Memory of Inflammation in Regulatory T Cells.

Eukaryotic cells can "remember" transient encounters with a wide range of stimuli, inducing lasting states of altered responsiveness. Regulatory T (Treg) cells are a specialized lineage of suppressive CD4 T cells that act as critical negative regulators of inflammation in various biological contexts. Treg cells exposed to inflammatory conditions acquire strongly enhanced suppressive function. Using inducible genetic tracing, we analyzed the long-term stability of activation-induced transcriptional, epigenomic, and functional changes in Treg cells. We found that the inflammation-experienced Treg cell population reversed many activation-induced changes and lost its enhanced suppressive function over time. The "memory-less" potentiation of Treg suppressor function may help avoid a state of generalized immunosuppression that could otherwise result from repeated activation.

A Distinct Function of Regulatory T Cells in Tissue Protection.

Regulatory T (Treg) cells suppress immune responses to a broad range of non-microbial and microbial antigens and indirectly limit immune inflammation-inflicted tissue damage by employing multiple mechanisms of suppression. Here, we demonstrate that selective Treg cell deficiency in 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 that, during infectious lung injury, 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.

Novel antigen-presenting cell imparts Treg-dependent tolerance to gut microbiota.

Establishing and maintaining tolerance to self-antigens or innocuous foreign antigens is vital for the preservation of organismal health. Within the thymus, medullary thymic epithelial cells (mTECs) expressing autoimmune regulator (AIRE) have a critical role in self-tolerance through deletion of autoreactive T cells and promotion of thymic regulatory T (Treg) cell development1-4. Within weeks of birth, a separate wave of Treg cell differentiation occurs in the periphery upon exposure to antigens derived from the diet and commensal microbiota5-8, yet the cell types responsible for the generation of peripheral Treg (pTreg) cells have not been identified. Here we describe the identification of a class of RORγt+ antigen-presenting cells called Thetis cells, with transcriptional features of both mTECs and dendritic cells, comprising four major sub-groups (TC I-TC IV). We uncover a developmental wave of Thetis cells within intestinal lymph nodes during a critical window in early life, coinciding with the wave of pTreg cell differentiation. Whereas TC I and TC III expressed the signature mTEC nuclear factor AIRE, TC IV lacked AIRE expression and was enriched for molecules required for pTreg generation, including the TGF-ß-activating integrin αvß8. Loss of either major histocompatibility complex class II (MHCII) or ITGB8 by Thetis cells led to a profound impairment in intestinal pTreg differentiation, with ensuing colitis. By contrast, MHCII expression by RORγt+ group 3 innate lymphoid cells (ILC3) and classical dendritic cells was neither sufficient nor required for pTreg generation, further implicating TC IV as the tolerogenic RORγt+ antigen-presenting cell with an essential function in early life. Our studies reveal parallel pathways for the establishment of tolerance to self and foreign antigens in the thymus and periphery, respectively, marked by the involvement of shared cellular and transcriptional programmes.

Corrigendum: Stability and function of regulatory T cells expressing the transcription factor T-bet.

This corrects the article DOI: 10.1038/nature22360.

Stability and function of regulatory T cells expressing the transcription factor T-bet.

Adaptive immune responses are tailored to different types of pathogens through differentiation of naive CD4 T cells into functionally distinct subsets of effector T cells (T helper 1 (TH1), TH2, and TH17) defined by expression of the key transcription factors T-bet, GATA3, and RORγt, respectively. Regulatory T (Treg) cells comprise a distinct anti-inflammatory lineage specified by the X-linked transcription factor Foxp3 (refs 2, 3). Paradoxically, some activated Treg cells express the aforementioned effector CD4 T cell transcription factors, which have been suggested to provide Treg cells with enhanced suppressive capacity. Whether expression of these factors in Treg cells-as in effector T cells-is indicative of heterogeneity of functionally discrete and stable differentiation states, or conversely may be readily reversible, is unknown. Here we demonstrate that expression of the TH1-associated transcription factor T-bet in mouse Treg cells, induced at steady state and following infection, gradually becomes highly stable even under non-permissive conditions. Loss of function or elimination of T-bet-expressing Treg cells-but not of T-bet expression in Treg cells-resulted in severe TH1 autoimmunity. Conversely, following depletion of T-bet- Treg cells, the remaining T-bet+ cells specifically inhibited TH1 and CD8 T cell activation consistent with their co-localization with T-bet+ effector T cells. These results suggest that T-bet+ Treg cells have an essential immunosuppressive function and indicate that Treg cell functional heterogeneity is a critical feature of immunological tolerance.

A mechanism for expansion of regulatory T-cell repertoire and its role in self-tolerance.

T-cell receptor (TCR) signalling has a key role in determining T-cell fate. Precursor cells expressing TCRs within a certain low-affinity range for complexes of self-peptide and major histocompatibility complex (MHC) undergo positive selection and differentiate into naive T cells expressing a highly diverse self-MHC-restricted TCR repertoire. In contrast, precursors displaying TCRs with a high affinity for 'self' are either eliminated through TCR-agonist-induced apoptosis (negative selection) or restrained by regulatory T (Treg) cells, whose differentiation and function are controlled by the X-chromosome-encoded transcription factor Foxp3 (reviewed in ref. 2). Foxp3 is expressed in a fraction of self-reactive T cells that escape negative selection in response to agonist-driven TCR signals combined with interleukin 2 (IL-2) receptor signalling. In addition to Treg cells, TCR-agonist-driven selection results in the generation of several other specialized T-cell lineages such as natural killer T cells and innate mucosal-associated invariant T cells. Although the latter exhibit a restricted TCR repertoire, Treg cells display a highly diverse collection of TCRs. Here we explore in mice whether a specialized mechanism enables agonist-driven selection of Treg cells with a diverse TCR repertoire, and the importance this holds for self-tolerance. We show that the intronic Foxp3 enhancer conserved noncoding sequence 3 (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. Our findings highlight the complementary roles of these two main mechanisms of self-tolerance.

TGF-ß signalling is required for CD4⁺ T cell homeostasis but dispensable for regulatory T cell function.

TGF-ß is widely held to be critical for the maintenance and function of regulatory T (T(reg)) cells and thus peripheral tolerance. This is highlighted by constitutive ablation of TGF-ß receptor (TR) during thymic development in mice, which leads to a lethal autoimmune syndrome. Here we describe that TGF-ß-driven peripheral tolerance is not regulated by TGF-ß signalling on mature CD4⁺ T cells. Inducible TR2 ablation specifically on CD4⁺ T cells did not result in a lethal autoinflammation. Transfer of these TR2-deficient CD4⁺ T cells to lymphopenic recipients resulted in colitis, but not overt autoimmunity. In contrast, thymic ablation of TR2 in combination with lymphopenia led to lethal multi-organ inflammation. Interestingly, deletion of TR2 on mature CD4⁺ T cells does not result in the collapse of the T(reg) cell population as observed in constitutive models. Instead, a pronounced enlargement of both regulatory and effector memory T cell pools was observed. This expansion is cell-intrinsic and seems to be caused by increased T cell receptor sensitivity independently of common gamma chain-dependent cytokine signals. The expression of Foxp3 and other regulatory T cells markers was not dependent on TGF-ß signalling and the TR2-deficient T(reg) cells retained their suppressive function both in vitro and in vivo. In summary, absence of TGF-ß signalling on mature CD4⁺ T cells is not responsible for breakdown of peripheral tolerance, but rather controls homeostasis of mature T cells in adult mice.

TCR signaling via Tec kinase ITK and interferon regulatory factor 4 (IRF4) regulates CD8+ T-cell differentiation.

CD8(+) T-cell development in the thymus generates a predominant population of conventional naive cells, along with minor populations of "innate" T cells that resemble memory cells. Recent studies analyzing a variety of KO or knock-in mice have indicated that impairments in the T-cell receptor (TCR) signaling pathway produce increased numbers of innate CD8(+) T cells, characterized by their high expression of CD44, CD122, CXCR3, and the transcription factor, Eomesodermin (Eomes). One component of this altered development is a non-CD8(+) T cell-intrinsic role for IL-4. To determine whether reduced TCR signaling within the CD8(+) T cells might also contribute to this pathway, we investigated the role of the transcription factor, IFN regulatory factor 4 (IRF4). IRF4 is up-regulated following TCR stimulation in WT T cells; further, this up-regulation is impaired in T cells treated with a small-molecule inhibitor of the Tec family tyrosine kinase, IL-2 inducible T-cell kinase (ITK). In contrast to WT cells, activation of IRF4-deficient CD8(+) T cells leads to rapid and robust expression of Eomes, which is further enhanced by IL-4 stimulation. In addition, inhibition of ITK together with IL-4 increases Eomeso up-regulation. These data indicate that ITK signaling promotes IRF4 up-regulation following CD8(+) T-cell activation and that this signaling pathway normally suppresses Eomes expression, thereby regulating the differentiation pathway of CD8(+) T cells.

NIP45 controls the magnitude of the type 2 T helper cell response.

Nuclear factor of activated T cell (NFAT) transcription factors are key regulators of gene transcription within immune cells. The NFAT-interacting protein, (NIP45), augments NFAT-driven IL-4 expression by a mechanism that relies on arginine methylation. To establish the function of NIP45 in vivo, we generated mice with a targeted deletion of the gene encoding this cofactor. NIP45-deficient T helper cells displayed profound defects in the expression of NFAT-regulated cytokine genes, including IL-4. Whereas NIP45 deficiency does not interfere with T helper cell NFAT activation or lineage-specific transcription-factor expression, NIP45 acts as an enhancer for the assembly of protein arginine methyltransferase 1 and the protein arginine methyltransferase 1-linked histone 4 arginine 3 methylation with the IL-4 promoter. Our study reveals an essential role for NIP45 in promoting robust cytokine expression in vivo, which is required for the efficient handling of parasites. We propose that NIP45 acts as a molecular rheostat serving to amplify the type-2 immune response.

T reg cell-intrinsic requirements for ST2 signaling in health and neuroinflammation.

ST2, the receptor for the alarmin IL-33, is expressed by a subset of regulatory T (T reg) cells residing in nonlymphoid tissues, and these cells can potently expand upon provision of exogenous IL-33. Whether the accumulation and residence of T reg cells in tissues requires their cell-intrinsic expression of and signaling by ST2, or whether indirect IL-33 signaling acting on other cells suffices, has been a matter of contention. Here, we report that ST2 expression on T reg cells is largely dispensable for their accumulation and residence in nonlymphoid organs, including the visceral adipose tissue (VAT), even though cell-intrinsic sensing of IL-33 promotes type 2 cytokine production by VAT-residing T reg cells. In addition, we uncovered a novel ST2-dependent role for T reg cells in limiting the size of IL-17A-producing γδT cells in the CNS in a mouse model of neuroinflammation, experimental autoimmune encephalomyelitis (EAE). Finally, ST2 deficiency limited to T reg cells led to disease exacerbation in EAE.

Building a zoo of mice for genetic analyses: a comprehensive protocol for the rapid generation of BAC transgenic mice.

Transgenic mice are highly valuable tools for biological research as they allow cell type-specific expression of functionally instrumental genes. In this protocol, the generation of bacterial artificial chromosome (BAC) transgenic constructs is described. We give an overview of different transgenic inserts, such as fluorescent proteins (alone or in combination with Cre variants), diphtheria toxin receptor, lacZ, and light-activated ion channels. The most reliable and versatile approach to express these genes is by using BACs, which allow "highjacking" of the expression pattern of a gene without characterizing its transcriptional control elements. Here, we describe the necessary cloning techniques compared with conventional transgenesis. With the provided "toolbox" of already available transgene constructs, the generation of the BAC transgenes is made easy and rapid. We provide a comprehensive outline how to insert the different transgenes into a chosen BAC by either ET cloning or recombineering. We also describe in detail the methods to identify the correct insertion and the integrity of the final BAC construct, and finally, the preparation of the BAC DNA for oocyte injection is described.

IL-2 production by self-reactive CD4 thymocytes scales regulatory T cell generation in the thymus.

Regulatory T (T reg) cells, a specialized subset of CD4+ T cells, are essential to prevent fatal autoimmunity. Expression of the T reg lineage-defining transcription factor Foxp3, and therefore their differentiation in the thymus, is dependent upon T cell receptor (TCR) and interleukin-2 (IL-2) signaling. Here, we report that the majority of IL-2-producing cells in the thymus are mature CD4 single-positive (CD4SP) thymocytes and that continuous IL-2 production sustained thymic T reg cell generation and control of systemic immune activation. Furthermore, single-cell RNA sequencing analysis of CD4 thymocyte subsets revealed that IL-2 was expressed in self-reactive CD4SP thymocytes, which also contain T reg precursor cells. Thus, our results suggest that the thymic T reg cell pool size is scaled by a key niche factor, IL-2, produced by self-reactive CD4SP thymocytes. This IL-2-dependent scaling of thymic T reg cell generation by overall self-reactivity of a mature post-selection thymic precursor pool may likely ensure adequate control of autoimmunity.

Nemo-like Kinase Drives Foxp3 Stability and Is Critical for Maintenance of Immune Tolerance by Regulatory T Cells.

The Foxp3 transcription factor is a crucial determinant of both regulatory T (TREG) cell development and their functional maintenance. Appropriate modulation of tolerogenic immune responses therefore requires the tight regulation of Foxp3 transcriptional output, and this involves both transcriptional and post-translational regulation. Here, we show that during T cell activation, phosphorylation of Foxp3 in TREG cells can be regulated by a TGF-ß activated kinase 1 (TAK1)-Nemo-like kinase (NLK) signaling pathway. NLK interacts and phosphorylates Foxp3 in TREG cells, resulting in the stabilization of protein levels by preventing association with the STUB1 E3-ubiquitin protein ligase. Conditional TREG cell NLK-knockout (NLKΔTREG) results in decreased TREG cell-mediated immunosuppression in vivo, and NLK-deficient TREG cell animals develop more severe experimental autoimmune encephalomyelitis. Our data suggest a molecular mechanism, in which stimulation of TCR-mediated signaling can induce a TAK1-NLK pathway to sustain Foxp3 transcriptional activity through the stabilization of protein levels, thereby maintaining TREG cell suppressive function.

Myeloid-Specific Deletion of Peptidylarginine Deiminase 4 Mitigates Atherosclerosis.

Increasing evidence suggests that neutrophil extracellular traps (NETs) may play a role in promoting atherosclerotic plaque lesions in humans and in murine models. The exact pathways involved in NET-driven atherogenesis remain to be systematically characterized. To assess the extent to which myeloid-specific peptidylarginine deiminase 4 (PAD4) and PAD4-dependent NET formation contribute to atherosclerosis, mice with myeloid-specific deletion of PAD4 were generated and backcrossed to Apoe-/- mice. The kinetics of atherosclerosis development were determined. NETs, but not macrophage extracellular traps, were present in atherosclerotic lesions as early as 3 weeks after initiating high-fat chow. The presence of NETs was associated with the development of atherosclerosis and with inflammatory responses in the aorta. Specific deletion of PAD4 in the myeloid lineage significantly reduced atherosclerosis burden in association with diminished NET formation and reduced inflammatory responses in the aorta. NETs stimulated macrophages to synthesize inflammatory mediators, including IL-1ß, CCL2, CXCL1, and CXCL2. Our data support the notion that NETs promote atherosclerosis and that the use of specific PAD4 inhibitors may have therapeutic benefits in this potentially devastating condition.

Suppression of lethal autoimmunity by regulatory T cells with a single TCR specificity.

The regulatory T cell (T reg cell) T cell receptor (TCR) repertoire is highly diverse and skewed toward recognition of self-antigens. TCR expression by T reg cells is continuously required for maintenance of immune tolerance and for a major part of their characteristic gene expression signature; however, it remains unknown to what degree diverse TCR-mediated interactions with cognate self-antigens are required for these processes. In this study, by experimentally switching the T reg cell TCR repertoire to a single T reg cell TCR, we demonstrate that T reg cell function and gene expression can be partially uncoupled from TCR diversity. An induced switch of the T reg cell TCR repertoire to a random repertoire also preserved, albeit to a limited degree, the ability to suppress lymphadenopathy and T helper cell type 2 activation. At the same time, these perturbations of the T reg cell TCR repertoire led to marked immune cell activation, tissue inflammation, and an ultimately severe autoimmunity, indicating the importance of diversity and specificity for optimal T reg cell function.

The Cell-Intrinsic Circadian Clock Is Dispensable for Lymphocyte Differentiation and Function.

Circadian rhythms regulate many aspects of physiology, ranging from sleep-wake cycles and metabolic parameters to susceptibility to infection. The molecular clock, with transcription factor BMAL1 at its core, controls both central and cell-intrinsic circadian rhythms. Using a circadian reporter, we observed dynamic regulation of clock activity in lymphocytes. However, its disruption upon conditional Bmal1 ablation did not alter T- or B-cell differentiation or function. Although the magnitude of interleukin 2 (IL-2) production was affected by the time of bacterial infection, it was independent of cell-intrinsic expression of BMAL1. The circadian gating of the IL-2 response was preserved in Bmal1-deficient T cells, despite a slight reduction in cytokine production in a competitive setting. Our results suggest that, contrary to the prevailing view, the adaptive immune response is not affected by the cell-intrinsic clock but is likely influenced by cell-extrinsic circadian cues operating across multiple cell types.

Regulatory T cell ablation causes acute T cell lymphopenia.

Regulatory T (Treg) cells enforce T cell homeostasis and maintain peripheral T cell tolerance. Here we report a previously unappreciated phenomenon of acute T cell lymphopenia in secondary lymphoid organs and non-lymphoid tissues triggered by Treg cell depletion that precedes the expansion of self-reactive T cells. Lymphopenia affects both neonates and adults indicating a dominant role of Treg cells in maintaining peripheral T cell numbers regardless of the developmental stage. The lymphopenia was neither triggered by caspase-dependent apoptosis nor macrophage-mediated clearance of T cells, nor diminished survival of naïve or recently activated T cells due to paucity of IL-7. It is possible that transient lymphopenia associated with congenital or acute Treg cell deficiency may contribute to the development of T cell mediated autoimmune disorders.

IL-2-dependent adaptive control of NK cell homeostasis.

Activation and expansion of T and B lymphocytes and myeloid cells are controlled by Foxp3(+) regulatory T cells (T reg cells), and their deficiency results in a fatal lympho- and myeloproliferative syndrome. A role for T reg cells in the homeostasis of innate lymphocyte lineages remained unknown. Here, we report that T reg cells restrained the expansion of immature CD127(+) NK cells, which had the unique ability to up-regulate the IL2Rα (CD25) in response to the proinflammatory cytokine IL-12. In addition, we observed the preferential accumulation of CD127(+) NK cells in mice bearing progressing tumors or suffering from chronic viral infection. CD127(+) NK cells expanded in an IL-2-dependent manner upon T reg cell depletion and were able to give rise to mature NK cells, indicating that the latter can develop through a CD25(+) intermediate stage. Thus, T reg cells restrain the IL-2-dependent CD4(+) T cell help for CD127(+) immature NK cells. These findings highlight the adaptive control of innate lymphocyte homeostasis.

Dendritic epidermal T cells regulate skin antimicrobial barrier function.

The epidermis, the outer layer of the skin, forms a physical and antimicrobial shield to protect the body from environmental threats. Skin injury severely compromises the epidermal barrier and requires immediate repair. Dendritic epidermal T cells (DETC) reside in the murine epidermis where they sense skin injury and serve as regulators and orchestrators of immune responses. Here, we determined that TCR stimulation and skin injury induces IL-17A production by a subset of DETC. This subset of IL-17A-producing DETC was distinct from IFN-γ producers, despite similar surface marker profiles. Functionally, blocking IL-17A or genetic deletion of IL-17A resulted in delayed wound closure in animals. Skin organ cultures from Tcrd-/-, which lack DETC, and Il17a-/- mice both exhibited wound-healing defects. Wound healing was fully restored by the addition of WT DETC, but only partially restored by IL-17A-deficient DETC, demonstrating the importance of IL-17A to wound healing. Following skin injury, DETC-derived IL-17A induced expression of multiple host-defense molecules in epidermal keratinocytes to promote healing. Together, these data provide a mechanistic link between IL-17A production by DETC, host-defense, and wound-healing responses in the skin. These findings establish a critical and unique role of IL-17A-producing DETC in epidermal barrier function and wound healing.