The transcription factor E4bp4 (Nfil3) is essential for natural killer (NK) cell production. Here, we show that E4bp4 is required at the NK lineage commitment point when NK progenitors develop from common lymphoid progenitors (CLPs) and that E4bp4 must be expressed at the CLP stage for differentiation toward the NK lineage to occur. To elucidate the mechanism by which E4bp4 promotes NK development, we identified a central core of transcription factors that can rescue NK production from E4bp4(-/-) progenitors, suggesting that they act downstream of E4bp4. Among these were Eomes and Id2, which are expressed later in development than E4bp4. E4bp4 binds directly to the regulatory regions of both Eomes and Id2, promoting their transcription. We propose that E4bp4 is required for commitment to the NK lineage and promotes NK development by directly regulating the expression of the downstream transcription factors Eomes and Id2.
Sepsis is a heterogeneous illness characterised by inflammation secondary to suspected or proven infection. A clinical and research challenge in this area is the ability to diagnose true sepsis, defined as inflammation secondary to infection. Infection is often indirectly confirmed using surrogates, whilst awaiting microbiological confirmation. microRNAs are novel molecules with a potential to be point of care rapid diagnostic test for true sepsis.
Obesity-associated insulin resistance is accompanied by an alteration in the Th1/Th2 balance in adipose tissue. T-bet (Tbx21) is an immune cell transcription factor originally described as the master regulator of Th1 cell development, although is now recognized to have a role in both the adaptive and innate immune systems. T-bet also directs T-cell homing to pro-inflammatory sites by the regulation of CXCR3 expression. T-bet(-/-) mice have increased visceral adiposity but are more insulin-sensitive, exhibiting reduced immune cell content and cytokine secretion specifically in the visceral fat depot, perhaps due to altered T-cell trafficking. Studies of T-bet deficiency on Rag2-- and IFN-?-deficient backgrounds indicate the importance of CD4(+) T cells and IFN-? in this model. This favorable metabolic phenotype, uncoupling adiposity from insulin resistance, is present in young lean mice yet persists with age and increasing obesity. We suggest a novel role for T-bet in metabolic regulation.
The complex relationship between Th1 and Th17 cells is incompletely understood. The transcription factor T-bet is best known as the master regulator of Th1 lineage commitment. However, attention is now focused on the repression of alternate T cell subsets mediated by T-bet, particularly the Th17 lineage. It has recently been suggested that pathogenic Th17 cells express T-bet and are dependent on IL-23. However, T-bet has previously been shown to be a negative regulator of Th17 cells. We have taken an unbiased approach to determine the functional impact of T-bet on Th17 lineage commitment. Genome-wide analysis of functional T-bet binding sites provides an improved understanding of the transcriptional regulation mediated by T-bet, and suggests novel mechanisms by which T-bet regulates Th cell differentiation. Specifically, we show that T-bet negatively regulates Th17 lineage commitment via direct repression of the transcription factor IFN regulatory factor-4 (IRF4). An in vivo analysis of the pathogenicity of T-bet-deficient T cells demonstrated that mucosal Th17 responses were augmented in the absence of T-bet, and we have demonstrated that the roles of T-bet in enforcing Th1 responses and suppressing Th17 responses are separable. The interplay of the two key transcription factors T-bet and IRF4 during the determination of T cell fate choice significantly advances our understanding of the mechanisms underlying the development of pathogenic T cells.
Originally described over a decade ago as a T cell transcription factor regulating T helper 1 cell lineage commitment, T-bet is now recognized as having an important role in many cells of the adaptive and innate immune system. T-bet has a fundamental role in coordinating type 1 immune responses by controlling a network of genetic programmes that regulate the development of certain immune cells and the effector functions of others. Many of these transcriptional networks are conserved across innate and adaptive immune cells and these shared mechanisms highlight the biological functions that are regulated by T-bet.
Immunotherapy with biological agents or small molecules is revolutionising the treatment of chronic inflammatory disease in humans; however, a significant proportion of patients fail to respond or lose responsiveness. This is particularly evident in inflammatory bowel disease (IBD), a group of chronic, immune-mediated disorders of the gastrointestinal tract. Different responsiveness to treatment in IBD can be explained by substantial disease heterogeneity, which is being increasingly recognised by genetic and immunological studies. The current enthusiasm for stratified medicine suggests that it may become possible to identify clinical, immunological, biochemical or genetic biomarkers to target immunotherapy to patients more likely to respond. Here, we identify and highlight the opportunities and the challenges of this strategy in the context of IBD.
Cell-based therapy with natural (CD4(+)CD25(hi)CD127(lo)) regulatory T cells to induce transplant tolerance is now technically feasible. However, regulatory T cells from hemodialysis patients awaiting transplantation may be functionally/numerically defective. Human regulatory T cells are also heterogeneous, and some are able to convert to proinflammatory Th17 cells. This study addresses the suitability of regulatory T cells from hemodialysis patients for cell-based therapy in preparation for the first clinical trials in renal transplant recipients (the ONE Study).
It has been 20 years since the first description of a rapidly progressive renal disease that is associated with the consumption of Chinese herbs containing aristolochic acid (AA) and is now termed aristolochic acid nephropathy (AAN). Recent data have shown that AA is also the primary causative agent in Balkan endemic nephropathy and associated urothelial cancer. Aristolochic acid nephropathy is associated with a high long-term risk for renal failure and urothelial cancer, and the potential worldwide population exposure is enormous. This evidence-based review of the diagnostic approach to and management of AAN draws on the authors experience with the largest and longest-studied combined cohort of patients with this condition. It is hoped that a better understanding of the importance of this underrecognized and severe condition will improve epidemiologic, preventive, and therapeutic strategies to reduce the global burden of this disease.
Low-grade inflammation in fat is associated with insulin resistance, although the mechanisms are unclear. We report that mice deficient in the immune cell transcription factor T-bet have lower energy expenditure and increased visceral fat compared with wild-type mice, yet paradoxically are more insulin sensitive. This striking phenotype, present in young T-bet(-/-) mice, persisted with high-fat diet and increasing host age and was associated with altered immune cell numbers and cytokine secretion specifically in visceral adipose tissue. However, the favorable metabolic phenotype observed in T-bet-deficient hosts was lost in T-bet(-/-) mice also lacking adaptive immunity (T-bet(-/-)xRag2(-/-)), demonstrating that T-bet expression in the adaptive rather than the innate immune system impacts host glucose homeostasis. Indeed, adoptive transfer of T-bet-deficient, but not wild-type, CD4(+) T cells to Rag2(-/-) mice improved insulin sensitivity. Our results reveal a role for T-bet in metabolic physiology and obesity-associated insulin resistance.
Sepsis is a common cause of death in the intensive care unit with mortality up to 70% when accompanied by multiple organ dysfunction. Rapid diagnosis and the institution of appropriate antibiotic therapy and pressor support are therefore critical for survival. MicroRNAs are small non-coding RNAs that play an important role in the regulation of numerous cellular processes, including inflammation and immunity.
Discovery of novel immune biomarkers for monitoring of disease prognosis and response to therapy in immune-mediated inflammatory diseases is an important unmet clinical need. Here, we establish a novel framework for immunological biomarker discovery, comparing a conventional (liquid) flow cytometry platform (CFP) and a unique lyoplate-based flow cytometry platform (LFP) in combination with advanced computational data analysis. We demonstrate that LFP had higher sensitivity compared to CFP, with increased detection of cytokines (IFN-? and IL-10) and activation markers (Foxp3 and CD25). Fluorescent intensity of cells stained with lyophilized antibodies was increased compared to cells stained with liquid antibodies. LFP, using a plate loader, allowed medium-throughput processing of samples with comparable intra- and inter-assay variability between platforms. Automated computational analysis identified novel immunophenotypes that were not detected with manual analysis. Our results establish a new flow cytometry platform for standardized and rapid immunological biomarker discovery with wide application to immune-mediated diseases.
Gene expression can be modulated depending on physiological and developmental requirements. A multitude of regulatory genes, which are organized in interdependent networks, guide development and eventually generate specific phenotypes. Transcription factors (TF) are a key element in the regulatory cascade controlling cell fate and effector functions. In this review, we discuss recent data on the diversity of TF that determine natural killer (NK) cell fate and NK cell function.
Heterozygous germline mutations of BMPR2 contribute to familial clustering of pulmonary arterial hypertension (PAH). To further explore the genetic basis of PAH in isolated cases, we undertook a candidate gene analysis to identify potentially deleterious variation. Members of the bone morphogenetic protein (BMP) pathway, namely SMAD1, SMAD4, SMAD5, and SMAD9, were screened by direct sequencing for gene defects. Four variants were identified in SMADs 1, 4, and 9 among a cohort of 324 PAH cases, each not detected in a substantial control population. Of three amino acid substitutions identified, two demonstrated reduced signaling activity in vitro. A putative splice site mutation in SMAD4 resulted in moderate transcript loss due to compromised splicing efficiency. These results demonstrate the role of BMPR2 mutation in the pathogenesis of PAH and indicate that variation within the SMAD family represents an infrequent cause of the disease.
Asymmetric cell division is an important mechanism for generating cellular diversity, however, techniques for measuring the distribution of fate-regulating molecules during mitosis have been hampered by a lack of objectivity, quantitation, and statistical robustness. Here we describe a novel imaging flow cytometric approach that is able to report a cells proliferative history and cell cycle position using dye dilution, pH3, and PI staining to then measure the spatial distribution of fluorescent signals during mitosis using CCD-derived imagery. Using Jurkat cells, resolution of the fluorescently labeled populations was comparable to traditional PMT based cytometers thus eliminating the need to sort cells with specific division histories for microscopy. Subdividing mitotic stages by morphology allowed us to determine the time spent in each cell cycle phase using mathematical modeling approaches. Furthermore high sample throughput allowed us to collect statistically relevant numbers of cells without the need to use blocking agents that artificially enrich for mitotic events. The fluorescent imagery was used to measure PKC? protein and EEA-1+ endosome distribution during different mitotic phases in Jurkat cells. While telophase cells represented the favorable population for measuring asymmetry, asynchronously dividing cells spent approximately 43 seconds in this stage, explaining why they were present at such low frequencies. This necessitated the acquisition of large cell numbers. Interestingly we found that PKC? was inherited asymmetrically in 2.5% of all telophasic events whereas endosome inheritance was significantly more symmetrical. Furthermore, molecular polarity at early mitotic phases was a poor indicator of asymmetry during telophase highlighting that, though rare, telophasic events represented the best candidates for asymmetry studies. In summary, this technique combines the spatial information afforded by fluorescence microscopy with the statistical wealth and objectivity of traditional flow cytometry, overcoming the key limitations of existing approaches for studying asymmetry during mitosis.
Natural killer (NK) cells play a major role in immunologic surveillance of cancer. Whether NK-cell subsets have specific roles during antitumor responses and what the signals are that drive their terminal maturation remain unclear. Using an in vivo model of tumor immunity, we show here that CD11b(hi)CD27(low) NK cells migrate to the tumor site to reject major histocompatibility complex class I negative tumors, a response that is severely impaired in Txb21(-/-) mice. The phenotypical analysis of Txb21-deficient mice shows that, in the absence of Txb21, NK-cell differentiation is arrested specifically at the CD11b(hi)CD27(hi) stage, resulting in the complete absence of terminally differentiated CD11b(hi)CD27(low) NK cells. Adoptive transfer experiments and radiation bone marrow chimera reveal that a Txb21(+/+) environment rescues the CD11b(hi)CD27(hi) to CD11b(hi)CD27(low) transition of Txb21(-/-) NK cells. Furthermore, in vivo depletion of myeloid cells and in vitro coculture experiments demonstrate that spleen monocytes mediate the terminal differentiation of peripheral NK cells in a Txb21- and IL-15R?-dependent manner. Together, these data reveal a novel, unrecognized role for Txb21 expression in monocytes in promoting NK-cell development and help appreciate how various NK-cell subsets are generated and participate in antitumor immunity.
It is uncertain whether NK cells modulate T cell memory differentiation. By using a genetic model that allows the selective depletion of NK cells, we show in this study that NK cells shape CD8(+) T cell fate by killing recently activated CD8(+) T cells in an NKG2D- and perforin-dependent manner. In the absence of NK cells, the differentiation of CD8(+) T cells is strongly biased toward a central memory T cell phenotype. Although, on a per-cell basis, memory CD8(+) T cells generated in the presence or the absence of NK cells have similar functional features and recall capabilities, NK cell deletion resulted in a significantly higher number of memory Ag-specific CD8(+) T cells, leading to more effective control of tumors carrying model Ags. The enhanced memory responses induced by the transient deletion of NK cells may provide a rational basis for the design of new vaccination strategies.
In the United Kingdom, many foundations and institutions and the government have made substantial investments in translational research. We examine the structures that surround this support and consider some of the results of this prodigious push toward enhancing translational research pursuits and thus improved clinical medicine.
In this study we demonstrate a new form of immunoregulation: engagement on CD4(+) T cells of the complement regulator CD46 promoted the effector potential of T helper type 1 cells (T(H)1 cells), but as interleukin 2 (IL-2) accumulated, it switched cells toward a regulatory phenotype, attenuating IL-2 production via the transcriptional regulator ICER/CREM and upregulating IL-10 after interaction of the CD46 tail with the serine-threonine kinase SPAK. Activated CD4(+) T cells produced CD46 ligands, and blocking CD46 inhibited IL-10 production. Furthermore, CD4(+) T cells in rheumatoid arthritis failed to switch, consequently producing excessive interferon-gamma (IFN-gamma). Finally, gammadelta T cells, which rarely produce IL-10, expressed an alternative CD46 isoform and were unable to switch. Nonetheless, coengagement of T cell antigen receptor (TCR) gammadelta and CD46 suppressed effector cytokine production, establishing that CD46 uses distinct mechanisms to regulate different T cell subsets during an immune response.
Identifying transplant recipients in whom immunological tolerance is established or is developing would allow an individually tailored approach to their posttransplantation management. In this study, we aimed to develop reliable and reproducible in vitro assays capable of detecting tolerance in renal transplant recipients. Several biomarkers and bioassays were screened on a training set that included 11 operationally tolerant renal transplant recipients, recipient groups following different immunosuppressive regimes, recipients undergoing chronic rejection, and healthy controls. Highly predictive assays were repeated on an independent test set that included 24 tolerant renal transplant recipients. Tolerant patients displayed an expansion of peripheral blood B and NK lymphocytes, fewer activated CD4+ T cells, a lack of donor-specific antibodies, donor-specific hyporesponsiveness of CD4+ T cells, and a high ratio of forkhead box P3 to alpha-1,2-mannosidase gene expression. Microarray analysis further revealed in tolerant recipients a bias toward differential expression of B cell-related genes and their associated molecular pathways. By combining these indices of tolerance as a cross-platform biomarker signature, we were able to identify tolerant recipients in both the training set and the test set. This study provides an immunological profile of the tolerant state that, with further validation, should inform and shape drug-weaning protocols in renal transplant recipients.
Using genome-wide association, we identify common variants at 2p12-p13, 6q26, 17q23 and 19q13 associated with serum creatinine, a marker of kidney function (P = 10(-10) to 10(-15)). Of these, rs10206899 (near NAT8, 2p12-p13) and rs4805834 (near SLC7A9, 19q13) were also associated with chronic kidney disease (P = 5.0 x 10(-5) and P = 3.6 x 10(-4), respectively). Our findings provide insight into metabolic, solute and drug-transport pathways underlying susceptibility to chronic kidney disease.
T-cell immunoglobulin, mucin domain-3 (Tim-3) is a membrane protein expressed at late stages of IFN-gamma secreting CD4(+) Th1 cell differentiation and constitutively on DC. Ligation of Tim-3 on Th1 cells terminates Th1 immune responses. In addition, Tim-3 plays a role in tolerance induction, although the mechanism by which this is accomplished has yet to be elucidated. While it is clear that Tim-3 plays an important role in the immune system, little is known regarding the molecular pathways that regulate Tim-3 expression. In the current study, we examine the role of Th1-associated transcription factors in regulating Tim-3 expression. Our experiments reveal that Tim-3 expression is regulated by the Th1-specific transcription factor T-bet. This introduces a novel paradigm into the generation of a Th1 response, whereby a transcription factor responsible for effector Th1 cell differentiation also increases the expression of a specific counter-regulatory molecule to ensure appropriate termination of pro-inflammatory Th1 immune responses.
Upon detection of antigen, CD4(+) T helper (Th) cells can differentiate into a number of effector types that tailor the immune response to different pathogens. Alternative Th1 and Th2 cell fates are specified by the transcription factors T-bet and GATA-3, respectively. Only a handful of target genes are known for these two factors and because of this, the mechanism through which T-bet and GATA-3 induce differentiation toward alternative cell fates is not fully understood. Here, we provide a genomic map of T-bet and GATA-3 binding in primary human T cells and identify their target genes, most of which are previously unknown. In Th1 cells, T-bet associates with genes of diverse function, including those with roles in transcriptional regulation, chemotaxis and adhesion. GATA-3 occupies genes in both Th1 and Th2 cells and, unexpectedly, shares a large proportion of targets with T-bet. Re-complementation of T-bet alters the expression of these genes in a manner that mirrors their differential expression between Th1 and Th2 lineages. These data show that the choice between Th1 and Th2 lineage commitment is the result of the opposing action of T-bet and GATA-3 at a shared set of target genes and may provide a general paradigm for the interaction of lineage-specifying transcription factors.
There is increasing evidence that activation of inflammatory responses in a variety of tissues is mediated co-operatively by the actions of more than one cell type. In particular, the monocyte has been implicated as a potentially important cell in the initiation of inflammatory responses to Toll-like receptor (TLR)-activating signals. To determine the potential for monocyte-regulated activation of tissue cells to underpin inflammatory responses in the vasculature, we established cocultures of primary human endothelial cells and monocytes and dissected the inflammatory responses of these systems following activation with TLR agonists. We observed that effective activation of inflammatory responses required bidirectional signalling between the monocyte and the tissue cell. Activation of cocultures was dependent on interleukin-1 (IL-1). Although monocyte-mediated IL-1beta production was crucial to the activation of cocultures, TLR specificity to these responses was also provided by the endothelial cells, which served to regulate the signalling of the monocytes. TLR4-induced IL-1beta production by monocytes was increased by TLR4-dependent endothelial activation in coculture, and was associated with increased monocyte CD14 expression. Activation of this inflammatory network also supported the potential for downstream monocyte-dependent T helper type 17 activation. These data define co-operative networks regulating inflammatory responses to TLR agonists, identify points amenable to targeting for the amelioration of vascular inflammation, and offer the potential to modify atherosclerotic plaque instability after a severe infection.
Despite advances in immunosuppression, allograft rejection remains a significant challenge to the long-term success of solid-organ transplantation. Whilst allorecognition pathways are clearly central to rejection, the effector mechanisms of this process are less defined. T helper (Th) type 17 cells are a recently described CD4 T-cell subset, and have been implicated in a range of autoimmune and inflammatory conditions that were previously thought to be Th1 mediated. In light of these developments, this review examines the relative roles of these subsets in allograft rejection.
Th17 cells are a recently defined subset of proinflammatory T cells that contribute to pathogen clearance and tissue inflammation by means of the production of their signature cytokine IL-17A (henceforth termed IL-17). Although the in vitro requirements for human Th17 development are reasonably well established, it is less clear what their in vivo requirements are. Here, we show that the production of IL-17 by human Th17 cells critically depends on both the activation status and the anatomical location of accessory cells. In vivo activated CD14+ monocytes were derived from the inflamed joints of patients with active rheumatoid arthritis (RA). These cells were found to spontaneously and specifically promote Th17, but not Th1 or Th2 responses, compared with resting CD14+ monocytes from the blood. Surprisingly, unlike Th17 stimulation by monocytes that were in vitro activated with lipopolysaccharide, intracellular IL-17 expression was induced by in vivo activated monocytes in a TNF-alpha- and IL-1beta-independent fashion. No role for IL-6 or IL-23 production by either in vitro or in vivo activated monocytes was found. Instead, in vivo activated monocytes promoted Th17 responses in a cell-contact dependent manner. We propose that, in humans, newly recruited memory CD4(+) T cells can be induced to produce IL-17 in nonlymphoid inflamed tissue after cell-cell interactions with activated monocytes. Our data also suggest that different pathways may be utilized for the generation of Th17 responses in situ depending on the site or route of accessory cell activation.
We previously described a mouse model of ulcerative colitis linked to T-bet deficiency in the innate immune system. Here, we report that the majority of T-bet(-/-)RAG2(-/-) ulcerative colitis (TRUC) mice spontaneously progress to colonic dysplasia and rectal adenocarcinoma solely as a consequence of MyD88-independent intestinal inflammation. Dendritic cells (DCs) are necessary cellular effectors for a proinflammatory program that is carcinogenic. Whereas these malignancies arise in the setting of a complex inflammatory environment, restoration of T-bet selectively in DCs was sufficient to reduce colonic inflammation and prevent the development of neoplasia. TRUC colitis-associated colorectal cancer resembles the human disease and provides ample opportunity to probe how inflammation drives colorectal cancer development and to test preventative and therapeutic strategies preclinically.
T-bet and GATA3 regulate the CD4+ T cell Th1/Th2 cell fate decision but little is known about the interplay between these factors outside of the murine Ifng and Il4/Il5/Il13 loci. Here we show that T-bet and GATA3 bind to multiple distal sites at immune regulatory genes in human effector T cells. These sites display markers of functional elements, act as enhancers in reporter assays and are associated with a requirement for T-bet and GATA3. Furthermore, we demonstrate that both factors bind distal sites at Tbx21 and that T-bet directly activates its own expression. We also show that in Th1 cells, GATA3 is distributed away from Th2 genes, instead occupying T-bet binding sites at Th1 genes, and that T-bet is sufficient to induce GATA3 binding at these sites. We propose these aspects of T-bet and GATA3 function are important for Th1/Th2 differentiation and for understanding transcription factor interactions in other T cell lineage decisions.
Mice lacking the transcription factor T-bet in the innate immune system develop microbiota-dependent colitis. Here, we show that interleukin-17A (IL-17A)-producing IL-7R?(+) innate lymphoid cells (ILCs) were potent promoters of disease in Tbx21(-/-)Rag2(-/-) ulcerative colitis (TRUC) mice. TNF-? produced by CD103(-)CD11b(+) dendritic cells synergized with IL-23 to drive IL-17A production by ILCs, demonstrating a previously unrecognized layer of cellular crosstalk between dendritic cells and ILCs. We have identified Helicobacter typhlonius as a key disease trigger driving excess TNF-? production and promoting colitis in TRUC mice. Crucially, T-bet also suppressed the expression of IL-7R, a key molecule involved in controlling intestinal ILC homeostasis. The importance of IL-7R signaling in TRUC disease was highlighted by the dramatic reduction in intestinal ILCs and attenuated colitis following IL-7R blockade. Taken together, these data demonstrate the mechanism by which T-bet regulates the complex interplay between mucosal dendritic cells, ILCs, and the intestinal microbiota.
Regulatory T cells (CD4(+)CD25(hi)CD127(lo)FOXP3(+) T cells [Tregs]) are a population of lymphocytes involved in the maintenance of self-tolerance. Abnormalities in function or number of Tregs are a feature of autoimmune diseases in humans. The ability to expand functional Tregs ex vivo makes them ideal candidates for autologous cell therapy to treat human autoimmune diseases and to induce tolerance to transplants. Current tests of Treg function typically take up to 120 hours, a kinetic disadvantage as clinical trials of Tregs will be critically dependent on the availability of rapid diagnostic tests before infusion into humans. Here we evaluate a 7-hour flow cytometric assay for assessing Treg function, using suppression of the activation markers CD69 and CD154 on responder T cells (CD4(+)CD25(-) [Tresp]), compared with traditional assays involving inhibition of CFSE dilution and cytokine production. In both freshly isolated and ex vivo expanded Tregs, we describe excellent correlation with gold standard suppressor cell assays. We propose that the kinetic advantage of the new assay may place it as the preferred rapid diagnostic test for the evaluation of Treg function in forthcoming clinical trials of cell therapy, enabling the translation of the large body of preclinical data into potentially useful treatments for human diseases.
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