Oral immunotherapy as a curative treatment for food‐allergic preschool children: Current evidence and potential underlying mechanisms

The worldwide rising prevalence of food allergy is a major public health concern. Standard care consists of allergen avoidance and rescue medication upon accidental exposure. Oral immunotherapy (OIT) is increasingly being studied as a treatment option. Although desensitization (an increased reaction threshold) is often achieved during OIT, sustained unresponsiveness (SU; clinical nonreactivity after finishing OIT) is not achieved in most patients. A few studies have investigated the effectiveness of OIT in children younger than 4 years of age (early = e‐OIT) and have shown a much more favorable outcome in terms of SU development. Together with food allergy prevention studies, which have demonstrated high efficacy of early oral allergen exposure, the outcomes of e‐OIT studies indicate an early‐life window of opportunity to achieve SU, allowing unrestricted dietary intake. However, the underlying mechanism of the high effectiveness of e‐OIT is not understood yet. Both cohort and OIT studies indicate early‐life immune plasticity. An immature food‐allergic response in the first years of life seems to be a major driver of this immune plasticity, along with a higher tolerogenic immunological state. Allergy maturation can likely be disrupted effectively by early intervention, preventing the development of persistent food allergy. Upcoming studies will provide important additional data on the safety, feasibility, and effectiveness of e‐OIT. Combined with immune mechanistic studies, this should inform the implementation of e‐OIT.

is not achieved in most patients.A few studies have investigated the effectiveness of OIT in children younger than 4 years of age (early = e-OIT) and have shown a much more favorable outcome in terms of SU development.Together with food allergy prevention studies, which have demonstrated high efficacy of early oral allergen exposure, the outcomes of e-OIT studies indicate an early-life window of opportunity to achieve SU, allowing unrestricted dietary intake.However, the underlying mechanism of the high effectiveness of e-OIT is not understood yet.Both cohort and OIT studies indicate early-life immune plasticity.An immature food-allergic response in the first years of life seems to be a major driver of this immune plasticity, along with a higher tolerogenic immunological state.Allergy maturation can likely be disrupted effectively by early intervention, preventing the development of persistent food allergy.
Upcoming studies will provide important additional data on the safety, feasibility, and effectiveness of e-OIT.Combined with immune mechanistic studies, this should inform the implementation of e-OIT.

K E Y W O R D S
allergen immunotherapy, food allergy, immune mechanisms, oral immunotherapy, preschoolers

| INTRODUC TI ON
Food allergies are an important public health concern.The prevalence of food allergic diseases continues to rise worldwide and carries a significant impact on the quality of life of both patients and those around them.Additionally, the economic burden is serious.The standard care for food allergy consists of strict dietary avoidance and timely administration of rescue medication in case of an allergic reaction upon accidental exposure.Driven by the rising prevalence and high burden of the disease, food allergy research has become more and more a public priority.Recent scientific advancements have led to exciting new insights into the development and management of food allergies.The long-held view that sensitization to food occurs through oral exposure and prevention of food allergy can be accomplished through elimination diets in early life is now obsolete.Several studies have placed the skin at the center of attention, as early cutaneous exposure to food through impaired barriers in eczematous skin can easily lead to allergic sensitization. 1,2Landmark studies, such as the LEAP and PETIT studies, have demonstrated that early oral exposure to food allergens is strongly associated with tolerance development, particularly in children at high risk of food allergy development because of an impaired skin barrier. 3,4This effectiveness of early oral allergen exposure regards not only primary prevention, aimed at preventing the onset of sensitization, but also secondary prevention, aimed at interrupting the development of food allergy and related symptoms in sensitized children.These observations are unified in the dual-allergen exposure hypothesis, stating that early-life cutaneous exposure to food allergens might lead to sensitization, whereas early oral exposure leads to immunological tolerance. 2sides prevention strategies, food allergen immunotherapy is increasingly being studied, with oral immunotherapy (OIT) leading the way.In OIT trials, the most commonly reported efficacy outcomes are desensitization and sustained unresponsiveness (SU). 5sensitization is described as a temporary increase of the allergic reaction threshold level during ongoing treatment, requiring continued treatment to maintain the effect.A patient reaches SU when clinical nonreactivity after a period of treatment cessation is observed, also referred to as "allergy remission," which allows unrestricted dietary intake of the specific food.However, the period of dietary avoidance after treatment that defines remission is currently not standardized, and it is uncertain how long remission persists and whether ad libitum intake affects the persistence of remission.

Studies on the effectiveness of OIT have primarily been conducted
in school-aged children and adults.In these patients, the therapy is shown to be effective in inducing desensitization, while SU is achieved in only a minority of patients. 6There is growing evidence that immunotherapeutic intervention in children under 4 years of age (hereafter referred to as preschoolers) leads to superior outcomes.
8][9] This review presents an overview of the current evidence on the safety and effectiveness of OIT in preschoolers (early life = e-OIT) and explores possible mechanisms underlying the observed higher effectiveness of e-OIT.In the final section, suggestions for future research are discussed.

| E VIDEN CE FOR E ARLY OR AL IMMUNOTHER APY SAFE T Y
Emerging clinical trials have assessed the safety of e-OIT.In the DEVIL study, the safety of peanut e-OIT was assessed in 37 children aged 9-36 months. 7In this study, allergic side effects were common (95% of subjects), but all were mild (85%) or moderate (15%) and only 1/37 subjects required epinephrine (in the high-dose group [3000 mg], not in the low-dose group [300 mg]).One subject in the low-dose group withdrew from the study because of biopsy-proven eosinophilic esophagitis (EoE), which persisted despite stopping e-OIT.As part of a Canada-wide quality improvement project, the safety of low-dose [300 mg] peanut e-OIT was assessed in 270 children aged 9-71 months. 10In this study, 68% of participants experienced allergic side effects, of which only 0.4% had a severe (grade 4) reaction.Of all allergic reactions, 2.2% were treated with epinephrine, representing 0.03% of all doses.EoE was diagnosed in one patient.In the same project, the safety of low-dose [300 mg] tree nut e-OIT was assessed in 92 children aged 9-70 months. 11Allergic reactions were described in 71% of participants.All reactions were mildmoderate (grade 1 or 2).Epinephrine was used by two participants both for a grade 2 reaction.No patients were diagnosed with EoE.In a cow's milk e-OIT trial, 29/68 (43%) patients aged <12 months experienced allergic side effects during treatment at home. 12Allergic reactions were mainly mild (87.5% class 1) and no participants

Key Message
This review adds an updated overview of the current state of knowledge on oral immunotherapy (OIT) for food allergy.Recent studies have shown that starting the therapy early in life is associated with developing sustained unresponsiveness, making OIT a promising food allergy treatment.The review delves deeply into the immunological aspects of OIT, discussing possible explanations for the observed clinical effects of OIT in preschool children versus older children and adults.
Important remaining knowledge gaps are being identified.
We believe that this manuscript is of interest to the readers of Pediatric Allergy and Immunology, because it represents an impactful contribution to the knowledge on a hot and highly relevant topic that may have great impact on future food allergy management.Many studies on OIT for food allergy are currently running or being designed and will be finalized in the coming years.This review invites researchers to address the important knowledge gaps to inform future clinical management of food allergy.
required epinephrine or hospital admission.In the IMPACT trial, 96 children aged 12-48 months with peanut allergy received high-dose [2000 mg] e-OIT, of which 98% experienced allergic side effects. 8mptoms were predominantly mild-moderate, with only five participants experiencing severe symptoms.Epinephrine was used in 22% of participants and 91% of the treated reactions were mildmoderate (grade 1 or 2), suggesting a low threshold for epinephrine administration.Biopsy-confirmed EoE was seen in 3 OIT-treated participants, of which 2/3 recovered after cessation of OIT.In the PPOIT study, the exposure-adjusted incidence rate of allergic reactions during peanut OIT (83 patients) and probiotic peanut OIT (PPOIT; 79 patients) was assessed in children aged 1-10 years with a subgroup analysis by age (1-5 years versus 6-10 years). 9The exposure-adjusted incidence rate was 10.1 in the 1-5 years OIT group versus 12.8 in the 6-10 years OIT group and 7.7 in the 1-5 years PPOIT group versus 14.1 in the 6-10 years PPOIT group.Overall, 93% of adverse events were mild, 6% were moderate, and only 1% were severe.Preliminary results of the POSEIDON study, as published on clini caltr ials.gov, show that 74/98 (75.5%) peanut-allergic children aged 1-4 years experienced treatment-related adverse events, of which two patients (2%) had a systemic allergic reaction. 13l treatment-related adverse events were mild-moderate with no severe/serious adverse events.None of the patients were diagnosed with EoE.
It can be concluded that e-OIT appears to be a safe treatment for most preschool-aged children.Although allergic side effects are common, almost all symptoms appear mild and self-limiting or treatable with oral medication.More research on the safety of e-OIT is recommended to confirm these observations, especially for allergies other than peanut and for multi-allergen OIT.

| E VIDEN CE FOR E ARLY OR AL IMMUNOTHER APY EFFIC AC Y
Over the past decade, studies on OIT have focused primarily on older children and adults.These studies have shown that the therapy is effective in inducing desensitization while on therapy, but SU after discontinuation of therapy is rarely achieved.The Consensus Report from the Food Allergy Research and Education OIT summit (2019) concludes that although OIT is an emerging treatment option potentially leading to desensitization in patients with persistent food allergy, this treatment is not curative. 14The European guideline on food allergy treatment shares this view on the effectiveness of OIT: "Most of patients treated with OIT will achieve desensitization; however, only a minority achieves post-discontinuation effectiveness." 15 This conclusion was also drawn by the authors of a recent systematic review and meta-analysis on the treatment of IgE-mediated food allergy including studies on school-aged children. 6limited amount of data is available on the effectiveness of OIT in preschoolers.Systematic reviews of OIT show that effectiveness trials have focused on peanut-, cow's milk-, and egg-allergic patients, mostly in mixed age groups. 6,16Some of these studies included preschool-aged children, but as part of a broad age range without subgroup analyses by age.3,[17][18][19][20] The endpoint of most of these studies is desensitization, as determined by an oral food challenge during maintenance therapy or by reaching a set target dose.
[9] The results of these studies suggest that early intervention greatly increases the likelihood of achieving SU.
The first randomized efficacy trial of peanut e-OIT (DEVIL) enrolled 37 children aged 9-36 months.Of the children who received OIT, 48% reached SU 8 weeks after completion of therapy, which was observed in only 5% of children who received placebo.In addition, SU was more often achieved in children aged 1-5 years (49/84 = 58%) compared with children aged 6-10 years (29/78 = 37%).Further details on age-related differences in effectiveness within the 1-5 years age group were not provided.
Twelve months after treatment, 99% of children who achieved SU were eating peanut ad libitum (median frequency 1-2 times per week) without allergic symptoms.
Taking the results of the above studies together, it can be concluded that there appears to be a significantly higher probability of achieving OIT-induced SU in preschoolers compared with older children and adults.Strengths of the e-OIT RCT's include entrance oral food challenges (OFC's) to diagnose peanut allergy, exit OFC's to determine SU, and the inclusion of a control group to correct for natural tolerance development.However, there is much heterogeneity regarding inclusion criteria, number of study participants, dosing, treatment length, and period off-treatment to determine SU, which complicates drawing a straightforward conclusion on the effectiveness of e-OIT.Interpretation of the results is further hampered by the afore-mentioned lack of consensus on the definition of SU and allergy remission.Nonetheless, these studies provide some important insights.The DEVIL and PPOIT-003 study had an endpoint of SU combined with eating peanut ad libitum.Although this raises the question of whether ongoing peanut consumption affects the immunological response to peanut, from a patient's (or parent's) perspective tolerance is defined as eating a product without restrictions and/or without allergic symptoms.This clinical endpoint was achieved in 27/32 (84%, DEVIL) and 48/84 (57%, PPOIT-003) children who received e-OIT. 7,9In addition, the results of the IMPACT trial suggest that OIT significantly improves the likelihood of remission in preschool children, as in this study 21% of participants in the peanut OIT group had a negative OFC after a 6-month period of strict peanut avoidance, compared with 2% in the placebo group. 8Furthermore, the e-OIT RCTs suggest that age matters, even within the group of preschool children.The DEVIL study reported the highest rate of SU (91%) and included children <36 months of age, of which 70% were <24 months of age. 7In the IMPACT trial, the highest SU rates were achieved by the youngest participants (<24 months), which is encouraging but should be interpreted with caution because of the small subgroup population. 8Differences in the rates of SU between the e-OIT RCT's may therefore (at least partial) be affected by differences in age at study entry.The results suggest that starting e-OIT before 24 months of age may increase the effectiveness in inducing SU.A caveat in comparing OIT outcomes in preschool children with those in older children and adults is that the probability of natural tolerance development is higher in preschool children, which may lead to an overestimation of the effect size of OIT in preschool children.3][24][25] Levels of sIgE can vary from slightly elevated (just above the cut-off point of 0.35 kU/L) to highly elevated (>100 kU/L).In an Australian cohort study of 267 peanut-allergic preschool children, participants with spontaneous tolerance development had a mean level of peanut sIgE of 2.0 kU/L, with only one child having a peanut sIgE ≥3 kU/L. 248][9] Spontaneous tolerance development is therefore not expected to be a confounding factor in interpreting the effectiveness of OIT in these studies.Baseline levels of sIgE have also been shown to correlate with allergy outcome after OIT, as lower levels were predictive of achieving SU. 8,[26][27][28][29][30] Therefore, the inclusion of participants with relatively high levels of peanut sIgE in the e-OIT RCT's may lead to an underestimation of the potency of e-OIT in inducing SU in the total population of peanut-allergic preschool children.
The available data on the efficacy of OIT suggest that the therapy is effective in inducing SU in preschool children, making it a highly promising food allergy treatment.If this endpoint proves equivalent to food allergy remission of true long-term tolerance, then e-OIT is a curative therapy for food allergy.This would be a major breakthrough in the management of food allergy.

| P OSS IB LE MECHANIS MS UNDERLYING THE ADDED B ENEFIT OF E-OIT
The question that arises is: "what are possible explanations for the observed higher effectiveness of OIT in preschoolers?" Both efficacy trials and a recent review on e-OIT hypothesize immunological plasticity in early life, that is, a greater capacity to undergo immunological changes in response to external factors. 7,8,31is plasticity may refer to a feature of the immature immune system itself or to a potency to reverse food allergy in young children before the allergy has fully matured.Another hypothesis discusses that our tolerogenic capacity is higher in early life and decreases over time, and therefore tolerance is easier to induce in young children.Below we will explore these possible mechanisms.

| Age-related immunological plasticity in food allergy development
Primary food allergy typically manifests in early life. 32The first food-allergic reaction is mostly seen with the first known oral exposure.In prevention studies, it is confirmed that food sensitization frequently starts during infancy, sometimes even before 6 months of age. 3,4,33The initial/early allergic response in infants seems to be immature, characterized by weak T-cell receptor affinity, unstable GATA-3 expression, and relatively low IL-4 production. 34Ripening of this response seems to be required for allergy persistence.A gradual increase of T helper 2 (Th2) cytokine production and concomitant IgE production is seen in the first years of life. 353][24][25] In addition, a substantial expansion of IgE epitope recognition is described after 2 years of age, which appears to be an emergence of sequential epitope recognition. 35,368][39][40][41] Taken together, these data show that the initially weak allergic response to food allergens matures during the preschool years.This early maturation seems to determine allergy persistence in later life, which provides a potential window of opportunity to influence the natural course of food allergy development.
Oral immunotherapy is the obvious route to influence the natural course of food allergy development, as allergenic stimulation in the gastro-intestinal tract (GI-tract) is known to induce tolerance via key regulatory pathways (Figure 1, Supplementary Material Box 1).Prevention studies have shown that this tolerogenic potential is maintained when sensitization has already occurred.In the LEAP trial, 89.4% of infants in the peanut consumption group with baseline peanut sensitization remained tolerant. 3In the EAT study, tolerance remained in 80.8% of sensitized infants in the early dietary introduction group. 42An immune mechanistic study on the LEAP cohort describes that, in contrast to peanut-sensitized children who avoided peanut and became peanut-allergic, peanut-sensitized children who consumed peanut and remained tolerant did not show IgE epitope spreading. 36This suggests that early oral allergen exposure inhibits IgE epitope spreading, a process known to be associated with persistent allergy.These observations show that food allergy development is reversible with appropriately timed oral allergen exposure, at least in sensitized nonallergic children.Currently, there is increasing consensus that this immunological effect of early oral allergen exposure also applies to e-OIT.Immune mechanistic data from OIT studies support the rationale of early OIT prior to full maturation of the allergic response to induce lasting tolerance.Allergy outcome after OIT has been repeatedly shown to correlate with baseline levels of IgE, as lower levels were predictive of achieving SU. 8,[26][27][28][29][30] In addition, SU was more frequently seen in patients with lower baseline diversity and lower binding intensity of sequential IgE. 30,43Together with the described increase of IgE in the first years of life and significant epitope spreading beyond 2 years of age, this supports the hypothesis of early immunological plasticity during food allergy development as an explanation for enhanced effectiveness of e-OIT.

| The maturing immune system: Age-dependent differences in tolerogenic capacity
Immediately after birth, our immune system faces several challenges.During the first years of life, we encounter a huge quantity and diversity of foreign antigens that may or may not be harmful.Accordingly, our immune system has to balance between a protective or tolerogenic response.This balancing act has to continue throughout life in a dynamic environment with continuously changing antigens.In case of food allergens, our body should develop tolerance.This is an active process of regulated immune responses (Figure 1, Supplementary Material Box 1).The question now is whether the capacity to develop tolerance to food allergens changes throughout life as our immune system matures, as this may affect the effectiveness of OIT.

| Maturation of the gastrointestinal tract
Before the immune system is exposed to food components, food is processed in the GI-tract and nutrients have to pass the intestinal barrier.The extent of digestion and absorption are affected by the maturity of the GI-tract.Digestion and absorption are known to play a role in determining the allergenic potential of foods. 44,45erefore, GI-tract maturation may affect the potential to develop tolerance.
Compared with adults, infants and young children have lower gastric acid secretion, resulting in a higher intragastric pH. 46This leads to a reduced activity of pepsin, the main protease in the stomach.Together with lower concentrations of other digestive enzymes, this may cause overall less proteolysis in the GI-tract of infants. 47 addition, until 3 years of age, gastric emptying occurs at a faster rate than in adults. 48After initiation of proteolysis in the stomach, further protein digestion takes place in the small intestine.The activity of trypsin, the most important protease in the small intestine, is not age-dependent. 46However, the activity of chymotrypsin and carboxypeptidase is lowest in newborns and increases with age. 49e motility of the small intestine also affects digestion.In infants, the contraction amplitude, propagation speed and propagation frequency are lower than in adults. 46Nevertheless, intestinal transit time is considered to be the same in the whole age range from 1 to 17 years as in adults. 48In general, protein digestion in infants and young children is considered to be relatively insufficient compared to adults, allowing more intact food proteins to contact the intestinal epithelium and underlying mucosal tissue.
After mechanical and chemical breakdown of food in the GItract, food proteins have to cross the intestinal epithelium to meet the intestinal immune system.The route of antigen passage is thought to affect the immune response.In early life, enhanced macromolecular transfer takes place, due to a high endocytic capacity of immature intestinal epithelial cells. 50This results in increased exposure of the intestinal mucosal immune system to food allergens. 513][4] Therefore, the enhanced macromolecular passage in early life is expected to result in a greater tendency to develop tolerance to food allergens.The endocytic capacity is largely lost with epithelial maturation and macromolecular transepithelial passage ceases at "gut closure."The exact time of "gut closure" in humans is unknown but has been described as being as early as 22 weeks after birth. 51This is too early to explain a difference in effectiveness of tolerance development between preschoolers and older children/adults.In addition, there are no known other important developmental changes in the intestinal epithelial antigen passage that occur in childhood.

| Maturation of dendritic cells
Dendritic cells (DCs) present in the intestinal mucosa capture food allergens from the intestinal lumen as well as allergens that have crossed the epithelial barrier.Several populations of DCs, such as monocyte-derived DCs, conventional DCs, and plasmacytoid DCs, inhabit the subepithelial region. 52In neonates, DCs are found in lower numbers and have an impaired capacity to present proteins to T cells, due to lower MHC-II expression levels. 53,54In addition, the presence of costimulatory receptors CD80, CD86, and CD40, which are necessary for proper naïve T-cell activation, is decreased in neonatal conventional DCs. 54Furthermore, secretion of T-helper 1-promoting cytokines, such as IL12p70 and IFNβ, upon LPS stimulation is severely impaired in monocyte-derived DCs from cord blood, 55 despite similar TLR4 expression in adults and neonates. 56Yet, fetal DCs secrete higher concentrations of regulatory cytokines compared with adults, thereby promoting the development of FoxP3+ regulatory T cells (Tregs). 57However, the development of intestinal Tregs depends on interactions with CD103+ DCs, which appear to be lacking in neonatal mice. 58A similar observation has not yet been described for humans.

F I G U R E 1
Immune mechanisms of natural oral tolerance development.After ingestion and digestive processing, food allergens can cross the gut epithelial barrier to interact with the underlying immune cells.This process might include paracellular diffusion, endocytosis via goblet cells and M cells or transepithelial sampling by DCs.Soluble (s)IgA in the gut lumen serves as an anchor, limiting the epithelial passage of food allergens.After epithelial passage, allergen-loaded DCs will express CD103 allowing them to migrate to the mesenteric lymph node (mLN).In the mLN, the allergen-loaded CD103 + DCs promote differentiation of naïve T cells into FOXP3+ Treg cells via secretion of TGF-β and retinoic acid (RA).The newly induced Tregs will then migrate to the lamina propria (LP) after upregulation of gut-homing factors CCR9 and α4β7 integrin.In the LP the Tregs undergo clonal expansion, which is driven by IL-10 production by CX3CR1+ macrophages.Major roles of FOXP3+ Tregs in tolerance development are promoting B-cell class switching to produce non-inflammatory IgA and IgG, induction of Th2 anergy and inhibition of downstream pro-inflammatory cells.Involvement of additional cell types in oral tolerance development have been implicated, including Bregs (suppressing Th2 cells and basophils/mast cells via production of suppressor cytokines and IgG) and Tr1 cells (production of suppressor cytokines).The gut environment (commensal intestinal bacteria) favors tolerance development.

| Maturation of adaptive immunity T-cell development
Functional T-cell maturation occurs after birth, it therefore seems likely that developmental changes in the human T-cell compartment affect the effectiveness of oral tolerance development.
First, the absolute number of naïve T cells strongly increases after birth to peak at 4 weeks of age.After this early increase, their number remains stable until 2 years of age, after which it gradually decreases to adult levels. 59,60Compared with adults, a higher frequency of naïve T cells is also seen in pediatric tissues, including the intestine. 61Second, phenotypical changes in the T-cell compartment take place.Neonatal T-cell responses to foreign antigens are skewed toward Th2 immunity, which is a result of generated tolerance to maternal alloantigens during pregnancy. 62At the same time, neonatal naïve CD4+ T cells are strongly predisposed to differentiate into Tregs, which persist for an extended period of time. 63Tregs have been identified to exert a key function in oral tolerance induction and maintenance (Figures 1 and 2, Supplementary Material Boxes 1 and 2).Human neonatal Tregs seem to preferentially migrate to the intestinal mucosa, while development of skin homing potential is delayed. 64,65Accordingly, the total number and frequency of Tregs are relatively high in infant blood, lymphoid tissues and the gut, but relatively sparse in the skin.This may lead to both a high susceptibility to sensitization via the skin and a great capacity to induce tolerance via the gut in early life.Infant and adult T-cell compartments are described to remain phenotypically distinct for at least the first 2 years of life.Antibody production (mainly IgM) initiates in utero as early as 9 weeks after gestation. 66However, isotype switching into IgG and IgA production is limited during the first 6 months after birth. 67In addition, affinity maturation via somatic hypermutation F I G U R E 2 Immune mechanisms of tolerance induction by OIT in food allergic individuals.In food allergic individuals, baseline Th2 skewing leads to B-cell IgE class switching and subsequent production of allergen-specific IgE, which binds to high-affinity Fcε receptors on the surface of mast cells and basophils.After renewed exposure to suprathreshold amounts of the allergen, mast cells and basophils rapidly degranulate, leading to allergic symptoms.This allergic state is manifested by an increase in skin prick test (SPT) wheel size and basophil activation test (BAT).OIT leads to an increase of IL-10 and IFN-α production by DCs and subsequent Th2 anergy and an increase in regulatory cells, including Tregs.This results in decreased production of sIgE and increased production of sIgG, including sIgG4, and sIgA.These changes lead to downstream suppression of the allergic response.In desensitized individuals, hypo-responsiveness of basophils and mast cells to the specific allergen is seen, characterized by decreased SPT wheel size and BAT, and an increase in the threshold level of reactivity.However, this effector cell suppression seems to be dependent on continuation of daily therapy.Persistent effector cell hyporesponsiveness after discontinuation of OIT occurs in individuals who achieve sustained unresponsiveness.This seems to be caused by changes at the T-cell and B-cell level: reprogramming of Th2 gene networks resulting in lasting suppression of inflammatory immune responses, epigenetic modifications within the FOXP3 locus of Tregs resulting in Tregs with enhanced suppressive function, and qualitative humoral changes (epitope specificity and avidity) resulting in an increase in diversified, affinity matured allergen-specific antibodies.Sustained unresponsiveness is characterized by a persistent decrease in SPT wheel size and BAT and persistent lack of clinical reactivity.
is attenuated in the first 2 years after birth. 62The effects of this lower degree of isotype switching and affinity maturation can be observed up to 6 years of age, while the number of naïve B cells in blood slowly decreases to adult-like levels at 10-15 years of age. 68e-dependent developmental changes in the composition of the peripheral blood B-cell pool are most evident in the first 5 years after birth.While the number of transitional and naïve B cells gradually decreases, the fractions of different memory B-cell populations gradually increases. 68In addition, neonatal B cells express lower levels of costimulatory receptors necessary to properly interact with T cells. 69Therefore, humoral responses are generally weaker in early life compared with adults. 62,67Both genetic and environmental factors such as pathogen exposure and intestinal microbial colonization shape this development of the humoral response in early life. 62,70

| Implications for the effectiveness of tolerance induction by e-OIT
The current knowledge on the mechanism of tolerance induction by OIT is summarized in Figure 2 and Supplementary Material Box 2.
Tolerance induction starts with inhibition of already established allergic inflammation.This inhibition appears to result from several sequential immune responses, starting with early hyporesponsiveness of effector cells, increased production of the suppressor cytokine IL-10 by DCs, and leading to induction of Th2 effector cell anergy.Recently, SU has been associated with rewiring of Th2-related gene networks, leading to lasting suppression of the Th2 cell response. 714][75] In addition, important qualitative and quantitative humoral changes are observed during OIT.Interestingly, all these immunological changes seem to be possible in older children and adults.OIT-induced persistent loss of Th2 upregulation was observed in children (age range 2.1-9.5 years) and the same mechanism was seen during house dust mite immunotherapy in adults. 71,76Epigenetic modifications in the FOXP3 locus of Tregs during OIT are described in both children and adults (age range 5-45 years). 72is implies that there is no age-specific difference in the mechanism of lasting tolerance development during OIT.However, there seems to be a strong age-specific difference in capacity to develop tolerance.
As described above, significant maturation takes place in our immune system during the first years of life.Characteristics of the GI-tract and immune system in early life seem to promote the development of tolerance.Insufficient protein digestion and enhanced macromolecular passage in the GI-tract allow more intact food proteins to contact the intestinal immune system.In addition, infant naïve T cells are predisposed to differentiate into Tregs, which preferentially migrate to the intestinal mucosa as skin homing potential is not yet well-developed.
At the same time, humoral responses are weaker in the first years of life, because affinity maturation via somatic hypermutation is limited and a limited number of costimulatory molecules are present on B cells for interaction with T cells.We hypothesize that during this developmental period, tolerance induction following "overexposure" of the GItract to food allergens (where many Tregs are present early on) can balance out further development of food allergy (increase and expansion of the humoral response) and that this balance is more difficult to shift from the time the B cells are functionally mature, making OIT less effective in school-aged children and adults.

| CON CLUS I ON AND FUTURE PER S PEC TIVE
Clinical studies have shown that application of OIT for food allergy early in life offers a great opportunity to develop long-term tolerance, while this is rarely achieved in older children and adults.The main explanation for this striking difference in susceptibility to developing long-term tolerance is the initial immaturity of the immunological response to the food allergen.After the initial development of food allergy, which occurs mainly in early infancy, the immunological response matures during the preschool years.This maturation is characterized by a gradual increase of IgE and significant IgE epitope spreading.Both immunological changes are associated with allergy persistence.Early oral allergen exposure has been repeatedly shown to effectively disrupt this allergy maturation and prevent the development of persistent food allergy, indicating the existence of a window of opportunity for e-OIT to induce long-term tolerance.
Clinical OIT-studies confirm that lower baseline levels of IgE and less baseline IgE epitope-diversity are predictive of achieving SU.In addition, early-life characteristics of the GI-tract and immune system seem to be tolerance-promoting, which may contribute to higher susceptibility to develop oral tolerance.Despite growing evidence on the added benefit of e-OIT, further exploration of the therapy is warranted, especially for allergens other than peanut and for multiplefood OIT, for which evidence is currently lacking.In addition, more research is needed to determine the appropriate dose and timing of OIT.Forthcoming studies will provide important additional data on the safety, feasibility, and effectiveness of e-OIT. 13,77,78It is recommended that immunological research be included in these studies, to further elucidate the underlying immune mechanism of e-OIT.
In addition, it is recommended that a randomized control group be included in efficacy trials to correct for natural food allergy resolution in order to determine the precise effect size of the therapy, both clinically and immunologically.Furthermore, it is advisable for future studies to objectify long-term tolerance.This knowledge will inform the clinical implementation of e-OIT and will be useful in determining which patients will benefit most from treatment, with respect to the likelihood of achieving SU.In addition, identification of more precise predictors of food allergy prognoses may be useful in determining whether selection of patients for e-OIT based on the likelihood of spontaneous tolerance development is appropriate.Furthermore, it will be interesting to study the mechanism of cutaneous sensitization in early life, which may provide insight into epitope recognition during food allergy development.

AUTH O R CO NTR I B UTI O N S
Lieke J. C. Barten was involved in conceptualization (equal), investigation (lead), and writing-original draft (lead).Marit Zuurveld was involved in conceptualization (supporting), investigation (supporting), writing-review and editing (equal), and visualization (lead).Joyce Faber and Johan Garssen were involved in conceptualization (supporting), writing-review and editing (equal), and supervision (supporting).Ted Klok was involved in conceptualization (equal), investigation (supporting), writing-review and editing (equal), and supervision (lead).All authors have read the manuscript and agreed to the submitted version.

CO N FLI C T O F I NTE R E S T S TATE M E NT
J.G. is partially employed as an advisor by Danone Nutricia Research B.V. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

PE E R R E V I E W
The peer review history for this article is available at https:// www.webof scien ce.com/ api/ gatew ay/ wos/ peer-review/ 10. 1111/ pai.14043 .

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Children were randomized between high-dose [3000 mg] and low-dose [300 mg] peanut protein for daily maintenance.The median level of peanut sIgE at baseline was 14.4 kU/L (IQR 3.4-48.6)and the median cumulative tolerated dose at baseline was 21.0 mg (IQR 21-171 mg).A matched standard-care control group (n = 154) was retrospectively collected.The primary endpoint, SU at four weeks after stopping e-OIT, was assessed by an oral food challenge in 32 children.SU was achieved in 91% (29/32) of these children.No difference in effectiveness was observed between high-dose and low-dose e-OIT.In the control group, tolerance development was assessed by the levels of peanut sIgE and, when indicated according to clinical guidelines, an oral food challenge.At the end of the study period, 4% (6/154) of children in the control group were tolerant for peanut.Five years after the completion of e-OIT, parents reported that 27/29 children (93%) continued dietary peanut consumption (86% at least once a week) of which only two reported mild, transient oropharyngeal symptoms within 2 hours after ingestion. 21Parents of two children reported peanut avoidance, because of confirmed EoE (n = 1) and extreme aversion to peanut (n = 1).A limitation of this study is that no placebo control group was included.The second study (IMPACT) is a recently published peanut e-OIT RCT. 8 In total, 146 children aged 12-48 months were assigned to receive either peanut OIT (daily 2000 mg peanut protein, n = 96) or oat flour placebo (n = 50).The median level of peanut sIgE at baseline was 53.1 kU/L (IQR 27.3-195.0)and the median cumulative tolerated dose at baseline was 25.0 mg (IQR 5.0-75.0mg).After 134 weeks of treatment, the desensitization rate was 71% (n = 68) in the OIT group versus 2% (n = 1) in the placebo group.After 26 weeks of avoidance, the SU rate was 21% (n = 20) in the OIT group versus 2% (n = 1) in the placebo group.Subgroup analysis showed a decline of SU rate with age at baseline: 71% (n = 5/7) in children aged 12-24 months, 35% (n = 7/20) in children aged 24-36 months and 19% (n = 8/43) in children aged 36-48 months.The third study (PPOIT-003) is a randomized placebo controlled trial of peanut OIT (maintenance dose 2000 mg peanut protein) in children aged 1-10 years with subgroup analysis by age. 9In this study, 201 children with a median age of 5.9 years (SD 2.8; 1-5 years n = 104, 6-10 years n = 97) were randomized to receive either peanut OIT or placebo.The total treatment duration was 18 months.The median baseline level of peanut sIgE was 12.0 kU/L (IQR 2.0-70.6).

B
-cell development B-cells are known as IgE-producing cells in the pathogenesis of food allergy, but emerging data show that they also play an important role in oral tolerance development as IgG-and IgA-producing regulatory B-cells.The functional capacity of B-cells changes throughout the first years of life, which may affect the development of food allergy and oral tolerance.