Cross-talk between ILC2 and Gata3high Tregs locally constrains adaptive type 2 immunity.

Regulatory T cells (Tregs) control adaptive immunity and restrain type 2 inflammation in allergic disease. Interleukin-33 promotes the expansion of tissue-resident Tregs and group 2 innate lymphoid cells (ILC2s); however, how Tregs locally coordinate their function within the inflammatory niche is not understood. Here, we show that ILC2s are critical orchestrators of Treg function. Using spatial, cellular, and molecular profiling of the type 2 inflamed niche, we found that ILC2s and Tregs engage in a direct (OX40L-OX40) and chemotaxis-dependent (CCL1-CCR8) cellular dialogue that enforces the local accumulation of Gata3high Tregs, which are transcriptionally and functionally adapted to the type 2 environment. Genetic interruption of ILC2-Treg communication resulted in uncontrolled type 2 lung inflammation after allergen exposure. Mechanistically, we found that Gata3high Tregs can modulate the local bioavailability of the costimulatory molecule OX40L, which subsequently controlled effector memory T helper 2 cell numbers. Hence, ILC2-Treg interactions represent a critical feedback mechanism to control adaptive type 2 immunity.

Intratumoral antigen signaling traps CD8+ T cells to confine exhaustion to the tumor site.

Immunotherapy advances have been hindered by difficulties in tracking the behaviors of lymphocytes after antigen signaling. Here, we assessed the behavior of T cells active within tumors through the development of the antigen receptor signaling reporter (AgRSR) mouse, fate-mapping lymphocytes responding to antigens at specific times and locations. Contrary to reports describing the ready egress of T cells out of the tumor, we find that intratumoral antigen signaling traps CD8+ T cells in the tumor. These clonal populations expand and become increasingly exhausted over time. By contrast, antigen-signaled regulatory T cell (Treg) clonal populations readily recirculate out of the tumor. Consequently, intratumoral antigen signaling acts as a gatekeeper to compartmentalize CD8+ T cell responses, even within the same clonotype, thus enabling exhausted T cells to remain confined to a specific tumor tissue site.

BACH2 restricts NK cell maturation and function, limiting immunity to cancer metastasis.

Natural killer (NK) cells are critical to immune surveillance against infections and cancer. Their role in immune surveillance requires that NK cells are present within tissues in a quiescent state. Mechanisms by which NK cells remain quiescent in tissues are incompletely elucidated. The transcriptional repressor BACH2 plays a critical role within the adaptive immune system, but its function within innate lymphocytes has been unclear. Here, we show that BACH2 acts as an intrinsic negative regulator of NK cell maturation and function. BACH2 is expressed within developing and mature NK cells and promotes the maintenance of immature NK cells by restricting their maturation in the presence of weak stimulatory signals. Loss of BACH2 within NK cells results in accumulation of activated NK cells with unrestrained cytotoxic function within tissues, which mediate augmented immune surveillance to pulmonary cancer metastasis. These findings establish a critical function of BACH2 as a global negative regulator of innate cytotoxic function and tumor immune surveillance by NK cells.

Senescence-induced endothelial phenotypes underpin immune-mediated senescence surveillance.

Senescence is a stress-responsive tumor suppressor mechanism associated with expression of the senescence-associated secretory phenotype (SASP). Through the SASP, senescent cells trigger their own immune-mediated elimination, which if evaded leads to tumorigenesis. Senescent parenchymal cells are separated from circulating immunocytes by the endothelium, which is targeted by microenvironmental signaling. Here we show that SASP induces endothelial cell NF-κB activity and that SASP-induced endothelial expression of the canonical NF-κB component Rela underpins senescence surveillance. Using human liver sinusoidal endothelial cells (LSECs), we show that SASP-induced endothelial NF-κB activity regulates a conserved transcriptional program supporting immunocyte recruitment. Furthermore, oncogenic hepatocyte senescence drives murine LSEC NF-κB activity in vivo. Critically, we show two distinct endothelial pathways in senescence surveillance. First, endothelial-specific loss of Rela prevents development of Stat1-expressing CD4+ T lymphocytes. Second, the SASP up-regulates ICOSLG on LSECs, with the ICOS-ICOSLG axis contributing to senescence cell clearance. Our results show that the endothelium is a nonautonomous SASP target and an organizing center for immune-mediated senescence surveillance.

The role of LPS and CpG in the farm effect against allergies, and beyond.

The prevalence of allergic disease has increased significantly over the past decades. Although allergies are inherently multifactorial and heterogenous; environmental, maternal, and early-life microbial exposures could strongly modify disease risk. The effects of environmental microbiota are illustrated by the "farm effect", showing protection against asthma when children grow up on traditional farms. Recent studies have further revealed an important role for early-life exposure to a microbe-rich environment imposing lung and gut microbiome maturation and immune education, preventing allergic disease in childhood. Advances are made in the field of immunology and microbiome research, which identified entire microbial taxa, as well as specific microbial metabolites and bacterial products associated with reducing disease risk. Here we discuss the cross-talk between the microbiota and the pathogenesis of allergic disease, including bacterial products as lipopolysaccharide and CpG, in the farm effect.

Early Neutrophilia Marked by Aerobic Glycolysis Sustains Host Metabolism and Delays Cancer Cachexia.

An elevated neutrophil-lymphocyte ratio negatively predicts the outcome of patients with cancer and is associated with cachexia, the terminal wasting syndrome. Here, using murine model systems of colorectal and pancreatic cancer we show that neutrophilia in the circulation and multiple organs, accompanied by extramedullary hematopoiesis, is an early event during cancer progression. Transcriptomic and metabolic assessment reveals that neutrophils in tumor-bearing animals utilize aerobic glycolysis, similar to cancer cells. Although pharmacological inhibition of aerobic glycolysis slows down tumor growth in C26 tumor-bearing mice, it precipitates cachexia, thereby shortening the overall survival. This negative effect may be explained by our observation that acute depletion of neutrophils in pre-cachectic mice impairs systemic glucose homeostasis secondary to altered hepatic lipid processing. Thus, changes in neutrophil number, distribution, and metabolism play an adaptive role in host metabolic homeostasis during cancer progression. Our findings provide insight into early events during cancer progression to cachexia, with implications for therapy.

Pulmonary Eosinophils at the Center of the Allergic Space-Time Continuum.

Eosinophils are typically a minority population of circulating granulocytes being released from the bone-marrow as terminally differentiated cells. Besides their function in the defense against parasites and in promoting allergic airway inflammation, regulatory functions have now been attributed to eosinophils in various organs. Although eosinophils are involved in the inflammatory response to allergens, it remains unclear whether they are drivers of the asthma pathology or merely recruited effector cells. Recent findings highlight the homeostatic and pro-resolving capacity of eosinophils and raise the question at what point in time their function is regulated. Similarly, eosinophils from different physical locations display phenotypic and functional diversity. However, it remains unclear whether eosinophil plasticity remains as they develop and travel from the bone marrow to the tissue, in homeostasis or during inflammation. In the tissue, eosinophils of different ages and origin along the inflammatory trajectory may exhibit functional diversity as circumstances change. Herein, we outline the inflammatory time line of allergic airway inflammation from acute, late, adaptive to chronic processes. We summarize the function of the eosinophils in regards to their resident localization and time of recruitment to the lung, in all stages of the inflammatory response. In all, we argue that immunological differences in eosinophils are a function of time and space as the allergic inflammatory response is initiated and resolved.

ILC2-driven innate immune checkpoint mechanism antagonizes NK cell antimetastatic function in the lung.

Metastasis constitutes the primary cause of cancer-related deaths, with the lung being a commonly affected organ. We found that activation of lung-resident group 2 innate lymphoid cells (ILC2s) orchestrated suppression of natural killer (NK) cell-mediated innate antitumor immunity, leading to increased lung metastases and mortality. Using multiple models of lung metastasis, we show that interleukin (IL)-33-dependent ILC2 activation in the lung is involved centrally in promoting tumor burden. ILC2-driven innate type 2 inflammation is accompanied by profound local suppression of interferon-γ production and cytotoxic function of lung NK cells. ILC2-dependent suppression of NK cells is elaborated via an innate regulatory mechanism, which is reliant on IL-5-induced lung eosinophilia, ultimately limiting the metabolic fitness of NK cells. Therapeutic targeting of IL-33 or IL-5 reversed NK cell suppression and alleviated cancer burden. Thus, we reveal an important function of IL-33 and ILC2s in promoting tumor metastasis via their capacity to suppress innate type 1 immunity.

Human Lung Conventional Dendritic Cells Orchestrate Lymphoid Neogenesis during Chronic Obstructive Pulmonary Disease.

Rationale: Emerging evidence supports a crucial role for tertiary lymphoid organs (TLOs) in chronic obstructive pulmonary disease (COPD) progression. However, mechanisms of immune cell activation leading to TLOs in COPD remain to be defined.Objectives: To examine the role of lung dendritic cells (DCs) in T follicular helper (Tfh)-cell induction, a T-cell subset critically implicated in lymphoid organ formation, in COPD.Methods: Myeloid cell heterogeneity and phenotype were studied in an unbiased manner via single-cell RNA sequencing on HLA-DR+ cells sorted from human lungs. We measured the in vitro capability of control and COPD lung DC subsets, sorted using a fluorescence-activated cell sorter, to polarize IL-21+CXCL13+ (IL-21-positive and C-X-C chemokine ligand type 13-positive) Tfh-like cells. In situ imaging analysis was performed on Global Initiative for Chronic Obstructive Lung Disease stage IV COPD lungs with TLOs.Measurements and Main Results: Single-cell RNA-sequencing analysis revealed a high degree of heterogeneity among human lung myeloid cells. Among these, conventional dendritic type 2 cells (cDC2s) showed increased induction of IL-21+CXCL13+ Tfh-like cells. Importantly, the capacity to induce IL-21+ Tfh-like cells was higher in cDC2s from patients with COPD than in those from control patients. Increased Tfh-cell induction by COPD cDC2s correlated with increased presence of Tfh-like cells in COPD lungs as compared with those in control lungs, and cDC2s colocalized with Tfh-like cells in TLOs of COPD lungs. Mechanistically, cDC2s exhibited a unique migratory signature and (transcriptional) expression of several pathways and genes related to DC-induced Tfh-cell priming. Importantly, blocking the costimulatory OX40L (OX40 ligand)-OX40 axis reduced Tfh-cell induction by control lung cDC2s.Conclusions: In COPD lungs, we found lung EBI2+ (Epstein-Barr virus-induced gene 2-positive) OX-40L-expressing cDC2s that induced IL-21+ Tfh-like cells, suggesting an involvement of these cells in TLO formation.

IL-33trap is a novel IL-33-neutralizing biologic that inhibits allergic airway inflammation.

BACKGROUND: The emergence of IL-33 as a key molecular player in the development and propagation of widespread inflammatory diseases, including asthma and atopic dermatitis, has established the need for effective IL-33-neutralizing biologics. OBJECTIVE: Here we describe the development and validation of a new antagonist of IL-33, termed IL-33trap, which combines the extracellular domains of the IL-33 receptor (ST2) and its coreceptor, IL-1 receptor accessory protein, into a single fusion protein. METHODS: We produced and purified recombinant IL-33trap from human cells and analyzed its IL-33-binding affinity and IL-33 antagonistic activity in cultured cells and mice. IL-33trap activity was also benchmarked with a recombinant soluble ST2 corresponding to the naturally occurring IL-33 decoy receptor. Finally, we studied the effect of IL-33trap in the Alternaria alternata mouse model of allergic airway inflammation. RESULTS: In vitro IL-33trap binds IL-33 and inhibits IL-33 activity to a much stronger degree than soluble ST2. Furthermore, IL-33trap inhibits eosinophil infiltration, splenomegaly, and production of signature cytokines in splenic lymphocytes and lung tissue on IL-33 injection. Finally, administration of IL-33trap at the time of allergen challenge inhibits inflammatory responses in a preclinical mouse model of acute allergic airway inflammation. CONCLUSIONS: IL-33trap is a novel IL-33 antagonist that outperforms the natural IL-33 decoy receptor and shows anti-inflammatory activities in a preclinical mouse model of acute allergic airway inflammation when administered at the time of allergen challenge.

Professional and 'Amateur' Antigen-Presenting Cells In Type 2 Immunity.

Dendritic cells (DCs) are critical for the activation of naïve CD4+ T cells and are considered professional antigen-presenting cells (APCs), as are macrophages and B cells. Recently, several innate type 2 immune cells, such as basophils, mast cells (MCs), eosinophils, and innate type 2 lymphocytes (ILC2), have also emerged as harboring APC behavior. Through surface expression or transfer of peptide-loaded MHCII, expression of costimulatory and co-inhibitory molecules, as well as the secretion of polarizing cytokines, these innate cells can extensively communicate with effector and regulatory CD4+ T cells. An exciting new concept is that the complementary tasks of these 'amateur' APCs contribute to shaping and regulating adaptive immunity to allergens and helminths, often in collaboration with professional APCs.

Isolated Schistosoma mansoni eggs prevent allergic airway inflammation.

Chronic helminth infection with Schistosoma (S.) mansoni protects against allergic airway inflammation (AAI) in mice and is associated with reduced Th2 responses to inhaled allergens in humans, despite the presence of schistosome-specific Th2 immunity. Schistosome eggs strongly induce type 2 immunity and allow to study the dynamics of Th2 versus regulatory responses in the absence of worms. Treatment with isolated S. mansoni eggs by i.p. injection prior to induction of AAI to ovalbumin (OVA)/alum led to significantly reduced AAI as assessed by less BAL and lung eosinophilia, less cellular influx into lung tissue, less OVA-specific Th2 cytokines in lungs and lung-draining mediastinal lymph nodes and less circulating allergen-specific IgG1 and IgE antibodies. While OVA-specific Th2 responses were inhibited, treatment induced a strong systemic Th2 response to the eggs. The protective effect of S. mansoni eggs was unaltered in µMT mice lacking mature (B2) B cells and unaffected by Treg cell depletion using anti-CD25 blocking antibodies during egg treatment and allergic sensitization. Notably, prophylactic egg treatment resulted in a reduced influx of pro-inflammatory, monocyte-derived dendritic cells into lung tissue of allergic mice following challenge. Altogether, S. mansoni eggs can protect against the development of AAI, despite strong egg-specific Th2 responses.

The Generation and Use of Allergen-Specific TCR Transgenic Animals.

The generation of allergen-specific TCR transgenic animals allows for the characterization of allergen-specific T-cell responses in vivo and in vitro and is a powerful tool to study adaptive immunity to allergens. Here we describe an approach starting from the isolation of antigen-specific T-cell hybridomas and using PCR, flow cytometric, and co-culture methods to obtain antigen-specific MHC class II-restricted CD4+ TCR transgenic mice on the Rag2-/- background.

Group 2 innate lymphocytes at the interface between innate and adaptive immunity.

Group 2 innate lymphoid cells (ILC2) are innate immune cells that respond rapidly to their environment through soluble inflammatory mediators and cell-to-cell interactions. As tissue-resident sentinels, ILC2 help orchestrate localized type 2 immune responses. These ILC2-driven type 2 responses are now recognized in diverse immune processes, different anatomical locations, and homeostatic or pathological settings. ILC2-derived cytokines and cell surface signaling molecules function as key regulators of innate and adaptive immunity. Conversely, ILC2 are governed by their environment. As such, ILC2 form an important nexus of the immune system and may present an attractive target for immune modulation in disease.

TNF-α-induced protein 3 levels in lung dendritic cells instruct TH2 or TH17 cell differentiation in eosinophilic or neutrophilic asthma.

BACKGROUND: It is currently unknown why allergen exposure or environmental triggers in patients with mild-to-moderate asthma result in TH2-mediated eosinophilic inflammation, whereas patients with severe asthma often present with TH17-mediated neutrophilic inflammation. The activation state of dendritic cells (DCs) is crucial for both TH2 and TH17 cell differentiation and is mediated through nuclear factor κB activation. Ablation of TNF-α-induced protein 3 (TNFAIP3), one of the crucial negative regulators of nuclear factor κB activation in myeloid cells and DCs, was shown to control DC activation. OBJECTIVE: In this study we investigated the precise role of TNFAIP3 in myeloid cells for the development of TH2- and TH17-cell mediated asthma. METHODS: We exposed mice with conditional deletion of the Tnfaip3 gene in either myeloid cells (by using the lysozyme M [LysM] promotor) or specifically in DCs (by using the Cd11c promotor) to acute and chronic house dust mite (HDM)-driven asthma models. RESULTS: We demonstrated that reduced Tnfaip3 gene expression in DCs in either Tnfaip3CD11c or Tnfaip3LysM mice dose-dependently controlled development of TH17-mediated neutrophilic severe asthma in both acute and chronic HDM-driven models, whereas wild-type mice had a purely TH2-mediated eosinophilic inflammation. TNFAIP3-deficient DCs induced HDM-specific TH17 cell differentiation through increased expression of the TH17-instructing cytokines IL-1ß, IL-6, and IL-23, whereas HDM-specific TH2 cell differentiation was hampered by increased IL-12 and IL-6 production. CONCLUSIONS: These data show that the extent of TNFAIP3 expression in DCs controls TH2/TH17 cell differentiation. This implies that reducing DC activation could be a new pharmacologic intervention to treat patients with severe asthma who present with TH17-mediated neutrophilic inflammation.

House dust mite-driven asthma and allergen-specific T cells depend on B cells when the amount of inhaled allergen is limiting.

BACKGROUND: Allergic asthma is a CD4 TH2-lymphocyte driven disease characterized by airway hyperresponsiveness and eosinophilia. B cells can present antigens to CD4 T cells and produce IgE immunoglobulins that arm effector cells; however, mouse models are inconclusive on whether B cells are necessary for asthma development. OBJECTIVES: We sought to address the role of B cells in a house dust mite (HDM)-driven TH2-high asthma mouse model. METHODS: Wild-type and B cell-deficient muMT mice were sensitized and challenged through the airways with HDM extracts. The antigen-presenting capacities of B cells were studied by using new T-cell receptor transgenic 1-DER mice specific for the Der p 1 allergen. RESULTS: In vitro-activated B cells from HDM-exposed mice presented antigen to 1-DER T cells and induced a TH2 phenotype. In vivo B cells were dispensable for activation of naive 1-DER T cells but necessary for full expansion of primed 1-DER T cells. At high HDM challenge doses, B cells were not required for development of pulmonary asthmatic features yet contributed to TH2 expansion in the mediastinal lymph nodes but not in the lungs. When the amount of challenge allergen was decreased, muMT mice had reduced asthma features. Under these limiting conditions, B cells contributed also to expansion of TH2 effector cells in the lungs and central memory T cells in the mediastinal lymph nodes. CONCLUSION: B cells are a major part of the adaptive immune response to inhaled HDM allergen, particularly when the amount of inhaled allergen is low, by expanding allergen-specific T cells.

Perinatal Activation of the Interleukin-33 Pathway Promotes Type 2 Immunity in the Developing Lung.

Allergic disease originates in early life and polymorphisms in interleukin-33 gene (IL33) and IL1RL1, coding for IL-33R and decoy receptor sST2, confer allergy risk. Early life T helper 2 (Th2) cell skewing and allergy susceptibility are often seen as remnants of feto-maternal symbiosis. Here we report that shortly after birth, innate lymphoid type 2 cells (ILC2s), eosinophils, basophils, and mast cells spontaneously accumulated in developing lungs in an IL-33-dependent manner. During the phase of postnatal lung alveolarization, house dust mite exposure further increased IL-33, which boosted cytokine production in ILC2s and activated CD11b+ dendritic cells (DCs). IL-33 suppressed IL-12p35 and induced OX40L in neonatal DCs, thus promoting Th2 cell skewing. Decoy sST2 had a strong preventive effect on asthma in the neonatal period, less so in adulthood. Thus, enhanced neonatal Th2 cell skewing to inhaled allergens results from postnatal hyperactivity of the IL-33 axis during a period of maximal lung remodeling.

The Helminth-Derived Immunomodulator AvCystatin Reduces Virus Enhanced Inflammation by Induction of Regulatory IL-10+ T Cells.

Respiratory Syncytial Virus (RSV) is a major pathogen causing low respiratory tract disease (bronchiolitis), primarily in infants. Helminthic infections may alter host immune responses to both helminths and to unrelated immune triggers. For example, we have previously shown that filarial cystatin (AvCystatin/Av17) ameliorates allergic airway inflammation. However, helminthic immunomodulators have so far not been tested in virus-induced disease. We now report that AvCystatin prevents Th2-based immunopathology in vaccine-enhanced RSV lung inflammation, a murine model for bronchiolitis. AvCystatin ablated eosinophil influx, reducing both weight loss and neutrophil recruitment without impairing anti-viral immune responses. AvCystatin also protected mice from excessive inflammation following primary RSV infection, significantly reducing neutrophil influx and cytokine production in the airways. Interestingly, we found that AvCystatin induced an influx of CD4+ FoxP3+ interleukin-10-producing T cells in the airway and lungs, correlating with immunoprotection, and the corresponding cells could also be induced by adoptive transfer of AvCystatin-primed F4/80+ macrophages. Thus, AvCystatin ameliorates enhanced RSV pathology without increasing susceptibility to, or persistence of, viral infection and warrants further investigation as a possible therapy for virus-induced airway disease.

A20 Deficiency in Lung Epithelial Cells Protects against Influenza A Virus Infection.

A20 negatively regulates multiple inflammatory signalling pathways. We here addressed the role of A20 in club cells (also known as Clara cells) of the bronchial epithelium in their response to influenza A virus infection. Club cells provide a niche for influenza virus replication, but little is known about the functions of these cells in antiviral immunity. Using airway epithelial cell-specific A20 knockout (A20AEC-KO) mice, we show that A20 in club cells critically controls innate immune responses upon TNF or double stranded RNA stimulation. Surprisingly, A20AEC-KO mice are better protected against influenza A virus challenge than their wild type littermates. This phenotype is not due to decreased viral replication. Instead host innate and adaptive immune responses and lung damage are reduced in A20AEC-KO mice. These attenuated responses correlate with a dampened cytotoxic T cell (CTL) response at later stages during infection, indicating that A20AEC-KO mice are better equipped to tolerate Influenza A virus infection. Expression of the chemokine CCL2 (also named MCP-1) is particularly suppressed in the lungs of A20AEC-KO mice during later stages of infection. When A20AEC-KO mice were treated with recombinant CCL2 the protective effect was abrogated demonstrating the crucial contribution of this chemokine to the protection of A20AEC-KO mice to Influenza A virus infection. Taken together, we propose a mechanism of action by which A20 expression in club cells controls inflammation and antiviral CTL responses in response to influenza virus infection.

A Generic Polymer-Protein Ligation Strategy for Vaccine Delivery.

Although the field of cancer immunotherapy is intensively investigated, there is still a need for generic strategies that allow easy, mild and efficient formulation of vaccine antigens. Here we report on a generic polymer-protein ligation strategy to formulate protein antigens into reversible polymeric conjugates for enhanced uptake by dendritic cells and presentation to CD8 T-cells. A N-hydroxypropylmethacrylamide (HPMA)-based copolymer was synthesized via RAFT polymerization followed by introduction of pyridyldisulfide moieties. To enhance ligation efficiency to ovalbumin, which is used as a model protein antigen, protected thiols were introduced onto lysine residues and deprotected in situ in the presence of the polymer. The ligation efficiency was compared for both the thiol-modified versus unmodified ovalbumin, and the reversibility was confirmed. Furthermore, the obtained nanoconjugates were tested in vitro for their interaction and association with dendritic cells, showing enhanced cellular uptake and antigen cross-presentation to CD8 T-cells.