Mechanisms of Oral Immunotherapy

Abstract

Food allergy presents a significant global health concern with up to 10% of the population affected in developed nations and a steadily increasing prevalence. In many cases, particularly with peanut, tree nut and shellfish, food allergy is a lifelong and potentially life-threatening diagnosis. While no “cure” for IgE-mediated food allergy exists, oral immunotherapy (OIT) is a promising treatment modality with the peanut OIT drug Palforzia (Aimmune Therapeutics) the only treatment for food allergy that is currently approved by the United States Food and Drug Administration. OIT primarily induces a state of desensitization with only a minority of subjects achieving sustained unresponsiveness, a state of limited clinical remission that appears to be immunologically distinct from natural tolerance. Early humoral changes during OIT include an initial increase in allergen-specific IgE, which eventually decreases to below baseline levels as OIT progresses, and a gradual increase in allergen-specific IgA and IgG4 that continues throughout the course of OIT. Basophil hyporesponsiveness and decreased skin prick test wheal size are observed within the first year of OIT, and persistence after completion of therapy has been associated with sustained unresponsiveness. In the T-cell compartment, there is an initial expansion followed by a decline in the number and activity of T helper 2 (T_H2) cells, the latter of which may be dependent on an expansion of IL-10 producing cells, including regulatory T-cells. Our understanding of the immunomodulatory effects of OIT continues to evolve, with new technologies such as single-cell transcriptional profiling and antibody epitope analysis allowing for more detailed study of T-cell and B-cell responses to OIT. In this review, we present evidence to illustrate what is currently known about the immunologic changes induced by OIT, explore potential mechanisms, and emphasize knowledge gaps where future research is needed.

Introduction:

Food allergy is a global health concern with a prevalence that has increased substantially over the past two decades. In developed nations, an estimated 5 to 10 percent of the population suffers from food allergy[1–5], a diagnosis which negatively impacts quality of life for patients and their caregivers and carries a significant economic burden[6–8]. While allergy to certain foods such as egg, milk, wheat and soy often resolves, allergy to peanuts, tree nuts, and seafood is usually lifelong. There is no cure for food allergy, and strict avoidance of the culprit food(s) with prompt medical management in the event of an allergic reaction is the standard of care[9].

Food allergen-specific immunotherapy, including oral immunotherapy (OIT), epicutaneous immunotherapy (EPIT), and sublingual immunotherapy (SLIT), has shown promise in the treatment of IgE-mediated food allergy with OIT the most thoroughly studied to date. The peanut OIT product Palforzia (Aimmune Therapeutics) is the only treatment for food allergy that has been approved by the United States Food and Drug Administration to date[10]. The approach to food allergy immunotherapy is perhaps most analogous to that of drug desensitization with continued exposure to an allergen achieved through an initial dose escalation (IDE) followed by a build-up and then longer maintenance phase. The majority of OIT trials have utilized single-allergen OIT, mainly peanut, milk, and egg, but multi-allergen OIT is an area of ongoing investigation[11–13]. OIT primarily induces desensitization, which is a temporary increase in clinical reaction threshold that protects from accidental ingestion during ongoing daily subthreshold allergen exposure. Some patients achieve sustained unresponsiveness (SU), or an increased clinical reaction threshold that persists despite discontinuation of daily dosing[14–20]. Long term trials suggest that SU may wane over time[21, 22]. It is unknown why some patients achieve SU, and the clinical and immunological phenotypes associated with SU are an area of active research[23–25].

The development of natural oral tolerance involves a complex interplay between tissues and cells resident in the intestinal mucosa. In a TGFβ and retinoic acid dependent mechanism, CD103+ dendritic cells (DCs) carry food antigens from the lamina propria to mesenteric lymph nodes where they interact with T-cells and promote the formation of forkhead box protein 3 (FOXP3) positive regulatory T (Treg) cells. These newly induced antigen-specific Tregs migrate to the lamina propria and undergo expansion, the latter of which is dependent on the production of IL-10 by CX3CR1+ macrophages[26–29]. Additional Treg subsets have been implicated in oral tolerance, including Th3 and Tr1 cells, but the role of these specific cell types is not clear. Other factors, such as the intestinal microbiome and dose and timing of antigen exposure, likely also contribute to oral tolerance[28]. Although the pathways above are postulated to play a role in the mechanism of action of OIT, this remains poorly understood.

OIT, through daily ingestion of allergen, may exploit these same cells and tissues that are involved in the development of oral tolerance. OIT protocols vary but typically start with an IDE resembling a rush desensitization protocol, where increasing doses of allergenic protein are administered over several hours under observation in a clinical setting. Starting with the highest tolerated dose, a build-up period follows where the first dose is ingested under observation and then continued daily at home for one to two weeks until the next escalation. Each dose escalation ranges from 25% to 100% and is again administered under clinical observation before continuing the new dose at home[30]. During this period, patients commonly have dose-related adverse effects, typically mild to moderate in severity[31–33]. In some subjects, these allergic symptoms may necessitate holding or decreasing dosage and, in some cases, cessation of therapy. The build-up phase typically occurs over 6-12 months until the maintenance phase is reached. Dosing during the maintenance phase is variable and ranges from 300mg to 4000mg of protein, which continues for weeks, years, or life-long. Response to treatment is determined using double-blind placebo-controlled food challenges (DBPCFC), which are administered at specified intervals during and post-treatment.

Our goal in this review is to summarize what is known about the immunomodulatory effects of OIT while highlighting areas where more research is needed. To increase clinical relevance, we will focus on literature primarily from human subjects with supplementation from animal models where warranted.

Baseline:

Aberrant activation of the innate immune system with T helper 2 (T_H2) skewing leads to sensitization in food allergic subjects. This is characterized by the decreased development of Tregs in favor of allergen-specific T_H2 cells, which promote IgE class-switching and the expansion of downstream allergic effectors[27]. The mechanism by which detectable levels of allergen-specific IgE (sIgE) are maintained are not well-understood, but memory B-cells and/or long-lived IgE+ plasma cells may be responsible. At baseline, food allergic subjects produce sIgE which binds to the high-affinity IgE receptor FcεRI on mast cells and basophils. Once primed with sIgE, mast cells and basophils rapidly degranulate upon subsequent exposure to suprathreshold amounts of the specific food allergen, resulting in allergic symptoms ranging from mild to life-threatening (Fig 1). Prior to beginning OIT, subjects have variably elevated sIgE and skin prick testing (SPT) with clinical reactivity documented by failure to pass a DBPCFC, which establishes the baseline reactivity threshold[34].

Figure 1: B-cell and Effector Cell Responses in OIT.

Baseline T_H2 skewing in food allergic subjects results in IgE class-switching and specific IgE (sIgE) production, which binds to the high-affinity IgE receptor FcεRI on mast cells and basophils. Once primed with sIgE, mast cells and basophils rapidly degranulate on subsequent exposure to suprathreshold amounts of allergen, leading to an allergic response. This allergic state is characterized by elevated skin prick (SPT) wheal diameter and basophil activation (BAT). Oral immunotherapy (OIT) leads to decreased T_H2 responses and increased regulatory responses, resulting in decreased production of sIgE and an increase in allergen-specific IgA (sIgA) and IgG4 (sIgG4). In desensitized subjects, sIgA and sIgG4 may function as blocking antibodies, preventing mast cell and basophil degranulation. It is unknown why some subjects experience loss of desensitization or remission, characterized by recurrence of clinical reactivity and loss of suppression of SPT and BAT, while some achieve sustained unresponsiveness (SU), characterized by the continued lack of clinical reactivity and suppression of SPT and BAT despite discontinuation of OIT.

During OIT:

Basophils and Mast Cells

While the majority of data is on peanut OIT, what has been published for milk and egg OIT has overall been consistent with peanut. Immunologic evidence of desensitization is seen within the first year of OIT and includes decreased SPT wheal diameter and decreased basophil activation in vitro[14, 17, 18, 35–39]. These findings are associated with early clinical desensitization and are likely secondary to the constant low-dose allergen exposure provided by OIT. Decreased basophil reactivity does not appear to be restricted to the allergen used in OIT and was noted even when basophils from subjects undergoing peanut OIT were non-specifically stimulated with an anti-IgE antibody. A subset of these patients had concomitant sensitization to egg, and a lack of basophil activation as measured by upregulation of CD63 was also seen when basophils were stimulated with egg allergen. There was no suppression in basophil activation when stimulated by the fMLP receptor pathway, which is distinct from the IgE receptor pathway[40]. These findings suggest that OIT induces an overall state of basophil hyporesponsiveness or anergy in a pathway-specific manner.

The mechanism by which basophil hyporesponsiveness occurs does not appear to be dependent on sIgE levels. There is an early and rapid increase in sIgE upon initiation of OIT, and sIgE levels remain elevated for months until they drop to baseline levels or lower[35, 36, 41]. Nevertheless, the monoclonal anti-IgE antibody omalizumab has shown benefit as adjunctive therapy in OIT trials, allowing participants to rapidly tolerate higher doses of OIT during dose escalation[42, 43]. In part, these beneficial effects of anti-IgE therapy may be due to a decrease in circulating IgE and downregulation of FcεRI on mast cells and basophils, but the full picture is probably more complex[44]. With regard to other antibodies, studies have shown that allergen-specific IgG and IgA may serve as blocking antibodies, likely explaining much of the clinical evidence of desensitization from OIT[45–47]. Plasma from subjects post-peanut OIT was able to suppress basophil activation by pre-OIT sera, and this inhibition was blocked by antibodies targeting FcγRIIb[48], the inhibitory receptor to which IgG4 binds with high affinity[49]. Similarly, depletion of peanut-specific IgG or IgG4 in plasma from subjects treated with peanut OIT failed to inhibit basophil and mast cell activation while the IgG-enriched fraction substantially blocked basophil activation[48].

Both allergen-specific and nonspecific basophil hyporesponsiveness persists through dose escalation and to some degree through maintenance but may not be long lasting. Several studies in human subjects undergoing peanut OIT have found a reduction from baseline in both peanut-specific and non-specific CD63 expression and histamine release during dose escalation and maintenance. However, this initial suppression appears to be temporary in many subjects. Non-specific suppression of basophil expression of the T_H2 cytokine IL-4 was similarly observed during peanut OIT but began increasing once maintenance therapy was discontinued, providing further evidence that OIT-induced basophil suppression is temporary[50]. More recently there is mounting evidence that persistent suppression of basophil activity is necessary to maintain remission after discontinuation of OIT[21, 48, 51].

While tissue-resident mast cells are also critical effector cells in food allergy, there is a paucity of data on their role in OIT, largely due to the difficulty in routinely sampling these cells in human subjects. There are a number of intrinsic differences between mast cells and basophils, and they should not be considered equivalent. While basophils live for several days, mast cells live for months to years, raising important questions about the contribution of each cell type to the kinetics of desensitization. Additionally, the inhibitory receptor FcγRIIb is present on mast cells that reside in the gastrointestinal (GI) tract but absent on dermal mast cells, and it is possible that these cells are differentially impacted by OIT[52]. This is an area of ongoing interest but methods to sample and track GI-resident mast cells are lacking. In a mouse model of passive anaphylaxis, in vitro rapid desensitization of peritoneal mast cells (PMCs) inhibited degranulation and led to decreased surface levels of IgE and increased IgE receptor internalization[53]. In contrast, a second study did not find a reduction in surface IgE or in the signal transduction ability of the high-affinity IgE receptor (FcεRI), but instead found that reorganization of the actin cytoskeleton in desensitized mast cells led to impaired Ca²⁺ signaling and prevention of mast cell degranulation[54]. A recent study using a cutaneous model of local anaphylaxis in dual-sensitized mice showed that desensitization to one allergen conferred protection when challenged to the second allergen[55]. This suggests that early subclinical degranulation of mast cells plays an important role in desensitization and, like basophils, probably provides bystander protection upon exposure to other allergens. Although the murine studies discussed above are compelling, there remains no consensus on the role of mast cells in OIT and more research is needed before these findings can be generalized to humans. It is feasible that rapid suppression of mast cells and basophils due to continued low-dose allergen exposure accounts for the clinical desensitization seen during early OIT, and that waning of this suppression correlates with the gradual recurrence of clinical reactivity that is seen in some subjects on discontinuation of OIT.

T-Cells

Aberrant T-cell activation with T_H2 skewing drives downstream effector responses in patients with food allergy (Fig 2), however it is not well understood what causative events or processes lead to this breakdown of oral tolerance. Epithelial cytokines, including thymic stromal lymphopoietin, IL-33, and IL-25 may be produced during an innate immune response to tissue inflammation or damage and can drive T_H2 responses[56, 57]. Mouse models of food allergy differ from humans in that oral sensitization typically requires the addition of adjuvants such as cholera toxin (CT), although several adjuvant-free models have been described[58, 59]. Notably, the development of food allergy in a CT-induced mouse model of peanut allergy was found to be dependent on IL-33. In fact, both IL-33 and CT cause the upregulation of OX40 ligand (OX40L) on dendritic cells, which potently signals naïve T-cells through ligation of OX40-OX40L to develop into T_H2 cells[60, 61].

Figure 2: T-cell Responses in OIT.

In food allergic subjects, a breakdown in natural oral tolerance leads to decreased development of regulatory T (Treg) cells in favor of allergen-specific T_H2 cells, including the allergen-specific T_H2 (T_H2A) subset. T_H2 and T_H2A cells produce the T_H2 cytokines IL-4, IL-5, IL-9 and IL-13, which promote IgE class-switching in addition to the activation and expansion of downstream allergic effectors, including mast cells, basophils, and eosinophils. During oral immunotherapy (OIT), continuous high-dose allergen exposure leads to T_H2 and T_H2A anergy and/or deletion and an increase in regulatory cells, including Tregs and IL-10 producing CD4+ T-cells. Together, these changes result in the suppression of downstream allergic responses as shown in Figure 1.

While T_H2 responses characterize the allergic state, Tregs are essential to the development of oral tolerance. In mice, the production of IL-4 by group 2 innate lymphoid cells (ILC2s) stimulated by IL-33 suppresses the development of allergen-specific Tregs and promotes food allergy[62]. Under normal conditions, it is thought that repeat exposure to low doses of antigen promote the development of Tregs[63]. In contrast, exposure to large amounts of antigen leads to T-cell anergy or FAS-mediated clonal deletion[64], both of which result in the secretion of TGF-β[65]. In mouse models of oral tolerance, TGF-β is necessary for inducible Treg (iTreg) cell differentiation[66]. It is likely that both low and high-dose responses play a role in the development of tolerance[28]. Natural Tregs (CD4+CD25+FOXP3+) develop in the thymus and are important in maintaining tolerance to self-antigens. In contrast, iTregs develop from naïve CD4+ T-cells in the periphery after exposure to antigen and are thought to govern response to exogenous antigens, including allergens. Experiments in mice have shown that FOXP3+ iTregs are necessary to develop mucosal tolerance on exposure to inhaled allergen[66]. There are at least two types of iTregs: T_R1 cells produce IL-10 while T_H3 cells produce TGF-β. Continued exposure to allergen leads to the development of T_R1 cells in humans, which is linked with clinical tolerance[67]. Additional IL-10 producing cells, including CD4+ T-cells and regulatory B-cells[68], may also influence the development and maintenance of peripheral tolerance.

Within the first weeks and months of OIT, there is a rapid increase in sIgE[17, 35, 36, 38, 41, 69, 70]. Clinically, this time period is associated with the highest rate of adverse reactions. Atopic features and presumably worse T_H2 skewing at baseline appear to predict the likelihood of allergic adverse events (AEs) during OIT. In a cohort of patients receiving peanut OIT, baseline allergic rhinitis and peanut SPT size were significantly associated with an increased risk of AEs[32]. During the build-up phase, this rapid increase in sIgE is likely driven by an expansion in allergen-specific T_H2 cells[71]. Single-cell transcriptional analysis of Ara h 2-specific CD4+ T-cells in human subjects undergoing peanut OIT has shown an increase in a cluster of cells expressing a T_H2 phenotype (IL4+ IL13+ CD27_low) within the first 3 months of OIT. Interestingly, during this same time period, there is a decrease in a cluster expressing a Treg phenotype (FOXP3+, IL-10+, CD25+) and a gradual increase in an anergic cluster (CD28_low K_i-67_low), the latter of which increases throughout OIT[72]. These observations suggest that constant exposure to low doses of allergen during the first few months of OIT may enhance pathogenic T_H2 responses and inhibit formation of Tregs.

In contrast, when omalizumab is used as adjunctive therapy, there is evidence of early suppression of T-effector (Teff) responses. In a cohort of children undergoing high-dose cow’s milk OIT with omalizumab, an almost absent CD4+ T-cell response to milk was found within a week of starting therapy[43]. More recently, it was shown that treatment with omalizumab prior to starting high-dose peanut OIT led to markedly decreased proliferation of allergen-specific CD4+ Teff and Treg cells. Interestingly, these Tregs had a T_H2-like phenotype at baseline and lacked the ability to suppress peanut-specific Teff cells. The in vitro addition of IL-2 or anti-IL-10 monoclonal antibodies failed to rescue proliferation of either peanut-specific Teff or Treg cells, providing evidence that omalizumab-mediated suppression may not be due to anergy or suppression via IL-10, although deletion is possible[73].

As OIT progresses, subjects are continually exposed to larger doses of allergen, which has been associated with a decline in the activity and expansion of T_H2 cells[72]. Notably, suppression of the T_H2 cytokines IL-13, IL-5 and IL-9 in subjects undergoing peanut OIT was recently found to be dose-independent, with no significant difference found between 300 and 3000mg maintenance dosing[74]. In support of these observations, a recent phase III clinical trial demonstrated tolerability and efficacy with a maintenance dose of just 300mg of peanut protein[10]. Although a shift from T_H2 to T_H1 responses has been proposed to occur during OIT, there was no increase in T_H1 or Tr1 cytokines during peanut OIT, suggesting that OIT does not skew away from T_H2 responses and restore natural tolerance[74]. A recently described allergic-specific T_H2 subset, termed T_H2A cells, are specifically upregulated in atopic patients including patients with food allergy[75]. These terminally differentiated CD4+ T-cells express CRT_H2, CD49d, and CD161 and, unlike canonical T_H2 cells, express multiple T_H2 cytokines simultaneously. Compared to T_H2 cells, T_H2A cells more highly express the epithelium-derived cytokine receptors for IL-25 (IL-17RB), IL-33 (IL1RL1), and thymic stromal lymphopoietin (CLRF2), providing evidence that T_H2A cells may be involved in the pathogenesis of allergic disorders. In subjects undergoing peanut OIT, the majority of peanut-reactive CD4+ T-cells at baseline were T_H2A cells, which decreased throughout the course of OIT. Importantly, the decrease in T_H2A cells after completion of OIT directly correlated with successful desensitization[75]. Recently, others have identified T_H2 cells with cell-surface marker expression and cytokine profiling similar to T_H2A cells[76].

How T_H2 responses are downregulated during OIT is unknown. This may be orchestrated by an expansion of IL-10 producing cells, whether these are T_H2[77], Treg[39], or Breg cells[68]. Tregs are critical in the successful response to immunotherapy in mice, but the role of Tregs in the human response to OIT is less clear. Nevertheless, several studies have observed an increase in FOXP3+ Tregs with OIT and Syed et al recently demonstrated an association with sustained unresponsiveness[39]. Furthermore, there is evidence that different subsets of Tregs are induced depending on the type of immunotherapy used, including OIT, SLIT and EPIT[78]. Additional cell types such as T follicular helper (Tfh) cells may be involved in the response to OIT, but this is largely unexplored. A unique cell subset of Tfh, termed Tfh13, was recently described that co-expresses BCL6 and GATA3 and has the cytokine profile IL-13^hi IL-4^hiIL-5^hiIL-21^lo. Tfh13 cells are present in mice and humans during allergic conditions and were found to be essential for the production of high-affinity IgE during anaphylaxis[79]. More research is needed to better characterize the T-cell changes associated with successful oral immunotherapy.

B-Cells and Antibodies

In patients with IgE-mediated food allergy, serum levels of sIgE and the ratio of specific to total IgE positively correlate with the likelihood of an allergic reaction during oral food challenge[80]. sIgE is critical to the pathogenesis of food allergy. In mice, sIgE is required for both humoral and T_H2 responses to ingestion of peanut and leads to the impairment of Treg induction. Furthermore, deletion of the FCεRI kinase Syk or Syk blockade prevents sensitization and, in mice with established peanut allergy, Syk blockade facilitates desensitization and Treg induction[81]. In addition to food-specific IgE, component-resolved diagnostics is commonly used both in clinical practice and research settings, and in many cases is more predictive than food-specific IgE alone[24]. In the case of peanut allergy, elevated sIgE to Ara h 1, Ara h 2, Ara h 3 and Ara h 6 is associated with clinical reactivity[82] with elevated sIgE to Ara h 2 having greater predictive value than sIgE to peanut alone[83, 84]. Epitope analysis, which assays IgE binding to individual regions on an allergen, is a newer diagnostic tool that provides further resolution. Linear epitope analysis in subjects undergoing peanut OIT showed associations with OFC outcomes, including eliciting dose at baseline OFC, and likelihood of attaining SU[85]. Recent publications using multiplex bead-based assays have demonstrated efficacy as a predictor of clinical peanut allergy when combined with peanut sIgE[86] and in distinguishing clinical phenotype in children with cow’s milk allergy[87]. Future studies are needed to better characterize the clinical utility of epitope sIgE in patients undergoing OIT for food allergy.

It is well established that there is an early increase in sIgE after initiation of OIT. Patil et al used fluorescent Ara h 2 multimers to demonstrate that peanut OIT induced a greater than 3-fold transient expansion of circulating oligoclonal Ara h 2-specific memory B-cells that peaked at week 7. This temporally aligned with an increase in Ara h 2 sIgE that peaked at almost twice baseline at 7 weeks and remained above baseline until after week 35. Ara h 2 sIgA, sIgG and sIgG4 all increased significantly above baseline by 7 weeks and continued to increase throughout the duration of peanut OIT[41]. Interestingly, analysis of these oligoclonal Ara h 2-specific memory B-cells in 3 unrelated patients showed significant sequence homology among immunoglobulin heavy chain sequences, which may represent shared or “public” clonotypes as recently described[88]. Providing further support for this notion, next generation sequencing of BCR repertoires similarly revealed an oligoclonal enrichment of specific immunoglobulin alpha and gamma heavy chain clones after peanut OIT[89].

Allergen-specific IgG and IgA increase throughout the course of OIT, and their appearance coincides with the formation of allergen-specific memory B-cells and plasmablasts[41]. IgG4 levels may increase by more than 10-fold throughout the course of OIT, and remain elevated years after therapy is completed[14, 17, 35–37]. The processes that lead to an increase in IgG4-producing B-cells are not known, but this could be explained by an expansion of IL-10 producing cells such as Tregs and Bregs [71]. sIgA levels correlate with food challenge outcome in subjects undergoing peanut SLIT[90], and elevated IgA and IgA2 are associated with response to egg OIT[91]. It is important to note that although sIgE eventually decreases below baseline during OIT it is not eliminated, and sIgE retains its functional abilities to induce mast cell activation despite OIT[46].

Sustained Unresponsiveness:

A significant limitation of OIT is that, while the majority of subjects can be effectively desensitized, many will experience loss of desensitization after stopping daily dosing, with a higher risk of loss of desensitization over time [17, 50]. A minority of subjects remain clinically unreactive, which has been termed sustained unresponsiveness (SU) to distinguish it from natural tolerance. It is not presently clear whether SU is simply a state of prolonged desensitization or if it is a state of induced tolerance that lies somewhere between desensitization and natural tolerance.

In support of the former hypothesis, basophil activation and SPT rarely completely normalize in patients who have achieved SU and may remain positive for years after OIT. Persistence of decreased SPT and basophil activation tests is associated with SU[17, 50, 51]. In contrast, loss of suppression of these tests is seen in patients who regain allergic reactivity, which has been reported as early as one week after completion of OIT[37]. Longer duration of maintenance therapy in addition to younger age[18] appear to be associated with an increased chance of achieving SU[15]. Studies have identified biomarkers that are predictive of SU, including lower baseline and end of maintenance SPT and sIgE in addition to an early and persistent decrease in basophil reactivity[21, 24, 51]. Interestingly, IgG4 levels alone do not appear to be predictive of SU. OIT may induce qualitative changes in antibodies themselves, such as epitope specificity and avidity[85, 92], and there is evidence that OIT modifies the B-cell repertoire itself through oligoclonal expansion of allergen-specific memory B-cells[89].

As described above, OIT also modulates the T-cell compartment through anergy or deletion of pathogenic CD4+ T-cells such as T_H2A cells. Epigenetic modification has been linked to SU, with hypomethylation of FOXP3 CpG sites in peanut-specific Tregs associated with SU and increased methylation found in subjects who later lost tolerance[39]. An expansion of IL-10 producing cells may be necessary for the downregulation of T_H2 responses as has been shown in other forms of immunotherapy[68, 93]. Recently, Yoneyama et al determined that the Notch-dependent expansion of IL-10 producing CD4+ T-cells and myeloid-derived suppressor cells (MDSCs) was critical to the development of SU in a mouse model of ovalbumin OIT. Importantly, depletion of Gr1+ monocytic MDSCs in this model abrogated SU[94]. MDSCs inhibit T-cell function and are involved in the pathogenesis of a wide variety of conditions, including malignancy and autoimmunity, but their role in food allergy immunotherapy has not previously been reported[95, 96].

Conclusions and Future Directions:

Our understanding of the immunologic mechanisms underlying OIT continues to evolve. It is clear that OIT impacts multiple cell compartments, which together orchestrate an adaptive immune response that culminates in desensitization or SU. It is still unclear why some subjects achieve SU while others experience loss of desensitization, and there is a need for biomarkers that are highly predictive of SU. Going forward, it will be important to clarify the role of suppressor cells and more fully characterize the adaptive and humoral changes that are associated with successful OIT. Additional cell types, such as dendritic cells, ILCs, Tfh cells, and MDSCs should be explored in the context of food allergy and immunotherapy. The microbiome[97] and other local factors in the gut may also impact the response to OIT. Future studies are needed to better elucidate the complex immunologic changes that occur during OIT, particularly those that are associated with SU.