The immune system has evolved to mount an effective defense against pathogens and 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 by our laboratory and by others of an essential role for X-chromosome–encoded transcription factor Foxp3 in the Treg cell lineage facilitated recent progress in understanding the biology of Treg cells.
Treg cells differentiate in the thymus (tTreg) and extrathymically (peripheral, pTreg) upon expression of Foxp3. We showed that Foxp3 is the Treg lineage–specifying factor, and its loss leads to fatal early-onset autoimmunity; paucity of Treg cells fully accounts for the disease. We also found that ablation of Treg cells upon diphtheria toxin (DT) treatment of adult Foxp3DTR mice harboring diphtheria toxin receptor (DTR) under control of the Foxp3 locus triggers a similar fatal disease.
Regulatory T Cell Differentiation
Through inducible genetic cell-fate mapping, we addressed a major outstanding issue: Do Treg cells represent a stable lineage or "plastic" state? 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). CNS3 was poised in Treg cell precursors and facilitated Foxp3 expression in both thymocytes and naïve peripheral T cells. In contrast, CNS1 was dispensable for thymic but essential for extrathymic transforming growth factor-β (TGFβ)-dependent Treg generation. Finally, CNS2 was dispensable for Foxp3 induction but required for its maintenance.
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 T cell receptor (TCR)-driven chromatin remodeling during Treg differentiation.
Thymic versus Extrathymic 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.
Regulatory T Cell Function
In higher organisms, commensal microbiota represent the largest source of nonself-antigens and -ligands for innate sensors. It is possible that, in the absence of Treg cells, unrestrained stimulation of innate immune cells by commensal microbiota drives devastating inflammation and autoimmunity. Alternatively, self-MHC (major histocompatibility complex)–restricted CD4+ T cell recognition poses an imminent threat of autoimmunity. Our finding that ablation of Treg cells in germ-free Foxp3DTR mice results in fatal inflammatory syndrome unequivocally supported the latter idea.
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.
Our genetic studies revealed an important role for microRNA (miRNA)-dependent regulation of gene expression in Treg function. We found that microRNA-155 (miR-155), highly expressed in Treg cells, conferred competitive fitness through targeting SOCS1 (suppressor of cytokine signaling 1), an important negative-feedback regulator of IL-2 (interleukin-2) signaling. We also found that miR-146a, also highly expressed in Treg cells, enables Treg cell–mediated suppression of type 1 inflammation. These and other functional studies emphasized the need for comprehensive identification of miRNA targets. The first unbiased transcriptome-wide identification of the targets of a single miRNA revealed pervasive noncanonical Argonaute binding. These sites often contained seed-like motifs and regulated gene expression, generating a continuum of targeting. Our studies suggested a parallel between regulation of gene expression by miRNAs and transcription factors.
Grants from the National Institutes of Health provided support for some of these studies.
As of July 9, 2013