Contribution of T cell subsets to different food allergic diseases.

Food allergies occur due to a lack of tolerance to the proteins found in foods. While IgE- and non-IgE-mediated food allergies have different clinical manifestations, epidemiology, pathophysiology, and management, they share dysregulated T cell responses. Recent studies have shed light on the contributions of different T cell subsets to the development and persistence of different food allergic diseases. This review discusses the role of T cells in both IgE- and non-IgE-mediated food allergies and considers the potential future investigations in this context.

Secreted IgD Amplifies Humoral T Helper 2 Cell Responses by Binding Basophils via Galectin-9 and CD44.

B cells thwart antigenic aggressions by releasing immunoglobulin M (IgM), IgG, IgA, and IgE, which deploy well-understood effector functions. In contrast, the role of secreted IgD remains mysterious. We found that some B cells generated IgD-secreting plasma cells following early exposure to external soluble antigens such as food proteins. Secreted IgD targeted basophils by interacting with the CD44-binding protein galectin-9. When engaged by antigen, basophil-bound IgD increased basophil secretion of interleukin-4 (IL-4), IL-5, and IL-13, which facilitated the generation of T follicular helper type 2 cells expressing IL-4. These germinal center T cells enhanced IgG1 and IgE but not IgG2a and IgG2b responses to the antigen initially recognized by basophil-bound IgD. In addition, IgD ligation by antigen attenuated allergic basophil degranulation induced by IgE co-ligation. Thus, IgD may link B cells with basophils to optimize humoral T helper type 2-mediated immunity against common environmental soluble antigens.

Immunology of Food Allergy.

Many consider food allergy as the "second wave" of the allergy epidemic following the "first wave" of respiratory allergy, i.e., asthma and allergic rhinitis, plaguing westernized countries, with up to 8% of young children and 2%-3% of adults in the United States now affected by hypersensitivity reactions to various foods. In the past decade, there have been great strides in our understanding of the underlying immunopathogenesis of these disorders, which have led to improved diagnostic techniques, management strategies, and therapeutic approaches. Here we will review the most recent understanding of basic mechanisms underlying IgE-mediated food allergies and novel therapeutic approaches under investigation for both the prevention and treatment of IgE-mediated food allergies.

Joint transcriptomic and cytometric study of children with peanut allergy reveals molecular and cellular cross talk in reaction thresholds.

BACKGROUND: Reaction thresholds in peanut allergy are highly variable. Elucidating causal relationships between molecular and cellular processes associated with variable thresholds could point to therapeutic pathways for raising thresholds. OBJECTIVE: The aim of this study was to characterize molecular and cellular systemic processes associated with reaction threshold in peanut allergy and causal relationships between them. METHODS: A total of 105 children aged 4 to 14 years with suspected peanut allergy underwent double-blind, placebo-controlled food challenge to peanut. The cumulative peanut protein quantity eliciting allergic symptoms was considered the reaction threshold for each child. Peripheral blood samples collected at 0, 2, and 4 hours after challenge start were used for RNA sequencing, whole blood staining, and cytometry. Statistical and network analyses were performed to identify associations and causal mediation between the molecular and cellular profiles and peanut reaction threshold. RESULTS: Within the cohort (N = 105), 81 children (77%) experienced allergic reactions after ingesting varying quantities of peanut, ranging from 43 to 9043 mg of cumulative peanut protein. Peripheral blood expression of transcripts (eg, IGF1R [false discovery rate (FDR) = 5.4e-5] and PADI4 [FDR = 5.4e-5]) and neutrophil abundance (FDR = 9.5e-4) were associated with peanut threshold. Coexpression network analyses revealed that the threshold-associated transcripts were enriched in modules for FcγR-mediated phagocytosis (FDR = 3.2e-3) and Toll-like receptor (FDR = 1.4e-3) signaling. Bayesian network, key driver, and causal mediation analyses identified key drivers (AP5B1, KLHL21, VASP, TPD52L2, and IGF2R) within these modules that are involved in bidirectional causal mediation relationships with neutrophil abundance. CONCLUSION: Key driver transcripts in FcγR-mediated phagocytosis and Toll-like receptor signaling interact bidirectionally with neutrophils in peripheral blood and are associated with reaction threshold in peanut allergy.

Differential Tfh cell phenotypes distinguish IgE-mediated milk allergy from eosinophilic esophagitis in children.

BACKGROUND: IgE-mediated food allergy and eosinophilic esophagitis (EoE) are diseases commonly triggered by milk. Milk-responsive CD4+ T cells producing type 2 cytokines are present in both diseases, yet the clinical manifestation of disease in milk allergy (MA) and EoE are distinct. OBJECTIVE: To identify CD4+ T cell differences between EoE and MA that may be responsible for distinct disease manifestations. METHODS: The total and milk-specific CD4+ T cell phenotype of children with milk allergy (MA), EoE (active or in remission) and controls was measured using spectral flow cytometry of peripheral blood (all groups) or esophageal biopsies (EoE and control). RESULTS: Circulating milk-responsive T cells could be identified in active (A)-EoE and MA. An increased frequency of Th2A cells was also noted in MA and EoE. In circulating T cells, type 2 cytokine production was elevated in MA, but not EoE. Within the milk-responsive Tfh subset, a dichotomy of phenotype was noted: Tfh13 cells predominated in MA, while IL-10-producing Tfh cells predominated in EoE. In the esophagus, CD4+ T cells were constitutively activated and expressed not only type 2 cytokines, but also IL-10 and IL-21 in A-EoE. There was production of IgG4 from CD38+ plasma cells in close proximity to CD4+ T cells. In vitro activation studies demonstrated that IL-10 and IL-21 elicited strong IgG4 responses in B lymphocytes, while IL-4 and IL-13 promoted IgE production. CONCLUSION: Our studies demonstrate a dichotomy of Tfh responses that may be the basis for the different clinical manifestations to milk in EoE and MA.

Untargeted serum metabolomic analysis reveals a role for purinergic signaling in FPIES.

BACKGROUND: Food protein-induced enterocolitis syndrome (FPIES) is a non-IgE-mediated food allergy with a typical onset in infancy. Its symptoms are distinct from those of IgE-mediated food allergies and include severe repetitive vomiting, lethargy, and pallor. FPIES reactions are associated with TH17 cytokines and a systemic innate immune activation; however, the link between immune activation and symptoms is poorly understood. OBJECTIVE: Our aim was to use an untargeted metabolomics approach to identify novel pathways associated with FPIES reactions. METHODS: Serum samples were obtained before, during, and after oral food challenge (OFC) (10 subjects with FPIES and 10 asymptomatic subjects), and they were analyzed by untargeted metabolomics. Two-way ANOVA with false discovery rate adjustment was used for analysis of metabolites. Stomach and duodenal biopsy specimens from non-FPIES donors were stimulated with adenosine in vitro and serotonin measured by immunoassay. RESULTS: The levels of a total of 34 metabolites, including inosine and urate of the purine signaling pathway, were increased during OFCs performed on the patients with symptomatic FPIES compared with the levels found for asymptomatic subjects. Expression of the purine receptors P2RX7 and P2RY10 and the ectonucleotidase CD73 in peripheral blood was significantly reduced after OFC of the patients with FPIES. The level of the serotonin metabolite 5-hydroxyindoleacetate was significantly elevated after reaction. Adenosine stimulation of gastric and duodenal biopsy specimens from FPIES-free donors induced a significant release of serotonin, suggesting a link between purinergic pathway activation and serotonin release. CONCLUSIONS: Activation of the purinergic pathway during FPIES reactions provides a possible mechanism connecting inflammation and vomiting by triggering serotonin release from gastric and duodenal mucosa.

Oral tolerance and oral immunotherapy for food allergy: Evidence for common mechanisms?

Food allergies affect up to 10% of the US population, can be life-threatening, and have a significant negative impact on quality of life. Delayed dietary introduction of foods in childhood can hinder the induction of oral tolerance, an active regulatory response to foods that prevents the development of food allergy. Some children outgrow their food allergies naturally, while many others have persistent, lifelong food allergy for which there are few therapeutic options. Oral immunotherapy (OIT) is a therapeutic approach of giving increasing amounts of food to attempt to desensitize the allergic individual. In this review, we focus on the immune mechanisms common to oral tolerance and response to oral immunotherapy, with the objective of determining whether true tolerance can be achieved after food allergy has been established.

Allergen recognition by specific effector Th2 cells enables IL-2-dependent activation of regulatory T-cell responses in humans.

Type 2 allergen-specific T cells are essential for the induction and maintenance of allergies to foods, and Tregs specific for these allergens are assumed to be involved in their resolution. However, it has not been convincingly demonstrated whether allergen-specific Treg responses are responsible for the generation of oral tolerance in humans. We observed that sustained food allergen exposure in the form of oral immunotherapy resulted in increased frequency of Tregs only in individuals with lasting clinical tolerance. We sought to identify regulatory components of the CD4+ T-cell response to food allergens by studying their functional activation over time in vitro and in vivo. Two subsets of Tregs expressing CD137 or CD25/OX40 were identified with a delayed kinetics of activation compared with clonally enriched pathogenic effector Th2 cells. Treg activation was dependent on IL-2 derived from effector T cells. In vivo exposure to peanut in the form of an oral food challenge of allergic subjects induced a delayed and persistent activation of Tregs after initiation of the allergen-specific Th2 response. The novel finding of our work is that a sustained wave of Treg activation is induced by the release of IL-2 from Th2 effector cells, with the implication that therapeutic administration of IL-2 could improve current OIT approaches.

The year in food allergy.

Research into food allergy continues to rapidly evolve, accompanying and driving real changes in the clinical approach to these diseases. The past year has seen the rollout of the first treatment approved for active management of food allergy, more data on alternative methods of treatment, the continued evolution of strategies for prevention of food allergy, a renewed interest in phenotyping food allergy subtypes, and, importantly, key new insights into the pathophysiology of food allergy. We expect that in the coming years, the therapies that are in preclinical or early clinical evaluation now will make their way to the clinic, finally allowing the possibility of safe and effective treatments for food allergy.

Allergen-specific T cells and clinical features of food allergy: Lessons from CoFAR immunotherapy cohorts.

BACKGROUND: Allergen-specific IL-4+ and IL-13+ CD4+ cells (type 2 cells) are essential for helping B cells to class-switch to IgE and establishing an allergic milieu in the gastrointestinal tract. The role of T cells in established food allergy is less clear. OBJECTIVE: We examined the food allergen-specific T-cell response in participants of 2 food allergen immunotherapy trials to assess the relationship of the T-cell response to clinical phenotypes, including response to immunotherapy. METHODS: Blood was obtained from 84 participants with peanut allergy and 142 participants with egg allergy who underwent double-blind placebo-controlled food challenges. Peanut- and egg-responsive T cells were identified by CD154 upregulation after stimulation with the respective extract. Intracellular cytokines and chemokine receptors were also detected. The response to peanut epicutaneous immunotherapy (Peanut Epicutaneous Phase II Immunotherapy Clinical Trial [CoFAR6]; 49 participants receiving epicutaneous immunotherapy) and egg oral immunotherapy or a baked egg diet (Baked Egg or Egg Oral Immunotherapy for Children With Egg Allergy [CoFAR7]; 92 participants) was monitored over time. RESULTS: Peanut-specific type 2 and CCR6+ T cells were negatively correlated with each other and differently associated with immune parameters, including specific IgE level and basophil activation test result. At baseline, type 2 cells, but not CCR6+ cells, were predictive of clinical parameters, including a successfully consumed dose of peanut and baked egg tolerance. Exposure to peanut or egg immunotherapy was associated with a decrease in type 2 cell frequency. At baseline, high egg-specific type 2 cell frequency was the immune feature most predictive of oral immunotherapy failure. CONCLUSION: Food-specific type 2 T cells at baseline are informative of threshold of reactivity and response to immunotherapy.

Acute FPIES reactions are associated with an IL-17 inflammatory signature.

BACKGROUND: Food protein-induced enterocolitis syndrome (FPIES) is a non-IgE-mediated food allergy characterized by profuse vomiting within hours of ingestion of the causative food. We have previously reported that FPIES is associated with systemic innate immune activation in the absence of a detectable antigen-specific antibody or T-cell response. The mechanism of specific food recognition by the immune system remains unclear. OBJECTIVE: Our aim was to identify immune mechanisms underlying FPIES reactions by proteomic and flow cytometric analysis of peripheral blood. METHODS: Children with a history of FPIES underwent supervised oral food challenge. Blood samples were taken at baseline, at symptom onset, and 4 hours after symptom onset. We analyzed samples from 23 children (11 reactors and 12 outgrown). A total of 184 protein markers were analyzed by proximity ligation assay and verified by multiplex immunoassay. Analysis of cell subset activation was performed by mass cytometry and spectral cytometry. RESULTS: Symptomatic FPIES challenge results were associated with significant elevation of levels of cytokines and chemokines, including IL-17 family markers (IL-17A, IL-22, IL-17C, and CCL20) and T-cell activation (IL-2), and innate inflammatory markers (IL-8, oncostatin M, leukemia inhibitory factor, TNF-α, IL-10, and IL-6). The level of the mucosal damage marker regenerating family member 1 alpha (REG1A) was also significantly increased. These biomarkers were not increased in asymptomatic challenges or IgE-mediated allergy. The level of phospho-STAT3 was significantly elevated in myeloid and T cells after challenge in individuals with symptoms. Mass cytometry indicated preferential activation of nonconventional T-cell populations, including γδ T cells and CD3+CD4-CD8-CD161+ cells; however, the potential sources of IL-17 in PBMCs were primarily CD4+ TH17 cells. CONCLUSIONS: These results demonstrate a unique IL-17 signature and activation of innate lymphocytes in FPIES.

Flow cytometric identification of Tfh13 cells in mouse and human.

Anaphylaxis is a life-threatening allergic reaction caused by cross-linking of high-affinity IgE antibodies on the surface of mast cells and basophils. Understanding the cellular mechanisms that lead to high-affinity IgE production is required to develop better therapeutics for preventing this severe reaction. A recently discovered population of T follicular helper Tfh13 cells regulates the production of high-affinity IgE in mouse models of allergy and can also be found in patients with allergies with IgE antibodies against food or aeroallergens. Here we describe optimized protocols for identifying Tfh13 cells in both mice and humans.

Epicutaneous immunotherapy for treatment of peanut allergy: Follow-up from the Consortium for Food Allergy Research.

BACKGROUND: Consortium for Food Allergy Research investigators previously reported 52-week outcomes from a randomized controlled trial of peanut epicutaneous immunotherapy, observing modest and statistically significant induction of desensitization, highest in children ages 4 to 11 years. OBJECTIVE: We sought to evaluate changes in efficacy, safety, and mechanistic parameters following extended open-label peanut epicutaneous immunotherapy. METHODS: Peanut-allergic participants (4-25 years) received 52 weeks of placebo (PLB), Viaskin Peanut 100 µg (VP100) or 250 µg (VP250), and then crossed over to VP250 for PLB (PLB-VP250) and VP100 (VP100-VP250) participants and continued treatment for VP250 participants (total = 130 weeks of active epicutaneous immunotherapy). Efficacy was assessed by double-blind, placebo-controlled food challenge (5044 mg peanut protein), and adherence, safety, and mechanistic parameters were evaluated. RESULTS: At week 130, desensitization success was achieved in 1 of 20 (5%) PLB-VP250, 5 of 24 (20.8%) VP100-VP250, and 9 of 25 (36%) VP250 participants, with median successfully consumed dose change from baseline of 11.5 mg, 141.5 mg, and 400 mg, respectively. Median age (years) for week 130 desensitization success was 6.2 years (interquartile range, 5.2-9.1) versus 9.4 years (interquartile range, 7.6-12.8) for failures (P < .001). Adherence was 96%. Adverse reactions were predominantly local patch-site reactions. Significant increases in peanut- and Ara h2-specific IgG4 observed at week 52 persisted to week 130. By a post hoc analysis, there were no statistically significant increases from week 52 to week 130 in either desensitization success or successfully consumed dose. CONCLUSIONS: Extended treatment with VP250 was well tolerated, and desensitization observed at week 52 persisted between weeks 52 and 130. Treatment success was observed predominantly in younger participants, with younger age at initiation of active therapy an important predictor of success.

Pathogenesis of IgE-mediated food allergy and implications for future immunotherapeutics.

Our understanding of the immune basis of food allergy has grown rapidly in parallel with the development of new immune-targeted interventions for the treatment of food allergy. Local tissue factors, including the composition of skin and gastrointestinal microbiota and production of Th2-inducing cytokines (TSLP, IL-33, and IL-25) from barrier sites, have been shown not only to contribute to the development of food allergy, but also to act as effective targets for treatment in mice. Ongoing clinical trials are testing the targeting of these factors in human disease. There is a growing understanding of the contribution of IL-13 to the induction of high-affinity IgE and the need for continual T-cell help in the maintenance of long-lived IgE. This provides a strong rationale to test biologics targeting both IL-4 and IL-13 in the treatment of established food allergy. Various forms of allergen immunotherapy for food allergy have clearly shown that low specific IgE and elevated specific IgG4 are predictive of sustained treatment effect. Treatments that mimic that immune response, for example, lowering IgE, with monoclonal antibodies such as omalizumab, or administering allergen-specific IgG, are in various stages of investigation. As we gain more opportunities to use immune-modifying treatments for the treatment of food allergy, studies of the immune and clinical response to those interventions will continue to rapidly advance our understanding of the immune basis of food allergy and tolerance.

Advances in understanding immune mechanisms of food protein-induced enterocolitis syndrome.

OBJECTIVE: This review provides an overview of our current understanding of the mechanisms of food protein-induced enterocolitis syndrome (FPIES). DATA SOURCES: To capture recent articles published since our previous comprehensive review on the pathophysiology of FPIES, we performed a literature search through PubMed database, using the search terms FPIES and food protein-induced enterocolitis syndrome from 2016 to the current year. STUDY SELECTIONS: Studies in English containing biomarker or immune data were reviewed and summarized. RESULTS: Studies of peripheral blood fail to exhibit evidence of antigen-specific humoral or cellular immunity underlying clinical reactivity to foods in FPIES. However, growing evidence suggests a robust systemic innate immune activation occurring during FPIES reactions and the activation of neuroendocrine pathways. CONCLUSION: FPIES reactions are associated with marked activation of innate immune and neuroendocrine pathways; however, the mechanism underlying the specific recognition of foods remains elusive.

Induction of sustained unresponsiveness after egg oral immunotherapy compared to baked egg therapy in children with egg allergy.

BACKGROUND: While desensitization and sustained unresponsiveness (SU) have been shown with egg oral immunotherapy (OIT), the benefits of baked egg (BE) therapy for egg allergy have not been well studied. OBJECTIVES: This study sought to evaluate the safety and efficacy of BE ingestion compared with egg OIT in participants allergic to unbaked egg but tolerant to BE. METHODS: Children who are BE-tolerant but unbaked egg reactive ages 3 to 16 years were randomized to 2 years of treatment with either BE or egg OIT. Double-blind, placebo-controlled food challenges were conducted after 1 and 2 years of treatment to assess for desensitization, and after 2 years of treatment followed by 8 to 10 weeks off of treatment to assess for SU. Mechanistic studies were conducted to assess for immune modulation. A cohort of participants who are BE-reactive underwent egg OIT and identical double-blind, placebo-controlled food challenges as a comparator group. RESULTS: Fifty participants (median age 7.3 years) were randomized and initiated treatment. SU was achieved in 3 of 27 participants assigned to BE (11.1%) versus 10 of 23 participants assigned to egg OIT (43.5%) (P = .009). In the BE-reactive comparator group, 7 of 39 participants (17.9%) achieved SU. More participants who are BE-tolerant withdrew from BE versus from egg OIT (29.6% vs 13%). Dosing symptom frequency in participants who are BE-tolerant was similar with BE and egg OIT, but more frequent in participants who are BE-reactive. Egg white-specific IgE, skin testing, and basophil activation decreased similarly after BE and egg OIT. CONCLUSIONS: Among children allergic to unbaked egg but tolerant to BE, those treated with egg OIT were significantly more likely to achieve SU than were children ingesting BE.

Mechanisms that define transient versus persistent food allergy.

Currently, we have a poor understanding of why some food allergies are outgrown and others are not. Deciphering the immune basis of the natural resolution of food allergy will likely provide critical information for developing new therapies for the treatment of persistent food allergies. There are limited cohort studies that have followed children with food allergy over time, but information generated from such cohorts points to features of innate and adaptive immunity, as well as environmental differences (microbiome) that discriminate those with persistent versus transient food allergy. Studies from mouse models highlight the importance of novel subsets of memory B cells rather than plasma cells combined with antigen re-exposure and T-cell help in the maintenance of IgE. In this review we discuss these findings from human cohorts and experimental systems and discuss existing gaps in our knowledge.

Food allergy and the microbiome: Current understandings and future directions.

Growing evidence points to an important role for the commensal microbiota in susceptibility to food allergy. Epidemiologic studies demonstrate associations between exposures known to modify the microbiome and risk of food allergy. Direct profiling of the gut microbiome in human cohort studies has demonstrated that individuals with food allergy have distinct gut microbiomes compared to healthy control subjects, and dysbiosis precedes the development of food allergy. Mechanistic studies in mouse models of food allergy have confirmed that the composition of the intestinal microbiota can imprint susceptibility or resistance to food allergy on the host and have identified a unique population of microbially responsive RORγt-positive FOXp3-positive regulatory T cells as critical for the maintenance of tolerance to foods. Armed with this new understanding of the role of the microbiota in food allergy and tolerance, therapeutics aimed at modifying the gastrointestinal microbiota are in development. In this article we review key milestones in the development of our current understanding of how the gastrointestinal microbiota contributes to food allergy and discuss our vision for the future of the field.

The Consortium for Food Allergy Research (CoFAR): The first generation.

The Consortium for Food Allergy Research (CoFAR) was established by the National Institute of Allergy and Infectious Diseases in 2005 as a collaborative research program bringing together centers focused on the study of food allergy. CoFAR was charged with developing studies to better understand the pathogenesis and natural history of food allergy, as well as potential approaches to the treatment of food allergy. In its first iteration an observational study of infants with milk and egg allergy was established, and studies of oral immunotherapy for egg allergy and sublingual immunotherapy for peanut allergy were initiated, as was a phase 1 study of a recombinant peanut protein vaccine. CoFAR was renewed in 2010 for an additional 5-year period during which the initial observational study was continued, a study of eosinophilic esophagitis was initiated, and new therapeutic trials were established to study epicutaneous immunotherapy for peanut allergy and to compare the safety and efficacy of egg oral immunotherapy to the ingestion of baked egg for the treatment of egg allergy. The results of these efforts will be reviewed in this rostrum, with a brief look to the future of CoFAR.

Single-cell profiling of peanut-responsive T cells in patients with peanut allergy reveals heterogeneous effector TH2 subsets.

BACKGROUND: The contribution of phenotypic variation of peanut-specific T cells to clinical allergy or tolerance to peanut is not well understood. OBJECTIVES: Our objective was to comprehensively phenotype peanut-specific T cells in the peripheral blood of subjects with and without peanut allergy (PA). METHODS: We obtained samples from patients with PA, including a cohort undergoing baseline peanut challenges for an immunotherapy trial (Consortium of Food Allergy Research [CoFAR] 6). Subjects were confirmed as having PA, or if they passed a 1-g peanut challenge, they were termed high-threshold subjects. Healthy control (HC) subjects were also recruited. Peanut-responsive T cells were identified based on CD154 expression after 6 to 18 hours of stimulation with peanut extract. Cells were analyzed by using flow cytometry and single-cell RNA sequencing. RESULTS: Patients with PA had tissue- and follicle-homing peanut-responsive CD4+ T cells with a heterogeneous pattern of TH2 differentiation, whereas control subjects had undetectable T-cell responses to peanut. The PA group had a delayed and IL-2-dependent upregulation of CD154 on cells expressing regulatory T (Treg) cell markers, which was absent in HC or high-threshold subjects. Depletion of Treg cells enhanced cytokine production in HC subjects and patients with PA in vitro, but cytokines associated with highly differentiated TH2 cells were more resistant to Treg cell suppression in patients with PA. Analysis of gene expression by means of single-cell RNA sequencing identified T cells with highly correlated expression of IL4, IL5, IL9, IL13, and the IL-25 receptor IL17RB. CONCLUSIONS: These results demonstrate the presence of highly differentiated TH2 cells producing TH2-associated cytokines with functions beyond IgE class-switching in patients with PA. A multifunctional TH2 response was more evident than a Treg cell deficit among peanut-responsive T cells.