Activated ß-catenin in Foxp3+ regulatory T cells links inflammatory environments to autoimmunity.

Foxp3+ regulatory T cells (Treg cells) are the central component of peripheral immune tolerance. Whereas a dysregulated Treg cytokine signature has been observed in autoimmune diseases, the regulatory mechanisms underlying pro- and anti-inflammatory cytokine production are elusive. Here, we identify an imbalance between the cytokines IFN-γ and IL-10 as a shared Treg signature present in patients with multiple sclerosis and under high-salt conditions. RNA-sequencing analysis on human Treg subpopulations revealed ß-catenin as a key regulator of IFN-γ and IL-10 expression. The activated ß-catenin signature was enriched in human IFN-γ+ Treg cells, as confirmed in vivo with Treg-specific ß-catenin-stabilized mice exhibiting lethal autoimmunity with a dysfunctional Treg phenotype. Moreover, we identified prostaglandin E receptor 2 (PTGER2) as a regulator of IFN-γ and IL-10 production under a high-salt environment, with skewed activation of the ß-catenin-SGK1-Foxo axis. Our findings reveal a novel PTGER2-ß-catenin loop in Treg cells linking environmental high-salt conditions to autoimmunity.

Impaired ketogenesis ties metabolism to T cell dysfunction in COVID-19.

Anorexia and fasting are host adaptations to acute infection, and induce a metabolic switch towards ketogenesis and the production of ketone bodies, including ß-hydroxybutyrate (BHB)1-6. However, whether ketogenesis metabolically influences the immune response in pulmonary infections remains unclear. Here we show that the production of BHB is impaired in individuals with SARS-CoV-2-induced acute respiratory distress syndrome (ARDS) but not in those with  influenza-induced ARDS. We found that BHB promotes both the survival of and the production of interferon-γ by CD4+ T cells. Applying a metabolic-tracing analysis, we established that BHB provides an alternative carbon source to fuel oxidative phosphorylation (OXPHOS) and the production of bioenergetic amino acids and glutathione, which is important for maintaining the redox balance. T cells from patients with SARS-CoV-2-induced ARDS were exhausted and skewed towards glycolysis, but could be metabolically reprogrammed by BHB to perform OXPHOS, thereby increasing their functionality. Finally, we show in mice that a ketogenic diet and the delivery of BHB as a ketone ester drink restores CD4+ T cell metabolism and function in severe respiratory infections, ultimately reducing the mortality of mice infected with SARS-CoV-2. Altogether, our data reveal that BHB is an alternative source of carbon that promotes T cell responses in pulmonary viral infections, and highlight impaired ketogenesis as a potential confounding factor in severe COVID-19.

NK cells link obesity-induced adipose stress to inflammation and insulin resistance.

An important cause of obesity-induced insulin resistance is chronic systemic inflammation originating in visceral adipose tissue (VAT). VAT inflammation is associated with the accumulation of proinflammatory macrophages in adipose tissue, but the immunological signals that trigger their accumulation remain unknown. We found that a phenotypically distinct population of tissue-resident natural killer (NK) cells represented a crucial link between obesity-induced adipose stress and VAT inflammation. Obesity drove the upregulation of ligands of the NK cell-activating receptor NCR1 on adipocytes; this stimulated NK cell proliferation and interferon-γ (IFN-γ) production, which in turn triggered the differentiation of proinflammatory macrophages and promoted insulin resistance. Deficiency of NK cells, NCR1 or IFN-γ prevented the accumulation of proinflammatory macrophages in VAT and greatly ameliorated insulin sensitivity. Thus NK cells are key regulators of macrophage polarization and insulin resistance in response to obesity-induced adipocyte stress.

Continuous requirement for the TCR in regulatory T cell function.

Foxp3(+) regulatory T cells (T(reg) cells) maintain immunological tolerance, and their deficiency results in fatal multiorgan autoimmunity. Although heightened signaling via the T cell antigen receptor (TCR) is critical for the differentiation of T(reg) cells, the role of TCR signaling in T(reg) cell function remains largely unknown. Here we demonstrated that inducible ablation of the TCR resulted in T(reg) cell dysfunction that could not be attributed to impaired expression of the transcription factor Foxp3, decreased expression of T(reg) cell signature genes or altered ability to sense and consume interleukin 2 (IL-2). Instead, TCR signaling was required for maintaining the expression of a limited subset of genes comprising 25% of the activated T(reg) cell transcriptional signature. Our results reveal a critical role for the TCR in the suppressor capacity of T(reg) cells.

IL-7 signaling must be intermittent, not continuous, during CD8⁺ T cell homeostasis to promote cell survival instead of cell death.

The maintenance of naive CD8(+) T cells is necessary for lifelong immunocompetence but for unknown reasons requires signaling via both interleukin 7 (IL-7) and the T cell antigen receptor (TCR). We now report that naive CD8(+) T cells required IL-7 signaling to be intermittent, not continuous, because prolonged IL-7 signaling induced naive CD8(+) T cells to proliferate, produce interferon-γ (IFN-γ) and undergo IFN-γ-triggered cell death. Homeostatic engagement of the TCR interrupted IL-7 signaling and thereby supported the survival and quiescence of CD8(+) T cells. However, CD8(+) T cells with insufficient TCR affinity for self ligands received prolonged IL-7 signaling and died during homeostasis. In this study we identified regulation of the duration of IL-7 signaling by homeostatic engagement of the TCR as the basis for in vivo CD8(+) T cell homeostasis.

Rapid effector function of memory CD8+ T cells requires an immediate-early glycolytic switch.

Antigen-experienced memory T cells acquire effector function with innate-like kinetics; however, the metabolic requirements of these cells are unknown. Here we show that rapid interferon-γ (IFN-γ) production of effector memory (EM) CD8(+) T cells, activated through stimulation mediated by the T cell antigen receptor (TCR) and the costimulatory receptor CD28 or through cognate interactions, was linked to increased glycolytic flux. EM CD8(+) T cells exhibited more glyceraldehyde-3-phosphate dehydrogenase (GAPDH) activity at early time points, before proliferation commenced, than did naive cells activated under similar conditions. CD28 signaling via the serine-threonine kinase Akt and the metabolic-checkpoint kinase mTORC2 was needed to sustain TCR-mediated immediate-early glycolysis. Unlike glycolysis in proliferating cells, immediate-early glycolysis in memory CD8(+) T cells was rapamycin insensitive. Thus, CD8(+) memory T cells have an Akt-dependent 'imprinted' glycolytic potential that is required for efficient immediate-early IFN-γ recall responses.

Human type 1 innate lymphoid cells accumulate in inflamed mucosal tissues.

Innate lymphoid cells (ILCs) are effectors of innate immunity and regulators of tissue modeling. Recently identified ILC populations have a cytokine expression pattern that resembles that of the helper T cell subsets T(H)2, T(H)17 and T(H)22. Here we describe a distinct ILC subset similar to T(H)1 cells, which we call 'ILC1'. ILC1 cells expressed the transcription factor T-bet and responded to interleukin 12 (IL-12) by producing interferon-γ (IFN-γ). ILC1 cells were distinct from natural killer (NK) cells as they lacked perforin, granzyme B and the NK cell markers CD56, CD16 and CD94, and could develop from RORγt(+) ILC3 under the influence of IL-12. The frequency of the ILC1 subset was much higher in inflamed intestine of people with Crohn's disease, which indicated a role for these IFN-γ-producing ILC1 cells in the pathogenesis of gut mucosal inflammation.

Targeting the CBM complex causes Treg cells to prime tumours for immune checkpoint therapy.

Solid tumours are infiltrated by effector T cells with the potential to control or reject them, as well as by regulatory T (Treg) cells that restrict the function of effector T cells and thereby promote tumour growth1. The anti-tumour activity of effector T cells can be therapeutically unleashed, and is now being exploited for the treatment of some forms of human cancer. However, weak tumour-associated inflammatory responses and the immune-suppressive function of Treg cells remain major hurdles to broader effectiveness of tumour immunotherapy2. Here we show that, after disruption of the CARMA1-BCL10-MALT1 (CBM) signalosome complex, most tumour-infiltrating Treg cells produce IFNγ, resulting in stunted tumour growth. Notably, genetic deletion of both or even just one allele of CARMA1 (also known as Card11) in only a fraction of Treg cells-which avoided systemic autoimmunity-was sufficient to produce this anti-tumour effect, showing that it is not the mere loss of suppressive function but the gain of effector activity by Treg cells that initiates tumour control. The production of IFNγ by Treg cells was accompanied by activation of macrophages and upregulation of class I molecules of the major histocompatibility complex on tumour cells. However, tumour cells also upregulated the expression of PD-L1, which indicates activation of adaptive immune resistance3. Consequently, blockade of PD-1 together with CARMA1 deletion caused rejection of tumours that otherwise do not respond to anti-PD-1 monotherapy. This effect was reproduced by pharmacological inhibition of the CBM protein MALT1. Our results demonstrate that partial disruption of the CBM complex and induction of IFNγ secretion in the preferentially self-reactive Treg cell pool does not cause systemic autoimmunity but is sufficient to prime the tumour environment for successful immune checkpoint therapy.

A defined commensal consortium elicits CD8 T cells and anti-cancer immunity.

There is a growing appreciation for the importance of the gut microbiota as a therapeutic target in various diseases. However, there are only a handful of known commensal strains that can potentially be used to manipulate host physiological functions. Here we isolate a consortium of 11 bacterial strains from healthy human donor faeces that is capable of robustly inducing interferon-γ-producing CD8 T cells in the intestine. These 11 strains act together to mediate the induction without causing inflammation in a manner that is dependent on CD103+ dendritic cells and major histocompatibility (MHC) class Ia molecules. Colonization of mice with the 11-strain mixture enhances both host resistance against Listeria monocytogenes infection and the therapeutic efficacy of immune checkpoint inhibitors in syngeneic tumour models. The 11 strains primarily represent rare, low-abundance components of the human microbiome, and thus have great potential as broadly effective biotherapeutics.

Aiolos promotes TH17 differentiation by directly silencing Il2 expression.

CD4(+) interleukin 17 (IL-17)-producing helper T cells (T(H)17 cells) are instrumental in the immune response to pathogens. However, an overactive T(H)17 response results in tissue inflammation and autoimmunity, and therefore it is important to identify the molecular mechanisms that control the development of T(H)17 cells. IL-2 suppresses such development, but how IL-2 production is actively suppressed during T(H)7 differentiation is not understood. Here we report that under T(H)17-polarizing conditions, the transcription factors STAT3 and AhR upregulated the expression of Aiolos, a member of the Ikaros family of transcription factors. Using Aiolos-deficient mice, we demonstrated that Aiolos silenced the Il2 locus, promoting T(H)17 differentiation in vitro and in vivo. Thus, we have identified a module in the transcriptional program of T(H)17 cells that actively limits IL-2 production and promotes their differentiation.

The transcription factor Sox4 is a downstream target of signaling by the cytokine TGF-ß and suppresses T(H)2 differentiation.

Sox4 is a transcription factor that regulates various developmental processes. Here we show that Sox4 was induced by TGF-ß and negatively regulated the transcription factor GATA-3, the master regulator of function of T helper type 2 (T(H)2) cells, by two distinct mechanisms. First, Sox4 bound directly to GATA-3, preventing its binding to GATA-3 consensus DNA sequences. Second, Sox4 bound to the promoter region of the gene encoding interleukin 5 (IL-5), a T(H)2 cytokine, and prevented binding of GATA-3 to this promoter. T(H)2 cell-driven airway inflammation was modulated by alterations in Sox4 expression. Thus, Sox4 acted as a downstream target of TGF-ß to inhibit GATA-3 function, T(H)2 differentiation and T(H)2 cell-mediated inflammation.

Interleukin-22 induces interleukin-18 expression from epithelial cells during intestinal infection.

T helper 1 (Th1) cell-associated immunity exacerbates ileitis induced by oral Toxoplasma gondii infection. We show here that attenuated ileitis observed in interleukin-22 (IL-22)-deficient mice was associated with reduced production of Th1-cell-promoting IL-18. IL-22 not only augmented the expression of Il18 mRNA and inactive precursor protein (proIL-18) in intestinal epithelial cells after T. gondii or Citrobacter rodentium infection, but also maintained the homeostatic amount of proIL-18 in the ileum. IL-22, however, did not induce the processing to active IL-18, suggesting a two-step regulation of IL-18 in these cells. Although IL-18 exerted pathogenic functions during ileitis triggered by T. gondii, it was required for host defense against C. rodentium. Conversely, IL-18 was required for the expression of IL-22 in innate lymphoid cells (ILCs) upon T. gondii infection. Our results define IL-18 as an IL-22 target gene in epithelial cells and describe a complex mutual regulation of both cytokines during intestinal infection.

DNA damage primes the type I interferon system via the cytosolic DNA sensor STING to promote anti-microbial innate immunity.

Dysfunction in Ataxia-telangiectasia mutated (ATM), a central component of the DNA repair machinery, results in Ataxia Telangiectasia (AT), a cancer-prone disease with a variety of inflammatory manifestations. By analyzing AT patient samples and Atm(-/-) mice, we found that unrepaired DNA lesions induce type I interferons (IFNs), resulting in enhanced anti-viral and anti-bacterial responses in Atm(-/-) mice. Priming of the type I interferon system by DNA damage involved release of DNA into the cytoplasm where it activated the cytosolic DNA sensing STING-mediated pathway, which in turn enhanced responses to innate stimuli by activating the expression of Toll-like receptors, RIG-I-like receptors, cytoplasmic DNA sensors, and their downstream signaling partners. This study provides a potential explanation for the inflammatory phenotype of AT patients and establishes damaged DNA as a cell intrinsic danger signal that primes the innate immune system for a rapid and amplified response to microbial and environmental threats.

Dynamic Number and Function of IL-10-Producing Regulatory B Cells in the Immune Microenvironment at Distinct Stages of Type 1 Diabetes.

The critical role of IL-10-producing B cells (B10 cells) with a unique CD1dhiCD5+ phenotype in suppressing autoimmune responses and relieving inflammation has been demonstrated in several models of autoimmune diseases. However, the regulatory role of B10 cells in T cell-mediated autoimmune responses during the natural history of type 1 diabetes is unclear. In this study, we used the NOD mouse model of autoimmune diabetes to clarify the changes and potential mechanisms of B10 cells for disease. Compared with B10 cells present in the 4-wk-old normoglycemic NOD mice, the frequency of B10 cells was increased in the insulitis and diabetic NOD mice, with the highest proportion in the insulitis NOD mice. The changes in the relative number of B10 cells were most pronounced in the pancreas-draining lymph nodes. The pathogenic T cells, including Th1 and Th17 cells, remarkably increased. The assays in vitro showed that B10 cells in the NOD mice did not inhibit the proliferation of CD4+CD25- T cells. They also had no regulatory effect on IFN-γ and IL-4 secretion or on Foxp3 expression of T cells. B10 cells suppressed T cell-mediated autoimmune responses via an IL-10-dependent pathway. In contrast, B10 cells in the NOD mice exhibited a significant reduction in IL-10 production. In summary, a defect in the number and function of B10 cells may participate in the development and progression of type 1 diabetes.

Regulation of Cutaneous Immunity In Vivo by Calcitonin Gene-Related Peptide Signaling through Endothelial Cells.

Calcitonin gene-related peptide (CGRP) can bias the outcome of Ag presentation to responsive T cells in vitro away from Th1-type immunity and toward the Th2 and Th17 poles through actions on endothelial cells (ECs). To test the in vivo significance of this observation, we engineered a mouse lacking functional CGRP receptors on ECs (EC receptor activity modifying protein 1 [RAMP1] knockout mice). On percutaneous immunization to 1-fluoro-2,4-dinitrobenzene, stimulated CD4+ T cells from draining lymph nodes showed significantly reduced IL-17A expression with significantly increased IFN-γ, IL-4, and IL-22 expression at the protein and mRNA levels compared with control mice. Retinoic acid receptor-related orphan receptor γ t mRNA was significantly reduced, while mRNAs for T-box expressed in T cells and GATA binding protein 3 were significantly increased. In addition, EC RAMP1 knockout mice had significantly reduced contact hypersensitivity responses, and systemic administration of a CGRP receptor antagonist similarly inhibited contact hypersensitivity in wild-type mice. These observations provide compelling evidence that CGRP is a key regulator of cutaneous immunity through effects on ECs and suggest a novel pathway for potential therapeutic manipulation.

The Purinergic Receptor P2X4 Promotes Th17 Activation and the Development of Arthritis.

Purinergic signaling plays a major role in T cell activation leading to IL-2 production and proliferation. However, it is unclear whether purinergic signaling contributes to the differentiation and activation of effector T cells. In this study, we found that the purinergic receptor P2X4 was associated with human Th17 cells but not with Th1 cells. Inhibition of P2X4 receptor with the specific antagonist 5-BDBD and small interfering RNA inhibited the development of Th17 cells and the production of IL-17 by effector Th17 cells stimulated via the CD3/CD28 pathway. Our results showed that P2X4 was required for the expression of retinoic acid-related orphan receptor C, which is the master regulator of Th17 cells. In contrast, inhibition of P2X4 receptor had no effect on Th1 cells and on the production of IFN-γ and it did not affect the expression of the transcription factor T-bet (T-box transcription factor). Furthermore, inhibition of P2X4 receptor reduced the production of IL-17 but not of IFN-γ by effector/memory CD4+ T cells isolated from patients with rheumatoid arthritis. In contrast to P2X4, inhibition of P2X7 and P2Y11 receptors had no effects on Th17 and Th1 cell activation. Finally, treatment with the P2X4 receptor antagonist 5-BDBD reduced the severity of collagen-induced arthritis in mice by inhibiting Th17 cell expansion and activation. Our findings provide novel insights into the role of purinergic signaling in T cell activation and identify a critical role for the purinergic receptor P2X4 in Th17 activation and in autoimmune arthritis.

IRE1α-XBP1 controls T cell function in ovarian cancer by regulating mitochondrial activity.

Tumours evade immune control by creating hostile microenvironments that perturb T cell metabolism and effector function1-4. However, it remains unclear how intra-tumoral T cells integrate and interpret metabolic stress signals. Here we report that ovarian cancer-an aggressive malignancy that is refractory to standard treatments and current immunotherapies5-8-induces endoplasmic reticulum stress and activates the IRE1α-XBP1 arm of the unfolded protein response9,10 in T cells to control their mitochondrial respiration and anti-tumour function. In T cells isolated from specimens collected from patients with ovarian cancer, upregulation of XBP1 was associated with decreased infiltration of T cells into tumours and with reduced IFNG mRNA expression. Malignant ascites fluid obtained from patients with ovarian cancer inhibited glucose uptake and caused N-linked protein glycosylation defects in T cells, which triggered IRE1α-XBP1 activation that suppressed mitochondrial activity and IFNγ production. Mechanistically, induction of XBP1 regulated the abundance of glutamine carriers and thus limited the influx of glutamine that is necessary to sustain mitochondrial respiration in T cells under glucose-deprived conditions. Restoring N-linked protein glycosylation, abrogating IRE1α-XBP1 activation or enforcing expression of glutamine transporters enhanced mitochondrial respiration in human T cells exposed to ovarian cancer ascites. XBP1-deficient T cells in the metastatic ovarian cancer milieu exhibited global transcriptional reprogramming and improved effector capacity. Accordingly, mice that bear ovarian cancer and lack XBP1 selectively in T cells demonstrate superior anti-tumour immunity, delayed malignant progression and increased overall survival. Controlling endoplasmic reticulum stress or targeting IRE1α-XBP1 signalling may help to restore the metabolic fitness and anti-tumour capacity of T cells in cancer hosts.

γδ T cell IFNγ production is directly subverted by Yersinia pseudotuberculosis outer protein YopJ in mice and humans.

Yersinia pseudotuberculosis is a foodborne pathogen that subverts immune function by translocation of Yersinia outer protein (Yop) effectors into host cells. As adaptive γδ T cells protect the intestinal mucosa from pathogen invasion, we assessed whether Y. pseudotuberculosis subverts these cells in mice and humans. Tracking Yop translocation revealed that the preferential delivery of Yop effectors directly into murine Vγ4 and human Vδ2+ T cells inhibited anti-microbial IFNγ production. Subversion was mediated by the adhesin YadA, injectisome component YopB, and translocated YopJ effector. A broad anti-pathogen gene signature and STAT4 phosphorylation levels were inhibited by translocated YopJ. Thus, Y. pseudotuberculosis attachment and translocation of YopJ directly into adaptive γδ T cells is a major mechanism of immune subversion in mice and humans. This study uncovered a conserved Y. pseudotuberculosis pathway that subverts adaptive γδ T cell function to promote pathogenicity.

Invariant natural killer T cells recognize glycolipids from pathogenic Gram-positive bacteria.

Natural killer T cells (NKT cells) recognize glycolipid antigens presented by CD1d. These cells express an evolutionarily conserved, invariant T cell antigen receptor (TCR), but the forces that drive TCR conservation have remained uncertain. Here we show that NKT cells recognized diacylglycerol-containing glycolipids from Streptococcus pneumoniae, the leading cause of community-acquired pneumonia, and group B Streptococcus, which causes neonatal sepsis and meningitis. Furthermore, CD1d-dependent responses by NKT cells were required for activation and host protection. The glycolipid response was dependent on vaccenic acid, which is present in low concentrations in mammalian cells. Our results show how microbial lipids position the sugar for recognition by the invariant TCR and, most notably, extend the range of microbes recognized by this conserved TCR to several clinically important bacteria.

Protein expression profiling suggests relevance of noncanonical pathways in isolated pulmonary embolism.

Patients with isolated pulmonary embolism (PE) have a distinct clinical profile from those with deep vein thrombosis (DVT)-associated PE, with more pulmonary conditions and atherosclerosis. These findings suggest a distinct molecular pathophysiology and the potential involvement of alternative pathways in isolated PE. To test this hypothesis, data from 532 individuals from the Genotyping and Molecular Phenotyping of Venous ThromboEmbolism Project, a multicenter prospective cohort study with extensive biobanking, were analyzed. Targeted, high-throughput proteomics, machine learning, and bioinformatic methods were applied to contrast the acute-phase plasma proteomes of isolated PE patients (n = 96) against those of patients with DVT-associated PE (n = 276) or isolated DVT (n = 160). This resulted in the identification of shared molecular processes between PE phenotypes, as well as an isolated PE-specific protein signature. Shared processes included upregulation of inflammation, response to oxidative stress, and the loss of pulmonary surfactant. The isolated PE-specific signature consisted of 5 proteins: interferon-γ, glial cell line-derived neurotrophic growth factor, polypeptide N-acetylgalactosaminyltransferase 3, peptidyl arginine deiminase type-2, and interleukin-15 receptor subunit α. These proteins were orthogonally validated using cis protein quantitative trait loci. External replication in an independent population-based cohort (n = 5778) further validated the proteomic results and showed that they were prognostic for incident primary isolated PE in individuals without history of VTE (median time to event: 2.9 years; interquartile range: 1.6-4.2 years), supporting their possible involvement in the early pathogenesis. This study has identified molecular overlaps and differences between VTE phenotypes. In particular, the results implicate noncanonical pathways more commonly associated with respiratory and atherosclerotic disease in the acute pathophysiology of isolated PE.