Transcriptomic changes associated with oral immunotherapy for food allergy

This review summarizes recent advances in characterizing the transcriptional pathways associated with outcomes following Oral Immunotherapy. Recent technological advances including single‐cell sequencing are transforming the ways in which the transcriptional landscape is understood. The application of these technologies is still in its infancy in food allergy but here we summarize current understanding of gene expression changes following oral immunotherapy for food allergy and specific signatures underpinning the different clinical outcomes of desensitization and remission (sustained unresponsiveness). T helper 2A cells have been identified as a cell type which correlates with disease activity and is modified by treatment. Molecular features at study entry may differentiate individuals who achieve more positive outcomes during OIT. Recent findings point to T cell anergy and Type 1 interferon pathways as potential mechanisms supporting redirection of the allergen‐specific immune response away from allergy towards remission. Despite these developments in our understanding of immune mechanisms following OIT, there are still significant gaps. Additional studies examining immune signatures associated with long term and well‐defined clinical outcomes are required to gain a more complete understanding of the pathways leading to remission of allergy, in order to optimize treatments and gain improved outcomes for patients.

remains centred on allergen avoidance and timely management of allergic reactions that result from accidental exposure to allergen, which often includes carrying an EpiPen for treatment of anaphylaxis.The constant vigilance required to adhere to allergen avoidance, unpredictability of reactions and ever-present fear of a fatal reaction, together have a severe impact on quality of life, similar to that of a child with diabetes. 6forts to identify effective treatments for food allergy that retrain the immune system away from allergy towards remission and ultimately tolerance have been hampered by a limited understanding of the key immune changes underpinning these outcomes, which in turn reflects the limited number of treatments that have been shown to successfully induce remission of allergy. 7Although a large number of oral immunotherapy trials have been completed to date, limitations in study design have hindered the ability to draw firm conclusions about mechanisms driving clinical outcomes.In particular there are major gaps in our understanding of immune pathways supporting lasting remission of allergy.Study limitations include small sample sizes,

G R A P H I C A L A B S T R A C T
In this review we summarise current understanding of immune changes associated with oral immunotherapy (OIT), as established through molecular profiling of the transcriptome.Changes observed during OIT include clonal anergy of T cells with a Th2 phenotype and decreased expression of Th2 genes in peanut reactive T cells.Demethylation of the FOXP3 locus in T regulatory cells has been demonstrated, correlating with treatment outcome.Decreased frequencies of gamma delta T cells have been observed early on in OIT, which may contribute to the loss of Th2 dominance.Multiple single cell studies of CD4 + T cell memory responses have identified a novel subset of a T-helper cells that express type I interferon stimulated genes and may also act to supress Th2.

Key message
This review summarizes the current understanding of immune changes associated with food oral immunotherapy (OIT), focusing on new discoveries gained from state of the art transcriptomic approaches.Recent computational and technological advances have transformed our understanding of immune mechanisms driving food allergy, desensitization and remission of food allergy.
There is compelling evidence that T cell anergy and type

| Food immunotherapy treatment outcomes
There are several clinical outcomes that can be achieved with food immunotherapy.A small subset of patients will not tolerate treatment, however the majority of those who are able to reach a target maintenance dose will achieve desensitization, defined as an increase in reaction threshold, and a smaller subset will achieve remission, defined as the absence of clinical reactivity after treatment has been discontinued for a period of time (Table 1).For the purposes of this review: Traditional approaches to investigate immune mechanisms have studied a few candidate genes or pathways at a time.Recent mechanistic studies in the last decade, however, have harnessed the power of RNA sequencing (RNAseq) to investigate mechanisms of food allergy and outcomes following OIT at a genome-wide scale.
The majority of RNAseq studies have focused on differential gene expression (DGE), however this approach is limited, because it does not account for changes in the structure of the underlying gene networks. 10nes do not exist nor act in isolation but must work together in a coordinated fashion to achieve complex immune functions.
Approaches to study genes within their full molecular context are necessary to understand the global organization and function of the gene expression program and unveil the dynamic molecular states that underpin clinical states.Towards this goal, systems biology methods including gene co-expression network analysis works backwards from g ene expression profiles to reconstruct the global connectivity structure and functional organization of the gene expression program.This approach is well suited to uncovering novel patterns of gene expression underlying complex diseases like food allergies, which are mediated by multiple cellular and molecular pathways acting together to mount a response.This systems-level approach clusters co-expressed genes into modules that are enriched for genes which are associated with specific biological functions and pathways.Once modules are defined, researchers can investigate the connectivity of genes within these modules, based on the sum of their pairwise co-expression relationships with all other genes.The degree of connectivity, or the number of edges (co-expression relationships) a gene has within a module, serves as a useful metric in prioritizing genes which are more strongly associated with the module and have potential regulatory functions, and accordingly are hypothesized to play pivotal TA B L E 1 Definitions of clinical outcomes following oral immunotherapy 9 for the purposes of this review.roles in regulating or driving the biological processes associated with the module.Identification of modules and highly connected genes (nodes) within modules thereby provides insights into the coordinated regulation of genes within the context of larger biological systems and can shed light on the molecular underpinnings of diseases.For example, by comparing networks from individuals with remission following OIT and those who fail to achieve remission, it may be possible to pinpoint key regulators of, and changes to, dysregulated pathways leading to this outcome.
Emerging technologies such as single cell RNA sequencing (scRNAseq) are now available that enable a much deeper understanding of complex disease mechanisms at single cell resolution.
For example, single cell data can be leveraged to infer cell-to-cell interactions that are mediated via ligand-receptor pairs. 11Moreover, gene regulatory networks can be constructed between transcription factors and target genes in a cell-type specific manner. 12Trajectory inference can be employed to study dynamic biological processes and map individual immune cells into a pseudotemporal order based on their progression through the activation process. 13While the application of this technology to samples from individuals undergoing OIT is still in its infancy, scRNAseq has been applied to study shifts in allergen-reactive CD4 + T cells in a few studies.

| FOOD ALLERGY A SSO CIATED IMMUNE REMODELING DURING OIT
Sequencing bulk cell populations such as whole blood or peripheral blood mononuclear cells (PBMC) to identify biomarkers of allergy or treatment success is an attractive endeavor due to the relative ease of collecting this type of specimen in clinical settings.
Blood is routinely collected in these settings and a marker measured directly with little to no need for cell culture would be practical for ease of implementation into clinical practice.This "baseline predicts outcome" approach has been successfully applied to predict the immune response to vaccination using systems biology methods. 14However, one important limitation to this approach is lack of resolution since allergen reactive cells make up only a tiny fraction of the overall whole blood or PBMC population and do not persist for very long in the peripheral circulation. 15A second challenge is that the cellular composition of these fractions can be highly variable between individuals and across different age groups, limiting the likelihood of identifying a universal biomarker. 16Despite these challenges, transcriptional studies have identified immune changes in whole blood and PBMC that are linked to peanut or egg allergy, acute allergic reactions, and OIT treatment effects.
In egg OIT, downregulation of genes involved in TREM1 signaling, IL-6 and IL-17 were identified in PBMC after eight months of treatment. 17 Because there is no longitudinal assessment of clinical outcome following treatment it is not possible to delineate whether molecular changes were linked to remission or desensitization without remission.
Several differentially regulated genes have been reported following OIT with omalizumab in whole blood. 18Of interest, 108 of the 680 differentially expressed genes (DEG) associated with peanut OIT + omalizumab overlapped with DEG linked to acute allergic reactions to peanut in another study. 19The common DEG between the two studies primarily had opposing direction of effect, indicating that treatment effects were in part mediated through modulation of downstream events that occur during an acute allergic reaction.
There were no consistent transcriptional changes linked to omalizumab alone prior to peanut OIT.However, the cohort was a mix of those who managed to tolerate the maintenance dose (n = 11), and those who failed to tolerate the maintenance dose (n = 6).
Transcriptional changes were not analyzed based on clinical outcomes.Instead, the analysis was adjusted to account for changes associated with specific outcomes.Therefore, the detected immune changes are not linked to treatment success but rather to treatment independent of outcome. 18Comparing DEG between the egg and peanut OIT transcriptional studies, there was little overlap between the early transcriptional signals detected in the egg study (3 months into OIT) compared to the genes associated with omalizumab + peanut OIT (measured after 2-3 years on treatment).Of the genes that did overlap between the two studies, the majority were not detected with the same direction of effect.This finding may be due to the considerably distant windows at which DEG was measured in each study, each possibly capturing distinct phases of immunological activity.Alternatively, differences may be due to other variable factors between the two populations, such as differences in treatment regime, genetic background of individuals, age or other demographic factors.Genes that were consistent between the two studies were not immune genes but rather genes associated with cellular processes such as transport proteins, protein synthesis, mitochondrial function.
Although there has been interest in the potential role of T follicular helper 2-like cells in allergy pathogenesis, 20 a study applying single-cell sequencing reported that OIT did not modify this population. 21The T follicular helper 2-like cell population was, however, positively associated with peanut sIgE production in the same OIT population.Another study identified that changes in gamma delta Tregs (γδTregs) may play a role in immunological mechanisms of OIT.
Reduced numbers, comparable to those of health controls were observed by week 6 of OIT. 22However, data are only available within a limited window of time and no food challenges were performed to determine clinical outcomes.
B and T cell receptor profiling may also provide further mechanistic insight.A novel method using gene expression profiling was recently developed and applied to study how OIT modifies the B cell repertoire in n = 17 peanut allergic individuals before and after treatment. 23Enrichment of selected immunoglobulin heavy chain alpha and gamma clones was identified following OIT.TCR betachain profiling showed that peanut allergy was associated with a pool of convergent activated clones, with 17% of the sequences shared between individuals.The frequency of these clones in the effector cell, but not regulatory cell compartment was higher in those with more reactive phenotypes. 24Application of methods to study allergen-specific B and T cell receptors, alongside gene expression profiling, in larger cohorts may reveal insights into selective clonal anergy or other patterns linked with clinical outcomes following OIT.
Key findings from studies applying techniques to study genes within co-expression networks have identified genes that are predicted to be drivers of the allergic response.By examining the transcriptional profile of whole blood samples (n = 19) across a time course (baseline, 2 h, 4 h) following food challenge, researchers were able to identify six genes LTB4R, PADI4, IL1R2, PPP1R3D, KLHL2, and ECHDC3 as having causal roles during peanut reactions. 19Using similar methods to examine whole blood collected from children with (n = 23) or without (n = 7) nut allergy, another group identified three gene drivers of nut allergy; IFIH1, DRAM1 and ZNF512B.A module enriched for type I interferon genes, with IFIH1 as the key driver, was most positively associated with nut allergy. 25

| MECHANIS MS OF DE S ENS ITIZ ATION IN OR AL IMMUNOTHER APY
Recent advances in our understanding of the immune changes associated with desensitization have arisen from gene expression profiling of the peanut-reactive T cell compartment.
These have included the identification of Th2A cells, also called peTh2, a pathogenic reservoir of Th2 cells elevated in allergic individuals. 24,26,27Th2A are a subset of cells that are distinctive from conventional Th2 cells.This subset is terminally differentiated, lacks CD27 expression, produces very high levels of Th2 effector cytokines, and is characterized by the markers CRTH2, CD49d, CD161.Transcriptomic analysis of Th2A cells by microarray in allergic individuals (n = 3 donors) revealed a unique immunological program that induces a pathogenic response to allergen.Using ex vivo pMHCII tetramer-based T cell profiling, the authors showed that this subset was reduced in frequency compared to baseline frequencies in patients who achieved desensitization following oral immunotherapy (n = 4). 26ofiling of peanut reactive T cells using scRNAseq has shed light on modifications to the Th2A population during OIT.Suppression of the Th2 transcriptional signals in Th2A-like cells was linked to desensitization following OIT in a study of cells from n = 12 participants at 0, 13, 58 and 70 weeks. 21However, it was supressed to a similar extent in both those who went on to fail a second food challenge after a period of 3 months of allergen avoidance following treatment (desensitized without SU), indicating no association with remission. 21Furthermore, desensitization was not linked to a complete elimination of these cells, which may point to a reservoir of Th2A cells, capable of expanding and contributing to a failure to achieve lasting protection.

| MECHANIS MS OF REMISS ION IN OR AL IMMUNOTHER APY
Interferons have diverse and often opposing roles in orchestrating immunological pathways.Recent studies from our group and others using a network analysis approach have found that type I interferons may also be important regulatory drivers in redirecting the allergic response towards remission of allergy following oral immunotherapy 28,29 and may be negative regulators of the allergic CD4 + T cells response both in and outside of the treatment setting. 30,31We examined gene co-expression networks in CD4 + T cells at study entry and following 18-months of Probiotic and Peanut Oral Immunotherapy in children who achieved remission following active treatment and placebo-treated controls who remained allergic.Results showed that remission following treatment was characterized by changes in the antigen-specific response to peanut, with loss of Th2 driven gene connectivity and replacement by a large integrated type I interferon network that incorporated Th2 genes within the module. 28In contrast, a Th2 dominant module persisted in allergic children who received placebo treatment.Notably, the role of type I interferons has also been highlighted in the setting of subcutaneous immunotherapy (SCIT) for allergic rhinitis in adults.Persistent reduction of house dust mite (HDM) associated symptoms after SCIT treatment was associated with progressive integration of previously distinct type I interferon and Th2 gene modules, resulting in a rewired module in which the top nodes, ranked by degree of connectivity, were made up of type I interferon genes. 29It is noteworthy that the same immune mechanisms were associated with disease remission across multiple allergen immunotherapy studies, despite major differences in the studies, including disease states and treatment modalities.This supports a role for type I interferon pathways as a fundamental mechanism through which lasting attenuation of allergen responsiveness is achieved following allergen immunotherapy.Both of these studies were conducted by profiling the total CD4 + T cell compartment, and IFNs can abrogate both the production of Th2 cytokines in antigen stimulated PBMC 33 and expression of the high affinity IgE receptor. 34Furthermore, type I interferons suppress the expression of GATA3 during differentiation of CD4 + T cells down a Th2 path and in fully committed CD4 + Th2 cells, and promotes epigenetic silencing of non-coding regions in the Th2 locus that controls Th2 cytokine expression. 35Interferon genes (type I and type II) have also been shown to regulate a cluster of genes associated with TA B L E 2 Transcriptomic changes associated with oral immunotherapy for food allergy.levels of component resolved egg sIgG4 and sIgA after 8-months of egg OIT. 17other regulatory mechanism that has been reported with OIT is T cell anergy.Expansion of the anergic (CD28⁻ /CD38⁻ / IFNγ/IL4 -/IL13 -/IL10 -non-cytokine secreting T cell subset, as well as non-allergic (IFNγ + ) T cell types has been detected in those who achieve remission/SU (defined as absence of clinical reactivity at 3 months post treatment).Conversely, in those desensitized without remission/SU CD4 + T cell subsets were scattered across multiple CD4 + T cell phenotypes with only slight increases in anergic and non-allergic T cell phenotypes. 36However, this finding was not replicated in a subsequent study when frequencies of peanutspecific T cells over the course of OIT were measured using the same approach. 37Although the frequency of the anergic cell cluster decreased over the course of OIT in both treated groups, this change was not specific to the treatment success group.While shifts in the frequency of peanut-reactive T cell clonotypes may not be clearly linked to outcomes following OIT, the application of scRNAseq to this population has demonstrated a link between dampened Th2 and Th1 signatures in effector cells and remission. 21Findings from this study were consistent with the findings that clonal anergy is linked with positive outcomes following OIT.
Conversely, baseline inflammatory signals in Th1 and Th17 effector cells were associated with poorer outcomes following OIT. 21other signal linked with clinical outcome following treatment is an early transient increase in TGFβ producing cells at one year into treatment. 37This signal was detected in a mixed group of patients who either avoided the allergen in their diet leading up to the 3 month remission/SU challenge or continued on 300 mg doses. 37erefore, it is unclear whether the finding relates to remission/SU or to desensitization without remission/SU.
T regulatory cells play an instrumental role in the development and maintenance of oral tolerance 38 and accordingly it is generally assumed that Treg must also play a role in remission of allergy in OIT. 39Notably, pathogenic Tregs with a Th2-like phenotype have been described in murine models of food allergy and were also found to be circulating in the periphery of children with food allergy. 40Functional studies from this work highlight that modulation of this population may promote the establishment of oral tolerance in food allergic individuals.Yet, evidence establishing Treg as primary mediators of remission following OIT in humans remains limited.Although two studies report changes to the antigen-specific Treg population following peanut OIT, 41,42 a more recent study profiling antigen-specific T cells with single-cell sequencing found no change in peanutreactive Treg with OIT. 21Moreover, a subset of peanut-reactive Treg with a Th2-like phenotype (Th2reg-like), matching a previously described population found in food allergic children, 40 was identified in this study.There is no evidence to support modulation of the frequency or phenotype of these cells with OIT, even in those who passed a food challenge 3-months after treatment and were classified as tolerant (remission). 21Induction of FOXP3 gene expression via demethylation of the FOXP3 has been linked with remission (defined as absence of clinical reactivity 3 months after treatment cessation, n = 7) compared to those who failed to achieve remission (n = 13), and this was reversed in those who lost their protection (n = 4 participants who failed a challenge at six months post-treatment regained methylation of the FOXP3 locus). 41Likewise, another study profiling gene expression and methylation of the Treg-specific Demethylated Region (TSDR) in PBMC from cow's milk allergic and resolved infants found significantly lower demethylation of TSDR in infants with active allergy when compared to those who outgrew their allergies and healthy controls. 43These findings suggest that persistent activation of FOXP3 may be a critical requirement for lasting persistence of remission.

| Summary
Studies have shown that OIT-induced remission of food allergy is associated with anergy of memory T cells and modest T regulatory cell activity that may be linked to the anergic T cell state.Type I interferons have recently emerged as candidate regulators of remission following OIT and may play a key role in suppression of the Th2 antigen response through regulatory action on GATA3 and the high affinity IgE receptor.In contrast, desensitization without remission/SU is associated with transient regulatory activity that may be insufficient to support longer-lasting redirection of the allergic response, suggesting that a more stable induction of regulatory activity is required to achieve durable redirection of immune responses towards a tolerant state.An overview of mechanisms highlighted in this review are presented in Figure 1 and Table 2.

I
interferons play important roles in determining clinical outcomes following OIT.Future studies with larger sample sizes, from individuals with well-defined clinical outcomes and longitudinal sampling are needed to provide high quality evidence of key mechanisms and further elucidate novel biomarkers of treatment response.highly variable treatment schedules with differing maintenance doses and treatment durations, and a wide divergence on how clinical end points are defined. 8Furthermore, most mechanistic studies have examined changes in treated vs untreated subjects, without taking account of varying clinical outcomes within the treated group, which prevents delineation of the mechanisms supporting short-term (desensitization) compared to longer-term (remission) benefits.Amongst the mechanistic studies that were designed to investigate immune changes linked to a clinical outcome, most have focused on the outcome of desensitization (a change in reaction threshold during food challenge); few have examined changes linked to remission (sustained unresponsiveness).Even fewer have examined long-term immune changes following cessation of active treatment.Here we discuss the current understanding of transcriptomic changes associated with food allergy and the specific clinical outcomes of desensitization or remission following oral immunotherapy.
Changes were linked to regulation of innate immunity and inflammation: Toll-like receptors (TLRs), interferons (IFNs), chemokines, cytokines, and adhesion molecules; and regulation of T cell genes including Th17 cells, regulatory T cells, and Th2 cells.Comparing outcome groups, expression profiles were largely overlapping between groups, however there was some evidence of delayed immunological remodeling in the group who did not reach the target dose (n = 23; partially desensitized) versus those who reached the target maintenance dose (n = 22).Characterized by persistence of Th2 responses and lack of upstream regulator engagement in the partially desensitized group.Interpretation of this study in the context of lasting outcomes following OIT was limited as outcomes were defined by the ability of participants to reach the target dose of 1 g following 8 months of build-up.

1
Transcriptomic changes associated with oral immunotherapy for food allergy.(A) The allergic immune state is skewed towards T helper 2 (Th2) cell immune responses.Th2 cytokines promote B class swiching and IgE production as well as further differentiation of Th2 type T cells.Production of IgE antibodies specific to the allergen prime mast cells and other granulocytes, leading to degranulation and allergic inflammation when cross linked to allergen.T follicular helper and gamma delta T cells can contribute to Th2 skew and IL-13 and IL-4 (hallmark Th2 cytokine) production.(B) Changes observed during OIT include clonal anergy of T cells with a Th2 phenotype, decreased expression of Th2 genes in peanut reactive T cells and decrease production of anitgen specific IgE antibodies.In parallel, increased production of sIgG4 functions to outcompete binding of sIgE.Demethylation of the FOXP3 locus in T regulatory cells has been demonstrated, correlating with treatment outcome.Decreased frequencies of gamma delta T cells have been observed early on in OIT, which may contibute to the loss of Th2 dominance.Multiple single cell studies of CD4 + T cell memory responses have identified a novel subset of a T-helper cells that express type I interferon stimulated genes (ISG).Although the function of these cells is not fully elucidated, T1-IFN plays important roles in T cell differentiation and the regulation of Tfh and may therefore regulate Th2 and antibody production.
future studies are needed to determine if the Th2 and IFN modules operate within a single CD4 + T cell population or whether these signals reflect changes in discrete cell subpopulations.In this context it is noteworthy that scRNAseq studies of CD4 + T cell memory responses to HDM have identified novel subsets of T-helper and T-regulatory cells, which express a type I interferon response signature, and are expanded in healthy non-allergic subjects, suggesting they may play a role in dampening allergic responses. 32Further studies applying similar systems biology approaches to compare the effects of different therapies and clinical outcomes following treatment will further elucidate the underlying processes.The precise mechanisms governing the interplay between type I IFN and Th2 responses in the context of allergen immunotherapy are unknown.Previous studies have demonstrated that type I Most studies have examined changes in CD4 + T cells and understanding of potential upstream factors or other cell types with pivotal roles in supporting stable remission remains limited.Systems biology approaches are well suited to provide a broader and much deeper understanding of OIT mechanisms and may facilitate development of more targeted and effective treatment approaches.For example, single cell profiling paired with cutting-edge computational tools holds great potential to unlock the immunological mechanisms underlying tolerance within the CD4 T cell compartment and beyond.There is also a need for larger studies including well-defined clinical outcomes and longitudinal sampling to enable high quality evidence of the key mechanisms driving redirection of the immune response in OIT.Such studies can unveil novel biomarkers of treatment response and associated therapeutic targets that can be leveraged to develop more effective treatments.