Th17 Lineage Research Articles

Transcriptional memory in T cells is maintained by priming elements which create active chromatin domains that enable rapid responses in memory T cells without influencing steady state transcription

We have defined epigenetic mechanisms whereby immune response genes are reprogrammed for rapid recall responses when naïve T cells are transformed via TCR signalling into activated T cells. Until recently, distal regulatory elements were typically defined in genome-wide studies as transcriptional enhancers based on the presence of histone H3 K4 methylation and K27 acetylation. However, we and others have found that this definition is too simplistic because many, if not most, such distal regulatory elements appear to lack classical transcriptional enhancer activity. By focusing on previously activated primed T cells, we found that distal regulatory elements can be further subdivided into two distinct sub-classes of gene regulatory elements. Firstly, there are classical enhancers which do indeed appear to function to activate transcription of a nearby gene, and/or the underlying enhancer sequence. Transcriptional enhancers have been observed to function up to a megabase away, most likely by looping to contact a promoter. In addition to these classical enhancers, we have now defined a distinct class of chromatin priming enhancers, which are also modified by H3 K4 methylation and K27 acetylation, but which do not result in any significant change in the levels of transcription of nearby genes or regulatory elements. In memory T cells, these priming elements function to maintain a transcriptional memory which allows inducible immune response genes to be rapidly reactivated. These highly specialised elements function locally to establish active accessible chromatin domains in the vicinity of promoters or transcriptional enhancers, allowing easy access for the recruitment of transcription factors induced by TCR signalling. We recently observed that these priming elements also support T cell differentiation to alternate Th1, Th2 and Th17 lineages by making lineage-specific loci permissive to the transcription factors that direct these lineage switches, such as T-bet, GATA3 and RORg. Rather than establishing new active loci, these factors are often recruited to open chromatin regions established in response to TCR signalling, prior to terminal differentiation.

Anemoside A3 ameliorates experimental autoimmune encephalomyelitis by modulating T helper 17 cell response.

Anemoside A3 (AA3) is a natural triterpenoid glycoside isolated from the root of Pulsatilla chinensis (Bunge) Regel. We previously showed that AA3 exhibits cognitive-enhancing and neuroprotective properties. In the present study, we demonstrated that AA3 modulates inflammatory responses by regulating prostaglandin E receptor 4 signaling. Because prostaglandin E receptor 4 is involved in the pathophysiology of experimental autoimmune encephalomyelitis (EAE), an animal model of human multiple sclerosis (MS), we assessed the beneficial effect of AA3 in EAE mice. AA3 treatment significantly reduced clinical severity and inflammatory infiltrates in the spinal cord of EAE mice. In vitro studies revealed that AA3 inhibited the T cell response toward the encephalitogenic epitope of myelin oligodendrocyte glycoprotein (MOG). AA3 significantly downregulated the expressions of certain Th1 and Th17 cytokines in activated T cells re-stimulated by MOG. Moreover, AA3 inhibited the activation of STAT4 and STAT3, which are the transcription factors pivotal for Th1 and Th17 lineage differentiation, respectively, in activated T cells. Pharmacological analysis further suggested that AA3 reduced Th17 cell differentiation and expansion. In conclusion, AA3 exerts an immunomodulatory effect in EAE, demonstrating its potential as a therapeutic agent for MS in humans.

Hidradenitis Suppurativa Is Characterized by Dysregulation of the Th17:Treg Cell Axis, Which Is Corrected by Anti-TNF Therapy

Hidradenitis suppurativa (HS) is a chronic, inflammatory, and debilitating disease of hair follicles with 1-4% prevalence and high morbidity. There is a dearth of information on the pathogenesis and immune dysregulation underlying HS; therefore, we carried out a detailed analysis of skin-infiltrating T cells. Cells isolated from skin biopsy samples and blood from HS patients and healthy control subjects were analyzed by 16-parameter flow cytometry to provide detailed profiles of CD4 T-cell subsets. We observed substantial infiltration of inflammatory T cells with a striking T helper (Th) type 17-skewed cytokine profile in HS skin; these cells expressed the Th17 lineage marker CD161 and IL-17, as well as proinflammatory cytokines GM-CSF, IL-22, IFN-γ, and tumor necrosis factor. Regulatory T cells were also enriched in HS lesional skin; however, the ratio of Th17 to regulatory T cells was nonetheless highly dysregulated in favor of Th17 cells. In contrast, lesional skin from anti-tumor necrosis factor-treated HS patients who showed substantial clinical improvement exhibited a significant reduction in the frequency of Th17 cells and normalization of the Th17 to regulatory T cell ratio. These data suggest that inhibition of pathogenic IL-17 via tumor necrosis factor blockade is associated with improvement in immune dysregulation in HS and may provide a rationale for targeting IL-17 in the disease.

A long-noncoding RNA, Gata3-AS1, is a positive transcriptional regulator of transcription factor Gata3 in TH2 cells.

Abstract Long non-coding RNAs (lncRNAs) are RNAs transcribed by the genome, are >200 base pairs long, but are not translated into protein due to presence of multiple translational stop codons. LncRNAs possess a diverse array of regulatory functions including activation and silencing of gene transcription, regulation of splicing, and coordinating epigenetic modifications. Divergent lncRNAs are noncoding genes, which produce a lncRNA in the antisense orientation to an mRNA gene, and may share a promoter region. These lncRNAs may regulate their neighbor mRNA by direct RNA interactions or through the process of their own transcription. Gata3-AS1 is a divergent lncRNA gene in humans neighboring Gata3. The GATA3 transcription factor initiates transcription of genes encoding cytokines IL-4, IL-5, and IL-13, the hallmarks of TH2 polarization. Gata3-AS1 and Gata3 are exclusively expressed in the TH2 lineage of lymphocytes. In this study, we show that GATA3-AS1 is selectively expressed in human TH2 cells, resides solely in the nucleus, and is required for expression of GATA3, IL5, and IL13. Further, GATA3-AS1 RNA is required to establish H3K4-methyation and H3K27-acetylation ‘marks’ at the GATA3 genomic locus under TH2 differentiation conditions and directly binds the WDR5 DNA binding subunit of the MLL methyltransferase. Thus, GATA3-AS1 is a functional lncRNA and positive transcriptional regulator of GATA3 and commitment to the TH2 lineage.

Pathogenic conversion of RORγt+/Foxp3+ double positive regulatory T cells involves β-catenin up-regulation

Abstract The nature of regulatory T cells (Tregs) in cancer is ambivalent. In Colon Cancer (CC) patients and animal models of CC, we previously described a distinct subset of Tregs that suppresses anti-tumor effector T cell functions but simultaneously maintains inflammation. These Tregs co-express the Th17 lineage transcription factor RORγt along with Foxp3. Moreover, we causatively linked the pro-inflammatory skewing of T cells to Wnt/β-catenin-signaling. To elucidate how Wnt/β-catenin signaling orchestrates the induction of pathologic properties in Tregs we analyzed inflammatory bowel disease (IBD) patients. As IBD can progress to CC, this disease presents a unique platform to study the mechanisms underlying the emergence of RORγt+/Foxp3+Tregs. Indeed, in a sub-population of IBD patients we were able to detect RORγt+/Foxp3+Tregs, expressing high levels of β-catenin in the patients’ blood and tissue. To further establish that activated Wnt/β-catenin signaling is responsible for the pro-inflammatory skewing of Foxp3+ Tregs we over-expressed β-catenin Treg-specifically in a conditional Foxp3-Cre driven mouse model. Mice expressing constitutively active β-catenin in Tregs show striking X-linked immune pathology. Males die 4 weeks after birth of a severe scurfy-like phenotype. Although their Tregs are highly activated, they fail to suppress the proliferation of effector T cells. In heterozygous Foxp3-Cre mice, representing natural chimeras harboring wildtype and β-catenin over-expressing Tregs, the relative fitness of genetically altered Tregs is decreased. By further elucidating how Wnt/β-catenin signaling mediates the pathogenic conversion of Tregs, we hope to reveal novel targets for cancer immuno-therapy.

O-GlcNAc Regulates CD4+ T Cell Differentiation

Abstract Chronic inflammation is a feature of obesity and enhances the risk of heart disease, cancer, and diabetes. Specifically, pro-inflammatory TH1 and TH17 CD4+ effector T cells are increased in metabolic diseases. However, a clear molecular mechanism linking metabolic changes with pro-inflammatory T cells is lacking. We hypothesize that elevated levels of O-linked β-N-acetylglucosamine (O-GlcNAc), a post-translational modification (PTM) of nuclear and cytoplasmic proteins, promotes pro-inflammatory CD4+ T cell differentiation. Since production of O-GlcNAc involves input from multiple metabolic pathways, the PTM acts as a nutrient sensor. To investigate the role of O-GlcNAc in a setting of metabolic disease, we analyzed O-GlcNAc levels in CD4+ T cells in mice fed a high fat diet. CD4+ T cells from obese mice have elevated O-GlcNAc levels and are primed to secrete more IL-17A, the eponymous TH17 cytokine, and IFNγ, the signature TH1 cytokine. We also cultured mouse splenic CD4+ T cells with Thiamet-G (TMG), a selective inhibitor of the enzyme that removes O-GlcNAc. We find a significant increase in protein and transcript levels of IL-17A and IFNγ in TMG treated cells. Acetyl CoA carboxylase (ACC1), the rate limiting enzyme in fatty acid synthesis, is known to enhance generation of TH17 cells by producing ligands that increase the activity of RORγt, the transcription factor defining the TH17 lineage. We find that ACC1 is O-GlcNAcylated in CD4+ T cells. Collectively, our data suggest that elevated O-GlcNAc levels prime TH17 CD4+ T cell differentiation by altering ACC1 activity. Further study into how O-GlcNAcylation promotes pro-inflammatory T cell differentiation will provide insight into how nutritional excess exacerbates inflammation.

Unexpected STAT3-mediated mechanisms of regulation of effector Th17 function in autoimmunity

Abstract Th17 cells have been linked to the emergence of many autoimmune inflammatory conditions as well as defense against fungal pathogens. Early differentiation and expansion of Th17 cells relies on key cytokines that signal through STAT3, including IL-6 and IL-21. Later STAT3-dependent signals from IL-23 are critical for gain of inflammatory function by effector Th17 cells. Thus, development of drugs that target STAT3 or its upstream receptors are a major research focus to alleviate disease. However, the requirements for STAT3 signaling in effector Th17 cells remain elusive. Here, we developed a model to study the effect of STAT3 deficiency specifically in effector Th17 cells in EAE, an animal model of multiple sclerosis. Our data show that mice lacking STAT3 in effector Th17 cells are resistant to EAE development. Unexpectedly, we demonstrate that in Th17 cells, STAT3 is not required for maintenance of Th17 lineage commitment or Th17 cytokine production in vivo. To determine mechanisms by which STAT3 regulates Th17 effector cells to promote development of autoimmune inflammation, we performed bioinformatics analysis of the transcriptome of effector Th17 cells isolated from lymph nodes (LN) at day 10 (onset) of EAE. This revealed that STAT3 regulates expression of genes associated with particular cell-death pathways, and pathways associated with proliferation and T cell mediated tissue damage. Corresponding to these changes in gene expression, we found that STAT3 deficient effector Th17 cells were reduced in numbers in LN and target tissue during EAE. Our data reveal a hitherto unappreciated mechanism for STAT3 in regulating effector Th17 cells, which have major implications for use of STAT3-targeted therapies.

IL-17A inhibits expression of IL-17-lineage cytokines through a negative feedback loop involving IL-24 and controls autoimmune uveitis

Abstract The Th17 response has been associated with autoimmune diseases in patients and in animal models. IL-17A is recognized as the Th17 signature cytokine and IL-17A-producing T cells are pathogenic effectors in models of autoimmunity, including experimental autoimmune uveitis (EAU). Paradoxically, however, injection of IL-17 ameliorates EAU (PMID: 19234216). Using a model of spontaneous uveitis in IRBP T cell receptor transgenic R161H mice, we investigated their susceptibility on the IL-17A−/− background. Surprisingly, IL-17A−/− R161H mice developed essentially undiminished uveitis and IL-17−/− R161H T cells, polarized to Th17 and infused into wild type recipients, induced similar disease to IL-17A sufficient R161H T cells. Interestingly, IL-17A−/− R161H T cells polarized under Th17 conditions produced elevated amounts of other Th17-related cytokines, i.e. IL-17F, GM-CSF and IL-22. Supplementing these cultures with recombinant IL-17A normalized the elevated production of these cytokines. RNAseq analysis revealed that IL-17A−/− T cells expressed lower levels of IL-24 compared to their IL-17A sufficient counterparts. Mechanistic studies indicated a negative feedback loop where IL-17A induces Th17 cells to produce IL-24, which subsequently suppresses production of Th17 lineage cytokines. Finally, injection of recombinant IL-24 ameliorated adoptive Th17-induced EAU, and conversely, silencing IL-24 expression in Th17 cells increased their pathogenicity and enhanced disease severity. Our data suggest that: (a) IL-17A exerts a negative feedback on uveitogenic Th17 cells via IL-24 production, and (b) IL-24 limits the expression of other Th17-related cytokines and controls their pathogenicity.

Identification of a role for Casz1 in the control of T helper differentiation and inflammation

Abstract While T helper (Th) cells constitute a critical arm of adaptive immune system and are important for host defense, unregulated expansion and imbalance in Th effector subsets contribute to inflammatory disorders. Here we show that Casz1, whose function is previously unknown in CD4+ T cells, determines the balance between various Th subsets. As systemic knockout mice are embryonically lethal, we generated conditional CD4 Casz1 knockout mice. Conditional deletion of Casz1 in CD4+ T cells significantly inhibits Th17 cell differentiation, but promotes Th1 differentiation. Loss of Casz1 in CD4+ T cells lowers susceptibility to experimental autoimmune encephalomyelitis, consistent with the reduced frequency of Th17 cells, despite an increase in Th1 cells. These results underscore the critical role of Casz1 in determining Th17 lineage differentiation in vivo. Transcriptome analyses of Casz1 deficient CD4+ T cells show a signature consistent with defective Th17 differentiation but enhanced Th1 differentiation. Taken together, these data reveal Casz1 as a new T helper cell regulator having important clinical implications for autoimmune inflammation.

Regulatory action of multiple AP-1 transcription factors balances Th17 lineage stability and plasticity

Abstract During an immune response, naïve CD4+ T cells encounter their cognate antigen and are induced to differentiate into one of several distinct T helper (Th) subsets. Although previously regarded as lineage-committed, these differentiated subsets are now appreciated to exhibit a degree of plasticity, whereby certain environmental stimuli permit inter-subset conversion. In particular, Th17 cells have been demonstrated to exhibit a high degree of intrinsic plasticity and are capable of “reprogramming” towards phenotypes typical of both pro- and anti-inflammatory T cell subsets. Despite the suggested importance of this phenomenon in the etiology of autoimmune and inflammatory disorders, the mechanisms underlying Th17 cell plasticity are poorly understood. We have recently identified the AP-1 transcription factor family as a central controller of both Th17 cell stability and plasticity, with different AP-1 family members exhibiting both overlapping and unique control of distinct aspects of the Th17 transcriptional program. Using a combined strategy employing transcriptomics and global analysis of TF occupancy for multiple AP-1 family members, we have identified novel regulatory modules defined by distinct modes of AP-1-mediated activation. These findings provide new insights into the mechanism by which environmentally-regulated transcription factors collaborate for the maintenance of T cell identity.

MicroRNA-448 promotes multiple sclerosis development through induction of Th17 response through targeting protein tyrosine phosphatase non-receptor type 2 (PTPN2)

Multiple sclerosis (MS) is an immune-mediated demyelinating disease of the central nervous system, and its pathogenesis remains largely unclear. Much attention has been paid to the role of microRNAs (miRs) in regulation of autoimmune disease. Here, we found, for the first time, that miR-448 expression was significantly increased in periphery blood mononuclear cells (PBMC) and cerebrospinal fluid (CSF) of patients with MS, and its expression positively correlated with the disease severity. We further demonstrated that CD4+ T cells, especially the Th17 lineage, were the major source of miR-448 expression. Using gain- and loss-of-function approaches, we further verified that miR-448 could enhance Th17 differentiation, characterized by up-regulated expression levels of IL-17A and RORγt. Interleukin (IL)-1β as a potent driver of pathogenic Th17 cells was able to strongly induce miR-448 expression in CD4+ T cells through activating NF-κB pathway. Additionally, we identified that miR-448 directly targeted protein tyrosine phosphatase non-receptor type 2 (PTPN2), which has been known as an anti-inflammatory player with capacity to suppress Th17 differentiation. We also observed markedly decreased expression of PTPN2 in PBMC and CSF of MS patients. Our results suggest that miR-448 might promote Th17 differentiation in MS and thus aggravate the disease through inhibiting PTPN2.

Regulation of Gastric Carcinogenesis by InflammatoryCytokines.

Chronic inflammation caused by infection with Helicobacter pylori and autoimmune gastritis increases an individual’s risk of developing gastric cancer. More than 90% of gastric cancers are adenocarcinomas, which originate from epithelial cells in the chronically inflamed gastric mucosa. However, only a small subset of chronic gastritis patients develops gastric cancer, implying a role for genetic and environmental factors in cancer development. A number of DNA polymorphisms that increase gastric cancer risk have mapped to genes encoding cytokines. Many different cytokines secreted by immune cells and epithelial cells during chronic gastritis have been identified, but a better understanding of how cytokines regulate the severity of gastritis, epithelial cell changes, and neoplastic transformation is needed. This review summarizes studies in both human and mouse models, describing a number of different findings that implicate various cytokines in regulating the development of gastric cancer.

Th22 Cells Form a Distinct Th Lineage from Th17 Cells In Vitro with Unique Transcriptional Properties and Tbet-Dependent Th1 Plasticity.

Th22 cells are a major source of IL-22 and have been found at sites of infection and in a range of inflammatory diseases. However, their molecular characteristics and functional roles remain largely unknown because of our inability to generate and isolate pure populations. We developed a novel Th22 differentiation assay and generated dual IL-22/IL-17A reporter mice to isolate and compare pure populations of cultured Th22 and Th17 cells. Il17a fate-mapping and transcriptional profiling provide evidence that these Th22 cells have never expressed IL-17A, suggesting that they are potentially a distinct cell lineage from Th17 cells under in vitro culture conditions. Interestingly, Th22 cells also expressed granzymes, IL-13, and increased levels of Tbet. Using transcription factor-deficient cells, we demonstrate that RORγt and Tbet act as positive and negative regulators of Th22 differentiation, respectively. Furthermore, under Th1 culture conditions in vitro, as well as in an IFN-γ-rich inflammatory environment in vivo, Th22 cells displayed marked plasticity toward IFN-γ production. Th22 cells also displayed plasticity under Th2 conditions in vitro by upregulating IL-13 expression. Our work has identified conditions to generate and characterize Th22 cells in vitro. Further, it provides evidence that Th22 cells develop independently of the Th17 lineage, while demonstrating plasticity toward both Th1- and Th2-type cells.

Adenovirus Serotype 5 Vaccination Results in Suboptimal CD4 T Helper 1 Responses in Mice.

Adenovirus serotype 5 (Ad5) is one of the most widely used viral vectors and is known to generate potent T cell responses. While many previous studies have characterized Ad5-induced CD8 T cell responses, there is a relative lack of detailed studies that have analyzed CD4 T cells elicited by Ad5 vaccination. Here, we immunized mice with Ad5 vectors encoding lymphocytic choriomeningitis virus (LCMV) glycoprotein (GP) and examined GP-specific CD4 T cell responses elicited by Ad5 vectors and compared them to those induced by an acute LCMV infection. In contrast to LCMV infection, where balanced CD4 T helper 1 (Th1) and T follicular helper (Tfh) responses were induced, Ad5 immunization resulted in a significantly reduced frequency of Th1 cells. CD4 T cells elicited by Ad5 vectors expressed decreased levels of Th1 markers, such as Tim3, SLAM, T-bet, and Ly6C, had smaller amounts of cytotoxic molecules like granzyme B, and produced less interferon gamma than CD4 T cells induced by LCMV infection. This defective CD4 Th1 response appeared to be intrinsic for Ad5 vectors and not a reflection of comparing a nonreplicating vector to a live viral infection, since immunization with a DNA vector expressing LCMV-GP generated efficient CD4 Th1 responses. Analysis at early time points (day 3 or 4) after immunization with Ad5 vectors revealed a defect in the expression of CD25 (interleukin-2 [IL-2] receptor alpha chain) on Ad5-elicited CD4 T cells, and administration of exogenous IL-2 following Ad5 immunization partially restored CD4 Th1 responses. These results suggest that impairment of Th1 commitment after Ad5 immunization could be due to reduced IL-2-mediated signaling.IMPORTANCE During viral infection, generating balanced responses of Th1 and Tfh cells is important to induce effective cell-mediated responses and provide optimal help for antibody responses. In this study, to investigate vaccine-induced CD4 T cell responses, we characterized CD4 T cells after immunization with Ad5 vectors expressing LCMV-GP in mice. Ad5 vectors led to altered effector differentiation of LCMV GP-specific CD4 T cells compared to that during LCMV infection. CD4 T cells following Ad5 immunization exhibited impaired Th1 lineage commitment, generating significantly decreased Th1 responses than those induced by LCMV infection. Our results suggest that suboptimal IL-2 signaling possibly plays a role in reduced Th1 development following Ad5 immunization.

Genetic variants alter T-bet binding and gene expression in mucosal inflammatory disease.

The polarization of CD4+ T cells into distinct T helper cell lineages is essential for protective immunity against infection, but aberrant T cell polarization can cause autoimmunity. The transcription factor T-bet (TBX21) specifies the Th1 lineage and represses alternative T cell fates. Genome-wide association studies have identified single nucleotide polymorphisms (SNPs) that may be causative for autoimmune diseases. The majority of these polymorphisms are located within non-coding distal regulatory elements. It is considered that these genetic variants contribute to disease by altering the binding of regulatory proteins and thus gene expression, but whether these variants alter the binding of lineage-specifying transcription factors has not been determined. Here, we show that SNPs associated with the mucosal inflammatory diseases Crohn’s disease, ulcerative colitis (UC) and celiac disease, but not rheumatoid arthritis or psoriasis, are enriched at T-bet binding sites. Furthermore, we identify disease-associated variants that alter T-bet binding in vitro and in vivo. ChIP-seq for T-bet in individuals heterozygous for the celiac disease-associated SNPs rs1465321 and rs2058622 and the IBD-associated SNPs rs1551398 and rs1551399, reveals decreased binding to the minor disease-associated alleles. Furthermore, we show that rs1465321 is an expression quantitative trait locus (eQTL) for the neighboring gene IL18RAP, with decreased T-bet binding associated with decreased expression of this gene. These results suggest that genetic polymorphisms may predispose individuals to mucosal autoimmune disease through alterations in T-bet binding. Other disease-associated variants may similarly act by modulating the binding of lineage-specifying transcription factors in a tissue-selective and disease-specific manner.

Ndfip1 restricts Th17 cell potency by limiting lineage stability and proinflammatory cytokine production

While Th17 cells can protect against colonization by pathogenic organisms, they also have the potential to become pathogenic and promote autoimmune and inflammatory diseases. Mechanisms that control their pathogenic potential remain poorly understood. Here we show that Ndfip1, a co-activator of the E3 ubiquitin ligase Itch, restricts the frequency and pathogenicity of Th17 cells. Mice lacking Ndfip1 have increased numbers of Th17 cells, and this increase is cell intrinsic. We found that Ndfip1 restricts production of the proinflammatory cytokines in Th17 cells. Increased cytokine production correlated with reduced degradation and accumulation of RORγT. When transferred in vivo, Th17 cells lacking Ndfip1 were more likely to maintain their ability to make IL-17, were more potent proinflammatory cytokine producers, and were powerful inducers of colitis. Together our data support an essential role for Ndfip1 in degrading RORγT and suppressing Th17 lineage stability, proinflammatory cytokine production, and pathogenicity.

Impairment of the Intrinsic Capability of Th1 Polarization in Irradiated Mice: A Close Look at the Imbalanced Th1/Th2 Response after Irradiation.

Two major CD4+ T-helper (Th) lineages are Th1 and Th2, and well balanced Th1/Th2 responses are essential for immune function. In previously published studies, it was reported that radiation induces a Th1/Th2 immune imbalance toward a Th2-dominant direction, and this imbalance may contribute to postirradiation immune dysfunction. The polarization of Th cells is driven by the cytokine milieu and controlled by intracellular regulatory pathways that respond to cytokine signaling. It is widely accepted that radiation induces cytokine aberration, however, the precise alterations of cytokines in various tissue environments have been difficult to evaluate. In addition, the effects of radiation on the intrinsic functions of Th cells remain uncharacterized. Therefore, how radiation affects Th1/Th2 balance remains somewhat unclear. To address this, we investigated the changes in the polarization capability of Th cells by isolating them from mice previously exposed to radiation and assessing the cells in an established in vitro Th polarization system. Our novel results demonstrate that prior exposure to radiation led to the persistent aberration of the inherent capability of Th cells to differentiate into Th1 and Th2 lineages. The parallel changes in expression of Th1-specific master transcription factors and the key genes in metabolic reprograming indicated that radiation affects the core components in Th1 polarization. While Th1 differentiation was impaired after irradiation, little adverse effect was observed in Th2 differentiation; both of these findings contribute to the known phenotypes of Th1/Th2 imbalance caused by radiation.

Atherosclerosis-Driven Treg Plasticity Results in Formation of a Dysfunctional Subset of Plastic IFNγ+ Th1/Tregs.

Forkhead box P3+ T regulatory cells (Tregs) are key players in maintaining immune homeostasis. Evidence suggests that Tregs respond to environmental cues to permit or suppress inflammation. In atherosclerosis, Th1-driven inflammation affects Treg homeostasis, but the mechanisms governing this phenomenon are unclear. Here, we address whether atherosclerosis impacts Treg plasticity and functionality in Apoe-/- mice, and what effect Treg plasticity might have on the pathology of atherosclerosis. We demonstrate that atherosclerosis promotes Treg plasticity, resulting in the reduction of CXCR3+ Tregs and the accumulation of an intermediate Th1-like interferon (IFN)-γ+CCR5+ Treg subset (Th1/Tregs) within the aorta. Importantly, Th1/Tregs arise in atherosclerosis from bona fide Tregs, rather than from T-effector cells. We show that Th1/Tregs recovered from atherosclerotic mice are dysfunctional in suppression assays. Using an adoptive transfer system and plasticity-prone Mir146a-/- Tregs, we demonstrate that elevated IFNγ+ Mir146a-/- Th1/Tregs are unable to adequately reduce atherosclerosis, arterial Th1, or macrophage content within Apoe-/- mice, in comparison to Mir146a+/+ Tregs. Finally, via single-cell RNA-sequencing and real-time -polymerase chain reaction, we show that Th1/Tregs possess a unique transcriptional phenotype characterized by coexpression of Treg and Th1 lineage genes and a downregulation of Treg-related genes, including Ikzf2, Ikzf4, Tigit, Lilrb4, and Il10. In addition, an ingenuity pathway analysis further implicates IFNγ, IFNα, interleukin-2, interleukin-7, CTLA-4 (cytotoxic T-lymphocyte-associated protein 4), T-cell receptor, and Csnk2b-related pathways in regulating Treg plasticity. Atherosclerosis drives Treg plasticity, resulting in the accumulation of dysfunctional IFNγ+ Th1/Tregs that may permit further arterial inflammation and atherogenesis.

T Helper 1 (Th1), Th2, and Th17 Responses to Leishmania major Lipophosphoglycan 3

ABSTRACTLeishmania major is the main causal agent of cutaneous leishmaniasis (CL) that remains a serious public health concern in many tropical and subtropical countries. A long-lasting protective vaccine against leishmaniasis remains as a medical unmet need. Lipophosphoglycan 3 (LPG3) is one of the class II LPG genes from HSP90 family involved in the host immune responses. The aim of the present study is to investigate the capability of recombinant LPG3 (rLPG3) to induce Th1, Th2, Th17 responses. The results showed that rLPG3 in moderate and high concentrations significantly induced expression of Th1 lineage-specific transcription factor (T-bet) and cytokine (IFN-γ)(P < 0.05). Moreover, the Th1-stimulating effect of rLPG3 was confirmed by significant induction of IFN-γ secretion from treated T cells (P < 0.01). However, no significant effect of rLPG3 on Th2 and Th17 lineage cells was observed even in high concentration. Our findings demonstrate that rLPG3 induces Th1, but not Th2 and Th17, lineage responses. Further studies are needed to investigate adjuvant properties of rLPG3 for leishmania therapy.

Generation of a Novel T Cell Specific Interleukin-1 Receptor Type 1 Conditional Knock Out Mouse Reveals Intrinsic Defects in Survival, Expansion and Cytokine Production of CD4 T Cells.

Interleukin-1 (IL-1) plays a crucial role in numerous inflammatory diseases via action on its only known signaling IL-1 receptor type 1 (IL-1R1). To investigate the role of IL-1 signaling in selected cell types, we generated a new mouse strain in which exon 5 of the Il1r1 gene is flanked by loxP sites. Crossing of these mice with CD4-Cre transgenic mice resulted in IL-1R1 loss of function specifically in T cells. These mice, termed IL-1R1ΔT, displayed normal development under steady state conditions. Importantly, isolated CD4 positive T cells retained their capacity to differentiate toward Th1 or Th17 cell lineages in vitro, and strongly proliferated in cultures supplemented with either anti-CD3/CD28 or Concanavalin A, but, as predicted, were completely unresponsive to IL-1β administration. Furthermore, IL-1R1ΔT mice were protected from gut inflammation in the anti-CD3 treatment model, due to dramatically reduced frequencies and absolute numbers of IL-17A and interferon (IFN)-γ producing cells. Taken together, our data shows the necessity of intact IL-1 signaling for survival and expansion of CD4 T cells that were developed in an otherwise IL-1 sufficient environment.