CD4+CD25+ regulatory T (TR) cells have been described in both humans and mice. In mice, TR are thymically derived, and lack of TR leads to organ-specific autoimmunity. Recently, the forkhead/winged helix transcription factor, FoxP3, has been shown to be important for the function of TR cells in mice. In this study, human TR cells were examined and, in results similar to those of studies done in mice, expression of FoxP3 was found exclusively in CD4+CD25+ T cells and correlated with the suppressive activity of these cells. In contrast to the mouse studies, activation of human CD4+CD25- T cells led to expression of FoxP3. Expression of FoxP3 in activated human CD4+CD25+ cells also correlated with suppression of proliferation by these cells in freshly isolated CD4+CD25- T cells from the same donor. This suppression was cell-contact dependent and cytokine independent. Thus, in humans, during activation of CD4+CD25- T cells in an immune response, two populations of cells may arise, effector CD4+CD25+ and regulatory CD4+CD25+ T cells, with expression of FoxP3 correlated with regulatory activity. These data also raise the possibility that a failure to generate peripheral TR cells properly may contribute to autoimmune disease and suggest a possible therapeutic role for FoxP3 in the treatment of such diseases.

Antigen-specificity is a hallmark of adaptive T cell-mediated immune responses. CD4 ؉ CD25 ؉ FOXP3 ؉ regulatory T cells (TR) also require activation through the T cell receptor for function. Although these cells require antigen-specific activation, they are generally able to suppress bystander T cell responses once activated. This raises the possibility that antigen-specific T R may be useful therapeutically by localizing generalized suppressive activity to tissues expressing select target antigens. Here, we demonstrate that T R specific for particular peptide-MHC complexes can be generated from human CD4 ؉ CD25 ؊ T cells in vitro and isolated by using HLA class II tetramers. Influenza hemagglutinin epitopes were used to generate hemagglutinin-specific T R, which required cognate antigen for activation but which subsequently suppressed noncognate bystander T cell responses as well. These findings have implications for the generation of therapeutic regulatory T cells in disease, and also suggest an important mechanism by which T cells may be regulated at the site of inflammation.autoimmunity ͉ T lymphocytes ͉ tolerance ͉ suppression ͉ anergy T he immune system has evolved a series of mechanisms to protect against autoimmunity or excessive inflammatory responses to pathogens. It has become increasingly clear that CD4 ϩ CD25 ϩ regulatory T cells (T R ) are an important component of this immune regulation in the periphery. In both humans and mice, CD4 ϩ CD25 ϩ T R have been shown to suppress T cell responses in a contact-dependent, cytokine-independent manner and to require activation via the T cell receptor to be functional. The forkhead-family transcription factor FoxP3 is essential for the development and function of CD4 ϩ CD25 ϩ T R , and spontaneous mutation of FoxP3 leads to widespread lymphocytosis and autoimmunity in the scurfy mouse and in humans with immune disregulation, polyendocrinopathy, enteropathy, X-linked syndrome (IPEX) (1, 2). Studies in animal models of autoimmunity have also shown an increased frequency or severity of autoimmunity in the absence of T R (3-7) and that transfer of T R is sufficient to protect from or reverse autoimmunity.Recent studies have suggested that T R may be antigen-specific. Freshly isolated unmodified CD4 ϩ CD25 ϩ T cells have been able to suppress proliferation in assays using peptides to stimulate cells rather than polyclonal anti-CD3 stimulation (7,8). Clones have also been generated that express CD25 and are suppressive when given cognate antigen (9, 10). Alloantigen stimulation of both human and mice CD4 ϩ CD25 ϩ T cells (11,12) or priming mice with alloantigen (13) has also resulted in antigen-specific T R . The induction of T R has now been described in both mouse and man. T R have been induced in vivo in mice by administration of oral or i.v. antigen (14), antigen emulsified in incomplete ϩ CD25 Ϫ FoxP3-T cells can differentiate into T R (23). In addition, studies in animal models of autoimmunity have demonstrated the therapeutic benefit of transfer of antigen-s...

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CD4 1 CD25 1 FOXP3 1 Treg cells require TCR engagement for suppressive function, thus ensuring that suppression occurs only in the presence of specific antigens; however, to date no studies have addressed the function of self-antigen-specific Treg in humans. These studies were designed to determine whether peripheral generation and function of islet antigen-specific adaptive Treg are defective in human subjects with type 1 diabetes (T1D). Islet antigen-specific adaptive Treg were induced in vitro by activation of CD4 1 FOXP3 À T cells with glutamic acid decarboxylase and islet-specific glucose-6-phosphate catalytic subunit-related protein peptides in the context of T1D-associated HLA-DRb alleles. Antigen-specific Treg were characterized using flow cytometry for FOXP3 and class II tetramer and assessed for the ability to inhibit proliferation. These adaptive Treg were then compared with influenza-specific Treg from the same study population. The function of tetramer 1 cells that expressed FOXP3 was similar for both influenza and islet antigens generated from control and T1D subjects. In fact, the potency of suppression correlated with FOXP3 expression, not antigen specificity. Thus, these data suggest that development of functional adaptive Treg can occur in response to islet antigens and activation of isletspecific Treg may potentially be used as a targeted immunotherapy in T1D.Key words: Autoimmunity . Human subjects . Treg . Type 1 diabetes Supporting Information available online Introduction CD4 1 CD25 1 FOXP3 1 T regulatory cells are vital for immune regulation. The lack of Treg results in severe autoimmunity in both mouse and man [1][2][3]. The importance of Treg in type 1 diabetes (T1D) has been demonstrated in murine models by the acceleration of disease upon depletion of Treg as well as the cure of diabetes by transfer of Treg to animals prior to and after the onset of disease [4][5][6][7]. Furthermore, adoptive transfer is more effective when the transferred Treg are specific for an islet antigen [8]. In humans, defects in polyclonal Treg have been proposed as one mechanism by which individuals develop T1D and this defect appears to be in the function [9][10][11], as compared with the number of Treg [12]. The specificity of Treg in animal models and the role of Treg in regulating disease raise the possibility that islet-specific Treg are inadequate in number or function in individuals with T1D.Although Treg were initially believed to originate only from the thymus [13], subsequent studies in mouse and man have 612shown that CD4 1 CD25 1 FOXP3 1 Treg can develop in the periphery under a variety of conditions and these cells are referred to as adaptive Treg [14,15]. Other types of adaptive Treg have been described including Tr1 and Th3 cells; however, these adaptive Treg are not associated with FOXP3 expression. For this study we focus on CD4 1 CD25 1 FOXP3 1 adaptive Treg and use the term adaptive Treg to refer to these cells in this paper. Adoptive transfer of polyclonal adaptive Treg can prevent devel...