Selective IL-27 production by intestinal regulatory T cells permits gut-specific regulation of TH17 cell immunity.

Regulatory T cells (Treg cells) are instrumental in establishing immunological tolerance. However, the precise effector mechanisms by which Treg cells control a specific type of immune response in a given tissue remains unresolved. By simultaneously studying Treg cells from different tissue origins under systemic autoimmunity, in the present study we show that interleukin (IL)-27 is specifically produced by intestinal Treg cells to regulate helper T17 cell (TH17 cell) immunity. Selectively increased intestinal TH17 cell responses in mice with Treg cell-specific IL-27 ablation led to exacerbated intestinal inflammation and colitis-associated cancer, but also helped protect against enteric bacterial infection. Furthermore, single-cell transcriptomic analysis has identified a CD83+CD62Llo Treg cell subset that is distinct from previously characterized intestinal Treg cell populations as the main IL-27 producers. Collectively, our study uncovers a new Treg cell suppression mechanism crucial for controlling a specific type of immune response in a particular tissue and provides further mechanistic insights into tissue-specific Treg cell-mediated immune regulation.

Selective IL-27 production by intestinal regulatory T cells permits gut-specific regulation of Th17 immunity.

Regulatory T (Treg) cells are instrumental in establishing immunological tolerance. However, the precise effector mechanisms by which Treg cells control a specific type of immune response in a given tissue remains unresolved. By simultaneously studying Treg cells from different tissue origins under systemic autoimmunity, here we show that IL-27 is specifically produced by intestinal Treg cells to regulate Th17 immunity. Selectively increased intestinal Th17 responses in mice with Treg cell-specific IL-27 ablation led to exacerbated intestinal inflammation and colitis-associated cancer, but also helped protect against enteric bacterial infection. Furthermore, single-cell transcriptomic analysis has identified a CD83+TCF1+ Treg cell subset that is distinct from previously characterized intestinal Treg cell populations as the main IL-27 producers. Collectively, our study uncovers a novel Treg cell suppression mechanism crucial for controlling a specific type of immune response in a particular tissue, and provides further mechanistic insights into tissue-specific Treg cell-mediated immune regulation.

Induction of Airway Hypersensitivity to Ovalbumin and Dust Mite Allergens as Mouse Models of Allergic Asthma.

Mouse models of allergic asthma have been utilized to establish the role of T helper type 2 (Th2) cells in driving lung inflammation, airway hyperresponsiveness, and obstruction. Here, we present the allergic asthma models, in which mice are hypersensitized to ovalbumin (OVA) and house dust mite (HDM). These models mimic the major characteristics of human asthma including the eosinophilic inflammation and hyperactivity of the airway, overproduction of Th2 cytokines in the lung, and elevated total and allergen-specific immunoglobulin E (IgE) in serum.

DNA methyltransferase inhibitor alleviates bleomycin-induced pulmonary inflammation.

Acute lung injury (ALI) is a severe disease characterized by several inflammatory responses in the lung with high mortality. We applied a mouse model of the pulmonary inflammation induced by the intratracheal instillation of bleomycin which is widely used to induce ALI and fibrosis in animal models. We hypothesized that DNA methyltransferase inhibitor, 5-azacytidine (5-Aza), with its anti-inflammatory benefits, might attenuate bleomycin-induced ALI through the alleviation of inflammation in the lung. We quantified white blood cells with cell blood count (CBC) test, lung inflammation by analyzing cells in the collected bronchoalveolar lavage fluid (BALF) and histological analysis of the lung tissues, and gene expression levels by real-time PCR. Intratracheal administration of bleomycin in mice induced pulmonary inflammation, characterized by increased neutrophil infiltration and inflammatory cytokine expression in the lungs. Mice treated with 5-Aza showed a significant reduction of lung neutrophilia, together with lower expressions of CXCL2 and MCP-1. Furthermore, 5-Aza treatment decreased the expression of proinflammatory cytokines in the lung tissue. Collectively, our data show that DNA methyltransferase inhibitor can alleviate the lung inflammation of bleomycin-induced ALI, indicating an alternative treatment option for the inflammation-triggered lung injury.

MiR-23~27~24-mediated control of humoral immunity reveals a TOX-driven regulatory circuit in follicular helper T cell differentiation.

Follicular helper T (TFH) cells are essential for generating protective humoral immunity. To date, microRNAs (miRNAs) have emerged as important players in regulating TFH cell biology. Here, we show that loss of miR-23~27~24 clusters in T cells resulted in elevated TFH cell frequencies upon different immune challenges, whereas overexpression of this miRNA family led to reduced TFH cell responses. Mechanistically, miR-23~27~24 clusters coordinately control TFH cells through targeting a network of genes that are crucial for TFH cell biology. Among them, thymocyte selection-associated HMG-box protein (TOX) was identified as a central transcription regulator in TFH cell development. TOX is highly up-regulated in both mouse and human TFH cells in a BCL6-dependent manner. In turn, TOX promotes the expression of multiple molecules that play critical roles in TFH cell differentiation and function. Collectively, our results establish a key miRNA regulon that maintains optimal TFH cell responses for resultant humoral immunity.

TOX and TOX2 transcription factors cooperate with NR4A transcription factors to impose CD8+ T cell exhaustion.

T cells expressing chimeric antigen receptors (CAR T cells) have shown impressive therapeutic efficacy against leukemias and lymphomas. However, they have not been as effective against solid tumors because they become hyporesponsive ("exhausted" or "dysfunctional") within the tumor microenvironment, with decreased cytokine production and increased expression of several inhibitory surface receptors. Here we define a transcriptional network that mediates CD8+ T cell exhaustion. We show that the high-mobility group (HMG)-box transcription factors TOX and TOX2, as well as members of the NR4A family of nuclear receptors, are targets of the calcium/calcineurin-regulated transcription factor NFAT, even in the absence of its partner AP-1 (FOS-JUN). Using a previously established CAR T cell model, we show that TOX and TOX2 are highly induced in CD8+ CAR+ PD-1high TIM3high ("exhausted") tumor-infiltrating lymphocytes (CAR TILs), and CAR TILs deficient in both TOX and TOX2 (Tox DKO) are more effective than wild-type (WT), TOX-deficient, or TOX2-deficient CAR TILs in suppressing tumor growth and prolonging survival of tumor-bearing mice. Like NR4A-deficient CAR TILs, Tox DKO CAR TILs show increased cytokine expression, decreased expression of inhibitory receptors, and increased accessibility of regions enriched for motifs that bind activation-associated nuclear factor κB (NFκB) and basic region-leucine zipper (bZIP) transcription factors. These data indicate that Tox and Nr4a transcription factors are critical for the transcriptional program of CD8+ T cell exhaustion downstream of NFAT. We provide evidence for positive regulation of NR4A by TOX and of TOX by NR4A, and suggest that disruption of TOX and NR4A expression or activity could be promising strategies for cancer immunotherapy.

OK-432 Acts as Adjuvant to Modulate T Helper 2 Inflammatory Responses in a Murine Model of Asthma.

Enhanced type 2 helper T (Th2) cell responses to inhaled harmless allergens are strongly associated with the development of allergic diseases. Antigen formulated with an appropriate adjuvant can elicit suitable systemic immunity to protect individuals from disease. Although much has been learned about Th1-favored immunomodulation of OK-432, a streptococcal preparation with antineoplastic activity, little is known about its adjuvant effect for allergic diseases. Herein, we demonstrate that OK-432 acts as an adjuvant to favor a systemic Th1 polarization with an elevation in interferon- (IFN-) γ and ovalbumin- (OVA-) immunoglobulin (Ig) G2a. Prior vaccination with OK-432 formulated against OVA attenuated lung eosinophilic inflammation and Th2 cytokine responses that were caused by challenging with OVA through the airway. This vaccination with OK-432 augmented the ratios of IFN-γ/interleukin- (IL-) 4 cytokine and IgG2a/IgG1 antibody compared to the formulation with Th2 adjuvant aluminum hydroxide (Alum) or antigen only. The results obtained in this study lead us to propose a potential novel adjuvant for clinical use such as prophylactic vaccination for pathogens and immunotherapy in atopic diseases.

In Vivo Target Gene Activation via CRISPR/Cas9-Mediated Trans-epigenetic Modulation.

Current genome-editing systems generally rely on inducing DNA double-strand breaks (DSBs). This may limit their utility in clinical therapies, as unwanted mutations caused by DSBs can have deleterious effects. CRISPR/Cas9 system has recently been repurposed to enable target gene activation, allowing regulation of endogenous gene expression without creating DSBs. However, in vivo implementation of this gain-of-function system has proven difficult. Here, we report a robust system for in vivo activation of endogenous target genes through trans-epigenetic remodeling. The system relies on recruitment of Cas9 and transcriptional activation complexes to target loci by modified single guide RNAs. As proof-of-concept, we used this technology to treat mouse models of diabetes, muscular dystrophy, and acute kidney disease. Results demonstrate that CRISPR/Cas9-mediated target gene activation can be achieved in vivo, leading to measurable phenotypes and amelioration of disease symptoms. This establishes new avenues for developing targeted epigenetic therapies against human diseases. VIDEO ABSTRACT.

miR-25/93 mediates hypoxia-induced immunosuppression by repressing cGAS.

The mechanisms by which hypoxic tumours evade immunological pressure and anti-tumour immunity remain elusive. Here, we report that two hypoxia-responsive microRNAs, miR-25 and miR-93, are important for establishing an immunosuppressive tumour microenvironment by downregulating expression of the DNA sensor cGAS. Mechanistically, miR-25/93 targets NCOA3, an epigenetic factor that maintains basal levels of cGAS expression, leading to repression of cGAS during hypoxia. This allows hypoxic tumour cells to escape immunological responses induced by damage-associated molecular pattern molecules, specifically the release of mitochondrial DNA. Moreover, restoring cGAS expression results in an anti-tumour immune response. Clinically, decreased levels of cGAS are associated with poor prognosis for patients with breast cancer harbouring high levels of miR-25/93. Together, these data suggest that inactivation of the cGAS pathway plays a critical role in tumour progression, and reveal a direct link between hypoxia-responsive miRNAs and adaptive immune responses to the hypoxic tumour microenvironment, thus unveiling potential new therapeutic strategies.

MicroRNA in Immune Regulation.

The immune system protects us from enormously diverse microbial pathogens but needs to be tightly regulated to avoid deleterious immune-mediated inflammation and tissue damage. A wide range of molecular determinants and cellular components work in concert to control the magnitude and duration of a given immune response. In the past decade, microRNAs (miRNAs), a major class of small non-coding RNA species, have been extensively studied as key molecular players in immune regulation. In this chapter, we will discuss how miRNAs function as negative regulators to restrict innate and adaptive immune responses. Moreover, we will review the current reports regarding miRNAs in human immunological diseases. Finally, we will also address the emerging roles of other non-coding RNAs, long non-coding RNAs (lncRNAs) in particular, in the regulation of the immune system.

A Novel miR-24-TCF1 Axis in Modulating Effector T Cell Responses.

miR-23∼27∼24 was recently implicated in restricting Th2 immunity, as well as the differentiation and function of other effector T cell lineages. Interestingly, miR-24, unlike other family members, actually promotes Th1 and Th17 responses. In this article, we show that miR-24 drives the production of IFN-γ and IL-17 in T cells at least in part through targeting TCF1, a transcription factor known for its role in limiting Th1 and Th17 immunity. Surprisingly, whereas TCF1 was previously shown to promote Th2 responses through inducing GATA3, enforced TCF1 expression in miR-24-overexpressing T cells led to further downregulation of IL-4 and GATA3 expression, suggesting miR-24-mediated inhibition of Th2 immunity cannot be attributed to TCF1 repression by miR-24. Together, our data demonstrate a novel miR-24-TCF1 pathway in controlling effector cytokine production by T cells and further suggest miR-24 could function as a key upstream molecule regulating TCF1-mediated immune responses.

Excessive expression of miR-27 impairs Treg-mediated immunological tolerance.

MicroRNAs (miRs) are tightly regulated in the immune system, and aberrant expression of miRs often results in hematopoietic malignancies and autoimmune diseases. Previously, it was suggested that elevated levels of miR-27 in T cells isolated from patients with multiple sclerosis facilitate disease progression by inhibiting Th2 immunity and promoting pathogenic Th1 responses. Here we have demonstrated that, although mice with T cell-specific overexpression of miR-27 harbor dysregulated Th1 responses and develop autoimmune pathology, these disease phenotypes are not driven by miR-27 in effector T cells in a cell-autonomous manner. Rather, dysregulation of Th1 responses and autoimmunity resulted from a perturbed Treg compartment. Excessive miR-27 expression in murine T cells severely impaired Treg differentiation. Moreover, Tregs with exaggerated miR-27-mediated gene regulation exhibited diminished homeostasis and suppressor function in vivo. Mechanistically, we determined that miR-27 represses several known as well as previously uncharacterized targets that play critical roles in controlling multiple aspects of Treg biology. Collectively, our data show that miR-27 functions as a key regulator in Treg development and function and suggest that proper regulation of miR-27 is pivotal to safeguarding Treg-mediated immunological tolerance.

DNA Methyltransferase Inhibitor Promotes Human CD4+CD25hFOXP3+ Regulatory T Lymphocyte Induction under Suboptimal TCR Stimulation.

The "master transcription factor" FOXP3 regulates the differentiation, homeostasis, and suppressor function of CD4+ regulatory T (Treg) cells, which are critical in maintaining immune tolerance. Epigenetic regulation of FOXP3 expression has been demonstrated to be important to Treg cell development, but the induction of human Treg cells through epigenetic modification has not been clearly described. We report that the combination of the DNA methyltransferase inhibitor 5-azacytidine (5-Aza) and suboptimal T cell receptor (TCR) stimulation promoted CD4+CD25hFOXP3+ T cell induction from human CD4+CD25- T cells. 5-Aza treatment enhanced the expression of Treg cell signature genes, such as CD25, FOXP3, CTLA-4, and GITR, in CD4+CD25h cells. Moreover, 5-Aza-treated CD4+CD25h T cells showed potent suppressive activity in a cell contact-dependent manner and reduced methylation in the Treg-specific demethylated region (TSDR) in the FOXP3 gene. The analysis of cytokine production revealed that CD4+CD25- T cells with 5-Aza treatment produced comparable levels of interferon (IFN)-γ and transforming growth factor (TGF)-ß, but less IL-10 and more IL-2, when compared to cells without 5-Aza treatment. The increased IL-2 was indispensible to the enhanced FOXP3 expression in 5-Aza-treated CD4+CD25h cells. Finally, 5-Aza-treated CD4+CD25h T cells could be expanded with IL-2 supplementation alone and maintained FOXP3 expression and suppressor function through the expansion. Our findings demonstrate that DNA demethylation can enhance the induction of human Treg cells and promise to solve one of the challenges with using Treg cells in therapeutic approaches.

A miR-155-Peli1-c-Rel pathway controls the generation and function of T follicular helper cells.

MicroRNA (miRNA) deficiency impairs the generation of T follicular helper (Tfh) cells, but the contribution of individual miRNAs to this phenotype remains poorly understood. In this study, we performed deep sequencing analysis of miRNAs expressed in Tfh cells and identified a five-miRNA signature. Analyses of mutant mice deficient of these miRNAs revealed that miR-22 and miR-183/96/182 are dispensable, but miR-155 is essential for the generation and function of Tfh cells. miR-155 deficiency led to decreased proliferation specifically at the late stage of Tfh cell differentiation and reduced CD40 ligand (CD40L) expression on antigen-specific CD4(+) T cells. Mechanistically, miR-155 repressed the expression of Peli1, a ubiquitin ligase that promotes the degradation of the NF-κB family transcription factor c-Rel, which controls cellular proliferation and CD40L expression. Therefore, our study identifies a novel miR-155-Peli1-c-Rel pathway that specifically regulates Tfh cell generation and function.

CD4 down regulation and raft dissociation by the non-depleting YTS177 antibody hinder murine T helper cell activities.

Non-depleting YTS177 anti-CD4 monoclonal antibody (MoAb) has been reported to lead to antigen-specific immunotolerance in allograft transplantation and autoimmune diabetes, as well as possibly to inhibition of allergic inflammation in mice. However, the molecular mechanisms underlying hyporesponsive T cell responses induced by YTS177 MoAb remain elusive. Herein, we demonstrate that the YTS177 MoAb increases the levels of anergy factors p27(kip1) and Cbl-b, inhibits IL-2 production, and impairs calcium mobilization in activated T cells in vitro. YTS177 MoAb suppresses OVA-driven proliferation of DO11.10 CD4(+) T cells in vivo as well. Mechanistically, YTS177 MoAb induces tolerance by causing CD4 down-regulation through clathrin-dependent and raft dissociation. The results obtained in this study lead us to propose novel protective or curative approaches to CD4 T cell-mediated diseases.

miR-23∼27∼24 clusters control effector T cell differentiation and function.

Coordinated repression of gene expression by evolutionarily conserved microRNA (miRNA) clusters and paralogs ensures that miRNAs efficiently exert their biological impact. Combining both loss- and gain-of-function genetic approaches, we show that the miR-23∼27∼24 clusters regulate multiple aspects of T cell biology, particularly helper T (Th) 2 immunity. Low expression of this miRNA family confers proper effector T cell function at both physiological and pathological settings. Further studies in T cells with exaggerated regulation by individual members of the miR-23∼27∼24 clusters revealed that miR-24 and miR-27 collaboratively limit Th2 responses through targeting IL-4 and GATA3 in both direct and indirect manners. Intriguingly, although overexpression of the entire miR-23 cluster also negatively impacts other Th lineages, enforced expression of miR-24, in contrast to miR-23 and miR-27, actually promotes the differentiation of Th1, Th17, and induced regulatory T cells, implying that under certain conditions, miRNA families can fine tune the biological effects of their regulation by having individual members antagonize rather than cooperate with each other. Together, our results identify a miRNA family with important immunological roles and suggest that tight regulation of miR-23∼27∼24 clusters in T cells is required to maintain optimal effector function and to prevent aberrant immune responses.

Prophylactic vaccination with adjuvant monophosphoryl lipid a prevents Th2-mediated murine asthmatic responses.

OBJECTIVE: Asthma is a chronic respiratory disorder characterized by airway hyperreactivity, eosinophilic infiltration, high titer of allergen-specific IgE, and overproduction of T helper 2 (Th2) cytokines. Antigen combined with an appropriate adjuvant and administrated through the proper route can elicit suitable immunological responses to protect humans and animals from diseases. Antigen formulated with monophosphoryl lipid A (MPLA) can produce priming of Th1-mediated immune responses. The purpose of this study was to examine the utility of MPLA as an adjuvant to prevent asthma. METHODS: BALB/c mice were immunized with ovalbumin (OVA) formulated with or without MPLA by intraperitoneal, footpad, or subcutaneous injection. Vaccinated mice were challenged with OVA aerosol to estimate the protective efficacy of MPLA in comparison to Th2-adjuvant aluminum hydroxide (Alum). Airway hyperresponsiveness to methacholine, eosinophilia in bronchoalveolar lavage fluid (BALF), circulating titers of OVA-specific antibodies, and stimulating levels of IFN-γ and IL-4 cytokines from splenocytes were evaluated. RESULTS: Mice immunized by all injection routes with OVA formulated with MPLA increased the ratio of Th1/Th2 responses compared to mice receiving antigen alone. For prophylactic vaccination purpose, MPLA reduced airway responsiveness and eosinophilic inflammation in the lung, decreased serum OVA-specific IgE level, and increased the serum ratio of OVA-specific IgG2a/IgG1 and the ratio of IFN-γ/IL4 from OVA-activated splenocytes compared with mice vaccinated with Alum. CONCLUSION: MPLA may be clinically useful in the vaccination of individuals predisposed to asthma.

The DNA methylation inhibitor 5-azacytidine increases regulatory T cells and alleviates airway inflammation in ovalbumin-sensitized mice.

BACKGROUND: Asthma is characterized as a chronic inflammatory disorder of the airways associated with an enhanced TH2 response to inhaled allergens. CD4+ T regulatory (Treg) cells are controlled by the master transcription factor FoxP3 and strictly maintain peripheral immunotolerance. Epigenetic regulation of FoxP3 by DNA methyltransferase inhibitors, such as 5-azacytidine (Aza), can generate a steady supply of functional Treg cells. Therefore, we propose that Aza can augment Treg cells in vivo to prevent the pathogenesis of asthma. METHODS: BALB/c mice were sensitized with chicken ovalbumin (OVA) and treated with different doses of Aza. Airway hyperresponsiveness to methacholine, eosinophilia in bronchoalveolar lavage fluid, circulating titers of OVA-specific IgG1 and IgE, and stimulating levels of TH2 cytokines from splenocytes were then determined. Cellular populations were examined by flow cytometry. PC61 antibody, which depletes CD25+ cells, was used to verify the role of CD25+ cells in Aza-induced tolerance. RESULTS: Administration of Aza to OVA-sensitized mice diminished airway hyperreactivity, pulmonary eosinophilia, levels of OVA-specific IgG1 and IgE in serum, and secretion of TH2 cytokines from OVA-stimulated splenocytes in a dose-dependent manner. Percentages of CD25+ and FoxP3+ cells in the CD4+ cell population were notably increased in Aza-treated mice compared to sensitized control mice. Furthermore, the major symptoms of asthma were exacerbated by depleting CD25+ cells in Aza-treated mice. CONCLUSIONS: Aza may have applications as a novel clinical strategy to increase the production of Treg cells in order to modulate the airway inflammation associated with asthma.

Evaluation of a common variant of the gene encoding clara cell 10 kd protein (CC10) as a candidate determinant for asthma severity and steroid responsiveness among Chinese children.

OBJECTIVE: The gene (SCGB1A1) encoding Clara cell 10-kDa protein (CC10), a steroid-inducible immune modulator, is a candidate gene for asthma, but the evidence is equivocal. The potential influence of a common variant on asthma severity and serum CC10 levels during acute exacerbation and after corticosteroid treatment in Chinese case-control children and its functional relevance was investigated. METHODS: Genotyping of a non-coding variant G+38A was performed in 489 children, of whom 277 had asthma with varying severity, and 212 healthy controls. Associations were tested for asthma, asthma severity, and responsiveness to steroid treatment. The transcriptional activity of this variant was examined in a Clara-like cell line (H358) using transient transfection assays. RESULTS: Significant association was observed for the combined GA and AA genotypes of the CC10 G+38A variant and an increased risk of asthma [odds ratio (OR), 2.62, p < .001]. This association was correlated with asthma severity (moderate: OR, 2.85, p < .001; near-fatal: OR, 4.81, p < .001). Also, patients with the GA and AA genotypes showed significantly lower serum CC10 (p < .01) and provocation concentration causing a 20% fall (PC(20)) in forced expiratory volume in 1 s (FEV(1)) (p < .0001) when compared with those with the GG. After glucocorticoid treatment, the CC10 levels were significantly increased in asthmatic patients with GG (p < .0001), but not those with the GA and AA genotypes. Moreover, a lower dexamethasone-induced reporter (luciferase) activity was observed for H358 cells transiently transfected with the G38A risk allele (A) compared with wild-type allele (G). CONCLUSIONS: These findings suggest that the CC10 G+38A variant may contribute to the severity of asthma and lower level of steroid responsiveness.

Distribution and clinical aspects of primary immunodeficiencies in a Taiwan pediatric tertiary hospital during a 20-year period.

Recent advances in immunologic techniques have lead to increased recognition of primary immunodeficiencies. A review of patients with suspected immunodeficiencies in a Taiwan tertiary hospital from January 1985 to October 2004 and molecular/genetic analyses done on some patients were investigated. Of the 403 patients selected based on the International Classification of Disease, Ninth Revision, 37 patients with PID (8 females and 29 males) were identified: 17 (46%) with antibody production deficiencies, nine (24%) with defective phagocyte function, four (11%) with combined B and T cell immunodeficiencies, seven (19%) with T cell deficiencies, but none with primary complement deficiencies. Those with secondary immunodeficiencies were excluded from the study. Recurrent sinopulmonary infections (62%) were the most common clinical manifestation, followed by sepsis (57%), severe skin infection (40%), splenomegaly/hepatomegaly (27%), central nervous system dysfunction (22%), chronic diarrhea (22%), and failure to thrive (19%). Seven (19%) patients died, five of infections, one of disseminated intravascular coagulopathy and one of hepatocellular carcinoma. Six novel mutations were found from 11 agreed patients. This is the first report on primary immunodeficiencies in Taiwan covering a 20-year period.