The principal mechanism of peripheral T-cell tolerance is sup pression by Tregs. In addition, functional unresponsiveness and cell death also contribute (Fig. 1). These mechanisms may be responsible for T-cell tolerance to tissue-specific self antigens, especially those that are not abundant in the thymus. We do not know if tolerance to different self antigens is maintained by one or another mechanism or if all of these mechanisms function cooperatively to prevent autoimmunity. The same mechanisms may also induce unresponsiveness to foreign antigens that are presented to the immune system under tolerogenic conditions.
Fig1. Mechanisms of peripheral T-cell tolerance. The signals involved in a normal immune response (A) and the three major mechanisms of peripheral T-cell tolerance (B) are illustrated. TCR, T-cell receptor.
Suppression by Regulatory T Cells
The concept that some lymphocytes could control the responses of other lymphocytes was proposed many years ago and was soon followed by experimental demonstrations of populations of T lymphocytes that suppressed immune responses. These initial findings led to enormous interest in suppressor T cells, which became one of the dominant topics of immunology research in the 1980s. However, this field has had a somewhat checkered history, mainly because attempts to define populations of sup pressor cells and their mechanisms of action were largely unsuccessful. In the 1990s, the idea had an impressive rebirth, with the application of better approaches to define, purify, and analyze populations of T lymphocytes that inhibit immune responses. These cells are called regulatory T cells (Tregs).
Regulatory T cells are a subset of CD4+ T cells whose principal function is to suppress immune responses (Fig. 2). Most Tregs are CD4+, and express high levels of the transcription factor FOXP3, the IL-2 receptor α chain (CD25), and CTLA 4. FOXP3 is a member of the forkhead family of transcription factors and is critical for the development and function of most Tregs. The essential role of FOXP3+ Tregs in maintaining self tolerance and preventing autoimmunity has been established by the analysis of inherited diseases. A rare systemic autoimmune disease in humans called IPEX (immune dysregulation, poly endocrinopathy, enteropathy, X-linked) syndrome is caused by loss-of-function mutations in the FOXP3 gene, which result in a deficiency of Tregs. This disease is fatal unless the children are given hematopoietic stem cell transplants, which restore normal Tregs. Mice with spontaneous or experimentally induced mutations in the foxp3 gene develop a similar multisystem autoimmune disease associated with an absence of CD25+ Tregs, and the disease can be prevented by transferring Tregs from normal mice. These observations have established the importance of Tregs for maintaining self-tolerance. The recent surge of interest in Tregs is because of an increasing appreciation of their physio logic roles, as well as the possibility that defects in these cells may result in various autoimmune diseases and, conversely, that Tregs can be administered or expanded to treat inflammatory diseases.
Fig2. Regulatory T cells (Tregs). Tregs are generated by self antigen recognition in the thymus (some times called natural regulatory cells) and (probably to a lesser extent) by antigen recognition in peripheral lymphoid organs (called inducible or adaptive regulatory cells). The development and survival of these Tregs require interleukin-2 and the transcription factor FOXP3. In peripheral tissues, Tregs suppress the activation and effector functions of other self-reactive and potentially pathogenic lymphocytes. DC, Dendritic cell; NK, natural killer.
Phenotypic Markers and Heterogeneity of Regulatory T Cells. Although numerous T-cell populations have been described as possessing suppressive activity, the cell type whose regulatory role is best established is CD4+ FOXP3+ CD25high. FOXP3 and CD25 are essential for the generation, maintenance, and function of these cells. These cells usually express low levels of the receptor for IL-7 (CD127) and, as predicted from this pattern of receptor expression, they use IL-2 but not IL-7 as their growth and survival factor. FOXP3+ Tregs typically express high levels of CTLA-4, which is required for their function. Demethylation of the FOXP3 gene locus and of other loci containing genes that are expressed in these cells serves to maintain a stable regulatory T-cell phenotype, and these epigenetic changes may be used to identify Tregs in basic and clinical research. FOXP3+ Tregs are classified as thymic or peripheral, based on where they develop. There also may be subsets of Tregs that are distinguished by history of antigen response (naive, activated, and memory) or tissue residence (lymphoid and nonlymphoid). Tregs that are located in peripheral tissues, such as the intestines, lungs, skin, and fat, may have functions that are unique to each tissue. Tregs have been described in the follicles of secondary lymphoid organs, where they may inhibit antibody responses. In different types of immune responses, Tregs may be induced to express the same chemokine receptors as effector T cells. This is one way in which Tregs colocalize in tissues with effector cells, enabling the Tregs to control diverse immune reactions in tissues.
Generation and Maintenance of Regulatory T Cells. Tregs are generated mainly by self antigen recognition in the thymus and also by recognition of self and foreign antigens in peripheral tissues. In the thymus, development of Tregs is one of the fates of T cells committed to the CD4 lineage that recognize self antigens (see Fig. 3). In peripheral tissues, antigen recognition in the absence of strong innate immune responses favors the generation of regulatory cells from naive CD4+ T lymphocytes; Tregs can also develop after inflammatory reactions. Although many markers have been proposed to distinguish thymic from peripheral Tregs, whether these markers can be used to reliably distinguish the two subsets in lymphoid organs or blood of mice and humans has not been established.
Fig3. Central T-cell tolerance. Recognition of self antigens by immature T cells in the thymus leads to the death of the cells (negative selection, or deletion) or to the development of regulatory T cells (Tregs) that enter peripheral tissues.
The generation, survival, and functional competence of Tregs are dependent on the cytokine IL-2. Patients with homozygous mutations in the IL-2 receptor α or β chain develop systemic autoimmunity and have few functional Tregs. Mice in which the gene for IL-2 or for the α or β chain of the IL-2 receptor is knocked out also develop autoimmunity, manifested by inflammatory bowel disease (IBD), autoimmune hemolytic anemia, and multiple autoantibodies (including anti-erythrocyte and anti-DNA antibodies). These mice have greatly reduced numbers of CD25+ FOXP3+ Tregs, and their disease can be corrected by injecting these cells. IL-2 is required for the maintenance of FOXP3+ Tregs in the periphery and may pro mote differentiation of T cells in the thymus into the regulatory subset. Because FOXP3+ Tregs do not produce IL-2, this growth factor is provided by the many conventional T cells that are normally responding to foreign antigens (Fig. 4). IL-2 activates the transcription factor STAT5, which may enhance expression of FOXP3 as well as other proteins that are involved in the function of Tregs.
Fig4. Role of interleukin-2 (IL-2) in the maintenance of regulatory T cells (Tregs). IL-2 produced by conventional T cells responding to antigens (typically foreign antigens) acts on Tregs recognizing self or foreign antigens on antigen-presenting dendritic cells and promotes the survival and function of the Tregs. The Tregs, in turn, inhibit the activation of activated effector cells, a classic negative feedback loop.
The generation of some Tregs requires the cytokine trans forming growth factor-β (TGF-β). The culture of naive T cells with activating anti-TCR antibodies together with TGF-β (and IL-2) can induce the development of regulatory cells in vitro. In mice, elimination of TGF-β or blocking of TGF-β signals in T cells leads to a systemic inflammatory disease because of uncontrolled leukocyte activation. TGF-β stimulates expression of FOXP3, and this could be its principal role in Treg development.
Mechanisms of Action of Regulatory T Cells. Tregs appear to suppress immune responses at multiple steps—at the induction of naive T-cell activation in lymphoid organs as well as the effector phase of these responses in tissues. They control the initiation of T-cell responses mainly by inhibiting the ability of dendritic cells (DCs) to stimulate naïve T cells, and by suppressing T-cell activation. Tregs may also suppress B-cell activation and inhibit the proliferation and differentiation of natural killer (NK) cells. Although numerous mechanisms of suppression have been proposed, the following are the best supported by available data.
• CTLA-4-mediated inhibition of costimulation. CTLA-4 on Tregs binds to B7 molecules on APCs and blocks and removes these molecules, resulting in reduced availability of B7 for CD28-mediated costimulation.
• Production of the immunosuppressive cytokines IL-10 and TGF-β. These cytokines are discussed below.
• Consumption of IL-2. Because of the high level of expression of the IL-2 receptor, these cells may absorb IL-2 and deprive other cell populations of this growth factor, resulting in reduced proliferation and differentiation of other IL-2 dependent cells.
Whether all regulatory cells work by all these mechanisms or if there are subpopulations that use different mechanisms to control immune responses has not been established.
Inhibitory Cytokines Produced by Regulatory T Cells. TGF-β and IL-10 are involved in the generation and functions of Tregs. These cytokines are produced by and act on many other cell types in addition to regulatory cells. Here we describe the properties and actions of these cytokines.
Transforming Growth Factor-β. TGF-β was discovered as a protein made by tumors that promoted the survival of cancer cells in vitro. It is actually a family of closely related molecules encoded by distinct genes, commonly designated TGF-β1, TGF β2, and TGF-β3. Cells of the immune system synthesize mainly TGF-β1. TGF-β1 is produced by CD4+ Tregs, activated macro phages, and many other cell types.
TGF-β has many important and diverse roles in the immune system:
• TGF-β inhibits the proliferation and effector functions of T cells and the activation of macrophages. TGF-β inhibits classical macrophage activation but is one of the cytokines secreted by alternatively activated macrophages. TGF-β also suppresses the activation of other cells, such as neutrophils and endothelial cells. By these inhibitory actions, TGF-β functions to control immune and inflammatory responses.
• TGF-β regulates the differentiation of functionally distinct subsets of T cells. As described earlier, TGF-β stimulates the development of peripheral FOXP3+ Tregs, best established in vitro. In combination with cytokines produced during innate immune responses, such as IL-1 and IL-6, TGF-β promotes the development of the Th17 subset of CD4+ T cells by virtue of its ability to induce the transcription factor RORγt. The ability of TGF-β to suppress immune and inflammatory responses, in part by generating Tregs, and to promote the development of proinflammatory Th17 cells in the presence of other cytokines, is an interesting example of how a single cytokine can have diverse and sometimes opposing actions depending on the context in which it is produced. TGF-β can also inhibit development of Th1 and Th2 subsets.
• TGF-β stimulates production of immunoglobulin A (IgA) antibodies by inducing B cells to switch to this isotype. IgA is the major antibody class required for mucosal immunity.
• TGF-β promotes tissue repair after local immune and inflammatory reactions subside. This function is mediated mainly by the ability of TGF-β to stimulate collagen synthesis and matrix-modifying enzyme production by macrophages and fibroblasts and by promoting angiogenesis. This cytokine may play a pathologic role in diseases in which fibrosis is an important component, such as pulmonary fibrosis and systemic sclerosis.
Interleukin-10. IL-10 is an inhibitor of activated macro phages and DCs and is thus involved in the control of innate immune reactions and cell-mediated immunity. It is a member of a family of heterodimeric cytokines that includes IL-22 and others. The IL-10 receptor belongs to the type II cytokine receptor family (similar to the receptor for IFNs) and consists of two chains that associate with the Janus family kinases JAK1 and TYK2 and activate STAT3. IL-10 is produced by many immune cell populations, including activated macrophages and DCs, Tregs, and Th1 and Th2 cells. Because it is both produced by and inhibits macrophages and DCs, it functions as a negative feedback regulator. IL-10 is also produced by some B lymphocytes, which have been shown to have immune suppressive functions and have been called regulatory B cells.
The biologic effects of IL-10 result from its ability to inhibit many of the functions of activated macrophages and DCs.
• IL-10 inhibits the production of IL-12 by activated DCs and macrophages. Because IL-12 is a critical stimulus for IFN-γ secretion, which plays an important role in innate and adaptive cell-mediated immune reactions against intracellular microbes, IL-10 suppresses all such reactions. In fact, IL-10 was first identified as a cytokine that inhibited IFN-γ production.
• IL-10 inhibits the expression of costimulators and MHC class II molecules on DCs and macrophages. Because of these actions, IL-10 inhibits T-cell activation and terminates cell-mediated immune reactions.
Infants who have homozygous loss-of-function mutations in the IL-10 gene or in the gene for the IL-10 receptor develop severe colitis before the age of 1 year. Knockout mice lacking IL-10 in all cells or only in Tregs also develop colitis, probably as a result of uncontrolled activation of lymphocytes and macro phages reacting to enteric microbes. Because of these findings, it is thought that this cytokine is especially important for control ling inflammatory reactions in mucosal tissues, particularly the gastrointestinal tract.
The Epstein-Barr virus contains a gene homologous to human IL-10, and viral IL-10 has the same activities as the natural cytokine. This raises the intriguing possibility that the virus has acquired the IL-10–like gene during its evolution, giving it the ability to inhibit host immunity and thus a survival advantage in the infected host.
Role of Regulatory T Cells in Self-Tolerance. The elucidation of the genetic basis of IPEX syndrome and the similar disease in mice caused by mutations in the foxp3 gene, described earlier, is convincing proof of the importance of Tregs in maintaining self-tolerance and homeostasis in the immune system. Numerous attempts are being made to identify defects in the development or function of Tregs in more common autoimmune and inflammatory diseases in humans such as IBD, type 1 diabetes, and multiple sclerosis (MS), as well as in allergic disorders. Defects in Tregs or resistance of effector cells to suppression by Tregs may contribute to the pathogenesis of these diseases. However, it has proved difficult to definitively establish the role of Treg abnormalities in these common diseases.
There is also potential for expanding Tregs in culture and injecting them back into patients to control pathologic immune responses. Clinical trials of Treg transfer are ongoing in attempts to treat transplant rejection, graft-versus-host disease, and auto immune and other inflammatory disorders. In addition, clinical trials are underway to expand these cells in patients by administering IL-2 in doses or forms that preferentially bind to CD25 and thus activate Tregs.
Tregs have been shown to serve roles in addition to maintaining self-tolerance and thus preventing autoimmunity. Subpopulations of Tregs with unique transcriptional signatures are present in many tissues and appear to perform functions that are especially beneficial for those tissues. Tregs that reside in the skin, muscles, and organs, such as the lungs, pro mote tissue repair and the proliferation and differentiation of stem cells, thus helping to restore tissue integrity after inflammatory reactions resolve. Adipose tissue Tregs control fat metabolism. Tregs are also involved in maintaining fetal tolerance and preventing the rejection of fetuses and play a role in preventing inflammatory responses to commensal microbes.
