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.

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.

Altered chromatin landscape in circulating T follicular helper and regulatory cells following grass pollen subcutaneous and sublingual immunotherapy.

BACKGROUND: Allergen-specific immunotherapy is a disease-modifying treatment that induces long-term T-cell tolerance. OBJECTIVE: We sought to evaluate the role of circulating CXCR5+PD-1+ T follicular helper (cTFH) and T follicular regulatory (TFR) cells following grass pollen subcutaneous immunotherapy (SCIT) and sublingual immunotherapy (SLIT) and the accompanying changes in their chromatin landscape. METHODS: Phenotype and function of cTFH cells were initially evaluated in the grass pollen-allergic (GPA) group (n = 28) and nonatopic healthy controls (NAC, n = 13) by mathematical algorithms developed to manage high-dimensional data and cell culture, respectively. cTFH and TFR cells were further enumerated in NAC (n = 12), GPA (n = 14), SCIT- (n = 10), and SLIT- (n = 8) treated groups. Chromatin accessibility in cTFH and TFR cells was assessed by assay for transposase-accessible chromatin sequencing (ATAC-seq) to investigate epigenetic mechanisms underlying the differences between NAC, GPA, SCIT, and SLIT groups. RESULTS: cTFH cells were shown to be distinct from TH2- and TH2A-cell subsets, capable of secreting IL-4 and IL-21. Both cytokines synergistically promoted B-cell class switching to IgE and plasma cell differentiation. Grass pollen allergen induced cTFH-cell proliferation in the GPA group but not in the NAC group (P < .05). cTFH cells were higher in the GPA group compared with the NAC group and were lower in the SCIT and SLIT groups (P < .01). Time-dependent induction of IL-4, IL-21, and IL-6 was observed in nasal mucosa following intranasal allergen challenge in the GPA group but not in SCIT and SLIT groups. TFR and IL-10+ cTFH cells were induced in SCIT and SLIT groups (all, P < .01). ATAC-seq analyses revealed differentially accessible chromatin regions in all groups. CONCLUSIONS: For the first time, we showed dysregulation of cTFH cells in the GPA group compared to NAC, SCIT, and SLIT groups and induction of TFR and IL-10+ cTFH cells following SCIT and SLIT. Changes in the chromatin landscape were observed following allergen-specific immunotherapy in cTFH and TFR cells.

Predictors of asthma relapse in patients who attended an emergency department.

BACKGROUND: Patients with asthma exacerbations and frequent relapses that require admission to the emergency department (ED) often have more severe disease, worse quality of life, and higher use of health care resources. OBJECTIVE: The aim of this study was to identify potential predictors of relapse after patients are treated in an ED for an asthma exacerbation. METHODS: A retrospective, noninterventional cohort study was conducted in adult patients who attended the ED of a tertiary hospital in 2014 for an asthma exacerbation. We analyzed the subpopulation who experienced at least one relapse (returned to the ED < 15 days after the previous event). RESULTS: Fifty-two of 831 patients experienced 66 relapses after going to the ED (mean ± standard deviation [SD] age, 58.5 ± 23.4 years). The average ± SD probability of a relapse was 6 ± 0.8%. The frequency of episodes was higher in May and November. Twenty-four patients had ≥260 blood eosinophils/µL, including 17 who had ≥400 eosinophils/µL. Only 15% of the patients were referred to an asthma specialist at discharge. Factors related to a higher probability of relapse were the following: having multiple visits to the ED in 1 year, uncontrolled asthma, wheezing in the pulmonary auscultation, peripheral eosinophilia with ≥400 eosinophils/ µL, and being discharged in the first visit to the ED (p < 0.01 for all). CONCLUSION: In this population, patients who had multiple ED visits in 1 year, those with uncontrolled asthma, wheezing, ≥400 blood eosinophils/µL, or who had been discharged at the first ED visit are at higher risk of relapse.

Bronchiectasis in severe asthma: Clinical features and outcomes.

BACKGROUND: Bronchiectasis is increasingly being identified in patients with severe asthma and could contribute to disease severity. OBJECTIVE: To determine the prevalence of bronchiectasis in a population of patients with severe asthma and to better characterize the clinical features of these patients and their outcomes. METHODS: We retrospectively reviewed the medical files of 184 subjects with confirmed severe asthma who had undergone high-resolution thoracic computed tomography and compared the characteristics and outcomes of subjects with and without bronchiectasis. RESULTS: Bronchiectasis was identified in 86 patients (47%). These patients had concomitant hypersensitivity to nonsteroidal anti-inflammatory drugs (odds ratio [OR] 2.24, 95% confidence interval [CI] 1.00-5.03) and gastroesophageal reflux disease (OR 1.89, 95% CI 1.05-3.41) more frequently than subjects without bronchiectasis, but had less atopic dermatitis (OR 0.188, 95% CI 0.04-0.88). Subjects with bronchiectasis were more frequently hospitalized for asthma exacerbations (OR 2.09, 95% CI 1.08-4.05) and had higher blood eosinophil levels (464 vs 338; P = .005) than subjects without bronchiectasis. CONCLUSION: Our study suggests that in subjects with severe asthma, the presence of bronchiectasis is associated with more frequent hospitalizations, concomitant gastroesophageal reflux disease, hypersensitivity to nonsteroidal anti-inflammatory drugs, and higher blood eosinophil counts. Bronchiectasis could represent an additional phenotypic feature of severe eosinophilic asthma.