Thymus-derived regulatory T lymphocytes of CD4 ؉ CD25 ؉ phenotype regulate a large variety of beneficial and deleterious immune responses and can inhibit lethal graft-versus-host disease in rodents. In vitro, CD4 ؉ CD25 ؉ T cells require specific major histocompatibility complex (MHC)/peptide ligands for their activation, but once activated they act in an antigen-nonspecific manner. In vivo, regulatory T cells are also activated in an antigen-specific fashion, but nothing is known about antigen specificity of their suppressor-effector function. Here we show that CD4 ؉ CD25 ؉ regulatory T lymphocytes isolated from naive mice and activated in vitro with allogeneic antigenpresenting cells (APCs) induced specific long-term tolerance to bone marrow grafts disparate for major and minor histocompatibility antigens; whereas "target" bone marrow was protected, third-party bone marrow was rejected. Importantly, in mice injected with a mix of target and thirdparty bone marrows, protection and rejection processes took place simultaneously. These results indicate that CD4 ؉ CD25 ؉ regulatory T cells can act in an antigenspecific manner in vivo. Our results suggest that CD4 ؉ CD25 ؉ regulatory T cells could in the future be used in clinical settings to induce specific immunosuppression. ( IntroductionDue to the random rearrangements of genes encoding T-and B-lymphocyte antigen receptors, a significant number of autospecific and potentially autoreactive lymphocytes develop in primary lymphoid organs. [1][2][3] Central tolerance (ie, induced in primary lymphoid organs) eliminates (by deletion) or functionally inactivates (by induction of anergy) such dangerous lymphocytes. In absence of central tolerance induction, a strongly self-reactive T-cell repertoire develops. 4,5 However, when central tolerance is partially defective, self-tolerance can be maintained by peripheral mechanisms. [6][7][8] Several types of peripheral tolerance mechanisms control lymphocytes having escaped central tolerance and are known to play a crucial role in preventing autoimmunity (for reviews see Sprent et al 6 and Stockinger 9 ).One of these peripheral tolerance mechanisms was discovered using the day-3 thymectomy model of multiorgan autoimmunity in mice. 10 The pathology can be prevented by injection of CD4 ϩ CD25 ϩ lymphocytes, which appear after day 3 of life in the peripheral lymphoid organs of normal mice. CD4 ϩ CD25 ϩ regulatory T cells do not only inhibit autoimmunity, they can also inhibit experimental inflammatory bowel disease induced by injection of CD4 ϩ CD45RB high cells into severe combined immunodeficiency (SCID) mice or recombination-activating gene (RAG)-deficient animals. 11 Moreover, they contribute to the fine control of immunity to infectious agents such as parasites and viruses. 12,13 An undesired side effect of the activity of CD4 ϩ CD25 ϩ regulatory T lymphocytes is the occasional incapacity of the immune system to eliminate tumor cells. 14,15 Therefore, regulatory T lymphocytes play a crucial role in the pathophysiologic mai...