Feedback mechanisms regulate immune activation and prevent excessive tissue damage. TNFAIP3, also known as A20, serves as a crucial brake on inflammation, and mutations or haploinsufficiency of this gene are linked to diseases characterized by inappropriate inflammation. In this study, we document highly conserved patterns of cell type-specific gene expression, regulation, and induction of TNFAIP3, and employ transgenic and gnotobiotic mouse models to investigate how adaptive immunity and the gut microbiome contribute to pathology arising from impaired A20 function. Contrary to our expectations, systemic inflammation resulting from Tnfaip3 deficiency in CD11c (Itgax)-expressing cells developed independently of autoreactive antibodies, B cells, and T cells. The microbiome also proved dispensable for disease manifestations in these models. These findings suggest that in diseases caused by insufficient TNFAIP3/A20 activity, autoantibodies may reflect a downstream consequence of disease rather than a causative driver, suggesting autoinflammatory rather than autoimmune pathology. These insights carry therapeutic implications for the treatment of TNFAIP3-associated diseases.

Fc receptors mediate antibody effector functions. Immunoglobulin G (IgG), the predominant antibody in circulation and in clinical use, engages diverse Fc gamma (Fcγ) receptors differentially expressed across cell types. Here, we provide a comprehensive overview of Fcγ receptor and neonatal Fc receptor (FcRn) expression in humans, macaques, and mice. This analysis revealed substantial differences in Fcγ receptor diversity, cell-specific expression, and regulatory mechanisms that compromise the translation of mouse and macaque models for antibody research. To improve preclinical modeling, we generated a mouse in which humanized Fcγ receptors (FcγRI/CD64, FcγRIIA/CD32A, FcγRIIB/CD32B, FcγRIIIA/CD16A, and FcγRIIIB/CD16B), expressed under control of human promotors, replace their murine counterparts. This model also incorporates human FcRn to improve antibody pharmacokinetics. Humanization resulted in more faithful Fcγ receptor expression. We validated receptor functionality and demonstrated how cytokines modulate their expression. Together, this cross-species Fcγ receptor atlas and humanized mouse model can improve the preclinical evaluation of antibody-based therapeutics.

Cytoskeletal remodelling is central to naive T cell fitness, organizing receptor-proximal signaling and mechanotransduction during TCR engagement. However, how cytoskeletal dynamics are coordinated with TCR signaling to preserve naive T cell fitness remains incompletely defined. Here, we identify the Sterile 20-family member Thousand and One Kinase 3 (TAOK3) as a kinase-dependent regulator of naive CD8+ T cell maintenance that couples TCR signal integration to cytoskeletal control. Genetic deletion or kinase inactivation of TAOK3 resulted in a profound, cell-intrinsic loss of naive CD8+ T cells. Despite enhanced sensitivity to TCR ligation and enhanced downstream signaling, proliferating CD8+ T cells did not survive in vitro and anti-viral CD8+ T cell immunity was compromised in vivo in the absence of TAOK3. Unbiased phospho-proteomic analysis of Taok3-deficient mice revealed altered phosphorylation of the Rac regulators Dedicator of Cytokinesis DOCK8 and DOCK10, alongside actin-membrane scaffolding proteins. Consistent with this, Taok3-deficient naive CD8+ T cells exhibited elevated basal actin polymerisation, excessive reactive oxygen species accumulation, mitochondrial hyperpolarisation, and reduced spare respiratory capacity. Pharmacologic Rac inhibition normalised cytoskeletal dynamics, corrected the heightened TCR sensitivity, and preferentially restored mitochondrial membrane potential. Collectively, these findings identify TAOK3 as a coordinator of membrane-proximal organisation and cytoskeletal regulation that calibrates Rac-dependent signaling, thereby linking TCR signal integration to mitochondrial fitness and long-term maintenance of the naive CD8+ T cell pool.

Secondary lymphoid organs (SLO) are prototypic sites of antibody production, yet mucosal sites also generate and maintain mucosal antibodies during continued inflammation. Using a murine model of house dust mite induced airway inflammation, we show that prolonged allergen exposure induces tertiary lymphoid organs (TLO) within para-bronchial adventitial cuffs. Within these regions, IL1 signaling instructs fibroblasts to form niches that recruit, retain, and sustain lung antibody-secreting cells (ASCs) through chemokine induction. These mucosal ASCs share immunoglobulin repertoires with TLO derived germinal center B cells. Thus, continued allergen exposure reshapes the lung microenvironment by converting para-bronchial fibroblasts into IL1 dependent supportive niches for non IgE antibody production, revealing a fibroblast mediated mechanism for local immune regulation in chronic inflammation. ### Competing Interest Statement B.N.L. received consulting fees from Sanofi and GSK and holds stock options from Argenx. PG has served as an advisor or speaker or has received research support from 3NT, Ablynx, ALK, ArgenX, AstraZeneca, Bekaert Textiles, Genentech, GSK, Hall Allergy, Medtronic, Novartis, Regeneron, Roche, Sanofi-Genzyme, Stallergenes-Greer, Teva, and Thermo Fisher. All other authors declare no competing interests. All other authors have no competing interests to declare. Research Foundation - Flanders, 11D9623N, 3F015119, 3E011619, 3E002820, 312ZZR22 Research Foundation - Flanders, https://ror.org/03qtxy027, S006825N, 3G0A7422, 3G062218W, 01IB0423 Ghent University, bof/baf/2y/2024/01/017, 01M01521, 01G03524, BOF21-DOC-105

Chitin is highly abundant in organisms with allergenic potential such as house dust mites, fungi, nematodes, and arthropods. Several observations suggest a role for chitin in allergic disease, although the mechanism of action is poorly understood. Humans and mice express highly conserved chitin-degrading chitinases: acidic mammalian chitinase (AMCase; encoded by CHIA in humans and Chia in mice) and chitotriosidase (Chit1; encoded by CHIT1 in humans and Chit1 in mice), which have been suggested to modulate allergic responses through enzymatic degradation of chitins. Humans with nasal polyposis and mice with type 2 immune responses had increased chitinolytic activity in their airways, emanating from two different enzymes. Due to redundancy between both enzymes and lack of a mouse model completely devoid of all chitinolytic activity, their precise role in regulating type 2 immunity to chitin remains controversial. Using transgenic mice with single or combined deficiency of Chia and Chit1, we investigated the impact of chitin and chitinases on type 2 immunity. Our data demonstrate that chitin is a potent adjuvant for type 2 inflammation by triggering increased activation and migration of type 2 dendritic cells, with consequent increased polarization of T helper 2 cells. This adjuvant potential of chitin is however unaffected by the absence of endogenous chitinolytic activity in different models of allergic airway disease. Taken together, we here highlight the role of chitin as an allergenic adjuvant and question if the strength of this response is influenced by endogenous chitinases.

Asthma is a chronic inflammatory disease of the airways characterized by variable airway obstruction. In some patients with severe disease there are frequent disease flares, and some individuals develop irreversible airway obstruction. The immune system has a predominant effect on many aspects of the disease. A large proportion of people with asthma have signs of type 2 immunity, rich in eosinophils, mast cells and basophils, and controlled by either type 2 helper T cells or type 2 innate lymphoid cells. Other patients have a more neutrophil-predominant disease, and some have little underlying immune dysfunction. Here we review the immunology of asthma by integrating data from mouse model studies with clinical intervention studies. In this Review, the authors update us on the immunology and clinical options for treating asthma.

Allergic asthma arises from complex genetic and environmental interactions. Analysis of a population-wide registry revealed that infants hospitalized for human respiratory syncytial virus (RSV) bronchiolitis who are born to asthmatic parents have a markedly increased risk of developing asthma. To model this interaction, neonatal mice infected with pneumonia virus of mice (PVM), an RSV analog, before house dust mite (HDM) exposure developed amplified type 2 inflammation and asthma-like pathology. Maternal, but not paternal, HDM allergy intensified disease, implicating vertical transmission of an immune risk factor. Mechanistically, neonatal viral infection up-regulated Fc receptors (FcRs) and promoted maturation of type 2 conventional dendritic cells (cDC2s). Maternal allergen-specific immunoglobulin G (IgG), transferred via neonatal Fc receptor (FcRn), enhanced Fc gamma receptor (FcγR)-mediated allergen uptake and T helper 2 (TH2) cell priming. Preventive RSV immunoprophylaxis blocked asthma development in this setting. These findings identify maternal allergy and neonatal RSV infection as converging FcR-dependent causal asthma risk factors, preventable through immunoprophylaxis.

Type 2high asthma, which accounts for the majority of asthma cases, is driven by Th2 cells that produce cytokines such as IL-4, IL-5, and IL-13. These cytokines promote several features of the disease, including eosinophilia, IgE production, bronchial hyperresponsiveness (BHR), mucus hypersecretion, and susceptibility to exacerbations. In contrast, type 2low asthma is characterized by the presence of neutrophils and reduced responsiveness to corticosteroids. In recent years, advances in our understanding of the distinct mechanisms at play in each asthma endotype have paved the way for the development of targeted therapies tailored to specific patient profiles. In this review, we first explore the underlying immunological mechanisms of various asthma endotypes. We also provide an overview of the different types of immunotherapies currently available to asthmatic patients and their clinical efficacy. Finally, we highlight emerging therapeutic strategies that hold promise for improving asthma management in the future.

Granulocyte-macrophage colony stimulating factor (GM-CSF) is a pleiotropic cytokine, able to promote both myelopoiesis and activation of immune cells. Particularly in the lung, GM-CSF plays an important homeostatic role in the development and maintenance of alveolar macrophages, and is therefore considered to play a role in respiratory virus infections such as influenza and SARS-CoV-2, although the benefits of GM-CSF treatment in clinical studies remain inconclusive. To address this, we tested inhaled GM-CSF treatment in the Pneumonia Virus of Mice (PVM) mouse model. Our findings show that local GM-CSF therapy during PVM disease increased local neutrophilia and monocyte-derived cell influx, but diminished CD8+ T cells responses. Despite this, the observed effects on T cells and myeloid cells did not result in an altered clinical outcome during PVM infection. We conclude that inhaled GM-CSF therapy cannot be considered as a universal protective therapy in respiratory virus infections.

Chronic infection with Schistosoma mansoni parasites is associated with reduced allergic sensitization in humans, while schistosome eggs protects against allergic airway inflammation (AAI) in mice. One of the main secretory/excretory molecules from schistosome eggs is the glycosylated T2-RNAse Omega-1 (ω1). We hypothesized that ω1 induces protection against AAI during infection. Peritoneal administration of ω1 prior to sensitization with Ovalbumin (OVA) reduced airway eosinophilia and pathology, and OVA-specific Th2 responses upon challenge, independent from changes in regulatory T cells. ω1 was taken up by monocyte-derived dendritic cells, mannose receptor (CD206)-positive conventional type 2 dendritic cells (CD206+ cDC2), and by recruited peritoneal macrophages. Additionally, ω1 impaired CCR7, F-actin, and costimulatory molecule expression on myeloid cells and cDC2 migration in and ex vivo, as evidenced by reduced OVA+ CD206+ cDC2 in the draining mediastinal lymph nodes (medLn) and retainment in the peritoneal cavity, while antigen processing and presentation in cDC2 were not affected by ω1 treatment. Importantly, RNAse mutant ω1 was unable to reduce AAI or affect DC migration, indicating that ω1 effects are dependent on its RNAse activity. Altogether, ω1 hampers migration of OVA+ cDC2 to the draining medLn in mice, elucidating how ω1 prevents allergic airway inflammation in the OVA/alum mouse model.

Asthma is characterized by lung eosinophilia, remodeling, and mucus plugging, controlled by adaptive Th2 effector cells secreting IL-4, IL-5, and IL-13. Inhaled house dust mite (HDM) causes the release of barrier epithelial cytokines that activate various innate immune cells like DCs and basophils that can promote Th2 adaptive immunity directly or indirectly. Here, we show that basophils play a crucial role in the development of type 2 immunity and eosinophilic inflammation, mucus production, and bronchial hyperreactivity in response to HDM inhalation in C57Bl/6 mice. Interestingly, conditional depletion of basophils during sensitization did not reduce Th2 priming or asthma inception, whereas depletion during allergen challenge did. During the challenge of sensitized mice, basophil-intrinsic IL-33/ST2 signaling, and not FcεRI engagement, promoted basophil IL-4 production and subsequent Th2 cell recruitment to the lungs via vascular integrin expression. Basophil-intrinsic loss of the ubiquitin modifying molecule Tnfaip3, involved in dampening IL-33 signaling, enhanced key asthma features. Thus, IL-33-activated basophils are gatekeepers that boost allergic airway inflammation by controlling Th2 tissue entry.

IntroductionDespite the new biologics to treat inflammation in severe asthma, targeting persistent obstruction of the airways remains challenging. Galectin-10 eosinophil derived crystals, also known as Charcot-Leyden crystals (CLCs) have been described to be present in the mucus plugs in the airways of patients with severe asthma. However, a direct role for CLCs in mucus production has not been established. We hypothesize that plugged airways constitute a unique niche where type 2 immune cells communicate with structural cells to perpetuate disease. We aimed to set up a new model using induced pluripotent stem cells (iPSCs).MethodsThree human iPSCs lines from type 2 severe asthma patients have been derived (MOSAIC study, University Hospital of Montpellier, NCT05616338) and differentiated into airway epithelium in air–liquid interface (i-ALI). The healthy iPSC line UHOMi002-A was used as a control. At day 21 of ALI culture, iPSC derived-airway epithelia were stimulated at the apical side with either IL-13 every two days (10ng/mL) during one week, acute stimulation (24h) with recombinant Gal10 crystals (100ng/mL), both IL-13 and Gal10 crystals or PBS (vehicle). We aimed to evaluate the effect on i-ALI differentiation at day 30.ResultsWe successfully differentiated the iPSC lines generated from the T2 severe asthma patients, and achieving a high purity rate at each developmental stages. The mean cell purity at the definitive endoderm for each cell line was>80% assessed by flow cytometry quantification of C-X-C Motif Chemokine Receptor 4 (CXCR4)/c-KIT double positive cells and immunolabelling of Forkhead Box A2 (FOXA2)+/SRY-box transcription factor 17 (SOX17)+. Purity for ventral anterior foregut endoderm (vAFE) stage was evaluated at 70%, through Transcription Factor NK2 Homeobox 1 (NKX2.1) expression, Carboxy Peptidase M (CPM) by flow cytometry. vAFE cells from the hiPSC lines differentiated into bronchial epithelium in air–liquid interface conditions. Chronic IL-13 challenging and CLC were both able to induce an increasing of MUC5AC+ cells and also an increase of neuroendocrine cells in asthmatic iPSC lines.ConclusioniALI bronchial epithelium can recapitulate T2 severe asthma features in vitro, and highlighted a possible direct effect of the CLC on the airway epithelium.

Sublingual allergen immunotherapy (SLIT) is an emerging treatment option for allergic asthma and a potential disease-modifying strategy for asthma prevention. The key cellular events leading to such long-term tolerance remain to be fully elucidated. We administered prophylactic SLIT in a mouse model of house dust mite (HDM)-driven allergic asthma. HDM extract was sublingually administered over 3 weeks followed by intratracheal sensitization and intranasal challenges with HDM. Prophylactic SLIT prevented allergic airway inflammation and hyperreactivity with a low lab-to-lab variation. The HDM-specific T helper (Th)2 (cluster of differentiation 4 Th) response was shifted by SLIT toward a regulatory and Th17 response in the lung and mediastinal lymph node. By using Derp1-specific cluster of differentiation 4+ T cells (1-DER), we found that SLIT blocked 1-DER T cell recruitment to the mediastinal lymph node and dampened IL-4 secretion following intratracheal HDM sensitization. Sublingually administered Derp1 protein activated 1-DER T cells in the cervical lymph node via chemokine receptor7+ migratory dendritic cells (DC). DCs migrating from the oral submucosa to the cervical lymph node after SLIT-induced Foxp3+ regulatory T cells. When mice were sensitized with HDM, prior prophylactic SLIT increased Derp1 specific regulatory T cells (Tregs) and lowered Th2 recruitment in the lung. By using Foxp3-diphtheria toxin receptor mice, Tregs were found to contribute to the immunoregulatory prophylactic effect of SLIT on type 2 immunity. These findings in a mouse model suggest that DC-mediated functional Treg induction in oral mucosa draining lymph nodes is one of the driving mechanisms behind the disease-modifying effect of prophylactic SLIT.

L’asthme est une maladie chronique inflammatoire des voies aériennes hétérogène. Parmi les patients atteints d’asthme sévère, un tiers présente une obstruction fonctionnelle persistante en lien avec une obstruction des voies aériennes par des bouchons de mucus contenant des mucines, de la fibrine et des cristaux de Charcot-Leyden dérivés d’éosinophiles. Les mécanismes moléculaires qui sous-tendent cet endotype ne sont pas clairement compris. Le développement de nouveaux outils afin de modéliser l’asthme chez l’Homme sont nécessaires, du fait notamment des différences considérables qu’il existe entre l’épithélium des voies aériennes de l’Homme et celui des rongeurs. Nous avons montré, avec d’autres équipes, qu’il est possible de reconstituer in vitro un épithélium bronchique fonctionnel et mature à partir de cellules souches humaines pluripotentes humaines (hiPSC). Notre objectif est de mettre au point un modèle in vitro humain d’asthme sévère à partir des hiPSC afin de modéliser les modifications de l’épithélium bronchique ainsi que les bouchons de mucus.

Since the precursor frequency of naive T cells is extremely low, investigating the early steps of antigen-specific T cell activation is challenging. To overcome this detection problem, adoptive transfer of a cohort of T cells purified from T cell receptor (TCR) transgenic donors has been extensively used but is not readily available for emerging pathogens. Constructing TCR transgenic mice from T cell hybridomas is a labor-intensive and sometimes erratic process, since the best clones are selected based on antigen-induced CD69 upregulation or IL-2 production in vitro, and TCR chains are polymerase chain reaction (PCR)-cloned into expression vectors. Here, we exploited the rapid advances in single-cell sequencing and TCR repertoire analysis to select the best clones without hybridoma selection, and generated CORSET8 mice ( COR ona S pike E pitope specific CD8 T cell), carrying a TCR specific for the Spike protein of SARS-CoV-2. Implementing newly created DALI software for TCR repertoire analysis in single-cell analysis enabled the rapid selection of the ideal responder CD8 T cell clone, based on antigen reactivity, proliferation, and immunophenotype in vivo. Identified TCR sequences were inserted as synthetic DNA into an expression vector and transgenic CORSET8 donor mice were created. After immunization with Spike/CpG-motifs, mRNA vaccination or SARS-CoV-2 infection, CORSET8 T cells strongly proliferated and showed signs of T cell activation. Thus, a combination of TCR repertoire analysis and scRNA immunophenotyping allowed rapid selection of antigen-specific TCR sequences that can be used to generate TCR transgenic mice.

Severe asthma patients with persistent airflow obstruction are characterized by functional obstruction due to mucus plugs containing mucins, fibrin, and eosinophil derived Charcot- Leyden crystals. The molecular mechanisms underlying this endotype are not clearly understood. Developing new models is crucial to respiratory research insofar as critical differences exist between human and rodent airway epithelium. We (and other teams) have shown that it is possible to reconstitute in vitro a complex and functional airway epithelium displaying all the features described in vivo from human-induced pluripotent stem cells (hiPSC). Our aim is to establish a human in vitro model of severe asthma that will recapitulate airway epithelium remodeling and mucus plugs. (c) 2024 SPLF. Published by Elsevier Masson SAS. All rights reserved.

Background: Asthma is often accompanied by type 2 immunity rich in IL-4, IL-5, and IL-13 cytokines produced by TH2 lymphocytes or type 2 innate lymphoid cells (ILC2s). IL-2 family cytokines play a key role in the differentiation, homeostasis, and effector function of innate and adaptive lymphocytes. Objective: IL-9 and IL-21 boost activation and proliferation of TH2 and ILC2s, but the relative importance and potential synergism between these g common chain cytokines are currently unknown. Methods: Using newly generated antibodies, we inhibited IL-9 and IL-21 alone or in combination in various murine models of asthma. In a translational approach using segmental allergen challenge, we recently described elevated IL-9 levels in human subjects with allergic asthma compared with nonasthmatic controls. Here, we also measured IL-21 in both groups. Results: IL-9 played a central role in controlling innate IL-33- induced lung inflammation by promoting proliferation and activation of ILC2s in an IL-21-independent manner. Conversely, chronic house dust mite-induced airway inflammation, mainly driven by adaptive immunity, was solely dependent on IL-21, which controlled TH2 activation, eosinophilia, total serum IgE, and formation of tertiary lymphoid structures. In a model of innate on adaptive immunity driven by papain allergen, a clear synergy was found between both pathways, as combined anti-IL-9 or anti-IL-21 blockade was superior in reducing key asthma features. In human bronchoalveolar lavage samples we measured elevated IL-21 protein within the allergic asthmatic group compared with the allergic control group. We also found increased IL21R transcripts and predicted IL-21 ligand activity in various disease-associated cell subsets. Conclusions: IL-9 and IL-21 play important and nonredundant roles in allergic asthma by boosting ILC2s and TH2 cells, revealing a dual IL-9 and IL-21 targeting strategy as a new and testable approach. (J Allergy Clin Immunol 2024;154:1129-45.)

Spontaneous protein crystallization is a rare event, yet protein crystals are frequently found in eosinophil-rich inflammation. In humans, Charcot-Leyden crystals (CLCs) are made from galectin-10 (Gal10) protein, an abundant protein in eosinophils. Although mice do not encode Gal10 in their genome, they do form pseudo-CLCs, made from the chitinase-like proteins Ym1 and/or Ym2, encoded by Chil3 and Chil4 and made by myeloid and epithelial cells respectively. Here, we investigated the biological effects of pseudo-CLCs since their function is currently unknown. We produced recombinant Ym1 crystals which were shown to have identical crystal packing and structure by X-ray crystallography as in vivo native crystals derived from murine lung. When administered to the airways of mice, crystalline but not soluble Ym1 stimulated innate and adaptive immunity and acted as a type 2 immune adjuvant for eosinophilic inflammation via triggering of dendritic cells (DCs). Murine Ym1 protein crystals found at sites of eosinophilic inflammation reinforce type 2 immunity and could serve as a surrogate model for studying the biology of human CLCs.

Respiratory viral infections represent one of the major causes of death worldwide. The recent coronavirus disease 2019 pandemic alone claimed the lives of over 6 million people around the globe. It is therefore crucial to understand how the immune system responds to these threats and how respiratory infection can be controlled and constrained. Dendritic cells (DCs) are one of the key players in antiviral immunity because of their ability to detect pathogens. They can orchestrate an immune response that will, in most cases, lead to viral clearance. Different subsets of DCs are present in the lung and each subset can contribute to antiviral responses through various mechanisms. In this review, we discuss the role of the different lung DC subsets in response to common respiratory viruses, with a focus on respiratory syncytial virus, influenza A virus and severe acute respiratory syndrome coronavirus 2. We also review how lung DC-mediated responses to respiratory viruses can lead to the worsening of an existing chronic pulmonary disease such as asthma. Throughout the review, we discuss results obtained from animal studies as well as results generated from infected patients.