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The immunosuppressive function of regulatory T (Treg) cells is dependent on continuous expression of the transcription factor Foxp3. Foxp3 loss of function or induced ablation of Treg cells results in a fatal autoimmune disease featuring all known types of inflammatory responses with every manifestation stemming from Treg cell paucity, highlighting a vital function of Treg cells in preventing fatal autoimmune inflammation. However, a major question remains whether Treg cells can persist and effectively exert their function in a disease state, where a broad spectrum of inflammatory mediators can either inactivate Treg cells or render innate and adaptive pro-inflammatory effector cells insensitive to suppression. By reinstating Foxp3 protein expression and suppressor function in cells expressing a reversible Foxp3 null allele in severely diseased mice, we found that the resulting single pool of rescued Treg cells normalized immune activation, quelled severe tissue inflammation, reversed fatal autoimmune disease and provided long-term protection against them. Thus, Treg cells are functional in settings of established broad-spectrum systemic inflammation and are capable of affording sustained reset of immune homeostasis.

This study examines fraud vulnerability in the food service industry; identifies underlying fraud vulnerability factors; and studies the differences in fraud vulnerability between casual dining restaurants, fine dining restaurants and mass caterers for four product groups. Vulnerability was assessed by an adapted SSAFE food fraud vulnerability assessment, tailored to the food service sector situation. The 15 food service operators rated high vulnerability for 40% of the fraud indicators. This is considerably more than food manufacturers, wholesalers and retailers did previously. In particular, more opportunities and fewer controls were noted. Overall fraud vulnerability was more determined by the type of food service operator than by the type of food product. Casual dining restaurants appeared most vulnerable, followed by fine dining restaurants. Mass caterers seemed the least vulnerable operators, because they had more adequate food fraud controls in place. Considering its high vulnerability, reinforcement of mitigation measures in the food service industry is urgently recommended.

FoxP3 is an essential transcription factor (TF) for immunologic homeostasis, but how it utilizes the common forkhead DNA-binding domain (DBD) to perform its unique function remains poorly understood. We here demonstrate that, unlike other known forkhead TFs, FoxP3 forms a head-to-head dimer using a unique linker (Runx1-binding region, RBR) preceding the forkhead domain. Head-to-head dimerization confers distinct DNA-binding specificity and creates a docking site for the cofactor Runx1. RBR is also important for proper folding of the forkhead domain, as truncation of RBR induces domain-swap dimerization of forkhead, which was previously considered the physiological form of FoxP3. Rather, swap-dimerization impairs FoxP3 function, as demonstrated with the disease-causing mutation R337Q, while a swap-suppressive mutation largely rescues R337Q-mediated functional impairment. Altogether, our findings suggest that FoxP3 can fold into two distinct dimerization states: head-to-head dimerization representing functional specialization of an ancient DBD and swap-dimerization with impaired functions.

Regulatory T (Treg) cells expressing the transcription factor Foxp3 are an essential suppressive T cell lineage of dual origin: Foxp3 induction in thymocytes and mature CD4

FoxP3 is an essential transcription factor (TF) for immunologic homeostasis, but how it utilizes the common forkhead DNA-binding domain (DBD) to perform its unique function remains poorly understood. We here demonstrated that unlike other known forkhead TFs, FoxP3 formed a head-to-head dimer using a unique linker (Runx1-binding region [RBR]) preceding the forkhead domain. Head-to-head dimerization conferred distinct DNA-binding specificity and created a docking site for the cofactor Runx1. RBR was also important for proper folding of the forkhead domain, as truncation of RBR induced domain-swap dimerization of forkhead, which was previously considered the physiological form of FoxP3. Rather, swap-dimerization impaired FoxP3 function, as demonstrated with the disease-causing mutation R337Q, whereas a swap-suppressive mutation largely rescued R337Q-mediated functional impairment. Altogether, our findings suggest that FoxP3 can fold into two distinct dimerization states: head-to-head dimerization representing functional specialization of an ancient DBD and swap dimerization associated with impaired functions.

The immunosuppressive function of regulatory T (Treg) cells is dependent on continuous expression of the transcription factor Foxp3. Foxp3 loss-of-function or induced ablation of Treg cells results in a fatal autoimmune disease featuring all known types of inflammatory responses with every manifestation stemming from Treg cell paucity, highlighting a vital function of Treg cells in preventing fatal autoimmune inflammation. However, a major question remains whether Treg cells can persist and effectively exert their function in a disease state, where broad spectrum of inflammatory mediators can either inactivate Treg cells or render innate and adaptive pro-inflammatory effector cells insensitive to suppression. By reinstating Foxp3 protein expression and suppressor function in cells expressing a reversible Foxp3 null allele in severely diseased mice, we found that the resulting single pool of “redeemed” Treg cells normalized immune activation, quelled severe tissue inflammation, reversed fatal autoimmune disease and provided long-term protection against them. Thus, Treg cells are functional in settings of established broad spectrum systemic inflammation and are capable of affording sustained reset of the immune homeostasis.

Significance Anorexia or decreased appetite is a common feature of infection. The effects of acute nutritional stress on effector T cells, which are required for pathogen-specific responses and protective immunological memory, are not fully understood. We show that reduced food intake during infection decreases the numbers of effector T cells in a manner dependent on the Farnesoid X Receptor (FXR). FXR was previously implicated in controlling hepatic responses to fasting. Our findings suggest that FXR functions in effector T cells to promote coherent physiological responses to decreased feeding, allowing organisms to scale their immune responses according to food availability., Reduced nutrient intake is a widely conserved manifestation of sickness behavior with poorly characterized effects on adaptive immune responses. During infectious challenges, naive T cells encountering their cognate antigen become activated and differentiate into highly proliferative effector T cells. Despite their evident metabolic shift upon activation, it remains unclear how effector T cells respond to changes in nutrient availability in vivo. Here, we show that spontaneous or imposed feeding reduction during infection decreases the numbers of splenic lymphocytes. Effector T cells showed cell-intrinsic responses dependent on the nuclear receptor Farnesoid X Receptor (FXR). Deletion of FXR in T cells prevented starvation-induced loss of lymphocytes and increased effector T cell fitness in nutrient-limiting conditions, but imparted greater weight loss to the host. FXR deficiency increased the contribution of glutamine and fatty acids toward respiration and enhanced cell survival under low-glucose conditions. Provision of glucose during anorexia of infection rescued effector T cells, suggesting that this sugar is a limiting nutrient for activated lymphocytes and that alternative fuel usage may affect cell survival in starved animals. Altogether, we identified a mechanism by which the host scales immune responses according to food intake, featuring FXR as a T cell-intrinsic sensor.

Summary CD8 T cells play an essential role in defense against viral and bacterial infections and in tumor immunity. Deciphering T cell loss of functionality is complicated by the conspicuous heterogeneity of CD8 T cell states described across experimental and clinical settings. By carrying out a unified analysis of over 300 assay for transposase-accessible chromatin sequencing (ATAC-seq) and RNA sequencing (RNA-seq) experiments from 12 studies of CD8 T cells in cancer and infection, we defined a shared differentiation trajectory toward dysfunction and its underlying transcriptional drivers and revealed a universal early bifurcation of functional and dysfunctional T cell states across models. Experimental dissection of acute and chronic viral infection using single-cell ATAC (scATAC)-seq and allele-specific single-cell RNA (scRNA)-seq identified state-specific drivers and captured the emergence of similar TCF1+ progenitor-like populations at an early branch point, at which functional and dysfunctional T cells diverge. Our atlas of CD8 T cell states will facilitate mechanistic studies of T cell immunity and translational efforts.

CD8 T cells play an essential role in defense against viral and bacterial infections and in tumor immunity. Deciphering T cell loss of functionality is complicated by the conspicuous heterogeneity of CD8 T cell states described across different experimental and clinical settings. By carrying out a unified analysis of over 300 ATAC-seq and RNA-seq experiments from twelve independent studies of CD8 T cell dysfunction in cancer and infection we defined a shared differentiation trajectory towards terminal dysfunction and its underlying transcriptional drivers and revealed a universal early bifurcation of functional and dysfunctional T cell activation states across models. Experimental dissection of acute and chronic viral infection using scATAC-seq and allele-specific scRNA-seq identified state-specific transcription factors and captured the early emergence of highly similar TCF1+ progenitor-like populations at an early branch point, at which epigenetic features of functional and dysfunctional T cells diverge. Our atlas of CD8 T cell states will facilitate mechanistic studies of T cell immunity and translational efforts.

Summary Regulatory T (Treg) cell identity is defined by the lineage-specifying transcription factor (TF) Foxp3. Here we examined mechanisms of Foxp3 function by leveraging naturally occurring genetic variation in wild-derived inbred mice, which enables the identification of DNA sequence motifs driving epigenetic features. Chromatin accessibility, TF binding, and gene expression patterns in resting and activated subsets of Treg cells, conventional CD4 T cells, and cells expressing a Foxp3 reporter null allele revealed that the majority of Foxp3-dependent changes occurred at sites not bound by Foxp3. Chromatin accessibility of these indirect Foxp3 targets depended on the presence of DNA binding motifs for other TFs, including TCF1. Foxp3 expression correlated with decreased TCF1 and reduced accessibility of TCF1-bound chromatin regions. Deleting one copy of the Tcf7 gene recapitulated Foxp3-dependent negative regulation of chromatin accessibility. Thus, Foxp3 defines Treg cell identity in a largely indirect manner by fine-tuning the activity of other major chromatin remodeling TFs such as TCF1.

Summary Stable changes in chromatin states and gene expression in cells of the immune system form the basis for memory of infections and other challenges. Here, we used naturally occurring cis-regulatory variation in wild-derived inbred mouse strains to explore the mechanisms underlying long-lasting versus transient gene regulation in CD8 T cells responding to acute viral infection. Stably responsive DNA elements were characterized by dramatic and congruent chromatin remodeling events affecting multiple neighboring sites and required distinct transcription factor (TF) binding motifs for their accessibility. Specifically, we found that cooperative recruitment of T-box and Runx family transcription factors to shared targets mediated stable chromatin remodeling upon T cell activation. Our observations provide insights into the molecular mechanisms driving virus-specific CD8 T cell responses and suggest a general mechanism for the formation of transcriptional and epigenetic memory applicable to other immune and non-immune cells.

The transcription factor Foxp3 is indispensable for the ability of regulatory T cells (Treg cells) to suppress fatal inflammation. Here we characterized the role of Foxp3 in chromatin remodeling and the regulation of gene expression in actively suppressive Treg cells in an inflammatory setting. Although genome-wide occupancy of regulatory elements in DNA by Foxp3 was similar in resting Treg cells and those activated in vivo, Foxp3-bound enhancer elements in the DNA were poised for repression only in activated Treg cells. Following activation, Foxp3-bound sites showed diminished accessibility of chromatin and selective deposition of histone H3 trimethylated at Lys27 (H3K27me3), which was associated with recruitment of the histone methyltransferase Ezh2 and downregulation 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.

T regulatory (Treg) cells expressing the forkhead box transcription factor (Foxp3) are important regulators of immunity and play a key role in cancer and metastasis by controlling the activity of effector T-cells. Lineage-specifying transcription factors (TFs) establish cell identity and maintain cell specific transcriptional programs. Foxp3 is a key master TF controlling various Treg cell developmental and functional modalities. Foxp3 deficiency in humans and mice causes a life threatening autoimmune disease, and the enforced expression of Foxp3 in CD4+ T-cells confers suppressor functions. However, our understanding of Treg cell regulation during differentiation in the thymus, and in mature Treg cells in the periphery, whether Foxp3 dependent or independent, is still lacking. Single-nucleotide polymorphism (SNPs) may occur within coding genes or noncoding genomic regulatory areas and can have severe phenotypic consequences. We have recently attempted to link SNPs with differential Treg transcriptomic and epigenetic features in humans. However, such studies are limited by their intrinsic correlative nature, and by the limited number of SNPs. Here we use evolutionally distant mice strains C57Bl/6 (B6), CAST/EiJ (Cast) and SPRET/EiJ (Spret), to identify allele-specific imbalances and functional differences in the transcriptional and epigenetic programs during differentiation and in various conditions of Treg cells and their precursors. Using Foxp3 reporter and conditional knockout (KO) systems combined with interbreeding of the evolutionary distant mice strains in vivo, we explore the genetic and molecular mechanisms of Treg cell differentiation and function, employing high-throughput genomic methods including RNA-seq, ATAC-seq and Chip-seq. Such studies may pave a way for the development of novel therapeutic approaches to treat cancer and metastasis. Citation Format: Ariella Glasner, Yi Zhong, Joris Van Der Veeken, Alexander Rudensky. Mechanisms governing Foxp3-dependent and -independent gene expression in regulatory T-cells in evolutionary distant mice [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr A190.

Intestinal microbes provide multicellular hosts with nutrients and confer resistance to infection. The delicate balance between pro- and anti-inflammatory mechanisms, essential for gut immune homeostasis, is affected by the composition of the commensal microbial community. Regulatory T cells (Treg cells) expressing transcription factor Foxp3 have a key role in limiting inflammatory responses in the intestine. Although specific members of the commensal microbial community have been found to potentiate the generation of anti-inflammatory Treg or pro-inflammatory T helper 17 (TH17) cells, the molecular cues driving this process remain elusive. 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 tested this hypothesis by exploring the effect of microbial metabolites on the generation of anti-inflammatory Treg 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, as the observed phenomenon was dependent on intronic enhancer CNS1 (conserved non-coding sequence 1), essential for extrathymic but dispensable for thymic Treg-cell differentiation. 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 (HDAC) inhibition, but not acetate, which lacks this HDAC-inhibitory activity. Our results suggest that bacterial metabolites mediate communication between the commensal microbiota and the immune system, affecting the balance between pro- and anti-inflammatory mechanisms.

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.

Regulatory T (Treg) cells, which suppress autoimmunity and other inflammatory states, are characterized by a distinct set of genetic elements controlling their gene expression. However, the extent of genetic and associated epigenetic variation in the Treg cell lineage and its possible relation to disease states in humans remain unknown. We explored evolutionary conservation of regulatory elements and natural human inter-individual epigenetic variation in Treg cells to identify the core transcriptional control program of lineage specification. Analysis of single nucleotide polymorphisms in core lineage-specific enhancers revealed disease associations, which were further corroborated by high-resolution genotyping to fine map causal polymorphisms in lineage-specific enhancers. Our findings 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. DOI: http://dx.doi.org/10.7554/eLife.07571.001, eLife digest The immune system protects the body from infection. Key to this protection is the ability to mount an immune response that is sufficient to deal with the threat, but is not so large that the damage it causes to the body exceeds its immediate benefit. Immune cells called regulatory T cells (or Treg cells for short) help to shut down the immune response after a threat has been successfully destroyed. They also prevent the immune system from attacking the body's own cells, a phenomenon known as autoimmunity. All cells in the body carry the same set of genes, but the activity of these genes varies between cell types to enable the cells to perform their different jobs. This is possible because our DNA contains regions called regulatory elements that can control the expression of particular genes. These regions can be activated in specific types of cells, which results in specific chemical modifications to DNA that only affect gene activity in those cells. The DNA sequences of these regulatory elements vary between individuals. This ‘genetic variation’ can lead to differences in the chemical modifications that occur to DNA, which is known as epigenetic variation. This means that Treg cells in one person may work in a different way to those in another individual, which could make some individuals more susceptible to autoimmune diseases than others. However, it was not clear how much genetic and epigenetic variation exists in Treg cells. Here, Arvey et al. examined Treg and other immune cells from human and mouse donors. The experiments show that some of the epigenetic modifications present in many individuals only in Treg cells, which indicates that they may be important for the activity of the Treg cells. Unexpectedly, most of the epigenetic modifications were specific to either mice or humans, but Arvey et al. identified a core set of genes that had been modified only in Treg cells in both species. In the human cells, Arvey et al. also identified genetic differences in regulatory elements that are associated with autoimmune diseases. Arvey et al.'s findings suggest that a small set of regulatory elements are crucial to the role of Treg cells in the immune system. Furthermore, genetic variation in these elements can lead to epigenetic changes in Treg cells that contribute to autoimmune diseases in humans. Further study may lead to the development of new treatments for these diseases. DOI: http://dx.doi.org/10.7554/eLife.07571.002

SummaryIn multicellular organisms, specialized functions are delegated to distinct cell types whose identity and functional integrity are maintained upon challenge. However, little is known about the mechanisms enabling lineage inheritance and their biological implications. Regulatory T (Treg) cells, which express the transcription factor Foxp3, suppress fatal autoimmunity throughout the lifespan of animals. Here, we show that a dedicated Foxp3 intronic element CNS2 maintains Treg cell lineage identity by acting as a sensor of the essential Treg cell growth factor IL-2 and its downstream target STAT5. CNS2 sustains Foxp3 expression during division of mature Treg cells when IL-2 is limiting and counteracts proinflammatory cytokine signaling that leads to the loss of Foxp3. CNS2-mediated stable inheritance of Foxp3 expression is critical for adequate suppression of diverse types of chronic inflammation by Treg cells and prevents their differentiation into inflammatory effector cells. The described mechanism may represent a general principle of the inheritance of differentiated cell states.

Regulatory T (Treg) cells are essential for maintaining peripheral tolerance and for limiting excessive inflammatory responses under various conditions. The lineage-specific transcription factor Foxp3 has a critical role in Treg-cell biology. Foxp3 forms large protein complexes and cooperates with environmentally induced transcription factors to shape the Treg-cell transcriptional program. Here, we discuss mechanisms of gene regulation that underlie Treg-cell differentiation and function with an emphasis on studies from our laboratory.

The epidermal growth factor receptor (EGFR) is a validated target for anti-cancer therapy and several EGFR inhibitors are used in the clinic. Over the years, an increasing number of studies have reported on the crosstalk between EGFR and other receptors that can contribute to accelerated cancer development or even acquisition of resistance to anti-EGFR therapies. Combined targeting of EGFR and insulin-like growth factor 1 receptor (IGF-1R) is a rational strategy to potentiate anti-cancer treatment and possibly retard resistance development. In the present study, we have pursued this by encapsulating the kinase inhibitor AG538 in anti-EGFR nanobody-liposomes. The thus developed dual-active nanobody-liposomes associated with EGFR-(over)expressing cells in an EGFR-specific manner and blocked both EGFR and IGF-1R activation, due to the presence of the EGFR-blocking nanobody EGa1 and the anti-IGF-1R kinase inhibitor AG538 respectively. AG538-loaded nanobody-liposomes induced a strong inhibition of tumor cell proliferation even upon short-term exposure followed by a drug-free wash-out period. Therefore, AG538-loaded nanobody-liposomes are a promising anti-cancer formulation due to efficient intracellular delivery of AG538 in combination with antagonistic and downregulating properties of the EGa1 nanobody-liposomes.

The epidermal growth factor receptor (EGFR) is a recognized target for tumor therapy and monoclonal antibodies (mAbs, e.g. cetuximab) have been developed to inhibit receptor activation. Besides blocking ligand (e.g. EGF) binding to the receptor, reports have shown that mAbs promote slow receptor internalization and degradation in lysosomes, i.e. downregulation. The efficacy of receptor downregulation was recently shown to depend on the size of receptor clusters formed at the cell surface. In this study, a multivalent platform is presented, consisting of nanobodies recognizing the ectodomain of EGFR (EGa1) coupled to PEG-liposomes, and the in vitro and in vivo effects of this system on EGFR internalization and downregulation were investigated. Nanobodies are the smallest functional antigen-binding immunoglobulin fragments and the EGa1 nanobody has been described as an EGFR-antagonist. EGa1-liposomes (EGa1-L) induced a more than 90% removal of EGFR from the cell surface, as a result of receptor internalization. Furthermore, this massive sequestration of EGFR mediated by EGa1-L lead to receptor degradation, while no degradation was detected with the monovalent nanobody. The downregulatory capacity here reported was found to be independent of the epitope on EGFR recognized by the grafted nanobody, and exclusive to the nanobody-liposomes, as anti-EGFR single chain variable fragments (scFv) coupled to liposomes were unable to induce this effect. Importantly, EGa1-L induced a significant inhibition of tumor cell proliferation, in vitro, an effect likely mediated by the combination of receptor downregulation and receptor antagonism. Also in vivo, EGFR downregulation was observed in tumors of mice intravenously injected with EGa1-L, indicating that this multivalent platform blocks ligand binding to the receptor and simultaneously induces the downregulation of EGFR.