Regulatory T Cell Specificity Directs Tolerance versus Allergy against Aeroantigens in Humans.

FOXP3+ regulatory T cells (Tregs) maintain tolerance against self-antigens and innocuous environmental antigens. However, it is still unknown whether Treg-mediated tolerance is antigen specific and how Treg specificity contributes to the selective loss of tolerance, as observed in human immunopathologies such as allergies. Here, we used antigen-reactive T cell enrichment to identify antigen-specific human Tregs. We demonstrate dominant Treg-mediated tolerance against particulate aeroallergens, such as pollen, house dust mites, and fungal spores. Surprisingly, we found no evidence of functional impairment of Treg responses in allergic donors. Rather, major allergenic proteins, known to rapidly dissociate from inhaled allergenic particles, have a generally reduced capability to generate Treg responses. Most strikingly, in individual allergic donors, Th2 cells and Tregs always target disparate proteins. Thus, our data highlight the importance of Treg antigen-specificity for tolerance in humans and identify antigen-specific escape from Treg control as an important mechanism enabling antigen-specific loss of tolerance in human allergy.

Induction of IL-10-producing type 2 innate lymphoid cells by allergen immunotherapy is associated with clinical response.

The role of innate immune cells in allergen immunotherapy that confers immune tolerance to the sensitizing allergen is unclear. Here, we report a role of interleukin-10-producing type 2 innate lymphoid cells (IL-10+ ILC2s) in modulating grass-pollen allergy. We demonstrate that KLRG1+ but not KLRG1- ILC2 produced IL-10 upon activation with IL-33 and retinoic acid. These cells attenuated Th responses and maintained epithelial cell integrity. IL-10+ KLRG1+ ILC2s were lower in patients with grass-pollen allergy when compared to healthy subjects. In a prospective, double-blind, placebo-controlled trial, we demonstrated that the competence of ILC2 to produce IL-10 was restored in patients who received grass-pollen sublingual immunotherapy. The underpinning mechanisms were associated with the modification of retinol metabolic pathway, cytokine-cytokine receptor interaction, and JAK-STAT signaling pathways in the ILCs. Altogether, our findings underscore the contribution of IL-10+ ILC2s in the disease-modifying effect by allergen immunotherapy.

Mechanisms for initiation of food allergy by skin pre-disposed to atopic dermatitis.

Food allergy can be life-threatening and often develops early in life. In infants and children, loss-of-function mutations in skin barrier genes associate with food allergy. In a mouse model with skin barrier mutations (Flakey Tail, FT+/- mice), topical epicutaneous sensitization to a food allergen peanut extract (PNE), an environmental allergen Alternaria alternata (Alt) and a detergent induce food allergy and then an oral PNE-challenge induces anaphylaxis. Exposures to these allergens and detergents can occur for infants and children in a household setting. From the clinical and preclinical studies of neonates and children with skin barrier mutations, early oral exposure to allergenic foods before skin sensitization may induce tolerance to food allergens and thus protect against development of food allergy. In the FT+/- mice, oral food allergen prior to skin sensitization induce tolerance to food allergens. However, when the skin of FT+/- pups are exposed to a ubiquitous environmental allergen at the time of oral consumption of food allergens, this blocks the induction of tolerance to the food allergen and the mice can then be skin sensitized with the food allergen. The development of food allergy in neonatal FT+/- mice is mediated by altered skin responses to allergens with increases in skin expression of interleukin 33, oncostatin M and amphiregulin. The development of neonate food allergy is enhanced when born to an allergic mother, but it is inhibited by maternal supplementation with α-tocopherol. Moreover, preclinical studies suggest that food allergen skin sensitization can occur before manifestation of clinical features of atopic dermatitis. Thus, these parameters may impact design of clinical studies for food allergy, when stratifying individuals by loss of skin barrier function or maternal atopy before offspring development of atopic dermatitis.

Whence and wherefore IgE?

Despite the near ubiquitous presence of Ig-based antibodies in vertebrates, IgE is unique to mammals. How and why it emerged remains mysterious. IgE expression is greatly constrained compared to other IgH isotypes. While other IgH isotypes are relatively abundant, soluble IgE has a truncated half-life, and IgE plasma cells are mostly short-lived. Despite its rarity, IgE is consequential and can trigger life-threatening anaphylaxis. IgE production reflects a dynamic steady state with IgG memory B cells feeding short-lived IgE production. Emerging evidence suggests that IgE may also potentially be produced in longer-lived plasma cells as well, perhaps as an aberrancy stemming from its evolutionary roots from an antibody isotype that likely functioned more like IgG. As a late derivative of an ancient systemic antibody system, the benefits of IgE in mammals likely stems from the antibody system's adaptive recognition and response capability. However, the tendency for massive, systemic, and long-lived production, common to IgH isotypes like IgG, were likely not a good fit for IgE. The evolutionary derivation of IgE from an antibody system that for millions of years was good at antigen de-sensitization to now functioning as a highly specialized antigen-sensitization function required heavy restrictions on antibody production-insufficiency of which may contribute to allergic disease.

The Allergen Der p3 from House Dust Mite Stimulates Store-Operated Ca2+ Channels and Mast Cell Migration through PAR4 Receptors.

The house dust mite is the principal source of perennial aeroallergens in man. How these allergens activate innate and adaptive immunity is unclear, and therefore, there are no therapies targeting mite allergens. Here, we show that house dust mite extract activates store-operated Ca2+ channels, a common signaling module in numerous cell types in the lung. Activation of channel pore-forming Orai1 subunits by mite extract requires gating by STIM1 proteins. Although mite extract stimulates both protease-activated receptor type 2 (PAR2) and PAR4 receptors, Ca2+ influx is more tightly coupled to the PAR4 pathway. We identify a major role for the serine protease allergen Der p3 in stimulating Orai1 channels and show that a therapy involving sub-maximal inhibition of both Der p3 and Orai1 channels suppresses mast cell activation to house dust mite. Our results reveal Der p3 as an important aeroallergen that activates Ca2+ channels and suggest a therapeutic strategy for treating mite-induced asthma.

DeepAlgPro: an interpretable deep neural network model for predicting allergenic proteins.

Allergies have become an emerging public health problem worldwide. The most effective way to prevent allergies is to find the causative allergen at the source and avoid re-exposure. However, most of the current computational methods used to identify allergens were based on homology or conventional machine learning methods, which were inefficient and still had room to be improved for the detection of allergens with low homology. In addition, few methods based on deep learning were reported, although deep learning has been successfully applied to several tasks in protein sequence analysis. In the present work, a deep neural network-based model, called DeepAlgPro, was proposed to identify allergens. We showed its great accuracy and applicability to large-scale forecasts by comparing it to other available tools. Additionally, we used ablation experiments to demonstrate the critical importance of the convolutional module in our model. Moreover, further analyses showed that epitope features contributed to model decision-making, thus improving the model's interpretability. Finally, we found that DeepAlgPro was capable of detecting potential new allergens. Overall, DeepAlgPro can serve as powerful software for identifying allergens.

The role of the skin in the atopic march.

Atopic diseases, including atopic dermatitis (AD), food allergy (FA), asthma, and allergic rhinitis (AR) are closely related to inflammatory diseases involving different body sites (i.e. the skin, airway, and digestive tract) with characteristic features including specific IgE to allergens (so-called "atopy") and Th2 cell-mediated inflammation. It has been recognized that AD often precedes the development of other atopic diseases. The progression from AD during infancy to FA or asthma/AR in later childhood is referred to as the "atopic march" (AM). Clinical, genetic, and experimental studies have provided evidence that allergen sensitization occurring through AD skin could be the origin of the AM. Here, we provide an updated review focusing on the role of the skin in the AM, from genetic mutations and environmental factors associated with epidermal barrier dysfunction in AD and the AM to immunological mechanisms for skin sensitization, particularly recent progress on the function of key cytokines produced by epidermal keratinocytes or by immune cells infiltrating the skin during AD. We also highlight the importance of developing strategies that target AD skin to prevent and attenuate the AM.

Reduction of circulating IgE and allergens by a pH-sensitive antibody with enhanced FcγRIIb binding.

Allergen-crosslinked IgE triggers allergy by interacting with its receptor on basophils and mast cells. The anti-IgE monoclonal antibody omalizumab can alleviate allergy by competing with the receptor for IgE binding. However, along with neutralization, omalizumab also inhibits IgE degradation, which is clinically associated with high-dose and total IgE accumulation problems. In this study, we have developed an IgE-eliminating antibody on the basis of omalizumab, which has pH-dependent Fabs and an Fc with high affinity for FcγRIIb. In mice, the antibody rapidly eliminated total serum IgE to baseline levels and caused lower free IgE levels than omalizumab. At low dosages, the antibody also exhibited favorable IgE elimination effects. In addition, the antibody can degrade the corresponding allergen with the removal of IgE, addressing the allergy from its source. Introduction of the M252Y/S254T/T256E (YTE) mutation into this antibody prolongs its serum half-life without reducing potency. Thus, this engineered antibody holds a promising therapeutic option for allergy patients. Mechanistic insights are also included in this study.

Subcutaneous immunotherapy for bee venom allergy induces epitope spreading and immunophenotypic changes in allergen-specific memory B cells.

BACKGROUND: Allergen immunotherapy (AIT) is the only disease-modifying treatment for allergic disorders. We have recently discovered that allergen-specific memory B cells (Bmem) are phenotypically altered after 4 months of sublingual AIT for ryegrass pollen allergy. Whether these effects are shared with subcutaneous allergen immunotherapy (SCIT) and affect the epitope specificity of Bmem remain unknown. OBJECTIVE: The study aimed to evaluate the phenotype and antigen receptor sequences of Bmem specific to the major bee venom (BV) allergen Api m 1 before and after ultra-rush SCIT for BV allergy. METHODS: Recombinant Api m 1 protein tetramers were generated to evaluate basophil activation in a cohort of individuals with BV allergy before and after BV SCIT. Comprehensive flow cytometry was performed to evaluate and purify Api m 1-specific Bmem. Immunoglobulin genes from single Api m 1-specific Bmem were sequenced and structurally modeled onto Api m 1. RESULTS: SCIT promoted class switching of Api m 1-specific Bmem to IgG2 and IgG4 with increased expression of CD23 and CD29. Furthermore, modeling of Api m 1-specific immunoglobulin from Bmem identified a suite of possible new and diverse allergen epitopes on Api m 1 and highlighted epitopes that may preferentially be bound by immunoglobulin after SCIT. CONCLUSIONS: AIT induces shifting of epitope specificity and phenotypic changes in allergen-specific Bmem.

Distinct proteomes and allergen profiles appear across the life-cycle stages of Alternaria alternata.

BACKGROUND: Alternaria alternata is associated with allergic respiratory diseases, which can be managed with allergen extract-based diagnostics and immunotherapy. It is not known how spores and hyphae contribute to allergen content. Commercial allergen extracts are manufactured by extracting proteins without separating the different forms of the fungus. OBJECTIVE: We sought to determine differences between spore and hyphae proteomes and how allergens are distributed in Aalternata. METHODS: Data-independent acquisition mass spectrometry was used to quantitatively compare the proteomes of asexual spores (nongerminating and germinating) with vegetative hyphae. RESULTS: We identified 4515 proteins in nongerminating spores, germinating spores, and hyphae; most known allergens are more abundant in nongerminating spores. On comparing significant protein fold-change differences between nongerminating spores and hyphae, we found that 174 proteins were upregulated in nongerminating spores and 80 proteins in hyphae. Among the spore proteins are ones functionally involved in cell wall synthesis, responding to cellular stress, and maintaining redox balance and homeostasis. On comparing nongerminating and germinating spores, 25 proteins were found to be upregulated in nongerminating spores and 54 in germinating spores. Among the proteins specific to germinating spores were proteases known to be virulence factors. One of the most abundant proteins in the spore proteome is sialidase, which has not been identified as an allergen but may be important in the pathogenicity of this fungus. Major allergen Alt a 1 is present at low levels in spores and hyphae and appears to be largely secreted into growth media. CONCLUSIONS: Spores and hyphae express overlapping but distinct proteomes. Most known allergens are found more abundantly in nongerminating spores.

A pediatric randomized, controlled trial of German cockroach subcutaneous immunotherapy.

BACKGROUND: Cockroach allergy contributes to morbidity among urban children with asthma. Few trials address the effect of subcutaneous immunotherapy (SCIT) with cockroach allergen among these at-risk children. OBJECTIVES: We sought to determine whether nasal allergen challenge (NAC) responses to cockroach allergen would improve following 1 year of SCIT. METHODS: Urban children with asthma, who were cockroach-sensitized and reactive on NAC, participated in a year-long randomized double-blind placebo-controlled SCIT trial using German cockroach extract. The primary endpoint was the change in mean Total Nasal Symptom Score (TNSS) during NAC after 12 months of SCIT. Changes in nasal transcriptomic responses during NAC, skin prick test wheal size, serum allergen-specific antibody production, and T-cell responses to cockroach allergen were assessed. RESULTS: Changes in mean NAC TNSS did not differ between SCIT-assigned (n = 28) versus placebo-assigned (n = 29) participants (P = .63). Nasal transcriptomic responses correlated with TNSS, but a treatment effect was not observed. Cockroach serum-specific IgE decreased to a similar extent in both groups, while decreased cockroach skin prick test wheal size was greater among SCIT participants (P = .04). A 200-fold increase in cockroach serum-specific IgG4 was observed among subjects receiving SCIT (P < .001) but was unchanged in the placebo group. T-cell IL-4 responses following cockroach allergen stimulation decreased to a greater extent among SCIT versus placebo (P = .002), while no effect was observed for IL-10 or IFN-γ. CONCLUSIONS: A year of SCIT failed to alter NAC TNSS and nasal transcriptome responses to cockroach allergen challenge despite systemic effects on allergen-specific skin tests, induction of serum-specific IgG4 serum production and down-modulation of allergen-stimulated T-cell responses.

Engineering nanoparticle therapeutics for food allergy.

Food allergy is a growing public health issue among children and adults that can lead to life-threatening anaphylaxis following allergen exposure. The criterion standard for disease management includes food avoidance and emergency epinephrine administration because current allergen-specific immunotherapy treatments are limited by adverse events and unsustained desensitization. A promising approach to remedy these shortcomings is the use of nanoparticle-based therapies that disrupt disease-driving immune mechanisms and induce more sustained tolerogenic immune pathways. The pathophysiology of food allergy includes multifaceted interactions between effector immune cells, including lymphocytes, antigen-presenting cells, mast cells, and basophils, mainly characterized by a TH2 cell response. Regulatory T cells, TH1 cell responses, and suppression of other major allergic effector cells have been found to be major drivers of beneficial outcomes in these nanoparticle therapies. Engineered nanoparticle formulations that have shown efficacy at reducing allergic responses and revealed new mechanisms of tolerance include polymeric-, lipid-, and emulsion-based nanotherapeutics. This review highlights the recent engineering design of these nanoparticles, the mechanisms induced by them, and their future potential therapeutic targets.

Gene editing in allergic diseases: Identification of novel pathways and impact of deleting allergen genes.

Gene editing technology has emerged as a powerful tool in all aspects of health research and continues to advance our understanding of critical and essential elements in disease pathophysiology. The clustered regularly interspaced short palindromic repeats (CRISPR) gene editing technology has been used with precision to generate gene knockouts, alter genes, and identify genes that cause disease. The full spectrum of allergic/atopic diseases, in part because of shared pathophysiology, is ripe for studies with this technology. In this way, novel culprit genes are being identified and allow for manipulation of triggering allergens to reduce allergenicity and disease. Notwithstanding current limitations on precision and potential off-target effects, newer approaches are rapidly being introduced to more fully understand specific gene functions as well as the consequences of genetic manipulation. In this review, we examine the impact of editing technologies of novel genes relevant to peanut allergy and asthma as well as how gene modification of common allergens may lead to the deletion of allergenic proteins.

Nonspecific lipid-transfer proteins trigger TLR2 and NOD2 signaling and undergo ligand-dependent endocytosis in epithelial cells.

BACKGROUND: Allergens can cross the epithelial barrier to enter the body but how this cellular passage affects protein structures and the downstream interactions with the immune system are still open questions. OBJECTIVE: We sought to show the molecular details and the effects of 3 nonspecific lipid transfer proteins (nsLTPs; Mal d 3 [allergenic nsLTP1 from apple], Cor a 8 [allergenic nsLTP1 from hazelnut], and Pru p 3 [allergenic nsLTP1 from peach]) on epithelial cell uptake and transport. METHODS: We used fluorescent imaging, flow cytometry, and proteomic and lipidomic screenings to identify the mechanism involved in nsLTP cellular uptake and signaling on selected epithelial and transgenic cell lines. RESULTS: nsLTPs are transported across the epithelium without affecting cell membrane stability or viability, and allergen uptake was largely impaired by inhibition of clathrin-mediated endocytosis. Analysis of the lipidome associated with nsLTPs showed a wide variety of lipid ligands predicted to bind inside the allergen hydrophobic cavity. Importantly, the internalization of nsLTPs was contingent on these ligands in the protein complex. nsLTPs were found to initiate cellular signaling via Toll-like receptor 2 but not the cluster of differentiation 1 protein receptor, despite neither being essential for nsLTP endocytosis. We also provide evidence that the 3 allergens induced intracellular stress signaling through activation of the NOD2 pathway. CONCLUSIONS: Our work consolidates the current model on nsLTP-epithelial cell interplay and adds molecular details about cell transport and signaling. In addition, we have developed a versatile toolbox to extend these investigations to other allergens and cell types.

Precision engineering for localization, validation, and modification of allergenic epitopes.

The allergen-IgE interaction is essential for the genesis of allergic responses, yet investigation of the molecular basis of these interactions is in its infancy. Precision engineering has unveiled the molecular features of allergen-antibody interactions at the atomic level. High-resolution technologies, including x-ray crystallography, nuclear magnetic resonance spectroscopy, and cryo-electron microscopy, determine allergen-antibody structures. X-ray crystallography of an allergen-antibody complex localizes in detail amino acid residues and interactions that define the epitope-paratope interface. Multiple structures involving murine IgG mAbs have recently been resolved. The number of amino acids forming the epitope broadly correlates with the epitope area. The production of human IgE mAbs from B cells of allergic subjects is an exciting recent development that has for the first time enabled an actual IgE epitope to be defined. The biologic activity of defined IgE epitopes can be validated in vivo in animal models or by measuring mediator release from engineered basophilic cell lines. Finally, gene-editing approaches using the Clustered Regularly Interspaced Short Palindromic Repeats technology to either remove allergen genes or make targeted epitope engineering at the source are on the horizon. This review presents an overview of the identification and validation of allergenic epitopes by precision engineering.

Indoor allergen exposure and its association to upper respiratory infections and pulmonary outcomes among children with asthma.

BACKGROUND: Certain environmental allergen exposures are more common in disadvantaged communities and may contribute to differences in susceptibility to upper respiratory infections (URIs). OBJECTIVES: We examined associations between indoor allergens and: (1) URI; (2) URI + cold symptoms; (3) URI + cold symptoms + pulmonary eosinophilic inflammation (fraction of exhaled nitric oxide ≥20 ppb); and (4) URI + cold symptoms + reduced lung function (percent predicted forced expiratory volume in 1 second of <80%). METHODS: We used data from the Environmental Control as Add-on Therapy for Childhood Asthma (ECATCh) study. Allergen concentrations were measured in air (mouse) and settled dust (mouse, cockroach, dog, and cat). URI was determined by testing nasal mucus for upper respiratory viruses. We evaluated associations between allergen concentrations and URI-associated outcomes accounting for age, sex, study month, season, health insurance, and household size. RESULTS: Ninety participants (92% Black, 92% public insurance) with 192 observations were included; 52 (27%) of observations were positive for URI. A doubling in cockroach allergen concentration increased the odds of a URI with cold symptoms by 18% (odds ratio [OR] = 1.18, 95% confidence interval [CI], 0.99-1.40), the odds of a URI + cold symptoms + pulmonary eosinophilic inflammation by 31% (OR = 1.31, 95% CI, 1.10-1.57), and the odds of a URI + cold symptoms + reduced lung function by 45% (OR = 1.45, 95% CI, 1.13-1.85). Mouse allergen concentrations were positively associated with all outcomes. Associations were suggestively stronger among children sensitized to pest allergens. CONCLUSIONS: Cockroach and mouse, but not dog or cat, allergen exposure may predispose children with asthma to URIs with colds and lower respiratory outcomes.

B regulatory cells in allergy.

B cells have classically been recognized for their unique and indispensable role in the production of antibodies. Their potential as immunoregulatory cells with anti-inflammatory functions has received increasing attention during the last two decades. Herein, we highlight pioneering studies in the field of regulatory B cell (Breg) research. We will review the literature on Bregs with a particular focus on their role in the regulation of allergic inflammation.

Cell-Specific Contribution of IL4 Receptor α Signaling Shapes the Overall Manifestation of Allergic Airway Disease.

IL-4 and IL-13 play a critical role in allergic asthma pathogenesis via their common receptor, i.e., IL4Rα. However, the cell-specific role of IL4Rα in mixed allergens (MA)-induced allergic asthma has remained unclear. Therefore, we aimed to identify the cell-specific contribution of IL4Rα signaling in the manifestation of various pathological outcomes in mice with allergic airway disease. We compared MA-induced pathological outcomes between hematopoietic progenitor cells (HPCs)- or non-HPCs-specific IL4Rα-deficient chimera, myeloid cell-specific IL4Rα-deficient (LysMcre+/+/IL4Rαfl/fl), and airway epithelial cell-specific IL4Rα-deficient (CCSP-Cre+ /IL4Rαfl/fl) mice. Chimeric mice with systemic IL4Rα sufficiency displayed hallmark features of allergic asthma, including eosinophilic and lymphocytic infiltration, type 2 (Th2) cytokine/chemokine production, IgE production, and lung pathology. These features were markedly reduced in chimeric mice with systemic IL4Rα deficiency. Non-HPCs-specific IL4Rα-deficient mice displayed typical inflammatory features of allergic asthma but with markedly reduced mucous cell metaplasia (MCM). Deletion of IL4Rα signaling on airway epithelial cells, a subpopulation within the non-HPC lineage, resulted in almost complete absence of MCM. In contrast, all features of allergic asthma except for MCM and mucin production were mitigated in HPCs-specific IL4Rα-deficient chimeric mice. Deleting IL4Rα signaling in myeloid cells, a subpopulation within the HPC lineage, significantly alleviated MA-induced allergic airway inflammatory responses, but similar to the HPCs-specific IL4Rα-deficient chimeric mice, these mice showed significant MCM and mucin production. Our findings demonstrate that the differential allergen responsiveness seen in mice with HPCs-specific and non-HPCs-specific IL4Rα deficiency is predominantly driven by the absence of IL4Rα in myeloid cells and airway epithelial cells, respectively. Our findings also highlight distinct and mutually exclusive roles of IL4Rα signaling in mediating pathological outcomes within the myeloid and airway epithelial cell compartments.

Biological Sex Is an Effect Modifier of Allergen-Mediated Alteration of the Lung Proteome.

Asthma exhibits a distinct sex bias in the disease prevalence, severity, and response to therapy. However, sex-related differences in alterations of the lung proteome mediated by aeroallergens critical in asthma, such as house dust mites (HDM), remain unknown. In this study, we define sex-related differences in the lung proteome using an HDM-challenged mouse model by 1D LC-MS/MS. Sex-disaggregated data analysis showed that 406 proteins were uniquely altered in females, 273 proteins were uniquely altered in males, and 414 proteins were altered in both females and males in response to HDM. In a linear mixed model analysis, sex modified the HDM exposure effect for 163 proteins, i.e., a significant sex:exposure interaction was identified in 84 proteins in females and 35 proteins in males. Of these, 12 proteins showed a significant sex effect in both female and male lungs. We further selected 3 proteins Tjp1, Lamtor1, and G3BP2 for independent confirmation studies. Our findings detail the sex-specific lung proteome in response to an aeroallergen critical in asthma and demonstrate that sex is a significant effect modifier of HDM response. These results will serve as a valuable resource for delineating sex-specific mechanisms in aeroallergen-driven responses in asthma research.

NMR residual dipolar couplings investigation in the topology of house dust mite Group V allergens.

Blo t 5 is an important major allergen protein from Blomia tropicalis mites, which are prevalent in tropical and subtropical regions, including Taiwan. It is a coiled-coil triple helical bundle, but there currently is ambiguity around its structural fold and packing of the three helices. We have relied on NMR residual dipolar coupling data collected from four different alignment media to confirm that Blo t 5 has left-handed helical topology and further used that data to refine its solution structure. Earlier we had described conformational epitope for a detection monoclonal antibody by exclusive use of TROSY NMR experiments that studied Blo t 5 binding with the antibody FAB' fragment. Here, we confirm those findings with an extensive mutagenesis and biophysical study to validate the NMR epitope mapping approach proposed by us.