IntroductionOral tolerance has long been recognized as a physiologic mechanism of immune unresponsiveness to dietary antigens and bacterial microflora antigens, which maintain tissue integrity by preventing harmful delayed-type hypersensitivity (DTH) responses in the intestine and may also limit the efficiency of oral vaccination. Indeed, antigen encounter in the intestine triggers an active inhibitory process preventing the onset of CD4 ϩ and CD8 ϩ T-cell antigen-specific immune responses to subsequent systemic immunization with the same antigen (reviewed in Garside and Mowat 1 ). Several mechanisms have been proposed to explain peripheral tolerance induced by antigen feeding. These include anergy 2,3 or deletion of antigen-specific T cells, 4,5 immune deviation to Th2-biased immune response, and induction of regulatory Th3 (transforming growth factor  [TGF]-producing) cells. 6,7 The naturally occurring regulatory subset of CD4 ϩ CD25 ϩ T cells accounting for 5% to 10% of peripheral CD4 ϩ T cells has been extensively reported to exert potent immunosuppressive function in vivo and in vitro toward CD4 ϩ T-cell effectors 8 and may represent regulatory T cells responsible for orally induced peripheral tolerance. Indeed, CD4 ϩ CD25 ϩ T cells, which arise from the thymus as early as day 3 of life, 9 are characterized by a memory phenotype; low proliferative capacity and interleukin-2 (IL-2) production; secretion of high levels of the immunosuppressive cytokines IL-10 and TGF-; and expression of cytotoxic Tlymphocyte antigen 4 (CTLA-4), 10-13 a molecule that contributes to orally induced tolerance. 14 These cells have been described in a variety of experimental models to protect from autoimmune diseases, as well as colitis and allograft rejection. 8 Reminiscent to these cells, IL-10-producing ovalbumin (OVA)-specific CD4 ϩ T clones (Tr1) generated in vitro after repeated stimulation with antigen in the presence of IL-10 were shown to prevent colitis when cotransferred with naive CD4 ϩ CD45RB high T cells in OVAfed immunocompromised severe combine immunodeficiency (SCID) or Nude mice. 15 Interestingly, mice genetically deficient for either IL-2, IL-2R, T-cell receptor ␣ (TcR␣), TGF, IL-10, or major histocompatibility complex (MHC) class II were shown to develop spontaneous colitis, [16][17][18][19][20] compatible with a shared physical or functional defect in the regulatory CD4 ϩ CD25 ϩ subset. Although recent studies in TcR transgenic models have reported that oral antigen delivery can induce activation and/or differentiation of regulatory CD4 ϩ CD25 ϩ T cells, 21,22 evidence that they are instrumental for in vivo induction of oral tolerance has not been provided. Moreover, evidence as to whether CD4 ϩ CD25 ϩ cells are responsible for peripheral suppression of antigen-specific CD8 ϩ cytotoxic T-lymphocyte (CTL) responses is still sparse. 23 In this study we examined whether CD4 ϩ CD25 ϩ T cells contribute to oral tolerance in normal nonlymphopenic hosts, using a pathophysiologic model of antigen-specific skin in...