Alexander Y. Rudensky

Foxp3 programs the development and function of CD4+CD25+ regulatory T cells

CD4+CD25+ regulatory T cells are essential for the active suppression of autoimmunity. Here we report that the forkhead transcription factor Foxp3 is specifically expressed in CD4+CD25+ regulatory T cells and is required for their development. The lethal autoimmune syndrome observed in Foxp3-mutant scurfy mice and Foxp3-null mice results from a CD4+CD25+ regulatory T cell deficiency and not from a cell-intrinsic defect of CD4+CD25− T cells. CD4+CD25+ regulatory T cells rescue disease development and preferentially expand when transferred into neonatal Foxp3-deficient mice. Furthermore, ectopic expression of Foxp3 confers suppressor function on peripheral CD4+CD25− T cells. Thus, Foxp3 is a critical regulator of CD4+CD25+ regulatory T cell development and function.

A function for interleukin 2 in Foxp3-expressing regulatory T cells.

Regulatory T cells (Treg cells) expressing the forkhead family transcription factor Foxp3 are critical mediators of dominant immune tolerance to self. Most Treg cells constitutively express the high-affinity interleukin 2 (IL-2) receptor α-chain (CD25); however, the precise function of IL-2 in Treg cell biology has remained controversial. To directly assess the effect of IL-2 signaling on Treg cell development and function, we analyzed mice containing the Foxp3gfp knock-in allele that were genetically deficient in either IL-2 (Il2−/−) or CD25 (Il2ra−/−). We found that IL-2 signaling was dispensable for the induction of Foxp3 expression in thymocytes from these mice, which indicated that IL-2 signaling does not have a nonredundant function in the development of Treg cells. Unexpectedly, Il2−/− and Il2ra−/− Treg cells were fully able to suppress T cell proliferation in vitro. In contrast, Foxp3 was not expressed in thymocytes or peripheral T cells from Il2rg−/− mice. Gene expression analysis showed that IL-2 signaling was required for maintenance of the expression of genes involved in the regulation of cell growth and metabolism. Thus, IL-2 signaling seems to be critically required for maintaining the homeostasis and competitive fitness of Treg cells in vivo.*Note: In the version of this article initially published, the GEO database accession number is missing. This should be the final subsection of Methods, as follows: Accession code. GEO: microarray data, GSE4179. The error has been corrected in the PDF version of the article.

Regulatory T cells prevent catastrophic autoimmunity throughout the lifespan of mice

Mice lacking the transcription factor Foxp3 (Foxp3−) lack regulatory T (Treg) cells and develop fatal autoimmune pathology. In Foxp3− mice, many activated effector T cells express self-reactive T cell receptors that are expressed in Treg cells in wild-type mice. Thus, in wild-type mice, most self-reactive thymocytes escaping negative selection are diverted into the Treg lineage, and whether Treg cells are critical in self-tolerance in wild-type mice remains unknown. Here, acute in vivo ablation of Treg cells demonstrated a vital function for Treg cells in neonatal and adult mice. We suggest that self-reactive T cells are continuously suppressed by Treg cells and that when suppression is relieved, self-reactive T cells become activated and facilitate accelerated maturation of dendritic cells.

Metabolites produced by commensal bacteria promote peripheral regulatory T-cell generation

Intestinal microbes provide multicellular hosts with nutrients and confer resistance to infection. The delicate balance between pro- and anti-inflammatory mechanisms, essential for gut immune homeostasis, is affected by the composition of the commensal microbial community. Regulatory T cells (Treg cells) expressing transcription factor Foxp3 have a key role in limiting inflammatory responses in the intestine. Although specific members of the commensal microbial community have been found to potentiate the generation of anti-inflammatory Treg or pro-inflammatory T helper 17 (TH17) cells, the molecular cues driving this process remain elusive. Considering the vital metabolic function afforded by commensal microorganisms, we reasoned that their metabolic by-products are sensed by cells of the immune system and affect the balance between pro- and anti-inflammatory cells. We tested this hypothesis by exploring the effect of microbial metabolites on the generation of anti-inflammatory Treg cells. We found that in mice a short-chain fatty acid (SCFA), butyrate, produced by commensal microorganisms during starch fermentation, facilitated extrathymic generation of Treg cells. A boost in Treg-cell numbers after provision of butyrate was due to potentiation of extrathymic differentiation of Treg cells, as the observed phenomenon was dependent on intronic enhancer CNS1 (conserved non-coding sequence 1), essential for extrathymic but dispensable for thymic Treg-cell differentiation. In addition to butyrate, de novo Treg-cell generation in the periphery was potentiated by propionate, another SCFA of microbial origin capable of histone deacetylase (HDAC) inhibition, but not acetate, which lacks this HDAC-inhibitory activity. Our results suggest that bacterial metabolites mediate communication between the commensal microbiota and the immune system, affecting the balance between pro- and anti-inflammatory mechanisms.

TGF-beta-induced Foxp3 inhibits T(H)17 cell differentiation by antagonizing RORgammat function.

T helper cells that produce IL-17 (TH17 cells) promote autoimmunity in mice and have been implicated in the pathogenesis of human inflammatory diseases. At mucosal surfaces, TH17 cells are thought to protect the host from infection, whereas regulatory T (Treg) cells control immune responses and inflammation triggered by the resident microflora. Differentiation of both cell types requires transforming growth factor-β (TGF-β), but depends on distinct transcription factors: RORγt (encoded by Rorc(γt)) for TH17 cells and Foxp3 for Treg cells. How TGF-β regulates the differentiation of T cells with opposing activities has been perplexing. Here we demonstrate that, together with pro-inflammatory cytokines, TGF-β orchestrates TH17 cell differentiation in a concentration-dependent manner. At low concentrations, TGF-β synergizes with interleukin (IL)-6 and IL-21 (refs 9–11) to promote IL-23 receptor (Il23r) expression, favouring TH17 cell differentiation. High concentrations of TGF-β repress IL23r expression and favour Foxp3+ Treg cells. RORγt and Foxp3 are co-expressed in naive CD4+ T cells exposed to TGF-β and in a subset of T cells in the small intestinal lamina propria of the mouse. In vitro, TGF-β-induced Foxp3 inhibits RORγt function, at least in part through their interaction. Accordingly, lamina propria T cells that co-express both transcription factors produce less IL-17 (also known as IL-17a) than those that express RORγt alone. IL-6, IL-21 and IL-23 relieve Foxp3-mediated inhibition of RORγt, thereby promoting TH17 cell differentiation. Therefore, the decision of antigen-stimulated cells to differentiate into either TH17 or Treg cells depends on the cytokine-regulated balance of RORγt and Foxp3.

CD4+ Regulatory T Cells Control TH17 Responses in a Stat3-Dependent Manner

Outfoxing Immune Excess Immune responses are kept in check by Foxp3-expressing CD4+-regulatory T cells (Tregs) through a variety of mechanisms. Expression of specific transcription factors directs Treg responses into distinct T helper cell lineages; however, the transcription factors that regulate particular helper lineages have not been completely characterized. Chaudhry et al. (p. 986, published online 1 October) show that the transcription factor Stat3, that is required for the initial differentiation of TH17-effector T cells, is also required for Treg cell-mediated suppression of TH17-mediated immune responses. Mice carrying a Treg cellspecific deletion in Stat3 succumb to an intestinal inflammatory disease driven by uncontrolled TH17 responses. Thus, different classes of immune responses can result from the expression of helper lineage–specific transcription factors. Suppressor T cells regulate different classes of immune responses through induction of specific transcription factors. Distinct classes of protective immunity are guided by activation of STAT transcription factor family members in response to environmental cues. CD4+ regulatory T cells (Tregs) suppress excessive immune responses, and their deficiency results in a lethal, multi-organ autoimmune syndrome characterized by T helper 1 (TH1) and T helper 2 (TH2) CD4+ T cell–dominated lesions. Here we show that pathogenic TH17 responses in mice are also restrained by Tregs. This suppression was lost upon Treg-specific ablation of Stat3, a transcription factor critical for TH17 differentiation, and resulted in the development of a fatal intestinal inflammation. These findings suggest that Tregs adapt to their environment by engaging distinct effector response–specific suppression modalities upon activation of STAT proteins that direct the corresponding class of the immune response.

Maintenance of the Foxp3-dependent developmental program in mature regulatory T cells requires continued expression of Foxp3

The transcription factor Foxp3 is required for the development of regulatory T cells (Treg cell). Here we report that induced ablation of a loxP-flanked Foxp3 allele in mature Treg cells resulted in the loss of their suppressive function in vivo and acquisition of the ability to produce interleukin 2 and T helper type 1 cytokines. Furthermore, after adoptive transfer in the absence of functional Treg cells into lymphopenic hosts, Treg cells with deletion of Foxp3 proliferated and were predominant among tissue-infiltrating T cells. In agreement with those results, we found deregulation of Foxp3 target gene expression after Foxp3 deletion. Thus, continued Foxp3 expression in mature Treg cells is needed to maintain the transcriptional and functional program established during Treg cell development.

Homeostasis and anergy of CD4 + CD25 + suppressor T cells in vivo

CD4+CD25+ suppressor T (TS) cells play a critical role in the maintenance of peripheral tolerance. We examined here proliferative and functional responses as well as differential gene expression in TS cells. We found that TS cells were hyporesponsive to antigenic stimuli in vivo and unable to flux Ca2+ upon T cell receptor (TCR) engagement. However, TS cells were not impaired in their proliferative response to lymphopenia, which was dependent on major histocompatibility complex class II expression. Homeostatic proliferation did not abolish TS cell anergy; rather, it substantially augmented TS cell function. DNA array analyses identified genes that may inhibit responsiveness at a number of levels in multiple signaling cascades in TS cells, as well as several anti-apoptotic genes that may mediate their survival.

Extrathymically generated regulatory T cells control mucosal TH2 inflammation

A balance between pro- and anti-inflammatory mechanisms at mucosal interfaces, which are sites of constitutive exposure to microbes and non-microbial foreign substances, allows for efficient protection against pathogens yet prevents adverse inflammatory responses associated with allergy, asthma and intestinal inflammation. Regulatory T (Treg) cells prevent systemic and tissue-specific autoimmunity and inflammatory lesions at mucosal interfaces. These cells are generated in the thymus (tTreg cells) and in the periphery (induced (i)Treg cells), and their dual origin implies a division of labour between tTreg and iTreg cells in immune homeostasis. Here we show that a highly selective blockage in differentiation of iTreg cells in mice did not lead to unprovoked multi-organ autoimmunity, exacerbation of induced tissue-specific autoimmune pathology, or increased pro-inflammatory responses of T helper 1 (TH1) and TH17 cells. However, mice deficient in iTreg cells spontaneously developed pronounced TH2-type pathologies at mucosal sites—in the gastrointestinal tract and lungs—with hallmarks of allergic inflammation and asthma. Furthermore, iTreg-cell deficiency altered gut microbial communities. These results suggest that whereas Treg cells generated in the thymus appear sufficient for control of systemic and tissue-specific autoimmunity, extrathymic differentiation of Treg cells affects commensal microbiota composition and serves a distinct, essential function in restraint of allergic-type inflammation at mucosal interfaces.

Stability of the regulatory T cell lineage in vivo

Self-Renewing T Cells The homeostasis of cell populations within an organism can be achieved through a variety of mechanisms, including the differentiation of precursor populations, self-renewal of terminally differentiated cells, or by programming cells to be extremely long-lived. Regulatory T cells that express the transcription factor Foxp3 are critical for maintaining immune tolerance by preventing excessive inflammation and autoimmunity. Rubtsov et al. (p. 1667) now use genetic fate mapping and cell transfer studies in vivo to demonstrate that Foxp3-expressing cells are remarkably stable under both basal and inflammatory conditions. Thus, regulatory T cells appear to be maintained through self-renewal and should maintain their identity if used in adoptive cell therapies for treatment of autoimmunity or other inflammatory disorders. A subset of T cells that suppress immune-mediated inflammation is maintained by self-renewal. Tissue maintenance and homeostasis can be achieved through the replacement of dying cells by differentiating precursors or self-renewal of terminally differentiated cells or relies heavily on cellular longevity in poorly regenerating tissues. Regulatory T cells (Treg cells) represent an actively dividing cell population with critical function in suppression of lethal immune-mediated inflammation. The plasticity of Treg cells has been actively debated because it could factor importantly in protective immunity or autoimmunity. By using inducible labeling and tracking of Treg cell fate in vivo, or transfers of highly purified Treg cells, we have demonstrated notable stability of this cell population under physiologic and inflammatory conditions. Our results suggest that self-renewal of mature Treg cells serves as a major mechanism of maintenance of the Treg cell lineage in adult mice.