Novel Reagents for the Diagnosis and Monitoring of Food Allergies and Methods of Use Thereof

Cross-Reference to Related Application

This application claims priority of US Provisional application number 63/384,030 filed November 16, 2022, the entire contents being incorporated herein by reference as though set forth in full.

Statement Regarding Government Funding

This invention was made with government support under Grant number K08DK116668 awarded by the National Institutes of Health. The government has certain rights in the invention.

Incorporation-by- Reference of Material Submitted in Electronic Form

The Contents of the electronic sequence listing (CHOP-143-PCT.xml; Size: 45,049 bytes; and Date of Creation: November 16, 2023) is herein incorporated by reference in its entirety.

Field of the Invention

The present invention relates to the fields of allergy testing and monitoring. More specifically, the invention provides minimally invasive methods for identifying food allergies by culturing PBMCs with synthetic allergens and, or, allergenic peptides (antigens) and analyzing levels of T cell activation.

Background of the Invention

Several publications and patent documents are cited throughout the specification in order to describe the state of the art to which this invention pertains. Each of these citations is incorporated by reference herein as though set forth in full.

Eosinophilic esophagitis (EoE) is a chronic, T-cell-mediated disease that is caused by specific foods and results in inflammation, odynophagia, and progressive esophageal dysfunction. Due to variable contribution of food-specific immunoglobulin E (IgE) to EoE immunopathology, and limited correlation between allergen patch, skin-prick, and serum allergen- specific IgE testing and clinical EoE food allergy, existing testing modalities are of low i utility when attempting to identify EoE-causal foods. As such, current clinical management consists of proton-pump inhibitor therapy, empiric elimination diets, biologies, and/or swallowed steroids with recurrent endoscopies to assess clinical response. Due to the considerable morbidity and time to inflammation resolution associated with current management practices, there is critical need for the development of minimally invasive assays to aid in the identification of EoE-causal foods.

In addition to clinical features including inflammation resolution with food avoidance ^{8 l 0} , there is ample experimental evidence that EoE is an antigen-driven process ¹¹ . Mouse models show correlations between antigen- specific T helper type 2 (Tn2) cell responses and esophageal eosinophilia ^{12 l4} . Esophageal eosinophilia in these models is dependent on CD4+ TH cells, but not CD8+ T cells, B cells, or IgE ^14,15 . In human studies, activated TH cells have been detected in the circulation of EoE subjects after polyclonal or allergen- specific stimulation ^7,16,17 , and in the esophagus during active disease ¹⁸ . We recently identified allergen- specific memory TH cells in the circulation of patients with EoE.

Additional evidence for antigen-driven inflammation in EoE comes from studies of foodspecific IgG4 levels. For example, adults with EoE have higher total and food-specific IgG4 levels in the serum and esophagus ¹⁹ , while children with EoE have higher food-specific IgG4 levels in the serum ²⁰ , as compared with healthy controls. In adults this phenomenon seems to be closely linked to consumption of the allergenic food, and as a result, EoE activity ^21,22 . It is not known whether a similar relationship exists in children. Taken together, these studies highlight the relevance of antigen-driven inflammation to EoE pathogenesis.

The ability to detect allergen- specific markers of immune activation in EoE raises the potential for the development of minimally invasive assays to aid in the identification of EoE- causal foods. Such assays would allow for the institution of directed elimination diets, and minimize the number of required endoscopies and time to inflammation resolution ⁷ . In addition, there is emerging evidence that EoE can resolve (clinical remission) in some children with prolonged food avoidance ²³ . As a result, there is also need for assays that can determine the presence of food-specific immune responses when an individual is avoiding known EoE-causal food(s). Such an assay would be valuable in determining if and when an allergenic food can be reintroduced into a patient’s diet (akin to skin prick testing in IgE-mediated food allergy). As bovine milk is the most common cause of EoE in children and adults ¹¹ , we sought to identify peripheral markers of milk-specific immune memory that are independent of allergenic food consumption, and arc indicative of clinical EoE milk allergy.

The ability to detect allergen- specific markers of immune activation in EoE raises the potential for the development of minimally invasive assays to aid in the identification of EoE- causal foods. Such assays would allow for the institution of directed elimination diets and minimize the number of required endoscopies and time to inflammation resolution.

There is also need for assays that can determine the presence of food-specific immune responses when an individual is avoiding known EoE-causal food(s). Such an assay would be valuable in determining whether and when an allergenic food can be reintroduced into a patient's diet (akin to skin prick testing in IgE-mediated food allergy).

Summary of the Invention

In accordance with the present invention, a method of detecting a food specific allergy in biological sample obtained from a patient is provided. An exemplary method comprises culturing the sample in the presence of one or more endotoxin-depleted food proteins or immunogenically active fragment thereof, measuring levels of allergy specific memory TH cells produced, and comparing the levels of allergy specific memory TH cells to an untreated control, wherein an increase in the level of allergy specific memory TH cells in the sample relative to the level of the allergy specific memory TH cells in the untreated control indicates the presence of a food specific allergy in said patient.

In one embodiment, the sample is selected from whole blood or a peripheral blood sample and elevated TH cell proliferation and cytokine production is indicative of clinical EoE allergy. In another embodiment, peripheral blood mononuclear cells (PBMCs) are isolated prior to the culturing of step a. In certain aspects the PBMCs are isolated by Ficoll gradient. In certain embodiments, the endotoxin-depleted food protein is selected from one or more of a milk protein, soybean protein, wheat protein, egg protein, or peanut protein or an immunologically active fragment thereof. In certain embodiments the endotoxin-depleted food protein is selected from one or more proteins in Table 1.

In another aspect of the invention, methods of detecting a milk-based food allergy in a biological sample obtained from a patient is provided. An exemplary method comprises culturing the sample in the presence of one or more milk based food proteins or immunogenically active fragment thereof, measuring levels of allergy specific memory TH cells produced, and comparing the levels of allergy specific memory TH cells to an untreated control, wherein an increase in the level of allergy specific memory TH cells in the sample relative to the level of the allergy specific memory TH cells in the untreated control indicates the presence of a milk-based food allergy in said patient.

In yet another aspect of the invention, methods of detecting a soy- based food allergy in a biological sample obtained from a patient is provided. An exemplary method comprises culturing the sample in the presence of one or more soy based food proteins or immunogenically active fragment thereof, measuring levels of allergy specific memory TH cells produced, and comparing the levels of allergy specific memory TH cells to an untreated control, wherein an increase in the level of allergy specific memory TH cells in the sample relative to the level of the allergy specific memory TH cells in the untreated control indicates the presence of a soy-based food allergy in said patient.

In one embodiment, the sample in the above methods is selected from whole blood or a peripheral blood sample and elevated TH cell proliferation and cytokine production is indicative of clinical EoE allergy. In another embodiment, peripheral blood mononuclear cells (PBMCs) are isolated prior to the culturing of step a. In certain aspects the PBMCs are isolated by Ficoll gradient.

In preferred embodiments of the above methods, the endotoxin-depleted food protein is a recombinant protein or an immunologically active fragment thereof. As noted above, the sample can be cultured in the presence of at least one additional endotoxin-depleted food protein.

The method also comprises measurement of one or more specific allergy specific memory TH cells. In yet another aspect, the method can comprise determining activation of other T cell populations including, but not limited to, T _ieg cells or CD8+ T cells.

A kit comprising reagents for practicing any of the aforementioned methods is also disclosed.

Brief Description of the Drawings

Fig. 1: Graphical representation of Example I. Example I shows that milk- activated memory TH2 cells circulate in the blood of milk- allergic EoE patients. Measuring the proliferation and cytokine production of these cells predicts clinical EoE allergy to milk with high sensitivity, high specificity, and minimal risk to the patient. Abbreviation: EoE, eosinophilic esophagitis Fig. 2A-2B: Reduction in human TLR4 signaling and T cell proliferation after milk protein endotoxin removal. (Fig. 2A) Human TLR4 (hTLR4) receptor signaling by milk proteins was measured before (pre) and after (post) endotoxin removal, and compared to LPS-mediated signaling. N=6 (technical replicates). Mean ±SEM shown. Statistics by t-test. *, P<0.05; **, P<0.01. (Fig. 2B) Flow cytometry of CFSE-labeled peripheral memory T cells from a control subject after culture in the absence (Unstim) or presence of tetanus toxoid (Tetanus) or milk proteins before (Pre) or after (Post) endotoxin depletion. Gated on live, CD8’, CD 19", CD3 ⁺ , CD4 ⁺ , CD45RA", CD45RO ⁺ . Representative of >3 experiments.

Fig. 3: Representative CD4+ memory T lymphocyte gating strategy. Memory TH cells were gated as follows: live, CD8‘, CD 19", CD3 ⁺ , CD4 ⁺ , CD45RA", CD45ROF

Fig. 4: Inclusion cohort. Patient samples with a difference between the % Pl tetanus toxoid stimulated (Tetanus) and % Pl unstimulated (Unstim) > 2 were included in subsequent ratio and ROC analyses.

Fig. 5A-5D: Plasma total and milk-specific IgG4 levels show non-significant correlations with clinical EoE milk allergy. (Fig. 5A) Total plasma IgG4 levels in control or clinical EoE milk allergy subjects. (Fig. 5B) Milk- specific plasma IgG4 levels in control or clinical EoE milk allergy subjects. (Fig. 5C) Ratio of milk-specific to total plasma IgG4 levels in control or clinical EoE milk allergy subjects. Clinical EoE milk allergy subjects consuming milk at time of assay in red. (Fig. 5D) Receiver operating characteristic (ROC) curve of the milk-specific to total IgG4 ratio for the clinical EoE milk allergy outcome. N=17. Mean ±SEM shown. Statistics by t-test. ns, not significant.

Fig. 6A-6D: Examination of the effect of active milk consumption on assay outcomes. Associations between assay outcomes including (Fig. 6A) Specific to Total IgG4 Ratio, (Fig. 6B) Milk to Tetanus Pl Ratio, (Fig. 6C) IgG4 x CFSE measure, and (Fig. 6D) %IL-4 ⁺ T Cells, and milk consumption at the time of assay, were determined by Pearson’s correlation. Fig. 7A-7G: Allergen-specific immune responses in subjects with IgE-mediated food allergy to milk. (Fig. 7A) Total IgG4 levels, (Fig. 7B) milk-specific IgG4 levels, and (Fig. 7C) ratio of milk-specific to total IgG4 levels in plasma of subjects with IgE-mediated food allergy to milk. (Fig. 7D) Flow cytometry of CFSE-labeled peripheral memory T cells from subjects with IgE- mediated food allergy to milk after culture in the absence (Unstim) or presence of tetanus toxoid (Tetanus) or milk proteins (Milk). (Fig. 7E) Number of CFSE dim (Pl) peripheral memory T cells after culture in the absence (Unstim) or presence of tetanus toxoid (Tetanus) or milk proteins (Milk). (Fig. 7F) Flow cytometry of intracellular IL-4 expression in PBMCs from subjects with IgE-mediated food allergy to milk after culture in the absence (Unstim) or presence of tetanus toxoid (Tetanus) or milk proteins (Milk). Gated on live, CD8’, CD 19", CD3 ⁺ , CD4 ⁺ , CD45RA’, CD45RO ⁺ . (Fig. 7G) Number of IL-4+ memory TH2 cells after culture in the absence (Unstim) or presence of tetanus toxoid (Tetanus) or milk proteins (Milk). Gated on live, CD8’, CD 19’, CD3 ⁺ , CD4 ⁺ , CD45RA’, CD45RO ⁺ . N=7-9. Mean ±SEM shown. Statistics by t-test. * indicate comparisons between treatment arms of a single experimental group. *, P<0.05; **, P<0.01.

Fig. 8A-8B: Presence of milk-reactive memory T cells in subjects with clinical EoE milk allergy. (Fig. 8A) Flow cytometry of CFSE-labeled peripheral memory T cells from control or clinical EoE milk allergy subjects after culture in the absence (Unstim) or presence of tetanus toxoid (Tetanus) or milk proteins (Milk). Gated on live, CD8’, CD19’, CD3 ⁺ , CD4 ⁺ , CD45RA’, CD45RO ⁺ . (Fig. 8B) Number of CFSE dim (Pl) peripheral memory T cells after culture in the absence (Unstim) or presence of tetanus toxoid (Tetanus) or milk proteins (Milk). N= 14-17. Mean +SEM shown. Statistics by t-test. * indicate comparisons between treatment arms of a single experimental group, # indicate comparisons between experimental groups of a single treatment arm. **. P<0.01; *«, P<0.001, ****. P<0.0001; #, P<0.05; ####, P<0.0001.

Fig. 9A-9C: Proliferation of milk- reactive memory T cells predicts clinical EoE milk allergy. (Fig. 9A) Variability among T cell proliferative responses. (Fig. 9B) Ratio of the percentage of Pl cells in cultures of milk-expanded as compared with tetanus-expanded PBMCs from control or clinical EoE milk allergy subjects. Clinical EoE milk allergy subjects consuming milk at time of assay in red. (Fig. 9C) Receiver operating characteristic (ROC) curve of the %P1 milk-stimulated to tetanus-stimulated ratio for the clinical EoE milk allergy outcome. N=17. Mean ±SEM shown. Statistics by t-tcst. ####, P<0.0001.

Fig. 10A-10C: Examination of milk-specific IgG4 and milk-reactive memory T cell proliferation improves identification of clinical EoE milk allergy. (Fig. 10A) Ratio of milkspecific to total plasma IgG4 levels in control or clinical EoE milk allergy subjects multiplied by the ratio of the percentage of Pl cells in cultures of milk-expanded as compared with tetanus- expanded PBMCs from control or clinical EoE milk allergy subjects (IgG4 x CFSE). Clinical EoE milk allergy subjects consuming milk at time of assay in red. (Fig. 10B) Receiver operating characteristic (ROC) curve of the IgG4 x CFSE measure for the clinical EoE milk allergy outcome. (Fig. IOC) Comparison of ROC curves of the IgG4, CFSE, and IgG4 x CFSE measures. N=17. Mean +SEM shown. Statistics by t-test. #, P<0.05.

Fig. 11A-11C: Milk-reactive memory TH2 cells are present in the circulation of children with clinical EoE milk allergy. (Fig. 11A) Flow cytometry of intracellular IL-4 expression in PBMCs from control or clinical EoE milk allergy subjects after culture in the absence (Unstim) or presence of tetanus toxoid (Tetanus) or milk proteins (Milk). Gated on live, CD8‘, CD 19", CD3 ⁺ , CD4 ⁺ , CD45RA’, CD45RO ⁺ . (Fig. 11B) Number of IL-4+ memory TH2 cells after culture in the absence (Unstim) or presence of tetanus toxoid (Tetanus) or milk proteins (Milk). (Fig. 11C) IL-4, IL-5, and IL-13 amounts in culture superannuates of PBMCs from control or clinical EoE milk allergy subjects after culture in the absence (Unstim) or presence of milk proteins (Milk). N=7-8. Mean ±SEM shown. Statistics by t-test. * indicate comparisons between treatment arms of a single experimental group, # indicate comparisons between experimental groups of a single treatment arm. *, P<0.05; **, P<0.001; ****, P<0.0001; #, P<0.05; ##, P<0.01 ; ####, P<0.0001.

Fig. 12A-12B: Examination of milk-reactive memory TH2 cells predicts clinical EoE milk allergy. (Fig. 12A) %IL-4 ⁺ CD45RO ⁺ TH2 cells from control or clinical EoE milk allergy subjects after stimulation with milk proteins. Clinical EoE milk allergy subjects consuming milk at time of assay in red. (Fig. 12B) Receiver operating characteristic (ROC) curve of the %IL4 ⁺ of the CD45RO ⁺ population for the clinical EoE milk allergy outcome. N=8-9. Mean +SEM shown. Statistics by t-tcst. ####, P<0.0001.

Fig. 13A-13B: Evaluation of Milk-Activated T cell blood test. (Fig. 13A) Performance characteristics of the milk-activated T cell blood test in all subjects (both EoE and non-EoE) of prospective cohort of children undergoing clinically-indicated endoscopy. (Fig. 13B) Performance characteristics of the milk-activated T cell blood test in the subset of subjects with EoE who had a determination of milk tolerance or milk allergy.

FIG. 14: Evaluation of Soy-Activated T cell blood test.

Detailed Description of the Invention

Existing allergy testing modalities are not helpful when attempting to identify EoE-causal foods necessitating empiric food elimination and recurrent endoscopy. The goal of this invention is to identify and compare allergen- specific immune features that can be assayed in a minimally invasive manner to predict clinical food allergy in EoE.

Described herein, we obtained blood samples from control subjects (n=17), subjects with clinical EoE milk allergy (n=17), and subjects with IgE-mediated milk allergy (n=9). We measured total and milk- specific plasma IgG4 levels and peripheral memory CD4+ T helper (TH) cell proliferation and cytokine production after stimulation with endotoxin-depleted milk proteins. Sensitivity and specificity for predicting clinical EoE milk allergy was calculated and compared between approaches.

Total and milk-specific IgG4 levels were not significantly different between control subjects and subjects with clinical EoE milk allergy. Stimulation with milk proteins caused TH lymphocytes from subjects with clinical EoE milk allergy to proliferate more (%P1 of 38.3+4.6 vs 12.7+2.8, P<0.0001), and produce more type 2 cytokines (%IL-4+ of 33.7+2.8 vs 6.9+1.6, P<0.0001), than cells from control subjects. Milk-dependent memory TH cell proliferation (sensitivity and specificity of 88 and 82%, respectively) and IL-4 production (sensitivity and specificity of 100%) most strongly predicted clinical EoE milk allergy.

Peripheral markers of allergen- specific immune activation may be useful in identifying EoE-causal foods. Assaying milk-dependent IL-4 production by circulating memory TH lymphocytes most accurately predicts clinical EoE milk allergy. Accordingly, the invention provides synthetic sequences encoding immunogenic epitopes and methods of use thereof in assays for identifying and controlling allergen- specific immune activation in patients in need.

Definitions:

Unless otherwise defined herein, scientific and technical terms used in connection with the present application shall have the meanings that are commonly understood by those of ordinary skill in the art. In addition to definitions included in this sub-section, further definitions of terms are interspersed throughout the text.

In this invention, “a“ or “an“ means “at least one“ or “one or more,“ etc., unless clearly indicated otherwise by context. The term “or“ means “and/or“ unless stated otherwise. In the case of a multiple-dependent claim, however, use of the term “or“ refers back to more than one preceding claim in the alternative only.

Furthermore, a compound "selected from the group consisting of" refers to one or more of the compounds in the list that follows, including mixtures (i.e. combinations) of two or more of the compounds. According to the present invention, an isolated, or biologically pure molecule is a compound that has been removed from its natural milieu. As such, "isolated" and "biologically pure" do not necessarily reflect the extent to which the compound has been purified. An isolated compound of the present invention can be obtained from its natural source, can be produced using laboratory synthetic techniques or can be produced by any such chemical synthetic route.

The phrase "consisting essentially of" when referring to a particular nucleotide or amino acid means a sequence having the properties of a given SEQ ID NO. For example, when used in reference to an amino acid sequence, the phrase includes the sequence per se and molecular modifications that would not affect the functional and novel characteristics of the sequence.

The terms “subject,” “individual,” “host,” “donor,” and “patient” are used interchangeably herein to refer to a vertebrate, for example, a mammal. Mammals include, but are not limited to, murine, simians, humans, farm animals, sport animals, and pets. Tissues, cells, and their progeny of a biological entity obtained in vivo or cultured in vitro are also encompassed. Designation as a “subject,” “individual,” “host,” “donor,” or “patient” does not necessarily entail supervision of a medical professional. “Sample” or “patient sample” or “biological sample” generally refers to a sample which may be tested for a particular molecule, preferably an allergen, such as a those described hereinbelow. Samples may include but arc not limited to cells, body fluids, including blood, serum, plasma, cerebral spinal fluid, urine, saliva, tears, pleural fluid and the like. In certain embodiments, the sample contains peripheral blood mononuclear cells (PBMCs). In certain embodiments, the PBMCs are isolated prior to culturing.

Culture

In certain embodiments, the sample is cultured in the presence of an allergen. The culture medium is suitable for survival of the cells in the sample. The culture medium contains specific components necessary to for the survival of the cell. These components may include, without limitation, growth factors, a carbon source, amino acids, vitamins, a pH buffer and ion buffer. In certain embodiments, the culture is maintained in conditions necessary for survival of the cells. Said conditions include, without limitation, temperature regulation, humidity regulation, and O2 level regulation. In certain embodiments, the medium can be changed at certain periods. The compositions and methods for producing said medium are known by those skilled in the art. In certain embodiments, the culture is grown in suspension or adherent culture.

In certain embodiments, the culture is endotoxin free. The term “endotoxin free” or “substantially endotoxin free” relates generally to compositions, solvents, and/or vessels that contain at most trace amounts (e.g., amounts having no clinically adverse physiological effects to a subject) of endotoxin, and preferably undetectable amounts of endotoxin. Endotoxins are toxins associated with certain micro-organisms, such as bacteria, typically gram-negative bacteria, although endotoxins may be found in gram-positive bacteria, such as Listeria monocytogenes. The most prevalent endotoxins are lipopolysaccharides (LPS) or lipo-oligosaccharides (LOS) found in the outer membrane of various Gram-negative bacteria, and which represent a central pathogenic feature in the ability of these bacteria to cause disease. Small amounts of endotoxin in humans may produce fever, a lowering of the blood pressure, and activation of inflammation and coagulation, among other adverse physiological effects.

Allergens In certain embodiments, the sample is cultured with at least one allergen. The term “allergen,” as used herein, includes any substance, chemical, particle or composition that is capable of stimulating an allergic response in a susceptible individual. The allergen may be naturally occurring or a recombinantly produced protein or substance. “Food specific allergens” may be contained within or derived from a food item such as, e.g., dairy products (e.g., cow's milk), egg, celery, sesame, wheat, soy, fish, shellfish, sugars (e.g., sugars present on meat such as alpha-galactose), peanuts, other legumes (e.g., beans, peas, soybeans, etc.), and tree nuts.

Alternatively, an allergen may be contained within or derived from a non-food item such as, e.g., dust (e.g., containing dust mite), pollen, insect venom (e.g., venom of bees, wasps, mosquitos, fire ants, etc.), mold, animal fur, animal dander, wool, latex, metals (e.g., nickel), household cleaners, detergents, medication, cosmetics (e.g., perfumes, etc.), drugs (e.g., penicillin, sulfonamides, salicylate, etc.), therapeutic monoclonal antibodies (e.g., cetuximab), ragweed, grass and birch. Exemplary pollen allergens include, e.g., tree pollens such as birch pollen, cedar pollen, oak pollen, alder pollen, hornbeam pollen, aesculus pollen, willow pollen, poplar pollen, plantanus pollen, tilia pollen, olea pollen, Ashe juniper pollen, and Alstonia scholaris pollen.

In certain embodiments, the sample is cultured in the presence of the complete allergen (e.g., in the presence of cow’s milk). In certain embodiments, the culture contains at least one isolated protein or polypeptide from the allergen that is capable of stimulating an allergic response (antigen) in a susceptible individual. In certain embodiments, multiple antigens may be synthetized in tandem to create a polypeptide that is not present in nature but has a maximal capacity to stimulate an allergic response. In certain embodiments, the allergen source (e.g. cow’s milk) has multiple EoE antigens spread across multiple proteins. In certain embodiments, the isolated protein or peptide is a recombinant protein that contains one or more antigens or immunogenic epitopes from each of the protein from a specific source. In certain embodiments, the recombinant peptide contains spacer amino acids separating these antigens.

In certain embodiments, the protein is an endotoxin-depleted protein. The phrase “endotoxin-depleted protein” refers to proteins which contain at most trace amounts (e.g., amounts having no clinically adverse physiological effects to a subject) of an endotoxin protein, and preferably undetectable amounts of an endotoxin protein such that its presence would not cause a reaction in a patient. In certain embodiments of the invention, the allergen is an endotoxin-depleted food protein.

The term “polypeptide” is used herein to refer to polymers of amino acids of any length. The polymer may be linear or branched, it may comprise modified amino acids, and it may be interrupted by non-amino acids. The term also encompasses an amino acid polymer that has been modified naturally or by intervention; for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, sulfation or any other manipulation or modification, such as conjugation with a labeling or half-life extending component. Also included within the term are, for example, polypeptides containing one or more analogs of an amino acid (including, for example, unnatural amino acids, etc.), as well as other modifications known in the art. The polypeptide may be in the form of a multimer of individual polypeptides.

The polypeptide may also be a variant of naturally-occurring polypeptide, for example a variant of one of the polypeptides described in Table 1. A variant may comprise fragments of said naturally-occurring polypeptide. Preferably, the fragments contain immunogenic epitopes.

In certain embodiments, the polypeptide may be fused to a heterologous amino acid sequence to facilitate production and separation of the polypeptide prior to contact with the T cells. Said heterologous amino acid sequence can comprise or consist of a polypeptide selected from the group consisting of immunoglobulin constant regions and portions thereof, e.g. the Fc fragment, transferrin and fragments thereof, the C-terminal peptide of human chorionic gonadotropin, solvated random chains with large hydrodynamic volume known as XTEN, homoamino acid repeats (HAP), proline-alanine- serine repeats (PAS), albumin, afamin, alphafetoprotein, Vitamin D binding protein, polypeptides capable of binding under physiological conditions to albumin or immunoglobulin constant regions, and combinations thereof. According to a preferred embodiment the polypeptide is fused to albumin or an Fc fragment, in particular to albumin. In preferred embodiments, the heterologous polypeptide is removed from the antigen containing peptide prior to contact with the T cells to avoid stimulating an unwanted immune response.

The polypeptide may alternatively or in addition be conjugated to a further moiety. Said moiety is selected from the group consisting of hydroxyethyl starch (HES), polyethylene glycol (PEG), polysialic acids (PSAs), elastin-like polypeptides, heparosan polymers, hyaluronic acid and albumin binding ligands, e.g. fatty acid chains or albumin binding peptides, and combinations thereof.

While not limiting to the inventive subject matter, endotoxin-depleted food proteins will typically be drawn from foods generally known or suspected to trigger signs or symptoms of EoE. Particularly suitable proteins may be identified by the experimental procedures outlined below. Thus, it should be appreciated that the proteins need not be limited to the items described herein, but that all items are contemplated that can be identified by the methods presented herein.

Therefore, exemplary endotoxin-depleted food proteins include proteins from at least one, two, at least three, at least four, or at least five of the foods identified in Table 1. The endotoxin-depleted food proteins can be anyone, or any combination, of the sequences listed in Table 1. The term “recombinant polypeptide” or “recombinant protein” is used herein to refer to a polypeptide that has been rccombinantly expressed. In particular, the polypeptide has been obtained from a transgenic organism genetically-engineered to express the polypeptide, or from a cell line recombinantly producing the polypeptide. Non-limiting examples of an organism include birds and mammals, such as, e.g. mice, rats, goats, sheep, horses, donkeys, cows, primates and humans. Non-limiting examples of a transgenic organism include organisms that have been genetically-engineered to express the polypeptide. A polypeptide from a transgenic organism may be obtained from a biological fluid, tissue or organ extract, or other source from an organism using routine methods known in the art. More typically, however, a prokaryote and/or eukaryotic expression system is used to recombinantly express the polypeptide. Expression systems can include any of a variety of characteristics including, without limitation, inducible expression, non-inducible expression, constitutive expression, tissue-specific expression, cell-specific expression, viral-mediated expression, stably-integrated expression, and transient expression. How to make and use such expression systems are known in the art.

CD4 T cells recognize antigenic peptides presented via MHC II (HLA-DR, -DP, -DQ) molecules to induce responses in peripheral tissues. Using computational (in silico) and experimental approaches it is possible to predict and determine the antigenic epitopes of allergenic food proteins based on established characteristics including but not limited to 13-25 amino acids in length, more surface exposed, less secondary structure elements, more coil content, low glycosylation, and high predicted affinity for MHC II (Jprgensen KW, Buus S, Nielsen M. Structural properties of MHC class II ligands, implications for the prediction of MHC class II epitopes. PLoS One. 2010 Dec 30;5(12):el5877. doi: 10.1371/joumal.pone.0015877. PMID: 21209859; PMCID: PMC3012731.). Accordingly, also contemplated herein is the synthesis of polypeptides that combine multiple antigenic epitopes spanning common food allergens (Table 1) from a single food to create an artificial reagent which maximizes both immune reactivity and antigenic diversity thereby facilitating use in the broadest population of patients. Using the sequences above, the skilled artisan would be capable of determining the antigenic epitopes for each antigen. (See, e.g. Schaap-Johansen A-L, Vujovic M, Borch A, Hadrup SR and Marcatili P (2021) T Cell Epitope Prediction and Its Application to Immunotherapy. Front. Immunol. 12:712488. doi: 10.3389/fimmu.2021.712488 and Jose L. Sanchez-Trincado, Marta Gomez-Perosanz, Pedro A. Reche, "Fundamentals and Methods for T- and B-Cell Epitope Prediction", Journal of Immunology Research, vol. 2017, Article ID 2680160, 14 pages, 2017. https://doi.org/10.1155/2017/2680160)

TH cells

The terms “memory CD4 ⁺ T helper cells”, “memory TH cells”, and “TH cells” refer to the set of T cells produced during a primary immunogenic challenge that persist and are capable of generating a recall response to secondary challenge. Upon exposure to a specific antigen or allergen, T cells specific to the antigen or allergen are activated, proliferate and differentiate into effector cells. The increased number of antigen specific cells with effector functions can act to clear the antigen, but then, however, the vast majority of these cells die. The surviving cells are memory TH cells. These cells can provide protection or an enhanced response upon re-exposure to the same allergen or antigen. After exposure, TH cells are present in higher frequencies than the original T cells. This higher frequency of TH cells increases the likelihood that any re- introduction of the allergen or antigen will be detected quickly, allowing the immune response to get underway before allergen or antigen has time to spread. In composition, TH cells can be distinguished from naive cells, and from each other, by the number and type of cell surface molecules. These alterations are often used to define memory cells

In certain embodiments, the TH cells of the present invention are specific to a particular allergen, food or protein. Nonlimiting examples of allergen specific memory TH cells include milk-specific TH cells, a-casein- specific TH cells, and SEQ ID NO: 1-specific TH cells. Allergenspecific TH cell populations can be identified by the presence/absence of specific cell surface markers.

In certain embodiments, T cells with similar antigen specificity have the capacity to differentiate into distinct functional subsets and may be used instead of TH cells. We have shown that milk-activated CD4+ T cells can express molecules associated with the TH2 subset (e.g. IL- 4). This indicates that milk-specific T cells with the same or similar antigen specificity can adopt other functional states including THI, THI7. and T regulatory status depending on disease state and context. As such, an assay for CD4+ T cell expression of cytokines and transcription factors characteristic of other CD4+ T cell functional states including but not limited to FoxP3, Gata3, IFNy, IL-2. IL- 10, IL- 17, IL-21, IL-22, T-bet, TGF£, TNFa is contemplated herein. Whether a cell or cell population is positive for a particular cell surface marker can he determined by flow cytometry using staining with a specific antibody for the surface marker and an isotype matched control antibody. A cell population negative for a marker refers to the absence of significant staining of the cell population with the specific antibody above the isotype control, positive refers to uniform staining of the cell population above the isotype control. In some alternatives, a decrease in expression of one or markers refers to loss of 1 log 10 in the mean fluorescence intensity and/or decrease of percentage of cells that exhibit the marker of at least 20% of the cells, 25% of-the cells, 30% of the cells, 35% of the cells, 40% of the cells, 45% of the cells, 50% of the cells, 55% of the cells, 60% of the cells, 65% of the cells, 70% of the cells, 75% of the cells, 80% of the cells, 85% of the cells, 90% of the cell, 95% of the cells, and 100% of the cells or any % between 20 and 100% when compared to a reference cell population. In some alternatives, a cell population positive for one or markers refers to a percentage of cells that exhibit the marker of at least 50% of the cells, 55% of the cells, 60% of the cells, 65% of the cells, 70% of the cells, 75% of the cells, 80% of the cells, 85% of the cells, 90% of the cell, 95% of the cells, or 100% of the cells or any % between 50 and 100% when compared to a reference cell population.

Diagnostic and Monitoring Methods

Provided herein are methods for diagnosing patient with an allergy. In certain embodiments, a sample is taken from the patient and cultured in the presence of an allergen. In certain embodiments, the sample is cultured for at least 1-10 days. In certain embodiments, the sample is cultured for at least 6 days. After the sample is cultured, the levels of an allergen specific memory TH cell. The levels of allergy specific memory TH cells are then compared to the levels of the same TH cells in an untreated control. In certain embodiments, the level of allergy specific memory TH cells in the sample is higher than the level of the allergy specific memory TH cells in the untreated control. In certain embodiments, the sample is cultured in the presence of multiple allergens and the levels of multiple allergy specific memory Tn cells are analyzed.

In certain embodiments, the patient is treated for an allergy after diagnosis. Allergy treatments are known by those skilled in the art. For example, treatments may involve diet changes, medications, or medical procedures. In certain embodiments, the diet changes involve eliminating the foods containing the allergen from the patient’s diet. In certain embodiments, the diet changes involve an element diet. This approach involves avoiding any food or drinks that contain proteins and drinking a liquid containing amino acids. In certain embodiments, the treatment involves taking at least one medication selected from proton pump inhibitors, topical corticosteroids and biologic medications. In certain embodiments, treatment may include Esophageal Endoscopy, Biopsy, or Dilation.

Also provided herein are methods for monitoring a patient with a previously diagnosed allergy. In certain embodiments, a sample is taken from the patient and cultured in the presence of an allergen. In certain embodiments, the sample is cultured for at least 1-10 days. In certain embodiments, the sample is cultured for at least 6 days. After the sample is cultured, the levels of an allergen specific memory TH cell. The levels of allergy specific memory TH cells are then compared to the levels of the same TH cells in an untreated control. In certain embodiments, this test is repeated regularly. This test may be performed once every day, every week, every two weeks, every month, every two months, every 6 months, or every year. Once the level of allergen specific memory TH cells in the sample is less than or equal to the level of the allergy specific memory TH cells in the untreated control, the patient’s allergy has resolved. In certain embodiments, the patient stops being treated and stops being monitored after the patient’s allergy has resolved.

Kits and Articles of Manufacture

The invention provides kits for performing the aforementioned methods. In particular embodiments, the kit comprises a sterile container which contains a culture comprising the allergen- specific proteins or fragments thereof; such containers can be boxes, ampoules, bottles, vials, tubes, bags, pouches, blister-packs, or other suitable container forms known in the art. Such containers can be made of plastic, glass, laminated paper, metal foil, or other materials suitable for holding the culture. The kit may also contain a device for obtaining a sample from the patient. The kit may also contain the TH cells described above and instructions for methods of use.

The kit preferably contains instructions that generally include information about the use of the kit for performing the aforementioned methods. The kit further contains precautions; warnings; indications; counter-indications; overdosage information; adverse reactions; animal pharmacology; clinical studies; and/or references. The instructions may be printed directly on the container (when present), or as a label applied to the container, or as a separate sheet, pamphlet, card, or folder supplied in or with the container.

The materials and methods below are provided to facilitate the practice of the present invention.

Subject recruitment and IRB approval:

Subjects with clinical EoE milk allergy, IgE-mediated milk allergy, or non-allergic controls were recruited at Children’s Hospital of Philadelphia (CHOP) between January 2019 and January 2021. Subjects were enrolled with subject or guardian consent, and subject assent (when applicable), via CHOP IRB protocol 18-015524.

PBMC isolation and culture:

Peripheral blood samples were obtained from subjects or controls. Peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll gradient, carboxyfluorescein succinimidyl ester (CFSE) labeled (Invitrogen, REF C34554), and cultured at 1 million cells/mL in OpTimizer SFM (Gibco, REF A 10221-01) for 6 days in a 96 well round-bottom plate (Coming Inc. Costar, REF 3799) in the presence or absence of endotoxin-depleted milk proteins (6.25 pg of each of a- lactalbumin, P-lactoglobulin, a-casein, P-casein, and K-casein per 200k cells; Figure 2) or tetanus toxoid (TT; Astarte Biologies, CAT 1002; 0.625 pg per 200k cells).

Flow Cytometry:

Cells were surface and/or intracellularly stained prior to being acquired on an LSR Fortessa (BD) within 24 hours of harvest. The cytometer was compensated using OneComp eBeads (Invitrogen), unstained cells, CFSE stained cells, or Live/Dead Fixable Blue (Invitrogen) stained cells. Fluorescence minus one (FMO) controls were used to establish lower gate bounds. A minimum of 250k events were acquired for each arm, with a range of up to 1 million events depending on initial PBMC recovery. Data was analyzed using FlowJo software (Becton Dickinson). Events were gated by Lymphocytes, Live/Dead negative, CD8-, CD19“ CD3 ⁺ ’ CD4 ⁺ ’ CD45RA” CD45RO ⁺ (Figure 4). Data Analysis and Statistics:

Intracellular IL-4 production by memory TH cells was measured by intracellular cytokine staining, and memory TH cell proliferation was determined by identifying CFSEbright and CFSEdim cells as PO and Pl gates (representing undivided and proliferating cells, respectively). Samples with a TT vs unstimulated % Pl difference of less than two (% Pl TT - % Pl unstimulated < 2) were excluded due to lack of adequate positive control response (Figure 4), and significant outliers within each group were determined by extreme studentized deviate method and excluded. Significant differences between experimental groups were determined by unpaired or paired parametric t test, depending on context and as indicated in figure legends. To reduce inter-subject variability, the % Pl milk proteins was normalized to the % Pl TT, for a given sample. Associations between assay outcomes and milk consumption at the time of assay were determined by Pearson’s correlation. The association between milk protein-activation of memory TH cells and clinical sensitivity to milk was measured using a Receiver Operating Characteristic (ROC) curve. A positive test threshold was selected which maximized sensitivity and specificity for correlation with clinical EoE milk allergy. Pairwise comparisons between ROCs were made via Delong’s test.

Disease definitions

A board-certified allergist reviewed all subject charts for 1) completed tetanus vaccine series; 2) clinical features of EoE or IgE-mediated milk allergy including meeting international diagnostic criteria (1); 3) presence of bovine milk as an established EoE-causal food; and 4) presence of other atopic diagnoses. Clinical EoE milk allergy was defined as a) prior upper endoscopy and biopsy showing at least 15 eosinophils per high-power field isolated to the esophagus while the subject was on a proton pump inhibitor and meeting the consensus diagnosis of EoE (1), and b) upper endoscopy and biopsy showing 0-10 eos/hpf isolated to the esophagus after a minimum of 6 weeks under milk-free diet.

Surface stain

After 6 days of culture, cells were harvested, enumerated, and stained with Live/Dead Fixable Blue (Invitrogen, REF L34962). Cells were surface stained for anti-human CD8 (SKI, BioLegend, 1: 100, BV510), CD3 (SK7, eBioscience, 1: 100, APC-Cy7), CD4 (OKT4, BioLegend, 2.5: 100, BV605), CD19 (HIB 19, BioLegend, 5: 100, AF700), CD45RA (HUGO, BD Bioscicnccs, 5:100, V450), and CD45RO (UCHL1, BioLcgcnd, 5: 100, BV650) for 30 minutes at 4°C in the dark. Surface-stained cells were fixed with fixation buffer (Invitrogen) prior to analysis.

Intracellular stain

Samples that received an intracellular stain were restimulated with phorbol myristate acetate (1 ng per 200k cells, Sigma- Aldrich Millipore Sigma) and ionomycin (0.2 pg per 200k cells, Sigma- Aldrich Millipore Sigma) for 5 hours prior to harvest, and Brefeldin A (10 pg per 200k cells, BioLegend) for the final 2 hours prior to harvest to amplify the endogenous cytokine signal ²⁴ . Cells were surface stained, fixed, and permeabilized (Invitrogen) prior to overnight staining for interleukin (IL)-4 (MP4-25D2, BioLegend, 5: 100, AF647).

IgG4 ELISA

ELISA was used to quantify total and food-specific IgG4 as previously described ^2,3 .

Briefly, for total plasma IgG4 measurements MicroIon® Flat Bottom High Binding Microplates (Greiner Bio-One, 655061) were coated with mouse anti-human Ig K light chain (clone G20-193; BD Biosciences; diluted 1:250). For food-specific IgG4 measurements, plates were coated with 20 pg/mL of a bovine milk peptide cocktail which contained a-lactalbumin, P-lactoglobulin, a- casein, P-casein, and K-casein (Sigma-Aldrich Millipore Sigma, C6780-250MG, C6905-250MG, C0406-100MG, L5385-1OOMG, L3908-250MG). A standard curve was created by coating wells with purified mouse anti-human IgG4 (clone G17-4; BD Biosciences; diluted 1:250) and using serial dilutions of native human IgG4 (Abeam, Ab 183266). Detection was performed using mouse anti-human IgG4-horseradish peroxidase antibody (clone HP6025; Southern Biotech; diluted 1: 1000) with tetramethylbenzidine substrate (Thermo Scientific, #34021). The reaction was halted at 5 minutes using ELISA Stop Solution (Invitrogen by ThermoFisher Scientific, #SS04) and the plate was read at 450nm. Food-specific IgG4 was normalized as a ratio of total plasma IgG4. Standards and samples were run in duplicate and averaged. For total IgG4 measurements, patient sera samples were diluted in a range from 1:25000-50000. For milkspecific IgG4 measurements, samples were diluted in ranges from 1:50-500 and 1:50-3000 for control and EoE groups, respectively. Endotoxin depletion and testing of milk proteins

Milk proteins (a/p/K-casein, a-lactalbumin, and P-lactoglobulin) were initially tested for endotoxin contamination by Limulus Amebocyte Lysate assay (Table 2).

Proteins were divided into groups based on estimated levels of endotoxin contamination. Endotoxin was depleted from high-endotoxin containing samples (a-lactalbumin and - lactoglobulin) using Proteus MIDI Endotoxin Removal High Capacity spin columns (Bio-Rad), while samples with lower levels of contamination (a/p/K-casein) were processed using Proteus Mini Endotoxin Removal spin columns (Bio-Rad). Protein amounts were measured by BCA (Pierce, REF 23227), and protein solutions were adjusted to 5 mg/mL in DPBS and stored at - 80°C.

Pre- and post-endotoxin depletion milk protein samples were examined for their ability to signal via human TLR4 (hTLR4) using the hTLR4 reporter cell line HEK-Blue hTLR4 (InvivoGen, CAT# hkb-htlr4). HEK-Blue cells were suspended in detection media when 80-90% confluent and seeded at -50,000 cells/well in a 96 well flat bottom plate. Pre- and post-endotoxin depletion milk protein samples were added at a concentration of 50 ug per well. Purified LPS (Sigma- Aldrich, 297-473-0) was serially diluted to create a standard curve. Color change was observed after 10 hours of incubation at 37°C, and the plate was read at 635 nm. Standards and samples were run in duplicate and averaged. A logarithmic best-fit line for the LPS standard curve was determined (R2=0.9993) and utilized to calculate LPS -equivalent hTLR4 signaling values.

Luminex Culture supernatant cytokine levels (IL-4, IL-5, IL-13) were assayed by high-sensitivity Human T cell Cytokine Lumincx assay (Milliporc). Levels below the detectable limit were considered 0, and samples with levels above the detectable limit were excluded.

5 The following example is provided to illustrate certain embodiments of the invention.

They are not intended to limit the invention in any way.

Example I

A graphical representation of Example I is provided as Figure 1.

We recruited 17 subjects with clinical EoE milk allergy and 17 non-milk-allergic 0 controls. For comparison, we recruited 9 subjects with IgE-mediated milk allergy. All subjects met international criteria for the diagnosis of EoE or IgE-mediated food allergy ⁶ , and the majority of clinical EoE milk allergy subjects and all IgE-mediated milk allergy subjects were avoiding milk at the time of enrollment. Subject characteristics are shown in Table 3. The average age of enrolled subjects was 13 years, and the clinical EoE milk allergy cohort was 5 predominantly male (65%) and had a high degree of allergic comorbidity, observations that are consistent with prior studies of demographic and clinical characteristics of EoE subjects. ²⁵

As food- specific IgG4 levels have been shown to correlate with clinical EoE food allergy ^{7 19 22} , we first examined total and milk-specific IgG4 levels in the plasma of control or clinical EoE milk allergy subjects. We did not observe a significant difference in total IgG4 levels (Figure 5A), but did observe a non-significant increase in milk-specific IgG4 levels in

5 clinical EoE milk allergy subjects compared with controls (Figure 5B). Similarly, we observed a non-significant increase in the ratio of milk-specific to total IgG4 in clinical EoE milk allergy subjects compared with controls (Figure 5C), an outcome that has been shown to correlate with clinical food allergy in active disease cohorts ^7,21 . Notably, the ratio of milk-specific to total IgG4 in clinical EoE milk allergy subjects was higher in individuals consuming milk at the time of 0 assay (Figure 6, P=0.05). Children with IgE-mediated milk allergy had slightly lower total serum IgG4 levels, and similar milk-specific IgG4 and specific to total IgG4 ratios, as compared with clinical EoE milk allergy subjects (Figure 7A-7C). When examining all the clinical EoE milk allergy subjects the relative overlap in milk-specific IgG4 levels between our control and experimental cohorts resulted in a moderate retrospective sensitivity and specificity for 5 predicting clinical allergy to milk (77% and 71%, respectively) (Figure 5D). While prior studies in animal models and human subjects have shown that allergenspecific TH cells circulate in the blood of patients with EoE ^{7 12 14 17} , examinations of memory TH cell populations have not been undertaken. We therefore sought to determine whether milkspecific memory TH cells are present in the circulation of clinical EoE milk allergy subjects. As proliferation in response to antigen- specific stimulation is a key feature of memory CD4 ⁺ TH cells ²⁶ , we labeled PBMCs from control or clinical EoE milk allergy subjects with CFSE prior to ex vivo culture in the absence or presence of tetanus toxoid (TT) or a solution of 5 endotoxin- depleted milk proteins (u/p/K-casein, a-lactalbumin, and P -lactoglobulin). Consistent with prior tetanus vaccination, we observed that CD4 ⁺ CD45RO ⁺ memory TH cells from both control and clinical EoE milk allergy subjects proliferated in response to stimulation with TT (Figure 8A). However, significantly more memory TH cells from clinical EoE milk allergy subjects proliferated in response to stimulation with milk proteins as compared with cells from controls (Figure 8 A, ,8B). Children with IgE-mediated milk allergy had higher baseline, but proportionally similar proliferative responses as compared with clinical EoE milk allergy subjects (Figure 7D,7E). Together, these results indicate that milk-reactive memory TH cells are present in the circulation of children with clinical EoE milk allergy.

We next sought to quantify the extent to which milk-dependent memory TH cell proliferation associated with clinical EoE milk allergy. We found that there was a high degree of variability in TH cell proliferative responses between subjects that complicated identification of a threshold that distinguished control from clinical EoE milk allergy subjects (Figure 9A). To account for this, we performed intra-sample normalization of milk-induced to tetanus-induced memory TH cell proliferation. This normalization minimized inter- sample variability and allowed more accurate determination of a threshold for assay positivity (Figure 9B). Notably, memory TH cells from clinical EoE milk allergy subjects consuming milk at the time of assay did not significantly differ in their proliferative response to milk stimulation as compared with TH cells from clinical EoE milk allergy subjects avoiding milk at the time of assay (Figure 6, P=0.5382). Using this approach, we found that a threshold of 0.86 (ratio of %P1 milk protein- stimulated to %P1 tetanus-stimulated) resulted in a retrospective assay sensitivity and specificity of 88% and 82% for clinical EoE milk allergy, respectively (Figure 9C). Together, these results indicate that milk-dependent proliferation of milk-specific memory TH cells significantly correlates with clinical EoE milk allergy irrespective of whether an individual is consuming milk at the time of the assay.

To test whether we could improve upon the sensitivity and specificity of our milkspecific IgG4 and milk-dependent memory TH cell proliferation assays, we examined whether combining these two assays improved our ability to identify clinical EoE milk allergy. To do so, we multiplied the milk-specific to total IgG4 ratio by the milk to tetanus Pl CFSE ratio (IgG4 x CFSE). The IgG4 x CFSE measure provided an outcome that was significantly different between the control and clinical EoE milk allergy subjects (Figure 10A), and resulted in a modest improvement in the overall specificity for identifying clinical EoE milk allergy (88%) (Figure 10B). Like the ratio of milk-specific to total IgG4 in clinical EoE milk allergy subjects, the combined IgG4 x CFSE measure was higher in individuals consuming milk at the time of assay (Figure 6, P=0.0495). A pairwise comparison of each of the ROC curves confirmed that the area under the IgG4 x CFSE curve was greater than that of the milk-specific to total IgG4 ratio curve (Figure IOC, P=0.0178 by DeLong’s test). However, the area under the IgG4 x CFSE curve was not significantly different from that of the milk to tetanus Pl CFSE ratio curve (P=0.6492 by DeLong’s test). Thus, our analysis of milk-dependent memory TH cell proliferation alone is roughly equivalent to our combined analysis of milk-specific IgG4 and milk-dependent memory TH cell proliferation.

Having established that milk-reactive memory TH cell populations are present in the circulation of children with clinical EoE milk allergy, we next sought to identify the activation state of these cells. To do so, we isolated PBMCs from control or clinical EoE milk allergy subjects and stimulated them ex vivo for six days in the absence or presence of TT or milk proteins, prior to assaying intracellular interleukin (IL)— 4 production. CD4 ⁺ CD45RO ⁺ memory TH cells from both control and clinical EoE milk allergy subjects produced IL-4 in response to stimulation with TT (Figure 5A). However, significantly more memory TH cells from clinical EoE milk allergy subjects produced IL-4 in response to stimulation with milk proteins as compared with cells from control subjects (Figure 11 A, 1 IB). Children with IgE-mediated milk allergy had higher baseline, but proportionally similar IL-4 production as compared with clinical EoE milk allergy subjects (Figure 7F,7G). To test whether milk-specific memory TH cells produced other canonical type 2 cytokines, we examined culture supernatant fractions. Milk- stimulated cultures of PBMCs from clinical EoE milk allergy subjects contained higher amounts of IL-4, IL-5, and IL- 13 as compared with PBMCs from control subjects (Figure 1 1C). These results indicate that milk-specific memory TH2 cells circulate in the periphery of children with clinical EoE milk allergy.

Finally, we sought to quantify the extent to which peripheral milk-specific memory Tu2 cell frequency associated with clinical EoE milk allergy. When examining the association between the percent of milk-specific memory TH cells that express IL-4 (Figure 12A), and clinical EoE milk allergy, a positive cutoff threshold of > 13.1% had a retrospective sensitivity and specificity of 100% for identifying clinical EoE milk allergy subjects (Figure 12B). Notably, TH cells from clinical EoE milk allergy subjects consuming milk at the time of assay did not significantly differ in their production of IL-4 in response to milk stimulation as compared with TH cells from clinical EoE milk allergy subjects avoiding milk at the time of assay (Figure 6, P=0.2896). In sum, these results indicate that assaying milk-dependent memory TH2 cell IL-4 production has the potential to identify clinical EoE milk allergy with a very high degree of accuracy, irrespective of whether an individual is consuming milk at the time of the assay. Discussion

A minimally-invasive assay that clinicians could use to guide food elimination would minimize the need for endoscopies and represent a significant step forward for EoE clinical care. Prior work has suggested that food-specific IgG4 levels in the serum and/or the esophagus may be a viable method to identify EoE-causal foods ^{19 22} . In our cohort, this approach had a relatively low sensitivity and specificity for distinguishing clinical EoE milk allergy. This could be due to the fact that we studied a pediatric cohort, assayed plasma antibody levels as opposed to those of esophageal tissue, or because most of the clinical EoE milk allergy subjects in this study were avoiding milk at the time of sample acquisition. Indeed, both disease activity and/or consumption of allergenic foods may influence total or antigen- specific IgG4 levels, as well as circulating memory TH2 cell responses ^21,22 . Consistent with this, we found a higher degree of correlation between food-specific IgG4 levels and milk allergy when examining the subset of subjects that were consuming milk at the time of assay. Larger studies of the correlation between foodspecific IgG4 levels, clinical allergens, and disease activity in children can be performed.

Efforts to combine examinations of esophageal allergen- specific IgG4 levels with total CD4 ⁺ allergen-dependent TH cell proliferation have also been undertaken, and have yielded a combined sensitivity and specificity for detecting milk-allergic EoE of 64% and 25%, respectively ⁷ . In our cohort, examination of milk-dependent memory TH cell proliferation alone has an 88% sensitivity and 82% specificity for identifying children with clinical EoE milk allergy. When we combine assays of milk-specific IgG4 and TH cell proliferation, we found that the overall specificity for identifying clinical EoE milk allergy of 88%, though the ROC curve for this approach was not significantly different from that of the TH cell proliferation assay alone.

There are three technical advances that have improved the sensitivity and specificity of our Tn cell proliferation assay over prior studies ⁷ . These include 1) depletion of endotoxin from milk proteins, 2) assaying CD4 ⁺ CD45RO ⁺ memory TH cells as opposed to total TH cell populations, and 3) intra-sample normalization of milk-dependent to TT-dependent proliferative responses (though this final technical advance limits potential use of this assay to individuals who have been adequately immunized against tetanus). The fact that we observe that all of the milk proteins tested are able to signal via hTLR4 highlights the importance of accounting for endotoxin contamination in TH cell activation assays. Further, while we observed significant reductions in hTLR4 signaling for all five proteins after endotoxin depletion some hTLR4 signaling capacity persisted. This was particularly relevant for P-lactoglobulin which was relatively resistant to endotoxin removal when compared to the other milk proteins. This could be the result of intrinsic ability for P-lactoglobulin to signal via hTLR4, ²⁷ or inherent endotoxin binding capacity of this protein as is the case with other mammalian milk-derived proteins. ^28,29

Our TH cell proliferation assay approach has the additional benefit of being able to be performed solely using a peripheral blood sample, and thus qualifies as a minimally-invasive assay that could be performed by most clinical laboratories. An important finding is that this approach is able to identify foods for which the immune system has an established memory TH cell response, irrespective of whether the individual is actively consuming the food in question. This is of particular interest as this or similar assays could assist in determining not only foods to avoid but also the timing of food reintroduction, a useful feature given emerging evidence that some children can outgrow EoE after periods of prolonged causal food avoidance. ²³

In an attempt to further improve our ability to identify clinical EoE milk allergy, we first established the existence of milk-specific memory TH2 cells in the circulation of children with clinical EoE milk allergy. The existence of allergen-reactive memory TH2 cells in the circulation of EoE patients is a novel finding, but also consistent with observations in mouse models that have shown that skin sensitization can lead to the development of antigen- specific TH2 cell responses and esophageal eosinophilia. ^{12 14} This finding is also consistent with our understanding of the role that cpicutancous sensitization plays in IgE-mcdiatcd food allergy ³⁰ , the high degree of clinical comorbidity between IgE-mediated food allergy and EoE, ³¹ and EoE being a member of the allergic march. ²⁵ Ultimately, the establishment of milk-specific memory Tu2 cells in the circulation of children with clinical EoE milk allergy improves our understanding of EoE immunopathology.

When examining the utility of measuring allergen-dependent memory Tn2 cell cytokine production in identifying EoE-causal foods we found that this approach has a very high sensitivity and specificity for identifying clinical EoE milk allergy. The high accuracy of this approach likely also derives from the depletion of endotoxin from milk proteins, and examination of memory TH cell populations. This approach can be performed using a peripheral blood sample, and also seems to be useful irrespective of whether an individual is actively consuming the allergic food in question lending itself to determining the timing of food reintroduction in the case of clinical remission. However, this approach has the disadvantage of intracellular IL-4 staining being a technically challenging protocol for some clinical labs.

Finally, it is notable that milk-specific immune responses in children with IgE-mediated milk allergy were grossly similar to those observed in children with clinical EoE milk allergy. The presence of circulating, allergen-activated TH cells in IgE-mediated milk allergy is consistent with both our understanding of the immunopathology of this condition, as well as our hypotheses as to the clinical and immunopathological relationship between IgE-mediated food allergy and EoE. ^31,32 Further, this observation may have implications for understanding EoE that occurs after outgrowing IgE-mediated food allergy, ³³ or initiation of oral immunotherapy. ³⁴ Notable differences in the immune responses between these two related conditions include the generally lower level of total IgG4 in children with IgE-mediated milk allergy, and the higher baseline proliferation and IL-4 production exhibited by circulating TH cells obtained from IgE-mediated milk allergy subjects. These latter observations could be indicative of important mechanistic differences between these two conditions. Regardless, the implications of these observations to the potential clinical assays described herein are likely inconsequential as IgE-mediated food allergy and EoE are easily distinguished clinically.

In sum, we report the identification of milk-reactive memory TH2 cells in the circulation of children with clinical EoE milk allergy. This approach provides a minimally-invasive assay that accurately predicts clinical EoE milk allergy with high sensitivity and specificity. Further, the success of these milk assays may indicate that the approach is generalizable to the development of assays for other common EoE allergens.

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Example II

Evaluation of Milk- Activated T Cell Blood Test

To prospectively evaluate the milk- activated T cell blood test in a ‘real world’ cohort, 41 subjects including, those with and without EoE, were recruited for an IRB -approved study in the endoscopy suite. Blood samples were drawn from each of the subjects and the milk-activated T cell test was performed as described in Example I. Using the criteria established in Example I, the patients were separated into two groups: “milk tolerant” and “milk allergic”. These subjects were then followed by chart review until one of three clinical determinations were made: 1) control (no EoE); 2) EoE, milk-tolerant; and 3) EoE; milk- allergic. The results from this study are shown in Fig. 13 A.

W found that the milk assay had a sensitivity of 100% and specificity of 70% in all subjects (control and EoE) in this prospective cohort (Figure 13A). However, when we limited our analysis to children with EoE (either milk allergic or milk tolerant), we found that a threshold of 0.92 resulted in a assay sensitivity and specificity of 100% for clinical EoE milk allergy (Figure 13B).

Example III Development of Soy-Activated T Cell Blood Test

To evaluate the soy-activated T cell blood test, 18 subjects, including subjects with an EoE soy allergy and without an EoE soy allergy (healthy controls) were recruited for an IRB- approved study in the endoscopy suite. Blood samples were drawn from each of the subjects and the soy-activated T cell test was performed using the methods established in Example I but using purified soy protein extract for PBMC stimulation. Due to the high variability in T cell proliferative responses, we performed intra-sample normalization of the subjects (Figure 14). Using this approach, wc found that the soy-activated T cell responses were significantly higher in children with confirmed EoE soy allergy, as compared with healthy controls.

Example IV

Diagnosis and Monitoring Method for Food Associated Allergies

The information herein above can be applied clinically to patients for therapeutic diagnosis and monitoring. A preferred embodiment of the invention comprises clinical application of the information described herein to a patient. This can occur after a patient arrives in the clinic and presents with allergy-associated diseases or symptoms. A sample may be taken from the patient. The sample can then be tested for one or more of the allergens identified in Table 1. In certain embodiments, the sample is tested for multiple allergens identified in Table 1. In certain embodiments, the sample is tested for multiple allergens using a recombinant protein or immune reaction stimulating fragments thereof. The sample is cultured for a suitable time period with any of the above allergens and tested for at least one of the TH cells. If an allergen is identified, the patient is treated. After an allergen is identified, the test may be repeated multiple times over time. In certain embodiments, the allergy resolves after monitoring.

While certain of the preferred embodiments of the present invention have been described and specifically exemplified above, it is not intended that the invention be limited to such embodiments. Various modifications may be made thereto without departing from the scope and spirit of the present invention, as set forth in the following claims.