Peanut oral immunotherapy transiently expands circulating Ara h 2–specific B cells with a homologous repertoire in unrelated subjects

Background Peanut oral immunotherapy (PNOIT) induces persistent tolerance to peanut in a subset of patients and induces specific antibodies that might play a role in clinical protection. However, the contribution of induced antibody clones to clinical tolerance in PNOIT is unknown. Objective We hypothesized that PNOIT induces a clonal, allergen-specific B-cell response that could serve as a surrogate for clinical outcomes. Methods We used a fluorescent Ara h 2 multimer for affinity selection of Ara h 2–specific B cells and subsequent single-cell immunoglobulin amplification. The diversity of related clones was evaluated by means of next-generation sequencing of immunoglobulin heavy chains from circulating memory B cells with 2x250 paired-end sequencing on the Illumina MiSeq platform. Results Expression of class-switched antibodies from Ara h 2–positive cells confirms enrichment for Ara h 2 specificity. PNOIT induces an early and transient expansion of circulating Ara h 2–specific memory B cells that peaks at week 7. Ara h 2–specific sequences from memory cells have rates of nonsilent mutations consistent with affinity maturation. The repertoire of Ara h 2–specific antibodies is oligoclonal. Next-generation sequencing–based repertoire analysis of circulating memory B cells reveals evidence for convergent selection of related sequences in 3 unrelated subjects, suggesting the presence of similar Ara h 2–specific B-cell clones. Conclusions Using a novel affinity selection approach to identify antigen-specific B cells, we demonstrate that the early PNOIT-induced Ara h 2–specific B-cell receptor repertoire is oligoclonal and somatically hypermutated and shares similar clonal groups among unrelated subjects consistent with convergent selection. Key words Immunotherapy; antigen-specific B cells; peanut allergy; food allergy; antibody repertoire Abbreviations used APC, Allophycocyanin; BCR, B-cell receptor; CDR, Complementarity-determining region; NGS, Next-generation sequencing; OIT, Oral immunotherapy; PNOIT, Peanut oral immunotherapyNational Institute of Allergy and Infectious Diseases (U.S.) (NIAID U19 AI087881)National Institute of Allergy and Infectious Diseases (U.S.) (NIAID U19 AI095261)United States. National Institutes of Health (1S10RR023440-01A1)National Institute of Allergy and Infectious Diseases (U.S.) (NIAID F32 AI104182)United States. National Institutes of Health (UL1 TR001102

Standardization and performance evaluation of mononuclear cell cytokine secretion assays in a multicenter study

BACKGROUND: Cryopreservation of peripheral blood mononuclear cells has been used to preserve and standardize immunologic measurements for multicenter studies, however, effects of cryopreservation on cytokine responses are incompletely understood. In designing immunologic studies for a new multicenter birth cohort study of childhood asthma, we performed a series of experiments to determine the effects of two different methods of cryopreservation on the cytokine responses of cord and peripheral blood mononuclear cells. RESULTS: Paired samples of PBMC were processed freshly, or after cryopreservation in a Nalgene container (NC) or a controlled-rate freezer (CRF). Although there were some differences between the methods, cryopreservation inhibited PHA-induced IL-10 secretion and Der f 1-induced IL-2 secretion, and augmented PHA-induced IL-2 secretion and spontaneous secretion of TNF-α. In separate experiments, NC cryopreservation inhibited secretion of several cytokines (IL-13, IL-10, IFN-γ, TNF-α) by PHA-stimulated cord blood mononuclear cells. With the exception of PHA-induced IL-13, results from fresh and cryopreserved cord blood samples were not significantly correlated. Finally, in reproducibility studies involving processing of identical cell samples in up to 4 separate laboratories, variances in cytokine responses of fresh cells stimulated at separate sites did not exceed those in cryopreserved cells stimulated at a central site. CONCLUSION: Collectively, these studies indicate that cryopreservation can affect mononuclear cell cytokine response profiles, and that IL-10 secretion and antigen-induced responses may be especially vulnerable. These studies also demonstrate that mononuclear cell responses can be standardized for performance in a small number of laboratories for multicenter studies, and underscore the importance of measuring reproducibility and of testing whether cryopreservation techniques alter specific immunologic outcomes

Epithelial-Associated Inflammatory Pathways Underlie Residual Asthma Exacerbations in Urban Children Treated with Mepolizumab Therapy

Rationale: Identification of airway inflammatory pathways in asthma has proven essential to understanding mechanisms of disease and has led to effective personalized treatment with biologic therapies. However, relatively little is known about patterns of airway inflammation at the time of respiratory illnesses and how such patterns relate to responsiveness to biologic therapies. Methods: The MUPPITS-1 (n=106) and MUPPITS-2 (n=290) studies investigated asthma exacerbations in urban children with exacerbation-prone asthma and ≥150/microliter blood eosinophils. Children in both studies received guidelines-based asthma care; in MUPPITS-2, participants were additionally randomized (1:1) to placebo or mepolizumab. Nasal lavage samples were collected during respiratory illnesses for RNA-sequencing and analyzed by modular analysis to assess genome-wide expression patterns associated with exacerbation illnesses. Results: Among 284 illnesses, exacerbations that occurred in the absence of mepolizumab therapy showed significantly higher upregulation of eosinophil associated inflammatory pathways (fold change values [FC]=1.27-1.43, p-values\u3c0.05), including a Type-2 inflammation module composed of eosinophil, mast cell, and IL-13 response genes. In contrast, exacerbations that occurred while on mepolizumab therapy showed significantly higher upregulation of several epithelial inflammatory pathways (FC=1.36-1.64, p-values\u3c0.05) including TGF-β/Smad3 signaling, extracellular matrix production, and epidermal growth factor receptor signaling. Conclusions: These results indicate that novel inflammatory pathways, likely originating from the airway epithelium and distinct from Type-2 or eosinophilic inflammation, drive residual exacerbations that occur in children treated with mepolizumab therapy added to guideline-based care. These findings identify likely mechanisms of persistent disease expression in these children despite significant depletion of eosinophils and can identify novel treatment targets for future studies

Mepolizumab Alters Regulation of Airway Type-2 Inflammation in Urban Children with Asthma by Disrupting Eosinophil Gene Expression but Enhancing Mast Cell and Epithelial Pathways

Rationale: Mepolizumab (anti-IL5) reduces asthma exacerbations in urban children. We previously utilized nasal transcriptomics to identify inflammatory pathways (gene co-expression modules) associated with exacerbations despite this therapy. To understand mepolizumab’s precise impact on these pathways, we assess gene co-expression and loss of correlation, “decoherence,” using differential co-expression network analyses. Methods: 290 urban children (6-17 years) with exacerbation-prone asthma and blood eosinophils ≥150/microliter were randomized (1:1) to q4 week placebo or mepolizumab injections added to guideline-based care for 52 weeks. Nasal lavage samples were collected before and during treatment for RNA-sequencing. Differential co-expression of gene networks was evaluated to assess interactions and regulatory aspects of type-2 and eosinophilic airway inflammation. Results: Mepolizumab, but not placebo, significantly reduced the overall expression of an established type-2 inflammation gene co-expression module (fold change=0.77, p=0.002) enriched for eosinophil, mast cell, and epithelial IL-13 response genes (242 genes). Mepolizumab uncoupled co-expression of genes in this pathway. During mepolizumab, but not placebo treatment, there was significant loss of correlation among eosinophil-specific genes including RNASE2 (EDN), RNASE3 (ECP), CLC, SIGLEC8, and IL5RA contrasting a reciprocal increase in correlation among mast cell-specific genes (TPSAB1, CPA3, FCER1A), T2 cytokines (IL4, IL5, and IL13), and POSTN. Conclusions: These results suggest mepolizumab disrupts the regulatory interactions of gene co-expression among airway eosinophils, mast cells and epithelium by interrupting transcription regulation in eosinophils with enhancement in mast cell and epithelial inflammation. This paradoxical effect may contribute to an incomplete reduction of asthma exacerbations and demonstrates how differential co-expression network analyses can identify targets for more precise therapies

Association of Childhood Obesity With Atopic and Nonatopic Asthma: Results From the National Health and Nutrition Examination Survey 1999–2006

Obesity and asthma prevalence have both risen among children over the last several decades, and research efforts increasingly suggest that obesity is associated with asthma. Some, but not all, studies have shown that the effect of obesity on asthma is stronger among non-atopic individuals than among those with atopy. Systemic inflammation may be a factor in this relationship

Association of Obesity with IgE and Allergy Symptoms in Children and Adolescents: Results from NHANES 2005–2006

The prevalence of both obesity and allergic disease has increased among children over the last several decades. Previous literature on the relationship between obesity and allergic disease has been inconsistent. It is not known whether systemic inflammation could be a factor in this relationship

The Effect of Subcutaneous German Cockroach Immunotherapy (SCIT) on Nasal Allergen Challenge (NAC) and Cockroach-specific Antibody Responses Among Urban Children and Adolescents

Rationale: Cockroach allergy contributes to asthma and rhinitis morbidity among many urban children. Treatment with cockroach SCIT could be beneficial. Methods: 8-17 year-old children with mild-moderate asthma from 11 urban sites participated in a randomized double-blind placebo-controlled SCIT trial using non-standardized, glycerinated German cockroach extract. Positive cockroach skin tests, cockroach-specific IgE, and nasal challenge response with total nasal symptom scores (TNSS) ≥6 or maximal sneeze scores of 3 during a graded NAC were required for enrollment. Following dose escalation, 0.4 ml of undiluted extract was targeted for maintenance dosing (∼7 mcg Bla g2/dose). The primary endpoint was change in NAC-induced mean TNSS from baseline to one year post randomization. Changes in cockroach-specific IgE (CRsIgE) and IgG4 (CRsIgG4) were also analyzed. Results: Mean TNSS did not significantly change from baseline in either group (placebo n=29, SCIT n=28). There was no significant difference in the change in mean TNSS between placebo and SCIT [−0.79±0.35 vs. −1.02±0.37, respectively, difference=0.2(−1.15, 0.70), p=0.63]. Baseline CRsIgE and CRsIgG4 didn’t differ between groups. Mean CRsIgE decreased in both groups following treatment: 3.6 to 2.3 kU/L (0.64 fold change), p=0.015 and 8.3 to 4.2 kU/L (0.51 fold change), p\u3c0.001 in placebo and SCIT respectively, but did not differ between groups [p=0.33]. Significant increases in CRsIgG4 post-treatment were observed among SCIT recipients only: 0.07 to 12.3 mg/L (176 fold change), p\u3c0.001. Conclusions: Cockroach SCIT increased CRsIgG4 levels but did not significantly alter NAC-induced TNSS responses. The extent to which NAC in these children may reflect clinical efficacy for rhinitis or asthma is uncertain

Multi-omic association study identifies DNA methylation-mediated genotype and smoking exposure effects on lung function in children living in urban settings

Impaired lung function in early life is associated with the subsequent development of chronic respiratory disease. Most genetic associations with lung function have been identified in adults of European descent and therefore may not represent those most relevant to pediatric populations and populations of different ancestries. In this study, we performed genome-wide association analyses of lung function in a multiethnic cohort of children (n = 1,035) living in low-income urban neighborhoods. We identified one novel locus at the TDRD9 gene in chromosome 14q32.33 associated with percent predicted forced expiratory volume in one second (FEV1) (p = 2.4x10-9; βz = -0.31, 95% CI = -0.41- -0.21). Mendelian randomization and mediation analyses revealed that this genetic effect on FEV1 was partially mediated by DNA methylation levels at this locus in airway epithelial cells, which were also associated with environmental tobacco smoke exposure (p = 0.015). Promoter-enhancer interactions in airway epithelial cells revealed chromatin interaction loops between FEV1-associated variants in TDRD9 and the promoter region of the PPP1R13B gene, a stimulator of p53-mediated apoptosis. Expression of PPP1R13B in airway epithelial cells was significantly associated the FEV1 risk alleles (p = 1.3x10-5; β = 0.12, 95% CI = 0.06–0.17). These combined results highlight a potential novel mechanism for reduced lung function in urban youth resulting from both genetics and smoking exposure

Prevalence of allergic sensitization in the U.S.: Results from the National Health and Nutrition Examination Survey (NHANES) 2005–2006

Allergic sensitization is an important risk factor for the development of atopic disease. The National Health and Nutrition Examination Survey (NHANES) 2005–2006 provides the most comprehensive information on IgE-mediated sensitization in the general US population

Heterogeneity of magnitude, allergen immunodominance, and cytokine polarization of cockroach allergen-specific T cell responses in allergic sensitized children.

Background: Characterization of allergic responses to cockroach (CR), a common aeroallergen associated with asthma, has focused mainly on IgE reactivity, but little is known about T cell responses, particularly in children. We conducted a functional evaluation of CR allergen-specific T cell reactivity in a cohort of CR allergic children with asthma. Methods: Peripheral blood mononuclear cells (PBMCs) were obtained from 71 children, with mild-to-moderate asthma who were enrolled in a CR immunotherapy (IT) clinical trial, prior to treatment initiation. PBMC were stimulated with peptide pools derived from 11 CR allergens, and CD4+ T cell responses assessed by intracellular cytokine staining. Results: Highly heterogeneous responses in T cell reactivity were observed among participants, both in terms of the magnitude of cytokine response and allergen immunodominance. Reactivity against Bla g 9 and Bla g 5 was most frequent. The phenotype of the T cell response was dominated by IL-4 production and a Th2 polarized profile in 54.9% of participants, but IFNγ production and Th1 polarization was observed in 25.3% of the participants. The numbers of regulatory CD4+ T cells were also highly variable and the magnitude of effector responses and Th2 polarization were positively correlated with serum IgE levels specific to a clinical CR extract. Conclusions: Our results demonstrate that in children with mild-to-moderate asthma, CR-specific T cell responses display a wide range of magnitude, allergen dominance, and polarization. These results will enable examination of whether any of the variables measured are affected by IT and/or are predictive of clinical outcomes