TGF-β is a pluripotent cytokine that is capable of inducing the expression of Foxp3 in naive T lymphocytes. TGF-β-induced cells are phenotypically similar to thymic-derived regulatory T cells in that they are anergic and suppressive. We have examined the cytokine and costimulatory molecule requirements for TGF-β-mediated induction and maintenance of Foxp3 by CD4+Foxp3− cells. IL-2 plays a non-redundant role in TGF-β-induced Foxp3 expression. Other common γ-chain-utilizing cytokines were unable to induce Foxp3 expression in IL-2-deficient T cells. The role of CD28 in the induction of Foxp3 was solely related to its capacity to enhance the endogenous production of IL-2. Foxp3 expression was stable in vitro and in vivo in the absence of IL-2. As TGF-β-induced T regulatory cells can be easily grown in vitro, they may prove useful for the treatment of autoimmune diseases, for the prevention of graft rejection, and graft versus host disease.

IntroductionCD4 ϩ CD25 ϩ regulatory/suppressor T cells were originally identified by their capacity to primarily control immune responses to autoantigens, 1-3 but recent studies have shown that they exert pleiotropic suppressive effects on immune responses to alloantigens, 4 tumor antigens, 5 and infectious agents. 6,7 The functional phenotype of CD4 ϩ CD25 ϩ T cells has been extensively characterized in vitro. CD4 ϩ CD25 ϩ T cells do not proliferate when stimulated via the TCR but are able to proliferate when costimulated with IL-2. 8,9 On activation via their TCR, they suppress the proliferation of both CD4 ϩ T cells 8 and CD8 ϩ T cells 10 by inhibiting the transcription of IL-2 mRNA. 8 In vitro studies have demonstrated that the suppression is mediated exclusively by a cell-contact-dependent, cytokine-independent mechanism. 4,9,10 However, suppression in some in vivo experimental models requires the cooperation of suppressive cytokines. [11][12][13] The nature of the target cells for CD4 ϩ CD25 ϩ T-cell-mediated suppression remains controversial. CD4 ϩ CD25 ϩ T cells can suppress the activation of CD8 ϩ T cells in the absence of professional antigenpresenting cells (APCs), 10 but this does not exclude the possibility that they are also capable of acting on other cell types. One recent study has suggested that some (or all) of the suppressive effects of mouse CD4 ϩ CD25 ϩ T cells on T-cell proliferation are secondary to CD4 ϩ CD25 ϩ T-cell-mediated killing of the responder cells by a perforin-independent, granzyme-B-dependent pathway. 14 In the day 3 thymectomy model of organ-specific autoimmune disease, CD4 ϩ CD25 ϩ T cells suppressed the activation of autoantigenspecific T cells and inhibited the production of autoantibodies to the target organ. 2,3 Similar results have been obtained in cell transfer studies using TCR transgenic T cells. [15][16][17][18] However, it remains unclear whether these results are secondary to a direct effect of CD4 ϩ CD25 ϩ T cells on B-cell function or whether the effects are mediated indirectly by an inhibition of T-helper function. One recent study has shown that activation of both human T regulatory 1 (Tr1)-like cells and CD4 ϩ CD25 ϩ T cells by cross-linking CD3 and CD46 induces potent lytic activity against T cells, monocytes, and dendritic cells. 19 In this study, we examined the effects of CD4 ϩ CD25 ϩ T cells on B-cell function. We demonstrate that CD4 ϩ CD25 ϩ T cells suppress B-cell proliferation in response to polyclonal B-cell activators by inducing death of the responding B cells. Most interestingly, B-cell death is not mediated by the Fas-Fas ligand (FasL) pathway, but instead is mediated by a granzyme-dependent, partially perforin-dependent pathway. The implications of these findings for the role of CD4 ϩ CD25 ϩ T cells in the control of B-cell function in vivo are discussed. Materials and methods MiceFemale BALB/c and C57BL/6 mice were obtained from the National Cancer Institute (Frederick, MD). Mice expressing a transgenic TCR specific for influenza hemagglutinin...

Numerous studies over the past 10 years have demonstrated the importance of naturally occurring CD4+ CD25+ Foxp3+ regulatory T cells (nTregs) in immune regulation. We analyzed the mechanism of action of nTregs in a well-characterized model of autoimmune gastritis and demonstrated that nTregs act at an early stage of disease progression to inhibit the differentiation of naïve T cells to pathogenic T-helper 1 effectors. The effects of nTregs in this model are not antigen-specific but are mediated by activation of the nTregs by ubiquitous self-peptide major histocompatibility complex class II complexes together with cytokines released by activated effector cells. Studies in vitro confirmed that some nTregs exist in an activated state in vivo and can be activated to exert non-specific suppressor effector function by stimulation with interleukin-2 in the absence of engagement of their T-cell receptor. Natural Tregs can differentiate in vitro to exhibit potent granzyme B-dependent, partially perforin-independent cytotoxic cells that are capable of specifically killing antigen-presenting B cells. Natural Treg-mediated killing of antigen-presenting cells may represent one pathway by which they can induce long-lasting suppression of autoimmune disease.