The immune system has evolved to mount an effective defense against pathogens and to minimize deleterious immune-mediated inflammation caused by commensal microorganisms, immune responses against self and environmental antigens, and metabolic inflammatory disorders. Regulatory T (Treg) cell–mediated suppression serves as a vital mechanism of negative regulation of immune-mediated inflammation and features prominently in autoimmune and autoinflammatory disorders, allergy, acute and chronic infections, cancer, and metabolic inflammation. The discovery that Foxp3 is the transcription factor that specifies the Treg cell lineage facilitated recent progress in understanding the biology of regulatory T cells. In this review, we discuss cellular and molecular mechanisms in the differentiation and function of these cells.

SummaryAs the premier model organism in biomedical research, the laboratory mouse shares the majority of protein-coding genes with humans, yet the two mammals differ in significant ways. To gain greater insights into both shared and species-specific transcriptional and cellular regulatory programs in the mouse, the Mouse ENCODE Consortium has mapped transcription, DNase I hypersensitivity, transcription factor binding, chromatin modifications, and replication domains throughout the mouse genome in diverse cell and tissue types. By comparing with the human genome, we not only confirm substantial conservation in the newly annotated potential functional sequences, but also find a large degree of divergence of other sequences involved in transcriptional regulation, chromatin state and higher order chromatin organization. Our results illuminate the wide range of evolutionary forces acting on genes and their regulatory regions, and provide a general resource for research into mammalian biology and mechanisms of human diseases.

Immune homeostasis is dependent on tight control over the size of a population of regulatory T (T reg ) cells capable of suppressing over-exuberant immune responses. The T reg cell subset is comprised of cells that commit to the T reg lineage by upregulating the transcription factor Foxp3 either in the thymus (tT reg ) or in the periphery (iT reg ) 1,2 . Considering a central role for Foxp3 in T reg cell differentiation and function 3,4 , we proposed that conserved non-coding DNA sequence (CNS) elements at the Foxp3 locus encode information defining the size, composition and stability of the T reg cell population. Here we describe the function of three Foxp3 CNS elements (CNS1-3) in T reg cell fate determination in mice. The pioneer element CNS3, which acts to potently increase the frequency of T reg cells generated in the thymus and the periphery, binds c-Rel in in vitro assays. In contrast, CNS1, which contains a TGF-β-NFAT response element, is superfluous for tT reg cell differentiation, but has a prominent role in iT reg cell generation in gut-associated lymphoid tissues. CNS2, although dispensable for Foxp3 induction, is required for Foxp3 expression in the progeny of dividing T reg cells. Foxp3 binds to CNS2 in a Cbf-β-Runx1 and CpG DNA demethylationdependent manner, suggesting that Foxp3 recruitment to this 'cellular memory module' facilitates the heritable maintenance of the active state of the Foxp3 locus and, therefore, T reg lineage stability.Together, our studies demonstrate that the composition, size and maintenance of the T reg cell population are controlled by Foxp3 CNS elements engaged in response to distinct cell-extrinsic orintrinsic cues.To determine cis-elements that are potentially involved in the control of T reg cell fate, we first examined permissive (mono-methylated histone H3 at Lys4 (H3K4me1), di-methylated H3K4 (H3K4me2), H3K4me3, H3K36me3, and acetylated H3K9/14 (H3K9/14Ac)) and nonpermissive (H3K9me2, H3K9me3 and H3K27me3) modifications of histone H3 bound to three Foxp3 CNS elements (CNS1-3; Fig. 1a) in CD4 + CD25 − Foxp3 − naive T cells (T N ),

Transcription factor Foxp3 (forkhead box P3), restricted in its expression to a specialized regulatory CD4+ T-cell subset (T(R)) with a dedicated suppressor function, controls T(R) lineage development. In humans and mice, Foxp3 deficiency results in a paucity of T(R) cells and a fatal breach in immunological tolerance, causing highly aggressive multi-organ autoimmune pathology. Here, through genome-wide analysis combining chromatin immunoprecipitation with mouse genome tiling array profiling, we identify Foxp3 binding regions for approximately 700 genes and for an intergenically encoded microRNA. We find that a large number of Foxp3-bound genes are up- or downregulated in Foxp3+ T cells, suggesting that Foxp3 acts as both a transcriptional activator and repressor. Foxp3-mediated regulation unique to the thymus affects, among others, genes encoding nuclear factors that control gene expression and chromatin remodelling. In contrast, Foxp3 target genes shared by the thymic and peripheral T(R) cells encode primarily plasma membrane proteins, as well as cell signalling proteins. Together, our studies suggest that distinct transcriptional sub-programmes implemented by Foxp3 establish T(R) lineage during differentiation and its proliferative and functional competence in the periphery.

A balance between pro- and anti-inflammatory mechanisms at mucosal interfaces, 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 inflammation1. 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 (iTreg cells) and their dual origin implies a division of labor between tTreg and iTreg cells in immune homeostasis. Here we demonstrate that a highly selective blockage in differentiation of iTreg cells did not lead to unprovoked multi-organ autoimmunity, exacerbation of induced tissue-specific autoimmune pathology (EAE), or increased pro-inflammatory Th1 and Th17 cell responses. However, iTreg cell-deficient mice 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 impacts commensal microbiota composition and serves a distinct, essential function in restraint of allergic type inflammation at mucosal interfaces.

Tissue maintenance and homeostasis can be achieved through replacement of dying cells by differentiating precursors, self-renewal of terminally differentiated cells or relies heavily on cellular longevity in poorly regenerating tissues. Regulatory T (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 as it could factor importantly in protective immunity or autoimmunity. Here, by using inducible labeling and tracking of Treg cell fate in vivo, or transfers of highly purified Treg cells, we demonstrate remarkable 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.

Summary Regulatory T (Treg) cells, whose differentiation and function are controlled by X-chromosome-encoded transcription factor Foxp3, are generated in the thymus (tTreg) and extrathymically (peripheral, pTreg) and their deficiency results in fatal autoimmunity. Here we demonstrate that a Foxp3 enhancer, conserved non-coding sequence 1 (CNS1), essential for pTreg, but dispensable for tTreg cell generation, is present only in placental mammals. CNS1 is largely composed of mammalian-wide interspersed repeats (MIR) that have undergone retrotransposition during early mammalian radiation. During pregnancy pTreg cells specific to a model paternal alloantigen were generated in a CNS1-dependent manner and accumulated in the placenta. Furthermore, when mated with allogeneic, but not syngeneic males, CNS1-deficient females showed increased fetal resorption accompanied by increased immune cell infiltration and defective remodeling of spiral arteries. Our results suggest that during evolution a CNS1-dependent mechanism of extrathymic differentiation of Treg cells emerged in placental animals to enforce maternal-fetal tolerance.

Summary Regulatory T (Treg) cells, whose identity and function are defined by the transcription factor Foxp3, are indispensable for immune homeostasis. It is unclear whether Foxp3 exerts its Treg lineage specification function through active modification of the chromatin landscape and establishment of new enhancers or by exploiting a pre-existing enhancer landscape. Analysis of the chromatin accessibility of Foxp3-bound enhancers in Treg and Foxp3-negative T cells showed that Foxp3 was bound overwhelmingly to pre-accessible enhancers occupied by its cofactors in precursor cells or a structurally related predecessor. Furthermore, the bulk of Foxp3- bound Treg cell enhancers lacking in Foxp3− CD4+ cells became accessible upon T cell receptor activation prior to Foxp3 expression with only a small subset associated with several functionally important genes being exclusively Treg cell-specific. Thus, in a late cellular differentiation process Foxp3 defines Treg cell functionality in an “opportunistic” manner by largely exploiting the preformed enhancer network instead of establishing a new enhancer landscape.