T cell epitope discovery and single cell technologies to advance food allergy research

Abstract

There is good evidence for a role of T cells in food allergy, but there is a lack of mechanistic understanding and phenotypic markers of the specific T cells contributing to pathology. Recent technological advancements have allowed for a new experimental paradigm where we can find and pull out rare antigen specific T cells and characterize them at a single cell level. However, studies in infectious disease and broader allergy have shown that these techniques benefit greatly from precisely defined T cell epitopes. Food allergy has fewer epitopes currently available, but it is growing and promises to overcome this gap. Using this experimental design, it will be important to unbiasedly map T cell phenotypes across food allergy and look for commonalities and contrasts to other allergic and infectious diseases. Once a pathological phenotype for T cells has been established, the frequencies of these cells can be monitored with simpler techniques that could be applied to the clinic and used in diagnosis, prediction of treatment responsiveness, and finding targets for new treatments.

Introduction

Food allergy is prevalent in developed countries (5–10% of the population) and incidence has risen in recent years^1–3. Food allergy is an atopic disorder resulting from an inappropriate immunological response to an otherwise harmless substance. Accidental ingestion of food allergens is the major cause of anaphylaxis in the U.S. where the most common food allergies are peanuts, milk, shellfish, tree nuts, egg, fin fish, wheat and soy^4–6. The gold standard diagnostic method for food allergy to date is double-blind placebo-controlled food challenge (DBPCFC), an oral food challenge (OFC) procedure which is potentially dangerous and must be performed by highly specialized professionals⁷. Other diagnostic methods include measurement of IgE antibodies and skin prick tests (SPT), which are not reliably predictive of clinical reactivity and may only reflect allergen sensitization⁸. The most common treatment plan is allergen avoidance, which comes with risk of accidental exposure. An oral immunotherapy (OIT) for peanut allergy was recently FDA approved; however, this promising treatment is limited to patients with milder forms of disease⁹. Thus, we need improved approaches to diagnose and treat food allergy, which will require a better understanding of the immunological mechanisms involved.

Food allergies are broadly characterized as IgE-mediated, non-IgE-mediated, or mixed IgE-and non-IgE-mediated¹. Antigen-specific T cells evidently play a role in both sensitization and tolerance to food allergens in both IgE and non-IgE mediated forms of disease¹⁰. During IgE-mediated food allergy, it has been shown that antigen-specific CD4+ T cells are skewed towards a T helper 2 (Th2) phenotype. These Th2 cells can produce cytokines (IL-4, IL5, IL-13) that lead to B cell production of IgE and additionally cause degranulation of mast cells, basophils, and eosinophils leading to allergy symptoms and in severe cases, anaphylaxis. Non-IgE mediated food allergy is less common, but the mechanisms are thought to be mediated by allergen-specific T cells^{11, 12}. The mechanisms behind the onset and downstream involvement of these T cell pathologies in any form of food allergy remain elusive. Understanding and characterizing the phenotypic markers of allergen specific T cells that contribute to pathology could create opportunities for the better diagnosis and treatment of food allergies. With this comes with a need for well-defined T cell epitopes for food antigens. While there are many other important contributors (i.e. ILCs^{13, 14}), this short review will focus on highlighting new technologies and recent advances in the study of T cells and T cell epitope discovery in food allergy and identifying areas for further research.

Single cell technologies and T cell epitope discovery in food allergy and OIT

The study of allergen-specific T cells is limited by the low frequencies of these cells in blood and lack of methods able to characterize them. The advent of a new experimental paradigm where antigen-specific cell selection assays^15–18 (Activation Induced Marker (AIM)/Antigen Reactive T cell Enrichment (ARTE)/MHC II tetramers/Cytokine secretion) are coupled with single cell genomic, epigenomic and immune repertoire sequencing has opened the door for new discoveries (Figure 1). These methods allow for the identification, isolation, and high-resolution characterization of low frequency T cell populations responsible for disease status based on their reactivity to well defined antigens. With this being said, the best molecularly defined antigens are specific peptide epitopes that can be reproducibly synthesized and delivered as individual peptides, peptide pools or as the payload of MHC-multimers. The benefit of using peptide epitopes over crude extracts when characterizing antigen specific T cell responses has been demonstrated in studies of infectious disease and non-food allergy, as it allows us to pull apart T cell populations reacting to different targets¹⁹. Complementing these well-established approaches with antigen-specific cell selection and downstream genomics applications allows for the generation of large datasets and a wealth of knowledge. Food allergens have fewer epitopes defined, but the field has been growing with 21 identified antigens and 496 T cell epitopes verified through a number of proliferation and cytokine production-based assays (Figure 2). The identification of more T cell epitopes for different food allergens will allow us to better characterize the profiles of antigen-specific T cells. Comparing antigen-specific T cell profiles with different specificities and further linking them to an individual’s allergic disease status will reveal commonalities and differences of T cell phenotypes in food allergies that can then be further compared to phenotypes from other allergic and infectious diseases.

Figure 1: Schematic representation of allergen-specific T cell identification/selection and downstream single cell sequencing applications.

Created using Biorender.com.

Figure 2: Identified T cell epitopes for food allergens.

Data obtained from the Immune Epitope Database (IEDB) https://www.iedb.org/ ⁵⁰. A) Table representing known antigens from food allergens, number of identified epitopes, number and type of assays performed to validate those epitopes and references. B) Assay type broken down into a pie chart.

Recent advances in studying T cells in food allergy and OIT

The combined availability of epitopes and advancements in assays have already led to the discovery of new T cell subsets in the context of food allergy (Table 1). Recent studies have identified new Th2 effector and follicular subtypes with potential functional consequences in the pathogenesis and severity of allergic disease^{59, 71, 73–75}. The identification and characterization of allergen-specific T regulatory populations for their roles in tolerance is also being explored^{76, 77}. Identifying these T cell subsets in different food allergies and tracking them longitudinally through disease exacerbations and tolerance development can help provide biomarkers for diagnosis, and targets for treatment.

Table 1:

Described T cell phenotypes in food allergy

T cell Subset	Protein Markers	Gene Expression Markers	Described Roles in Food Allergy
CD8+ T	CD8, IFNγ	CD8A	• Production of IFNγ protects against peanut-induced anaphylaxis51 • Increased allergen-specific CD8 T cells with peanut allergy52
Gamma-delta T	TCRgamma/delta, IL-9	TCRG, TCRD	• Conflicting studies in mice showing that activation leads to breakdown of tolerance, but blocking activation leads to increased food allergy53, 54 • Evidence that gamma-delta T cells can produce IL-955
iNKT	Va24, VB11		• Increased frequency during OIT for milk allergy56
Tfh	CXCR5, IL-21, BCL6, IL-4, IL-21	CXCR5	• Alternate source of IL-4, contribute to IgE production57
Tfh13	BCL6, GATA-3, IL-13hi, IL-4hi, IL-5hi, IL-21lo		• Tfh13 cells are important in production of high-affinity IgE and allergen-induced anaphylaxis58, 59
Th1	IFNγ, CXCR3, T-bet, TNFα, IL-2	IFNG, GZMB, IL26, SLC4A10, CXCR3, NFATC1, FASLG, XCL1, KLRG1	• Decreased ratio of Th1/Th2 known to be important in food allergy60
Th17	IL-17A/F, RORγT, IL-22, IL-6, IL-21	RORC, IL17A, IL17F, ZEB2, IL26, CCR6, IL22, CTSH, CCL20	• Defect in Th17 responses in food allergic individuals with lower IL-17 production61 • Skewing towards Th2/Th17 cytokine profiles with cashew allergy20
Th2	IL-4, IL-5, IL-13, IL-9, CCR4, CRTH2, GATA-3	IL-4, IL-5, IL-13, GATA3, IL4R, IL17RB, IL9, IL1RL1, ICOS	• Produce cytokines IL-4, IL-5,IL-13 that lead to downstream IgE production and mast cell/basophil/eosinophil degranulation • Heterogeneous population of peanut-specific Th2 cells with peanut allergy62 • During OIT, suppression of antigen-specific Th2 subsets is associated with better outcomes63
Th22	AHR, IL-22, CCR10, TNFα, Granzyme B, IL-13, IL-26		• Increased plasma IL-22 observed in infants with CMA64 • IL-22 is important for the development of atopic dermatitis in mice65
Th2A	CRTh2, CD49d, CD161	KLRB1, PTGDR2	• Pathogenic Th2 population in peanut allergy66
Th9	IL-9, PU.1		• Correlation between IL-9 and eosinophilia in infants with CMA64, 67 • Peanut-specific IL-9 production important in peanut allergy68, 69
Treg	IL-10, TGFβ, CD25, FOXP3, CD127	IL2RA, FOXP3, TIGIT, IKZF2	• Important for the development of tolerance70 • Several subsets have been identified71 • In mice, Th2-like reprogramming of Tregs72
peTh2	CRTH2, HPGDS, CD161, IL-5hi, IL-13hi, IL25, IL-33	IL2RA, TIGIT, IL5, IL13, HPGDS, GATA3, IL17RB, FFAR3, IL1RL1	• Primary Th2 subpopulation responsible for allergic eosinophilic inflammation73 • Home to the esophagus during EoE74

Aiming to induce similar mechanisms to natural tolerance, OITs for different food allergens are coming to the clinic, most notably for peanut allergy⁹. These treatments involve the introduction of increasing oral doses of the allergen in order to induce desensitization and protect against accidental ingestion⁷⁸. Recent studies on OIT have identified changes in the transcriptional and phenotypic signatures of antigen-specific T cells over the course of OIT. They suggest that successful OIT alters the phenotype of Th2 cells, shifting them towards a more anergic or regulatory state²⁸. Further, food antigen specific T cells at baseline can be informative in predicting responsiveness to the treatment^{63, 79, 80}. One proposed mechanism has been an increase in allergen-specific IgG4 antibodies that block the inflammatory response cascade, and which are increasingly produced after Treg-mediated tolerogenic environment^{81, 82}. As OIT treatments become available for more food allergies, studies examining allergen-specific T cell populations (Th2, Treg) over the course of these treatments can improve our understanding of T cell driven immunological mechanisms behind food allergy as well as to predict treatment outcomes.

Advancements in single cell TCR sequencing in combination with transcriptome analysis provide exciting opportunities for identification and characterization of the TCR repertoires of allergen-specific T cells. A recent study showed that Th subsets were clonally distinct but converged with peanut allergy, suggesting that antigen interactions skew TCR sequences⁶³. More studies and analysis tools are needed, but this field opens the door to our understanding of T cell epitope interactions and better characterization of antigen-specific cells.

Concluding remarks and directions for future studies

In this short review, we have highlighted recent discoveries on the topic of T cells in food allergy and pointed to gaps in knowledge. New assay techniques that have been pioneered in the infectious disease setting are paving the way to identifying food allergen-specific T cells and characterizing their phenotype. This is facilitated by an increase in the availability of clearly molecularly defined T cell epitopes in food allergens, which allow to find these antigen-specific cells. Combining these methods with single cell sequencing technologies will allow us to characterize the phenotype and immune repertoire of these disease-associated cell populations and identify pathological phenotypes for T cells in food allergy. These cells can then be monitored using simpler techniques in the clinic for diagnosis as well as tracked longitudinally through natural tolerance and OIT to be used as both predictive marks of treatment responsiveness as well as markers of desensitization, and potentially provide targets for new treatment approaches.

ABBREVIATIONS:

DBPCFC

Double-blind placebo-controlled food challenge

OFC

Oral Food Challenge

SPT

Skin prick test

OIT

Oral immunotherapy

AIM

Activation Induced Marker

MHC

Major Histocompatibility Complex

Th2

T Helper 2

TCR

T Cell Receptor

ILC

Innate Lymphoid Cells