A Novel Homozygous Germline Mutation in Transferrin Receptor 1 (TfR1) Leads to Combined Immunodeficiency and Provides New Insights into Iron-Immunity Axis.

A homozygous missense mutation in the transferrin receptor 1 (TfR1), also known as CD71, leads to a rare inborn error of immunity (IEI) characterized by the impaired lymphocyte activation and proliferation due to defective iron uptake of cells. However, only one causative mutation (c.58T > C, p.Y20H) in the TFRC gene coding for TfR1 has been reported so far. We herein identified a new disease-causing homozygous germline mutation in the TFRC gene (c.64C > T, p.R22W) (referred to as TfR1R22W from now on) in a Turkish patient with combined immunodeficiency (CID). TfR1R22W results in impaired TfR1 internalization similar to previously defined TfR1Y20H mutation. We found that TfR1R22W is associated with severely restricted B and T lymphocyte clonal diversity and impaired T cell activation and cytokine production as well as defective mitochondrial oxidative phosphorylation in helper T cells. In addition, circulating NK, Treg, and MAIT cell populations were significantly decreased in the patient. Using whole transcriptome analysis, we found dysregulated immune homeostasis and novel biological processes associated with TfR1R22W. We also identified a considerable expansion of circulating low-density neutrophils (LDNs) in patient's PBMCs. Overall, TfR1R22W mutation expands the current understanding of the IEI associated with TfR1 dysfunction and provides new insights underlying impaired immune function, lymphocyte diversity, and granulocyte homeostasis.

Dual inhibition of airway inflammation and fibrosis by common ß cytokine receptor blockade.

BACKGROUND: Patients with severe asthma can present with eosinophilic type 2 (T2), neutrophilic, or mixed inflammation that drives airway remodeling and exacerbations and represents a major treatment challenge. The common ß (ßc) receptor signals for 3 cytokines, GM-CSF, IL-5, and IL-3, which collectively mediate T2 and neutrophilic inflammation. OBJECTIVE: To determine the pathogenesis of ßc receptor-mediated inflammation and remodeling in severe asthma and to investigate ßc antagonism as a therapeutic strategy for mixed granulocytic airway disease. METHODS: ßc gene expression was analyzed in bronchial biopsy specimens from patients with mild-to-moderate and severe asthma. House dust mite extract and Aspergillus fumigatus extract (ASP) models were used to establish asthma-like pathology and airway remodeling in human ßc transgenic mice. Lung tissue gene expression was analyzed by RNA sequencing. The mAb CSL311 targeting the shared cytokine binding site of ßc was used to block ßc signaling. RESULTS: ßc gene expression was increased in patients with severe asthma. CSL311 potently reduced lung neutrophils, eosinophils, and interstitial macrophages and improved airway pathology and lung function in the acute steroid-resistant house dust mite extract model. Chronic intranasal ASP exposure induced airway inflammation and fibrosis and impaired lung function that was inhibited by CSL311. CSL311 normalized the ASP-induced fibrosis-associated extracellular matrix gene expression network and strongly reduced signatures of cellular inflammation in the lung. CONCLUSIONS: ßc cytokines drive steroid-resistant mixed myeloid cell airway inflammation and fibrosis. The anti-ßc antibody CSL311 effectively inhibits mixed T2/neutrophilic inflammation and severe asthma-like pathology and reverses fibrosis gene signatures induced by exposure to commonly encountered environmental allergens.

IgE receptor of mast cells signals mediator release and inflammation via adaptor protein 14-3-3ζ.

BACKGROUND: Mast cells (MCs) are tissue-resident immune cells that mediate IgE-dependent allergic responses. Downstream of FcεRI, an intricate network of receptor-specific signaling pathways and adaptor proteins govern MC function. The 14-3-3 family of serine-threonine phosphorylation-dependent adapter proteins are known to organize intracellular signaling. However, the role of 14-3-3 in IgE-dependent activation remains poorly defined. OBJECTIVE: We sought to determine whether 14-3-3 proteins are required for IgE-dependent MC activation and whether 14-3-3 is a viable target for the treatment of MC-mediated inflammatory diseases. METHODS: Genetic manipulation of 14-3-3ζ expression in human and mouse MCs was performed and IgE-dependent mediator release assessed. Pharmacologic inhibitors of 14-3-3 and 14-3-3ζ knockout mice were used to assess 14-3-3ζ function in a MC-dependent in vivo passive cutaneous anaphylaxis (PCA) model of allergic inflammation. Expression and function of 14-3-3ζ were assessed in human nasal polyp tissue MCs. RESULTS: IgE-dependent mediator release from human MCs was decreased by 14-3-3ζ knockdown and increased by 14-3-3ζ overexpression. Deletion of the 14-3-3ζ gene decreased IgE-dependent activation of mouse MCs in vitro and PCA responses in vivo. Furthermore, the 14-3-3 inhibitor, RB-11, which impairs dimerization of 14-3-3, inhibited cultured MC and polyp tissue MC activation and signaling downstream of the FcεRI receptor and dose-dependently attenuated PCA responses. CONCLUSION: IgE/FcεRI-mediated MC activation is positively regulated by 14-3-3ζ. We identify a critical role for this p-Ser/Thr-binding protein in the regulation of MC FcεRI signaling and IgE-dependent immune responses and show that this pathway may be amenable to pharmacologic targeting.

Translating the biology of ß common receptor-engaging cytokines into clinical medicine.

The family of cytokines that comprises IL-3, IL-5, and GM-CSF was discovered over 30 years ago, and their biological activities and resulting impact in clinical medicine has continued to expand ever since. Originally identified as bone marrow growth factors capable of acting on hemopoietic progenitor cells to induce their proliferation and differentiation into mature blood cells, these cytokines are also recognized as key mediators of inflammation and the pathobiology of diverse immunologic diseases. This increased understanding of the functional repertoire of IL-3, IL-5, and GM-CSF has led to an explosion of interest in modulating their functions for clinical management. Key to the successful clinical translation of this knowledge is the recognition that these cytokines act by engaging distinct dimeric receptors and that they share a common signaling subunit called ß-common or ßc. The structural determination of how IL-3, IL-5, and GM-CSF interact with their receptors and linking this to their differential biological functions on effector cells has unveiled new paradigms of cell signaling. This knowledge has paved the way for novel mAbs and other molecules as selective or pan inhibitors for use in different clinical settings.

Do more with Less: Improving High Parameter Cytometry Through Overnight Staining.

Recent advances in flow cytometry have allowed high-dimensional characterization of biological phenomena, enabling breakthroughs in a multitude of fields. Despite the appreciation of the unique properties of antigens and fluorophores in high-parameter panel design, staining conditions are often standardized for short surface stains, regardless of antibody affinity or antigen accessibility. Here, we demonstrate how increasing antibody incubation times can lead to substantial improvements in sensitivity, maintaining specificity, and reducing background, while also significantly reducing the costs of high-parameter cytometry panels. Furthermore, overnight staining reduces the influence of interexperimental variability, assisting accurate pooling over experiments over extended time courses. We provide guidance on how to optimize staining conditions for diverse antigens, including how different fixation strategies can affect epitope accessibility. Overnight staining can thus substantially improve the resolution, repeatability, and cost-effectiveness of high-parameter cytometry. © 2022 The Authors. Current Protocols published by Wiley Periodicals LLC.

Blocking the human common beta subunit of the GM-CSF, IL-5 and IL-3 receptors markedly reduces hyperinflammation in ARDS models.

Acute respiratory distress syndrome (ARDS) is triggered by various aetiological factors such as trauma, sepsis and respiratory viruses including SARS-CoV-2 and influenza A virus. Immune profiling of severe COVID-19 patients has identified a complex pattern of cytokines including granulocyte macrophage-colony stimulating factor (GM-CSF) and interleukin (IL)-5, which are significant mediators of viral-induced hyperinflammation. This strong response has prompted the development of therapies that block GM-CSF and other cytokines individually to limit inflammation related pathology. The common cytokine binding site of the human common beta (ßc) receptor signals for three inflammatory cytokines: GM-CSF, IL-5 and IL-3. In this study, ßc was targeted with the monoclonal antibody (mAb) CSL311 in engineered mice devoid of mouse ßc and ßIL-3 and expressing human ßc (hßcTg mice). Direct pulmonary administration of lipopolysaccharide (LPS) caused ARDS-like lung injury, and CSL311 markedly reduced lung inflammation and oedema, resulting in improved oxygen saturation levels in hßcTg mice. In a separate model, influenza (HKx31) lung infection caused viral pneumonia associated with a large influx of myeloid cells into the lungs of hßcTg mice. The therapeutic application of CSL311 potently decreased accumulation of monocytes/macrophages, neutrophils, and eosinophils without altering lung viral loads. Furthermore, CSL311 treatment did not limit the viral-induced expansion of NK and NKT cells, or the tissue expression of type I/II/III interferons needed for efficient viral clearance. Simultaneously blocking GM-CSF, IL-5 and IL-3 signalling with CSL311 may represent an improved and clinically applicable strategy to reducing hyperinflammation in the ARDS setting.

Immunisation with the BCG and DTPw vaccines induces different programs of trained immunity in mice.

In addition to providing pathogen-specific immunity, vaccines can also confer nonspecific effects (NSEs) on mortality and morbidity unrelated to the targeted disease. Immunisation with live vaccines, such as the BCG vaccine, has generally been associated with significantly reduced all-cause infant mortality. In contrast, some inactivated vaccines, such as the diphtheria, tetanus, whole-cell pertussis (DTPw) vaccine, have been controversially associated with increased all-cause mortality especially in female infants in high-mortality settings. The NSEs associated with BCG have been attributed, in part, to the induction of trained immunity, an epigenetic and metabolic reprograming of innate immune cells, increasing their responsiveness to subsequent microbial encounters. Whether non-live vaccines such as DTPw induce trained immunity is currently poorly understood. Here, we report that immunisation of mice with DTPw induced a unique program of trained immunity in comparison to BCG immunised mice. Altered monocyte and DC cytokine responses were evident in DTPw immunised mice even months after vaccination. Furthermore, splenic cDCs from DTPw immunised mice had altered chromatin accessibility at loci involved in immunity and metabolism, suggesting that these changes were epigenetically mediated. Interestingly, changing the order in which the BCG and DTPw vaccines were co-administered to mice altered subsequent trained immune responses. Given these differences in trained immunity, we also assessed whether administration of these vaccines altered susceptibility to sepsis in two different mouse models. Immunisation with either BCG or a DTPw-containing vaccine prior to the induction of sepsis did not significantly alter survival. Further studies are now needed to more fully investigate the potential consequences of DTPw induced trained immunity in different contexts and to assess whether other non-live vaccines also induce similar changes.

Targeting the Human ßc Receptor Inhibits Contact Dermatitis in a Transgenic Mouse Model.

Allergic contact dermatitis (ACD) is a prevalent and poorly controlled inflammatory disease caused by skin infiltration of T cells and granulocytes. The beta common (ßc) cytokines GM-CSF, IL-3, and IL-5 are powerful regulators of granulocyte function that signal through their common receptor subunit ßc, a property that has made ßc an attractive target to simultaneously inhibit these cytokines. However, the species specificity of ßc has precluded testing of inhibitors of human ßc in mouse models. To overcome this problem, we developed a human ßc receptor transgenic mouse strain with a hematopoietic cell‒specific expression of human ßc instead of mouse ßc. Human ßc receptor transgenic cells responded to mouse GM-CSF and IL-5 but not to IL-3 in vitro and developed tissue pathology and cellular inflammation comparable with those in wild-type mice in a model of ACD. Similarly, Il3-/- mice developed ACD pathology comparable with that of wild-type mice. Importantly, the blocking anti-human ßc antibody CSL311 strongly suppressed ear pinna thickening and histopathological changes typical of ACD and reduced accumulation of neutrophils, mast cells, and eosinophils in the skin. These results show that GM-CSF and IL-5 but not IL-3 are major mediators of ACD and define the human ßc receptor transgenic mouse as a unique platform to test the inhibitors of ßc in vivo.

ACC1-expressing pathogenic T helper 2 cell populations facilitate lung and skin inflammation in mice.

T cells possess distinguishing effector functions and drive inflammatory disorders. We have previously identified IL-5-producing Th2 cells as the pathogenic population predominantly involved in the pathology of allergic inflammation. However, the cell-intrinsic signaling pathways that control the pathogenic Th2 cell function are still unclear. We herein report the high expression of acetyl-CoA carboxylase 1 (ACC1) in the pathogenic CD4+ T cell population in the lung and skin. The genetic deletion of CD4+ T cell-intrinsic ACC1 dampened eosinophilic and basophilic inflammation in the lung and skin by constraining IL-5 or IL-3 production. Mechanistically, ACC1-dependent fatty acid biosynthesis induces the pathogenic cytokine production of CD4+ T cells via metabolic reprogramming and the availability of acetyl-CoA for epigenetic regulation. We thus identified a distinct phenotype of the pathogenic T cell population in the lung and skin, and ACC1 was shown to be an essential regulator controlling the pathogenic function of these populations to promote type 2 inflammation.

Understanding mast cell heterogeneity at single cell resolution.

Mast cells (MC)s are evolutionarily conserved, tissue-resident immune cells with diverse roles in allergy, cancer, and protection from infection by helminths and microorganisms. The significant diversity in MC development and tissue-specific functional characteristics has recently begun to be understood. Exciting developments in single-cell-based RNA, protein, and chromatin profiling technologies offer new opportunities to characterize MC heterogeneity and to uncover novel MC functions and subtypes; these developments might lead to new and clinically effective therapies for certain pathologies. In this review, we provide an overview of the current understanding of MC development and heterogeneity and discuss new insights gained from single-cell-based studies that may lead to future research directions and therapeutic opportunities.

A deep convolutional neural network for segmentation of whole-slide pathology images identifies novel tumour cell-perivascular niche interactions that are associated with poor survival in glioblastoma.

BACKGROUND: Glioblastoma is the most aggressive type of brain cancer with high-levels of intra- and inter-tumour heterogeneity that contribute to its rapid growth and invasion within the brain. However, a spatial characterisation of gene signatures and the cell types expressing these in different tumour locations is still lacking. METHODS: We have used a deep convolutional neural network (DCNN) as a semantic segmentation model to segment seven different tumour regions including leading edge (LE), infiltrating tumour (IT), cellular tumour (CT), cellular tumour microvascular proliferation (CTmvp), cellular tumour pseudopalisading region around necrosis (CTpan), cellular tumour perinecrotic zones (CTpnz) and cellular tumour necrosis (CTne) in digitised glioblastoma histopathological slides from The Cancer Genome Atlas (TCGA). Correlation analysis between segmentation results from tumour images together with matched RNA expression data was performed to identify genetic signatures that are specific to different tumour regions. RESULTS: We found that spatially resolved gene signatures were strongly correlated with survival in patients with defined genetic mutations. Further in silico cell ontology analysis along with single-cell RNA sequencing data from resected glioblastoma tissue samples showed that these tumour regions had different gene signatures, whose expression was driven by different cell types in the regional tumour microenvironment. Our results further pointed to a key role for interactions between microglia/pericytes/monocytes and tumour cells that occur in the IT and CTmvp regions, which may contribute to poor patient survival. CONCLUSIONS: This work identified key histopathological features that correlate with patient survival and detected spatially associated genetic signatures that contribute to tumour-stroma interactions and which should be investigated as new targets in glioblastoma. The source codes and datasets used are available in GitHub: https://github.com/amin20/GBM_WSSM .

Short-term Oral Steroids Significantly Improves Chronic Rhinosinusitis Without Nasal Polyps.

OBJECTIVES/HYPOTHESIS: The efficacy of short-term oral corticosteroids in chronic rhinosinusitis without nasal polyps (CRSsNP) is unknown. The aim of this controlled study was to assess the immediate and long-term outcomes from a short course of a commonly used oral corticosteroid, prednisolone, in well-defined CRSsNP patients. STUDY DESIGN: Prospective, observational controlled study. METHODS: A prospective-controlled study of CRSsNP patients treated with prednisolone at 0.5 mg/kg tapered over 10 days and non-prednisolone treated CRSsNP patients (controls) and follow-up at 2, 6, and 12 months. Baseline and follow-up SinoNasal Outcome Test (SNOT)-22, nasal endoscopy (Lund-Kennedy), and sinus CT scan scores (Lund-Mackay) were compared. RESULTS: At 2 months, there was a significant improvement in the SNOT-22, nasal endoscopy, and sinus CT scan scores in the prednisolone group (P < .0001) compared with controls (p = ns, Mann-Whitney U test). 52.5% of prednisolone-treated CRSsNP patients had improved symptoms and did not require sinus surgery at 12 months compared with 14.3% of controls (P < .001). Side-effects were reported in 8.9% of prednisolone-treated patients. Patients who benefited from prednisolone had a median symptom duration of 7.25 (99% confidence, upper limit of 11) months compared with 18 months in those requiring surgery. CONCLUSIONS: Short-term oral prednisolone significantly improved all three clinical measures of disease in CRSsNP patients and avoided surgical intervention in 52.5% patients in the first 12 months. Patients with symptoms for less than 11 months were most likely to benefit. The side-effects of oral steroids require careful consideration and further studies are needed to ascertain appropriate dosage and treatment duration. LEVEL OF EVIDENCE: 3 Laryngoscope, 131:E2618-E2626, 2021.

The immunotoxicity, but not anti-tumor efficacy, of anti-CD40 and anti-CD137 immunotherapies is dependent on the gut microbiota.

Immune agonist antibodies (IAAs) are promising immunotherapies that target co-stimulatory receptors to induce potent anti-tumor immune responses, particularly when combined with checkpoint inhibitors. Unfortunately, their clinical translation is hampered by serious dose-limiting, immune-mediated toxicities, including high-grade and sometimes fatal liver damage, cytokine release syndrome (CRS), and colitis. We show that the immunotoxicity, induced by the IAAs anti-CD40 and anti-CD137, is dependent on the gut microbiota. Germ-free or antibiotic-treated mice have significantly reduced colitis, CRS, and liver damage following IAA treatment compared with conventional mice or germ-free mice recolonized via fecal microbiota transplant. MyD88 signaling is required for IAA-induced CRS and for anti-CD137-induced, but not anti-CD40-induced, liver damage. Importantly, antibiotic treatment does not impair IAA anti-tumor efficacy, alone or in combination with anti-PD1. Our results suggest that microbiota-targeted therapies could overcome the toxicity induced by IAAs without impairing their anti-tumor activity.

Essential Role for CD30-Transglutaminase 2 Axis in Memory Th1 and Th17 Cell Generation.

Memory helper T (Th) cells are crucial for secondary immune responses against infectious microorganisms but also drive the pathogenesis of chronic inflammatory diseases. Therefore, it is of fundamental importance to understand how memory T cells are generated. However, the molecular mechanisms governing memory Th cell generation remain incompletely understood. Here, we identified CD30 as a molecule heterogeneously expressed on effector Th1 and Th17 cells, and CD30hi effector Th1 and Th17 cells preferentially generated memory Th1 and Th17 cells. We found that CD30 mediated signal induced Transglutaminase-2 (TG2) expression, and that the TG2 expression in effector Th cells is essential for memory Th cell generation. In fact, Cd30-deficiency resulted in the impaired generation of memory Th1 and Th17 cells, which can be rescued by overexpression of TG2. Furthermore, transglutaminase-2 (Tgm2)-deficient CD4 T cells failed to become memory Th cells. As a result, T cells from Tgm2-deficient mice displayed impaired antigen-specific antibody production and attenuated Th17-mediated allergic responses. Our data indicate that CD30-induced TG2 expression in effector Th cells is essential for the generation of memory Th1 and Th17 cells, and that CD30 can be a marker for precursors of memory Th1 and Th17 cells.

CD103hi Treg cells constrain lung fibrosis induced by CD103lo tissue-resident pathogenic CD4 T cells.

Tissue-resident memory T cells (TRM cells) are crucial mediators of adaptive immunity in nonlymphoid tissues. However, the functional heterogeneity and pathogenic roles of CD4+ TRM cells that reside within chronic inflammatory lesions remain unknown. We found that CD69hiCD103lo CD4+ TRM cells produced effector cytokines and promoted the inflammation and fibrotic responses induced by chronic exposure to Aspergillus fumigatus. Simultaneously, immunosuppressive CD69hiCD103hiFoxp3+ CD4+ regulatory T cells were induced and constrained the ability of pathogenic CD103lo TRM cells to cause fibrosis. Thus, lung tissue-resident CD4+ T cells play crucial roles in the pathology of chronic lung inflammation, and CD103 expression defines pathogenic effector and immunosuppressive tissue-resident cell subpopulations in the inflamed lung.

The role of invariant T cells in inflammation of the skin and airways.

Invariant and semi-invariant T cells are emerging as important regulators of host environment interactions at barrier tissues such as the airway and skin. In contrast to conventional T cells, invariant natural killer T (iNKT) cells and mucosal associated invariant T (MAIT) cells express T cell receptors of very limited diversity. iNKT and MAIT cells recognise antigens presented by the MHC class 1-like monomorphic molecules CD1d and MR1, respectively. Both iNKT cells and MAIT cells have been identified in the skin and airways and can rapidly produce cytokines after activation. Numerous studies have implicated iNKT cells in the pathology of both skin and airway disease, but conflicting evidence in human disease means that more studies are necessary to resolve the exact roles of iNKT in inflammation. The functions of MAIT cells in skin and lung inflammation are even less well defined. We herein describe the current literature on iNKT and MAIT cells in allergic and non-allergic skin diseases (dermatitis and psoriasis) and airway diseases (asthma, chronic obstructive pulmonary disease, rhinitis, and chronic rhinosinusitis).

Enhanced Cell Division Is Required for the Generation of Memory CD4 T Cells to Migrate Into Their Proper Location.

CD4 T cell memory is fundamental for long-lasting immunity and effective secondary responses following infection or vaccination. We have previously found that memory CD4 T cells specific for systemic antigens preferentially reside in the bone marrow (BM) and arise from splenic CD49b+T-bet+ CD4 T cells. However, how BM-homing memory precursors are generated during an immune reaction is unknown. We show here that BM memory precursors are generated via augmented rates of cell division throughout a primary immune response. Treatment with the cytostatic drug cyclophosphamide or blockade of the CD28/B7 co-stimulatory pathway at the beginning of the contraction phase abrogates the generation of BM memory precursors. We determine that, following a critical number of cell divisions, memory precursors downregulate CCR7 and upregulate IL-2Rß, indicating that loss of CCR7 and gain of IL-2 signal are required for the migration of memory precursors toward the BM.