Mechanisms that define transient versus persistent food allergy

Abstract

We currently have a poor understanding of why some food allergies are outgrown while 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 food allergic children 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.

Introduction

Food allergy has its origins in early life and affects 6–8% of the pediatric population. Some food allergies are commonly outgrown, while others are more likely to persist into adulthood. Milk and egg allergies, which are the most common allergies of childhood, are two allergies that are commonly outgrown in childhood. A multi-center observational Consortium for Food Allergy Research (CoFAR) study of 3–15 month old infants enrolled with milk or egg allergy and/or eczema showed that approximately 50% of infants with egg allergy outgrew their egg allergy at a median age of 6 years¹. The same cohort showed that 52.6% of milk allergic children had outgrown their milk allergy at a median of 63 months². This is a slower rate of resolution than has been described in population-based (not selected as high-risk) cohorts. The population-based Australian HealthNuts cohort showed that 47% of egg allergic infants outgrew their egg allergy at 2 years of life³, and prevalence of egg allergy dropped from 9.5% at one year of life to 1.2% at 4 years of life⁴. In the EuroPrevall birth cohort of >12 000 children from countries throughout Europe, 57% of children with IgE-mediated milk allergy had outgrown their milk allergy by 2 years of life⁵. Unlike milk and egg allergy, peanut and tree nut allergies are commonly persistent. A 4-year follow-up of peanut allergy in the HealthNuts cohort found that 22% of children diagnosed with peanut allergy at one year of life had resolved their peanut allergy by 4 years of life⁶. This is consistent with previous reports on the resolution of peanut allergy⁷. There is limited information on tree nut allergy, with one report of a resolution rate of 9%. The occurrence of spontaneous resolution, even with peanut and tree nut allergies, demonstrates that true tolerance can be achieved in food allergy, and there is a need to understand the immune basis of this resolution in order to develop more rational therapies for the treatment of persistent food allergy.

Predictors of Food Allergy Resolution

Food specific immunoglobulins:

In the CoFAR natural history cohort, milk-specific IgE, skin prick test size to milk, and severity of atopic dermatitis (AD) at baseline were the three parameters that were the most important factors predicting resolution of milk allergy². Similarly, egg-specific IgE and severity of AD at baseline were the two most important parameters predicting resolution of egg allergy¹. A calculator incorporating these factors was generated to predict resolution of milk and egg allergy^{1, 2}. A limitation of the CoFAR natural history study was the absence of protocol-driven food challenges. The use of IgE levels to categorize individuals introduces bias toward finding a role for IgE in clinical outcomes. However, the CoFAR study also analyzed a subset of the cohort with confirmed food allergy or tolerance (by patient report of reaction or tolerance after exposure, or by challenges performed as part of clinical care). Predictors of persistent peanut allergy (defined as persistent at 4 years of age) in the HealthNuts cohort included skin test size and peanut-specific IgE measured at 1 year of age⁶. Eczema in the first year of life was not predictive of peanut allergy persistence. Allergen-specific IgG4 levels have not been found to be predictive of natural resolution, but levels of IgG4 are increased by allergen exposure.

In addition to the magnitude of the specific IgE response, the epitope specificity of the IgE response appears to be an important factor determining transience versus persistence. Studies examining persistent milk and egg allergy highlighted the importance of binding to linear epitopes in predicting allergy persistence^9–12. In the HealthNuts cohort, the magnitude of egg white-specific IgE was predictive of egg allergy persistence¹³. Measurement of IgE binding to egg components (ovomucoid, ovalbumin, ovotransferrin from egg white and alpha-livetin from egg yolk) did not improve prediction of egg allergy persistence compared to egg white specific-IgE, but binding to all 4 allergens increased the risk of persistent egg allergy by a factor of 4. Thus, the diversity or breadth of the IgE response appears to contribute to persistence. Using a microarray-based approach to profile linear epitope binding, Caubet et al found that a children with persistent milk allergy had stronger and more diverse binding to a-S and b caseins in the context of milk allergy¹⁴. Furthermore, those who outgrew their milk allergy were more likely to have IgE and IgG4 antibodies recognizing the same epitopes. The magnitude and quality (diversity) of the IgE response to foods appears to be critical to allergy persistence.

Food-specific T cell responses:

There is a lack of information on T cell phenotypes associated with the natural history of food allergy. In the CoFAR natural history cohort, food-specific T cell responses were examined by culturing PBMCs with food extracts, and purifying CD25+ T cells after 48 h of stimulation. Gene expression on the CD25+ fraction, including activated T cells and Tregs, was examined by semi-quantitative RT-PCR. At baseline, milk- and peanut-induced IL-4 expression was associated with milk and peanut allergy, respectively¹⁵. Baseline or serial measures of milk-induced IL-4, IFN-g, IL-10, or Foxp3 were not associated with milk allergy persistence versus resolution². Egg-induced IL-4 expression had a small (hazard ratio 1.04) but significant (p<0.05) association with egg allergy persistence, but this was a minor contribution compared to IgE levels¹.

Peanut-specific T cell responses were examined in a small cohort (n=4) of children who had outgrown their peanut allergy¹⁶. Intracellular cytokines produced by proliferating cells were described as more Th1 skewed in those who outgrew, although this was presented as a ratio between IFN-g or TNFa and Th2 cytokines so it is not clear if this was due to a drop in Th2 cytokines, an increase in Th1 cytokines, or both. Qamar et al studied 11 children who had recently developed tolerance to egg or peanut, in comparison with 22 children with egg or peanut allergy¹⁷. Tolerance was associated with an increase in frequency of Foxp3⁺CD25⁺CD127^low Tregs, and an increase in IL-10-expressing CD4+ T cells and Tregs. IL-10 production from the Tregs was elicited by the tolerated antigen upon stimulation in vitro, as had previously been reported using detection of IL-10 in culture supernatants¹⁸.

Innate immunity:

The innate immune system functions to integrate cues from environmental triggers (infection, stress, damage) and relay that information to the adaptive immune system. For example, dendritic cells (DCs) in tissues respond to microbial patterns, damage associated patterns, or alarmins with a change in phenotype that determines the function of the T cell response that is generated by antigen presentation. Tissue macrophages also respond to environmental cues and release cytokines that can shape the immune response to antigens derived from those tissues. Peripheral blood DCs from individuals with food allergy have an altered phenotype compared to healthy controls, with increased production of pro-inflammatory cytokine production in response to allergen stimulation¹⁹. Such responses are likely linked to signaling through FcεRI on DCs. However, alternations in innate immunity have also been shown to precede development of food allergy. Increased frequency of monocytes in cord blood and increased proinflammatory cytokine production in response to LPS was associated with development of food allergy at one year of life. This elevated responsiveness of the myeloid compartment to LPS was also identified by Neeland et al in children with egg allergy compared to non-allergic controls²¹. Furthermore, levels of TNFα and IL-8 production in response to LPS (measured at one year of age) were significantly higher in children with persistent versus transient egg allergy as determined at follow-up. Factors that have modulatory effects on the innate immune system may therefore promote allergy resolution in an allergen non-specific manner.

Microbiome:

There is growing evidence that the microbiome plays a key role in food allergy. Factors that alter the composition of microbiome (caesarean section birth, early life pet exposure) alter the risk of development of food allergy. Dysbiosis precedes the development of food allergy^{22, 23}. Work in mouse models has identified protective mechanisms induced by the intestinal microbiota (improved barrier, IgA production, Tregs) that prevent food allergy^24–26. There is a great deal of interest in harnessing this information for microbial-based therapies, but it is not known if the microbiome can be used to facilitate the resolution of food allergy once it has been established. Bunyavanich and colleagues used the CoFAR natural history cohort to examine the relationship between fecal microbial composition and milk²⁷ or egg allergy²⁸ resolution. They found that the microbial composition at 3–6 months of age differed significantly in infants with milk allergy who resolved their milk allergy compared to those with persistent milk allergy. They found enrichment of Clostridia species and Firmicutes in fecal samples of those who resolved their food allergy. Interestingly, Clostridia species have been identified as particularly potent inducers of regulatory cell pathways in the gastrointestinal tract^{24, 29, 30}. Stool samples collected at later time-points (6–15 months) did not show any difference between persistent and transient milk allergy, highlighting a critical window in early life in which microbiota influences disease outcome. In the context of egg allergy, no significant microbial predictors were identified for resolution of egg allergy although a unique microbial composition was observed in those with egg allergy compared to those with no food allergy²⁸. However, the study of egg allergy included stool samples collected from 3–15 months, reducing the likelihood to observe a signal that was unique to the 3–6 month collection time in milk allergy. It is not known if modulation of the intestinal microbiota after the window of early infancy will be useful for enhancing resolution of food allergy. It is also not known if altering the microbiome by itself could facilitate natural resolution of food allergy, or whether administration together with allergen, as has been done with probiotics and OIT³¹, would be required for microbial-facilitated resolution of food allergy.

Resolution of Food Allergy: Lessons from Animal Models

Animal models have been used to study mechanisms of sensitization and tolerance to foods, but there are few studies on the mechanisms of allergy persistence. Jimenez-Saiz et al focused on the maintenance of IgE responses over time in a mouse model of peanut induced anaphylaxis³². Mice were orally sensitized with peanut and the mucosal adjuvant cholera toxin, and followed over time. Susceptibility to anaphylaxis was maintained for 6 months, but was lost by 12 months after sensitization which coincided with a loss of detectable peanut-specific IgE. They observed that loss of peanut-specific IgE was preceded by a loss of peanut-specific germinal center memory cells. Allergen re-exposure and T cell help were required to maintain germinal center memory cells, which upon activation replenish the IgE plasma cell compartment and facilitate susceptibility to peanut-induced anaphylaxis.

It has been shown that IgE can be generated through sequential class-switch from IgG1 memory B cells, and that sequential IgG to IgE class-switch results in pathogenic (higher affinity) IgE compared to low-affinity IgE generated by direct class-switch³³. Recent work by He et al demonstrated that a unique subset of IgG1+ memory B cells housed allergen-specific IgE memory³⁴. Using a B cell transfer model, they showed that IgG1+ B memory cells that expressed the markers CD80 and CD73 were able to transfer production of high-affinity IgE to a naive mouse with T cell help. In contrast, IgG1+ memory B cells not expressing these two markers were unable to support high-affinity IgE, and supported production of low-affinity IgE. At high ratios of low affinity to high affinity IgE, the low affinity IgE could inhibit pathogenicity of the high affinity IgE. This work identifies unique IgG1 B cell subsets that house IgE memory, and also emphasizes the importance of measuring the affinity of IgE to determine its pathogenicity.

These few studies on IgE memory in animal models highlights the importance of allergen exposure and T cell help to maintain IgE plasma cells from a pool of memory IgG1+ B cells. It is not clear if these requirements also exist in humans. Allergy can persist for years in the absence of any known exposures, and strict avoidance does not appear to facilitate food allergy resolution. It is not clear if there is non-oral exposure (perhaps through ubiquitous antigens in household dust), or antigenic mimicry, or if indeed the mechanisms of IgE maintenance are different in mice or humans. Studies are needed to understand the cellular basis of elimination of high affinity IgE in humans during resolution of their food allergy.

Future Directions

Central to the forward progress of our field is a better understanding of the heterogeneity of food allergy, including how immune and clinical parameters relate to one another. If we understand the immune basis of the natural resolution of food allergy, we are much more likely to understand how to interfere with persistent food allergy. It is not clear if persistent food allergy is a phenotype that can encompass different foods. For example, is persistent milk allergy more similar to peanut allergy than it is to milk allergy that is outgrown in early childhood? Are there common features that unite these persistent food allergies, such as characteristics of the IgE epitopes or antigenic mimicry that maintains the IgE response despite careful food avoidance? Does persistent food allergy, whether it is peanut, milk, or egg, share a common site of sensitization? Do microbial adjuvants (such as Staphylococcal enterotoxin B) play a role in sensitization in persistent food allergy?

Local factors in the gastrointestinal tract have remained mostly inaccessible in our study of human food allergy. There may be local factors that maintain persistent food allergy. For example, we know that highly differentiated (pathogenic-effector) Th2 cells acquire responsiveness to innate cytokines such as TSLP, IL-33, and IL-25³⁵, similar to ILC2s. Persistence of food-specific IgE may be maintained by lymphocytes with innate responsiveness that respond to local elevated cytokine production. Can interventions that target the gastrointestinal tract, such as modern probiotics³⁰ or dietary fiber²⁶ alter the intestinal immune milieu and interrupt the maintenance of long-lived IgE? Questions about the contribution of the gastrointestinal mucosa in food allergy require a method to non-invasively monitor the status of the gut.

There may be other clinical parameters that are predictive of resolution or persistence. For example, we do not know if another phenotype of food allergy, threshold of reactivity, is associated with resolution or persistence. Are individuals with a high threshold of reactivity more likely to naturally outgrow their food allergy, caught in a state between allergy and tolerance? Are type of allergy manifestations (site or severity) predictive of persistence or resolution? Lastly, how do we define tolerance, clinically and immunologically? Under what conditions is an absence of symptoms synonymous with tolerance? For example, someone with food-dependent exercise-induced anaphylaxis would not be considered tolerant, despite the fact that they can eat the food without symptoms in the absence of co-factors such as exercise or alcohol. Is there an immune signature of a healthy response to foods in someone who was never allergic and how does that compare to someone with resolved food allergy? The barrier to answering all of these open questions is the establishment of large cohort(s) where food allergy and tolerance are carefully defined and assessed, and where longitudinal biospecimens are available for application of rigorous immune monitoring and biomarker discovery. The HealthNuts and CoFAR cohorts are two cohorts that have made significant contributions to our understanding of food allergy resolution, but fundamental questions remain to be addressed.

Figure 1:

Immune mechanisms of persistence and resolution of food allergy. Low allergen-specific IgE is associated with and predictive of food allergy resolution. IgE memory has been shown to be derived not from IgE plasma cells, but IgG1+ memory cells bearing the markers CD73 and CD80. Allergen-specific T cell responses associated with food allergy are Th2-biased, with IL-10-producing Tregs associated with natural resolution of food allergy. It is not known how or if T follicular helper (Tfh) cells contribute to the maintenance of IgE memory. Persistent food allergy is associated with elevated inflammatory responsiveness to TLR stimuli by the myeloid compartment, pointing to a role for innate immunity in persistence. The innate immune system responds to cues from the microbiota, which also differs in composition between persistent and resolved food allergy.

Abbreviations:

AD

Atopic dermatitis

CoFAR

Consortium for Food Allergy Research

DCs

dendritic cells

Glossary JACI-D-18-01523

CD25

also known as interleukin 2 receptor alpha (IL2RA), a cell marker that is constitutively expressed by T regulatory cells that respond to IL-2 which is generated by T cells during an immune response for their immunoregulatory functions.

CD73

5’-nucleotidase (5’-NT) an ecto enzyme that catalyzes the conversion of AMP to adenosine. CD73 has many physiological roles, such as regulation of barrier function, adaptation to hypoxia, ischemic preconditioning, anti-inflammation, leukocyte extravasation.

CD80

a co-stimulatory molecule found on antigen presenting cells such as dendritic cells, activated B cells and monocytes that binds to CD28 or CTLA-4 on T cells.

CD127

(interleukin-7 receptor-α) a protein that is one subunit of the heterodimer interleukin- 7 receptor found on the surface of various cells types. Signaling of CD127 is essential for T-cell development and regulation of naive and memory T-cell homeostasis.

Germinal cell memory cells

a sub type of B cells that are formed within germinal centers following a primary immune response and are critical for generating a robust antibody-mediated immune response in the upon re-exposure.

IFNγ

(Interferon-gamma) a cytokine that plays a critical role in inducing and modulating an array of innate and adaptive immune responses. Cellular responses to IFN-γ are mediated by its heterodimeric cell-surface receptor (IFN-γR), leading to the regulation of gene expression. Aberrant IFNγ expression is associated with various inflammatory and autoimmune diseases.

IL-8

a chemokine known as neutrophil chemotactic factor, is produced by a variety of cell types and known to induce chemotaxis and phagocytosis in neutrophils and other granulocytes, causing them to migrate toward the site of infection.

Linear epitope

a protein epitope that is recognized by specific antibodies by its linear sequence of amino acids (primary structure). Antibodies can recognize three dimensional shapes formed by amino acids brought together by protein folding (called a conformational epitope) or by amino acids that are adjacent to each other in the protein sequence (linear epitope).

Microarray

microarray refers to a multiplexing technology which allows high throughput analysis of DNA, RNA, or proteins from biological materials. Microarrays can be used to measure gene expression, gene methylation, or as referred to here, to measure antibody binding to peptides from allergens.

Myeloid compartment

a heterogeneous population of cells including granulocytes, monocytes, and dendritic cells that originate from the bone marrow. Bone marrow cells can differentiate into myeloid or lymphoid lineages.

PBMCs

(peripheral blood mononuclear cell) any peripheral blood cell having a round nucleus, which consists of lymphocytes (T cells, B cells, NK cells) and monocytes, whereas erythrocytes and platelets have no nuclei, and granulocytes (neutrophils, basophils, and eosinophils) have lobed nuclei.

RT PCR

(Real Time Polymerase Chain Reaction) a technology that utilizes a fluorescent reporter molecule to monitor the PCR as it progresses. The fluorescence emitted by the reporter molecule quantifies the product as it accumulates with each cycle of PCR amplification, as it makes thousands to millions of copies of specific gene sequences.

TNF α

(tumor necrosis factor α) a cytokine produced primarily by macrophages that is involved in systemic inflammation and acute phase reaction. TNF α is a chief regulator of cell proliferation, survival, differentiation and apoptosis while its aberrant production and receptor signaling have been implicated in the pathogenesis of several inflammatory diseases.