Abstract Introduction Intranasal house dust mite (HDM) exposure elicits allergen-peptide (p):MHCII-specific Th2 and Tregs in the airways of mice. The balance between opposing pro- and anti-inflammatory functions of these cell populations may influence the outcome of airway re-exposure to HDM. However, the number of cells specific for known HDM derived epitopes in B6 mice is low, making longitudinal studies of these cell populations difficult. Methods To overcome this obstacle, we developed an approach using adoptive transfer of a physiologically relevant number of CD4+ T cells from a fixed TCR beta chain transgenic (1-DERβ) mouse and corresponding HDM p:MHCII tetramer to simultaneously interrogate the trafficking, memory formation, and functional properties of HDM-specific Th2 and Treg cells. Results Naïve 1-DERβ CD4+ T cells generated similar frequencies of Th2 and Treg fates as endogenous HDM-specific cells. Parabiosis experiments demonstrated HDM-specific Tregs form stable tissue-resident memory (Trm) cells in the lungs, similar to their previously described Th2 counterparts. Single cell RNA sequencing of these cell populations revealed shared expression of Trm genes, including the chemokine receptors CXCR6 and CCR8. Co-adoptive transfer of wild type and chemokine receptor deficient 1-DERβ cells revealed differential roles for CXCR6 and CCR8 in the generation and maintenance in the lung. Depletion of Tregs in the memory phase led to increased Th2 responses in the lung upon allergen rechallenge. Conclusion Thus, our findings are consistent with a model where lung-resident allergen-specific Tregs persist long term and are capable of suppressing resident Th2 cells following allergen rechallenge. Funding Source NIH K08 KAI171176 Topic Categories Immediate Hypersensitivity, Asthma, and Allergic Responses (HYP)

Our understanding of the airway epithelium's role in driving asthma pathogenesis has evolved over time. From being regarded primarily as a physical barrier that could be damaged via inflammation, the epithelium is now known to actively contribute to asthma development through interactions with the immune system. The airway epithelium contains multiple cell types with specialized functions spanning barrier action, mucociliary clearance, immune cell recruitment, and maintenance of tissue homeostasis. Environmental insults may cause direct or indirect injury to the epithelium leading to impaired barrier function, epithelial remodelling, and increased release of inflammatory mediators. In severe asthma, the epithelial barrier repair process is inhibited and the response to insults is exaggerated, driving downstream inflammation. Genetic and epigenetic mechanisms also maintain dysregulation of the epithelial barrier, adding to disease chronicity. Here, we review the role of the airway epithelium in severe asthma and how targeting the epithelium can contribute to asthma treatment.

BACKGROUND:House dust mites (HDMs) such as Dermatophagoides pteronyssinus are major allergy elicitors worldwide, yet their gene expression across developmental stages remains underexplored. Herein, we report a comprehensive RNAseq analysis of larvae, nymphs, and adult males and females, mapped to a recently published high-quality genome with extended functional annotations. RESULTS:Analysis of differentially expressed genes (DEG) revealed that female-biased expression was the most prevalent profile (16% of genes), while males exhibited the highest fold-change differences. DEG data, combined with network clustering and functional enrichment analysis, highlighted distinct genes and biological processes for each stage and sex: females showed upregulation of genes related to cell division and oogenesis, with vitellogenins among the most abundant transcripts; males exhibited increased expression of genes encoding putative seminal fluid proteins (e.g. endopeptidases, serpins, antimicrobial peptides), and those involved in reproductive regulation (e.g. testis-specific serine kinases); while juveniles displayed enhanced expression of genes related to energy metabolism and growth. Further analysis of endocrine pathways revealed non-canonic mechanisms compared to insect models, particularly in ecdysteroid and sesquiterpenoid biosynthesis and regulation. Expression patterns in genes involved in cuticle formation were also identified, reflecting their role in developmental transitions and sexual differentiation. Allergen and allergen-related gene expression showed an overall increase in feeding juveniles, as well as sex-biased expression, with Der p 27 upregulated in females. These findings provide insight into the physiological roles of allergens in digestion, immunity, and muscle formation, among other functions. Additionally, seven new horizontally transferred genes, including a DNA-repair photolyase linked to females, and novel multigene families (e.g. 119 male-specific beta-propeller proteins, 70 hypothetical cuticular proteins, 23 tetraspanin-like proteins, 5 female-associated putative odorant-binding proteins) were identified. CONCLUSIONS:This study provides the first genome-wide transcriptomic analysis of a HDM across life stages and sexes, expanding our understanding of the molecular mechanisms underlying mite development, sexual reproduction, and allergen expression. The generated data, fully available via supplementary spreadsheet and the ORCAE online platform, provide a valuable foundation for future allergy research and the development of new mite control strategies.

RIPK1 is a crucial regulator of cell survival, inflammation and cell death. Human RIPK1 deficiency leads to early-onset intestinal inflammation and peripheral T cell imbalance, though its role in αβT cell-mediated intestinal homeostasis remains unclear. In this study, we demonstrate that mice with RIPK1 ablation in conventional αβT cells (Ripk1ΔCD4) developed a severe small intestinal pathology characterized by small intestinal elongation, crypt hyperplasia, and duodenum-specific villus atrophy. Using mixed bone marrow chimeras reveals a survival disadvantage of αβT cells compared to γδT cells in the small intestine. Broad-spectrum antibiotic treatment ameliorates crypt hyperplasia and prevents intestinal elongation, though villus atrophy persists. Conversely, crossing Ripk1ΔCD4 with TNF receptor 1 Tnfr1-/- knockout mice rescues villus atrophy but not intestinal elongation. Finally, combined ablation of Ripk1∆CD4 and Casp8∆CD4 fully rescues intestinal pathology, revealing that αβT cell apoptosis in Ripk1∆CD4 drives the enteropathy. These findings demonstrate that RIPK1-mediated survival of αβT cells is essential for proximal small intestinal homeostasis. In Ripk1∆CD4 mice, the imbalanced T cell compartment drives microbiome-mediated intestinal elongation and TNF-driven villus atrophy.

Antibody-recruiting molecules (ARMs) are bivalent molecules that contain a cell-binding domain and an antibody-binding domain. ARMs are designed to redirect circulating endogenous antibodies from the bloodstream to the surface of cancer cells and thereby trigger innate immune-mediated killing of the latter. The current generation of clinically explored ARMs relies on synthetic small molecule haptens. However, their effectiveness is restricted by the low affinity of the available repertoire of endogenous anti-hapten antibodies. Utilizing endogenous high-affinity allergen-specific antibodies could potentially circumvent this issue. In this study, a genetically encoded antibody-recruiting strategy that utilizes lipid nanoparticles (LNPs) to deliver mRNA encoding the house dust mite allergen Der p 2, fused to a cell membrane anchor, to induce cell surface display and enable the recruitment of anti-Der p 2 antibodies, is presented. Der p 2 mRNA LNP-treated cancer cells cause greatly reduced pulmonary tumor burden in Der p 2 immunized mice, compared to untreated cells or nonimmunized mice. Reduced tumor growth is dependent on circulating antibodies, and neutrophils are identified as a key immune cell subset recognizing and eliminating Der p 2-displaying cancer cells. These findings emphasize the effectiveness of mRNA LNPs as a powerful tool for generating a genetically encoded ARM strategy, with potential applications in cancer immunotherapy.

Therapeutic monoclonal antibodies can prevent severe disease in SARS-CoV-2 exposed individuals. However, currently circulating virus variants have evolved to gain significant resistance to nearly all neutralizing human immune system-derived therapeutic monoclonal antibodies that had previously been emergency-authorized for use in the clinic. Here, we describe the discovery of a panel of single-domain antibodies (VHHs) directed against the spike protein S2 subunit that broadly neutralize SARS-CoV-1 and -2 with unusually high potency. One of these VHHs tightly clamps the spike's monomers at a highly conserved, quaternary epitope in the membrane proximal part of the trimeric Heptad Repeat 2 (HR2) coiled-coil, thereby locking the HR2 in its prefusion conformation. Low dose systemic administration of a VHH-human IgG1 Fc fusion prevented SARS-CoV-2 infection in two animal models. Pseudovirus escape selection experiments demonstrate that the very rare escape variants are rendered almost non-infectious. This VHH-based antibody with a highly potent mechanism of antiviral action forms the basis for a new class of pan-sarbecovirus neutralizing biologics, which are currently under development. In addition, the unique quaternary binding mode of the VHHs to the prefusion HR2 could be exploited for other class I fusion proteins.

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.

Rationale Blood eosinophil counts (BEC) are frequently used to monitor eosinophilic inflammation in asthma, but do not always correspond with bronchial eosinophilic inflammation (Tang, M. et al. 2024, J Allergy Clin Immunol). The latter can be measured in sputum or by (immuno)histochemical staining on bronchial biopsies or lung tissue. However, there is no consensus on how to quantify peribronchial eosinophilia. We aimed to compare Hematoxylin & Eosin (H&E) and Congo Red histochemical staining methods with immunohistochemical staining for eosinophilic cationic protein (ECP) and Galectin-10 (GAL10), two markers for eosinophilic inflammation in asthma (Persson, E. K. et al. 2019, Science). Methods Paraffin-embedded lung tissue samples from patients with elevated BEC (190-330/µl) (n = 5, 2/5 with COPD, 4/5 history of asthma, 3/5 female, mean age ± SD = 63 ± 12), who underwent tumor resection surgery, were selected from our human lung tissue biobank. Lung tissue was collected as far as possible from the tumor and showed no abnormalities. Serial sections (3 µm) were stained by four methods: H&E, Congo Red, anti-ECP, and anti-GAL10. Staining for ECP and GAL10 was combined with staining for myeloperoxidase (anti-MPO, neutrophil marker). Airways were manually matched on images of the four different stainings. Eosinophil numbers (EOS) were scored between the basal membrane of the bronchial epithelium and the outer airway wall to calculate peribronchial EOS/mm² and compared between matched airways. Also, mean-per-patient EOS/mm² for the four staining methods was compared. Results The number of peribronchial EOS/mm2 was significantly lower in H&E-stained airways compared to ECP and GAL10-stained airways, with more EOS-negative airways in the H&E staining (Figure 1A). In contrast, no difference in EOS/mm2 was observed between the ECP and GAL10 staining. Of the 43 airways matched for H&E, MPO-ECP, and GAL10-MPO, only 19 could be matched with Congo Red. Congo Red staining showed a significantly lower EOS/mm2 compared to GAL10. H&E and Congo Red showed a trend to a lower mean-per-patient EOS/mm2 compared to GAL10 (Figure 1B). Furthermore, more peribronchial neutrophils were detected in the GAL10-MPO staining compared to MPO-ECP (data not shown), possibly due to the difference in antibodies and staining methods. Conclusions Chemical staining protocols on human lung tissue may underestimate peribronchial eosinophils in patients with airway eosinophilia. In contrast, immunohistochemistry methods such as our MPO-ECP and GAL10-MPO protocols show similar numbers of peribronchial eosinophils, indicating the specificity of those assays. We are currently validating these findings in a larger patient cohort.

Given the ongoing evolution of SARS-CoV-2 and the historical emergence of other highly transmissible coronaviruses like SARS-CoV and HCoV-NL63, in vivo coronavirus research remains crucial even when the COVID-19 pandemic is receding. Due to restricted tropism of SARS-CoV-2 and other coronaviruses for mouse cells, model systems of infection rely on transgenic expression of the entry receptor human ACE2 (hACE2). Available hACE2 transgenic models using the Krt18 promotor express the receptor across multiple cell types and organ systems, leading to multiple disease features including pneumonia, vascular compromise and neuro-inflammation, that reflect the multi-organ nature of COVID-19. To disentangle the role of cell tropism in driving the clinical manifestations of the disease, we generated two new transgenic mouse models, Sftpa1 -hACE2 and Cdh5 -hACE2 transgenic mice, with hACE2 restricted to lung epithelium or endothelial cells respectively. In Sftpa1 -hACE2 mice, with high expression of the hACE2 receptor in lung alveolar type 2 cells, SARS-CoV-2 infection led to rapidly progressing disease, characterised by a strong neutrophilic innate immune response in the lung, followed by viral neuro-invasion and early death. Krt18 -hACE2 mice additionally recruited various dendritic cell subsets and gradually developed adaptive immunity. In Cdh5 -hACE2 Tg mice with exclusive endothelial tropism of the virus, viral inoculation via the lung or systemic circulation did not lead to viral propagation or disease manifestations, despite endothelial expression of hACE2 in the lung. These results suggest that tropism for alveolar epithelial cells increases disease severity, while endothelial cell tropism per se does not drive the vascular consequences often seen in COVID-19 patients. Our new transgenic mouse models will be helpful to dissect how cell tropism contributes to the clinical manifestations of coronavirus infection. ### Competing Interest Statement The authors have declared no competing interest.

IntroductionRegulatory T-cells (Tregs) are characterized by the expression of Foxp3, a master regulator involved in the development and function of Tregs. Foxp3 expression is dependent on activity of the Treg specific demethylated site (TSDR), which contains a CREB binding site. We aimed to find out how Foxp3 specific CREB deletion affects Treg expression and function.MethodsTregs from Foxp3creCREBfl/fl mice and wild type (CREBfl/fl) mice were analyzed by flow cytometry. Cytokine analysis was performed by flow cytometry, ELISA and RT-qPCR. Gene expression analysis was performed using Affymetrix HTA2 assays, ATAC-sequencing, and Methylation-assays. For functional relevance, a CD4 T cell mediated transfer colitis was performed.Results and discussionFoxp3creCREBfl/fl mice showed increased frequencies of Tregs (CD25+/Foxp3+) in thymus, spleen and peripheral lymph nodes and in nonlymphoid organs including lung and colon, but decreased Foxp3 expression at the single cell level. Despite decreased Foxp3 expression, enhanced expression of the IL- 33 receptor (ST-2), IL-10, IL-13, and CREM was observed. CREB deficient Tregs were highly suppressive in vitro and prevented disease activity in a CD4 T cell mediated transfer colitis in an IL-10 dependent way. Mechanistically CREB fulfils dual roles in Tregs: (1) it promotes Foxp3 expression under Steady state conditions and (2) in cooperation with CREM, CREB restricts chromatin accessibility at the ST2 locus, thereby modulating IL-33 driven immune responses. This dual regulation balances FoxP3-dependent Treg stability with IL-10 mediated suppression of inflammation.

While much is known about the functional effects of type 2 cytokines interleukin (IL)-4, IL-5 and IL-13 in homeostasis and disease, we still poorly understand the functions of IL-9. Chronic inflammation seen in allergic diseases, autoimmunity and cancer is however frequently accompanied by overproduction of this elusive type 2 cytokine. Initially identified as a T cell and mast cell growth factor, and later as the hallmark cytokine defining TH9 cells, we now know that IL-9 is produced by multiple innate and adaptive immune cells. Recent evidence suggests that IL-9 controls discrete aspects of the allergic cascade, cellular responses of immune and stromal cells, cancer progression, tolerance and immune escape. Despite functioning as a pleiotropic cytokine in mucosal environments, like the lungs, the direct and indirect cellular targets of IL-9 are still not well characterized. Here, we discuss IL-9's cellular senders and receivers, focusing on asthma and cancer. Moreover, we review current research directions and the outlook of targeted therapy centered around the biology of IL-9.

Over the recent decades the market potential of biologics has substantially expanded, and many of the top-selling drugs worldwide are now monoclonal antibodies or antibody-like molecules. The common gamma chain (γc) cytokines, Interleukin (IL-)2, IL-4, IL-7, IL-9, IL-15, and IL-21, play pivotal roles in regulating immune responses, from innate to adaptive immunity. Dysregulation of cell signaling by these cytokines is strongly associated with a range of immunological disorders, which includes cancer as well as autoimmune and inflammatory diseases. Given the essential role of γc cytokines in maintaining immune homeostasis, the development of therapeutic interventions targeting these molecules poses unique challenges. Here, we provide an overview of current biologics targeting either single or multiple γc cytokines or their respective receptor subunits across a spectrum of diseases, primarily focusing on antibodies, antibody-like constructs, and antibody-cytokine fusions. We summarize therapeutic biologics currently in clinical trials, highlighting how they may offer advantages over existing therapies and standard of care, and discuss recent advances in this field. Finally, we explore future directions and the potential of novel therapeutic intervention strategies targeting this cytokine family.

Messenger RNA (mRNA) has emerged as a promising therapeutic modality for various diseases. However, efficient delivery of mRNA into target cells remains a significant challenge. In this study, the combinatorial synthesis and characterization of a novel series of ionizable biscarbamate lipids (IBLs) for mRNA lipid nanoparticle (LNP) delivery are reported. A simplified and scalable method is developed, resulting in IBLs suitable for formulating mRNA into stable LNPs. Two generations of IBLs are synthesized and evaluated for their mRNA transfection capacity in vitro, using eGFP as a reporter protein, leading to the identification of S-Ac7-DOG as a lead IBL. Upon intramuscular vaccination, S-Ac7-DOG LNPs instigated robust antigen-specific CD8+ T cell responses against an mRNA encoded viral oncoprotein and a tumor neo-antigen. In comparison to MC3 LNPs, which are used as a benchmark, S-Ac7-DOG LNPs exhibit low reactogenicity, robust mRNA transfection, and a distinct biodistribution, with higher accumulation in draining lymph nodes and spleen. These findings highlight the potential of IBLs as a novel and promising class of ionizable lipids for mRNA delivery in vaccines and beyond. This study reports on combinatorial synthesis of ionizable biscarbamate lipids (IBLs) for mRNA lipid nanoparticle (LNP) delivery. In vitro screenings identify S-Ac7-DOG as a lead IBL, with high mRNA transfection capacity. In vivo, S-Ac7-DOG mRNA LNPs combine low reactogenicity with high local transfection. S-Ac7-DOG LNPs encapsulating mRNA encoding for tumor antigen can mount protective T cell responses against a tumor challenge. image

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.

Self-amplifying mRNA (saRNA) is witnessing increased interest as a platform technology for protein replacement therapy, gene editing, immunotherapy, and vaccination. saRNA can replicate itself inside cells, leading to a higher and more sustained production of the desired protein at a lower dose. Controlling innate immune activation, however, is crucial to suppress unwanted inflammation upon delivery and self-replication of RNA in vivo. In this study, we report on a class of beta-aminoester lipids (beta AELs) synthesized through the Michael addition of an acrylate to diethanolamine, followed by esterification with fatty acids. These lipids possessed one or two ionizable amines, depending on the use of nonionic or amine-containing acrylates. We utilized beta AELs for encapsulating saRNA in lipid nanoparticles (LNPs) and evaluated their transfection efficiency in vitro and in vivo in mice, while comparing them to LNPs containing ALC-0315 as an ionizable lipid reference. Among the tested lipids, OC7, which comprises two unsaturated oleoyl alkyl chains and an ionizable azepanyl motif, emerged as a beta AEL with low cytotoxicity and immunogenicity relative to ALC-0315. Interestingly, saRNA delivered via the OC7 LNP exhibited a distinct in vivo transfection profile. Initially, intramuscular injection of OC7 LNP resulted in low protein expression shortly after administration, followed by a gradual increase over a period of up to 7 days. This pattern is indicative of successful self-amplification of saRNA. In contrast, saRNA delivered via ALC-0315 LNP demonstrated high protein translation initially, which gradually declined over time and lacked the amplification seen with OC7 LNP. We observed that, in contrast to saRNA OC7 LNP, saRNA ALC-0315 LNP induced potent innate immune activation by triggering cytoplasmic RIG-I-like receptors (RLRs), likely due to the highly efficient endosomal membrane rupturing properties of ALC-0315 LNP. Consequently, the massive production of type I interferons quickly hindered the amplification of the saRNA. Our findings highlight the critical role of the choice of ionizable lipid for saRNA formulation in LNPs, particularly in shaping the qualitative profile of protein expression. For applications where minimizing inflammation is desired, the use of ionizable lipids, such as the beta AEL reported in this study, that elicit a low type I interferon response in saRNA LNP is crucial.

Acute systemic inflammation critically alters the function of the immune system, often promoting myelopoiesis at the expense of lymphopoiesis. In the thymus, systemic inflammation results in acute thymic atrophy and, consequently, impaired T-lymphopoiesis. The mechanism by which systemic inflammation impacts the thymus beyond suppressing T-cell development is still unclear. Here, we describe how the synergism between TL1A and IL-18 suppresses T-lymphopoiesis to promote thymic myelopoiesis. The protein levels of these two cytokines were elevated in the thymus during viral-induced thymus atrophy infection with murine cytomegalovirus (MCMV) or pneumonia virus of mice (PVM). In vivo administration of TL1A and IL-18 induced acute thymic atrophy, while thymic neutrophils expanded. Fate mapping with Ms4a3-Cre mice demonstrated that thymic neutrophils emerge from thymic granulocyte-monocyte progenitors (GMPs), while Rag1-Cre fate mapping revealed a common developmental path with lymphocytes. These effects could be modeled ex vivo using neonatal thymic organ cultures (NTOCs), where TL1A and IL-18 synergistically enhanced neutrophil production and egress. NOTCH blockade by the LY411575 inhibitor increased the number of neutrophils in the culture, indicating that NOTCH restricted steady-state thymic granulopoiesis. To promote myelopoiesis, TL1A, and IL-18 synergistically increased GM-CSF levels in the NTOC, which was mainly produced by thymic ILC1s. In support, TL1A- and IL-18-induced granulopoiesis was completely prevented in NTOCs derived from Csf2rb-/- mice and by GM-CSFR antibody blockade, revealing that GM-CSF is the essential factor driving thymic granulopoiesis. Taken together, our findings reveal that TL1A and IL-18 synergism induce acute thymus atrophy while promoting extramedullary thymic granulopoiesis in a NOTCH and GM-CSF-controlled manner.

BackgroundDysregulated innate immune responses underlie multiple inflammatory diseases, but clinical translation of preclinical innate immunity research in mice is hampered by the difficulty of studying human inflammatory reactions in an in vivo context. We therefore sought to establish in vivo human inflammatory responses in NSG-QUAD mice that express four human myelopoiesis transgenes to improve engraftment of a human innate immune system.MethodsWe reconstituted NSG-QUAD mice with human hematopoietic stem and progenitor cells (HSPCs), after which we evaluated human myeloid cell development and subsequent human responses to systemic and local lipopolysaccharide (LPS) challenges.ResultsNSG-QUAD mice already displayed engraftment of human monocytes, dendritic cells and granulocytes in peripheral blood, spleen and liver at 6 weeks after HSPC reconstitution, in which both classical, intermediate and non-classical monocytes were present. These huNSG-QUAD mice responded to intraperitoneal and intranasal LPS challenges with production of NF-κB-dependent human cytokines, a human type I interferon response, as well as inflammasome-mediated production of human IL-1β and IL-18. The latter were specifically abrogated by the NLRP3 inhibitor MCC950, while LPS-induced human monocyte death was not altered. Besides providing proof-of-principle for small molecule testing of human inflammatory reactions in huNSG-QUAD mice, this observation suggests that LPS-induced in vivo release of human NLRP3 inflammasome-generated cytokines occurs in a cell death-independent manner.ConclusionHuNSG-QUAD mice are competent for the NF-κB, interferon and inflammasome effectors of human innate immunity, and can thus be utilized to investigate signaling mechanisms and pharmacological targeting of human inflammatory responses in an in vivo setting.