The immune system of vertebrate animals has evolved to mount an effective defense against a diverse set of pathogens while minimizing transient or lasting impairment in tissue function resulting from inflammation caused by the immune response to infectious agents. Furthermore, misguided immune responses to “self” and dietary antigens and commensal microorganisms, can lead to a variety of inflammatory disorders including autoimmunity, metabolic syndrome, allergies, and cancer. Regulatory T (Treg) cell–mediated suppression of inflammatory responses in diverse biological settings serves as a vital mechanism of negative regulation of immune-mediated inflammation and features prominently in autoimmune and auto-inflammatory disorders. The discovery by our laboratory and by others of an essential role for the X-chromosome–encoded transcription factor Foxp3 in the Treg cell lineage greatly facilitated understanding the biology of these critically important cells. Loss-of-function mutations in the Foxp3 gene in humans result in a highly aggressive fatal autoimmune and inflammatory syndrome known as IPEX (immune dysregulation polyendocrinopathy entheropaty X-linked). We observed a similarly severe disease upon ablation of the Foxp3 gene in mice. We showed that Foxp3 serves a role of the late-acting Treg cell lineage–specifying factor, and its continuous expression in Treg cells is required for their ability to prevent autoimmune and inflammatory syndrome. Furthermore, we demonstrated that the paucity of Treg cells fully accounts for the fatal disease associated with the Foxp3 deficiency. We also demonstrated a continuous requirement for the suppressor function of Treg cells throughout the lifespan of an organism – acute ablation of Treg cells upon diphtheria toxin (DT) treatment of healthy adult Foxp3DTR mice harboring diphtheria toxin receptor (DTR) under control of the endogenous Foxp3 locus triggers a similarly rapid and devastating disease regardless of the presence of commensal microbiota, the largest source of “non-self” antigens and ligands for innate immune sensors. Thus, the mechanism of Treg cell mediated suppression likely evolved to meet the threat of fatal self-reactivity posed by the emergence of self-MHC restricted T cell recognition in vertebrates.
Treg cell differentiation commences in the thymus (tTreg) and extrathymically (peripheral, pTreg) upon expression of Foxp3 in response to T-cell receptor (TCR) and interleukin-2 (IL-2) receptor signaling in precursor cells. Through inducible genetic cell-fate mapping, we addressed a major outstanding issue whether Treg cells represent a stable lineage or a "plastic" reversible state of activation. Our studies showed remarkable stability of differentiated Treg cells and heritable maintenance of Foxp3 expression over the lifespan of animals under physiologic and inflammatory conditions. Stable Foxp3 expression was acquired following a period of instability in newly generated Treg cells. These studies provided unequivocal proof that Treg cells represent a dedicated lineage.
Building on our findings of a key role Foxp3 plays in the biology of Treg cells, we explored regulation of the Foxp3 gene expression to gain understanding of Treg lineage commitment and function. Using computational, biochemical, and gene-targeting approaches, we identified three proximal regulatory elements in the Foxp3 locus (conserved noncoding sequences, CNS1–3) each with a distinct and non-redundant function in Treg cell biology. CNS3 was poised in Treg cell precursors and facilitated Foxp3 expression in both thymocytes and naïve peripheral T cells. In contrast to CNS3 contributing to both thymic and extrathymic differentiation of Treg cells, we found that CNS1 is dispensable for former, but essential for generation of Treg cells from naïve peripheral CD4 T cells. Our studies demonstrated that CNS3 acts as an epigenetic switch that confers a poised state to the Foxp3 promoter in precursor cells to make Treg cell lineage commitment responsive to a broad range of TCR stimuli, particularly to suboptimal ones. CNS3-dependent expansion of the TCR repertoire enables Treg cells to control self-reactive T cells effectively, especially when thymic negative selection is genetically impaired. The observations highlight the complementary roles of these two main mechanisms of “self”-tolerance.
Unlike CNS1 and CNS3 roles in promoting Treg cell differentiation, we found that CNS2 is dispensable for Foxp3 induction but is required for its maintenance. CNS2, inactive in precursors and recently generated Treg cells, is activated upon CpG demethylation in fully differentiated Treg cells. We found that a sole function of CNS2 is to enable heritable maintenance of Treg cell identity during cell division when a key factor inducing Treg cell lineage commitment, IL-2, is limiting and when pro-inflammatory cytokines, promoting alternative cell fate, are present. Thus, CNS2 enforces stability of the differentiated cell state in the Treg cell lineage in fluctuating environments and this unique function of CNS2 is important in diverse chronic inflammation types including autoimmunity, chronic infection, metabolic inflammation and cancer.
Considering a role for Foxp3 as Treg lineage specifying factor we asked whether Foxp3 exerts its function by establishing a new set of enhancers or by utilizing an enhancer landscape prepared in precursor cells by their developmental history. Genome-wide analysis of chromatin accessibility, using DNase I hypersensitivity, and of Foxp3 binding, using chromatin immunoprecipitation combined with transcriptome analysis, showed that Foxp3 binds predominantly to preformed enhancers. Further studies showed that Foxp3 exploits preformed enhancers occupied by its partners or a structurally related predecessor or formed in response to TCR-driven chromatin remodeling during Treg differentiation. In contrast to an “opportunistic” mode of action by Foxp3 during Treg cell differentiation, upon Treg cell activation under inflammatory conditions Foxp3 bound sites showed diminished accessibility of chromatin and deposition of “non-permissive” histone marks, a likely consequence of PRC2 recruitment, and down-regulation of the expression of nearby genes. Thus, Foxp3 poises its targets for repression by facilitating the formation of repressive chromatin in Treg cells upon their activation in response to inflammatory cues.
Our genome-wide gene expression studies identified sets of genetic elements operating in a Treg cell-specific and non-specific manner to control gene expression. The analysis of evolutionary conservation of these elements and natural human inter-individual epigenetic variation in Treg cells identified the core transcriptional control program of Treg cell lineage specification. Furthermore, analysis of single nucleotide polymorphisms in core lineage-specific enhancers revealed disease associations, most prominently with type I diabetes, which were further corroborated by high-resolution mapping of causal polymorphisms in lineage-specific enhancers. These studies suggest that a small set of regulatory elements specify the Treg lineage and that genetic variation in Treg cell-specific enhancers may alter Treg cell function contributing to polygenic disease.
Thymic vs. Extrathymic Differentiation of Regulatory T Cells
Our discovery of a selective requirement for CNS1 for pTreg differentiation suggested that biological functions of tTreg and pTreg cells are different. Since CNS1-deficient mice were devoid of frank autoimmune lesions characteristic of pan-Treg deficiency, we hypothesized that pTreg differentiation might have emerged to mitigate maternal-fetal allogeneic conflict. We found that CNS1 is present only in eutherian mammals, but not in monotremes or lower vertebrates. CNS1 contained a mammalian-wide interspersed repeat (MIR) retrotransposon, which emerged at a time of placental mammal radiation. pTreg cells specific for fetal alloantigens were generated in a CNS1-dependent manner and impairment in pTreg generation led to embryo loss during allogeneic pregnancy. The surviving placentas exhibited hallmarks of preeclampsia, a major complication of human pregnancy. Our results suggest that pTreg differentiation emerged during evolution of placental mammals upon exaptation of a retrotransposon to mitigate maternal-fetal allogenenic conflict.
Evolutionary acquisitions frequently find uses beyond the original ones driving their emergence. Indeed, we found that under physiologic conditions, pTreg cells have a nonredundant role in restraining allergic-type inflammation in the gut and asthma-like pathology in the lungs and influence the composition of the gut microbial community. 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 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. 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 inhibition. Our results suggest that bacterial metabolites mediate communication between the commensal microbiota and the immune system, affecting the balance between pro- and anti-inflammatory forces.
Regulatory T Cell Function
Considering a key role for TCR and IL-2R signaling pathways in Treg cell differentiation we explored their role in the suppressor function. We demonstrated that continuous signaling through TCR and IL-2R is required not only for differentiation, but also for the proliferative fitness and functional capability of Treg cells. These two signaling pathways control largely non-overlapping sets of genes responsible for different aspects of suppressor function. Acute ablation of IL-2 receptor subunits or TCR in differentiated Treg cells results in severe Treg cell dysfunction that could not be attributed solely to impaired expression of Foxp3. Furthermore, our gene targeting studies revealed that instead of employing a single unique suppressor molecule, Treg cell suppressor function in mediated by a range of secreted and cell-bound molecules.
Furthermore, we hypothesized that instead of employing a universal program of suppression, Treg cells adjust their homeostatic, migratory, and functional features in response to environmental cues. We further assumed that this is accomplished by maintaining an optimal threshold of activation of transcription factors involved in regulation of the corresponding type of inflammatory responses. We provided proof for this idea by demonstrating that activation of certain STAT (signal transducer and activator of transcription) family members in response to inflammatory cues in Treg cells is required for restraint of the inflammatory effector T cell responses these factors promote. Excessive activation of STAT family members in Treg cells also causes a failure to control the corresponding inflammatory response. Limiting STAT activity in Treg cells prevents their deviation into cytokine-producing effector cells and thereby safeguards their function and identity in inflammatory settings.
In addition to their key immunosuppressive role, we found that differentiated Treg cells residing in non-lymphoid organs can carry additional essential non-immune function by promoting tissue maintenance and repair. In support of this idea we showed that selective Treg cell deficiency in an EGF family member amphiregulin leads to severe acute lung damage and decreased blood oxygen concentration during influenza virus infection without any measureable alterations in Treg cell suppressor function, antiviral immune responses, or viral load. This tissue repair modality is mobilized in Treg cells in response to inflammatory mediator IL-18 or alarmin IL-33, but not by TCR signaling that is required for suppressor function. These results suggest Treg cells have a major direct and non-redundant role in tissue repair and maintenance-distinct from their role in suppression of immune responses and inflammation-and that these two essential Treg cell functions are invoked by separable cues.
In addition to lymphoid organs and barrier sites such as skin, intestine, lung and liver, Treg cells densely populate solid tumors and may promote progression through both their immunosuppressive and tissue repair promoting functions. We observed a major tumor-promoting role of Treg cells in poorly immunogenic oncogene-driven cancers and they reveal the potential therapeutic value of combining transient T reg cell ablation with radiotherapy for the management of poorly immunogenic, aggressive malignancies.
Grants from the National Institutes of Health provided support for some of these studies.
As of June 14, 2016