Methods and materials to distinguish between active eosinophilic esophagitis (EOE), EOE in remission, and non-EOE states

Abstract

The invention provides methods for diagnosing eosinophilic esophagitis (EoE) in a subject and methods for determining an EoE state (active or remission) in a subject. The methods include detecting elevated expression levels of GPR15 protein or a fragment thereof and / or CD38 protein or a fragment thereof in a biological sample from the subject. Panels, compositions, and kits are also provided.

Description

[0001] METHODS AND MATERIALS TO DISTINGUISH BETWEEN ACTIVE EOSINOPHILIC ESOPHAGITIS (EOE), EOE IN REMISSION, AND NON-EOE STATES

[0002] Field of the Invention

[0003] The invention relates to methods and compositions for diagnosing and monitoring eosinophilic esophagitis (EoE) and differentiating between EoE states (e.g., active EoE, EoE in remission, and non- EoE) by applying flow cytometry to quantify the expression levels of GPR15 and CD38.

[0004] Cross-Reference to Related Applications

[0005] This application claims benefit of U.S. Provisional Application No. 63 / 555,945 filed February 21 , 2024, the content of which is incorporated by reference.

[0006] Statement As To Federally Funded Research

[0007] This invention was made with government support under 5T32HL116275-10 and 2T32HL116275- 11 A1 awarded by the National Institutes of Health. The government has certain rights in the invention.

[0008] Background of the Invention

[0009] Eosinophilic esophagitis (EoE) is a chronic, antigen-mediated disease affecting more than 150,000 Americans. Suppression of eosinophil-predominant esophageal inflammation is necessary to prevent long-term consequences such as fibrosis and strictures. Eosinophil-targeting medications, however, have not met clinical trial endpoints, suggesting that other cell types (likely with the ability to recognize and respond to antigens) are critical to EoE pathogenesis.

[0010] Currently, the only way to diagnose EoE or to determine the efficacy of treatment for EoE is to perform an endoscopy and obtain biopsies for pathology. Reliable and scalable blood biomarkers have not been identified. The best non-invasive biomarker identified previously is a panel of plasma proteins that need to be measured individually with assays that are not currently available clinically (Wechsler et al., Allergy. 76(12):3755-3765, 2021 ). Other techniques requiring stimulation of T cells in culture have been reported in a small number of subjects (Cianferoni et al., Ann. Allergy Asthma Immunol. 120(2) :177- 183.e2, 2017; Dilollo et al., Allergy. 76(11 ):3470-3478, 2021 ). A T cell marker that does not require stimulation of cells and could be readily measured ex vivo in a flow cytometry assay has not been described.

[0011] Accordingly, there is a need for methods to diagnose EoE. The present invention addresses these needs.

[0012] Summary of the Invention

[0013] Currently, accurate methods for diagnosis of EoE are lacking. It is important to be able to accurately diagnose EoE in patients in order to provide them with appropriate and effective therapies.

[0014] In one aspect, the invention provides a method of diagnosing eosinophilic esophagitis (EoE) in a subject. The method involves the steps of: (a) providing a biological sample from a subject; and (b) detecting in the biological sample an elevated expression level of GPR15 protein or a fragment thereof compared to a control expression level, wherein an elevated expression level of GPR15 protein or a fragment thereof compared to a control expression level indicates that the subject has EoE.

[0015] In some embodiments, the method further detects CD38 expression in the biological sample.

[0016] In some embodiments, the biological sample is a sample of peripheral blood mononuclear cells (PBMCs).

[0017] In some embodiments, the biological sample is a biopsy. In some embodiments, the biopsy is an esophageal biopsy or a liquid biopsy.

[0018] In some embodiments, the elevated expression level of GPR15 protein or a fragment thereof is on pathogenic effector T helper 2 cells (peTh2s).

[0019] In some embodiments, the elevated expression level of GPR15 protein or a fragment thereof is on memory CD4+ T cells.

[0020] In some embodiments, the method further detects in the biological sample an increased percentage of CD4+ T cells that are GPR15 peTh2s to a control percentage of CD4+ T cells.

[0021] In some embodiments, the method further detects in the biological sample an increased percentage of CD38 expression compared to a control percentage of CD38 expression. In some embodiments, the increased percentage of CD38 expression is on GPR15+ peTh2s. In some embodiments, the increased percentage of CD38 expression is on memory CD4+ T cells that are GPR15+ peTh2s.

[0022] In some embodiments, the method further detects in the biological sample an increased number of CD38+GPR15+ cells out of 100,000 memory CD4+ T cells compared to a control number.

[0023] In some embodiments, the control expression level, control percentage, and control number are detected in a subject without EoE.

[0024] In some embodiments, the detection is with flow cytometry. In some embodiments, the detection includes the use of an anti-GPR15 antibody. In some embodiments, the detection includes the use of an anti-CD38 antibody.

[0025] In some embodiments, the subject is between 3 and 30 years of age.

[0026] In some embodiments, expression of GPR15 is detected by measuring expression of a gene encoding GPR15.

[0027] In some embodiments, expression of CD38 is detected by measuring expression of a gene encoding CD38.

[0028] In another aspect, the invention provides a method of determining an EoE status in a subject. The method involves the steps of: (a) providing a biological sample from a subject; and (b) detecting in the biological sample an increased percentage of CD38 expression compared to a control percentage, wherein an increased percentage of CD38 expression compared to a control percentage indicates that the subject has active EoE

[0029] In some embodiments, the biological sample is a sample of PBMCs.

[0030] In some embodiments, the biological sample is a biopsy. In some embodiments, the biopsy is an esophageal biopsy or a liquid biopsy.

[0031] In some embodiments, the increased percentage of CD38 expression is on GPR15+ peTh2s. In some embodiments, the increased percentage of CD38 expression is on memory CD4+ T cells that are GPR15+ peTh2s.

[0032] In some embodiments, the control percentage is detected in a subject with EoE in remission.

[0033] In some embodiments, the detection is with flow cytometry. In some embodiments, the detection includes the use of an anti-GPR15 antibody. In some embodiments, the detection includes the use of an anti-CD38 antibody.

[0034] In some embodiments, the subject is between 3 and 30 years of age.

[0035] In some embodiments, expression of CD38 is detected by measuring expression of a gene encoding CD38.

[0036] Yet in another aspect, the invention provides a panel including an antibody capable of binding GPR15 or CD38.

[0037] In some embodiments, the panel includes an antibody capable of binding GPR15.

[0038] In some embodiments, the panel includes an antibody capable of binding CD38.

[0039] In some embodiments, the panel includes an antibody capable of binding GPR15 and an antibody capable of binding CD38.

[0040] In some embodiments, the panel further includes an antibody capable of binding CRTH2 and an antibody capable of binding CD161 .

[0041] In some embodiments, the panel further includes an antibody capable of binding CD3, an antibody capable of binding CD4, and an antibody capable of binding CD45RA.

[0042] Yet in another aspect, the invention provides a composition including a biological sample and detectably labeled antibodies to identify GPR15 and CD38.

[0043] In some embodiments, the biological sample is a sample of PBMCs.

[0044] In some embodiments, the biological sample is a biopsy. In some embodiments, the biopsy is an esophageal biopsy or a liquid biopsy.

[0045] In some embodiments, the composition further includes an antibody that detects CRTH2 and an antibody that detects CD161 .

[0046] In some embodiments, the composition further includes an antibody that detects CD3, an antibody that detects CD4, and an antibody that detects CD45RA.

[0047] In some embodiments, the composition includes an antibody that detects GPR15 and an antibody that detects CD38.

[0048] In yet another aspect, the invention provides a kit including detectably labeled antibodies to identify GPR15 and CD38.

[0049] In some embodiments, the kit further includes an antibody that detects CRTH2 and an antibody that detects CD161 .

[0050] In some embodiments, the kit further includes an antibody that detects CD3, an antibody that detects CD4, and an antibody that detects CD45RA.

[0051] The invention is particularly advantageous in providing non-invasive means for diagnosing EoE.

[0052] Other features and advantages of the invention will be apparent from the following detailed description and figures, and from the claims. Definitions

[0053] To facilitate the understanding of the invention, a number of terms are defined below. Terms defined herein have meanings as commonly understood by those of ordinary skill in the areas relevant to the invention.

[0054] As used in this specification and the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the content clearly dictates otherwise.

[0055] As used herein, the term “antibody” refers to a molecule that specifically binds to or is immunologically reactive with a particular antigen and includes at least the variable domain of a heavy chain, and normally includes at least the variable domains of a heavy chain and of a light chain of an immunoglobulin. The term antibody further includes monoclonal antibodies, polyclonal antibodies, multivalent antibodies, multispecific antibodies, single chain antibodies, camelids, and antibody fragments so long as they exhibit the desired binding specificity.

[0056] As used herein, the term “biological sample” refers to any specimen (e.g., blood such as whole blood or peripheral blood), blood component (e.g., serum, plasma, or cells), tissue (e.g., biopsy such as an esophageal biopsy), mucus (e.g., liquid biopsy), urine, saliva, amniotic fluid, cerebrospinal fluid, pancreatic fluid, chorionic villus sample, and cells) taken from a subject. Preferably, the sample is peripheral blood (e.g., whole blood or peripheral blood mononuclear cells (PBMCs)), tissue (e.g., esophageal biopsy), or mucus (e.g., liquid biopsy).

[0057] As used herein, the terms “eosinophilic esophagitis state” and “EoE state” describes the presence or absence of EoE or the activity of EoE based on the gold standard of esophageal biopsy histology in a subject. An EoE state can currently only be determined with upper endoscopy with biopsy or an esophageal string test.

[0058] As used herein, the terms “subject” and “patient” refer to any animal, such as a mammal (e.g., a human) that receives treatment for a particular disease or condition as described herein (e.g., EoE). A subject to be assessed with the method and composition described herein may be one who has been diagnosed by a medical practitioner as having EoE or one at risk for developing EoE.

[0059] As used herein, the terms “a subject without EoE” and a subject in “non-EoE” state refers to a subject, for example, undergoing endoscopic evaluation for clinical indications but for whom EoE is excluded. In some embodiments, the subject without EoE has < 15 eosinophils per high-power field (eos / hpf). In some embodiments, the subject without EoE may present one or more of the following categories of symptoms: (1 ) Achalasia, (2) Functional dyspepsia, gastritis, (3) Food protein-induced enterocolitis syndrome (FPIES), food protein-induced allergic proctocolitis (FPIAP), (4) Cyclic Vomiting Syndrome, (5) Duodenitis, irritable bowel syndrome (IBS), lactose intolerance, small intestinal bacterial overgrowth (SIBO), (6) “mild chronic inflammation, non-specific,” (7) Diagnosis not clear, symptoms (sx) resolved, (8) Gastroesophageal reflux disease (GERD), cecal inflammation, (9) GERD, behavioral, sx improved, (10) GERD, and / or (11 ) Feeding difficulty. In some embodiments, a subject without EoE may present upper gastrointestinal (UGI) symptoms but test negative for esophagogastroduodenoscopy (EGD). In some embodiments, a subject without EoE may have known IgE-mediated food allergy without a history of EoE. A fragment of the GPR15 protein contains less than 350 or fewer, 300 of fewer, 250 or fewer, 200 or fewer, 150 or fewer, 100 or fewer, 90 or fewer, 80 or fewer, 70 or fewer, 60 or fewer, 50 or fewer, 40 or fewer, 30 or fewer, 20 or fewer, or 10 or fewer amino acids. Sequences of GPR15 and its variants are known in the art. See, e.g., Uniprot.org.

[0060] A fragment of the CD38 protein contains less than 275 or fewer, 250 or fewer, 200 or fewer, 150 or fewer, 100 or fewer, 90 or fewer, 80 or fewer, 70 or fewer, 60 or fewer, 50 or fewer, 40 or fewer, 30 or fewer, 20 or fewer, or 10 or fewer amino acids. Sequences of CD38 and its variants are known in the art. See, e.g., Uniprot.org.

[0061] Brief Description of the Figures

[0062] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application with color drawings will be provided by the Office upon request and payment of the necessary fee.

[0063] FIG. 1 shows the gating strategy for selecting CD38+GPR15+ peTh2s using FlowJo.

[0064] FIGS. 2A shows the number of convTh2s and peTh2s per 105memory CD4+ T cells. Square=Not EoE, triangle=Remission, circle=Active.

[0065] FIG. 2B shows the number of GPR15+ convTh2s and GPR15+ peTh2s per 105memory CD4+ T cells. Square=Not EoE, triangle=Remission, circle=Active.

[0066] FIG. 2C shows the number of a4p7+, CCR9+, CLA+, or GPR15+ peTh2s 105memory CD4+ T cells. Square=Not EoE, triangle=Remission, circle=Active.

[0067] FIG. 3A shows that GPR15 MFI is the highest on peTh2s in EoE. Square=Not EoE, triangle=Remission, circle=Active.

[0068] FIG. 3B shows a4p7, CCR9, or CLA expression in different cell populations. Square=Not EoE, triangle=Remission, circle=Active.

[0069] FIG. 4A shows the number of CD38+ convTh2s and CD38+ peTh2s per 105memory CD4+ T cells. Square=Not EoE, triangle=Remission, circle=Active.

[0070] FIG. 4B shows the number of GPR15+CD38+ convTh2s and GPR15+CD38+ peTh2s per 105memory CD4+ T cells. Square=Not EoE, triangle=Remission, circle=Active.

[0071] FIG. 4C shows the percentage of peTh2s and GPR15+ peTh2s that are CD38+. Square=Not EoE, triangle=Remission, circle=Active.

[0072] FIG. 5 shows the longitudinal change in CD38 expression on GPR15+ peTh2s. Triangle=Remission, circle=Active.

[0073] FIG. 6 shows that CD38 and GPR15 MFI are related in peTh2s in EoE. For the left panel (GPR15+ convTh2s), left=Not EoE, middle=Remission, right=Active. For the right panel (GPR15+ peTh2s), left=Not EoE, middle=Remission, right=Active.

[0074] FIG. 7A shows the discriminatory power between active EoE and non-EoE.

[0075] FIG. 7B shows examples of cut points for discriminating between active EoE and non-EoE.

[0076] FIG. 7C shows the discriminatory power between active EoE and non-EoE when multiple parameters are considered together.

[0077] FIG. 8A shows the discriminatory power between active EoE and EoE in remission. FIG. 8B shows examples of cut points for discriminating between active EoE and EoE in remission.

[0078] FIGS. 9A-9C show GPR15 and CD38 on esophageal peTh2s. For FIG. 9A, left histogram=Active, middle histogram=Remission, right histogram (single point)=Not EoE. For FIGS. 9B and 9C, circle=Active, triangle=Remission.

[0079] Detailed Description

[0080] The present invention provides methods and compositions for diagnosing eosinophilic esophagitis (EoE) and differentiating between EoE disease states by applying flow cytometry to quantify expression levels of GPR15 and CD38.

[0081] The present inventors discovered that GPR15 and CD38 are T cell markers that can be measured, for example, in a flow cytometry assay to diagnose and monitor EoE in a subject. Specifically, the inventors discovered that in EoE, there are populations of pathogenic effector T helper 2 cells (peTh2s) and memory CD4+ T cells that express GPR15 and CD38. The increased percentage of cells that are GPR15+CD38+ and the increased level of GPR15 expression in these cells can be used to distinguish between subjects with EoE (either active EoE or EoE in remission) and subjects without EoE. Further, the inventors discovered that increased percentage of cells that are CD38+ is particularly useful for distinguishing between subjects with active EoE and subjects with EoE in remission.

[0082] Without being bound by a particular theory, CD38 status of GPR15+ peTh2s appears to be a peripheral biomarker of both EoE presence and EoE in remission. GPR15 and CD38 may mark diseasecausing cells that could be isolated for further study or tracked longitudinally. Activation of GPR15+ peTh2s by antigens or EoE triggers may enhance migration to the esophagus through increased GPR15 expression in combination with expression of the surface marker CD38.

[0083] Methods of detection, diagnosis, and monitoring

[0084] The present invention features methods of detecting or diagnosing EoE in a subject (e.g., a human). The methods described herein are less invasive than currently available methods of detecting or diagnosing EoE. The subject, for example and without limitation, may be suffering from a food allergy, present one or more symptoms of allergic disorders (e.g., food allergy asthma, allergic rhinitis, or atopic dermatitis), and / or present one or more symptoms of upper gastrointestinal disorders (e.g., Achalasia, Cyclic Vomiting Syndrome, or Gastroesophageal reflux disease (GERD)). The method involves: (i) providing a biological sample (e.g., peripheral blood, esophageal biopsy, or liquid biopsy) from a subject and (ii) detecting an increased level of GPR15 expression with flow cytometry. GPR15 may be expressed on peTh2s and / or memory CD4+ T cells. An increased percentage of GPR15+ peTh2s, and increased mean fluorescence intensity (MFI) of GPR15 in GPR15+ peTh2s and GPR15+ memory CD4+ T cells indicate that the subject has EoE. The method further detects the level of CD38 expression with flow cytometry. An increased percentage of GPR15+ peTh2s that are CD38+ and an increased percentage of memory CD4+ T cells that are CD38+GPR15+ peTh2s indicate that the subject has active EoE.

[0085] The methods described herein are advantageous for diagnosing EoE in a subject (e.g., a human). The subject may be younger than 3 years old (e.g., 1 month old, 2 months old, 3 months old, 4 months old, 5 months old, 6 months old, 9 months old, 12 months old, 15 months old, 18 months old, 2 years old, or 3 years old) or between 3 and 30 years old (3 years old, 4 years old, 5 years old, 6 years old, 7 years old, 8 years old, 9 years old, 10 years old, 15 years old, 20 years old, 25 years old, or 30 years old).The methods described herein are also advantageous since they involve non-invasive methods for diagnosing EoE, allowing subjects to avoid procedures involving administration of anesthesia and endoscopies.

[0086] The present invention also features methods of determining the EoE state in a subject (e.g., a human) and monitoring a subject with EoE on a treatment. The methods provided herein can detect changes in the expression levels of GPR15 and CD38 on peTh2s as EoE goes into remission with a treatment and / or as EoE becomes active with the cease or pause of the treatment. In some embodiments, the treatment is an elimination diet. In some embodiments, the treatment is dupilumab. In some embodiments, the treatment is a swallowed topical steroid. In some embodiments, the treatment is a proton pump inhibitor. The method involves: (i) providing a biological sample (e.g., peripheral blood, esophageal biopsy, or liquid biopsy) from a subject and (ii) detecting an increased level of CD38+GPR15+ peTh2s with flow cytometry. An increased percentage of GPR15+ peTh2s that are CD38+ and an increased percentage of memory CD4+ T cells that are CD38+GPR15+ peTh2s indicate that the subject has EoE.

[0087] Standard methods of measuring protein levels that are available in the art can be used. Techniques for measuring protein levels include, but are not limited to, immunohistochemistry (IHC), enzyme linked immunosorbent assay (ELISA), cytometry (e.g., flow cytometry), Western blot, enzyme activity assays, spectrophotometry, protein gel staining methods, immunoprecipitation, and other methods utilizing antibodies for detection.

[0088] In some embodiments, the detecting step includes incubating the biological sample with an antibody or a fragment thereof that binds to the GPR15 protein or a fragment thereof and CD38 protein or a fragment thereof. For example, the detecting step includes using standard methods of measuring protein levels available in the art, e.g., IHC, ELISA, Western blot, spectrophotometry (e.g., UV-visible spectroscopy, mass spectrometry, and / or liquid chromatography-mass spectrometry), protein gel staining methods, immunoprecipitation, and other methods (e.g., utilizing antibodies for detection).

[0089] In some embodiments, the measurement of protein levels is performed on an intact tissue sample (e.g., from a biopsy or a liquid sample). In other embodiments, the measurement of protein levels is performed on a protein sample from a biopsy, where the protein sample is obtained from a biopsy after disruption of the tissue (e.g., lysate of a biopsy) and, optionally, extraction of protein. In other embodiments, the measurement of protein levels is performed on a serum, plasma, blood, urine, mucus (e.g., liquid biopsy), or saliva sample from the subject.

[0090] In some embodiments, the control level is obtained from a biological sample of a subject having normal esophageal mucosa (for example, a subject without EoE). In some embodiments, the control level is obtained from a biological sample of a subject after treatment for EoE (for example, a subject in remission).

[0091] In some embodiments, the method includes measuring the expression of at least two genes or proteins or fragments thereof in the biological sample compared to a control level and where: (a) expression of GPR15 in the biological sample is higher than a control level, and (b) expression of CD38 in the biological sample is higher than a control level.

[0092] In some embodiments, the expression of a gene encoding GPR15 is detected. In some embodiments, the expression of a gene encoding CD38 is detected. In some embodiments, the expression of a gene encoding GPR15 and a gene encoding CD38 is detected.

[0093] In some embodiments, the measuring step includes methods for analyzing nucleic acids. For measuring nucleic acid level, standard methods available in the art can be utilized. Techniques for measuring nucleic acid levels include, but are not limited to, polymerase chain reaction (PCR), real-time PCR (including quantitative real-time reverse-transcription PCR), reverse transcription, microarray, gene chips, in situ hybridization, Northern blot, and nuclease protection assays.

[0094] Methods known in the art can be used to quantitatively measure the amount of mRNA transcribed by cells present in a sample. Examples of such methods include quantitative PCR, Northern blots, Southern blots, and next generation sequencing. PCR allows for the detection and measurement of very low quantities of mRNA using an amplification process. Genes may either be up-regulated or down- regulated in any particular biological state, and hence mRNA levels shift accordingly.

[0095] Alternatively, the measuring step includes using IHC or ELISA. In some cases, the biological sample comprises a cell or cell extract from a tissue or bodily fluid. For example, the biological sample is isolated from an esophageal biopsy and the control level is obtained from a biological sample from an esophageal biopsy. In other cases, the biological sample is isolated from an esophageal luminal sample, and the control level is obtained from a biological sample from an esophageal luminal sample. In other cases, the biological sample contains a serum, plasma, blood, urine, mucus (e.g., liquid biopsy), or saliva sample of the subject, and the control level is obtained from a biological sample containing a serum, plasma, blood, urine, mucus (e.g., liquid biopsy), or saliva sample.

[0096] The invention accordingly provides a method of diagnosing EoE including the steps of: (a) providing a biological sample from a subject; and (b) measuring in the biological sample the concentration of at least one (e.g., at least two) of GPR15 and CD38, where an increased concentration of GPR15 and CD38 compared to a control concentration indicates that the subject has EoE.

[0097] Diagnostic panel

[0098] The present invention also features an EoE molecular diagnostic panel for the detection of at least one marker selected from GPR15 and CD38. In some embodiments, the panel includes GPR15 alone. In other embodiments, the panel includes both GPR15 and CD38. The diagnostic panel involves the use of fluorescently labeled antibodies that target markers such as GPR15 and CD38. Additional antibodies that can be included in the panel are antibodies against CD3, CD4, CD45RA or CD45RO, CD161 , and CRTH2 (CD294). Memory CD4+ T cells are CD3+CD4+CD45RA- or CD3+CD4+CD45RO+, “conventional” Th2 cells are CRTH2+CD161 -, and peTh2s are CRTH2+CD161 +. A 6-8 color flow panel with the antibodies described herein may be clinically translatable for the diagnosis and monitoring of EoE in a subject.

[0099] In addition, the combination of fluorescently-labeled antibodies and an appropriate detector can be used to determine whether memory CD4+ T cells and peTh2s express GPR15 and / or CD38, and the level of GPR15 expression (as mean fluorescence intensity, MFI) by cells in these groups expressing GPR15. Further, such a panel may be used to determine the GPR15 MFI of GPR15-expressing CD3+CD4-CD45RA- or CD3+CD4-CD45RO+ cells to be used as a control to normalize MFI of the other cell groups.

[0100] Such panels, for example, a chip including appropriate antibodies as described above, are useful for monitoring response to therapy.

[0101] Further, one embodiment of what is disclosed is the measurement of the panel of biomarkers with the specificity and sensitivity required for managing and diagnosing subjects that have or may have a predisposition to EoE.

[0102] Kits

[0103] The present invention is also directed to a kit for performing the above-described methods. The kit may comprise at least one reagent capable of specifically binding GPR15 and one reagent capable of specifically binding CD38 to quantify the levels of GPR15 and CD38 in the biological sample of a subject; and a reference standard indicating reference levels of GPR15 and CD38. This kit may also contain a set of instructions for use thereof.

[0104] The kit may contain a reagent capable of specifically binding to GPR15 and a reagent capable of specifically binding to CD38 to quantify the levels of GPR15 and CD38 in the biological sample of a subject. For example, the kit may contain at least one reagent that comprises an antibody capable of specifically binding GPR15 and at least one reagent that comprises an antibody capable of specifically binding CD38. The kit may further contain one or more antibodies that are labeled with a detectable label. For example, the kit may contain one antibody labeled with a detectable label, two antibodies labeled with a detectable label, three antibodies labeled with a detectable label, four antibodies labeled with a detectable label, five antibodies labeled with a detectable label, six antibodies labeled with a detectable label, etc.

[0105] An exemplary reagent capable of specifically binding GPR15 or CD38 is a detection antibody that includes a detection agent (e.g., reporter enzymes, bioluminescent labels, fluorophores, radioisotopes, nanoparticles, etc.) For example, the antibody is labeled with a detectable label that binds to an epitope on GPR15 or CD38 as is described herein.

[0106] An exemplary kit may include a container for a sample from a subject, a preservative, instruments for collecting the sample and / or placing the sample in the container, and instructions for collecting and preserving the sample. The kit may further contain the antibodies necessary to perform flow cytometry using the 6-8 color flow panel described above to detect GPR15 and CD38 in peTh2s and memory CD4+ T cells (e.g., an anti-CD3 antibody, an anti-CD4 antibody, an anti-CD45RA antibody, an anti-CRTH2 antibody, an anti-CD161 antibody, an anti-GPR15 antibody, and / or an anti-CD38 antibody). The instructions in the kits may explain how a practitioner (e.g., a physician, a nurse, or a laboratory assistant) may use the sample from the subject to determine the diagnosis of EoE or EoE state of the subject. The kits may further contain an implement for collection and transfer of the sample into the container (e.g., a needle or a catheter), components for keeping the samples at a defined temperature (e.g., between about 4 °C and about 27 °C), such as a coolant (e.g., an ice pack, dry ice, cooling pouch, or cooling plates). The kit may also include a cold box or insulated carrier for transport of the biological sample, if necessary.

[0107] The kit may further include reagents for detecting live cells as is described herein. For example, Live / Dead Blue can be used to detect dead cells.

[0108] The kit may further include reagents for detecting memory CD4+ T cells as is described herein. For example, an anti-CD3 antibody, an anti-CD4 antibody, and an anti-CD45RA antibody can be used to detect memory CD4+ T cells.

[0109] The kit may further include reagents for detecting peTh2s as is described herein. For example, an anti-CRTH2 antibody and an anti-CD161 antibody can be used to detect peTh2s.

[0110] Examples

[0111] The following examples are put forth so as to provide those of ordinary skill in the art with a description of how the compositions and methods described herein can be used and evaluated and are intended to be purely exemplary of the invention and are not intended to limit the scope of what the inventors regard as their invention.

[0112] Example 1. Differences in pathogenic effector Th2 cells (peTh2s) from subjects with active EoE, EoE in remission, and without EoE.

[0113] Objective

[0114] The objective of this study was to investigate whether there are any differences between blood T cells from subjects with active eosinophilic esophagitis (EoE) and subjects with inactive EoE.

[0115] Materials and Methods

[0116] Peripheral blood mononuclear cells (PBMCs) and esophageal biopsies were collected from subjects with active EoE (“Active”), subjects with EoE in remission (“Remission”), and subjects without EoE (“Not EoE”), and analyzed with flow cytometry.

[0117] Results

[0118] There are no significant differences in the percentage of T cells that are peTh2s among the three groups of subjects.

[0119] The expression level of GPR15 (GPR15 MFI) is significantly elevated in peTh2s of subjects with active or remission EoE when compared to peTh2s of subjects without EoE. The expression level of GPR15 on memory CD4+ cells and conventional Th2 cells is also higher in subjects with EoE, but not to the same degree as on peTh2s.

[0120] There are no significant differences in the percentage of CD38+ peTh2s among the three groups of subjects. However, the percentage of CD38+GPR15+ peTh2s is significantly higher in subjects with active EoE than in subjects with EoE in remission and subjects without EoE. Conclusion

[0121] These results show that peTh2s are different between subjects with active EoE and subjects with EoE in remission, and between subjects with active EoE and subjects without EoE.

[0122] Example 2. Protocol development

[0123] 10-25 million PBMCs collected from subjects were stained with marker-specific antibodies that are listed in Table 1 . This protocol is applicable to frozen PBMCs and freshly collected specimens. If using freshly collected specimens, the Live / Dead reagent is not required. Memory CD4+ T cells are Live, CD3+CD4+CD45RA-. Control cells used for data normalization are Live, CD+CD4-CD45RA-. “Conventional” Th2 cells are CRTH2+CD161 - and peTh2s are CRTH2+CD161 +.

[0124] Table 1 . Antibodies for surface staining

[0125] Surface staining was performed at 4 °C for 30 minutes with 0.5 pL of antibody in a total volume of 50 pL of antibodies and staining buffer (PBS + 0.5% BSA + 2 mM EDTA) per 5 million cells in the sample. This was followed by intracellular staining using the Thermofisher / eBioscience FoxP3 / Transcription Factor Buffer Set (none of the above markers are intracellular or require this step, but other markers in the larger panel required this).

[0126] Data were acquired on a Cytek Aurora 5-laser spectral flow cytometer. Acquisition was typically done the day after staining because the fix / perm buffer fixes the cells, but this could be done on the same day if cells are not fixed. 1 -5 million events (excluding events with very small FSC and SSC) were acquired per sample.

[0127] Cell populations of interest were generated analyzed using FlowJo as follows (FIG. 1 ):

[0128] 1 . CD3+ cells were selected on CD3-BUV395 vs SSC-A;

[0129] 2. Singlets were selected on FSC-A vs FSC-H;

[0130] 3. Lymphocytes were selected on FSC-A vs SSC-A;

[0131] 4. Live cells were selected on Live / Dead vs SSC-A;

[0132] 5. Memory CD4+ T cells (CD4+CD45RA-) were selected on CD4-BUV496 vs CD45RA-BV650;

[0133] 6. pathogenic effector T helper 2 cells (peTh2s; CRTH2+CD161 +) were selected on CD161 - BV480 vs CRTH2-PE-Cy5;

[0134] 7. peTh2s that were GPR15+ were selected on GPR15-APC vs SSC-A; and 8. GPR15+ peTh2s that were CD38+ were selected on CD38-APC-R700 vs SSC-A.

[0135] The following measures were determined:

[0136] 1 . The mean fluorescence intensity (MFI) of GPR15 on GPR15+ peTh2s and memory CD4 cells. MFI was normalized to Live CD3+CD4-CD45RA- cells;

[0137] 2. The percentage of GPR15+ peTh2s that were CD38+; and

[0138] 3. The percentage of memory CD4+ T cells that were GPR15+CD38+peTh2s.

[0139] Example 3. Percentage of peripheral blood GPR15+ pathogenic effector T helper 2 cells that are CD38+ may identify active EoE

[0140] Objective

[0141] The objective of this study was to investigate the peTh2s in EoE.

[0142] Materials and Methods

[0143] Cryopreserved PBMCs and fresh esophageal biopsies from subjects between the ages of 3 and 30 (the majority was less than 25 years of age) were collected. The breakdown of the subjects was as follows:

[0144] Those in “Not EoE” group had one or more of the following categories of symptoms:

[0145] 1 . Achalasia;

[0146] 2. Functional dyspepsia, gastritis;

[0147] 3. Food protein-induced enterocolitis syndrome (FPIES), food protein-induced allergic proctocolitis (FPIAP);

[0148] 4. Cyclic vomiting syndrome;

[0149] 5. Duodenitis, irritable bowel syndrome (IBS), lactose intolerance, small intestinal bacterial overgrowth (SIBO);

[0150] 6. “mild chronic inflammation, non-specific”;

[0151] 7. Diagnosis not clear, symptoms (sx) resolved;

[0152] 8. Gastroesophageal reflux disease (GERD), cecal inflammation;

[0153] 9. GERD, behavioral, sx improved;

[0154] 10. GERD; and / or

[0155] 11 . Feeding difficulty.

[0156] For the Not EoE group, some samples were from subjects who tested negative for esophagogastroduodenoscopy (EGD) but presented upper gastrointestinal (UGI) symptoms. Others were from subjects with known IgE-mediated food allergy but without a history of EoE. Age, sex, ethnic and racial background, and prevalence of allergic comorbidities did not differ between groups.

[0157] The PBMC and esophageal biopsy samples were analyzed with flow cytometry and FlowJo using the method described in Example 2. Samples with fewer than 5 GPR15+ peTh2s and samples that were stained with CRTH2 at room temperature instead of at 4 °C were excluded from the analysis. The final number of samples for the different parameters measured was:

[0158] GraphPad Prism and R 4.4.1 were used for statistical analyses (Mann-Whitney, Wilcoxon signed- rank, ROC curve).

[0159] Results

[0160] The number of conventional Th2s (CRTH2+CD161 -; convTh2) or peTh2s (CRTH2+CD161 +) per 105memory CD4+ T cells (CD4+CD45RA-) is not significantly different among the groups (FIG. 2A). While the number of GPR15+ convTh2s is not different between the groups, the number of GPR15+ peTh2s is significantly higher in samples from the active EoE group than in samples from the non-EoE group (FIG. 2B). No difference in the number of a4p7+, CCR9+, or CLA+ peTh2s among the three groups is observed (FIG. 2C).

[0161] The normalized GPR15 MFI is significantly higher in memory CD4+ T cells of the active EoE group or the EoE in remission group than in memory CD4+ T cells of the non-EoE group (Active EoE vs. non-EoE ROC AUC=0.89; FIG. 3A). Similarly, the normalized GPR15 MFI is significantly higher in peTh2s from the active EoE group or the EoE in remission group than in peTh2s from the non-EoE group (FIG. 3). The normalized MFI of a4p7, CCR9, or CLA+ was not significantly different in any other cell types among the three groups (FIG. 3B)

[0162] The number of CD38+ convTh2s or CD38+ peTh2s per 105memory CD4+ T cells is not significantly different among the groups (FIG. 4A). While the number of GPR15+CD38+ convTh2s is not different between the groups, the number of GPR15+CD38+ peTh2s is significantly higher in samples from the active EoE group than in samples from the EoE in remission group or the non-EoE group (FIG. 4B). The percentage of GPR15+ peTh2s that are CD38+ is significantly higher in the samples from the active EoE group than in samples from the EoE in remission or non-EoE group (FIG. 4C).

[0163] Within EoE subjects (either from the active EoE group or EoE in remission group), the number of GPR15+CD38+ peTh2s per 105memory CD4+ T cells is increased when a subject has active EoE compared to when the same subject has EoE in remission (FIG. 5). Within the active EoE group, the normalized GPR15 MFI is significantly higher in CD38+GPR15+ peTh2s than in CD38-GPR15+ peTh2s (FIG. 6).

[0164] The parameters with significant discriminatory power between active EoE and non-EoE are shown in FIG. 7A. The normalized GPR15 MFI on GPR15+ peTh2s can be used to discriminate between active EoE and non-EoE (FIG. 7B). For example, a subject can be determined to have active EoE, rather than no EoE, when a sample from the subject has a normalized GPR15 MFI value that is greater than 1 .636 (FIG. 7B, left) or between 1 .141 and 2.073 (FIG. 7B, right). The parameters with significant discriminatory power between active EoE and non-EoE, when multiple parameters are considered together, are shown in FIG. 7C.

[0165] The parameters with significant discriminatory power between active EoE and EoE in remission are shown in FIG. 8A. The percentage of GPR15+ peTh2s that are CD38+ is can be used to discriminate between active EoE and EoE in remission (FIG. 8B). For example, a subject can be determined to have active EoE, rather than EoE in remission, when more than 26.9% of the GPR15+ peTh2s from a sample from the subject are CD38+ (FIG. 8B, left) or when between 12.9% and 39.1% of the GPR15+ peTh2s from the sample are CD38+ (FIG. 8B, right).

[0166] In esophageal biopsies, the percentage of memory CD4+ T cells that are peTh2s is higher in the active EoE group than the EoE in remission group (FIG. 9A). Additionally, the active EoE group has a significantly higher percentage of CD38+, GPR15+, and CD38+GPR15+ peTh2s and a significantly higher percentage of GPR15+ peTh2s that are CD38+ than the EoE in remission group (FIG. 9B). Within the active EoE group, the normalized GPR15 MFI is significantly higher in peTh2s than in convTh2s or memory CD4+ T cells (FIG. 9C). Within the EoE in remission group, the normalized GPR15 MFI is significantly higher in peTh2s than in memory CD4+ T cells (FIG. 9C).

[0167] Conclusion

[0168] These results collectively suggest that the factors that are significantly different between active EoE and not EoE are:

[0169] 1 . Normalized GPR15 MFI on GPR15+ peTh2s;

[0170] 2. Normalized GPR15 MFI on GPR15+ memory CD4+ T cells;

[0171] 3. Percentage of memory CD4+ T cells that are GPR15+CD38+ peTh2s;

[0172] 4. Percentage of GPR15+ peTh2s that are CD38+; and

[0173] 5. Percentage of memory CD4+ T cells that are GPR15+ peTh2s.

[0174] The factors that are significantly different between active EoE and EoE in remission are:

[0175] 1 . Percentage of memory CD4+ T cells that are GPR15+CD38+ peTh2s; and

[0176] 2. Percentage of GPR15+ peTh2s that are CD38+.

[0177] In addition, these results suggest that in subjects with EoE, some triggers cause peTh2s to express GPR15. The GPR15+ peTh2s are then activated by an antigen, express CD38, and effectively migrate to the esophagus via a combination of CD38-CD31 interactions and GPR15-GPR15L interactions.

[0178] Other Embodiments All publications, patents, and patent applications mentioned in this specification are incorporated herein by reference to the same extent as if each independent publication or patent application was specifically and individually indicated to be incorporated by reference.

[0179] While the invention has been described in connection with specific embodiments thereof, it will be understood that it is capable of further modifications and this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the invention that come within known or customary practice within the art to which the invention pertains and may be applied to the essential features hereinbefore set forth, and follows in the scope of the claims. Other embodiments are within the claims. What is claimed is:

Claims

Claims1 . A method of diagnosing eosinophilic esophagitis (EoE) in a subject, the method comprising:(a) providing a biological sample from a subject; and(b) detecting in said biological sample an elevated expression level of GPR15 protein or a fragment thereof compared to a control expression level, wherein an elevated expression level of GPR15 protein or a fragment thereof compared to a control expression level indicates that the subject comprises EoE.

2. The method of claim 1 , further detecting CD38 expression in said biological sample.

3. The method of claim 1 , wherein said biological sample is a sample of peripheral blood mononuclear cells (PBMCs).

4. The method of claim 1 , wherein said biological sample is a biopsy.

5. The method of claim 4, wherein said biopsy is an esophageal biopsy or a liquid biopsy.

6. The method of any one of claims 1 -5, wherein said elevated expression level of GPR15 protein or a fragment thereof is on pathogenic effector T helper 2 cells (peTh2s).

7. The method of any one of claims 1 -6, wherein said elevated expression level of GPR15 protein or a fragment thereof is on memory CD4+ T cells.

8. The method of any one of claims 1 -7, further detecting in said biological sample an increased percentage of CD4+ T cells that are GPR15 peTh2s to a control percentage of CD4+ T cells.

9. The method of any one of claims 1 -8, further detecting in said biological sample an increased percentage of CD38 expression compared to a control percentage of CD38 expression.

10. The method of claim 9, wherein said increased percentage of CD38 expression is on GPR15+ peTh2s.11 . The method of claim 9 or 10, wherein said increased percentage of CD38 expression is on memory CD4+ T cells that are GPR15+ peTh2s.

12. The method of any one of claims 1 -11 , further detecting in said biological sample an increased number of CD38+GPR15+ cells out of 100,000 memory CD4+ T cells compared to a control number.

13. The method of any one of claims 1 -12, wherein said control expression level, control percentage, and control number are detected in a subject without EoE.

14. The method of any one of claims 1 -13, wherein said detection is with flow cytometry.

15. The method of claim 14, wherein said detection comprises the use of an anti-GPR15 antibody.

16. The method of claim 14, wherein said detection comprises the use of an anti-CD38 antibody.

17. The method of claim 1 , wherein said subject is between 3 and 30 years of age.

18. The method of claim 1 , wherein expression of GPR15 is detected by measuring expression of a gene encoding GPR15.

19. The method of claim 2, wherein expression of CD38 is detected by measuring expression of a gene encoding CD38.

20. A method of determining an EoE status in a subject, the method comprising:(a) providing a biological sample from a subject; and(b) detecting in said biological sample an increased percentage of CD38 expression compared to a control percentage, wherein an increased percentage of CD38 expression compared to a control percentage indicates that the subject comprises active EoE21 . The method of claim 20, wherein said biological sample is a sample of PBMCs.

22. The method of claim 20, wherein said biological sample is a biopsy.

23. The method of claim 22, wherein said biopsy is an esophageal biopsy or a liquid biopsy.

24. The method of any one of claims 20-23, wherein said increased percentage of CD38 expression is onGPR15+ peTh2s.

25. The method of any one of claims 20-24, wherein said increased percentage of CD38 expression is on memory CD4+ T cells that are GPR15+ peTh2s.

26. The method of any one of claims 20-25, wherein said control percentage is detected in a subject with EoE in remission.

27. The method of any one of claims 20-26, wherein said detection is with flow cytometry.

28. The method of claim 27, wherein said detection comprises the use of an anti-GPR15 antibody.

29. The method of claim 27, wherein said detection comprises the use of an anti-CD38 antibody.

30. The method of claim 20, wherein said subject is between 3 and 30 years of age.31 . The method of claim 20, wherein expression of CD38 is detected by measuring expression of a gene encoding CD38.

32. A panel comprising an antibody capable of binding GPR15 or CD38.

33. The panel of claim 32, wherein said panel comprises an antibody capable of binding GPR15.

34. The panel of claim 32, wherein said panel comprises an antibody capable of binding CD38.

35. The panel of claim 32, wherein said panel comprises an antibody capable of binding GPR15 and an antibody capable of binding CD38.

36. The panel of any one of claims 32-35, wherein said panel further comprises an antibody capable of binding CRTH2 and an antibody capable of binding CD161 .

37. The panel of any one of claims 32-36, wherein said panel further comprises an antibody capable of binding CD3, an antibody capable of binding CD4, and an antibody capable of binding CD45RA.

38. A composition comprising a biological sample and detectably labeled antibodies to identify GPR15 and CD38.

39. The composition of claim 38, wherein said biological sample is a sample of PBMCs.

40. The composition of claim 38, wherein said biological sample is a biopsy.41 . The composition of claim 40, wherein said biopsy is an esophageal biopsy or a liquid biopsy.

42. The composition of any one of claims 38-41 , further comprising an antibody that detects CRTH2 and an antibody that detects CD161 .

43. The composition of any one of claims 38-42, further comprising an antibody that detects CD3, an antibody that detects CD4, and an antibody that detects CD45RA.

44. The composition of any one of claims 38-43, wherein said composition comprises an antibody that detects GPR15 and an antibody that detects CD38.

45. A kit comprising detectably labeled antibodies to identify GPR15 and CD38.

46. The kit of claim 45, further comprising an antibody that detects CRTH2 and an antibody that detects CD161.

47. The kit of claim 45 or 46, further comprising an antibody that detects CD3, an antibody that detects CD4, and an antibody that detects CD45RA.

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