Nasal and blood transcriptomic pathways underpinning the clinical response to grass pollen immunotherapy.

BACKGROUND: Allergen immunotherapy (AIT) is a well-established disease-modifying therapy for allergic rhinitis, yet the fundamental mechanisms underlying its clinical effect remain inadequately understood. Gauging Response in Allergic Rhinitis to Sublingual and Subcutaneous Immunotherapy was a randomized, double-blind, placebo-controlled trial of individuals allergic to timothy grass who received 2 years of placebo (n = 30), subcutaneous immunotherapy (SCIT) (n = 27), or sublingual immunotherapy (SLIT) (n = 27) and were then followed for 1 additional year. OBJECTIVE: We used yearly biospecimens from the Gauging Response in Allergic Rhinitis to Sublingual and Subcutaneous Immunotherapy study to identify molecular mechanisms of response. METHODS: We used longitudinal transcriptomic profiling of nasal brush and PBMC samples after allergen provocation to uncover airway and systemic expression pathways mediating responsiveness to AIT. TRIAL REGISTRATION: ClinicalTrials.gov Identifier: NCT01335139, EudraCT Number: 2010-023536-16. RESULTS: SCIT and SLIT demonstrated similar changes in gene module expression over time. In nasal samples, alterations included downregulation of pathways of mucus hypersecretion, leukocyte migration/activation, and endoplasmic reticulum stress (log2 fold changes -0.133 to -0.640, false discovery rates [FDRs] <0.05). We observed upregulation of modules related to epithelial development, junction formation, and lipid metabolism (log2 fold changes 0.104 to 0.393, FDRs <0.05). In PBMCs, modules related to cellular stress response and type 2 cytokine signaling were reduced by immunotherapy (log2 fold changes -0.611 to -0.828, FDRs <0.05). Expression of these modules was also significantly associated with both Total Nasal Symptom Score and peak nasal inspiratory flow, indicating important links between treatment, module expression, and allergen response. CONCLUSIONS: Our results identify specific molecular responses of the nasal airway impacting barrier function, leukocyte migration activation, and mucus secretion that are affected by both SCIT and SLIT, offering potential targets to guide novel strategies for AIT.

Single-cell RNA sequencing identifies precise tolerogenic cellular and molecular pathways induced by depigmented-polymerized grass pollen allergen extract.

BACKGROUND: Immunologic mechanism of action of allergoids remains poorly understood. Previous models of allergenicity and immunogenicity have yielded suboptimal knowledge of these immunotherapeutic vaccine products. Novel single-cell RNA sequencing technology offers a bridge to this gap in knowledge. OBJECTIVE: We sought to identify the underpinning tolerogenic molecular and cellular mechanisms of depigmented-polymerized Phleum pratense (Phl p) extract. METHODS: The molecular mechanisms underlying native Phl p, depigmented Phl p (DPG-Phl p), and depigmented-polymerized (DPG-POL-Phl p) allergoid were investigated by single-cell RNA sequencing. Allergen-specific TH2A, T follicular helper (Tfh), and IL-10+ regulatory B cells were quantified by flow cytometry in peripheral blood mononuclear cells from 16 grass pollen-allergic and 8 nonatopic control subjects. 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-facilitated allergen binding assay. RESULTS: Analysis revealed that DPG-POL-Phl p downregulated genes associated with TH2 signaling, induced functional regulatory T cells 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 toward an IgA1 and IgG responses. In grass pollen-allergic subjects, DPG-POL-Phl p exhibited reduced capacity to elicit proliferation of TH2A, IL-4+ Tfh and IL-21+ Tfh cells while being the most prominent at inducing IL-10+CD19+CD5hi and IL-10+CD19+CD5hiCD38intCD24int regulatory B-cell subsets compared to Phl p (all P < .05). Furthermore, DPG-POL-Phl p demonstrated a hypoallergenic profile through basophil activation and histamine release compared to Phl p (31.54-fold, P < .001). CONCLUSIONS: Single-cell RNA sequencing provides an in-depth resolution of the mechanisms underlying the tolerogenic profile of DPG-POL-Phl p.

Technical standards in allergen exposure chambers worldwide - an EAACI Task Force Report.

Allergen exposure chambers (AECs) can be used for controlled exposure to allergenic and non-allergenic airborne particles in an enclosed environment, in order to (i) characterize the pathological features of respiratory diseases and (ii) contribute to and accelerate the clinical development of pharmacological treatments and allergen immunotherapy for allergic disease of the respiratory tract (such as allergic rhinitis, allergic rhinoconjunctivitis, and allergic asthma). In the guidelines of the European Medicines Agency for the clinical development of products for allergen immunotherapy (AIT), the role of AECs in determining primary endpoints in dose-finding Phase II trials is emphasized. Although methodologically insulated from the variability of natural pollen exposure, chamber models remain confined to supporting secondary, rather than primary, endpoints in Phase III registration trials. The need for further validation in comparison with field exposure is clearly mandated. On this basis, the European Academy of Allergy and Clinical Immunology (EAACI) initiated a Task Force in 2015 charged to gain a better understanding of how AECs can generate knowledge about respiratory allergies and can contribute to the clinical development of treatments. Researchers working with AECs worldwide were asked to provide technical information in eight sections: (i) dimensions and structure of the AEC, (ii) AEC staff, (iii) airflow, air processing, and operating conditions, (iv) particle dispersal, (v) pollen/particle counting, (vi) safety and non-contamination measures, (vii) procedures for symptom assessments, (viii) tested allergens/substances and validation procedures. On this basis, a minimal set of technical requirements for AECs applied to the field of allergology is proposed.

Differential induction of allergen-specific IgA responses following timothy grass subcutaneous and sublingual immunotherapy.

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 < .01). TGP-IgA1 level in serum was elevated in SLIT compared with SCIT at years 1, 2, and 3 (4.6-, 5.1-, and 4.7-fold, respectively; all P < .001). Serum TGP-IgG level was higher in SCIT compared with SLIT (2.8-fold) at year 2. Serum TGP-IgG4 level was higher in SCIT compared with SLIT at years 1, 2, and 3 (10.4-, 27.4-, and 5.1-fold, respectively; all P < .01). Serum IgG4 levels to Phl p1, 2, 5b, and 6 were increased at years 1, 2, and 3 in SCIT and SLIT compared with placebo (Phl p1: 11.8- and 3.9-fold; Phl p2: 31.6- and 4.4-fold; Phl p5b: 135.5- and 5.3-fold; Phl p6: 145.4- and 14.7-fold, respectively, all at year 2 when levels peaked; P < .05). IgE to TGP in nasal fluid increased in the SLIT group at year 2 but not at year 3 compared with SCIT (2.8-fold; P = .04) and placebo (3.1-fold; P = .02). IgA to TGP and IgE and IgG4 to TGP components stratified participants according to treatment group and clinical response. CONCLUSIONS: The observed induction of IgA1/2 in SLIT and IgG4 in SCIT suggest key differences in the mechanisms of action.

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.

Repetitive nasal allergen challenge in allergic rhinitis: Priming and Th2-type inflammation but no evidence of remodelling.

BACKGROUND: Local tissue eosinophilia and Th2 cytokines are characteristic features of seasonal allergic rhinitis. Airway remodelling is a feature of asthma whereas evidence for remodelling in allergic rhinitis (AR) is conflicting. OBJECTIVE: By use of a novel human repetitive nasal allergen challenge (RAC) model, we evaluated the relationship between allergic inflammation and features of remodelling in AR. METHODS: Twelve patients with moderate-severe AR underwent 5 alternate day challenges with diluent which after 4 weeks were followed by 5 alternate day challenges with grass pollen extract. Nasal symptoms, Th1/Th2 cytokines in nasal secretion and serum were evaluated. Nasal biopsies were taken 24 hours after the 1st and 5th challenges with diluent and with allergen. Sixteen healthy controls underwent a single challenge with diluent and with allergen. Using immunohistochemistry, epithelial and submucosal inflammatory cells and remodelling markers were evaluated by computed image analysis. RESULTS: There was an increase in early and late-phase symptoms after every allergen challenge compared to diluent (both P < .05) with evidence of both clinical and immunological priming. Nasal tissue eosinophils and IL-5 in nasal secretion increased significantly after RAC compared to corresponding diluent challenges (P < .01, P = .01, respectively). There was a correlation between submucosal mast cells and the early-phase clinical response (r = 0.79, P = .007) and an association between epithelial eosinophils and IL-5 concentrations in nasal secretion (r = 0.69, P = .06) in allergic rhinitis. No differences were observed after RAC with regard to epithelial integrity, reticular basement membrane thickness, glandular area, expression of markers of activation of airway remodelling including α-SMA, HSP-47, extracellular matrix (MMP7, 9 and TIMP-1), angiogenesis and lymphangiogenesis for AR compared with healthy controls. CONCLUSION: Novel repetitive nasal allergen challenge in participants with severe persistent seasonal allergic rhinitis resulted in tissue eosinophilia and increases in IL-5 but no structural changes. Our data support no link between robust Th2-inflammation and development of airway remodelling in AR.

EAACI Allergen Immunotherapy User's Guide.

Allergen immunotherapy is a cornerstone in the treatment of allergic children. The clinical efficiency relies on a well-defined immunologic mechanism promoting regulatory T cells and downplaying the immune response induced by allergens. Clinical indications have been well documented for respiratory allergy in the presence of rhinitis and/or allergic asthma, to pollens and dust mites. Patients who have had an anaphylactic reaction to hymenoptera venom are also good candidates for allergen immunotherapy. Administration of allergen is currently mostly either by subcutaneous injections or by sublingual administration. Both methods have been extensively studied and have pros and cons. Specifically in children, the choice of the method of administration according to the patient's profile is important. Although allergen immunotherapy is widely used, there is a need for improvement. More particularly, biomarkers for prediction of the success of the treatments are needed. The strength and efficiency of the immune response may also be boosted by the use of better adjuvants. Finally, novel formulations might be more efficient and might improve the patient's adherence to the treatment. This user's guide reviews current knowledge and aims to provide clinical guidance to healthcare professionals taking care of children undergoing allergen immunotherapy.

Pollen season is reflected on symptom load for grass and birch pollen-induced allergic rhinitis in different geographic areas-An EAACI Task Force Report.

BACKGROUND: The effectiveness of allergen immunotherapy (AIT) in seasonal and perennial allergic rhinitis (AR) depends on the definition of pollen exposure intensity or time period. We recently evaluated pollen and symptom data from Germany to examine the new definitions of the European Academy of Allergy and Clinical Immunology (EAACI) on pollen season and peak pollen period start and end. Now, we aim to confirm the feasibility of these definitions to properly mirror symptom loads for grass and birch pollen-induced allergic rhinitis in other European geographical areas such as Austria, Finland and France, and therefore their suitability for AIT and clinical practice support. METHODS: Data from twenty-three pollen monitoring stations from three countries in Europe and for 3 years (2014-2016) were used to investigate the correlation between birch and grass pollen concentrations during the birch and grass pollen season defined via the EAACI criteria, and total nasal symptom and medication scores as reported with the aid of the patient's hay-fever diary (PHD). In addition, we conducted a statistical analysis, together with a graphical investigation, to reveal correlations and dependencies between the studied parameters. RESULTS: The analysis demonstrated that the definitions of pollen season as well as peak pollen period start and end as proposed by the EAACI are correlated to pollen-induced symptom loads reported by PHD users during birch and grass pollen season. A statistically significant correlation (slightly higher for birch) has been found between the Total Nasal Symptom and Medication Score (TNSMS) and the pollen concentration levels. Moreover, the maximum symptom levels occurred mostly within the peak pollen periods (PPP) following the EAACI criteria. CONCLUSIONS: Based on our analyses, we confirm the validity of the EAACI definitions on pollen season for both birch and grass and for a variety of geographical locations for the four European countries (including Germany from a previous publication) analyzed so far. On this basis, the use of the EAACI definitions is supported in future clinical trials on AIT as well as in daily routine for optimal patient care. Further evaluation of the EAACI criteria in other European regions is recommended.

Allergen-specific IgG+ memory B cells are temporally linked to IgE memory responses.

BACKGROUND: 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. OBJECTIVE: The cellular and clonal origin of IgE memory responses following mucosal allergen exposure by sublingual immunotherapy (SLIT) were investigated. METHODS: 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. RESULTS: 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 IgE+ plasmablasts and IgG+ memory B cells. Mucosal grass pollen allergen exposure by SLIT resulted in highly diverse IgE and IgGE repertoires. These were extensively mutated and appeared relatively stable as per heavy chain isotype, somatic hypermutations, and clonal composition. Single IgGE+ memory B-cell and IgE+ preplasmablast transcriptomes encoded antibodies that were specific for major grass pollen allergens and able to elicit basophil activation at very low allergen concentrations. CONCLUSION: For the first time, we have shown that on mucosal allergen exposure, human IgE memory resides in allergen-specific IgG+ memory B cells. These cells rapidly switch isotype, expand into short-lived IgE+ plasmablasts, and serve as a potential target for therapeutic intervention.

Immunologic mechanisms of a short-course of Lolium perenne peptide immunotherapy: A randomized, double-blind, placebo-controlled trial.

BACKGROUND: A 3-week short-course of adjuvant-free hydrolysates of Lolium perenne peptide (LPP) immunotherapy for rhinoconjunctivitis with or without asthma over 4 physician visits is safe, well tolerated, and effective. OBJECTIVE: We sought to investigate immunologic mechanisms of LPP immunotherapy in a subset of patients who participated in a phase III, multicenter, randomized, double-blind, placebo-controlled trial (clinical.govNCT02560948). METHODS: Participants were randomized to receive LPP (n = 21) or placebo (n = 11) for 3 weeks over 4 visits. Grass pollen-induced basophil, T-cell, and B-cell responses were evaluated before treatment (visit [V] 2), at the end of treatment (V6), and after the pollen season (V8). RESULTS: Combined symptom and rescue medication scores (CSMS) were lower during the peak pollen season (-35.1%, P = .03) and throughout the pollen season (-53.7%, P = .03) in the LPP-treated group compared with those in the placebo-treated group. Proportions of CD63+ and CD203cbrightCRTH2+ basophils were decreased following LPP treatment at V6 (10 ng/mL, P < .0001) and V8 (10 ng/mL, P < .001) compared to V2. No change in the placebo-treated group was observed. Blunting of seasonal increases in levels of grass pollen-specific IgE was observed in LPP-treated but not placebo-treated group. LPP immunotherapy, but not placebo, was associated with a reduction in proportions of IL-4+ TH2 (V6, P = .02), IL-4+ (V6, P = .003; V8, P = .004), and IL-21+ (V6, P = .003; V8, P = .002) follicular helper T cells. Induction of FoxP3+, follicular regulatory T, and IL-10+ regulatory B cells were observed at V6 (all P < .05) and V8 (all P < .05) in LPP-treated group. Induction of regulatory B cells was associated with allergen-neutralizing IgG4-blocking antibodies. CONCLUSION: For the first time, we demonstrate that the immunologic mechanisms of LPP immunotherapy are underscored by immune modulation in the T- and B-cell compartments, which is necessary for its effect.

Nasal allergen-neutralizing IgG4 antibodies block IgE-mediated responses: Novel biomarker of subcutaneous grass pollen immunotherapy.

BACKGROUND: Grass pollen subcutaneous immunotherapy (SCIT) is associated with induction of serum IgG4-associated inhibitory antibodies that prevent IgE-facilitated allergen binding to B cells. OBJECTIVE: We sought to determine whether SCIT induces nasal allergen-specific IgG4 antibodies with inhibitory activity that correlates closely with clinical response. METHODS: In a cross-sectional controlled study, nasal fluid and sera were collected during the grass pollen season from 10 SCIT-treated patients, 13 untreated allergic patients (with seasonal allergic rhinitis [SAR]), and 12 nonatopic control subjects. Nasal and serum IgE and IgG4 levels to Phleum pratense components were measured by using the Immuno Solid Allergen Chip microarray. Inhibitory activity was measured by IgE-facilitated allergen binding assay. IL-10+ regulatory B cells were quantified in peripheral blood by using flow cytometry. RESULTS: Nasal and serum Phl p 1- and Phl p 5-specific IgE levels were increased in patients with SAR compared to nonatopic control subjects (all, P < .001) and SCIT-treated patients (nasal, P < .001; serum Phl p 5, P = .073). Nasal IgG4 levels were increased in the SCIT group compared to those in the SAR group (P < .001) during the pollen season compared to out of season. IgG-associated inhibitory activity in nasal fluid and serum was significantly increased in the SCIT group compared to that in the SAR (both, P < .01). The magnitude of the inhibitory activity was 93% (P < .001) in nasal fluid compared to 66% (P < .001) in serum and was reversed after depletion of IgG. Both nasal fluid (r = -0.69, P = .0005) and serum (r = -0.552, P = .0095) blocking activity correlated with global symptom improvement. IL-10+ regulatory B cells were increased in season compared to out of season in the SCIT group (P < .01). CONCLUSION: For the first time, we show that nasal IgG4-associated inhibitory activity correlates closely with the clinical response to allergen immunotherapy in patients with allergic rhinitis with or without asthma.

Role of IL-35 in sublingual allergen immunotherapy.

BACKGROUND: Grass pollen-specific immunotherapy involves immunomodulation of allergen-specific TH2 responses and induction of IL-10+ and/or TGF-ß+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 < .001) and NACs (all, P < .001) compared with patients with SAR. 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.

Synchronous immune alterations mirror clinical response during allergen immunotherapy.

BACKGROUND: Three years of treatment with either sublingual or subcutaneous allergen immunotherapy has been shown to be effective and to induce long-term tolerance. The Gauging Response in Allergic Rhinitis to Sublingual and Subcutaneous Immunotherapy (GRASS) trial demonstrated that 2 years of treatment through either route was effective in suppressing the response to nasal allergen challenge, although it was insufficient for inhibition 1 year after discontinuation. OBJECTIVE: We sought to examine in the GRASS trial the time course of immunologic changes during 2 years of sublingual and subcutaneous immunotherapy and for 1 year after treatment discontinuation. METHODS: We performed multimodal immunomonitoring to assess allergen-specific CD4 T-cell properties in parallel with analysis of local mucosal cytokine responses induced by nasal allergen exposure and humoral immune responses that included IgE-dependent basophil activation and measurement of serum inhibitory activity for allergen-IgE binding to B cells (IgE-facilitated allergen binding). RESULTS: All 3 of these distinct arms of the immune response displayed significant and coordinate alterations during 2 years of allergen desensitization, followed by reversal at 3 years, reflecting a lack of a durable immunologic effect. Although frequencies of antigen-specific TH2 cells in peripheral blood determined by using HLA class II tetramer analysis most closely paralleled clinical outcomes, IgE antibody-dependent functional assays remained inhibited in part 1 year after discontinuation. CONCLUSION: Two years of allergen immunotherapy were effective but insufficient for long-term tolerance. Allergen-specific TH2 cells most closely paralleled the transient clinical outcome, and it is likely that recurrence of the T-cell drivers of allergic immunity abrogated the potential for durable tolerance. On the other hand, the persistence of IgE blocking antibody 1 year after discontinuation might be an early indicator of a protolerogenic mechanism.

Antiapoptotic serine protease inhibitors contribute to survival of allergenic TH2 cells.

BACKGROUND: The mechanisms that regulate maintenance of persistent TH2 cells and potentiate allergic inflammation are not well understood. OBJECTIVE: The function of serine protease inhibitor 2A (Spi2A) was studied in mouse TH2 cells, and the serine protease inhibitor B3 (SERPINB3) and SERPINB4 genes were studied in TH2 cells from patients with grass pollen allergy. METHODS: Spi2A-deficient TH2 cells were studied in in vitro culture or in vivo after challenge of Spi2A knockout mice with ovalbumin in alum. Expression of SERPINB3 and SERPINB4 mRNA was measured in in vitro-cultured TH2 cells and in ex vivo CD27-CD4+ cells and innate lymphoid cell (ILC) 2 from patients with grass pollen allergy by using quantitative PCR. SERPINB3 and SERPINB4 mRNA levels were knocked down in cultured CD27-CD4+ cells with small hairpin RNA. RESULTS: There were lower levels of in vitro-polarized TH2 cells from Spi2A knockout mice (P < .005) and in vivo after ovalbumin challenge (P < .05), higher levels of apoptosis (Annexin V positivity, P < .005), and less lung allergic inflammation (number of lung eosinophils, P < .005). In vitro-polarized TH2 cells from patients with grass pollen allergy expressed higher levels of both SERPINB3 and SERPINB4 mRNA (both P < .05) compared with unpolarized CD4 T cells. CD27-CD4+ from patients with grass pollen allergy expressed higher levels of both SERPINB3 and SERPINB4 mRNA (both P < .0005) compared with CD27+CD4+ cells. ILC2 expressed higher levels of both SERPINB3 and SERPINB4 mRNA (both P < .0005) compared with ILC1. Knockdown of either SERPINB3 or SERPINB4 mRNA (both P < .005) levels resulted in decreased viability of CD27-CD4+ compared with control transduced cells. CONCLUSION: The Serpins Spi2A in mice and SERPINB3 and SERPINB4 in allergic patients control the viability of TH2 cells. This provides proof of principle for a therapeutic approach for allergic disease through ablation of allergic memory TH2 cells through SERPINB3 and SERPINB4 mRNA downregulation.

A Recombinant Fragment of Human Surfactant Protein D Suppresses Basophil Activation and T-Helper Type 2 and B-Cell Responses in Grass Pollen-induced Allergic Inflammation.

RATIONALE: Recombinant fragment of human surfactant protein D (rfhSP-D) has been shown to suppress house dust mite- and Aspergillus fumigatus-induced allergic inflammation in murine models. OBJECTIVES: We sought to elucidate the effect of rfhSP-D on high-affinity IgE receptor- and CD23-mediated, grass pollen-induced allergic inflammatory responses. METHODS: rfhSP-D, containing homotrimeric neck and lectin domains, was expressed in Escherichia coli BL21(λDE3)pLysS cells. Peripheral blood mononuclear cells and sera were obtained from individuals with grass pollen allergy (n = 27). The effect of rfhSP-D on basophil activation and histamine release was measured by flow cytometry. IgE-facilitated allergen binding and presentation were assessed by flow cytometry. T-helper cell type 2 (Th2) cytokines were measured in cell culture supernatants. The effect of rfhSP-D on IgE production by B cells when stimulated with CD40L, IL-4, and IL-21 was also determined. MEASUREMENTS AND MAIN RESULTS: rfhSP-D bound to Phleum pratense in a dose- and calcium-dependent manner. Allergen-induced basophil responsiveness and histamine release were inhibited in the presence of rfhSP-D, as measured by CD63, CD203c (P = 0.0086, P = 0.04205), and intracellularly labeled diamine oxidase (P = 0.0003, P = 0.0148). The binding of allergen-IgE complexes to B cells was reduced by 51% (P = 0.002) in the presence of rfhSP-D. This decrease was concomitant with reduction in CD23 expression on B cells (P < 0.001). rfhSP-D suppressed allergen-driven CD27-CD4+CRTh2+ T-cell proliferation (P < 0.01), IL-4, and IL-5 levels (all P < 0.01). Moreover, rfhSP-D inhibited CD40L/IL-4- and IL-21-mediated IgE production (77.12%; P = 0.02) by B cells. CONCLUSIONS: For the first time, to our knowledge, we show that rfhSP-D inhibited allergen-induced basophil responses at a single-cell level and suppressed CD23-mediated facilitated allergen presentation and Th2 cytokine production. In addition, rfhSP-D inhibited IgE synthesis by B cells, which is also a novel observation.

Effect of 2 Years of Treatment With Sublingual Grass Pollen Immunotherapy on Nasal Response to Allergen Challenge at 3 Years Among Patients With Moderate to Severe Seasonal Allergic Rhinitis: The GRASS Randomized Clinical Trial.

Importance: Sublingual immunotherapy and subcutaneous immunotherapy are effective in seasonal allergic rhinitis. Three years of continuous treatment with subcutaneous immunotherapy and sublingual immunotherapy has been shown to improve symptoms for at least 2 years following discontinuation of treatment. Objective: To assess whether 2 years of treatment with grass pollen sublingual immunotherapy, compared with placebo, provides improved nasal response to allergen challenge at 3-year follow-up. Design, Setting, and Participants: A randomized double-blind, placebo-controlled, 3-parallel-group study performed in a single academic center, Imperial College London, of adult patients with moderate to severe seasonal allergic rhinitis (interfering with usual daily activities or sleep). First enrollment was March 2011, last follow-up was February 2015. Interventions: Thirty-six participants received 2 years of sublingual immunotherapy (daily tablets containing 15 µg of major allergen Phleum p 5 and monthly placebo injections), 36 received subcutaneous immunotherapy (monthly injections containing 20 µg of Phleum p 5 and daily placebo tablets) and 34 received matched double-placebo. Nasal allergen challenge was performed before treatment, at 1 and 2 years of treatment, and at 3 years (1 year after treatment discontinuation). Main Outcomes and Measures: Total nasal symptom scores (TNSS; range; 0 [best] to 12 [worst]) were recorded between 0 and 10 hours after challenge. The minimum clinically important difference for change in TNSS within an individual is 1.08. The primary outcome was TNSS comparing sublingual immunotherapy vs placebo at year 3. Subcutaneous immunotherapy was included as a positive control. The study was not powered to compare sublingual immunotherapy with subcutaneous immunotherapy. Results: Among 106 randomized participants (mean age, 33.5 years; 34 women [32.1%]), 92 completed the study at 3 years. In the intent-to-treat population, mean TNSS score for the sublingual immunotherapy group was 6.36 (95% CI, 5.76 to 6.96) at pretreatment and 4.73 (95% CI, 3.97 to 5.48) at 3 years, and for the placebo group, the score was 6.06 (95% CI, 5.23 to 6.88) at pretreatment and 4.81 (95% CI, 3.97 to 5.65) at 3 years. The between-group difference (adjusted for baseline) was -0.18 (95% CI, -1.25 to 0.90; [P = .75]). Conclusions and Relevance: Among patients with moderate to severe seasonal allergic rhinitis, 2 years of sublingual grass pollen immunotherapy was not significantly different from placebo in improving the nasal response to allergen challenge at 3-year follow-up. Trial Registration: clinicaltrials.gov Identifier: NCT01335139; EudraCT Number: 2010-023536-16.