Antigen specific next-generation screening and therapeutics for the treatment of eosinophilic esophagitis and other food allergies

Abstract

Compositions for methods for antigen specific next generation screening approaches, diagnostics, biomarkers and therapeutics for the treatment and management of eosinophilic esophagitis and other inflammatory diseases are disclosed.

Description

[0001] ANTIGEN SPECIFIC NEXT-GENERATION SCREENING APPROACHES, DIAGNOSTICS, BIOMARKERS, AND THERAPEUTICS FOR THE TREATMENT AND MANAGEMENT OF EOSINOPHILIC ESOPHAGITIS (EOE) AND OTHER FOOD ALLERGIES By Julianna Dilollo Karen Cerosaletti David Hill CROSS-REFERENCE TO RELATED APPLICATION This application claims priority to US Provisional Application No. 63 / 692,328, filed September 9, 2024, which is incorporated by reference as though set forth in full. FIELD OF THE INVENTION This invention relates to the fields of chronic inflammatory disease and biomarker identification for screening, diagnosis, and management of the same. INCORPORATION-BY-REFERENCE OF MATERIAL SUBMITTED IN ELECTRONIC FORM The contents of the electronic sequence listing (CHOP-159-PCT.xml; Size: 516,836 bytes; and Date of Creation: September 9, 2025) is herein incorporated by reference in its entirety. 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 herein by reference as though set forth in full. First described in 1978,1eosinophilic esophagitis (EoE) is a chronic inflammatory disease and one of the leading causes of dysphagia, odynophagia, and esophageal food impaction.2,3Since the early 1980s,4EoE has been managed as a clinical food allergy through the use of empiric food elimination diets.5This practice was supported by investigations which revealed that EoE has a strong predominance of type 2 (T2) allergic inflammation.6,7Despite this, immunologic evidence of food antigen recognition by the immune system in EoE was initially elusive, confounding clinical practice and impairing diagnostic and therapeutic advances. In the early 2000s, the first data emerged for a central role for both CD4+T helper type 2 (TH2) cells8and antigen-specific immune responses9in animal models of EoE. More recently, translational studies have identified food allergen-activated T cells in the circulation of patients with EoE, but not in healthy controls.10–12In addition, single cell RNA sequencing (scRNA-seq) of patient biopsies has elucidated the effector phenotype13and identified clonal expansion14,15of T cells. Despite these findings, alternative mechanistic explanations for EoE have been proposed including an epithelial cell–intrinsic alteration in TGFβ signaling that is independent of T cells.16As such, there is an active debate in the field as to the immunopathologic etiology of EoE. Knowledge of the molecular basis of antigen presentation and recognition has clinical application in the diagnosis and management of inflammatory diseases. For example, the identification of specific autoantigens presented by MHC class II molecules to CD4+T cells and utilization of medications that interfere with T cell co-stimulation during antigen presentation has revolutionized the diagnosis and management of rheumatoid arthritis.17,18In the case of IgE- mediated anaphylactic food allergy, knowledge of the specific antigenic components has increased diagnostic accuracy, allowed development of personalized anaphylaxis risk assessments for patients, and holds promise for targeted immunotherapy.19,20Summary of the invention In accordance with one embodiment of the invention, an isolated immunogenic peptide of between 10 and 100 amino acids in length comprising a MHC class II T cell epitope of a milk antigen associated with eosinophilic esophagitis (EoE) present in a milk protein selected from β- casein, α-lactalbumin, β-lactoglobulin, α-casein, and ĸ- casein said immunogenic peptide inducing expansion of TCR clonotypes is disclosed. In certain embodiments, the isolated immunogenic milk peptide is 10 to 25 amino acids in length. In yet another embodiment, the isolated immunogenic peptide is a milk antigen comprising amino acids 59-78 from β- casein or an immunogenic peptide fragment thereof which effectively stimulates EoE TCR4+T cells. Also disclosed is a method for obtaining a population CD4+T cells which are stimulated when contacted with a milk antigen, comprising the steps of providing peripheral blood cells; contacting the cells in vitro with an immunogenic peptide described above comprising an MHC class II T cell epitope of the antigen; and expanding said cells in the presence of IL-2. The invention also comprises a population of T cells obtainable by the method described above. In certain aspects, the T cell can be selected from the group consisting of a primary T cell, an ex vivo cultured T cell, a tumor-infiltrating T cell, and an engineered T cell. In other aspects, the T cell is isolated via single-cell sorting. The invention also provides a method for identifying TCR sequences of a milk antigen- specific T cell. An exemplary method comprises contacting a sample with a particle comprising an agent having affinity for said T cell under conditions sufficient for a T cell to bind the particle; isolating an antigen-specific T cell; and obtaining the TCR sequences. TCR alpha and TCR beta sequences associated with milk induced EOS are provided in Table 1 and in Figures 1, 8, 9, and 10. The method can optionally further comprise amplifying at least one TCR alpha gene sequence and at least one TCR beta gene sequence. Also disclosed is a method for identifying a subject at increased risk for milk allergy induced eosinophilic esophagitis (EoE). An exemplary method comprises detecting in a nucleic acid obtained from said subject, an HLA-DR allele, or an HLA-DRB1*07.01 allele, the presence of the allele being associated with an increased risk of developing milk allergy induced EoE. A method for treating a subject having EoE comprising administration of at least one agent that selectively deletes milk reactive T cells, thereby reducing EoE symptoms is also disclosed. In certain embodiments, the agent is a CAR-T system. In yet another embodiment, an isolated peptide-loaded MHC class II molecule, comprising an MHC class II alpha chain, an MHC class II beta chain, and a MHC-class II binding peptide is provided. In a preferred embodiment, the isolated peptide-loaded MHC class II molecule comprises a DRB1 beta chain and / or is encoded by an HLA-DRB1*07.01 allele. In other embodiments, the isolated peptide-loaded MHC class II molecule comprises a tagged peptide of SEQ ID NO: 530 of β-casein or an immunogenic fragment thereof. In other embodiments, the isolated peptide-loaded MHC class II molecule above is linked to a ligand of a multivalent binding molecule. Also disclosed is an isolated MHC class II multimer, comprising a multivalent binding molecule, an isolated milk antigen peptide-loaded MHC class II molecule, further comprising a beta chain encoded by a DRB1 allele, and bound to a milk antigen peptide, wherein said peptide comprising at least 10 to 25 contiguous amino acids of β-casein, and / or wherein the MHC class II molecule is linked to a ligand of the multivalent binding molecule, and at least one additional MHC class II molecule linked to a ligand of the multivalent binding molecule, wherein each of the at least one additional MHC class II molecule is optionally peptide-loaded, and wherein the ligands bind to the multivalent binding molecule. In certain embodiments the multimer is a tetramer, comprising three additional MHC class II molecules linked to a ligand of the multivalent binding molecule. In a preferred embodiment, the MHC class II molecule of the multimer or the tetramer is loaded with a β-casein peptide comprising at least 10-25 contiguous amino acid sequence between residue 59 and residue 224 and is optionally labeled with a detectable label. In yet another aspect of the invention, a peTH2 genetic signature associated with the EoE phenotype comprising EoE biomarker genes IL4, IL5, IL13, PTGDR2, PLA2G16, PTGS2, HPGDS, PPARG, ACADVL, ACSL4, SLC27A2, LPCAT2, DGKE, GK, CHDH, ALOX5AP, GPR15, ICAM2, GATA3, IL17RB, FFAR3, IL1RL1, said genes exhibiting altered expression levels in EOE subjects when compared to healthy control subjects is disclosed. The invention also provides a method for diagnosing the presence of sensitivity to a milk protein in a human subject. An exemplary method comprising contacting PBLs obtained from the subject with multimers or oligomers of HLA-DRB1 monomers or modified monomers, having a bound HLA-binding peptide comprising an amino acid sequence as set forth in SEQ ID NO: 530, detecting binding of the multimers or oligomers to the PBLs, wherein the binding to PBLs is indicative of the presence of milk antigen sensitivity or previous exposure to milk antigen in the subject. The method can further comprise contacting the PBLs with additional multimers of HLA-DRB1 monomers or modified monomers, having a bound HLA-binding peptide comprising an amino acid sequence as set forth in any one of SEQ ID NOS: 531 to 538. In certain embodiments, the detecting comprises high throughput screening. Also disclosed is a method for determining efficacy of a drug for treatment or prevention of milk induced EoE in a human subject, comprising: obtaining a sample comprising PBLs from a patient with an EoE associated HLA allelle undergoing treatment for EoE using the drug; contacting the sample under suitable binding conditions with a solid support having bound tetramers of HLA monomers or modified monomers, wherein said monomers are HLA allele monomers or modified monomers, having a bound HLA- binding immunogenic milk peptide amino acid sequence; detecting the amount of binding of the tetramers to the PBLs; and repeating the aforementioned steps after a suitable interval of treatment time, wherein a decrease in the amount of the binding after the suitable interval of treatment time indicates efficacy of the drug and a lack of decrease indicates lack of efficacy of the drug for treatment or reduction of EoE symptoms in the human.. The treatment method can comprise administration of a steroid or CAR-T therapy. In preferred embodiments, the drug is any chemical compound that decreases or arrests peTH2 biomarker expression in said subject. In yet another aspect, the invention provides a method for contacting PBLs obtained from a subject with tetramers of HLA monomers or modified monomers, having a bound HLA-binding peptide, wherein the subject has an HLA- DRB1 allelle and the binding peptide comprises an amino acid sequence as set forth in SEQ ID NO: 530, and detecting binding of the tetramers to the PBLs to identify the presence of milk associated EoE or previous exposure to milk antigen in the subject. The methods described above focus on treatment of milk antigen induced EoE. However, the methods can also be used to advantage for diagnosis and treatment of other food allergies. Accordingly, the invention also provides isolated immunogenic peptides of between 10 and 100 amino acids in length comprising a MHC class II T cell epitope of a food antigen associated with allergic responses present in a food protein selected from a food protein listed in Table 4, said immunogenic peptide inducing expansion of TCR clonotypes. The immunogenic food peptide can be 10 to 25 amino acids in length. In certain aspects, the immunogenic food antigen stimulates reactive T cells Also disclosed is a method for obtaining a population CD4+T cells which are stimulated when contacted with a food antigen peptide listed in Table 4. A method for diagnosing the presence of sensitivity to a food protein in a human subject is also disclosed and comprises contacting PBLs obtained from the subject with multimers or oligomers of HLA-allele monomers or modified monomers, having a bound HLA-binding peptide comprising an amino acid sequence selected from the sequence listed in Table 4, and detecting binding of the multimers or oligomers to the PBLs wherein the binding to PBLs is indicative of the presence of food antigen sensitivity or previous exposure to food antigen in the subject. In another embodiment, an isolated MHC class II multimer is disclosed which comprises the following components: a multivalent binding molecule, an isolated milk antigen peptide- loaded MHC class II molecule, comprising an MHC class II molecule, comprising a beta chain encoded by an HLA allele, and bound to a milk antigen peptide, the said peptide comprising at least 10 to 25 contiguous amino acids of β-casein, and / or wherein the MHC class II molecule is linked to a ligand of the multivalent binding molecule, and at least one additional MHC class II molecule linked to a ligand of the multivalent binding molecule, wherein each of the at least one additional MHC class II molecule is optionally peptide-loaded, and wherein the ligands bind to the multivalent binding molecule, wherein said multimers specifically bind and detect one or more of the clonotypes listed in Figures 8, 9 or 10. The invention also provides an oral immunotherapy method of treatment (OIT) of food allergy in a subject in need thereof. An exemplary method comprising gradually introducing increasing doses of modified allergens or peptide fragments thereof that retain immunogenicity while exhibiting reduced allergenicity, and / or facilitate the delivery or effectiveness of the OIT into said subject, under conditions that desensitize said subject to said allergen. In certain embodiments the food is selected from milk, gluten, wheat, egg, soy, and peanut. Brief Description of the Drawings Figures 1A – 1K. Identification of milk-expanded TCR clonotypes from a child with EoE milk allergy. Extended Data (FIG. 1A) EoE Subject 1 demographic and clinical characteristics with timeline. EoE Subject 1 demographic, clinical characteristics, and clinical timeline. SPT = Skin prick test. (FIG. 1B) PBMC from EoE Subject 1 were unstimulated (Unstim.) or stimulated with a cocktail of α−casein1, α−casein2, β−casein, κ−casein, α−lactalbumin, and β−lactoglobulin (Milk) for 6 days. CD4+memory T cells were bead-purified and subjected to single cell RNA sequencing with linked TCR sequencing. (FIG. 1C) Carboxyfluorescein succinimidyl ester (CFSE) fluorescence intensity of unstimulated or milk- stimulated T cells from EoE Subject 1. (FIG. 1D) Interleukin 4 (IL-4) expression by unstimulated or milk-stimulated T cells from EoE Subject 1. (FIG. 1E) UMAP projection of singleton or expanded (≥ 2) CD4+T cell clones from unstimulated (unstim.) or milk-stimulated (milk) cells. Symbols represent individual cells. (FIG. 1F) TCR expansion and diversity (by inverse Simpson’s score) between unstimulated and milk-stimulated cultures from EoE Subject 1. Data represent the mean of 100 down-samples. (FIG. 1G Expanded TCR clonotypes in unstimulated vs. milk-stimulated samples for EoE Subject 1. (FIG. 1H) Distribution of the peTH2 gene signature per cell and by expansion state and stimulation condition in EoE Subject 1. Data shown as mean z-score of log-normalized expression. All experimental arms are significantly statistically different from one another (p < 10-16). (FIG. 1I) Mean scaled, log-normalized (norm.) expression of the peTH2 gene signature per cell by TCR pairs that were most expanded upon milk-stimulation in EoE Subject 1. Boxplot elements are defined as the median (center line), upper and lower quartiles (box limits), and whiskers (1.5x interquartile range). T cell clones expressing more than one alpha or beta chain are shown in clones 1 and 2. (FIG. 1J) Type 2 gene expression in the 20 most expanded clones from the milk-stimulated CD4+memory T cell cultures. peTH2 signature genes in the top 20 most expanded CD4+memory T cells clonotypes from the milk-stimulated cultures. Scaled log expression, FDR ≤ 0.05. (FIG. 1K) HLA-DRB1 is the predominant HLA responsible for milk protein presentation in EoE. Percent divided CD4+memory T cells in PBMC cultures from subjects with EoE milk allergy that were either unstimulated (Unstim.), or stimulated with tetanus toxoid (Tetanus) or a cocktail of α-casein, β- casein, κ-casein, α-lactalbumin, and β−lactoglobulin (Milk) in the absence or presence of antibodies blocking HLA-DR (αDR), HLA-DP (αDP), or HLA-DQ (αDQ) (n=8, ANOVA with Bonferroni’s multiple comparisons, * = p < 0.05, ** = p < 0.01, **** = p <0.0001). Figures 2A – 2G. Identification of a β−casein-specific TCR clonotype from a child with EoE milk allergy. (FIG. 2A) CD4+conventional T (T) cells were sort-purified from a HLA-DR- matched subject, transduced with lentiviral vectors encoding the TCR clonotypes of interest, and co-cultured with autologous, protein-pulsed monocyte-derived dendritic cells (moDCs) for three days prior to flow-cytometric evaluation. (FIG. 2B) Evaluation of eoeTCR-ε. Carboxyfluorescein succinimidyl ester (CFSE) fluorescence intensity of HLA-DR-matched conventional T cells (T) transduced with eoeTCR-ε and cultured alone or in the presence of anti-CD3 and anti-CD28 beads (αCD3 / 28), autologous monocyte-derived dendritic cells (moDCs), or moDCs pulsed with a cocktail of α-casein, β-casein, κ-casein (Caseins) or β-lactoglobulin (βLac). (FIG. 2C) Carboxyfluorescein succinimidyl ester (CFSE) fluorescence intensity of T cells transduced with eoeTCR-4 and cultured with moDCs alone or moDCs pulsed with α-casein subunit 1 and α- casein subunit 2 (αCas), β-casein (βCas), κ-casein (κCas), β-lactoglobulin (βLac), soy proteins (soy), or tetanus toxoid (Tetanus). (FIG. 2D) Milk antigen prediction. DRB1 binding site prediction for α-casein1 (αCas1), α-casein2 (αCas2), β-casein (βCas), κ-casein (κCas), α- lactalbumin (αLac), or β-lactoglobulin (βLac) by HLA-DR allele (% Rank score normalized to a set of randomly generated peptides, 0-1 “strong bind”, 1-5 “weak bind”, >5 “no bind” as determined by NetMHCIIpan). (FIG. 2E) Mass spectrometry of purified milk protein preparations. (FIG. 2F) Cell division of T cells transduced with eoeTCR-4 and cultured with moDCs pulsed with α, β, or κCas. (FIG. 2G) CFSE fluorescence intensity of T cells transduced with eoeTCR-4 and cultured with moDCs alone or moDCs pulsed with βCas or synthetic βCas (sβCas). Data representative of 2 or more independent experiments. Figures 3A -3B. eoeTCR-4 is restricted to HLA-DR. (FIG. 3A) Carboxyfluorescein succinimidyl ester (CFSE) fluorescence intensity of CD4+conventional T (T) cells transduced with eoeTCR-4 and cultured with autologous, protein-pulsed monocyte-derived dendritic cells (moDCs) alone or moDCs pulsed with synthetic βCas (sβCas) in the absence or presence of HLA-DR (αDR), -DP (αDP), or -DQ (αDQ) blocking antibodies. Data representative of 2 or more independent experiments. (FIG. 3B) CFSE fluorescence intensity of unstimulated, milk, or sβCas-stimulated T cells from EoE Subject 1 cultured in the absence or presence of αDR. Data representative of 1 experiment. Figures 4A – 4C. Milk protein presentation via eoeTCR-4 is restricted to HLA-DRB1 07:01. (FIG. 4A) Carboxyfluorescein succinimidyl ester (CFSE) fluorescence intensity of conventional T cells (T) from an individual with the 07:01, 13:01 HLA-DRB1 haplotype transduced with eoeTCR-4 and cultured alone or in the presence of anti-CD3 and anti-CD28 beads (αCD3 / 28), autologous monocyte-derived dendritic cells (moDCs), or moDCs pulsed with β-casein (βCas) or β−lactoglobulin (βLac). (FIG. 4B) CFSE fluorescence intensity of T cells from an individual with the 11:03, 15:01 HLA-DRB1 haplotype transduced with eoeTCR-4 and cultured alone or in the presence of αCD3 / 28, autologous antigen presenting cells (APCs), or APCs pulsed with a cocktail of α-casein, β-casein, κ-casein (Casein) or βLac. (FIG. 4C) CFSE fluorescence intensity of T cells from an individual with the 04:01, 04:04 HLA-DRB1 haplotype transduced with eoeTCR-4 and cultured alone or in the presence of αCD3 / 28, APCs, or APCs pulsed with Casein or βLac. Figures 5A – 5F. Identification of the cognate antigen for eoeTCR-4. (FIG. 5A) β−casein protein with predicted HLA-DR*07:01 binding sites (blue, as determined by NetMHCIIpan, A- C) and peptide library (red, 1-9). (FIG. 5B) Carboxyfluorescein succinimidyl ester (CFSE) fluorescence intensity of T cells transduced with eoeTCR-4 and cultured with moDCs alone, anti-CD3 anti-CD28 activating beads (beads), or moDCs pulsed with β−casein peptides 1, 6, or 9. Data representative of 2 or more independent experiments. β−casein peptide library screen for eoeTCR-4. (FIG. 5C) Carboxyfluorescein succinimidyl ester (CFSE) fluorescence intensity of T cells transduced with eoeTCR-4 and cultured with moDCs alone, anti-CD3 anti-CD28 activating beads (beads), or moDCs pulsed with β-casein peptides 2, 3, or 4. (FIG. 2D) CFSE fluorescence intensity of T cells transduced with eoeTCR-4 and cultured with moDCs alone, beads, or moDCs pulsed with β-casein peptides 5, 7, or 8. Data representative of 2 or more independent experiments. (FIG. 5E) Relative frequency of TCR distance scores. Histograms illustrate the distance scores (x-axis) computed with tcrdist3 for (FIG. 5E) TCRA and (FIG. 5F) TCRB CDR3 sequences against the arcsine square root transformed relative frequency of each score. Orange bars depict transformed distance scores between CDR3s derived from a composite dataset comprising 21 esophageal tissue and PBMC samples obtained from patients with EoE milk allergy. Blue bars depict distances. Figures 6A- 6G. Milk-expanded T cell clonotypes are enriched for interferon response gene signatures. (FIG. 6A) Minimally-biased clustering of CD4+memory T cells clonotypes from unstimulated and milk-stimulated culture conditions. (FIG.6B) CD4+memory T cell clonotype groups from unstimulated and milk-stimulated culture conditions. (FIG. 6C) UMAP cluster defining genes. Scaled log expression, FDR ≤ 0.05. (FIG. 6D) Individual UMAPs of CD4+memory T cell clonotype groups from unstimulated and milk-stimulated culture conditions. (FIG. 6E) Clonotype group proportions in each CD4+memory T cell cluster. (FIG. 6F) Mean z-score log expression of the top 20 enriched Hallmark gene sets by p-value in the CD4+memory T cell clusters. FDR < 0.05. (FIG. 6G) Transcriptional comparison of eoeTCR-4 vs T cell clonotype groups. Scaled log expression of differentially expressed genes between cells expressing eoeTCR-4 and unstimulated unexpanded cells, displayed in random subsamples of 100 cells per experimental group. FDR ≤ 0.05. Figure 7. The eoeTCR-4 expressing T cell clone show a unique transcriptional profile when compared with clones expressing non-milk-specific TCR clonotypes. Differential gene expression analysis of eoe-TCR4 expressing T cell clones and T cell clones expressing non-milk- specific TCR clonotypes (eoeTCR-6 and eoeTCR-7). Scaled log expression, FDR ≤ 0.05. Figures 8A – 8D. Isolated TCR α CDR3 and TCRβ CDR3 sequences and level of milk antigen stimulation in subject 1. SEQ ID NOS: 1 to 186 are shown. Figures 9A -9D. Isolated TCR α CDR3 and TCRβ CDR3 sequences and level of milk antigen stimulation in subject 154. SEQ ID NOS: 186 to 346 are shown. Figures 10A -10D. Isolated TCR α CDR3 and TCRβ CDR3 sequences and level of milk antigen stimulation in subject 51. SEQ ID NOS: 347 to 528 are shown. Figure 11: Validation of an additional, milk-specific TCR from a child with EoE milk allergy. CFSE fluorescence intensity of TN cells transduced with eoe1TCR-5 and cultured with moDCs alone or moDCs antigen loaded with Soy, Milk proteins, or synthetic β-casein (sβCas). Data representative of ≥2 independent experiments. Figure 12A-12B: Validation of an additional, milk-specific TCR from a different child with EoE milk allergy. (Fig.12A) CFSE fluorescence intensity of TN cells transduced with eoe2TCR-1 and cultured with moDCs alone or moDCs antigen loaded with α-lactalbumin (αLac), β-lactoglobulin (βLac), or α-casein (αCas). (Fig. 12B) Limiting dilution of milk proteins and their effects on eoe2TCR-1-transfected cell activation. Data representative of ≥1 independent experiments. Detailed Description Eosinophilic esophagitis (EoE) is a chronic, type 2 inflammatory disease that is increasing in incidence and has substantial morbidity. Despite being clinically defined as a food allergy, the molecular details of food antigen presentation and recognition by the immune system are unknown. The objective of this study was to identify and characterize the molecular basis of milk antigen presentation and T cell recognition in a patient with EoE milk allergy by identifying milk-expanded TCR clonotypes using ex vivo stimulation followed by single cell RNA with linked, paired TRA and TRB sequencing. TCR clonotype validation, HLA restriction, and antigen identification were performed using a combination of lentiviral expression, HLA sequencing, antibody blockade, and a peptide library screen. A β-casein specific TCR clonotype (eoeTCR-4) was isolated and its HLA restriction (HLA-DRB1*07:01) and cognate antigen determined. While eoeTCR-4 was not detected among a larger group of subjects, genotyping of 27 individuals revealed that HLA-DRB1*07:01 was significantly (P=0.0195) enriched in individuals with EoE milk allergy over the general population. This data provides the first molecular identity of food antigen presentation and immune recognition in EoE. In addition to supporting decades of clinical practice, our findings provide foundational knowledge for the development of novel antigen specific diagnostics, therapeutics, and screening approaches. Definitions The phrase “T cell clonotype” or “TCR clonotype” refers to a TCR clonotype comprising unique nucleotide sequence that arises during the gene rearrangement process for that receptor. The combination of nucleotide sequences for the surface expressed receptor pair would define the T cell clonotype. A milk expanded TCR clonotype is observed which subject having a milk allergy are exposed to milk antigens. “Clonotyping” is a process to identify the unique nucleotide CDR3 sequences of a TCR chain. This generally involves PCR amplification of the cDNA using V-region-specific primers and either constant region (C) specific or J-region-specific primer pairs, followed by nucleotide sequencing of the amplicon. “Eosinophilic esophagitis (EoE)” is an inflammatory disorder of the esophagus histologically characterized by accumulation of eosinophils in the esophageal epithelium. Clinical symptoms of EoE include dysphagia, failure to thrive, vomiting and epigastric or chest pain. A diagnosis of EoE is made following endoscopy and biopsy upon finding isolated eosinophils in the esophagus having ruled out gastroesophageal reflux. EoE is considered a food allergy-related disorder based on the high rate of food allergen sensitization and a higher rate of food anaphylaxis in cases compared with the general population. The term "control" refers to a standard or reference condition. An "immune response" refers to any cellular or humoral response against an antigen. "Proteins or molecules of the major histocompatibility complex (MHC)," "MHC molecules," "MHC proteins" or "HLA proteins" are to be understood as meaning, in particular, proteins capable of binding peptides resulting from the proteolytic cleavage of protein antigens and representing potential T cell epitopes, transporting them to the cell surface and presenting them to specific cells there, in particular naive T cells, cytotoxic T-lymphocytes or T-helper cells. The major histocompatibility complex in the genome comprises the genetic region whose gene products are expressed on the cell surface and are important for binding and presenting endogenous and / or foreign antigens, and thus for regulating immunological processes. The major histocompatibility complex is classified into two gene groups coding for different proteins: molecules of MHC class I and MHC class II. The molecules of the two MHC classes are specialized for different antigen sources. The molecules of MHC class I typically present but are not restricted to endogenously synthesized antigens, for example viral proteins and tumor antigens. The molecules of MHC class II present protein antigens originating from exogenous sources, for example bacterial products. The cellular biology and the expression patterns of the two MHC classes are adapted to these different roles. MHC molecules of class I consist of a heavy chain and a light chain and are capable of binding a peptide of about 8 to 11 amino acids, but usually 9 or 10 amino acids, if this peptide has suitable binding motifs, and presenting it to naive and cytotoxic T- lymphocytes. The peptide bound by the MHC molecules of class I typically but not exclusively originates from an endogenous protein antigen. The heavy chain of the MHC molecules of class I is preferably an HLA-A, HLA-B or HLA-C monomer, and the light chain is β-2-microglobulin. MHC molecules of class II consist of an α-chain and a β-chain and are capable of binding a peptide of about 15 to 24 amino acids if this peptide has suitable binding motifs and presenting it to T-helper cells. The peptide bound by the MHC molecules of class II usually originates from an extracellular or exogenous protein antigen. The α-chain and the β-chain are in particular HLA-DR, HLA-DQ and HLA-DP monomers. The present invention relates to methods for profiling subject specific T cell receptor (TCR) repertoires. More particularly, the present invention relates to methods for determining binding of T cell receptors to milk antigens and for determining cytolytic activity targeting the individual subject's esophagus using a single-cell sequencing method. The techniques described herein enable, for the first time, a transformative and general method to profile the T cell repertoire and to quantify the relative abundance of each T cell clone within a population. In addition, the techniques herein identify the antigenic targets of T cell receptors in the context of milk allergy associated EoE. Additionally, the present disclosure enables the discovery of T cell targets in numerous inflammatory diseases, with implications for understanding the basic mechanisms of the mammalian immune response and for developing antigen-specific diagnostic markers and therapies (whether immunizing or tolerizing). Finally, cloned TCRs can be used to formulate personalized immunotherapies for those inflicted with EoE and other inflammatory disorders. T cells play an important role in numerous diseases, and yet in most cases, the critical TCR heterodimers and their cognate antigens have not been identified. Furthermore, the principles underlying thymic positive and negative selection, generation of regulatory T cells, and induction of peripheral anergy are not well understood. Accordingly, there is a great need to identify the TCRs within single T cells and monitor the dynamics of the TCR repertoire during these processes. Using such an approach, it should be feasible to derive the rules of self vs, non- self antigen recognition and the evolution of the repertoire and to identify the TCRs that drive disease. For example, the ability to systematically discover the functional CD8 T cell TCRs that target milk antigens may make it possible to develop more rational treatments for milk allergy induced EoE. Moreover, T cells expressing TCRs associated with disease can be targeted for deletion. The highly polymorphic TCR is generated by joining of non-contiguous gene segments (Vβ, Dβ, Jβ for TCRβ and Vα, Jα for TCRα) together with deletion / insertion of random sequences at junctions and Recombination Signal Sequences (RSS) to form the highly variable CDR3 regions. The recognition of MHC-bound peptide by the combined TCRβ and TCRα proteins occurs primarily by the CDR3 regions (see e.g., FIG. 1; Robins et al., 2010 Sci Transl Med. 2:47ra64; Krogsgaard et al., 2005; Nicholson et al., 2005). Although there is a theoretical possibility of forming as many as 5x1011unique TCRβ chains, the actual number of unique TCRβ genes found in humans is closer to 0.1% of this estimate (Robins et al., 2010). Without being bound by theory, this reduction in complexity may be due to thymic education (positive / negative selection) and antigen exposure (e.g. pathogens, milk antigens, self antigens), processes that select specific T cell clones. T cells can be obtained from a number of sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, spleen tissue, and esophageal tissues. In certain embodiments of the present invention, T cells can be obtained from a unit of blood collected from a subject using any number of techniques known to the skilled artisan, such as Ficoll separation. In one preferred embodiment, cells from the circulating blood of an individual are obtained by apheresis or leukapheresis. The apheresis product typically contains lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated white blood cells, red blood cells, and platelets. In one embodiment, the cells collected by apheresis may be washed to remove the plasma fraction and to place the cells in an appropriate buffer or media for subsequent processing steps. In one embodiment of the invention, the cells are washed with phosphate buffered saline (PBS). In an alternative embodiment, the wash solution lacks calcium and may lack magnesium or may lack many if not all divalent cations. Initial activation steps in the absence of calcium lead to magnified activation. As those of ordinary skill in the art would readily appreciate a washing step may be accomplished by methods known to those in the art, such as by using a semi-automated "flow-through" centrifuge (for example, the Cobe 2991 cell processor) according to the manufacturer's instructions. After washing, the cells may be resuspended in a variety of biocompatible buffers, such as, for example, Ca-free, Mg-free PBS. Alternatively, the undesirable components of the apheresis sample may be removed and the cells directly resuspended in culture media. In another embodiment, T cells are isolated from peripheral blood lymphocytes by lysing the red blood cells and depleting the monocytes, for example, by centrifugation through a PERCOLL™ gradient. A specific subpopulation of T cells, such as CD28+, CD4+, CDC, CD45RA+, and CD45RO+T cells, can be further isolated by positive or negative selection techniques. For example, in one preferred embodiment, T cells are isolated by incubation with anti-CD3 / anti-CD28 conjugated beads, such as DYNABEADS® M-450 CD3 / CD28 T, or XCYTE DYNABEADS™ for a time period sufficient for positive selection of the desired T cells. In one embodiment, the time period is about 30 minutes. In a further embodiment, the time period ranges from 30 minutes to 36 hours or longer and all integer values there between. In a further embodiment, the time period is at least 1, 2, 3, 4, 5, or 6 hours. In yet another preferred embodiment, the time period is 10 to 24 hours. In one preferred embodiment, the incubation time period is 24 hours. For isolation of T cells from patients with leukemia, use of longer incubation times, such as 24 hours, can increase cell yield. Longer incubation times may be used to isolate T cells in any situation where there are few T cells as compared to other cell types, such in isolating infiltrating lymphocytes (TIL) from immunocompromised individuals. Further, use of longer incubation times can increase the efficiency of capture of CD8+T cells. Enrichment of a T cell population by negative selection can be accomplished with a combination of antibodies directed to surface markers unique to the negatively selected cells. A preferred method is cell sorting and / or selection via negative magnetic immunoadherence or flow cytometry that uses a cocktail of monoclonal antibodies directed to cell surface markers present on the cells negatively selected. For example, to enrich for CD4+cells by negative selection, a monoclonal antibody cocktail typically includes antibodies to CD14, CD20, CD11b, CD16, HLA-DR, and CDS. Further, monocyte populations (i.e., CD14+cells) may be depleted from blood preparations by a variety of methodologies, including anti-CD14 coated beads or columns, or utilization of the phagocytotic activity of these cells to facilitate removal. Accordingly, in one embodiment, the invention uses paramagnetic particles of a size sufficient to be engulfed by phagocytotic monocytes. In certain embodiments, the paramagnetic particles are commercially available beads, for example, those produced by Life Technologies under the trade name Dynabeads™. In one embodiment, other non-specific cells are removed by coating the paramagnetic particles with "irrelevant" proteins (e.g., serum proteins or antibodies). Irrelevant proteins and antibodies include those proteins and antibodies or fragments thereof that do not specifically target the T cells to be isolated. In certain embodiments the irrelevant beads include beads coated with sheep anti-mouse antibodies, goat anti-mouse antibodies, and human serum albumin. In brief, such depletion of monocytes is performed by preincubating T cells isolated from whole blood, apheresed peripheral blood, etc. with one or more varieties of irrelevant or non- antibody coupled paramagnetic particles at any amount that allows for removal of monocytes (approximately a 20:1 bead:cell ratio) for about 30 minutes to 2 hours at 22 to 37° C., followed by magnetic removal of cells which have attached to or engulfed the paramagnetic particles. Such separation can be performed using standard methods available in the art. For example, any magnetic separation methodology may be used including a variety of which are commercially available, (e.g., DYNAL® Magnetic Particle Concentrator (DYNAL MPC®)). Assurance of requisite depletion can be monitored by a variety of methodologies known to those of ordinary skill in the art, including flow cytometric analysis of CD14 positive cells, before and after depletion In one embodiment cells are directly labeled with an epitope-specific reagent for isolation by flow cytometry followed by characterization of phenotype and TCRs. In one embodiment antigen-specific T cells are isolated by contacting the T cells with antibodies specific for T cell activation markers. Antibodies that can be used with the methods of the present invention include, but are not limited to, anti-CD25, anti-CD54, anti-CD69, anti-CD38, anti-CD45RO, anti-CD49d, anti-CD40L, anti-CD137, anti-IFN-γ, IL-2, IL-4, and other activation induced cytokines, and anti-CD134 antibodies. Sorting of antigen-specific T cells, or generally any cells of the present invention, can be carried out using any of a variety of commercially available cell sorters, including, but not limited to, MoFlo sorter (DakoCytomation, Fort Collins, Colo.), FACSAria™, FACSArray™, FACSVantage™, BD™ LSR II, and FACSCalibur™ (BD Biosciences, San Jose, Calif). In a preferred embodiment, the method comprises selecting cells that also express CD3. The method may comprise specifically selecting the cells in any suitable manner. Preferably, the selecting is carried out using flow cytometry. The flow cytometry may be carried out using any suitable method known in the art. The flow cytometry may employ any suitable antibodies and stains. Preferably, the antibody is chosen such that it specifically recognizes and binds to the particular biomarker being selected. For example, the specific selection of CD3, CD8, TIM-3, LAG-3, 4-1BB, or PD-1 may be carried out using anti-CD3, anti-CD8, anti-TIM-3, anti-LAG-3, anti-4-1BB, or anti-PD-1 antibodies, respectively. The antibody or antibodies may be conjugated to a bead (e.g., a magnetic bead) or to a fluorochrome. Preferably, the flow cytometry is fluorescence-activated cell sorting (FACS). TCRs expressed on T cells can be selected based on reactivity to milk antigens. Additionally, T cells that are reactive to such antigens can be selected for based on markers using the methods described in patent publication Nos. WO2014133567 and WO2014133568. Additionally, activated T cells can be selected for based on surface expression of CD107a. In one embodiment of the invention, the method further comprises expanding the numbers of T cells in the enriched cell population. Such methods are described in U.S. Patent No. 8,637,307 The numbers of T cells may be increased at least about 3-fold (or 4-, 5-, 6-, 7-, 8-, or 9-fold), more preferably at least about 10-fold (or 20-, 30-, 40-, 50-, 60-, 70-, 80-, or 90-fold), more preferably at least about 100-fold, more preferably at least about 1,000 fold, or most preferably at least about 100,000-fold. The numbers of T cells may be expanded using any suitable method known in the art. Exemplary methods of expanding the numbers of cells are described in patent publication No. WO 2003057171, U.S. Patent No. 8,034,334, and U.S. Patent Application Publication No. 2012 / 0244133. In one embodiment, ex vivo T cell expansion can be performed by isolation of T cells and subsequent stimulation or activation followed by further expansion. In one embodiment of the invention, the T cells may be stimulated or activated by a single agent. In another embodiment, T cells are stimulated or activated with two agents, one that induces a primary signal and a second that is a co-stimulatory signal. Ligands useful for stimulating a single signal or stimulating a primary signal and an accessory molecule that stimulates a second signal may be used in soluble form. Ligands may be attached to the surface of a cell, to an Engineered Multivalent Signaling Platform (EMSP), or immobilized on a surface. In a preferred embodiment both primary and secondary agents are co-immobilized on a surface, for example a bead or a cell. In one embodiment, the molecule providing the primary activation signal may be a CD3 ligand, and the co-stimulatory molecule may be a CD28 ligand or 4-1BB ligand. Once T cells are isolated the TCRs present on individual cells are determined: by single- cell sequencing. Single cells may be sequenced by any method known in the art. The present invention utilizes single cell sequencing to identify TCR pairs. In one embodiment T cells are sorted into single wells of a plate and each well is sequenced individually. As described herein, specific sets of primers may be used to amplify TCR pairs for sequencing. The T cells may be sorted by FACS. The T cells may be sorted based on IFNγ or any other cell surface marker. In another embodiment, single cell analysis is performed by digital polymerase chain reactions (PCR), e.g., Fluidigm C. Digital polymerase chain reaction (digital PCR, DigitalPCR, dPCR, or dePCR) is a refinement of conventional polymerase chain reaction methods that can be used to directly quantify and clonally amplify nucleic acids including DNA, cDNA or RNA. The key difference between dPCR and traditional PCR lies in that PCR carries out one reaction per single sample and dPCR carries out a single reaction within samples separated into a large number of partitions wherein the reactions are carried out in each partition individually. A sample is partitioned so that individual nucleic acid molecules within the sample are localized and concentrated within many separate regions. The capture or isolation of individual nucleic acid molecules may be effected in micro well plates, capillaries, the dispersed phase of an emulsion, and arrays of miniaturized chambers, as well as on nucleic acid binding surfaces. In a preferred embodiment single cell sequencing is performed using microfluidics. Microfluidics involves micro-scale devices that handle small volumes of fluids. Because microfluidics may accurately and reproducibly control and dispense small fluid volumes, in particular volumes less than 1 µl, application of microfluidics provides significant cost-savings. The use of microfluidics technology reduces cycle times, shortens time-to-results, and increases throughput. Furthermore, incorporation of microfluidics technology enhances system integration and automation. Microfluidic reactions are generally conducted in microdroplets. The ability to conduct reactions in microdroplets depends on being able to merge different sample fluids and different microdroplets. See, e.g., US Patent Publication No. 20120219947 and PCT publication No. WO2014085802 A1. The term "solid matrix" as used herein refers to any format, such as beads, microparticles, a microarray, the surface of a microtitration well or a test tube, a dipstick or a filter. The material of the matrix may be polystyrene, cellulose, latex, nitrocellulose, nylon, polyacrylamide, dextran or agarose. EoE associated nucleic acids may be affixed or immobilized to a solid matrix. Affixed or immobilized as used herein refers to a linkage that is stable in solution, such that the nucleic acids remain attached to the solid matrix under different processing or experimental conditions. 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. "Target nucleic acid" as used herein refers to a previously defined region of a nucleic acid present in a complex nucleic acid mixture wherein the defined region contains at least one known nucleotide sequence present in a TCR clonotype associated with milk allergy, and, or EoE which informative of the risk of EoE. The nucleic acid molecule may be isolated from a natural source by cDNA cloning or subtractive hybridization or synthesized manually. The nucleic acid molecule may be synthesized manually by the triester synthetic method or by using an automated DNA synthesizer. When cloning a target nucleic acid comprising a deletion, the skilled artisan is well aware of methods for selecting nucleic acids of a sufficient length flanking the affected region to facilitate cloning the region into a vector of choice. The term "probe" as used herein refers to an oligonucleotide, polynucleotide or nucleic acid, either RNA or DNA, whether occurring naturally as in a purified restriction enzyme digest or produced synthetically, which is capable of annealing with or specifically hybridizing to a nucleic acid with sequences complementary to the probe. A probe may be either single-stranded or double-stranded. The exact length of the probe will depend upon many factors, including temperature, source of probe and use of the method. For example, for diagnostic applications, depending on the complexity of the target sequence, the oligonucleotide probe typically contains 15-25 or more nucleotides, although it may contain fewer nucleotides. The probes herein are selected to be complementary to different strands of a particular target nucleic acid sequence. This means that the probes must be sufficiently complementary so as to be able to "specifically hybridize" or anneal with their respective target strands under a set of pre-determined conditions. Therefore, the probe sequence need not reflect the exact complementary sequence of the target. For example, a non-complementary nucleotide fragment may be attached to the 5' or 3' end of the probe, with the remainder of the probe sequence being complementary to the target strand. Alternatively, non-complementary bases or longer sequences can be interspersed into the probe, provided that the probe sequence has sufficient complementarity with the sequence of the target nucleic acid to anneal therewith specifically. The term "primer" as used herein refers to an oligonucleotide, either RNA or DNA, either single-stranded or double-stranded, either derived from a biological system, generated by restriction enzyme digestion, or produced synthetically which, when placed in the proper environment, is able to functionally act as an initiator of template-dependent nucleic acid synthesis. When presented with an appropriate nucleic acid template, suitable nucleoside triphosphate precursors of nucleic acids, a polymerase enzyme, suitable cofactors and conditions such as a suitable temperature and H, the primer may be extended at its 3' terminus by the addition of nucleotides by the action of a polymerase or similar activity to yield a primer extension product. The primer may vary in length depending on the particular conditions and requirement of the application. For example, in diagnostic applications, the oligonucleotide primer is typically 15-25 or more nucleotides in length. The primer must be of sufficient complementarity to the desired template to prime the synthesis of the desired extension product, that is, to be able anneal with the desired template strand in a manner sufficient to provide the 3' hydroxyl moiety of the primer in appropriate juxtaposition for use in the initiation of synthesis by a polymerase or similar enzyme. It is not required that the primer sequence represent an exact complement of the desired template. For example, a non-complementary nucleotide sequence may be attached to the 5' end of an otherwise complementary primer. Alternatively, non- complementary bases may be interspersed within the oligonucleotide primer sequence, provided that the primer sequence has sufficient complementarity with the sequence of the desired template strand to functionally provide a template-primer complex for the synthesis of the extension product. Polymerase chain reaction (PCR) has been described in US Patents 4,683,195, 4,800,195, and 4,965,188, the entire disclosures of which are incorporated by reference herein. The term "vector" relates to a single or double stranded circular nucleic acid molecule that can be infected, transfected or transformed into cells and replicate independently or within the host cell genome. A circular double stranded nucleic acid molecule can be cut and thereby linearized upon treatment with restriction enzymes. An assortment of vectors, restriction enzymes, and the knowledge of the nucleotide sequences that are targeted by restriction enzymes are readily available to those skilled in the art, and include any replicon, such as a plasmid, cosmid, bacmid, phage or virus, to which another genetic sequence or element (either DNA or RNA) may be attached so as to bring about the replication of the attached sequence or element. A nucleic acid molecule of the invention can be inserted into a vector by cutting the vector with restriction enzymes and ligating the two pieces together. Vectors engineered to express nucleic acids encoding proteins having deletions can be generated by providing altered sequence along with flanking sequences of a sufficient length such that cloning into a vector is possible. Such flanking sequences can be between 10, 20, 50, 100, or 200 nucleotides in length. Many techniques are available to those skilled in the art to facilitate transformation, transfection, or transduction of the expression construct into a prokaryotic or eukaryotic organism. The terms "transformation", "transfection", and "transduction" refer to methods of inserting a nucleic acid and / or expression construct into a cell or host organism. These methods involve a variety of techniques, such as treating the cells with high concentrations of salt, an electric field, or detergent, to render the host cell outer membrane or wall permeable to nucleic acid molecules of interest, microinjection, PEG-fusion, and the like. As used herein, the terms "reporter," "reporter system", "reporter gene," or "reporter gene product" shall mean an operative genetic system in which a nucleic acid comprises a gene that encodes a product that when expressed produces a reporter signal that is a readily measurable, e.g., by biological assay, immunoassay, radio immunoassay, or by colorimetric, fluorogenic, chemiluminescent or other methods. The nucleic acid may be either RNA or DNA, linear or circular, single or double stranded, antisense or sense polarity, and is operatively linked to the necessary control elements for the expression of the reporter gene product. The required control elements will vary according to the nature of the reporter system and whether the reporter gene is in the form of DNA or RNA, but may include, but not be limited to, such elements as promoters, enhancers, translational control sequences, poly A addition signals, transcriptional termination signals and the like. The term "selectable marker gene" refers to a gene that when expressed confers a selectable phenotype, such as antibiotic resistance, on a transformed cell. The term "operably linked" means that the regulatory sequences necessary for expression of the coding sequence are placed in the DNA molecule in the appropriate positions relative to the coding sequence so as to effect expression of the coding sequence. This same definition is sometimes applied to the arrangement of transcription units and other transcription control elements (e.g. enhancers) in an expression vector. A "specific binding pair" comprises a specific binding member (sbm) and a binding partner (bp) which have a particular specificity for each other and which in normal conditions bind to each other in preference to other molecules. Examples of specific binding pairs are antigens and antibodies, ligands and receptors and complementary nucleotide sequences. The skilled person is aware of many other examples. Further, the term "specific binding pair" is also applicable where either or both of the specific binding member and the binding partner comprise a part of a large molecule. In embodiments in which the specific binding pair comprises nucleic acid sequences, they will be of a length to hybridize to each other under conditions of the assay, preferably greater than 10 nucleotides long, more preferably greater than 15 or 20 nucleotides long. "Sample" or "patient sample" or "biological sample" generally refers to a sample which may be tested for a particular molecule, preferably an EoE specific marker molecule, such as a marker described hereinbelow. Samples may include but are not limited to cells, body fluids, including blood, serum, plasma, cerebral spinal fluid, urine, saliva, tears, pleural fluid and the like. The terms "agent" and "compound" are used interchangeably herein and denote a chemical compound, a mixture of chemical compounds, a biological macromolecule, or an extract made from biological materials such as bacteria, plants, fungi, or animal (particularly mammalian) cells or tissues. Biological macromolecules include siRNA, shRNA, antisense oligonucleotides, peptides, peptide / DNA complexes, and any nucleic acid based molecule which exhibits the capacity to modulate the activity of the CNV or SNP-containing nucleic acids described herein or their encoded proteins. Agents and compounds may also be referred to as "test agents" or "test compounds" which are evaluated for potential biological activity by inclusion in screening assays described herein below. The term "modulate" as used herein refers to increasing / promoting or decreasing / inhibiting a particular cellular, biological or signaling function associated with the normal activities of the genetic alteration containing molecules described herein or the proteins encoded thereby. For example, the term modulate refers to the ability of a test compound or test agent to interfere with signaling or activity of a gene or protein of the present invention. A "pharmaceutically acceptable excipient, carrier or diluent" refers to an excipient, carrier or diluent that can be administered to a subject, together with an agent, and which does not destroy the pharmacological activity thereof and is nontoxic when administered in doses sufficient to deliver a therapeutic amount of the agent. The following materials and methods are provided to facilitate the practice of the invention. Study design and oversight. The objective of this study was to identify and characterize the molecular basis of milk antigen presentation and recognition in patients with EoE milk allergy. This study complied with research protocols approved by the Institutional Review Board (IRB) for Human Subject Research at the Children’s Hospital of Philadelphia (IRB protocols 18-015524, DAH & 16-013278, HH), the Partners HealthCare at Massachusetts General Hospital (IRB protocol 2011P001159, WS), or the Human Immunology Core at the University of Pennsylvania. Informed consent and subject assent, where applicable, were obtained for all subjects. Study participants and sample acquisition. A subject with biopsy-confirmed, clinically diagnosed EoE milk allergy was identified and recruited for TCR and scRNA sequencing at the Children’s Hospital of Philadelphia. A board-certified pediatric allergist reviewed the subject chart prior to recruitment. EoE was defined in accordance with international diagnostic criteria throughout the manuscript.21Specifically, a subject was determined to have EoE milk allergy if they had a normal upper endoscopy and esophageal biopsy (0-14 eosinophils / hpf) while avoiding milk only and / or an upper endoscopy and esophageal biopsy displaying esophageal eosinophilia (≥15 eos / hpf) having introduced only milk into their diet. A peripheral blood sample (10-20 mL) was obtained from EoE Subject 1 in sodium heparin tubes, transported at room temperature to the lab, and immediately processed for culture. Similar clinical criteria and sample acquisition and processing was used for EoE Subjects 2-16, 51, 154. For lentiviral transfections, peripheral blood was obtained from healthy donors (Controls 1 and 2) and similarly processed. Additional control PBMCs were obtained from an apheresis donor via the Human Immunology Core of the University of Pennsylvania (Control 3). PBMC isolation, stimulation, and blockade. PBMCs were isolated by Ficoll gradient, and the sample from EoE Subject 1, 51 and 154 were divided. Cells destined for flow cytometric analysis were carboxyfluorescein succinimidyl ester (CFSE) labeled (Invitrogen) and cultured at 1 million cells / mL in OpTimizer SFM (Gibco) for 6 days in a 96 well round-bottom plate (Corning) in the absence or presence of Tetanus toxoid (0.625 μg per 200 k cells, Astarte Biologics) or a cocktail of five endotoxin-depleted, purified milk protein preparations (6.25 μg of each of α-lactalbumin, β-lactoglobulin, α-casein, β-casein, and κ-casein per 200 k cells, Millipore Sigma), as previously described.12In HLA restriction assays of 8 patients with EoE milk allergy, blocking antibodies to HLA-DR (L243, BioLegend, 0.5 µg per 200 k cells), HLA-DP (B7 / 21, abcam, 1 µg per 200 k cells), HLA-DQ (SPVL3, Beckman Coulter, 1 µg per 200 k cells), all 3 blocking antibodies, or isotype control antibody to HLA-DR (MOPC-173, BioLegend, 1 µg per 200 k cells) were added to plated PBMCs 30 minutes prior to stimulation with antigens. Cells destined for sequencing from EoE subject 1 were cultured in parallel, without CFSE labeling. PBMCs from controls were isolated by Ficoll gradient or by apheresis prior to sorting of T cells for transduction or differentiation of monocyte derived dendritic cells. Flow cytometric analyses of stimulated PBMCs. After 6 days of culture, CFSE labeled PBMCs were harvested and enumerated. Cells were treated with Live / Dead Fixable Blue (Invitrogen) for 15 minutes at 4°C in the dark, washed, and stained with anti-human CD8 (SK1, BioLegend, 4:100, BV510), CD3 (SK7, eBioscience, 3:100, APC-Cy7, or BD Biosciences, 5:100, BUV395), CD4 (OKT4, BioLegend, 4:100, BV605), CD19 (HIB19, BioLegend, 4:100, AF700, or BioLegend, 4:100, BV711), CD45RA (HI100, BD Biosciences, 4:100, V450, or BioLegend, 4:100, BV786), and CD45RO (UCHL1, BioLegend, 5:100, BV650) for 30 minutes at 4°C in the dark. Cells were washed, fixed (Invitrogen), and permeabilized (Invitrogen), prior to overnight staining for interleukin (IL)-4 (MP4-25D2, BioLegend, 3:100, AF647). Data were acquired on a core maintained LSR Fortessa (BD) within 24 h of harvest. The cytometer was compensated using OneComp eBeads (Invitrogen), unstained cells, CFSE stained cells, and Live / Dead Fixable Blue stained dead cells, as appropriate.1 million events were acquired for each arm. Data were analyzed using FlowJo v8.1 software (Becton Dickinson). Memory CD4+T cells were gated as forward and side scatter low (lymphocytes), Live / Dead negative, CD8−, CD19−, CD3+, CD4+, CD45RA−, CD45RO+. scRNA / TCR-Seq library preparation. After 6 days of culture, PBMCs from EoE Subject 1, EoE Subject 154, and EoE Subject 51 were harvested, enumerated, and the fraction destined for scRNA / TCR-seq analysis were subjected to a bead purification for CD4 and CD45RO (StemCell Technologies; REF 19157). CD4+ / CD45RO+cells from each experimental arm (unstimulated, milk stimulated) were loaded onto separate channels of the 10X Chromium Controller (10X Genomics) according to the manufacturer’s protocol, with a target capture of 10,000 cells per channel. Sequencing libraries were generated using the NextGEM Single Cell 5’ Kit v1.1 and the Chromium Single Cell Human TCR Amplification Kit. Gene expression and feature barcoding libraries were pooled at a ratio of 4:1. Sequencing of pooled libraries was carried out on a NovaSeq 6000 system (Illumina) using one NovaSeq 6000 S1 Reagent Kit v1.5 (100 cycles) with target depth of 25,000 raw reads per cell. scRNA / TCR-Seq Analyses. Sequencing data was quantified using 10x Genomics Cell Ranger 6.1.1,22and aligned to the GRCh38 reference genome. Cell calling was performed using Cell Ranger’s default parameters. Genes detected in fewer than 10% of the cells were removed, and cells that contained fewer than 500 read counts, expression of fewer than 250 genes, or contained more than 25% mitochondrial reads were removed. Counts were normalized using the deconvolution method in the R package scran.23TCR sequence assembly, annotation, and clonotyping was performed using the cellranger VDJ pipeline with default parameters. Clonotypes were defined as a group of cells whose alpha and beta CDR3 regions were sufficiently similar to be considered part of the same lineage (plus cells that only have an alpha or a beta chain), according to the Cell Ranger algorithm. Cells that shared a clonotype with at least one other cell were considered expanded. TCR sequence diversity was computed using the R package immunarch24after downsampling to the same number of cells in milk-stimulated vs unstimulated cultures. Dimensionality reduction and clustering analysis were performed in R using the monocle3 package.25–31Differential expression analysis between clusters and between other experimental groups was also performed using the monocle3 package. The peTH2 signature for each cell was defined as the mean z-score of log-transformed normalized expression of the following 22 genes: IL4, IL5, IL13, PTGDR2, PLA2G16, PTGS2, HPGDS, PPARG, ACADVL, ACSL4, SLC27A2, LPCAT2, DGKE, GK, CHDH, ALOX5AP, GPR15, ICAM2, GATA3, IL17RB, FFAR3, IL1RL1.14Differential expression analysis between cells expressing eoeTCR-4 and cells expressing milk- insensitive TCRs was performed using the LIMMA R package (doi:10.1093 / nar / gkv007). Enrichment analysis on the Hallmark gene sets (10.1016 / j.cels.2015.12.004) between clusters was performed using the roast (10.1093 / bioinformatics / btq401) approach in the LIMMA R package (doi:10.1093 / nar / gkv007). Enrichment was based on a model containing all clusters, using separate comparisons for each cluster vs. the mean gene expression in the remaining clusters. Lentiviral vector generation. Plasmids were synthesized (GenScript) encoding the rearranged TRAV and TRBV sequences cloned upstream of the murine Trac and Trbc constant regions in the lentiviral plasmid, pRLL-MND-GFP (Addgene plasmid #36247), replacing the GFP gene.32Insert sequences were synthesized and cloned using engineered Notl and BstZ17I restriction enzyme sites. Correct sequence and orientation were confirmed Sanger sequencing, gel electrophoresis, and UV spec. The murine Trbc region was utilized for flow-cytometric detection. The CHOP Research Vector core amplified all plasmids and packaged all vectors. Vectors were manufactured at the pilot scale with a third-generation packaging system and concentrated by centrifugation. Viral tiers were assessed in HEK293 cells using a duplexed droplet digital PCR (ddPCR) with the viral packaging signal psi (Ψ) normalized to a housekeeping gene (RPP30), yielding the reported titers in transduction units (TU). Direct inoculation of bioburden plates was used to evaluate sterility. Lentiviral T cell transduction. PBMCs from control subjects were stained with Live / Dead Fixable Aqua (Invitrogen), CD8 (BV510), CD19 (BV711), CD3 (APC-Cy7), CD4 (BV605), CD11c (3.9, Biolegend, 5:100, BV650), CD45RA (BV786), and CD25 (BC96, BioLegend, 5:100, PE). Non-regulatory CD4+T cells were sort purified with a FACSAria Fusion flow cytometer (BD) using the following strategy: Live / Dead Fixable Aqua / CD8 negative, CD19-, CD11c-, CD3+, CD4+, CD25-. The resulting population was majority positive for the naïve marker CD45RA. T cells were suspended at 1 million cells per mL in complete OpTimizer SFM supplemented with rhIL-2 (50 ng / mL, PeproTech) and protamine sulfate (10 µg / mL, MP Biomedicals) and stimulated with anti-CD3 / 28 beads (5 µL per 200 k cells, Gibco). The Multiplicity of Infection (MOI) was determined for each TCR vector by titration. The optimal MOI for all vectors was 20 µL per 200 k cells, resulting in 50-75% transduction efficiency, depending on the clone. rLV was added to culture medium at the same time as anti-CD3 / 28 beads and peak transduction was achieved within 48 hours. Transduced cells were expanded for 10 days, subcultured with fresh media containing rhIL-2 every 2-3 days, after which beads were removed by magnetic separation and cells were enumerated. Cells were rested in the absence of beads and rhIL-2 for 48 hours, then frozen at 10 million cells per mL of freeze medium (Fetal Bovine Serum, 10% Dimethylsulfoxide). Generation of monocyte derived dendritic cells. Control PBMCs were suspended at 3 million cells per mL of OpTimizer SFM supplemented with 10% autologous serum, GM-CSF (55 ng / mL, R&D Systems), and rhIL-4 (0.2 µg / mL, PeproTech), and plated in a 12 well flat-bottom plate. After 48 hours, nonadherent cells were discarded with the culture medium and fresh media with cytokines was added to the monolayer of adherent cells. After 3 days of culture, media was discarded again and fresh media with cytokines was added. After 24 hours, maturation cytokines IFNy (5 ng / mL, R&D Systems) and CL075 (0.4 µg / mL, InvivoGen) were added directly to culture medium. After 24 hours of maturation, monocyte derived dendritic cells (moDC) were harvested by vigorous pipetting, enumerated, and frozen at 1 million cells per mL of freeze medium. Stimulation and flow cytometric analyses of TCR-transduced T cells. Transduced T cells and autologous moDCs were rapidly thawed, resuspended in complete OpTimizer SFM, and enumerated. moDCs were adjusted to 1 million cells per mL of media and evenly distributed to sterile FACS tubes with caps. moDCs were left untreated or antigen loaded by treating with 100 µg / mL of Soy protein extract (Stallergenes Greer), 100 µg / mL α-casein, 100 µg / mL β-casein, 100 µg / mL κ-casein, 100 µg / mL β-lactoglobulin, a cocktail of equal parts of the 3 caseins, 2 µg / mL Tetanus toxoid, 0.1 µg / mL of synthetic β-casein (KanPro Research, Inc, no post-translational modifications), or 0.1 µg / mL of synthetic β-casein peptides (Alan Scientific) for 2 hours at 37°C prior to coculture with transduced T cells. Concurrently, T cells were CFSE labeled, adjusted to 1 million cells per mL of OpTimizer, and plated at 170 k cells per well in a 96 well round bottom plate. Antigen loaded moDCs were washed, resuspended at 1 million cells per mL OpTimizer, and added to wells containing T cells at 30 k cells per well. Some T cells were cultured in the absence of moDCs and left unstimulated or stimulated with 5 µL / well of anti-CD3 / 28 beads as negative and positive controls, respectively. Cultures were incubated at 37°C for 3 days, then harvested and stained with Live / Dead Fixable Blue, CD19 (BV711), CD3 (BUV395), CD4 (BV605), CD8 (BV510), and anti-mouse TCR beta (H57-597, Invitrogen, 5:100, PE). Data were acquired with an LSR Fortessa, analyzed using FlowJo, and gated by Live / Dead Fixable Blue negative, CD19-, CD8-, CD3+, CD4+based on FMO controls. Synthesis of β-casein. Bovine β-casein A2 was cloned and expressed by KanPro Research, Inc. using E. coli. Plasmids were amplified by Inverse PCR. Synthesized gBLOCK was inserted directly between the NdeI and BamHI, linearized by PCR / Restriction digestion, using the one step plasmid by Infusion Cloning. Plasmids harboring β-casein protein were transformed into E coli Bl21(DE3)pRARE. Protein was expressed in LB medium in the presence of ampicillin and chloramphenicol with 0.4 mM IPTG at 370C for 3-5 hours. Cells were pelleted and disrupted by freeze / thaw and sonication. Whole cell, soluble and insoluble fractions were obtained by centrifugation at 195000 rpm for 30 minutes. Fractions of cells were loaded on 10-20% gradient Tris-Glycine SDS-PAGE gel and stained with Instant Blue. β-casein A2 was over-expressed in the form of insoluble fraction as inclusion bodies. The purity of the inclusion bodies was about 80%. Insoluble inclusion bodies of β-casein A2 were dissolved in 50 mM Tris-HCl (pH 8.0) and 6 M urea. The solubilized proteins were loaded on a 5 mL Q HP (GE Healthcare) equilibrated with the same buffer. The protein was eluted from the column with 0-100% gradient Buffer of 1 M NaCl, 50 mM Tris-HCl (pH 8.0) without urea. Fractions of cells were loaded on 10-20% gradient Tris-Glycine SDS-PAGE gel and stained with Instant Blue. β-casein A2 was over-expressed in the form of insoluble fraction, as inclusion bodies can be purified from Q column. The purity of the inclusion bodies was >95%. Recombinant β- casein A2 purified in 50 mM Tris-HCl (pH 8.0) and about 150 mM NaCl by the ion exchange chromatography were further purified with Superdex S75 size exclusion column equilibrated with 20 mM Tris-HCl (pH 8.0) and 150 mM NaCl. β-casein A2 purified from Q column can be eluted from S75 as a single peak. The purity of the inclusion bodies was >95%. Fractions of first peak eluted from 5 runs were pooled, flash frozen in liquid nitrogen, and stored at -800C. >95% pure, recombinant bovine β-casein A2 fractions were delivered at 1.1-3.5 mg / mL in 20 mM Tris-HCl (pH 8.0) and 150 mM NaCl. Synthesis of β-casein peptides. Peptides spanning the length of bovine β-casein from amino acid residue 59-224, encompassing the HLA-DRB1*07:01 binding sites predicted by NetMHCIIpan- 4.0, were generated via Solid-Phase Peptide Synthesis by Alan Scientific Inc. Synthesis of a large peptide spanning the first 56 residues of β-casein was attempted unsuccessfully, and because it did not contain any predicted binding sites, no further attempts to synthesize the region were made. Purity of all delivered peptides was 95%, and no N or C terminal modifications were added. Lyophilized peptides were reconstituted in DPBS -Ca / -Mg at 1 µg / µL and stored at -80°C. HLA restrictions. To test the HLA restriction of eoe-TCR4, moDC were incubated with synthetic β-casein at 0.1 µg / mL following 30 minutes of preincubation with either anti-HLA-DR (100 µg / mL), anti-HLA-DP (100 µg / mL), anti-HLA-DQ (100 µg / mL), all 3 blocking antibodies, or isotype control to anti-HLA-DR (100 µg / mL). After 2 hours of incubation with antigen in the absence or presence of blocking antibodies, moDCs were washed and cocultured with CFSE labeled T cells transduced with eoeTCR-4. To test the restriction of synthetic β-casein presentation by EoE Subject 1 PBMCs, anti-HLA-DR (L243, BioLegend, 0.5 µg per 200 k cells) was added to an arm of the synthetic β-casein (6.25 μg per 200 k cells) culture condition. Antigen binding prediction. Binding sites were predicted for EoE Subject 1’s HLA-DRB1 alleles, 07:01 and 11:04, for each of the five major milk proteins (including both subunits of α- casein) using the Technical University of Denmark’s NetMHCIIpan-4.0 tool. NetMHCIIpan-4.0 is an HLA class II bind prediction server freely available online.33Milk protein sequences were submitted in FASTA format, peptide length was set to 15 amino acids, the threshold for a strong bind was set to <1 %Rank, and the threshold for a weak bind was set to <5 %Rank. Server output was displayed as a heatmap for each protein in order of predicted bind position. Mass spectrometry data acquisition. Each of the five Millipore Sigma purified bovine milk proteins, endotoxin depleted and adjusted to 5 µg / µL in DPBS -Ca / -Mg, were submitted to the CHOP Proteomics Core for compositional analysis by Mass Spectrometry. Proteins were solubilized and digested with the iST kit (PreOmics GmbH, Martinsried, Germany) per manufacturers protocol.34The resulting peptides were desalted, dried by vacuum centrifugation and reconstituted in 0.1% TFA containing iRT peptides (Biognosys Schlieren, Switzerland). Peptides were analyzed on a QExactive HF mass spectrometer (ThermoFisher Scientific San Jose, CA) coupled with an Ultimate 3000 nano UPLC system and an EasySpray source using data dependent acquisition (DDA). Peptides were loaded onto an Acclaim PepMap 10075um x 2cm trap column (Thermo) at 5uL / min and separated by reverse phase (RP)-HPLC on a nanocapillary column, 75 μm id × 50cm 2um PepMap RSLC C18 column (Thermo). Mobile phase A consisted of 0.1% formic acid and mobile phase B of 0.1% formic acid / acetonitrile. Peptides were eluted into the mass spectrometer at 300 nL / min with each RP-LC run comprising a 90-minute gradient from 3% B to 45% B. The mass spectrometer was set to repetitively scan m / z from 300 to 1400 (R = 240,000) followed by data-dependent MS / MS scans on the twenty most abundant ions, minimum AGC 1e4, dynamic exclusion with a repeat count of 1, repeat duration of 30s, and resolution of 15000. The AGC target value was 3e6 and 1e5, for full and MSn scans, respectively. MSn injection time was 160 ms. Rejection of unassigned and 1+,6-8 charge states was set. Mass spectrometry system suitability, quality control, and data analysis. The suitability of Q Exactive HF instrument was monitored using QuiC software (Biognosys, Schlieren, Switzerland) for the analysis of the spiked-in iRT peptides. Meanwhile, as a measure for quality control, we injected standard E. coli protein digest prior to and after injecting sample set and collected the data in the Data Dependent Acquisition (DDA) mode. The collected DDA data were analyzed in MaxQuant35and the output was subsequently visualized using the PTXQC36package to track the quality of the instrumentation. MS / MS raw files were searched against a bos taurus protein sequence database including isoforms from the Uniprot Knowledgebase (taxonomy:9913, 37501 entries) using MaxQuant version 1.6.14.0 with the following set parameters: Fixed modifications, Carbamidomethyl (C); Decoy mode, revert; MS / MS tolerance FTMS 20 ppm; false discovery rate for both peptides and proteins, 1.0; Minimum peptide Length, 7; Modifications included in protein quantification, Acetyl (Protein N-term), Oxidation (M); Peptides used for protein quantification, Razor and unique. Scaffold (version Scaffold_5.1.0, Proteome Software Inc., Portland, OR) was used to validate MS / MS based peptide and protein identifications. Peptide identifications were accepted if they could be established at greater than 95.0% probability by the Percolator posterior error probability calculation.37Protein identifications were accepted if they could be established at greater than 99.0% probability and contained at least 1 identified peptide. Protein probabilities were assigned by the Protein Prophet algorithm.38Proteins that contained similar peptides and could not be differentiated based on MS / MS analysis alone were grouped to satisfy the principles of parsimony. Proteins sharing significant peptide evidence were grouped into clusters. Total Spectrum Counts were exported from Scaffold and the 218 identified proteins across all five samples were grouped into categories: one of the expected milk proteins (α-casein subunits 1 or 2, β-casein, κ-casein, α-lactalbumin, or β-lactoglobulin), other bovine proteins, the mass spectrometry control protein, or non-bovine contaminants. HLA genotyping and enrichment analysis. EoE Subjects 1-27, Control 1, and Control 2’s HLA types were determined by 11-loci Next Generation Sequencing at the American Red Cross HLA Laboratory in Philadelphia, PA. Control 3 was HLA typed by the same method at The Clinical Immunology and HLA Immunogenetics Laboratory at Hospital of the University of Pennsylvania. Enrichment analysis was performed by One Sample Proportion Test analysis with the following parameters: n=27, ƒ=12, H0: P=0.2684, Hα: Positive one-tail, α = 0.05. Characterization of CDR3 regions by high-throughput sequencing. Genomic DNA was extracted from whole blood using a standard protocol. Whole exome sequencing was performed at the Center for Applied Genomics of the Children’s Hospital of Philadelphia. Paired-end sequencing was performed on the Illumina NovaSeq 6000 platform (Illumina, San Diego, CA), using an S4 flowcell with run parameters of 101 x 10 x 10 x101 [Read 1 x Index 1 (i7) x Index 2 (i5) x Read 2]. All sequencing experiments adhered to quality control standards of depth of coverage, read quality, and error rates. The characterization of TRA and TRB CDR3 regions was performed using raw sequencing reads in FASTQ format with miTCR.39A threshold of a minimum quality score of 25 for Illumina sequencing reads was used to ensure that only high-quality reads are used for inferring TCRs. Comparison of CDR3 regions by scTCR-seq. We computed pairwise amino acid sequence similarity of 43,282 TCRB and 27,534 TCRA sequences from esophageal tissue and PBMC samples obtained from patients with EoE milk allergy. This was achieved by generating Levenshtein distance matrices of the CDR3 sequences using the stringdist package in R.40The results were confirmed by computing weighted multi-CDR distances between TCRs using tcrdist3, a Python3 package for TCR repertoire analysis, following the procedure described in Mayer- Blackwell et al., 2021.41Statistics: In HLA restriction assays of PBMCs from EoE Subjects 2-9, ANOVA with Bonferroni’s correction for multiple comparisons was performed for the unstimulated versus Tetanus Toxoid stimulated conditions, the unstimulated versus milk antigen stimulated with isotype control antibody conditions, the milk stimulated with isotype control antibody versus milk stimulated with anti-HLA-DR antibody conditions, and the milk stimulated with isotype control antibody versus milk stimulated with anti-HLA-DQ conditions. Data availability. Data will be deposited in a public repository and accession code will be made available before final publication. The following examples are provided to illustrate certain embodiments of the invention. They are not intended to limit the invention in any way. Example I – Identification of TCR clonotypes associated with EoE milk allergy To begin our search for a food specific T cell, we collected peripheral blood samples from a subject with clinically confirmed EoE caused by exposure to cow’s milk (EoE milk allergy) (EoE Subject 1, Fig. 1A). EoE Subject 1 had a “classic” presentation having developed EoE in early childhood with multiple subsequent periods of milk removal and reintroduction with corresponding remission and reactivation of his EoE (Fig. 1A). We then performed scRNA-seq with linked, paired TRA and TRB sequencing (scTCR-seq) to determine the activation states and TCR clonotypes of TH cells that expanded upon milk protein stimulation (Fig. 1B and Fig. 8) The same was carried out using PBMCs from EoE Subject 154 (Fig. 9) and EoE Subject 51 (Fig. 10). Milk protein stimulation of peripheral blood mononuclear cells (PBMCs) from EoE Subject 1 caused a subset of CD4+CD45RO+memory THcells to proliferate and produce IL-4 (Fig. 1C, 1D and data not shown).12THcell expansion was also demonstrated by our scTCR-seq analysis (Fig. 1E). To quantitate clonal expansion, we downsampled to equivalent numbers of cells per condition and determined the proportion of cells expanded and their TCR diversity using the inverse Simpson’s diversity index. This analysis confirmed increased clonal expansion and reduced TCR diversity upon milk stimulation (Fig. 1F). In addition, more unique TCRs were enriched in milk-stimulated cultures than in unstimulated cell cultures (FDR ≤ 0.05, Fig. 1G). Previous scRNA-seq analysis of esophageal biopsies from milk allergic EoE patients identified clonally expanded pathogenic effector TH2 cells (peTH2).14We therefore examined expression of peTH2 genes in our datasets. As anticipated, clonally expanded cells from milk- stimulated cultures had the highest expression of peTH2 genes compared with other conditions (Fig. 1H). To maximize our likelihood of identifying a milk-specific TCR from a pathogenic cell, we concentrated our validation efforts on milk-expanded clonotypes that expressed the peTH2 gene signature. Among the 20 most expanded TCR pairs in milk-stimulated culture from EoE Subject 1 (Table 1), we detected high expression of the peTH2 gene signature in several clonotypes (Fig. 1I and Fig. 1J). Of these, we chose eoeTCR-2 (TRA CVVVGRTGGFKTIF (SEQ ID NO: 6)), 4, 6, and 7 for functional testing based on their uniqueness from one-another, absence of predicted specificities, and the peTH2 gene signature of the related T cell clones. TABLE I TRA AND TRB GENE USAGE FOR THE TOP 20 MILK EXPANDED CLONOTYPES To create an expression system to test the specificities of TCRs, we had to determine the HLA class II molecule(s) responsible for milk protein presentation. To do so, we recruited eight additional subjects with EoE milk allergy (EoE Subjects 2-9, (Table 2) and determined HLA restrictions by adding HLA-blocking antibodies (anti-HLA-DR, DQ, and -DP) to PBMC milk stimulation cultures.12HLA-DR blockade abrogated milk-dependent T cell proliferation for most subjects (Fig. 1K), suggesting that HLA-DR is responsible for a majority of milk peptide presentation to T cells in EoE milk allergy. We next determined the HLA-DRB1 genotype of EoE Subject 1 (07:01, 11:04) and found a control donor with a near HLA-DRB1 match (07:01, 11:01) (Control 1, Table 2). CD4+THcells and autologous monocyte-derived dendritic cells (moDCs) from Control 1 were used as a TCR expression and testing system to establish the specificity of TCR clonotypes of interest.

[0002] TABLE 2- DEMOGRAPHICS AND HLA GENOTYPING OF SUBJECTS TCR clonotypes were transduced into sort purified CD25- conventional (Tconv) cells from Control 1 PBMCs using lentiviral vectors in which the recombined TRA and TRB V- CDR3-J region was cloned upstream of the murine Trac and Trbc region gene segments (Fig. 2A).32We then tested transduced Tconv cells for proliferation when cultured in the presence of protein pulsed autologous moDCs. Tconvcells expressing eoeTCR-2 (TRA CVVVGRTGGFKTIF), 6, and 7 were not reactive to milk proteins (Fig. 2B and not shown). However, Tconv cells expressing eoeTCR-4 showed robust proliferation when cultured in the presence of moDCs pulsed with α, β, or κ-casein, but not β-lactoglobulin, soy protein extract, or tetanus toxoid (Fig. 2C). These results indicate that eoeTCR-4 is casein-specific. We considered if the observed cross-reactivity of eoeTCR-4 to α, β, and κ-casein was due to epitope conservation across the proteins. However, machine learning predictions of potential binding sites for EoE Subject 1’s HLA-DRB1 alleles suggested that epitopes from κ-casein were unlikely to be presented by either DRB1*07:01 or 11:04 (Fig. 2D). Alternatively, we considered the presence of cross-contamination of the caseins in the protein preparations we were using in culture. Indeed, all three casein products contained α-casein subunit 1 and 2, β-casein, and κ- casein, while the whey protein β-lactoglobulin was not contaminated with casein (Fig. 2E). We next performed a limiting dilution of each of the casein products and found that β-casein caused proliferation of eoeTCR-4+T cells at low concentrations while α and κ-casein did not (Fig. 2F). Together, these data identify β-casein as the most likely target of eoeTCR-4. To confirm this hypothesis, we obtained synthetic β-casein without post-translational modifications (sβ-casein) and compared stimulation of eoeTCR-4+T cells with purified β-casein to sβ-casein. Similar responses from transduced cells to both native and synthetic β-casein established that β-casein contains the cognate antigen of eoeTCR-4 (Fig. 2G). We next sought to determine the HLA-DR allelic restriction for eoeTCR-4. To do so, we first confirmed HLA-DR restriction of eoeTCR-4 using sβ-casein and HLA class II blocking antibodies, showing that only anti-DR antibody blocked the response of eoeTCR-4 to sβ-casein (Fig. 3A). In addition, we confirmed that memory TH cells isolated from EoE Subject 1 were reactive to synthetic β-casein, and that the synthetic β-casein proliferative response of these TH cells was dependent on HLA-DR (Fig. 3B). Finally, we recruited three additional control subjects that shared either the HLA-DRB1*07:01 allele of EoE Subject 1 (Control 2), approximated the HLA-DRB1*11:04 allele of EoE Subject 1 and was positive for HLA- DRB3*02:02 (Control 3), or was positive for the HLA-DRB4*01:03 allele in the absence of HLA-DRB1*07:01 (Control 4) (Table 2). Culture of eoeTCR-4 transduced Tconv cells with β- casein pulsed autologous moDCs from Control 2 (07:01, 13:01) resulted in T cell proliferation (Fig. 4A), while culture of eoeTCR-4 transduced Tconvcells with β-casein pulsed autologous antigen presenting cells (APCs) from Control 3 (DRB1*11:03, DRB3*02:02) or Control 4 (DRB4*01:03) did not (Fig. 4B, 4C). Together, these data indicate that eoeTCR-4 is restricted to HLA-DRB1*07:01. Having established that eoeTCR-4 is specific for β-casein and restricted to HLA- DRB1*07:01, we next sought to determine its cognate antigen. To do so, we attempted to synthetize a peptide library that spanned the entirety of β-casein (Fig. 5A). Unfortunately, we were unable to synthesize the first 56 amino acids of the protein. However, we were able to synthesize an overlapping peptide library of 9 peptides the spanned amino acids 59-224 and contained all three of the predicted β-casein binding sites (Fig. 5A and Table 3). We then tested reactivity for each of these peptides with eoeTCR-4 using our lentiviral transduction system. We found that peptide 1, which contained the DRB1*07:01 predicted binding site 1, caused specific proliferation of eoeTCR-4 transduced T cells (Fig. 5B), while the remaining peptides did not (Fig. 5B and Fig. 5C). Together, these data identify β-casein peptide 1 as containing the cognate antigen for eoeTCR-4. TABLE 3 β-CASEIN PEPTIDE LIBRARY AND REACTIVITY WITH EoE TCR-4 Peptide Amino Sequence (SEQ ID NO) Length Notes (SEQ ID NO) Reactivity acids with 150 FTESQSLTL (540) e nex soug o de erm ne e ex en o w c eoe C - and - 0 :0 are shared among patients with EoE milk allergy. For our eoeTCR-4 analysis, we used a two- pronged approach. First, we inferred TRA and TRB sequences from whole exome sequencing data of 132 unique patients with EoE milk allergy (Figs. 8, 9 and 10).42–44Among these patients, we did not observe a nucleotide or amino acid sequence match for either the TRA or TRB gene sequence of eoeTCR-4. Second, we examined a set of single cell TCR sequences from 21 patients, enriched for potential specificity (e.g., CD154+following in vitro stimulation, expansion in tissue during active EoE, or expression of CD161+CRTH2+in circulation) from subjects with EoE milk allergy.14Using homology searching (Levenshtein distance, not shown; tcrdist3, Fig. 5D), we did not detect eoeTCR-4 or related sequences. For our HLA-DRB1*07:01 analysis, we HLA genotyped EoE Subjects 2-9 plus an additional 19 patients with EoE milk allergy (EoE Subjects 10-27, Table 2). Via this analysis we found that HLA-DRB1*07:01 was significantly enriched in patients with EoE milk allergy (P=0.0195), being present in 12 of 27 (44%) of subjects. Finally, we sought to understand the extent to which there were unique transcriptional features of our milk-expanded clonotypes. We first performed unbiased clustering of CD4+T cells from both unstimulated and milk stimulated cultures (Fig. 6A and Fig. 6C). We then overlayed clone groups based on their culture condition and expansion state, specifically noting the 20 most expanded clones in milk-stimulated culture as well as eoeTCR-4 clones (Fig. 6B and Fig. 6D). We noted that cluster 5 contained the highest proportion of milk-expanded clones, including eoeTCR-4. Cluster Hallmark gene set enrichment analysis revealed enrichment for interferon alpha and gamma response genes in cluster 5 as well as clusters 3 and 8, along with a generally pro-inflammatory transcriptome in the latter clusters (Fig. 6E, Fig. 6F). In addition, we performed a transcriptional analysis of cells expressing eoeTCR-4 as compared with cells from other conditions and expansion states. We found that cells expressing eoeTCR-4 were most similar to other expanded cells from the milk condition (Fig. 6G). Finally, we performed a transcriptional analysis of cells expressing eoeTCR-4 as compared with milk-expanded cells expressing TCR clonotypes that we established to be non-milk-specific (eoeTCR-6 and 7). Of note, cells expressing the eoeTCR-2 clonotype were not included in this analysis as we only tested one of the two expressed TRA chains. This final analysis revealed that cells expressing eoeTCR-4 had a unique transcriptome, with notably elevated expression of genes related to activation (TMSB4X, S100A4), cytotoxicity (SRGN), exhaustion (CAPG, S100A10), and regulation (NDFIP1) (Fig. 7). DISCUSSION The data provided herein provide the first molecular details of food allergen presentation and recognition by T cells in EoE, including the molecular identity of an EoE antigenic epitope. Building from seminal studies of the EoE T cell repertoire from the past few years,12–15and taken in context with early studies indicating a very high response rate of EoE to elemental and milk elimination diets,45–47our findings suggest that a large majority of EoE is likely caused by food antigen-specific activation of T cells. Our findings support decades of clinical practice focused on food allergen identification and elimination in EoE. Perhaps most notably, we find that the peTH2 transcriptional signature was preserved in milk-expanded circulating memory T cells, including cells expressing eoeTCR- 4. Our work also identifies novel features of the EoE T cell. For example, we find that circulating T cells that expanded upon milk stimulation were more likely to show transcriptional evidence of having responded to interferons. This finding is consistent with our recent observation that there is a conserved interferon (IFN) response gene signature in inflamed mucosa of children and adults with EoE.48While it is tempting to conclude that this finding is a result of IFNγ producing T cells in our culture system,48it is notable that the IFN signature was specifically enriched in milk-expanded cells and therefore not a general feature of T cells in culture. An alternative hypothesis is that presence of this IFN response gene signature is indicative of these T cells having circulated through the inflamed esophageal mucosa. We also observe specific upregulation of genes related to activation (TMSB4X, S100A4), cytotoxicity (SRGN), exhaustion (CAPG, S100A10), and regulation (NDFIP1) in eoeTCR-4 clones compared to the non-milk-specific clones eoeTCR-6 and 7. Of these, S100A4 expression may be of particular relevance as a means to identify food-specific T cell clones as it has been shown to promote allergic T cell responses and be elevated in EoE.49,50Similarly, Ndfip1 is induced when T cells respond to innocuous foreign or self-antigen that should normally be tolerated.51Ndfip1 has also been shown to play a role in suppressing IL-4 production in TH2 cells, limiting their effector function,52and to force TH2 cell exit from the cell cycle and therefore may be a critical counterpart to T cell anergy (which is potentially consistent with the observed upregulation of CAPG and S100A10).51It is therefore reasonable to hypothesize that this constellation of findings is consistent with a (failed) attempt at promoting tolerance by limiting peTH2 expansion and effector function. Our findings also have broad implications for the development of antigen specific next- generation diagnostics, biomarkers, and therapeutics for EoE. For example, we found that milk antigen presentation in EoE milk allergy is mediated by HLA-DRB1 in 8 out of 9 subjects tested, and specifically by HLA-DRB1*07:01 in EoE Subject 1. Further, we found that HLA- DRB1*07:01 is present in 44% of patients with EoE milk allergy. While HLA-DRB1*07:01 is common in people of self-identified white race (20-30%), it is less common in non-white populations (13-18%).53These findings simultaneously raise the potential future utility of HLA genotyping as a risk-stratification approach for EoE patients and highlight the need for additional research aimed at defining HLA restrictions of other EoE food-specific TCRs. Though efforts are underway to develop and implement functional T cell assays to detect food allergen-activated T cell responses in the circulation of EoE patients,11,12clinical diagnostics to aid in the identification of EoE-causal foods are not currently available. Understanding the TCR repertoire of food antigen recognition in EoE holds the potential to facilitate the development of tetramer-based assays that simultaneously and directly detect the presence of pathogenic T cell populations for multiple foods.54In addition to aiding the development of personalized elimination diets, the ability to detect and monitor allergen-specific T cell responses may be a useful prognostic tool to guide risk assessments and help with timing of food allergen reintroduction. The present data enable characterization of the different T cell functional profiles (pathogenic effector vs regulatory) which appear to have distinct TCR repertoires and antigen epitope specificities—information directly relevant to applications of oral immunotherapy designed to impair pathogenic and / or boost regulatory immune responses. Further, identification of the molecular details of antigen specificity in food allergy facilates the development of cell- specific therapeutics designed to delete peTH2 cells while leaving food-specific regulatory T cells and unrelated THcells intact. In summary, we provide the first report of a molecular basis for food allergen presentation and recognition in EoE. While not designed to provide an exhaustive evaluation of the EoE milk allergy TCR repertoire, our study provides a clear “proof of concept”. EXAMPLE 2 – Tetramer-based diagnostics Tetramer-based and similar diagnostics utilize tetramers (or related molecules), which are complexes formed by identical molecules, typically biotinylated peptide-MHC (major histocompatibility complex) molecules bound to a streptavidin scaffold. These tetramers are designed to bind specifically to T-cell receptors (TCRs) on the surface of T cells, allowing for the precise identification and quantification of antigen-specific T cells. The development of a tetramer requires detailed knowledge of the human leukocyte antigen (HLA) allele, the peptide antigen that is presented by the HLA molecule, and the specificity of the TCR that recognizes this peptide-HLA complex. This specificity is crucial for ensuring that the tetramer binds only to the intended T cells, thereby providing accurate diagnostic information. A tetramer can be designed by identifying the relevant HLA allele, which is the genetic variant of the MHC molecule that presents the peptide antigen. Next, the peptide antigen, (typically a short sequence of amino acids derived from a pathogen or allergen), is selected. This peptide is then loaded onto the HLA molecule. Finally, the specificity of the TCR that recognizes the peptide-HLA complex is determined. This involves understanding the unique interaction between the TCR and the peptide-HLA complex, ensuring that the tetramer will bind specifically to T cells expressing the corresponding TCR. This precise binding allows for accurate detection and analysis of antigen-specific T cells in various diagnostic applications. Tetramer-based diagnostics have significant applications in infectious disease, cancer immunotherapy, and autoimmune disease, where they can be used to advantage for tracking the immune response to pathogens, tumors, or self-antigens, respectively. The same applies to the field of food allergy, where they can be used to identify T cells specific to food allergens, aiding in diagnosis and monitoring of allergic responses. Beyond food allergy, these diagnostics and applications can be extended to personalized medicine, where tetramers could be used to tailor food allergy treatments based on an individual’s specific T-cell repertoire, thereby enhancing the efficacy and safety of therapeutic interventions. EXAMPLE 3 – Oral immunotherapy and next-generation strategies Oral immunotherapy (OIT) is a medical treatment designed to desensitize individuals with food allergies by gradually introducing small, controlled amounts of the allergenic food into their diet. The process begins with very low doses of the allergen, which are incrementally increased over time under medical supervision. This gradual exposure aims to retrain the immune system to tolerate the allergen, thereby reducing the severity of allergic reactions or potentially eliminating the allergy altogether. OIT works by modulating the immune response, promoting the development of regulatory T cells (Tregs) that suppress the allergic response and increasing the threshold at which an allergic reaction occurs. While milk antigens are exemplified herein, the present technology can be used to diagnose and treat most food allergies, e.g., allergies to gluten, wheat, egg, soy, peanut, etc. Useful peptide antigens for diagnosis of a variety of different food allergies are provided in Table 4 below and can be found in PCT / US2023 / 080121 which is incorporated by reference herein. Next-generation OIT applications leverage the advanced knowledge of food cognate antigens and antigen-specific T cell responses to enhance the safety and efficacy of the treatment. By identifying specific epitopes, e.g., the precise parts of the allergen recognized by the immune system, targeting in OIT protocols can be improved. Modified allergens or peptide fragments that retain immunogenicity but have reduced allergenicity are administered, minimizing the risk of adverse reactions during therapy. Additionally, understanding the T cell responses involved in food allergies allows for the monitoring and modulation of these responses, potentially through the use of adjuvants or immune modulators that enhance the induction of Tregs and other protective immune mechanisms. Finally, next-generation OIT can incorporate personalized approaches based on an individual’s unique immune profile. By analyzing the specific T cell receptors (TCRs) and HLA alleles involved in their allergic response as described in Example 1, tailored OIT regimens can now be developed to optimize desensitization. This personalized approach could also involve the use of biomarkers to monitor the effectiveness of the therapy (e.g., presence or absence of food specific TCRs, such as EOE-TCR4, eoe1TCR-5, and eoe2TCR-1; the peTh2 biomarkers present in the genetic signature associated with milk induced EOE described in Example 1) and the treatment protocol adjusted as needed. Such advancements in OIT should significantly improve the safety and success rates of food allergy treatments, offering a more precise and individualized method for managing and potentially curing food allergies. Table 4 F P t i E ID NO: κ-casein MMKSFFLVVTILALTLPFLGAQEQNQEQPIRCEKD 545 ERFFSDKIAKYIPIQYVLSRYPSYGLNYYQQKPVAL EEVIQHTFNLKSQQARQIKNNNPFKFLVPPQESQK RAVA Glycine (Gy4) MGKPFTLSLSSLCLLLLSSACFAISSSKLNECQLNN 551 LNALEPDHRVESEGGLIQTWNSQHPELKCAGVTVS VAEQGGEQGLEYVVFKTHHNAVSSYIKDVFRVIPS EVLSNSYNLGQSQVRQLKYQGNSGPLVNP PGSAQDVERLLKKQRESYFVDAQPQQKEEGSKGR KGPFPSILGALY Ĵ7 Wheat Gliadin MKTFLILALLAIVATTARIAVRVPVPQLQPQNPSQQ 559 (Alpha / Beta) QPQEQVPLVQQQQFPGQQQPFPPQQPYPQPQPFPS QGQQGQQPGQGQQGQQPGQGQPGYYPTSPQQSG QGQQPGQWQQPGQGQPGYYPTSPLQPGQGQPGY RGQGEREEEQGRGRGRRGEGERDEEHGDGRRPYV FGPRSFRRIIRSDHGFVKALRPFDEVSRLLRGIRNY SGISSAESLKISQAVHAAHAEINEAGREVVGSAEA GVDAASVSEEFRADHPFLFCIKHIATNAVLFFGRC YQDFNIKIRRSDKNSHLIYFTVTTDGVILEVKETGIT VNGNQIPLPFSLKSILIEDTCAYFQVTSKLGLTLKW PQGKCGNILYSLEGCYPECSPDKPYFDEERRECVSL PDCTSCNPEEKLCTEDSKDCLCCYNGKTYPLNETI MWNNEDYLMVLTEKKYMGKTCGMCGNYDGYEL NDFVSEGKLLDTYKFAALQKMDDPSEICLSEEISIP Alpha-livetin MKWVTLISFIFLFSSATSRNLQRFARDAEHKSEIAH 571 RYNDLKEETFKAVAMITFAQYLQRCSYEGLSKLV MVIVVINKGTGNLELVAVRKEQQQRGRREQEWEE EEEDEEEEGSNREVRRYTARLKEGDVFIMPAAHPV EXAMPLE 4 - HLA-based screens Disease screening techniques that utilize information on specific HLA risk alleles involve identifying genetic markers associated with an increased risk of developing certain diseases. Current applications of these techniques include screening for autoimmune diseases such as type 1 diabetes, celiac disease, and rheumatoid arthritis. For example, individuals with the HLA-DQ2 or HLA-DQ8 alleles are at a higher risk for celiac disease. Screening involves genotyping these alleles to identify individuals who may benefit from further diagnostic testing or early intervention. This approach allows for the identification of at-risk individuals before clinical symptoms appear, enabling proactive management and potentially preventing disease progression. Applications of HLA-based screening in the context of food allergy involve identifying individuals with specific HLA alleles that predispose them to allergic reactions to certain foods. Understanding and characterizing the genetic basis of food allergies, enables the development of personalized screening protocols that identify at-risk individuals early in life. Such information should facilitate implementation of preventive measures, such as dietary modifications or early introduction of allergenic foods under medical supervision, to reduce the likelihood of developing severe allergies. Additionally, HLA-based screening could be used to identify individuals who may respond favorably to oral immunotherapy (OIT), thereby optimizing treatment outcomes. In the context of eosinophilic esophagitis (EoE), HLA-based screening could identify individuals with genetic susceptibility to the condition, including individuals who are at risk of EoE as a side-effect of OIT. Screening for EoE risk HLA alleles facilitates early diagnosis and intervention. Such applications include the development of targeted therapies that modulate the immune response based on an individual’s HLA genotype, thereby improving treatment efficacy, and reducing disease burden. As shown in Example 1, understanding the HLA-related genetic predisposition to EoE leads to the identification of novel biomarkers for disease monitoring and the development of personalized treatment strategies. EXAMPLE 5 – Validation of milk-expanded TCR clonotypes To further establish the generalizability and feasibility of the approach described above, additional milk-expanded TCR clonotypes from EoE Subject 1 were examined. The data indicates that eoe1TCR-5 (α-chain / β-chain)(CALSDQGAQKLVF(SEQ ID NO: 576) / CASSWVLGGSYEQYF(SEQ ID NO: 577)) is also specific for β-casein and restricted to HLA-DRB1*07:01 (Fig. 11). In addition, milk-expanded, peTH2 gene-expressing, TCR clonotypes from an unrelated individual with EoE milk allergy that lacked DRB1*07:01 (EoE Subject 2) was examined. The data indicates that TN cells expressing eoe2TCR-1 (α-chain / β- chain) (CAVEKDPRTGGFKTIF(SEQ ID NO: 578) / CASSYGERGGNEQFF(SEQ ID NO: 579)) proliferated in response to moDCs antigen loaded with α-casein, but not α-lactalbumin or β- lactoglobulin (Fig. 12A). Subsequent limiting dilutions established eoe2TCR-1 specificity for α- casein (as opposed to β- or κ-casein, Fig. 12B). Of note, eoe2TCR-1 showed no homology to eoe1TCR-4 or eoe1TCR-5. These findings establish the generalizability of our approach by identifying two additional milk-specific TCRs, one from an unrelated EoE patient. We next assessed whether eoe1TCR-4, eoe1TCR-5, or eoe2TCR-1 were conserved among EoE milk allergy patients by inferring TCR sequences from whole-exome data across 132 unique individuals with EoE milk allergy.42-44While no identical matches were found, near- matches to the α-chain of eoe1TCR-5 (CALSDQGAQKLVF (SEQ ID NO: 576)) were observed in three subjects, each differing by 2–3 amino acids (similarity 0.86–0.88). Further, the β-chain of eoe1TCR-5 (CASSWVLGGSYEQYF(SEQ ID NO: 577)) exhibited near-matches in two additional subjects, also with 2–3 amino acid differences (similarity 0.86–0.87). No matches were observed in a control cohort (n = 200). These homology relationships indicate that eoe1TCR-5 is a semi-public clonotype, with sequence variants recurring across individuals and potential conservation of antigen specificity. REFERENCES 1. Landres, R. T., Kuster, G. G. & Strum, W. B. Eosinophilic esophagitis in a patient with vigorous achalasia. Gastroenterology 74, 1298–1301 (1978). 2. Straumann, A., Spichtin, H. P., Bernoulli, R., Loosli, J. & Vögtlin, J. Idiopathic Eosinophilic Esophagitis: A Frequently Overlooked Disease with Typical Clinical Aspects and Discrete Endoscopic Findings. Schweiz Med Wochenschr 124, 1419–1429 (1994). 3. Furuta, G. T. & Katzka, D. A. Eosinophilic Esophagitis. N Engl J Med 373, 1640–1648 (2015). 4. Münch, R., Kuhlmann, U., Makek, M., Ammann, R. & Siegenthaler, W. [Eosinophilic esophagitis, a rare manifestation of eosinophilic gastroenteritis]. Schweiz Med Wochenschr 112, 731–734 (1982). 5. Chang, J. W. et al. Development of a Practical Guide to Implement and Monitor Diet Therapy for Eosinophilic Esophagitis. Clin Gastroenterol Hepatol 21, 1690–1698 (2023). 6. Nicholson, A. G., Li, D., Pastorino, U., Goldstraw, P. & Jeffery, P. K. Full thickness eosinophilia in oesophageal leiomyomatosis and idiopathic eosinophilic oesophagitis. A common allergic inflammatory profile? J Pathol 183, 233–236 (1997). 7. Straumann, A., Bauer, M., Fischer, B., Blaser, K. & Simon, H. U. Idiopathic eosinophilic esophagitis is associated with a T(H)2-type allergic inflammatory response. J.Allergy Clin.Immunol. 108, 954–961 (2001). 8. Mishra, A., Schlotman, J., Wang, M. & Rothenberg, M. E. Critical role for adaptive T cell immunity in experimental eosinophilic esophagitis in mice. J Leukoc Biol 81, 916–924 (2007). 9. Noti, M. et al. Thymic stromal lymphopoietin-elicited basophil responses promote eosinophilic esophagitis. Nat Med 19, 1005–1013 (2013). 10. Cianferoni, A. et al. Elevated expression of activated TH2 cells and milk-specific TH2 cells in milk-induced eosinophilic esophagitis. Ann Allergy Asthma Immunol 120, 177-183.e2 (2018). 11. Dellon, E. S. et al. A Novel Allergen-Specific Immune Signature-Directed Approach to Dietary Elimination in Eosinophilic Esophagitis. Clin Transl Gastroenterol 10, e00099 (2019). 12. Dilollo, J. et al. Peripheral markers of allergen-specific immune activation predict clinical allergy in eosinophilic esophagitis. Allergy 76, 3470–3478 (2021). 13. Wen, T. et al. Single-cell RNA sequencing identifies inflammatory tissue T cells in eosinophilic esophagitis. J Clin Invest 129, 2014–2028 (2019). 14. Morgan, D. M. et al. Clonally expanded, GPR15-expressing pathogenic effector TH2 cells are associated with eosinophilic esophagitis. Sci Immunol 6, eabi5586 (2021). 15. Janarthanam, R. et al. Bulk T-cell receptor sequencing confirms clonality in pediatric eosinophilic esophagitis and identifies a food-specific repertoire. Allergy 78, 2487–2496 (2023). 16. Laky, K. et al. Epithelial-intrinsic defects in TGFβR signaling drive local allergic inflammation manifesting as eosinophilic esophagitis. Sci Immunol 8, eabp9940 (2023). 17. Buch, M. H., Vital, E. M. & Emery, P. Abatacept in the treatment of rheumatoid arthritis. Arthritis Res Ther 10 Suppl 1, S5 (2008). 18. Reyes-Castillo, Z. et al. Comparative analysis of autoantibodies targeting peptidylarginine deiminase type 4, mutated citrullinated vimentin and cyclic citrullinated peptides in rheumatoid arthritis: associations with cytokine profiles, clinical and genetic features. Clin Exp Immunol 182, 119–131 (2015). 19. Nilsson, C. et al. Accuracy of component-resolved diagnostics in peanut allergy: Systematic literature review and meta-analysis. Pediatr Allergy Immunol 31, 303–314 (2020). 20. Barber, D. et al. Molecular allergology and its impact in specific allergy diagnosis and therapy. Allergy 76, 3642–3658 (2021). 21. Dellon, E. S. et al. Updated International Consensus Diagnostic Criteria for Eosinophilic Esophagitis: Proceedings of the AGREE Conference. Gastroenterology 155, 1022-1033.e10 (2018). 22. Zheng, G. X. Y. et al. Massively parallel digital transcriptional profiling of single cells. Nat Commun 8, 14049 (2017). 23. Lun, A. T. L., McCarthy, D. J. & Marioni, J. C. A step-by-step workflow for low-level analysis of single-cell RNA-seq data with Bioconductor. F1000Res 5, 2122 (2016). 24. immunarch. 25. Trapnell, C. et al. The dynamics and regulators of cell fate decisions are revealed by pseudotemporal ordering of single cells. Nat Biotechnol 32, 381–386 (2014). 26. Qiu, X. et al. Reversed graph embedding resolves complex single-cell trajectories. Nat Methods 14, 979–982 (2017). 27. Cao, J. et al. The single-cell transcriptional landscape of mammalian organogenesis. Nature 566, 496–502 (2019). 28. Haghverdi, L., Lun, A. T. L., Morgan, M. D. & Marioni, J. C. Batch effects in single-cell RNA-sequencing data are corrected by matching mutual nearest neighbors. Nat Biotechnol 36, 421–427 (2018). 29. Mclnnes, L; Healy, J; Melville, J. Uniform Manifold Approximation and Projection for dimension reduction. Preprint at https: / / arxiv.org / abs / 1802.03426. 30. Traag, V. A., Waltman, L. & van Eck, N. J. From Louvain to Leiden: guaranteeing well- connected communities. Sci Rep 9, 5233 (2019). 31. Levine JH, Simonds EF, Bendall SC, Davis KL, Amir el-AD, Tadmor MD, Litvin O, Fienberg HG, Jager A, Zunder ER, Finck R, Gedman AL, Radtke I, Downing JR, Pe’er D, Nolan GP. Data-Driven Phenotypic Dissection of AML Reveals Progenitor-like Cells that Correlate with Prognosis. Cell 162, 184–97 (2015). 32. Linsley, P. S. et al. Autoreactive T cell receptors with shared germline-like α chains in type 1 diabetes. JCI Insight 6, e151349 (2021). 33. Reynisson, B. et al. Improved Prediction of MHC II Antigen Presentation through Integration and Motif Deconvolution of Mass Spectrometry MHC Eluted Ligand Data. J Proteome Res 19, 2304–2315 (2020). 34. Minimal, encapsulated proteomic-sample processing applied to copy-number estimation in eukaryotic cells - PubMed. https: / / pubmed.ncbi.nlm.nih.gov / 24487582 / . 35. The MaxQuant computational platform for mass spectrometry-based shotgun proteomics - PubMed. https: / / pubmed.ncbi.nlm.nih.gov / 27809316 / . 36. Proteomics Quality Control: Quality Control Software for MaxQuant Results - PubMed. https: / / pubmed.ncbi.nlm.nih.gov / 26653327 / . 37. Non-parametric estimation of posterior error probabilities associated with peptides identified by tandem mass spectrometry - PubMed. https: / / pubmed.ncbi.nlm.nih.gov / 18689838 / . 38. A statistical model for identifying proteins by tandem mass spectrometry - PubMed. https: / / pubmed.ncbi.nlm.nih.gov / 14632076 / . 39. Bolotin, D. A. et al. MiTCR: software for T-cell receptor sequencing data analysis. Nat Methods 10, 813–814 (2013). 40. Van Der Loo, M. The stringdist Package for Approximate String Matching. The R Journal 6, 111–122 (2014). 41. Mayer-Blackwell, K. et al. TCR meta-clonotypes for biomarker discovery with tcrdist3 enabled identification of public, HLA-restricted clusters of SARS-CoV-2 TCRs. Elife 10, e68605 (2021). 42. Rothenberg, M. E. et al. Common variants at 5q22 associate with pediatric eosinophilic esophagitis. Nat.Genet. 42, 289–291 (2010). 43. Sleiman, P. M. et al. GWAS identifies four novel eosinophilic esophagitis loci. Nat.Commun. 5, 5593 (2014). 44. Chang, X. et al. A genome-wide association meta-analysis identifies new eosinophilic esophagitis loci. J Allergy Clin Immunol 149, 988–998 (2022). 45. Kelly, K. J. et al. Eosinophilic esophagitis attributed to gastroesophageal reflux: improvement with an amino acid-based formula. Gastroenterology 109, 1503–1512 (1995). 46. Peterson, K. A. et al. Elemental diet induces histologic response in adult eosinophilic esophagitis. Am.J.Gastroenterol. 108, 759–766 (2013). 47. Kliewer, K. L. et al. One-food versus six-food elimination diet therapy for the treatment of eosinophilic oesophagitis: a multicentre, randomised, open-label trial. Lancet Gastroenterol Hepatol 8, 408–421 (2023). 48. Ruffner, M. A. et al. Conserved IFN Signature between Adult and Pediatric Eosinophilic Esophagitis. J Immunol 206, 1361–1371 (2021). 49. Bruhn, S. et al. A generally applicable translational strategy identifies S100A4 as a candidate gene in allergy. Sci Transl Med 6, 218ra4 (2014). 50. Babble, J. et al. S100A4 Levels in Pediatric Eosinophilic Esophagitis Cohort. Journal of Allergy and Clinical Immunology 149, AB157. 51. Altin, J. A. et al. Ndfip1 mediates peripheral tolerance to self and exogenous antigen by inducing cell cycle exit in responding CD4+ T cells. Proc Natl Acad Sci U S A 111, 2067–2074 (2014). 52. Oliver, P. M. et al. Ndfip1 protein promotes the function of itch ubiquitin ligase to prevent T cell activation and T helper 2 cell-mediated inflammation. Immunity 25, 929–940 (2006). 53. Gonzalez-Galarza, F. F. et al. Allele frequency net database (AFND) 2020 update: gold- standard data classification, open access genotype data and new query tools. Nucleic Acids Res 48, D783–D788 (2020). 54. Sarna, V. K. et al. HLA-DQ-Gluten Tetramer Blood Test Accurately Identifies Patients With and Without Celiac Disease in Absence of Gluten Consumption. Gastroenterology 154, 886-896.e6 (2018). 55. Ruiter, B. et al. Expansion of the CD4+ effector T-cell repertoire characterizes peanut- allergic patients with heightened clinical sensitivity. J Allergy Clin Immunol 145, 270–282 (2020). While certain features of the invention have been described herein, many modifications, substitutions, changes, and equivalents will now occur to those of ordinary skill in the art. It is, therefore, to be understood that the appended claims are intended to cover all such modifications and changes as fall within the scope of the invention.

Claims

What is claimed is:

1. An isolated immunogenic peptide of between 10 and 100 amino acids in length comprising a MHC class II T cell epitope of a milk antigen associated with eosinophilic esophagitis (EoE) present in a milk protein selected from β-casein, α-lactalbumin, β-lactoglobulin, α-casein, and ĸ- casein said immunogenic peptide inducing expansion of TCR clonotypes.

2. The isolated immunogenic milk peptide of claim 1 being 10 to 25 amino acids in length.

3. The isolated immunogenic peptide of claim 1, wherein said milk antigen comprises amino acids 59-78 from β- casein or an immunogenic peptide fragment thereof and EoE TCR4+T cells are stimulated.

4. A method for obtaining a population CD4+T cells which are stimulated when contacted with a milk antigen, the method comprising the steps of: a) providing peripheral blood cells; b) contacting said cells in vitro with an immunogenic peptide of claim 1 comprising an MHC class II T cell epitope of the antigen; and c) expanding said cells in the presence of IL-2.

5. A population of T cells obtainable by the method of claim 4.

6. The method of claim 4, wherein the T cell is selected from the group consisting of a primary T cell, an ex vivo cultured T cell, a tumor-infiltrating T cell, and an engineered T cell.

7. The method of claim 6, wherein said T cell is isolated via single-cell sorting.

8. A method for identifying TCR sequences of a milk antigen-specific T cell, the method comprising: a) contacting a sample with a particle comprising an agent having affinity for said T cell under conditions sufficient for a T cell to bind the particle; b) isolating an antigen-specific T cell; andc) obtaining the TCR sequences.

9. The method of claim 8 further comprising amplifying at least one TCR alpha gene sequence and at least one TCR beta gene sequence.

10. The method of claim 8, wherein the T cell is isolated in a single-cell reaction vessel.

11. A method for identifying a subject at increased risk for milk allergy induced eosinophilic esophagitis (EoE), comprising: a) detecting in a nucleic acid obtained from said subject, one or more of an HLA-DR allele and an HLA-DRB1*07.01 allele, said allele being associated with an increased risk of developing milk allergy induced EoE.

12. A method for treating a subject having EoE comprising administration of at least one agent that selectively deletes milk reactive T cells, thereby reducing EoE symptoms.

13. The method of claim 12, wherein said agent is a CAR-T system targeting T cells selected from T cells expressing EoETCR-4, and one or more T cell clonotypes in figures 8, 9, and 10.

14. An isolated peptide-loaded MHC class II molecule, comprising an MHC class II alpha chain, an MHC class II beta chain, and a MHC-class II binding peptide.

15. The isolated peptide-loaded MHC class II molecule of claim 14, wherein the MHC class II protein comprises a DRB1 beta chain and / or is encoded by an HLA-DRB1*07.01 allele.

16. The isolated peptide-loaded MHC class II molecule of claim 15, wherein the peptide is tagged and comprises SEQ ID NO: 530 of β-casein or an immunogenic fragment thereof.

17. The isolated peptide-loaded MHC class II molecule of claim 12, wherein the MHC class II molecule is linked to a ligand of a multivalent binding molecule.

18. The isolated peptide-loaded MHC class II molecule of claim 17, wherein the ligand binds to a multivalent binding molecule, and, optionally, wherein the multivalent binding molecule binds at least one additional MHC class II molecule, wherein each additional MHC class II molecule is optionally peptide-loaded.

19. An isolated MHC class II multimer, comprising a multivalent binding molecule, an isolated milk antigen peptide-loaded MHC class II molecule, comprising an MHC class II molecule, comprising a beta chain encoded by a DRB1 allele, and bound to a milk antigen peptide, the said peptide comprising at least 10 to 25 contiguous amino acids of β-casein, and / or wherein the MHC class II molecule is linked to a ligand of the multivalent binding molecule, and at least one additional MHC class II molecule linked to a ligand of the multivalent binding molecule, wherein each of the at least one additional MHC class II molecule is optionally peptide-loaded, and wherein the ligands bind to the multivalent binding molecule.

20. The isolated MHC class II multimer of claim 19, wherein the multimer is a tetramer, comprising three additional MHC class II molecules linked to a ligand of the multivalent binding molecule.

21. The isolated MHC class II multimer of claim 20, wherein the ligand is biotin and the multivalent binding molecule is streptavidin or avidin.

22. The isolated MHC class II multimer of claim 18, wherein a MHC class II molecule of the multimer or the tetramer is loaded with a β-casein peptide comprising at least 10-25 contiguous amino acid sequence between residue 59 and residue 224.

23. The isolated MHC class II multimer of claim 18, wherein the multimer or tetramer is labeled with a detectable label.

24. A peTH2 genetic signature associated with the EoE phenotype comprising EoE biomarker genes IL4, IL5, IL13, PTGDR2, PLA2G16, PTGS2, HPGDS, PPARG, ACADVL, ACSL4, SLC27A2, LPCAT2, DGKE, GK, CHDH, ALOX5AP, GPR15, ICAM2, GATA3, IL17RB, FFAR3, IL1RL1, said genes exhibiting altered expression levels in EoE subjects when compared to healthy control subjects.

25. A method for diagnosing the presence of sensitivity to a milk protein in a human subject, comprising: a) contacting PBLs obtained from the subject with multimers or oligomers of HLA- DRB1 monomers or modified monomers, having a bound HLA-binding peptide comprising an amino acid sequence as set forth in SEQ ID NO: 530, b) detecting binding of the multimers or oligomers to the PBLs wherein the binding to PBLs is indicative of the presence of milk antigen sensitivity or previous exposure to milk antigen in the subject.

26. The method of claim 25, further comprising contacting the PBLs with additional multimers of HLA-DRB1 monomers or modified monomers, having a bound HLA-binding peptide comprising an amino acid sequence as set forth in any one of SEQ ID NOS: 531 to 538.

27. The method of claim 25, wherein the detecting comprises high throughput screening.

28. The method of claim 25, wherein the multimers are tetramers and said method further comprises labeling the tetramers with a fluorophore, and the detecting comprises measuring the signal produced by the fluorophore.

29. The method of claim 25, wherein the oligomers are tetramers that comprise streptavidin or another multivalent entity and the fluorophore is attached to the streptavidin.

30. The method of claim 25, wherein the method further comprises biotinylating the monomers and binding the monomers to the streptavidin via biotin.

31. A method for determining efficacy of a drug for treatment or prevention of milk induced EoE in a human subject, comprising: a) obtaining a sample comprising PBLs from a patient with an EoE associated HLA allelle undergoing treatment for EOE using the drug; b) contacting the sample under suitable binding conditions with a solid support having bound tetramers of HLA monomers or modified monomers, wherein said monomers are HLA allele monomers or modified monomers, having a bound HLA- binding immunogenic milk peptide amino acid sequence c) detecting the amount of binding of the tetramers to the PBLs; and d) repeating a), b) and c) after a suitable interval of treatment time, wherein a decrease in the amount of the binding after the suitable interval of treatment time indicates efficacy of the drug and a lack of decrease indicates lack of efficacy of the drug for treatment or reduction of EoE symptoms in the human.

32. The method of claim 31, wherein the drug is a steroid or CAR-T therapy.

33. The method of claim 31, wherein the drug is any chemical compound that decreases or arrests peTH2 biomarker expression in said subject.

34. A method comprising: a) contacting PBLs obtained from a subject with tetramers of HLA monomers or modified monomers, having a bound HLA-binding peptide, wherein the subject has an HLA- DRB1 allelle and the binding peptide comprises an amino acid sequence as set forth in SEQ ID NO: 530, and b) detecting binding of the tetramers to the PBLs to identify the presence of milk associated EoE or previous exposure to milk antigen in the subject.

35. An isolated immunogenic peptide of between 10 and 100 amino acids in length comprising a MHC class II T cell epitope of a food antigen associated with allergic responses present in a food protein selected from a food protein listed in Table 4, said immunogenic peptide inducing expansion of TCR clonotypes.

36. The isolated immunogenic food peptide of claim 35 being 10 to 25 amino acids in length.

37. The isolated immunogenic peptide of claim 35, wherein said milk antigen comprises an immunogenic peptide and food antigen reactive T cells are stimulated.

38. A method for obtaining a population CD4+T cells which are stimulated when contacted with a food antigen peptide listed in Table 4, the method comprising the steps of: a) providing peripheral blood cells; b) contacting said cells in vitro with an immunogenic peptide of claim 35 comprising an MHC class II T cell epitope of the antigen; and c) expanding said cells in the presence of IL-2.

39. A method for diagnosing the presence of sensitivity to a food protein in a human subject, comprising: a) contacting PBLs obtained from the subject with multimers or oligomers of HLA-allele monomers or modified monomers, having a bound HLA-binding peptide comprising an amino acid sequence selected from the sequence listed in Table 4, b) detecting binding of the multimers or oligomers to the PBLs wherein the binding to PBLs is indicative of the presence of food antigen sensitivity or previous exposure to food antigen in the subject.

40. A population of T cells obtainable by the method of claim 38.

41. An isolated MHC class II multimer, comprising a multivalent binding molecule, an isolated milk antigen peptide-loaded MHC class II molecule, comprising an MHC class II molecule, comprising a beta chain encoded by an HLA allele, and bound to a milk antigen peptide, the said peptide comprising at least 10 to 25 contiguous amino acids of β-casein, and / or wherein the MHC class II molecule is linked to a ligand of the multivalent binding molecule, and at least one additional MHC class II molecule linked to a ligand of the multivalent binding molecule, wherein each of the at least one additional MHC class II molecule is optionally peptide-loaded,and wherein the ligands bind to the multivalent binding molecule, wherein said multimers specifically bind and detect one or more of the clonotypes listed in Figures 8, 9 or 10.

42. An oral immunotherapy method of treatment (OIT) of food allergy in a subject in need thereof, comprising gradually introducing increasing doses of modified allergens or peptide fragments thereof that retain immunogenicity while exhibiting reduced allergenicity into said subject, or facilitate the delivery or effectiveness of the OIT, under conditions that desensitize said subject to said allergen.

43. The method of claim 42, wherein said food is selected from milk, gluten, wheat, egg, soy, and peanut.

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