripheral immune tolerance. This study aims to test whether immunotherapy using in vitro-expanded Treg (iTregs) could suppress allograft rejection in corneal transplantation model. Methods: Natural CD4(+)CD25(+) T cells were freshly purified from naive mice and expanded in vitro by culturing with anti-CD3/CD28-coated Dynabeads, interleukin (IL)-2 and transforming growth factor (TGF-beta 1). Suppression ability of iTregs was assayed by co-culturing with CD4(+)CD25(-) T cells (Teff) in vitro and by targeting corneal allograft rejection in vivo. Tracking of iTreg after adoptive transfer in vivo were examined by CFSE labeling. Results: Natural Treg cells were expanded by culturing with anti-CD3/CD28-coated Dynabeads in the presence of IL-2 and TGF-beta 1. Compared with nTregs, iTregs had similar expression of CD62L, and PD-L1, lower expression of CD69, higher levels of PD-1, CD25, and Foxp3. iTreg cells exerted stronger suppression function than natural Treg cells when cocultured with CD4(+)CD25(-) T cells in vitro and prevented fully MHC-mismatched corneal allograft rejection. Survival of iTreg cells could suppress alloimmune reaction and most prone to migrate to graft draining LNs and spleens. Moreover, maintaining CD25 expression on iTregs was indicative for preservation of allosuppression. Conclusion: Therapeutic use of in vitro-expanded CD4(+)CD25(+) T cells may be a effective and safe tool for controlling allograft rejection and may help induce allograft tolerance. (c) 2012 American Society for Histocompatibility and Immunogenetics. Published by Elsevier Inc. All rights reserved.