Helper Lineage Research Articles

Distinctive features of CD4+ T cell dysfunction in chronic viral infections.

To describe recent advances in the understanding of virus-specific CD4 T cell dysfunction in chronic viral infections, with an emphasis on HIV disease. We highlight features that are distinctive for CD4 T cells, as opposed to their CD8 T cell counterparts. CD4 T cell activation and differentiation are tightly controlled. Regulation of these processes depends on the context of initial encounter of the naïve CD4 T cell with the cognate antigen and on ongoing external cues to the antigen-experienced CD4 T cell, in particular the inflammatory environment, which is prominent in HIV infection. Virus-specific CD4 T cell dysfunction results from a combination of an exhaustion program and skewing in T helper lineage differentiation which impact function. The CD4 and CD8 T cell exhaustion programs present similarities and distinct features. The sets of inhibitory coreceptors expression differ, although programmed-death 1 (PD-1) and T cell immunoglobulin mucin-3 (Tim-3) are upregulated on both HIV-specific CD4 and CD8 T cells, cytotoxic T-lymphocyte antigen 4 (CTLA-4) is largely specific to CD4 T cells, whereas 2B4 and CD160 are biased toward CD8 T cells. Understanding the molecular basis of HIV-specific CD4 T cell exhaustion and identifying key differences with CD8 T cell impairment will be critical to design effective therapeutic and preventive immunotherapies against HIV.

The second touch hypothesis: T cell activation, homing and polarization.

The second touch hypothesis states that T cell activation, proliferation, induction of homing receptors and polarization are distinguishable and, at least in part, sequential. The second touch hypothesis maintains that full T cell polarization requires T cell interaction with antigen-presenting cells (DCs, macrophages, B cells and certain activated stromal cells) in the non-lymphoid tissue where the antigen resides. Upon initial antigen encounter in peripheral lymph nodes (PLN), T cells become activated, proliferate and express homing receptors that enable them to recirculate to the (inflamed) tissue that contains the antigen. Differentiation into the T helper lineages Th1, Th2, Th17 and induced regulatory T cells (iTreg) requires additional antigen presentation by tissue macrophages and other antigen presenting cells (APCs) in the inflamed tissue. Here, I present a conceptual framework for the importance of peripheral (non-lymphoid) antigen presentation to antigen-experienced T cells.

TH17 cells in human recurrent pregnancy loss and pre-eclampsia.

T helper 17 (TH17) cells have been identified as a new lineage of helper T cells and have been shown to be important in host defense against extracellular infectious agents, autoimmune disease and chronic inflammatory diseases. Recently, TH17 cells have also been shown to participate in successful pregnancy, as well as in the pathogenesis of diseases of pregnancy, such as recurrent spontaneous abortion (RSA) and pre-eclampsia (PE). Here, we review our current knowledge of TH17 cells in human RSA and PE. We also discuss how the local uterine microenvironment affects the differentiation of TH17 cells and the mechanisms that regulate TH17 cells during pregnancy. Research into TH17 cells will not only advance our understanding of TH17-related pregnancy complications, but will also facilitate the design of novel therapies for reproductive diseases.

A silencer-proximal intronic region is required for sustained CD4 expression in postselection thymocytes.

It has been proposed that differential kinetics of CD4/CD8 coreceptors regulate fate choice of selected thymocytes. Sustained signals by interaction between MHC class II and TCR/CD4 is required for commitment to the CD4 helper lineage. Although prematurely terminated MHC-TCR/CD4 interaction in transgenic mouse models results in lineage redirection, it is unclear whether CD4 expression is actively maintained by endogenous cis-elements to facilitate prolonged signaling under physiological conditions. In this article, we show that sustained CD4 expression in postselection thymocytes requires an intronic sequence containing an uncharacterized DNase I hypersensitivity (DHS) site located 3' to the silencer. Despite normal CD4 expression before selection, thymocytes lacking a 1.5-kb sequence in intron 1 including the 0.4-kb silencer and the DHS, but not the 0.4-kb silencer alone, failed to maintain CD4 expression upon positive selection and are redirected to the CD8 lineage after MHC class II-restricted selection. Furthermore, CpG dinucleotides adjacent to the DHS are hypermethylated in CD8(+) T cells. These results indicate that the 1.5-kb cis-element is required in postselection thymocytes for helper lineage commitment, presumably mediating the maintenance of CD4 expression, and suggest that inactivation of the cis-element by DNA methylation may contribute to epigenetic Cd4 silencing.

The second touch hypothesis: T cell activation, homing and polarization

The second touch hypothesis states that T cell activation, proliferation, induction of homing receptors and polarization are distinguishable and, at least in part, sequential. The second touch hypothesis maintains that full T cell polarization requires T cell interaction with antigen-presenting cells (DCs, macrophages, B cells and certain activated stromal cells) in the non-lymphoid tissue where the antigen resides. Upon initial antigen encounter in peripheral lymph nodes (PLN), T cells become activated, proliferate and express homing receptors that enable them to recirculate to the (inflamed) tissue that contains the antigen. Differentiation into the T helper lineages Th1, Th2, Th17 and induced regulatory T cells (iTreg) requires additional antigen presentation by tissue macrophages and other antigen presenting cells (APCs) in the inflamed tissue. Here, I present a conceptual framework for the importance of peripheral (non-lymphoid) antigen presentation to antigen-experienced T cells.

Negative regulation of Hif1a expression and TH17 differentiation by the hypoxia-regulated microRNA miR-210.

MicroRNA-210 (miR-210) is a signature microRNA of hypoxia. We found robust increase (>100-fold) of miR-210 abundance in activated T cells, especially in the TH17 lineage. Hypoxia synergized with T cell receptor (TCR)–CD28 stimulation to accelerate and increase the magnitude of Mir210 expression. Mir210 was directly regulated by HIF-1α, a key regulator of TH17 polarization. Surprisingly, Hif1a was identified as a miR-210-target, suggesting negative-feedback by miR-210 to inhibit HIF-1α protein expression. Deletion of Mir210 promoted TH17 differentiation under conditions with limited oxygen. In experimental colitis, miR-210 reduced Hif1a transcript abundance, reduced the proportion of cells producing inflammatory cytokines and controlled disease severity. Our study identifies miR-210 as an important regulator of T cell differentiation in hypoxia, which can limit immunopathology.

The second touch hypothesis: T cell activation, homing and polarization

The second touch hypothesis states that T cell activation, proliferation, induction of homing receptors and polarization are distinguishable and, at least in part, sequential. The second touch hypothesis maintains that full T cell polarization requires T cell interaction with antigen-presenting cells (DCs, macrophages, B cells and certain activated stromal cells) in the non-lymphoid tissue where the antigen resides. Upon initial antigen encounter in peripheral lymph nodes (PLN), T cells become activated, proliferate and express homing receptors that enable them to recirculate to the (inflamed) tissue that contains the antigen. Differentiation into the T helper lineages Th1, Th2, Th17 and induced regulatory T cells (iTreg) requires additional antigen presentation by tissue macrophages and other antigen presenting cells (APCs) in the inflamed tissue. Here, I present a conceptual framework for the importance of peripheral (non-lymphoid) antigen presentation to antigen-experienced T cells.

Regulation of Germinal Center Reactions by B and T Cells

Break of B cell tolerance to self-antigens results in the development of autoantibodies and, thus, leads to autoimmunity. How B cell tolerance is maintained during active germinal center (GC) reactions is yet to be fully understood. Recent advances revealed several subsets of T cells and B cells that can positively or negatively regulate GC B cell responses in vivo. IL-21-producing CXCR5+ CD4+ T cells comprise a distinct lineage of helper T cells—termed follicular helper T cells (TFH)—that can provide help for the development of GC reactions where somatic hypermutation and affinity maturation take place. Although the function of TFH cells is beneficial in generating high affinity antibodies against infectious agents, aberrant activation of TFH cell or B cell to self-antigens results in autoimmunity. At least three subsets of immune cells have been proposed as regulatory cells that can limit such antibody-mediated autoimmunity, including follicular regulatory T cells (TFR), Qa-1 restricted CD8+ regulatory T cells (CD8+TREG), and regulatory B cells (BREG). In this review, we will discuss our current understanding of GC B cell regulation with specific emphasis on the newly identified immune cell subsets involved in this process.

Role of Aryl hydrocarbon receptor and the effect of selective AhR modulators on T cell differentiation (P1201)

Abstract The physiological role of AhR, particularly in immune response remains a key question after identification of AhR as an essential transcription factor for Th17 differentiation. Selective AhR modulators (SAhRMs) are a recently identified class of compounds that are capable of binding to AhR and repress cytokine- driven gene expression. These compounds are interesting therapeutically as they can be used to inhibit Dioxin Response Element (DRE)-mediated while inducing anti-inflammatory responses. Under non-skewing conditions, AhR agonist beta-naphthoflavone (BNF) enhances IL-17A production whereas SGA-360 an inhibitor suppresses it. Under Th17 skewing condition, AhR agonist BNF enhanced Th17 differentiation whereas AhR antagonist GNF and partial antagonist SGA-360 suppressed it. Interestingly, under Treg skewing condition the antagonists enhanced the Foxp3 expression, suggesting that such compounds have therapeutic potential in autoimmune disease by modulating the Th17/Treg balance. We have also examined AhR antagonists after disease development in a murine model of Th17 mediated disease, hypersensitivity pneumonitis mediated by Staphylopolyspora rectivirgula (SR). We find that these antagonists suppress AhR activity and induce IL-4, suggestive of a shift in T helper lineage. This suggests that SAhRMs may be useful in modulating AhR activation to regulate Th17/Treg balance, and that AhR selective ligands have potential for use as therapeutics in Th17 mediated diseases.

Role of Th17 cells in the pathogenesis of CNS inflammatory demyelination

Multiple sclerosis (MS) is an autoimmune disease of the central nervous system (CNS). The etiology of MS is not well understood, but it is believed that myelin-specific CD4+ T cells play a central role in initiating and orchestrating CNS inflammation. In this scenario, CD4+ T cells, activated in the periphery, infiltrate the CNS, where, by secreting cytokines and chemokines, they start an inflammatory cascade. Given the central role of CD4+ T cells in CNS autoimmunity, they have been studied extensively, principally by using experimental autoimmune encephalomyelitis (EAE), an animal model of MS. In the late 1980s, CD4+ T cells, based on their cytokine production, were divided into two helper lineages, Th1 and Th2 cells. It was postulated that Th1 cells, which produce IFN-γ, mediate inflammation of the CNS in MS/EAE, while Th2 cells, which produce IL-4, have a beneficial effect in disease, because of their antagonistic effect on Th1 cells. The Th1/Th2 paradigm remained the prevailing view of MS/EAE pathogenesis until 2005, when a new lineage, Th17, was discovered. In a relatively short period of time it became apparent that Th17 cells, named after their hallmark cytokine, IL-17A, play a crucial role in many inflammatory diseases, including EAE, and likely in MS as well. The Th17 paradigm developed rapidly, initiating the debate of whether Th1 cells contribute to EAE/MS pathogenesis at all, or if they might even have a protective role due to their antagonistic effects on Th17 cells. Numerous findings support the view that Th17 cells play an essential role in autoimmune CNS inflammation, perhaps mainly in the initial phases of disease. Th1 cells likely contribute to pathogenesis, with their role possibly more pronounced later in disease. Hence, the current view on the role of Th cells in MS/EAE pathogenesis can be called the Th17/Th1 paradigm. It is certain that Th17 cells will continue to be the focus of intense investigation aimed at elucidating the pathogenesis of CNS autoimmunity.

Epigenetic Thpok silencing limits the time window to choose CD4+ helper-lineage fate in the thymus

CD4(+) helper and CD8(+) cytotoxic T cells differentiate from common precursors in the thymus after T-cell receptor (TCR)-mediated selection. Commitment to the helper lineage depends on persistent TCR signals and expression of the ThPOK transcription factor, whereas a ThPOK cis-regulatory element, ThPOK silencer, represses Thpok gene expression during commitment to the cytotoxic lineage. Here, we show that silencer-mediated alterations of chromatin structures in cytotoxic-lineage thymocytes establish a repressive state that is epigenetically inherited in peripheral CD8(+) T cells even after removal of the silencer. When silencer activity is enhanced in helper-lineage cells, by increasing its copy number, a similar heritable Thpok silencing occurs. Epigenetic locking of the Thpok locus may therefore be an independent event from commitment to the cytotoxic lineage. These findings imply that long-lasting TCR signals are needed to establish stable Thpok expression activity to commit to helper T-cell fate and that full commitment to the helper lineage requires persistent reversal of silencer activity during a particular time window.

Detection of TCD4+ subsets in human carotid atheroma

Activated TCD4+ cells are detected in human atherosclerotic plaques which indicate their participation in disease progression and destabilization. Among these cells, IFN-γ-producing T cells (TH1) are recognized as having a pro-atherogenic role. Recently, the IL-17-producing T helper lineage of cells (TH17) has been identified in atherosclerotic lesions. They have been linked to atheroma development through the production of pro-inflammatory mediators present in these lesions. Furthermore, IL-22 producing TCD4+ cells (TH22) have been identified in the atheromatous environment, but their presence and function has not been investigated. The aim of this study was to analyze the immune response mediated by pro-inflammatory subtypes of TCD4+ cells in atheromatous lesions. Atherosclerotic plaques of 57 patients with critical stenosis of carotid submitted to endarterectomy were evaluated. Three carotid fragments from organ donors were used as control. mRNA analysis showed expression of TH1 (IFN-γ, T-bet, IL-2, IL-12p35, TNF-α and IL-18); TH2 (GATA-3); TH17 (IL-17A, IL-17RA, Rorγt, TGF-β, IL-6, IL-1β, IL-23p19, CCL20, CCR4 and CCR6) and TH22 (IL-22 and Ahr) related markers. Asymptomatic patients showed higher expression of mRNA of IL-10, TGF-β, CCR4 and GATA-3 when compared to symptomatic ones. Immunohistochemistry analysis showed higher levels of IL-23, TGF-β, IL-1β and IL-18 in macrophages and foam cells in unstable lesions compared to stable and control ones. In vitro stimulation of atheroma cells induced IL-17 and IFN-γ production. Finally we were able to detect, the following subpopulations of TCD3+ cells: TCD4+ IFN-γ+, TCD4+IL-17+, TCD4+IL-4+, TCD4+IL-22+ and double positive cells (IFN-γ/IL-17+, IFN-γ/IL-22+ or IL-17/IL-22+). Our results showed the presence of distinct TCD4+ cells subsets in human carotid lesions and suggest that interactions among them may contribute to the atheroma progression and destabilization.

Cutting Edge: Fine-Tuning of Thpok Gene Activation by an Enhancer in Close Proximity to Its Own Silencer

Differentiation of MHC class II-selected thymocytes toward the CD4(+) helper lineage depends on function of the transcription factor ThPOK, whose expression is repressed in CD8(+) cytotoxic lineage cells by a transcriptional silencer activity within the distal regulatory element (DRE) in the Thpok gene. Interestingly, the DRE also functions as a transcriptional enhancer. However, how the DRE exerts such dual functionality remains obscure. In this study, we dissected the DRE and identified DNA sequences specifically responsible for enhancer activity, and designated this as the thymic enhancer. Removal of the thymic enhancer from the murine Thpok locus resulted in inefficient ThPOK induction, thereby inducing a redirection toward alternative CD8(+) cytotoxic lineage in a proportion of MHC class II-selected cells, even when they express monoclonal MHC class II-restricted transgenic TCR. Thus, regulation of contiguous but separable sequences with opposite function in the DRE plays an important role in precise coupling of TCR signaling with the selection process of two opposite lineages.

Prevalence and clinical relevance of Th17 cells in patients with gastric cancer

BackgroundTh17 cells have recently been identified as a distinct T helper (Th) lineage in a cancer animal model and in human cancers. Their specific role in tumor immunity is unclear. We, therefore, sought to evaluate the role of Th17 cells in gastric cancer. MethodsThe prevalence of Th1, Th2, Treg, and Th17 cells in both peripheral blood mononuclear cells (PBMC) and gastric tissue was evaluated by multicolor flow cytometry. The concentration of interleukin (IL)-17 in sera was quantitated by enzyme-linked immunosorbent assay. ResultsWe observed a clear difference in the prevalence of Th17 cells in PBMC (0.34 ± 0.24%) versus gastric cancer tissues (19.4 ± 12.1) (P = 0.0002). Subset-specific phenotypic analysis of CD4+ T cells in both PBMC and gastric cancer tissue showed that Th1 and Treg cells predominate in PBMC, whereas Th17 cells are the most abundant CD4+ T cell subset in cancerous tissue. The concentrations of IL-17, a hallmark of Th17, in gastric cancer patients and normal controls were 0.6 ± 0.67 and 0.16 ± 0.19 pg/mL (P = 0.0032). Five-year survival rates of patients with high IL-17 and low IL-17 concentration were 47.1% and 83.9% (P = 0.0075). Multivariate analysis indicated that IL-17 concentration was an independent prognostic indicator, as well as lymph node metastasis. ConclusionsThere was a significant skewing toward a Th17 phenotype in gastric cancer tissue. IL-17 seems to play an important role in the progression of gastric cancer.

Elevated serum levels of interleukin-17A in children with autism

BackgroundThe T-helper (Th)1/Th2 dichotomy dominated the field of immune regulation until interleukin (IL)-17-expressing T cells (Th17) were proposed to be a third lineage of helper T cells, the key players in the pathogenesis of autoimmune disorders. Autoimmunity to brain tissue may play a pathogenic role in autism. IL-17A is a pro-inflammatory cytokine that has been shown to play an important role in various autoimmune neuroinflammatory diseases. The aim of this study was to measure serum levels of IL-17A in relation to the degree of the severity of autism.MethodsSerum IL-17A levels were measured by ELISA in 45 children with autism and 40 matched healthy controls.ResultsChildren with autism had significantly higher serum IL-17A levels than healthy controls (P <0.001), with increased serum levels of IL-17A found in 48.9% of the autism group. Patients with severe autism had significantly higher serum IL-17A levels than those with mild to moderate autism (P = 0.01), and raised serum IL-17A levels were significantly more common in children with severe autism (67.9%) than in those with mild to moderate autism (17.6%), P = 0.001.ConclusionsSerum IL-17A levels were raised in the group with autism, and the levels correlated significantly with the severity of autism. This is the first study to measure levels of IL-17A in relation to the severity of autism, to our knowledge. Further research, with a larger subject population, is warranted to determine whether the increase of serum IL-17A levels plasma has a pathogenic role in autism, and whether anti- IL-17A therapy could be useful

Conserved Intergenic Elements and DNA Methylation Cooperate to Regulate Transcription at the il17 Locus

Naive CD4(+) T cells can differentiate into distinct lineages with unique immune functions. The cytokines TGFβ and IL-6 promote the development of Th17 cells that produce IL-17, an inflammatory cytokine not expressed by other T helper lineages. To further understand how IL-17 production is controlled, we studied an ~120-kb genomic region containing the murine il17a and il17f genes and seven evolutionarily conserved, intergenic noncoding sequences. We show that the +28-kb noncoding sequence cooperates with STAT3, RORγt, and Runx1 to enhance transcription from both il17a and il17f promoters. This enhancer and both promoters exhibited Th17 lineage-specific DNA demethylation, accompanied by demethylation of lysine 27 of histone H3 (H3K27) and increased H3K4 methylation. Loss of DNA methylation tended to occur at STAT3 consensus elements, and we show that methylation of one of these elements in the il17a promoter directly inhibits STAT3 binding and transcriptional activity. These results demonstrate that TGFβ and IL-6 synergize to epigenetically poise the il17 loci for expression in Th17 cells, and suggest a general mechanism by which active STAT3 may be epigenetically excluded from STAT3-responsive genes in non-Th17 lineages.

De Novo DNA Methylation Is Required to Restrict T Helper Lineage Plasticity

Naïve CD4+ T cells are highly plastic and can differentiate into discrete lineages with unique functions during an immune response. Once differentiated, helper T cells maintain a stable transcriptional memory of their initial lineage choice and resist redifferentiation. During embryogenesis, de novo DNA methylation operates on the hypomethylated genome of the blastocyst to achieve tissue-specific patterns of gene expression. Similarly, the ifnγ promoter is hypomethylated in naïve T cells, but Th2, Th17, and iTreg differentiation is accompanied by substantial de novo DNA methylation at this locus. To determine whether de novo DNA methylation is required to restrict T helper lineage plasticity, we used mice with T cell-specific deletion of the methyltransferase DNMT3a. Induction of lineage-specific cytokines occurred normally in the absence of DNMT3a, however, DNMT3a-deficient Th2, Th17, and iTreg completely failed to methylate the ifnγ promoter. This was accompanied by an increase in the transcriptionally permissive trimethyl H3K4 mark, and a reduction in inhibitory H3K27 methylation at the ifnγ locus. Failed de novo methylation resulted in failed silencing of the ifnγ gene, as DNMT3a-deficient Th2, Th17, and iTreg cells produced significant levels of IFNγ following restimulation in the presence of IL-12. Therefore, DNMT3a-mediated DNA methylation restricts T helper plasticity by establishing an epigenetically silent chromatin structure at regulatory regions of the ifnγ gene.

Memory CD4+ T Cells: fate determination, positive feedback and plasticity

Naïve CD4(+) T cells undergo massive cell proliferation upon encountering their cognate ligand. This proliferation depends upon appropriate cues from the antigen-presenting cells that have processed the antigen and present the peptide to the T cells, and requires the establishment of a cytokine environment that can support such proliferation. Expansion of antigen-specific CD4(+) T cells needs to be coupled with differentiation into one of several effector/regulatory phenotypes if the priming event is to result in cells that can initially act to control the particular pathogen that elicited the response, and later to serve as memory cells to insure an appropriate response upon reintroduction of the pathogen. Here, we discuss the initiation of T helper lineage commitment, the positive feedback regulation by the cytokine environment to enhance and stabilize the differentiation into distinct T helper subsets, and the biological significance of CD4(+) T cell plasticity and long-term CD4(+) T cell memory.

Transcriptional and Epigenetic Control of T Helper Cell Specification: Molecular Mechanisms Underlying Commitment and Plasticity

T helper cell differentiation occurs in the context of the extracellular cytokine milieu evoked by diverse microbes and other pathogenic stimuli along with T cell receptor stimulation. The culmination of these signals results in specification of T helper lineages, which occurs through the combinatorial action of multiple transcription factors that establish distinctive transcriptomes. In this manner, inducible, but constitutively active, master regulators work in conjunction with factors such as the signal transducer and activator of transcriptions (STATs) that sense the extracellular environment. The acquisition of a distinctive transcriptome also depends on chromatin modifications that impact key cis elements as well as the changes in global genomic organization. Thus, signal transduction and epigenetics are linked in these processes of differentiation. In this review, recent advances in understanding T helper lineage specification and deciphering the action of transcription factors are summarized with emphasis on comprehensive views of the dynamic T cell epigenome.

Early Th1 Cell Differentiation Is Marked by a Tfh Cell-like Transition

Follicular helper T (Tfh) cells comprise an important subset of helper T cells; however, their relationship with other helper lineages is incompletely understood. Herein, we showed interleukin-12 acting via the transcription factor STAT4 induced both Il21 and Bcl6 genes, generating cells with features of both Tfh and Th1 cells. However, STAT4 also induced the transcription factor T-bet. With ChIP-seq, we defined the genome-wide targets of T-bet and found that it repressed Bcl6 and other markers of Tfh cells, thereby attenuating the nascent Tfh cell-like phenotype in the late phase of Th1 cell specification. Tfh-like cells were rapidly generated after Toxoplasma gondii infection in mice, but T-bet constrained Tfh cell expansion and consequent germinal center formation and antibody production. Our data argue that Tfh and Th1 cells share a transitional stage through the signal mediated by STAT4, which promotes both phenotypes. However, T-bet represses Tfh cell functionalities, promoting full Th1 cell differentiation.