Steven Z. Josefowicz

Regulatory T Cells: Mechanisms of Differentiation and Function

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.

Role of conserved non-coding DNA elements in the Foxp3 gene in regulatory T-cell fate

Immune homeostasis is dependent on tight control over the size of a population of regulatory T (Treg) cells capable of suppressing over-exuberant immune responses. The Treg cell subset is comprised of cells that commit to the Treg lineage by upregulating the transcription factor Foxp3 either in the thymus (tTreg) or in the periphery (iTreg). Considering a central role for Foxp3 in Treg cell differentiation and function, we proposed that conserved non-coding DNA sequence (CNS) elements at the Foxp3 locus encode information defining the size, composition and stability of the Treg cell population. Here we describe the function of three Foxp3 CNS elements (CNS1–3) in Treg cell fate determination in mice. The pioneer element CNS3, which acts to potently increase the frequency of Treg 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 tTreg cell differentiation, but has a prominent role in iTreg cell generation in gut-associated lymphoid tissues. CNS2, although dispensable for Foxp3 induction, is required for Foxp3 expression in the progeny of dividing Treg cells. Foxp3 binds to CNS2 in a Cbf-β–Runx1 and CpG DNA demethylation-dependent manner, suggesting that Foxp3 recruitment to this ‘cellular memory module’ facilitates the heritable maintenance of the active state of the Foxp3 locus and, therefore, Treg lineage stability. Together, our studies demonstrate that the composition, size and maintenance of the Treg cell population are controlled by Foxp3 CNS elements engaged in response to distinct cell-extrinsic or -intrinsic cues.

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.

Extrathymic Generation of Regulatory T Cells in Placental Mammals Mitigates Maternal-Fetal Conflict

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 noncoding 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.

Cutting Edge: TCR Stimulation Is Sufficient for Induction of Foxp3 Expression in the Absence of DNA Methyltransferase 1

TCR signaling is important for regulatory T cell (Tr) development. Using a genetic model of DNA methyltransferase 1 (Dnmt1) deficiency, we observed highly efficient Foxp3 induction following TCR stimulation, suggesting a dominant role for TCR signaling in Foxp3 induction. In the absence of Dnmt1, Foxp3 induction in thymic and peripheral Foxp3-negative T cells was maximized upon TCR engagement, and the provision of TGF-β was dispensable for Foxp3 expression. In addition, CD4-Cre × dnmt1fl/fl mice harbored sizeable thymic and peripheral populations of CD8+Foxp3+ cells, suggesting that Dnmt1 activity is required for restricting Foxp3 expression to the CD4 T cell lineage. Our results suggest that the TCR signal is sufficient for transcriptional activation of Foxp3 in the absence of maintenance DNA methylation and that TGF-β facilitates Foxp3 induction in part by opposing cell cycle-dependent Dnmt1 recruitment, leading to locus inactivation.

Mouse regulatory DNA landscapes reveal global principles of cis-regulatory evolution

To study the evolutionary dynamics of regulatory DNA, we mapped >1.3 million deoxyribonuclease I–hypersensitive sites (DHSs) in 45 mouse cell and tissue types, and systematically compared these with human DHS maps from orthologous compartments. We found that the mouse and human genomes have undergone extensive cis-regulatory rewiring that combines branch-specific evolutionary innovation and loss with widespread repurposing of conserved DHSs to alternative cell fates, and that this process is mediated by turnover of transcription factor (TF) recognition elements. Despite pervasive evolutionary remodeling of the location and content of individual cis-regulatory regions, within orthologous mouse and human cell types the global fraction of regulatory DNA bases encoding recognition sites for each TF has been strictly conserved. Our findings provide new insights into the evolutionary forces shaping mammalian regulatory DNA landscapes. Mouse-to-human genomic comparisons illuminate conserved transcriptional programs despite regulatory rewiring. Rewiring the gene regulatory landscape DNAse I hypersensitive sites (DHSs) correlate with genomic locations that control where messenger RNA is to be produced. DHSs differ, depending on the cell type, developmental stage, and species. Viestra et al. compared mouse and human genome-wide DHS maps. Approximately one-third of the DHSs are conserved between the species, which separated approximately 550 million years ago. Most DHSs fell into tissue-specific cohorts; however, these were generally not conserved between the human and mouse. It seems that the majority of DHSs evolve because of changes in the sequence that gradually change how the region is regulated. Science, this issue p. 1007

An Interactive Database for the Assessment of Histone Antibody Specificity

Access to high-quality antibodies is a necessity for the study of histones and their posttranslational modifications (PTMs). Here we debut the Histone Antibody Specificity Database (http://www.histoneantibodies.com), an online and expanding resource cataloging the behavior of widely used, commercially available histone antibodies by peptide microarray. This interactive web portal provides a critical resource to the biological research community that routinely uses these antibodies as detection reagents for a wide range of applications.

Greater Than the Sum of Parts: Complexity of the Dynamic Epigenome

Information encoded in DNA is interpreted, modified, and propagated as chromatin. The diversity of inputs encountered by eukaryotic genomes demands a matching capacity for transcriptional outcomes provided by the combinatorial and dynamic nature of epigenetic processes. Advances in genome editing, visualization technology, and genome-wide analyses have revealed unprecedented complexity of chromatin pathways, offering explanations to long-standing questions and presenting new challenges. Here, we review recent findings, exemplified by the emerging understanding of crossregulatory interactions within chromatin, and emphasize the pathologic outcomes of epigenetic misregulation in cancer.

Regulators of chromatin state and transcription in CD4 T-cell polarization.

Mature naive CD4 T‐cells possess the potential for an array of highly specialized functions, from inflammatory to potently suppressive. This potential is encoded in regulatory DNA elements and is fulfilled through modification of chromatin and selective activation by the collaborative function of diverse transcription factors in response to environmental cues. The mechanisms and strategies employed by transcription factors for the programming of CD4 T‐cell subsets will be discussed. In particular, the focus will be on co‐operative activity of environmental response factors in the initial activation of regulatory DNA elements and chromatin alteration, and the subsequent role of ‘master regulator’ transcription factors in defining the fidelity and environmental responsiveness of different CD4 T‐cell subsets.