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\begin{document}

\title{Transcriptomic changes associated with oral immunotherapy for food
allergy}



\author[1]{Sarah Ashley}%
\author[2]{Anthony Bosco}%
\author[1]{Mimi Tang}%
\affil[1]{Murdoch Children's Research Institute}%
\affil[2]{The University of Arizona Asthma \& Airway Disease Research Center}%


\vspace{-1em}



  
  \date{May 24, 2024}


\begingroup
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\selectlanguage{english}
\begin{abstract}
This review summarises recent advances in characterising 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 summarise current
understanding of gene expression changes following oral immunotherapy
for food allergy and specific signatures underpinning the different
clinical outcomes of desensitisation 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 optimise treatments and gain
improved outcomes for patients.%
\end{abstract}%



\sloppy


\textbf{Manuscript Title:Transcriptomic changes associated with oral
immunotherapy for food allergy}

Sarah E. Ashley\textsuperscript{1,2,3}\emph{,} Anthony
Bosco\textsuperscript{4,5}, Mimi L.K. Tang\textsuperscript{1,2,3}

\textsuperscript{1}Allergy Immunology, Murdoch Children's Research
Institute, Melbourne, Australia

\textsuperscript{2}Department of Paediatrics, The University of
Melbourne, Melbourne, Australia

\textsuperscript{3}The Royal Children's Hospital, Melbourne, Australia

\textsuperscript{4}Asthma and Airway Disease Research Center, University
of Arizona, Tucson, USA

\textsuperscript{5}Department of Immunobiology, The University of
Arizona College of Medicine, Tucson, USA

\emph{Correspondence}

\emph{Mimi L. K. Tang, Allergy Immunology,}

\emph{Murdoch Children's Research Institute,}

\emph{Melbourne, VIC, Australia.}

\emph{Email: mimi.tang@rch.org.au}

Word count: 3,795, number of tables and figures 1\emph{}

Ashley, S.E., Bosco, A., Tang, M.L.K. \textbf{Transcriptomic changes
associated with oral immunotherapy for food allergy} . Pediatr Allergy
Immunol.

Abstract

This review summarises recent advances in characterising 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 summarise current understanding of
gene expression changes following oral immunotherapy for food allergy
and specific signatures underpinning the different clinical outcomes of
desensitisation 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 optimise
treatments and gain improved outcomes for patients.

\textbf{Key words} : Food allergy; Transcriptomics; Systems biology;
Oral Immunotherapy; Gene expression; Remission; Desensitisation

\textbf{Key Message:} This review summarises 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,
desensitisation and remission of food allergy. There is compelling
evidence that T cell anergy and type 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.

\emph{}

Review

\emph{Introduction}

IgE-mediated food allergy affects up to 8\% of
children\textsuperscript{1} and 10\% of infants\textsuperscript{2} in
Westernised countries, which means there is likely to be at least one
food allergic child in every school classroom\textsuperscript{3}.
Currently there is no curative therapy for food allergy. The recent
approval of Palforzia, a desensitising oral immunotherapy, offers
patients temporary protection against accidental exposure to small
amounts of peanut\textsuperscript{4}, however drawbacks include the need
for continued maintenance treatment alongside allergen avoidance,
frequent treatment-related allergic reactions, and a lack of improvement
in quality of life\textsuperscript{5}. Consequently, management 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\textsuperscript{6}.

Efforts 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\textsuperscript{7}. Although 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, highly variable
treatment schedules with differing maintenance doses and treatment
durations, and a wide divergence on how clinical end points are
defined\textsuperscript{8}. Furthermore, 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
(desensitisation) 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
desensitisation (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 desensitisation or remission following
oral immunotherapy.

\emph{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 desensitisation, 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 (\textbf{Table 1} ). For the purposes of this review:\selectlanguage{english}
\begin{longtable}[]{@{}llll@{}}
\toprule
& Definition & Benefit to patient & Immune mechanisms\tabularnewline
\midrule
\endhead
Desensitisation & A temporary increase in reaction threshold. Maintained
by continuing daily treatment. & Patient is protected from accidental
reactions but ongoing maintenance dosing must be continued life-long. &
Temporary suppression of basophil and mast cell (effector cell)
responses. Increased sIgG4; no change in serum levels of sIgE or modest
reduction after prolonged treatment.\tabularnewline
Remission & Also referred to as sustained unresponsiveness. The absence
of clinical reactivity that persists weeks or months after stopping
treatment. & Individual may stop treatment and consume the allergen
freely in the diet. Some level of allergen intake is considered
important to consolidate the immune changes supporting remission. &
Remission/SU is thought to result from redirection of the underlying
allergy towards tolerance, however the precise immune changes involved
remain poorly understood.\tabularnewline
Long-term remission & Lasting persistence of remission/SU years after
treatment discontinuation. & As it is not possible to demonstrate
permanence of a treatment effect in the clinical trial setting,
long-term remission/SU may offer a surrogate measure of true immune
tolerance/resolution of allergy. & Mechanistic studies with data years
after treatment remain rare and the immune drivers leading to long-term
remission remain unidentified.\tabularnewline
\bottomrule
\end{longtable}

\textbf{Table 1} . Definitions of clinical outcomes following oral
immunotherapy\textsuperscript{9} 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\textsuperscript{10}.

Genes 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 gene 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 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\textsuperscript{11}. Moreover, gene regulatory
networks can be constructed between transcription factors and target
genes in a cell-type specific manner\textsuperscript{12}. Trajectory
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\textsuperscript{13}. While
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.

\textbf{Food allergy associated immune remodelling 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 endeavour 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\textsuperscript{14}. However, 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\textsuperscript{15}. A 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 \textsuperscript{16}. Despite 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\textsuperscript{17}. 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 remodelling in the group who did not reach the target dose
(n=23; partially desensitised) versus those who reached the target
maintenance dose (n=22). Characterised by persistence of Th2 responses
and lack of upstream regulator engagement in the partially desensitised
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 1g following 8 months of
build-up. 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 desensitisation without
remission.

Several differentially regulated genes have been reported following OIT
with omalizumab in whole blood\textsuperscript{18}. Of 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\textsuperscript{19}. The 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 analysed
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\textsuperscript{18}. Comparing 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\textsuperscript{20}, a study
applying single-cell sequencing reported that OIT did not modify this
population\textsuperscript{21}. The 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 (\selectlanguage{greek}γδ\selectlanguage{english}Tregs) may play a role in immunological
mechanisms of OIT. Reduced numbers, comparable to those of health
controls were observed by week 6 of OIT\textsuperscript{22}. However,
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\textsuperscript{23}. Enrichment of selected immunoglobulin
heavy chain alpha and gamma clones was identified following OIT. TCR
beta-chain 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\textsuperscript{24}. Application 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,
2hr, 4hr) following food challenge, researchers were able to identify
six genes LTB4R, PADI4, IL1R2, PPP1R3D, KLHL2, and ECHDC3 as having
causal roles during peanut reactions\textsuperscript{19}. Using 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\textsuperscript{25}.

\emph{Mechanisms of desensitisation~in oral immunotherapy}

Recent advances in our understanding of the immune changes associated
with desensitisation 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\textsuperscript{24,26,27}.
Th2A 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
characterised 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 \emph{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 desensitisation following oral immunotherapy
(n=4)\textsuperscript{26}.

Profiling 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 desensitisation
following OIT in a study of cells from n=12 participants at 0, 13, 58
and 70 weeks\textsuperscript{21}. However, 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
(desensitised without SU), indicating no association with
remission\textsuperscript{21}. Furthermore, desensitisation 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.

\emph{Mechanisms of remission in oral immunotherapy}

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\textsuperscript{28,29} and may be negative regulators of
the allergic CD4+ T cells response both in and outside of the treatment
setting\textsuperscript{30,31}. We 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 characterised 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\textsuperscript{18}. In 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\textsuperscript{19}. It 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 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\textsuperscript{32}. Further 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 IFNs can abrogate both
the production of Th2 cytokines in antigen stimulated
PBMC\textsuperscript{33} and expression of the high affinity IgE
receptor\textsuperscript{34}. Furthermore, 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\textsuperscript{35}. Interferon genes (type I and
type II) have also been shown to regulate a cluster of genes associated
with levels of component resolved egg sIgG4 and sIgA after 8-months of
egg OIT\textsuperscript{17}.

Another regulatory mechanism that has been reported with OIT is T cell
anergy. Expansion of the anergic (CD28-/CD38-/IFN-\selectlanguage{greek}γ/\selectlanguage{english}IL4-/IL13-/IL10-)
non-cytokine secreting T cell subset, as well as non-allergic (IFN-\selectlanguage{greek}γ\selectlanguage{english}+) 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 desensitised 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\textsuperscript{36}. However, this finding was not replicated
in a subsequent study by the same group when the frequency of
peanut-specific T cells over the course of OIT was measured using
Dextramers\textsuperscript{37}. 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\textsuperscript{21}. Findings 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\textsuperscript{21}.

Another signal linked with clinical outcome following treatment is an
early transient increase in TGF-\selectlanguage{greek}β \selectlanguage{english}producing cells at one year into
treatment\textsuperscript{37}. This 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 300mg
doses\textsuperscript{37}. Therefore, it is unclear whether the finding
relates to remission/SU or to desensitisation without remission/SU.

T regulatory cells play an instrumental role in the development and
maintenance of oral tolerance\textsuperscript{38} and accordingly it is
generally assumed that Treg must also play a role in remission of
allergy in OIT\textsuperscript{39}. Notably, 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\textsuperscript{40}. Functional 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\textsuperscript{41,42}, a more recent study profiling
antigen-specific T cells with single-cell sequencing found no change in
peanut-reactive Treg with OIT\textsuperscript{21}. Moreover, a subset of
peanut-reactive Treg with a Th2-like phenotype (Th2reg-like), matching a
previously described population found in food allergic
children\textsuperscript{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)\textsuperscript{21}. Induction of \emph{FOXP3} gene
expression via demethylation of the \emph{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
\emph{FOXP3}locus)\textsuperscript{41}. These findings suggest that
persistent activation of \emph{FOXP3} may be a critical requirement for
lasting persistence of remission.

\emph{Conclusions}

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, desensitisation 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. 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.

\textbf{References}

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