Regulatory T Cells and Human Disease.

Naturally occurring CD4+ regulatory T cells (Tregs), which specifically express the transcription factor FoxP3 in the nucleus and CD25 and CTLA-4 on the cell surface, are a functionally distinct T cell subpopulation actively engaged in the maintenance of immunological self-tolerance and homeostasis. Recent studies have facilitated our understanding of the cellular and molecular basis of their generation, function, phenotypic and functional stability, and adaptability. It is under investigation in humans how functional or numerical Treg anomalies, whether genetically determined or environmentally induced, contribute to immunological diseases such as autoimmune diseases. Also being addressed is how Tregs can be targeted to control physiological and pathological immune responses, for example, by depleting them to enhance tumor immunity or by expanding them to treat immunological diseases. This review discusses our current understanding of Treg immunobiology in normal and disease states, with a perspective on the realization of Treg-targeting therapies in the clinic.

Distinct Foxp3 enhancer elements coordinate development, maintenance, and function of regulatory T cells.

The transcription factor Foxp3 plays crucial roles for Treg cell development and function. Conserved non-coding sequences (CNSs) at the Foxp3 locus control Foxp3 transcription, but how they developmentally contribute to Treg cell lineage specification remains obscure. Here, we show that among Foxp3 CNSs, the promoter-upstream CNS0 and the intergenic CNS3, which bind distinct transcription factors, were activated at early stages of thymocyte differentiation prior to Foxp3 promoter activation, with sequential genomic looping bridging these regions and the promoter. While deletion of either CNS0 or CNS3 partially compromised thymic Treg cell generation, deletion of both completely abrogated the generation and impaired the stability of Foxp3 expression in residual Treg cells. As a result, CNS0 and CNS3 double-deleted mice succumbed to lethal systemic autoimmunity and inflammation. Thus, hierarchical and coordinated activation of Foxp3 CNS0 and CNS3 initiates and stabilizes Foxp3 gene expression, thereby crucially controlling Treg cell development, maintenance, and consequently immunological self-tolerance.

Characterization of a TNFR2-Selective Agonistic TNF-α Mutant and Its Derivatives as an Optimal Regulatory T Cell Expander.

Regulatory T cells (Tregs) are a subpopulation of lymphocytes that play a role in suppressing and regulating immune responses. Recently, it was suggested that controlling the functions and activities of Tregs might be applicable to the treatment of human diseases such as autoimmune diseases, organ transplant rejection, and graft-versus-host disease. TNF receptor type 2 (TNFR2) is a target molecule that modulates Treg functions. In this study, we investigated the role of TNFR2 signaling in the differentiation and activation of mouse Tregs. We previously reported the generation of a TNFR2-selective agonist TNF mutant, termed R2agoTNF, by using our unique cytokine modification method based on phage display. R2agoTNF activates cell signaling via mouse TNFR2. In this study, we evaluated the efficacy of R2agoTNF for the proliferation and activation of Tregs in mice. R2agoTNF expanded and activated mouse CD4+CD25+ Tregs ex vivo. The structural optimization of R2agoTNF by internal cross-linking or IgG-Fc fusion selectively and effectively enhanced Treg expansion in vivo. Furthermore, the IgG-Fc fusion protein suppressed skin-contact hypersensitivity reactions in mice. TNFR2 agonists are expected to be new Treg expanders.

Epigenetic conversion of conventional T cells into regulatory T cells by CD28 signal deprivation.

Foxp3-expressing regulatory T cells (Tregs) can be generated in vitro by antigenic stimulation of conventional T cells (Tconvs) in the presence of TGF-ß and IL-2. However, unlike Foxp3+ naturally occurring Tregs, such in vitro induced Tregs (iTregs) are functionally unstable mainly because of incomplete Treg-type epigenetic changes at Treg signature genes such as Foxp3 Here we show that deprivation of CD28 costimulatory signal at an early stage of iTreg generation is able to establish Treg-specific DNA hypomethylation at Treg signature genes. It was achieved, for example, by TCR/TGF-ß/IL-2 stimulation of CD28-deficient Tconvs or CD28-intact Tconvs without anti-CD28 agonistic mAb or with CD80/CD86-blocked or -deficient antigen-presenting cells. The signal abrogation could induce Treg-type hypomethylation in memory/effector as well as naive Tconvs, while hindering Tconv differentiation into effector T cells. Among various cytokines and signal activators/inhibitors, TNF-α and PKC agonists inhibited the hypomethylation. Furthermore, CD28 signal deprivation significantly reduced c-Rel expression in iTregs; and the specific genomic perturbation of a NF-κB binding motif at the Foxp3 CNS2 locus enhanced the locus-specific DNA hypomethylation even in CD28 signaling-intact iTregs. In addition, in vitro maintenance of such epigenome-installed iTregs with IL-2 alone, without additional TGF-ß or antigenic stimulation, enabled their expansion and stabilization of Treg-specific DNA hypomethylation. These iTregs indeed stably expressed Foxp3 after in vivo transfer and effectively suppressed antigen-specific immune responses. Taken together, inhibition of the CD28-PKC-NF-κB signaling pathway in iTreg generation enables de novo acquisition of Treg-specific DNA hypomethylation at Treg signature genes and abundant production of functionally stable antigen-specific iTregs for therapeutic purposes.

Therapeutic Strategy for Rheumatoid Arthritis by Induction of Myeloid-Derived Suppressor Cells with High Suppressive Potential.

Combination treatment using fingolimod (FTY720), an immunomodulator, and a pathogenic antigen prevents the progression of glucose-6-phosphate isomerase (GPI)325-339-induced arthritis. In this study, we focused on myeloid-derived suppressor cells (MDSCs; CD11b+Gr-1+ cells) and investigated the effects of the combination treatment on these cells. DBA/1J mice with GPI325-339-induced arthritis were treated using FTY720 and/or GPI325-339 for five days. The expanded CD11b+Gr-1+ cell population and its inhibitory potential were examined. The percentage of CD369+CD11b+Gr-1+ cells effectively increased in the combination-treated mice. The inhibitory potential of CD369+CD11b+Gr-1+ cells was higher than that of cells not expressing CD369. Among bone marrow cells, the expression of CD369 in CD11b+Gr-1+ cells increased following stimulation with granulocyte-macrophage colony-stimulating factor, and the expression of CD11c increased accordingly. The increased CD11c expression indicated a decrease in the potential to suppress T cell proliferation based on the results of the suppression assay. The percentage of CD11c-CD369+ cells in CD11b+Gr-1+ cells that were induced by the combination treatment also increased, and these cells tended to have a higher capacity to inhibit T cell proliferation. In conclusion, the combination treatment using FTY720 and the pathogenic antigen effectively induces MDSC, which demonstrates a high potential for suppressing T cell proliferation in the lymph nodes, thereby establishing an immune-tolerant state.

Characterization of an Expanded IL-10-Producing-Suppressive T Cell Population Associated with Immune Tolerance.

An increase in the number of glucocorticoid-induced tumor necrosis factor receptor-family related gene/protein (GITR)+CD25- (or fork-head box protein 3: Foxp3-) CD4+ T cells, after treating a mouse model of arthritis with fingolimod (FTY720), and a pathogenic antigen may play a key role in the establishment of immune tolerance. In this study, we characterized a specific expanded T cell subset in this population. Mice with glucose-6-phosphate isomerase peptide (GPI325-339)-induced arthritis were treated with FTY720 (1 mg/kg, per os) and GPI325-339 (10 µg/mouse, intravenously) for five days, starting from the onset of symptoms. The expanded GITR+CD25- (or Foxp3-) CD4+ T cell population and its cytokine production were examined using flow cytometry. Furthermore, time-dependent changes in T-bet and/or early growth response gene 2 (Egr-2) expression in this T cell subset were examined. The density of T cell immunoreceptors with immunoglobulin (Ig) and immunoreceptor tyrosine-based inhibition motif domains (TIGIT)+CD39+ cell subset in the GITR+Foxp3-CD4+ T cell population was significantly increased only in the combined treatment group, compared to that in the untreated and single-treatment groups. In the TIGIT+CD39+GITR+Foxp3-CD4+ T cell population, T-bet+Egr-2+/T-bet+Egr-2- cell ratio increased in the latter stage of the treatment. Furthermore, this T cell subset, which corresponded to a T helper 1 (Th1) response, produced high levels of both interleukin (IL)-10 and interferon (IFN)-γ. In conclusion, expanded TIGIT+CD39+GITR+Foxp3-CD4+ T cells shifted from an effector Th1 to IL-10-producing-suppressor T cell phenotype, which may promote an immune-tolerant state.

Lamtor1 Is Critically Required for CD4+ T Cell Proliferation and Regulatory T Cell Suppressive Function.

Mechanistic target of rapamycin complex (mTORC)1 integrates intracellular sufficiency of nutrients and regulates various cellular functions. Previous studies using mice with conditional knockout of mTORC1 component proteins (i.e., mTOR, Raptor, and Rheb) gave conflicting results on the roles of mTORC1 in CD4+ T cells. Lamtor1 is the protein that is required for amino acid sensing and activation of mTORC1; however, the roles of Lamtor1 in T cells have not been investigated. In this article, we show that Lamtor1-deficient CD4+ T cells exhibited marked reductions in proliferation, IL-2 production, mTORC1 activity, and expression of purine- and lipid-synthesis genes. Polarization of Th17 cells, but not Th1 and Th2 cells, diminished following the loss of Lamtor1. Accordingly, CD4-Cre-driven Lamtor1-knockout mice exhibited reduced numbers of CD4+ and CD8+ T cells at rest, and they were completely resistant to experimental autoimmune encephalomyelitis. In contrast, genetic ablation of Lamtor1 in Foxp3+ T cells resulted in severe autoimmunity and premature death. Lamtor1-deficient regulatory T cells survived ex vivo as long as wild-type regulatory T cells; however, they exhibited a marked loss of suppressive function and expression of signature molecules, such as CTLA-4. These results indicate that Lamtor1 plays essential roles in CD4+ T cells. Our data suggest that Lamtor1 should be considered a novel therapeutic target in immune systems.

Immuno-Navigator, a batch-corrected coexpression database, reveals cell type-specific gene networks in the immune system.

High-throughput gene expression data are one of the primary resources for exploring complex intracellular dynamics in modern biology. The integration of large amounts of public data may allow us to examine general dynamical relationships between regulators and target genes. However, obstacles for such analyses are study-specific biases or batch effects in the original data. Here we present Immuno-Navigator, a batch-corrected gene expression and coexpression database for 24 cell types of the mouse immune system. We systematically removed batch effects from the underlying gene expression data and showed that this removal considerably improved the consistency between inferred correlations and prior knowledge. The data revealed widespread cell type-specific correlation of expression. Integrated analysis tools allow users to use this correlation of expression for the generation of hypotheses about biological networks and candidate regulators in specific cell types. We show several applications of Immuno-Navigator as examples. In one application we successfully predicted known regulators of importance in naturally occurring Treg cells from their expression correlation with a set of Treg-specific genes. For one high-scoring gene, integrin ß8 (Itgb8), we confirmed an association between Itgb8 expression in forkhead box P3 (Foxp3)-positive T cells and Treg-specific epigenetic remodeling. Our results also suggest that the regulation of Treg-specific genes within Treg cells is relatively independent of Foxp3 expression, supporting recent results pointing to a Foxp3-independent component in the development of Treg cells.

CD28 signals the differential control of regulatory T cells and effector T cells.

One possible means of driving antigen-specific immune suppression is to expand or induce antigen-specific FoxP3-expressing Treg cells. One way of activating and expanding these specialized cells, both in vitro and in vivo, is by strong costimulation via CD28 with an agonistic anti-CD28 monoclonal antibody, called anti-CD28 superagonist (CD28SA). However, CD28SA also strongly activates conventional T (Tconv) cells to secrete proinflammatory cytokines and, under certain conditions, causes serious cytokine release syndrome. In this issue of European Journal of Immunology, Tabares et al. [Eur. J. Immunol. 2014. 44: 1225-1236] address how CD28SA can be used for the differential control of human Treg and Tconv cells to suppress immune responses without serious adverse effects. They show that, depending on the dose of the antibody or by comedication of cortico-steroid, the selective expansion of Treg cells can be achieved without significantly activating Tconv cells to produce inflammatory cytokines. This difference in CD28 signal sensitivity between the two populations can be exploited for better control of immune responses.

Calcitonin gene-related peptide and cyclic adenosine 5'-monophosphate/protein kinase A pathway promote IL-9 production in Th9 differentiation process.

Th9 cells are a novel Th cell subset that produces IL-9 and is involved in type I hypersensitivity such as airway inflammation. Although its critical roles in asthma have attracted interest, the physiological regulatory mechanisms of Th9 cell differentiation and function are largely unknown. Asthma is easily affected by psychological factors. Therefore, we investigated one of the physiological mediators derived from the nervous system, calcitonin gene-related peptide (CGRP), in asthma and Th9 cells because CGRP and activation of the cAMP/protein kinase A (PKA) pathway by CGRP are known to be important regulators in several immune responses and allergic diseases. In this study, we demonstrated that the CGRP/cAMP/PKA pathway promotes IL-9 production via NFATc2 activation by PKA-dependent glycogen synthase kinase-3ß inactivation. Moreover, CGRP also induces the expression of PU.1, a critical transcriptional factor in Th9 cells, which depends on PKA, but not NFATc2. Additionally, we demonstrated the physiological importance of CGRP in IL-9 production and Th9 differentiation using an OVA-induced airway inflammation model and T cell-specific CGRP receptor-deficient mice. The present study revealed a novel regulatory mechanism comprising G protein-coupled receptor ligands and nervous system-derived substances in Th9 cell differentiation and type I hypersensitivity.

Functional Mechanism(s) of the Inhibition of Disease Progression by Combination Treatment with Fingolimod Plus Pathogenic Antigen in a Glucose-6-phosphate Isomerase Peptide-Induced Arthritis Mouse Model.

We previously reported that combination treatment with fingolimod (FTY720) plus antigenic peptide of glucose-6-phosphate isomerase (residues 325-339) (GPI325-339) from the onset of symptoms significantly inhibited disease progression in a mouse model of GPI325-339-induced arthritis. In this study, we investigated the mechanism(s) involved. The model mice were treated from arthritis onset with FTY720 alone, GPI325-339 alone, or the combination of FTY720 plus GPI325-339. At the end of treatment, inguinal lymph nodes (LNs) were excised and examined histologically and in flow cytometry. Levels of apoptotic cells, programmed death-1-expressing CD4(+)forkhead box P3(-) nonregulatory T cells (non-Tregs), and cytotoxic T-lymphocyte antigen 4-expressing non-Tregs in inguinal LNs were markedly increased in the combination treatment group mice. Regulatory T cells (Tregs) were also increased. These results indicate that combination treatment with FTY720 plus GPI325-339 inhibits the progression of arthritis by inducing clonal deletion and anergy of pathogenic T cells and also by immune suppression via Tregs.

Single-cell transcriptome landscape of circulating CD4+ T cell populations in autoimmune diseases.

CD4+ T cells are key mediators of various autoimmune diseases; however, their role in disease progression remains unclear due to cellular heterogeneity. Here, we evaluated CD4+ T cell subpopulations using decomposition-based transcriptome characterization and canonical clustering strategies. This approach identified 12 independent gene programs governing whole CD4+ T cell heterogeneity, which can explain the ambiguity of canonical clustering. In addition, we performed a meta-analysis using public single-cell datasets of over 1.8 million peripheral CD4+ T cells from 953 individuals by projecting cells onto the reference and cataloging cell frequency and qualitative alterations of the populations in 20 diseases. The analyses revealed that the 12 transcriptional programs were useful in characterizing each autoimmune disease and predicting its clinical status. Moreover, genetic variants associated with autoimmune diseases showed disease-specific enrichment within the 12 gene programs. The results collectively provide a landscape of single-cell transcriptomes of CD4+ T cell subpopulations involved in autoimmune disease.

Calcitonin gene-related peptide enhances experimental autoimmune encephalomyelitis by promoting Th17-cell functions.

T(h)17 cells, an inflammatory T helper cell subset, are involved in the pathogenesis of various inflammatory, autoimmune and allergic diseases. Recent evidence supports the idea that immune cell functions and the inflammatory response are finely regulated by various physiological substances. Calcitonin gene-related peptide (CGRP), a neuropeptide released from the sensory nerve endings, is one of these mediators. By binding to its receptor composed of receptor activity-modifying protein 1 (RAMP1) and calcitonin receptor-like receptor, CGRP modulates various immune cell functions, but the function of CGRP in T(h)17 cells is largely unknown. Here, we investigated the effect of CGRP signaling on T(h)17 cells and T(h)17 cell-mediated inflammation and observed that CGRP activates nuclear factor of activated T cells c2 through cAMP/PKA to increase IL-17 production in vitro. In vivo, IL-17 production is suppressed in RAMP1-deficient mice in the experimental autoimmune encephalomyelitis (EAE) model and RAMP1-deficient mice are completely resistant to EAE. Furthermore, T(h)17 cell function and EAE induction are also suppressed in T cell-specific RAMP1-deficient mice. Taken together, our findings indicate that CGRP promotes T(h)17 cell-mediated autoimmune inflammation through the regulation of IL-17 expression.

Neuronal derivative mediators that regulate cutaneous inflammations.

Allergic diseases accompanied by skin inflammation are intricately regulated by several mediators. Among these molecules, neurotransmitters are known to affect several immune cells such as T cells, B cells, dendritic cells, and mast cells. Neurotransmitters are released from nerve endings, and most of them work through specific G-protein coupled receptors. In this review, we discuss the interactions of representative neurotransmitters, calcitonin gene-related peptide (CGRP), substance P (SP), neuropeptide Y (NPY), vasoactive intestinal peptide (VIP)/pituitary adenylate cyclase-activating polypeptide (PACAP), and prostaglandins (PGs), as well as their receptor signaling systems such as the cAMP/protein kinase A (PKA) pathway, phospholipase C (PLC) activation, and calcium ion channel activation, in cutaneous immunity. Although many studies have proposed that these neurotransmitters play several roles in mouse contact hypersensitivity (CHS) models, human allergic contact dermatitis (ACD), and atopic dermatitis (AD), their physiological effects are controversial due to the diverse and complex mechanisms of skin inflammation. Both Th1- and Th2-mediated skin inflammation are well known, and neurotransmitters affect the kinds of inflammatory cells and subsequent immune reactions in them. In addition, the characteristics of antigen-presenting cells are also different in Th1- or Th2-mediated immune responses. Therefore, we take particular note of the type of skin inflammation, Th1- or Th2-mediated, and review the physiological roles of the neurotransmitters in skin inflammation.

Calcitonin gene-related peptide is an important regulator of cutaneous immunity: effect on dendritic cell and T cell functions.

Some cutaneous inflammations are induced by percutaneous exposure to foreign Ags, and many chemical mediators regulate this inflammation process. One of these mediators, calcitonin gene-related peptide (CGRP), is a neuropeptide released from nerve endings in the skin. CGRP binds to its receptors composed of receptor activity-modifying protein 1 and calcitonin receptor-like receptor to modulate immune cell function. We show that CGRP regulates skin inflammation under physiological conditions, using contact hypersensitivity (CHS) models of receptor activity-modifying protein 1-deficient mice. CGRP has different functions in CHS responses mediated by Th1 or Th2 cells; it inhibits Th1-type CHS, such as 2,4,6-trinitrochlorobenzene-induced CHS, but promotes Th2-type CHS, such as FITC-induced CHS. CGRP inhibits the migration of Langerin(+) dermal dendritic cells to the lymph nodes in 2,4,6-trinitrochlorobenzene-induced CHS, and upregulates IL-4 production of T cells in the draining lymph nodes in FITC-CHS. These findings suggest that CGRP regulates several types of CHS reactions under physiological conditions and plays an important role in cutaneous immunity.

Regulatory T cells in autoimmune kidney diseases and transplantation.

Regulatory T (Treg) cells that express the transcription factor forkhead box protein P3 (FOXP3) are naturally present in the immune system and have roles in the maintenance of immunological self-tolerance and immune system and tissue homeostasis. Treg cells suppress T cell activation, expansion and effector functions by various mechanisms, particularly by controlling the functions of antigen-presenting cells. They can also contribute to tissue repair by suppressing inflammation and facilitating tissue regeneration, for example, via the production of growth factors and the promotion of stem cell differentiation and proliferation. Monogenic anomalies of Treg cells and genetic variations of Treg cell functional molecules can cause or predispose patients to the development of autoimmune diseases and other inflammatory disorders, including kidney diseases. Treg cells can potentially be utilized or targeted to treat immunological diseases and establish transplantation tolerance, for example, by expanding natural Treg cells in vivo using IL-2 or small molecules or by expanding them in vitro for adoptive Treg cell therapy. Efforts are also being made to convert antigen-specific conventional T cells into Treg cells and to generate chimeric antigen receptor Treg cells from natural Treg cells for adoptive Treg cell therapies with the aim of achieving antigen-specific immune suppression and tolerance in the clinic.

Treg-based immunotherapy for antigen-specific immune suppression and stable tolerance induction: a perspective.

FoxP3-expressing regulatory T cells (Tregs), whether naturally generated in the immune system or unnaturally induced from conventional T cells (Tconvs) in the laboratory, have much therapeutic value in treating immunological diseases and establishing transplantation tolerance. Natural Tregs (nTregs) can be selectively expanded in vivo by administration of low-dose IL-2 or IL-2 muteins for immune suppression. For adoptive Treg cell therapy, nTregs can be expanded in vitro by strong antigenic stimulation in the presence of IL-2. Synthetic receptors such as CAR can be expressed in nTregs to equip them with a particular target specificity for suppression. In addition, antigen-specific Tconvs can be converted in vitro to functionally stable Treg-like cells by a combination of antigenic stimulation, FoxP3 induction, and establishment of the Treg-type epigenome. This review discusses current and prospective strategies for Treg-based immune suppression and the issues to be resolved for achieving stable antigen-specific immune suppression and tolerance induction in the clinic by targeting Tregs.

Suppression of ovalbumin-induced allergic diarrhea by diminished intestinal peristalsis in RAMP1-deficient mice.

Recent studies have revealed that various neurotransmitters regulate the immune system via their receptors expressed on the immune cells. Calcitonin gene-related peptide (CGRP), a sensory nerve C-fiber neuropeptide, is also known to have the ability to modulate the functions of immune cells in vitro. However, the contribution of CGRP to the immune regulation in vivo remains to be fully elucidated. Here we report that mice deficient in receptor activity-modifying protein 1 (RAMP1), which is a subunit of the CGRP receptor, showed a significantly lower incidence of diarrhea compared with wild-type (WT) mice in the ovalbumin (OVA)-induced food allergic model. Serum OVA-specific IgE levels and the differentiation of T helper cells was comparable in WT mice and RAMP1-deficient mice. Moreover, there were no significant differences between recruitment and degranulation of mast cells in the small intestine of these mice. In contrast, significantly diminished intestinal peristalsis was observed by the allergy induction in RAMP1-deficient mice compared with WT mice. These results suggest that this suppression of allergic diarrhea is due to the diminished intestinal peristalsis in RAMP1-deficient mice.

Brazilian green propolis promotes TNFR2 expression on regulatory T cells.

FoxP3+ regulatory T cells (Tregs) are needed to suppress inflammatory diseases and maintain immune homeostasis. The suppressive function of Tregs can be used to control autoimmune or inflammatory diseases; therefore, it is well studied how Tregs can be artificially up- or downregulated in vitro and in vivo, by using antibodies, chemical compounds, foods, and natural resources. Propolis is a famous functional food that has an anti-inflammatory effect. However, the influences of propolis on Treg function have not been fully evaluated so far. Here, we demonstrated that Brazilian green propolis increases TNFR2 expression in Tregs via the IRF4/cMyc axis, and artepillin C was a major effective component of propolis on Tregs. These results indicate that propolis and artepillin C have the potential as Treg activators via TNFR2 expression and may be useful for the prevention and/or therapy of autoimmune or inflammatory diseases.

New Treg cell-based therapies of autoimmune diseases: towards antigen-specific immune suppression.

Naturally occurring FoxP3+CD4+ regulatory T (Treg) cells indispensable for the maintenance of immunological self-tolerance and homeostasis are instrumental in treating autoimmune and other immunological disorders. Stable function of natural Treg cells requires not only the expression of Foxp3 and other Treg signature genes such as CD25 and CTLA-4 but also the generation of Treg-specific epigenetic changes, especially Treg-specific DNA hypomethylation, at these gene loci. Recent studies have shown that the Treg-specific transcriptional and epigenetic changes can be induced in antigen-specific conventional T cells in vivo and in vitro, converting them to functionally stable Treg cells. Such natural or induced Treg cells bear the potential to achieve stable antigen-specific immune suppression and reestablish immunological self-tolerance in treating and preventing autoimmune diseases.