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Background: Immunological mechanism of action of allergoids remains poorly understood. Previous models of allergenicity and immunogenicity have yielded sub-optimal knowledge of these immunotherapeutic vaccine products. Novel single-cell RNA-seq technology offers a bridge to this gap in knowledge. Objective: To identify the underpinning tolerogenic molecular and cellular mechanisms of depigmented-polymerized Phleum pratense extract. Methods The molecular mechanisms underlying native Phleum pratense (Phl p), depigmented Phl p (DPG-Phl p), and depigmented-polymerized (DPG-POL-Phl p) allergoid were investigated using scRNA-seq. Allergen-specific Th2A, Tfh and IL-10+ Breg cells were quantified by flow cytometry in PBMCs from 16 grass pollen allergics (GPA) and 8 non-atopic controls (NAC). The ability of Phl p, DPG-Phl p and DPG-POL-Phl p to elicit FcεRI and FcεRII-mediated IgE responses was measured by basophil activation test and IgE-FAB assay. Results: ScRNA-seq analysis revealed that DPG-POL-Phl p downregulated genes associated with Th2 signaling, induced functional Tregs exhibiting immunosuppressive roles through CD52 and Siglec-10, modulated genes encoding immunoproteasome that dysregulate the processing and presentation of antigens to T cells and promoted a shift from IgE towards an IgA1 and IgG responses. In GPA, DPG-POL-Phl p exhibited reduced capacity to elicit proliferation of Th2A, IL-4+ Tfh and IL-21+ Tfh cells whilst being the most prominent at inducing CD19+CD5hiIL-10+ and CD19+CD5hiCD38intCD24intIL-10+ Breg cell subsets compared to Phl p (all, P

Background: Mechanisms causing the onset and perpetuation of inflammation in severe allergic patients remain unknown. Our previous studies suggested that severe allergic inflammation is linked to platelet dysfunction. Methods: Platelet-rich plasma (PRP) and platelet-poor plasma (PPP) samples were obtained by platelet-apheresis from severe (n = 7) and mild (n = 10) allergic patients and nonallergic subjects (n = 9) to perform platelet lipidomics by liquid chromatography coupled to mass spectrometry (LC–MS) and RNA-seq analysis. Significant metabolites and transcripts were used to identify compromised biological pathways in the severe phenotype. Platelet and inflammation-related proteins were quantified by Luminex. Results: Platelets from severe allergic patients were characterized by high levels of ceramides, phosphoinositols, phosphocholines, and sphingomyelins. In contrast, they showed a decrease in eicosanoid precursor levels. Biological pathway analysis performed with the significant lipids revealed the alteration of phospholipases, calcium-dependent events, and linolenic metabolism. RNAseq confirmed mRNA overexpression of genes related to platelet activation and arachidonic acid metabolism in the severe phenotypes. Pathway analysis indicated the alteration of NOD, MAPK, TLR, TNF, and IL-17 pathways in the severe phenotype. P-Selectin and IL-17AF proteins were increased in the severe phenotype. Conclusions: This study demonstrates that platelet lipid, mRNA, and protein content is different according to allergy severity. These findings suggest that platelet load is a potential source of biomarkers and a new chance for therapeutic targets in severe inflammatory pathologies. Acuerdo Transformativo - 2023

Rationale: Sensitization to Fel d 1 (Felis domesticus allergen 1) contributes to persistent allergic rhinitis and asthma. Existing treatment options for cat allergy, including allergen immunotherapy, are only moderately effective, and allergen immunotherapy has limited use because of safety concerns. Objectives: To explore the relationship among the pharmacokinetic, clinical, and immunological effects of anti–Fel d 1 monoclonal antibodies (REGN1908–1909) in patients after treatment. Methods: Patients received REGN1908–1909 (n = 36) or a placebo (n = 37) in a phase 1b study. Fel d 1–induced basophil and IgE-facilitated allergen binding responses were evaluated at baseline and Days 8, 29, and 85. Cytokine and chemokine concentrations in nasal fluids were measured, and REGN1908–1909 inhibition of allergen–IgE binding in patient serum was evaluated. Measurements and Main Results: Peak serum drug concentrations were concordant with maximal observed clinical response. The anti–Fel d 1 IgE/cat dander IgE ratio in pretreatment serum correlated with Total Nasal Symptom Score improvement. The allergen-neutralizing capacity of REGN1908–1909 was observed in serum and nasal fluid and was detected in an inhibition assay. Type 2 cytokines (IL-4, IL-5, and IL-13) and chemokines (CCL17/TARC, CCL5/RANTES [regulated upon activation, normal T-cell expressed and secreted]) in nasal fluid were inhibited in REGN1908–1909–treated patients compared with placebo (P < 0.05 for all); IL-13 and IL-5 concentrations correlated with Total Nasal Symptom Score improvement. Ex vivo assays demonstrated that REGN1908 and REGN1909 combined were more potent than each alone for inhibiting FcεRI- and FcεRII (CD23)–mediated allergic responses and subsequent T-cell activation. Conclusions: A single, passive-dose administration of Fel d 1–neutralizing IgG antibodies improved nasal symptoms in cat-allergic patients and was underscored by suppression of FcεRI-, FcεRII-, and T-helper cell type 2–mediated allergic responses. Clinical trial registered with www.clinicaltrials.gov (NCT02127801)

Long-term efficacy that occurs with allergen immunotherapy of proven value is associated with decreases in IgE-dependent activation of mast cells and tissue eosinophilia. This suppression of type 2 immunity is accompanied by early induction of regulatory T cells, immune deviation in favor of TH1 responses, and induction of local and systemic IgG, IgG4, and IgA antibodies. These "protective" antibodies can inhibit allergen-IgE complex formation and consequent mast cell triggering and IgE-facilitated TH2-cell activation. Recent studies have highlighted the importance of innate responses mediated by type 2 dendritic cells and innate lymphoid cells in allergic inflammation. These cell types are under the regulation of cytokines such as thymic stromal lymphopoietin and IL-33 derived from the respiratory epithelium. Novel subsets of regulatory cells induced by immunotherapy include IL-35–producing regulatory T cells, regulatory B cells, a subset of T follicular regulatory cells, and IL-10–producing group 2 innate lymphoid cells. These mechanisms point to biomarkers that require testing for their ability to predict clinical response to immunotherapy and to inform novel approaches for better efficacy, safety, and long-term tolerance.

Food allergy is an increasingly prevalent disease driven by uncontrolled type 2 immune response. Currently, knowledge about the underlying mechanisms that initiate and promote the immune response to dietary allergens is limited. Patients with food allergy are commonly sensitized through the skin in their early life, later on developing allergy symptoms within the gastrointestinal tract. Food allergy results from a dysregulated type 2 response to food allergens, characterized by enhanced levels of IgE, IL-4, IL-5, and IL-13 with infiltration of mast cells, eosinophils, and basophils. Recent studies raised a possible role for the involvement of innate lymphoid cells (ILCs) in driving food allergy. Unlike lymphocytes, ILCs lack They represent a group of lymphocytes that lack specific antigen receptors. ILCs contribute to immune responses not only by releasing cytokines and other mediators but also by responding to cytokines produced by activated cells in their local microenvironment. Due to their localization at barrier surfaces of the airways, gut, and skin, ILCs form a link between the innate and adaptive immunity. This review summarizes recent evidence on how skin and gastrointestinal mucosal immune system contribute to both homeostasis and the development of food allergy, as well as the involvement of ILCs toward inflammatory processes and regulatory mechanisms.

There are currently no biomarkers that can accurately predict clinical outcomes and segregate responders from nonresponders in allergen immunotherapy (AIT). Therefore, identifying a reliable predictive biomarker is essential to enable clinicians to tailor personalized therapy. New developments in AIT biomarkers are currently being explored, and it would be important to identify key areas of development and their feasibility for use in the clinic. Biomarkers can be categorized broadly into seven domains: (i) Immunoglobulin E (IgE), (ii) IgG and IgA responses, (iii) IgE -facilitated allergen binding/blocking factor, (iv) basophil activation, (v) cytokines and chemokines, (vi) cellular markers, and (vii) in vivo biomarkers. Despite their potential, most biomarkers remain infeasible to be translated to the clinical setting due to requirements of complex instruments such as flow cytometry. The identification of suitable biomarkers remains key in predicting outcomes of AIT and requires more research. Additional exploration into integrative biomarkers may be required.

Allergic rhinitis is an IgE-mediated inflammation that remains a clinical challenge, affecting 40% of the UK population with a wide range of severity from nasal discomfort to life-threatening anaphylaxis. It can be managed by pharmacotherapeutics and in selected patients by allergen immunotherapy (AIT), which provides long-term clinical efficacy, especially during peak allergy season. However, there are no definitive biomarkers for AIT efficacy. Here, we aim to summarize the key adaptive, innate, humoral, and metabolic advances in biomarker identification in response to AIT. Mechanisms of efficacy consist of an immune deviation towards T

Copyright © 2022 The Authors. Allergen immunotherapy (AIT) is an effective treatment for allergic rhinitis, inducing long-term clinical tolerance to the sensitizing allergen. Clinical tolerance induction can be achieved when AIT is administered for at least 3 years. AIT is associated with the modulation of innate and adaptive immune systems. This comprises inhibition of IgE-dependent activation of mast cells and basophils in the local target organ, suppression of TH2 cells, immune deviation toward TH1 cells, induction of T and B regulatory cells, and production of allergen-neutralizing antibodies. However, recent developments in their underpinning mechanisms have revealed that AIT, administered subcutaneously or sublingually, induces immune regulation through novel cell targets and molecular mechanisms. This comprehensive review discusses how immune tolerance driven by subcutaneous immunotherapy and sublingual immunotherapy is associated with the induction of a novel regulatory subset of innate lymphoid cells and suppression of proinflammatory TH2, allergen-specific TH2 (TH2A), and T follicular helper cells. Moreover, they are associated with exhaustion of TH2A cells and differential expression of nasal and systemic IgA antibodies. Uncovering the underpinning mechanisms of a successful AIT and immune tolerance induction will allow the development of targeted therapeutics for allergic rhinitis with and without asthma.

BACKGROUND: Unsupervised clustering of biomarkers derived from non-invasive samples such as nasal fluid is less evaluated as a tool for describing asthma endotypes. OBJECTIVE: To evauate whether protein expression in nasal fluid would identify distinct clusters of asthmatics with specific lower airway molecular phenotypes. METHODS: Unsupervised clustering of 168 nasal inflammatory and immune proteins and Shapley values was used to stratify 43 severe asthmatic patients (ENDANA) using a two 'modelling blocks' machine learning (ML) approach. This algorithm was also applied to nasal brushings transcriptomics from U-BIOPRED. Feature reduction and functional gene analysis were used to compare proteomic and transcriptomic clusters. Gene set variation analysis (GSVA) provided enrichment scores (ESs) of the ENDANA protein signature within U-BIOPRED sputum and blood. RESULTS: The nasal protein ML model identified two severe asthma endotypes, which were replicated in U-BIOPRED nasal transcriptomics. Cluster 1 patients had significant airway obstruction, small airways disease, air trapping, decreased diffusing capacity and increased oxidative stress, although only 4/18 were current smokers. Shapley identified 20 cluster-defining proteins. Forty-one proteins were significantly higher in Cluster 1. Pathways associated with proteomic and transcriptomic clusters were linked to Th1, Th2, neutrophil, JAK-STAT, TLR and infection activation. GSVA analysis of the nasal protein and gene signatures were enriched in subjects with sputum neutrophilic/mixed granulocytic asthma and in subjects with a molecular phenotype found in sputum neutrophil-high subjects. CONCLUSIONS: Protein or gene analysis may indicate molecular phenotypes within the asthmatic lower airway and provide a simple, non-invasive test for non-T2 asthma that is currently unavailable.

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.

Purpose of Review Allergic rhinitis (AR) is a chronic inflammatory immunoglobulin (Ig) E-mediated disease of the nasal mucosa that can be triggered by the inhalation of seasonal or perennial allergens. Typical symptoms include sneezing, rhinorrhea, nasal itching, nasal congestion and symptoms of allergic conjunctivitis. AR affects a quarter of the population in the United States of America and Europe. Recent Findings AR has been shown to reduce work productivity in 36–59% of the patients with 20% reporting deteriorated job attendance. Moreover, 42% of children with AR report reduced at-school productivity and lower grades. Most importantly, AR impacts the patient’s quality of life, due to sleep deprivation. However, a proportion of patients fails to respond to conventional medication and opts for the allergen immunotherapy (AIT), which currently is the only disease-modifying therapeutic option. AIT can be administered by either subcutaneous (SCIT) or sublingual (SLIT) route. Both routes of administration are safe, effective, and can lead to tolerance lasting years after treatment cessation. Both innate and adaptive immune responses that contribute to allergic inflammation are suppressed by AIT. Innate responses are ameliorated by reducing local mast cell, basophil, eosinophil, and circulating group 2 innate lymphoid cell frequencies which is accompanied by decreased basophil sensitivity. Induction of allergen-specific blocking antibodies, immunosuppressive cytokines, and regulatory T and B cell phenotypes are key pro-tolerogenic adaptive immune responses. Conclusion A comprehensive understanding of these mechanisms is necessary for optimal selection of AIT-responsive patients and monitoring treatment efficacy. Moreover, it could inspire novel and more efficient AIT approaches.

Introduction There is no detailed comparison of allergen-specific immunoglobulin responses following sublingual immunotherapy (SLIT) and subcutaneous immunotherapy (SCIT). Objective We sought to compare nasal and systemic timothy grass pollen (TGP)-specific antibody responses during 2 years of SCIT and SLIT and 1 year after treatment discontinuation in a double-blind, double-dummy, placebo-controlled trial. Methods Nasal fluid and serum were obtained yearly (per-protocol population, n = 84). TGP-specific IgA1, IgA2, IgG4, IgG, and IgE were measured in nasal fluids by ELISA. TGP-specific IgA1, IgA2, and Phleum pratense (Phl p)1, 2, 4, 5b, 6, 7, 11, and 12 IgE and IgG4 were measured in sera by ELISA and ImmunoCAP, respectively. Results At years 2 and 3, TGP-IgA1/2 levels in nasal fluid were elevated in SLIT compared with SCIT (4.2- and 3.0-fold for IgA1, 2.0- and 1.8-fold for IgA2, respectively; all P Conclusions The observed induction of IgA1/2 in SLIT and IgG4 in SCIT suggest key differences in the mechanisms of action.

For decades, the approach to clinical care has involved the assessment of a fairly small set of patients' signs and symptoms, sampled over a short period of time with limited attention given to individual variations in aetiology and pathophysiology. Subsequently, a therapy is assigned predominantly with a "one shoe fits all" approach. Precision medicine approaches are now starting to change ways to test, treat and develop new therapies for allergic and immunological disorders. (Figure 1).

Background and purpose: The Gi‐coupled, ADP‐activated P2Y12 receptor is well characterised as playing a key role in platelet activation via crosstalk with P2Y1 in ADP‐evoked intracellular Ca2+ response. There is limited knowledge on the role of P2Y12 in ADP‐evoked Ca2+ responses in other blood cells. Here we investigate the role of P2Y12 receptor activation in modulation of ADP‐evoked Ca2+ responses in human THP‐1 monocytic cells. Experimental approach: A combination of intracellular Ca2+ measurements, RT‐PCR, immunocytochemistry, leukocyte isolation and siRNA‐mediated gene knockdown were used to identify the role of P2Y12 receptor activation. Key results: ADP‐evoked intracellular Ca2+ responses (EC50 2.7 M) in THP‐1 cells were abolished by inhibition of phospholipase C (U73122) or sarco/endoplasmic reticulum Ca2+‐ATPase (thapsigargin). Loss of ADP‐evoked Ca2+ responses following treatment with MRS2578 (IC50 200 nM) revealed a major role for P2Y6 in mediating ADP‐evoked Ca2+ responses. ADP‐evoked responses were attenuated either with pertussis toxin treatment, or P2Y12 inhibition with two chemically distinct antagonists (ticagrelor, IC50 5.3 M; PSB‐0739, IC50 5.6 M). ADP‐evoked responses were suppressed following siRNA‐mediated P2Y12 gene knockdown. The inhibitory effects of P2Y12 antagonists were fully reversed following adenylate cyclase inhibition (SQ22536). P2Y12 receptor expression was confirmed in freshly isolated human CD14+ monocytes. Conclusion and implications: Taken together, these data suggest that P2Y12 activation positively regulates P2Y6‐mediated intracellular Ca2+ signalling through suppression of adenylate cyclase activity in human monocytic cells.

Leukocytes sense extracellular ATP, a danger-associated molecular pattern, released during cellular stress and death, via activation of cell surface P2X and P2Y receptors. Here, we investigate P2 receptor expression in primary human monocyte-derived macrophages and receptors that mediate ATP-evoked intracellular [Ca2+]i signals and cytokine production in response to ATP concentrations that exclude P2X7 receptor activation. Expression of P2X1, P2X4, P2X5, P2X7, P2Y1, P2Y2, P2Y4, P2Y6, P2Y11, and P2Y13 was confirmed by quantitative RT-PCR and immunocytochemistry. ATP elicited intracellular Ca2+ responses in a concentration-dependent fashion (EC50 = 11.4 ± 2.9 μM, n = 3). P2Y11 and P2Y13 activations mediated the amplitude of [Ca2+]i response, whereas P2X4 activation, but not P2X1 or P2X7, determined the duration of Ca2+ response during a sustained phase. ATP mediated gene induction of CXCL5, a proinflammatory chemokine. P2X4 antagonism (PSB-12062 or BX430) inhibited ATP-mediated induction of CXCL5 gene expression and secretion of CXCL5 by primary macrophage. Inhibition of CXCL5 secretion by P2X4 antagonists was lost in the absence of extracellular Ca2+. Reciprocally, positive allosteric modulation of P2X4 (ivermectin) augmented ATP-mediated CXCL5 secretion. P2X7, P2Y11, or P2Y13 receptor did not contribute to CXCL5 secretion. Together, the data reveals a role for P2X4 in determining the duration of ATP-evoked Ca2+ responses and CXCL5 secretion in human primary macrophage.

BACKGROUND: Immunoglobulin E (IgE) are least abundant, tightly regulated and IgE producing B cells are rare. The cellular origin and evolution of IgE responses are poorly understood.OBJECTIVE: To investigate the cellular and clonal origin of IgE memory responses following mucosal allergen exposure by sublingual immunotherapy (SLIT).METHODS: In a randomized double-blind, placebo-controlled, time-course SLIT study, peripheral blood mononuclear cells (PBMCs) and nasal biopsies were collected from forty adults with seasonal allergic rhinitis at baseline, 4, 8, 16, 28 and 52 weeks. RNA was extracted from PBMCs, sorted B cells and nasal biopsies for VH repertoire sequencing. Moreover, monoclonal antibodies were derived from single B cell transcriptomes.RESULTS: Combining VH repertoire sequencing and single cell transcriptomics yielded direct evidence of a parallel boost of two clonally and functionally related B cell subsets of short-lived IgE+ plasmablasts and IgG+ memory B cells (termed IgGE). Mucosal grass pollen allergen exposure by SLIT resulted in highly diverse IgE and IgGE repertoires. These were extensively mutated and appeared relative stable as per heavy chain isotype, somatic hypermutations and clonal composition. Single IgGE + memory B cell and IgE+ pre-plasmablast transcriptomes encoded antibodies that were specific for major grass pollen allergens and were able to elicit basophil activation at very low allergen concentrations.CONCLUSION: For the first time, we have shown that upon mucosal allergen exposure, human IgE memory resides in allergen-specific IgG+ memory B cells. These rapidly switch isotype and expand into short-lived IgE+ plasmablasts and serve as a potential target for therapeutic intervention.

Tissues differentially secrete multiple colony stimulating factors under conditions of homeostasis and inflammation, orientating recruited circulating monocytes to differentiate to macrophage with differing functional phenotypes. Here, we investigated ATP-evoked intracellular Ca2+ responses in human macrophage differentiated with macrophage colony-stimulating factor (M-CSF). Extracellular ATP evoked (EC50 13.3 ± 1.4 μM) robust biphasic intracellular Ca2+ responses that showed a dependency on both metabotropic (P2Y) and ionotropic (P2X) receptors. qRT-PCR and immunocytochemistry revealed the expression of P2Y1, P2Y2, P2Y6, P2Y11, P2Y13, P2X1, P2X4, P2X5, and P2X7. Pharmacological analysis revealed contribution of only P2X4 and P2Y11 to the Ca2+ response evoked by maximal ATP concentrations (100 µM). This study reveals the contribution of P2X4 and P2Y11 receptor activation to ATP-evoked intracellular Ca2+ responses, and makes comparison with macrophage differentiated using granulocyte colony-stimulating factor (GM-CSF).

IgE is the least abundant immunoglobulin and tightly regulated, and IgE-producing B cells are rare. The cellular origin and evolution of IgE responses are poorly understood.The cellular and clonal origin of IgE memory responses following mucosal allergen exposure by sublingual immunotherapy (SLIT) were investigated.In a randomized double-blind, placebo-controlled, time course SLIT study, PBMCs and nasal biopsy samples were collected from 40 adults with seasonal allergic rhinitis at baseline and at 4, 8, 16, 28, and 52 weeks. RNA was extracted from PBMCs, sorted B cells, and nasal biopsy samples for heavy chain variable gene repertoire sequencing. Moreover, mAbs were derived from single B-cell transcriptomes.Combining heavy chain variable gene repertoire sequencing and single-cell transcriptomics yielded direct evidence of a parallel boost of 2 clonally and functionally related B-cell subsets of short-lived IgEFor the first time, we have shown that on mucosal allergen exposure, human IgE memory resides in allergen-specific IgG

IgE is the less abundant immunoglobulin isotype in serum and displays higher affinity for its cognate Fc receptor (FceRI) than the rest of antibody isotypes. Moreover, the class switch recombination and the generation of memory responses remarkably differ between IgE and other isotypes. Importantly, class switch recombination to IgE can occur in the mucosae, preferentially through the sequential switching from IgG. Therefore, resident effector cells get rapidly sensitized, and free IgE can be found in mucosal secretions. All these aspects explain the involvement of IgE in respiratory diseases. In allergic rhinitis and allergic asthma, the IgE-sensitization to environmental allergens triggers an eosinophilic inflammation of the airway mucosa of atopic patients. In recent years, growing evidence indicates that some non-atopic patients with nasal reactivity to allergens display nasal eosinophilic inflammation, which could be triggered by the local production of allergen-specific IgE. This phenotype has been termed local allergic rhinitis. Mucosal IgE is also implicated in the pathophysiology of chronic rhinosinusitis with nasal polyps, even though the mechanisms for IgE synthesis might differ in this case. The role of IgE as mediator of airway diseases identify this marker as a therapeutic target. Some biologicals antagonize IgE-mediated inflammation of the airway mucosa, but they have not shown a beneficial long-term effect after discontinuation. In contrast, allergen immunotherapy does not only control the symptoms of airway allergy, but it also induces a long-lasting effect after discontinuation, thus modifying the natural course of the disease.

Purpose of review Sublingual allergen immunotherapy (SLIT), a disease-modifying treatment for allergic rhinitis, can induce long-term clinical benefits which are mediated by immune responses that include generation of regulatory B (Breg) and T (Treg) cells. The newest member of the IL-12 superfamily, IL-35, is an anti-inflammatory cytokine known to be produced by Breg and Treg cells. Limited studies are available on the role of IL-35 on allergic rhinitis and during SLIT. This review summarizes recent findings relevant to the topic of IL-35 and their role in SLIT. Recent findings Recombinant IL-35 protein can induce the generation of IL-35-producing Breg and Treg cells with immunosuppressive capacity. Levels of IL-35 and IL-35-inducible Treg (iTR35) cells are dysregulated in allergic rhinitis patients, which can be restored with SLIT. Mechanism of IL-35-mediated tolerance to allergens includes suppressions of T cell proliferation, Th2 cytokine production, and B cell production of IgE antibodies. Summary Emerging evidence supports a potential role for IL-35 and iTR35 cells in tolerance maintenance during SLIT. A better understanding for the role of IL-35 and iTR35 cells could provide new avenues for the development of clinical biomarker to assess efficacy of allergen immunotherapy and novel therapeutic strategies for allergic rhinitis.

Background Several studies have demonstrated the time course of inflammatory mediators in nasal fluids following nasal allergen challenge (NAC), whereas the effects of NAC on cells in the periphery are unknown. We examined the time course of effector cell markers (for basophils, dendritic cells and T cells) in peripheral blood after nasal grass pollen allergen challenge. Methods Twelve participants with seasonal allergic rhinitis underwent a control (diluent) challenge followed by NAC after an interval of 14 days. Nasal symptoms and peak nasal inspiratory flow (PNIF) were recorded along with peripheral basophil, T-cell and dendritic cell responses (flow cytometry), T-cell proliferative responses (thymidine incorporation), and cytokine expression (FluoroSpot assay). Results Robust increases in nasal symptoms and decreases in PNIF were observed during the early (0–1 h) response and modest significant changes during the late (1–24 h) response. Sequential peaks in peripheral blood basophil activation markers were observed (CD107a at 3 h, CD63 at 6 h, and CD203cbright at 24 h). T effector/memory cells (CD4+CD25lo) were increased at 6 h and accompanied by increases in CD80+ and CD86+ plasmacytoid dendritic cells (pDCs). Ex vivo grass antigen-driven T-cell proliferative responses and the frequency of IL-4+CD4+ T cells were significantly increased at 6 h after NAC when compared to the control day. Conclusion Basophil, T-cell, and dendritic cell activation increased the frequency of allergen-driven IL-4+CD4+ T cells, and T-cell proliferative responses are detectable in the periphery after NAC. These data confirm systemic cellular activation following a local nasal provocation.

Rationale: rfhSP-D has been shown to suppress house dust mite and Aspergillus 74 fumigatus-induced allergic inflammation in murine models. 75 Objectives: We sought to elucidate the effect of rfhSP-D on FcεRI and CD23- 76 mediated grass pollen induced allergic inflammatory responses. 77 Methods: rfhSP-D, containing homotrimeric neck and lectin domains, was 78 expressed in Escherichia coli BL21 (λDE3) pLysS. PBMCs and sera were obtained 79 from grass pollen allergic individuals (n=27). The effect of rfhSP-D on basophil 80 activation and histamine release was measured by flow cytometry. IgE-facilitated 81 allergen binding and presentation was assessed by flow cytometry. Th2 cytokines 82 were measured in cell culture supernatants. The effect of rfhSP-D on IgE production 83 by B cells when stimulated with CD40L, IL-4 and IL-21 was also determined. 84 Results: rfhSP-D bound to Phleum pratense in a dose- and calcium-dependent 85 manner. Allergen-induced basophil responsiveness and histamine release was 86 inhibited in the presence of rfhSP-D, as measured by CD63, CD203c 87 (P=0.0086,P=0.04205), and intracellular-labelled DAO (P=0.0003,P=0.0148). The 88 binding of allergen-IgE complexes to B cells was reduced by 51%(P=0.002) in the 89 presence of rfhSP-D. This decrease was concomitant with reduction in CD23 90 expression on B cells (P

Background Grass pollen–specific immunotherapy involves immunomodulation of allergen-specific TH2 responses and induction of IL-10+ and/or TGF-β+CD4+CD25+ regulatory T cells (induced Treg cells). IL-35+CD4+CD25+ forkhead box protein 3–negative T (IL-35–inducible regulatory T [iTR35]) cells have been reported as a novel subset of induced Treg cells with modulatory characteristics. Objective We sought to investigate mechanisms underlying the induction and maintenance of immunologic tolerance induced by IL-35 and iTR35 cells. Methods The biological effects of IL-35 were assessed on group 2 innate lymphoid cells (ILC2s); dendritic cells primed with thymic stromal lymphopoietin, IL-25, and IL-33; and B and TH2 cells by using flow cytometry and quantitative RT-PCR. Grass pollen–driven TH2 cell proliferation and cytokine production were measured by using tritiated thymidine and Luminex MagPix, respectively. iTR35 cells were quantified in patients with grass pollen allergy (seasonal allergic rhinitis [SAR] group, n = 16), sublingual immunotherapy (SLIT)–treated patients (SLIT group, n = 16), and nonatopic control subjects (NACs; NAC group, n = 16). Results The SAR group had increased proportions of ILC2s (P = .002) and IL-5+ cells (P = .042), IL-13+ cells (P = .042), and IL-5+IL-13+ ILC2s (P = .003) compared with NACs. IL-35 inhibited IL-5 and IL-13 production by ILC2s in the presence of IL-25 or IL-33 (P = .031) and allergen-driven TH2 cytokines by effector T cells. IL-35 inhibited CD40 ligand–, IL-4–, and IL-21–mediated IgE production by B cells (P = .015), allergen-driven T-cell proliferation (P = .001), and TH2 cytokine production mediated by primed dendritic cells. iTR35 cells suppressed TH2 cell proliferation and cytokine production. In addition, allergen-driven IL-35 levels and iTR35 cell counts were increased in patients receiving SLIT (all, P Conclusion IL-35 and iTR35 cells are potential novel immune regulators induced by SLIT. The clinical relevance of SLIT can be underscored by restoration of protective iTR35 cells.

Monocytes and macrophages express a repertoire of cell surface P2 receptors for adenosine 5′-triphosphate (ATP) a damage-associated molecular pattern molecule (DAMP), which are capable of raising cytoplasmic calcium when activated. This is achieved either through direct permeation (ionotropic P2X receptors) or by mobilizing intracellular calcium stores (metabotropic P2Y receptors). Here, a side-by-side comparison to investigate the contribution of P2X4 receptor activation in ATP-evoked calcium responses in model human monocytes and macrophages was performed. The expression of P2X1, P2X4, P2X5 and P2X7 was confirmed by qRT-PCR and immunocytochemistry in both model monocyte and macrophage. ATP evoked a concentration-dependent increase in intracellular calcium in both THP-1 monocyte and macrophages. The sarco/endoplasmic reticulum Ca2+-ATPase inhibitor thasigargin (Tg) responses to the maximal ATP concentration (100 μM) in THP-1 monocytes, and responses in macrophage were significantly attenuated. Tg-resistant ATP-evoked calcium responses in the model macrophage were dependent on extracellular calcium, suggesting a requirement for calcium influx. Ivermectin (IVM) potentiated the magnitude of Tg-resistant component and slowed the decay of response in the model macrophage. The Tg-resistant component was attenuated by P2X4 antagonists 5-BDBD and PSB-12062 but not by the P2X1 antagonist Ro0437626 or the P2X7 antagonist A438079. shRNA-mediated P2X4 knockdown resulted in a significant reduction in Tg-resistant ATP-evoked calcium response as well as reduced sensitivities towards P2X4-specific pharmacological tools, IVM and PSB-12062. Inhibition of endocytosis with dynasore significantly reduced the magnitude of Tg-resistant component but substantially slowed decay response. Inhibition of calcium-dependent exocytosis with vacuolin-1 had no effect on the Tg-resistant component. These pharmacological data suggest that P2X4 receptor activation contributed significantly towards the ionotropic calcium response evoked by ATP of the model human macrophage.

Leukocytes sense extracellular ATP, a danger-associated molecular pattern, released during cellular stress and death, via activation of cell surface P2X and P2Y receptors. Here, we investigate P2 receptor expression in primary human monocyte-derived macrophages and receptors that mediate ATP-evoked intracellular [Ca2+]i signals and cytokine production in response to ATP concentrations that exclude P2X7 receptor activation. Expression of P2X1, P2X4, P2X5, P2X7, P2Y1, P2Y2, P2Y4, P2Y6, P2Y11 and P2Y13 was confirmed by qRT-PCR and immunocytochemistry. ATP elicited intracellular Ca2+ responses in a concentration-dependent fashion (EC50 = 11.4 ± 2.9 μM, N=3). P2Y11 and P2Y13 activations mediated the amplitude of [Ca2+]i response, whilst P2X4 activation, but not P2X1 or P2X7, determined the duration of Ca2+ response during a sustained phase. ATP-mediated gene induction of CXCL5, a pro-inflammatory chemokine. P2X4 antagonism (PSB-12062 or BX-430) inhibited ATP-mediated induction of CXCL5 gene expression and secretion of CXCL5 by primary macrophage. Inhibition of CXCL5 secretion by P2X4 antagonists was lost in the absence of extracellular Ca2+. Reciprocally, positive allosteric modulation of P2X4 (ivermectin) augmented ATP-mediated CXCL5 secretion. P2X7, P2Y11 or P2Y13 receptor did not contribute to CXCL5 secretion. Together, the data reveals a role for P2X4 in determining the duration of ATP-evoked Ca2+ responses and CXCL5 secretion in human primary macrophage.

Mechanisms controlling endoplasmic reticulum (ER) Ca

Allergen-specific immunotherapy is the only allergen-specific and disease-modifying treatment for allergy. The construction and characterization of a vaccine for birch pollen allergy is reported. Two nonallergenic peptides, PA and PB, derived from the IgE-reactive areas of the major birch pollen allergen Bet v 1 were fused to the hepatitis B surface protein, PreS, in four recombinant fusion proteins containing different numbers and combinations of the peptides. Fusion proteins expressed in Escherichia coli and purified to homogeneity showed a lack of IgE reactivity and allergenic activity when tested with sera and basophils from patients allergic to birch pollen. Compared to Bet v 1 allergen, peptides PA and PB showed reduced T cell activation in PBMCs from allergic patients, whereas PreS fusion proteins induced less IL-5 and more IL-10 and IFN-γ. Immunization of rabbits with the fusion proteins, in particular with a PreS fusion protein 2PAPB-PreS, containing two copies of each peptide, induced high levels of IgG Abs against the major IgE-reactive site on Bet v 1 and related allergens. These IgG Abs inhibited allergic patients’ IgE binding to Bet v 1 better than did IgG induced by immunization with complete Bet v 1. Furthermore, 2PAPB-PreS–induced IgG inhibited Bet v 1–induced basophil activation in allergic patients and CD23-facilitated allergen presentation. Our study exemplifies novel beneficial features for a PreS carrier–based peptide vaccine for birch pollen, which, in addition to the established reduction in allergenic activity, include the enhanced focusing of blocking Ab responses toward IgE epitopes, immunomodulatory activity, and reduction of CD23-facilitated allergen presentation.

Background: In quiescent endothelial cells, the transcription factor Erg regulates cell homeostasis by repressing expression of proinflammatory genes. Results: Erg represses NF-κB p65-dependent transcription of ICAM-1, partly by inhibiting p65 binding to DNA. Conclusion: Erg acts as a gatekeeper in quiescent endothelial cells to inhibit basal NF-κB activity. Significance: Novel pathway controlling endothelial cell activation and inflammation., The interaction of transcription factors with specific DNA sequences is critical for activation of gene expression programs. In endothelial cells (EC), the transcription factor NF-κB is important in the switch from quiescence to activation, and is tightly controlled to avoid excessive inflammation and organ damage. Here we describe a novel mechanism that controls the activation of NF-κB in EC. The transcription factor Erg, the most highly expressed ETS member in resting EC, controls quiescence by repressing proinflammatory gene expression. Focusing on intercellular adhesion molecule 1(ICAM)-1 as a model, we identify two ETS binding sites (EBS −118 and −181) within the ICAM-1 promoter required for Erg-mediated repression. We show that Erg binds to both EBS −118 and EBS −181, the latter located within the NF-κB binding site. Interestingly, inhibition of Erg expression in quiescent EC results in increased NF-κB-dependent ICAM-1 expression, indicating that Erg represses basal NF-κB activity. Erg prevents NF-κB p65 from binding to the ICAM-1 promoter, suggesting a direct mechanism of interference. Gene set enrichment analysis of transcriptome profiles of Erg and NF-κB-dependent genes, together with chromatin immunoprecipitation (ChIP) studies, reveals that this mechanism is common to other proinflammatory genes, including cIAP-2 and IL-8. These results identify a role for Erg as a gatekeeper controlling vascular inflammation, thus providing an important barrier to protect against inappropriate endothelial activation.

Background Grass pollen immunotherapy is associated with reduction in symptoms, the need for rescue medication and improvement of quality of life in patients with severe seasonal pollinosis. Although, the suppression of the early allergic response following in vivo cutaneous allergen challenge is associated with inhibition of basophil histamine release, the effects of immunotherapy on basophil reactivity is yet to be fully determined. We hypothesized that basophil reactivity as measured by increased expression of surface activation markers CD203c, CD63 and CD107a on CRTH2+ basophils is increased in grass pollen allergic individuals following in vitro allergen stimulation. We further hypothesized that this hypereactivity is reduced in immunotherapy-treated patients. Methods Heparinized blood obtained from grass pollen allergics (n = 7), immunotherapy treated patients (n = 6) and non-atopic controls (n = 9) was incubated with 0, 0.1, 1, 10 and 100 ng/mL of P. Pratense extract at 37°C for 15 minutes. Cells were stained with anti-CD3, CRTh2, CD123, CD303, CD203c, CD63 and CD107a. Additionally, whole blood basophil histamine release was measured pre/post immunotherapy by ELISA (n = 6). Results A dose-dependent increase in the proportion of CD203c+, CD63+ and CD107a+ CRTH2+ basophils was observed following in vitro grass pollen stimulation in allergics but not in non-atopic controls. At 100 ng/mL of P. Pratense extract, CD203c+, CD63+ and CD107a+CRTH2+ basophils were significantly elevated in allergics compare to non-topics (P < 0.001, P < 0.001 and P < 0.009). This increase in CD203c+, CD63+CRTH2+ basophils in allergic individuals significantly correlated with timothy-specific IgE (r = 0.84, P < 0.0001; r = 0.85, P < 0.0001). Interestingly, 10- to 100-fold more allergen was required for CRTH2+ basophils to express CD203c, CD63 and CD107a in immunotherapy-treated patients compare to grass pollen allergics. At suboptimal allergen-concentration (10 ng/mL), CD203c+, CD63+ and CD107a+CRTH2+ basophils were significantly reduced in immunotherapy treated subjects compare to allergics (P < 0.001, P < 0.001 and P < 0.002). Basophil histamine release measured after treatment was significantly reduced compared to pre-treatment levels (P < 0.03). Conclusions Basophil reactivity and histamine release is significantly reduced following grass pollen immunotherapy. The use of surface activation markers CD203c, CD63 and CD107a on basophils for monitoring clinical efficacy requires further investigations.