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.

Distinct trajectories distinguish antigen-specific T cells in peanut-allergic individuals undergoing oral immunotherapy.

BACKGROUND: Despite similar clinical symptoms, peanut-allergic (PA) individuals may respond quite differently to the same therapeutic interventions. OBJECTIVE: This study aimed to determine whether inherent qualities of cell response at baseline could influence response to peanut oral immunotherapy (PnOIT). METHODS: We first performed ex vivo T-cell profiling on peanut-reactive CD154+CD137+ T (pTeff) cells from 90 challenge-confirmed PA individuals. We developed a gating strategy for unbiased assessment of the phenotypic distribution of rare pTeff cells across different memory CD4+ T-cell subsets to define patient immunotype. In longitudinal samples of 29 PA participants enrolled onto the IMPACT trial of PnOIT, we determined whether patient immunotype at baseline could influence response to PnOIT. RESULTS: Our data emphasize the heterogeneity of pTeff cell responses in PA participants with 2 mutually exclusive phenotypic entities (CCR6-CRTH2+ and CCR6+CRTH2-). Our findings lead us to propose that peanut allergy can be classified broadly into at least 2 discrete subtypes, termed immunotypes, with distinct immunologic and clinical characteristics that are based on the proportion of TH2A pTeff cells. PnOIT induced elimination of TH2A pTeff cells in the context of the IMPACT clinical trial. Only 1 PA patient with a low level of TH2A pTeff cells at baseline experienced long-lasting benefit of remission after PnOIT discontinuation. CONCLUSION: Dividing PA patients according to their individual peanut-specific T-cell profile may facilitate patient stratification in clinical settings by identifying which immunotypes might respond best to different therapies.

Effects of combination treatment with tezepelumab and allergen immunotherapy on nasal responses to allergen: A randomized controlled trial.

BACKGROUND: Thymic stromal lymphopoietin (TSLP) has been shown to play a central role in the initiation and persistence of allergic responses. OBJECTIVE: We evaluated whether tezepelumab, a human monoclonal anti-TSLP antibody, improved the efficacy of subcutaneous allergen immunotherapy (SCIT) and promoted the development of tolerance in patients with allergic rhinitis. METHODS: We conducted a double-blind parallel design trial in patients with cat allergy. A total of 121 patients were randomized to receive either intravenous tezepelumab plus subcutaneous cat SCIT, cat SCIT alone, tezepelumab alone, or placebo for 52 weeks, followed by 52 weeks of observation. Nasal allergen challenge (NAC), skin testing, and blood and nasal samples were obtained throughout the study. RESULTS: At week 52, the NAC-induced total nasal symptom scores (TNSS) (calculated as area under the curve [AUC0-1h] and as peak score [Peak0-1h] during the first hour after NAC) were significantly reduced in patients receiving tezepelumab/SCIT compared to SCIT alone. At week 104, one year after stopping treatment, the primary end point TNSS AUC0-1h was not significantly different in the tezepelumab/SCIT group compared to SCIT alone, while TNSS Peak0-1h was significantly lower in those receiving combination treatment versus SCIT. Transcriptomic analysis of nasal epithelial samples demonstrated that treatment with the combination of SCIT/tezepelumab, but neither monotherapy, caused persistent downregulation of a gene network related to type 2 inflammation that was associated with improvement in NAC responses. CONCLUSIONS: Inhibition of TSLP augments the efficacy of SCIT during therapy and may promote tolerance after a 1-year course of treatment. (ClinicalTrials.gov NCT02237196).

Peanut-Specific IgG4 and IgA in Saliva Are Modulated by Peanut Oral Immunotherapy.

BACKGROUND: Antigen-specific immunoglobulin responses have yet to be studied at the oral mucosal surface during peanut oral immunotherapy (PnOIT). OBJECTIVE: We aimed to quantify salivary peanut-specific IgG4 (PNsIgG4) and IgA (PNsIgA) and total IgG4 and IgA in participants from the Immune Tolerance Network's IMPACT study, a phase 2 PnOIT trial. METHODS: Peanut-allergic children, aged 12 months to younger than 48 months at screening, were enrolled and randomized to PnOIT or placebo oral immunotherapy (OIT) for 134 weeks. Per-protocol analysis included 69 PnOIT and 23 placebo participants. Double-blind, placebo-controlled food challenges were conducted at weeks 134 and 160 to assess desensitization and remission, respectively. Saliva samples were collected at baseline and 30, 82, 134, and 160 weeks to quantify PNsIgG4, PNsIgA, and total IgG4 and IgA. RESULTS: Participants who received PnOIT experienced significant increases in PNsIgG4 in saliva, whereas participants on placebo did not (P < .01 at all time points). The PNsIgA/total IgA ratio was also significantly increased in participants treated with PnOIT when compared with those receiving placebo at 30 and 82 weeks (P < .05). During PnOIT, desensitized participants had increased PNsIgA that plateaued, whereas the not desensitized/no remission group did not change over time. Interestingly, when the PnOIT group was evaluated by clinical outcome, PNsIgA was higher at baseline in the not desensitized/no remission group than in the desensitized/remission group (P < .05). CONCLUSIONS: PnOIT induces substantial increases in allergen-specific IgG4 and IgA in saliva. These data provide insight into OIT-induced mucosal responses and suggest the utility of these easily obtained samples for biomarker development.

Efficacy and safety of oral immunotherapy in children aged 1-3 years with peanut allergy (the Immune Tolerance Network IMPACT trial): a randomised placebo-controlled study.

BACKGROUND: For young children with peanut allergy, dietary avoidance is the current standard of care. We aimed to assess whether peanut oral immunotherapy can induce desensitisation (an increased allergic reaction threshold while on therapy) or remission (a state of non-responsiveness after discontinuation of immunotherapy) in this population. METHODS: We did a randomised, double-blind, placebo-controlled study in five US academic medical centres. Eligible participants were children aged 12 to younger than 48 months who were reactive to 500 mg or less of peanut protein during a double-blind, placebo-controlled food challenge (DBPCFC). Participants were randomly assigned by use of a computer, in a 2:1 allocation ratio, to receive peanut oral immunotherapy or placebo for 134 weeks (2000 mg peanut protein per day) followed by 26 weeks of avoidance, with participants and study staff and investigators masked to group treatment assignment. The primary outcome was desensitisation at the end of treatment (week 134), and remission after avoidance (week 160), as the key secondary outcome, were assessed by DBPCFC to 5000 mg in the intention-to-treat population. Safety and immunological parameters were assessed in the same population. This trial is registered on ClinicalTrials.gov, NCT03345160. FINDINGS: Between Aug 13, 2013, and Oct 1, 2015, 146 children, with a median age of 39·3 months (IQR 30·8-44·7), were randomly assigned to receive peanut oral immunotherapy (96 participants) or placebo (50 participants). At week 134, 68 (71%, 95% CI 61-80) of 96 participants who received peanut oral immunotherapy compared with one (2%, 0·05-11) of 50 who received placebo met the primary outcome of desensitisation (risk difference [RD] 69%, 95% CI 59-79; p<0·0001). The median cumulative tolerated dose during the week 134 DBPCFC was 5005 mg (IQR 3755-5005) for peanut oral immunotherapy versus 5 mg (0-105) for placebo (p<0·0001). After avoidance, 20 (21%, 95% CI 13-30) of 96 participants receiving peanut oral immunotherapy compared with one (2%, 0·05-11) of 50 receiving placebo met remission criteria (RD 19%, 95% CI 10-28; p=0·0021). The median cumulative tolerated dose during the week 160 DBPCFC was 755 mg (IQR 0-2755) for peanut oral immunotherapy and 0 mg (0-55) for placebo (p<0·0001). A significant proportion of participants receiving peanut oral immunotherapy who passed the 5000 mg DBPCFC at week 134 could no longer tolerate 5000 mg at week 160 (p<0·001). The participant receiving placebo who was desensitised at week 134 also achieved remission at week 160. Compared with placebo, peanut oral immunotherapy decreased peanut-specific and Ara h2-specific IgE, skin prick test, and basophil activation, and increased peanut-specific and Ara h2-specific IgG4 at weeks 134 and 160. By use of multivariable regression analysis of participants receiving peanut oral immunotherapy, younger age and lower baseline peanut-specific IgE was predictive of remission. Most participants (98% with peanut oral immunotherapy vs 80% with placebo) had at least one oral immunotherapy dosing reaction, predominantly mild to moderate and occurring more frequently in participants receiving peanut oral immunotherapy. 35 oral immunotherapy dosing events with moderate symptoms were treated with epinephrine in 21 participants receiving peanut oral immunotherapy. INTERPRETATION: In children with a peanut allergy, initiation of peanut oral immunotherapy before age 4 years was associated with an increase in both desensitisation and remission. Development of remission correlated with immunological biomarkers. The outcomes suggest a window of opportunity at a young age for intervention to induce remission of peanut allergy. FUNDING: National Institute of Allergy and Infectious Disease, Immune Tolerance Network.

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.

Nasal allergen challenge and environmental exposure chamber challenge: A randomized trial comparing clinical and biological responses to cat allergen.

BACKGROUND: The direct-instillation nasal allergen challenge (NAC) and the environmental exposure chamber (EEC) are 2 methods of conducting controlled allergen provocations. The clinical and biological comparability of these methods has not been thoroughly investigated. OBJECTIVE: We sought to compare clinical and immunologic responses to cat allergen in NAC versus EEC. METHODS: Twenty-four participants were randomized to receive either NAC followed by a 2-day challenge in an EEC or a 2-day challenge in an EEC followed by NAC. Challenges were separated by 28-day washout periods. We measured total nasal symptom scores, peak nasal inspiratory flow, nasal (0-8 hours) and serum cytokines, serum antibodies, peripheral blood antigen-specific T lymphocytes, and gene expression in nasal scrapings. The primary outcome was the total nasal symptom score area under the curve for the first 3 hours after allergen exposure in NAC or after initiation of exposure in EEC. RESULTS: Both challenges increased IL-5 and IL-13 in nasal fluids and serum and resulted in altered nasal cell expression of gene modules related to mucosal biology and transcriptional regulation. Changes in gene modules, more so than cytokine measurements, showed significant associations with total nasal symptom score and peak nasal inspiratory flow. Overall, EEC exposure generated larger responses and more early terminations compared with NAC. Although the 2 challenges did not correlate in symptom magnitude or temporality, striking correlations were observed in cytokine levels. CONCLUSIONS: Although clinical outcomes of NAC and EEC were temporally different and nonequivalent in magnitude, immunologic responses were similar. Selection of a particular allergen challenge method should depend on considerations of study objectives and cost.

Abatacept Targets T Follicular Helper and Regulatory T Cells, Disrupting Molecular Pathways That Regulate Their Proliferation and Maintenance.

Abatacept is a CTLA-4-Ig fusion protein that binds to the costimulatory ligands CD80 and CD86 and blocks their interaction with the CD28 and CTLA-4 receptors expressed by T cells, therefore inhibiting T cell activation and function. Abatacept has shown clinical efficacy in treating some autoimmune diseases but has failed to show clinical benefit in other autoimmune conditions. The reasons for these disparate results are not clear and warrant further investigation of abatacept's mode of action. Longitudinal specimens from the Immune Tolerance Network's A Cooperative Clinical Study of Abatacept in Multiple Sclerosis trial were used to examine the effects of abatacept treatment on the frequency and transcriptional profile of specific T cell populations in peripheral blood. We found that the relative abundance of CD4+ T follicular helper (Tfh) cells and regulatory T cells was selectively decreased in participants following abatacept treatment. Within both cell types, abatacept reduced the proportion of activated cells expressing CD38 and ICOS and was associated with decreased expression of genes that regulate cell-cycle and chromatin dynamics during cell proliferation, thereby linking changes in costimulatory signaling to impaired activation, proliferation, and decreased abundance. All cellular and molecular changes were reversed following termination of abatacept treatment. These data expand upon the mechanism of action of abatacept reported in other autoimmune diseases and identify new transcriptional targets of CD28-mediated costimulatory signaling in human regulatory T and Tfh cells, further informing on its potential use in diseases associated with dysregulated Tfh activity.

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.

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.

Highly variable population-based prevalence rates of unilateral hearing loss after the application of common case definitions.

OBJECTIVES: This study shows how population-based estimates of the prevalence of unilateral hearing loss (UHL) in children aged 6 to 19 yrs can differ considerably with various applications of commonly accepted case definitions. It also examines demographic variables and risk factors related to UHL. DESIGN: The Third National Health and Nutrition Examination Survey, conducted from 1988 to 1994, is a national population-based, cross-sectional survey. This study examined results of audiometric testing at 0.5 to 8 kHz and demographic data from in-person examination interviews. Three definitions of UHL were used: (1) 0.5, 1, and 2 kHz > or = 15 dB pure-tone average (PTA); (2) 0.5, 1, 2, and 4 kHz > or = 15 dB PTA; and (3) 0.5, 1, and 2 kHz > or = 20 dB or PTA >25 dB at two or more frequencies above 2 kHz (3, 4, 6, and 8 kHz). Case definitions 2 and 3 are not merely subsets of case definition 1. Some overlap exists between the groups, but each case definition classifies a proportion of children who fall uniquely under that case definition. Inclusion of participants based on tympanometry results (test of middle ear function) was also examined as were demographic characteristics and risk factors associated with UHL. RESULTS: Overall, the weighted proportion of children with UHL using case definition 1 was 6.3% (approximately 3,213,000 children nationally); using case definition 2, it was 5.8% (approximately 2,958,000 nationally); using case definition 3, it was 3.0% (approximately 1,530,000 nationally). For all three case definitions, children who failed tympanometry were at higher risk for UHL than children who passed. For case definition 2, children from rural areas were at higher risk for UHL than were children from urban areas. CONCLUSIONS: This study demonstrates that different applications of well-accepted case definitions of UHL can influence population-based prevalence estimates, in this study by as much as a factor of 2. These findings highlight the importance of controlling for tympanometry status as a risk factor in such estimates. Which demographic characteristics and risk factors are significantly associated with hearing loss seem to vary depending on the case definition. These findings have implications for the interpretation of prevalence rates and risk factors in the literature on hearing loss in general. Prevalence rate estimates require careful consideration of the case definition of hearing loss, tympanometry status, and demographic characteristics.

Delta activity independent of its activity as a ligand of Notch.

BACKGROUND: Delta, Notch, and Scabrous often function together to make different cell types and refine tissue patterns during Drosophila development. Delta is known as the ligand that triggers Notch receptor activity. Scabrous is known to bind Notch and promote Notch activity in response to Delta. It is not known if Scabrous binds Delta or Delta has activity other than its activity as a ligand of Notch. It is very difficult to clearly determine this binding or activity in vivo as all Notch, Delta, and Scabrous activities are required simultaneously or successively in an inter-dependent manner. RESULTS: Using Drosophila cultured cells we show that the full length Delta promotes accumulation of Daughterless protein, fringe RNA, and pangolin RNA in the absence of Scabrous or Notch. Scabrous binds Delta and suppresses this activity even though it increases the level of the Delta intracellular domain. We also show that Scabrous can promote Notch receptor activity, in the absence of Delta. CONCLUSION: Delta has activity that is independent of its activity as a ligand of Notch. Scabrous suppresses this Delta activity. Scabrous also promotes Notch activity that is dependent on Delta's ligand activity. Thus, Notch, Delta, and Scabrous might function in complex combinatorial or mutually exclusive interactions during development. The data reported here will be of significant help in understanding these interactions in vivo.