Methods relating to Crohn's disease associated T cell receptors

Abstract

Methods are provided for evaluating T cell receptor beta chain complementarity determining region 3 (TCRβ CDR3) sequences. In certain embodiments, prior to evaluating, the subject is identified as having or suspected of having inflammatory bowel disease (IBD). According to some embodiments, at the time of evaluating, the subject has one or more non-specific symptoms consistent with Crohn's disease. Also provided are methods that include administering Crohn's disease therapy to a subject identified as comprising T cells expressing a T cell receptor beta chain (TCRβ) comprising a TCRβ CDR3 sequence described in the present disclosure. Computer-readable media and systems for evaluating TCRβ CDR3 sequences are also provided.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Patent Application No. 63 / 160,213, filed March 12, 2021, which is incorporated herein by reference in its entirety. [Background technology]

[0002] Introduction Antigen-specific cellular immune responses are mediated by diverse populations of T cells and B cells, each of which possesses an immune cell receptor (TCR and BCR, respectively) capable of recognizing a specific antigen (in the case of T cells, an antigenic peptide bound to a specific major histocompatibility complex (MHC) molecule on the surface of a host cell). Antigen encounter leads to clonal expansion, activation, and maturation of T cells and B cells, respectively, which mediate cytotoxic (CD8 + CTL) and helper (CD4 + ) T cells, or effector populations of antibodies and memory B cells. The presence of antigen-specific effector cells is diagnostic of an immune response specific for that antigen.

[0003] Activated T cells proliferate by clonal expansion and reside for many years within the memory T cell compartment as clonal populations of cells (clones) with homoeologous rearranged TCR genes (Arstila TP, et al. A direct estimate of the human alpha / beta T cell receptor diversity, Science 286: 958-961).

[0004] Most of the TCR diversity resides in the beta chain of the TCR alpha / beta heterodimer. A very large diversity is generated by combining non-contiguous TCRβ variable (V), diversity (D), and joining (J) region gene segments, which collectively code for the CDR3 region, the primary region of the TCRβ locus for determining antigen specificity. Nucleotide deletions and template-independent insertions during rearrangements at the Vβ-Dβ and Dβ-Jβ junctions further increase the potential diversity of receptors that can be encoded (Cabaniols JP, et al. Most alpha / beta T cell receptor diversity is due to terminal deoxynucleotidyl transferase, J Exp Med 194: 1385-1390, 2001). Typically, at a given time, an adult with a healthy immune system will have approximately 100,000 T cell receptors in their 1000+ T cell receptors. 12 Each circulating T cell expresses approximately 10 million unique TCR beta chains (Robins HS, et al. (2009) Comprehensive assessment of T-cell receptor beta-chain diversity in alpha / beta T cells, Blood 114:4099-4107).

[0005] Therefore, the human T cell repertoire dynamically encodes exposure to disease-associated antigens through rearrangements of genes encoding their receptors, providing an excellent basis for diagnostic prediction. It has been demonstrated that TCRβ receptors in peripheral blood samples from human subjects can be used to predict disease exposure status, i.e., based on the presence and abundance of these receptors in a training cohort (Emerson et al., Immunosequencing identifies signatures of cytomegalovirus exposure history and HLA-mediated effects on the T cell repertoire, Nature Genetics April 2017; doi:10.1038 / ng.3822).

[0006] Inflammatory bowel disease (IBD) is an immune-mediated chronic relapsing inflammatory condition. IBD is thought to result from a pronounced immune response in genetically susceptible individuals, usually due to environmental factors such as gut commensals. IBD can be diagnosed at any age, but the majority of diagnoses are made between the ages of 20 and 30, with a second peak in IBD diagnoses occurring during the sixth or seventh decade. Due to the early onset of IBD, the severe symptoms associated with IBD, the unstable natural history of the disease, the number of hospitalizations, and the lack of a cure, a diagnosis of IBD significantly impacts the quality of life of patients.

[0007] IBD is a general term used to describe disorders involving chronic inflammation of the digestive tract. Types of IBD include Crohn's disease (CD) and ulcerative colitis (UC). Crohn's disease is characterized by inflammation of the lining of the digestive tract, often involving the deeper layers of the digestive tract. Ulcerative colitis involves inflammation and sores (ulcers) along the surface lining of the large intestine (colon) and rectum.

[0008] Both Crohn's disease and ulcerative colitis are characterized by diarrhea, fatigue, abdominal pain and cramps, rectal bleeding (blood in the stool), and unintentional weight loss. Distinguishing between Crohn's disease and ulcerative colitis is particularly difficult when IBD primarily involves the colon. Inaccurate diagnosis is estimated to occur in 30% of IBD patients. In the majority of cases, diagnostic uncertainty arises from overlapping clinical and histologic features, causing Crohn's disease to appear as ulcerative colitis. Distinguishing between Crohn's disease and ulcerative colitis depends on an often imprecise compilation of clinical, radiologic, endoscopic, and histopathologic interpretations.

[0009] An estimated 15% of IBD patients are indistinguishable after one or more clinical, radiological, serological, and pathological tests and are labeled "unclassifiable colitis" (IC). Another 15% of colonic IBD cases undergoing pouch surgery with an initial diagnosis of ulcerative colitis (based on the pathologist's initial designation of the endoscopic biopsy and colectomy specimens) will have their ulcerative colitis diagnosis changed to Crohn's disease based on postoperative follow-up if clinical and histopathological changes indicate the development of Crohn's disease in the ileal pouch. Half of these patients will require pouch resection or diversion.

[0010] Differentiating between Crohn's disease and ulcerative colitis is important to inform appropriate therapy. For example, renal-sparing proctocolectomy (RPC) should be contraindicated in patients with Crohn's disease, whereas ileal pouch-anal anastomosis (IPAA) is the standard acceptable treatment for patients with ulcerative colitis and for those with unclassified colitis who are predicted to develop ulcerative colitis.

[0011] There is considerable interest in identifying biomarkers that can accurately distinguish between Crohn's disease and ulcerative colitis. Studies to identify biomarkers with potential relevance for distinguishing between Crohn's disease and ulcerative colitis have been largely unsuccessful. In serum, these biomarkers include placental growth factor-1 (PLGF-1), IL-7, TQRb1, and IL-12P40. In biopsies obtained from mucosa, they include Rho GD1a, desmoglein, pleckstrin, VDAC (voltage-dependent anion channel), 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMG-CoA), and C10orf76. In stool, biomarkers include calprotectin, PMN elastase, lactoferrin, and S100A12. Although the identification of these biomarkers represents an advance in the field, they have not been shown to precisely form the basis for diagnosing Crohn's disease and / or delineating between Crohn's disease and ulcerative colitis.

[0012] Current diagnostic deficiencies and the potential morbidity due to inaccurate diagnosis (e.g., unnecessary and / or inappropriate surgical interventions) highlight the need for new diagnostic approaches with improved sensitivity and specificity for Crohn's disease or its subtypes, allowing for a more accurate diagnosis of Crohn's disease and / or a differential diagnosis between Crohn's disease and ulcerative colitis. Summary of the Invention

[0013] overview Methods are provided for evaluating T cell receptor beta chain complementarity determining region 3 (TCRβ CDR3) sequences. In certain embodiments, prior to evaluating, the subject is identified as having or suspected of having inflammatory bowel disease (IBD). According to some embodiments, at the time of evaluating, the subject has one or more non-specific symptoms consistent with Crohn's disease. Also provided are methods that include administering Crohn's disease therapy to a subject identified as comprising T cells expressing a T cell receptor beta chain (TCRβ) comprising a TCRβ CDR3 sequence described in the present disclosure. Computer-readable media and systems for evaluating TCRβ CDR3 sequences are also provided. [Brief description of the drawings]

[0014] [Figure 1] Data showing the identification of Crohn's disease associated TCRβ sequences that distinguish Crohn's disease patients from controls (healthy adults). [Diagram 2] Data showing model performance on the holdout dataset where a strong signal was observed only for Crohn's disease patients and not for other healthy / disease groups tested. [Diagram 3] FIG. 1 shows a schematic and data showing that Crohn's disease-associated TCRβ sequences cluster and show convergent recombination in Crohn's disease patients but not controls. [Figure 4] 13 shows data showing overlap in Crohn's disease-associated TCR β sequences between blood and tissue samples. [Diagram 5]Data showing the performance of a classification model trained on a large Crohn's disease dataset, including high sensitivity for Crohn's disease, high specificity over unrelated conditions, as well as significant differences in signal in more complex disease (stricture / fistula formation) and in ileal / ileocolonic locations versus colonic locations. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0015] Before describing the methods of the present disclosure in more detail, it should be understood that the method is not limited to particular embodiments described, as such may, of course, vary. It should also be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the method will be limited only by the appended claims.

[0016] Where a range of values ​​is provided, it is understood that each intervening value between the upper and lower limits of that range, to one-tenth of the unit of the lower limit, and any other stated or intervening value in that stated range, is encompassed within the method, unless the context clearly indicates otherwise. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and are also encompassed within the method, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the method.

[0017] Certain ranges are presented herein with numerical values ​​preceded by the term "about." The term "about" is used herein to provide literal support for the exact number it precedes, as well as a number that is near or approximately the number it precedes. In determining whether a number is near or approximately a specifically recited number, the near or approximately unrecited number may be a number that provides a substantial equivalent to the specifically recited number in the context in which it is presented.

[0018] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the method belongs. Although any methods similar or equivalent to those described herein may also be used in the practice or testing of the method, representative exemplary methods are now described.

[0019] All publications and patents cited herein are incorporated by reference as if each individual publication or patent was specifically and individually indicated to be incorporated by reference, and are incorporated by reference herein to disclose and describe the materials and / or methods in connection with which the publications are cited. The citation of any publication is for its disclosure prior to the filing date, and should not be construed as an admission that the method is not entitled to antedate such publication, since the publication dates provided may be different from the actual publication dates which may need to be independently confirmed.

[0020] It should be noted that, as used herein and in the appended claims, the singular forms "a," "an," and "the" include plural references unless the context clearly indicates otherwise. It should be further noted that the claims may be drafted to exclude any optional element. As such, this statement is intended to serve as a predicate to the use of exclusive terminology, such as "solely," "only," and the like, in connection with the recitation of claim elements or the use of a "negative" limitation.

[0021] It should be understood that certain features of the method described in the context of separate embodiments for clarity may also be provided in combination in a single embodiment. Conversely, various features of the method described in the context of a single embodiment for brevity may also be provided separately or in any suitable subcombination. All combinations of the embodiments are specifically encompassed by the present disclosure and are disclosed herein as if each and every combination were individually and expressly disclosed, to the extent that such combinations encompass operable processes and / or compositions. In addition, all subcombinations listed in the embodiments describing such variables are also specifically encompassed by the present disclosure and are disclosed herein as if each and every such subcombination were individually and expressly disclosed, to the extent that such combinations encompass operable processes and / or compositions.

[0022] As will be apparent to one of ordinary skill in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has distinct components and features which may be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the method. Any described method can be carried out in the order of events described or in any other order which is logically possible.

[0023] Methods for evaluating TCRβ CDR3 sequences

[0024] The present disclosure provides methods for evaluating T cell receptor beta chain complementarity determining region 3 (TCRβ CDR3) sequences. In certain embodiments, the methods include evaluating TCRβ CDR3 sequences determined from a sample obtained from a subject for the presence or absence of one or more TCRβ CDR3 sequences set forth in SEQ ID NOs: 1-1996 herein, e.g., SEQ ID NOs: 1-1281 herein. The inventors have determined that TCRs comprising such TCRβ CDR3 sequences are associated with Crohn's disease by being statistically more prevalent in individuals with Crohn's disease than in individuals without Crohn's disease. Thus, the methods of the present disclosure find use, for example, in predicting whether a subject has or does not have Crohn's disease. Prior to the evaluating, the subject may have been identified as having or is suspected of having inflammatory bowel disease (IBD). In certain embodiments, for subjects identified as having or suspected of having IBD, the methods find use in diagnosing the subject as having Crohn's disease. Such a diagnosis may be a differential diagnosis in which a subject presenting with one or more non-specific symptoms consistent with Crohn's disease is diagnosed with Crohn's disease rather than another condition characterized by symptoms overlapping with those of Crohn's disease, including but not limited to ulcerative colitis, irritable bowel syndrome, and / or celiac disease. Alternatively or additionally, such a diagnosis may be a differential diagnosis in which a subject is diagnosed with Crohn's disease with fistulization or structuring rather than Crohn's disease without fistulization or ileal / ileocolonic Crohn's disease rather than colonic Crohn's disease. Details regarding the methods of the present disclosure will now be described.

[0025] According to some embodiments, the methods of the present disclosure are computer-implemented. By "computer-implemented" it is meant that at least one step of the method is performed using one or more processors and one or more non-transitory computer-readable media. For example, in certain embodiments, a computer-implemented method for evaluating TCRβ CDR3 sequences is provided, the method being performed using one or more processors and one or more non-transitory computer-readable media comprising instructions stored thereon, the instructions, when executed by the one or more processors, causing the one or more processors to evaluate TCRβ CDR3 sequences determined from a sample obtained from the subject for the presence or absence of one or more TCRβ CDR3 sequences set forth in SEQ ID NOs: 1-1996 herein, e.g., SEQ ID NOs: 1-1281 herein. The computer-implemented method of the present disclosure may further include one or more steps that are not computer-implemented, such as obtaining a sample (e.g., a blood sample, an intestinal tissue sample, etc.) from the subject, preparing a sample for immune repertoire nucleic acid sequencing, administering a Crohn's disease therapy to a subject diagnosed with Crohn's disease based on the evaluation, and / or the like.

[0026] According to some embodiments, the subject has one or more non-specific symptoms consistent with Crohn's disease at the time of evaluation. Examples of such non-specific symptoms include, but are not limited to, diarrhea, fatigue, abdominal pain, abdominal cramps, rectal bleeding, unintentional weight loss, and any combination thereof. As mentioned above, the method of the present disclosure finds use in providing a differential diagnosis based on the evaluation, for example, where a subject who exhibits one or more non-specific symptoms consistent with Crohn's disease is diagnosed as having Crohn's disease rather than another condition characterized by symptoms that overlap with those of Crohn's disease, including, but not limited to, ulcerative colitis, irritable bowel syndrome, and / or celiac disease.

[0027] According to some embodiments, the methods of the present disclosure find use in providing a differential diagnosis of a subject as having Crohn's disease with fistulization or structuring, as opposed to Crohn's disease without fistulization (e.g., abnormal passages between affected intestinal loops) or structuring (narrowing of the intestine that can result in intestinal obstruction or changes in stool diameter). See, e.g., Gasche et al., Inflamm. Bowel Dis., 6:8-15 (2000). In certain embodiments, the methods of the present disclosure find use in providing a differential diagnosis of a subject as having ileal / ileocolonic Crohn's disease, as opposed to colonic Crohn's disease. More information regarding ileal / ileocolonic Crohn's disease and colonic Crohn's disease may be obtained, for example, from Atreya and Siegmund (2021) Nat Rev Gastroenterol Hepatol 18, 544-558.

[0028] As summarized above, the methods of the disclosure include evaluating a determined TCRβ CDR3 sequence from a sample obtained from a subject for the presence or absence of one or more TCRβ CDR3 sequences set forth in SEQ ID NOs: 1-1996 as described herein, such as SEQ ID NOs: 1-1281 as described herein. As noted above, in certain embodiments, the evaluating step may be computer-implemented, such as performed using one or more processors and one or more non-transitory computer-readable media comprising instructions stored thereon, which when executed by the one or more processors, cause the one or more processors to evaluate the determined TCRβ CDR3 sequence for the presence or absence of one or more TCRβ CDR3 sequences set forth in SEQ ID NOs: 1-1996 (e.g., SEQ ID NOs: 1-1281). For example, the instructions may cause the one or more processors to compare each of the determined TCR β CDR3 sequences (e.g., each determined TCR β CDR3 sequence or each unique determined TCR β CDR3 sequence) stored on a computer-readable medium to a database comprising one or more TCR β CDR3 sequences set forth in SEQ ID NOs: 1-1996 (e.g., SEQ ID NOs: 1-1281), stored on the same or a different computer-readable medium. According to some embodiments, the number of TCR β CDR3 sequences determined from a sample obtained from a subject is between 1,000 and 2,000,000. For example, in certain embodiments, the number of determined TCR β CDR3 sequences is between 1,000 and 2,000,000. The number of CDR3 sequences is 2,000,000 or less (e.g., 1,500,000 or less, 1,250,000 or less, 1,000,000 or less, 750,000 or less, or 500,000 or less), but can be 1,000 or more, 5,000 or more, 10,000 or more, 15,000 or more, 20,000 or more, 25,000 or more, 30,000 or more, 35,000 or more, 40,000 or more, 45,000 or more, 50,000 or more, 55,000 or more, 60,000 or more, 65,000 or more, 70,000 or more, 75,000 or more, 80,000 or more, 85,000 or more, 90,000 or more, 95,000 or more, or 100,000 or more.The number of TCR β CDR3 sequences set forth in SEQ ID NOs: 1 to 1996 (e.g., SEQ ID NOs: 1 to 1281) to which the determined TCR β CDR3 sequence is compared may vary. For example, the determined TCR β CDR3 sequence may be a TCR β CDR3 sequence set forth in SEQ ID NOs: 1 to 1996 (e.g., SEQ ID NOs: 1 to 1281) or each of the CDR3 sequences. When the determined TCR β CDR3 sequence is compared with fewer than all of the TCR β CDR3 sequences set forth in SEQ ID NOs: 1-1996 (e.g., SEQ ID NOs: 1-1281), the determined TCR β CDR3 sequence may be compared with any desired number (e.g., as described above) of, and any desired combination of, the TCR β CDR3 sequences set forth in SEQ ID NOs: 1-1996, e.g., SEQ ID NOs: 1-1281.

[0029] The methods of the present disclosure may include one or more additional steps based on the results of the evaluation step. For example, if it is determined from the evaluation step that none of the TCRβ CDR3 sequences set forth in SEQ ID NOs: 1-1996 (e.g., SEQ ID NOs: 1-1281) are present in the TCRβ CDR3 sequences determined from a sample obtained from a subject (e.g., a subject identified as having IB or suspected of having IBD, including, but not limited to, a subject exhibiting one or more non-specific symptoms consistent with Crohn's disease), the method may further include, for example, identifying the subject as not having Crohn's disease. Also by way of example, if the evaluation step determines that one or more (e.g., two or more, three or more, four or more, five or more, or ten or more) of the TCR β CDR3 sequences set forth in SEQ ID NOs: 1-1996 (e.g., SEQ ID NOs: 1-1281) are present in the TCR β CDR3 sequences determined from a sample obtained from a subject (e.g., a subject identified as having IB or suspected of having IBD, including, but not limited to, a subject exhibiting one or more non-specific symptoms consistent with Crohn's disease), the method may further include, for example, predicting that the subject has Crohn's disease, diagnosing the subject as having Crohn's disease, identifying the subject as one who should be administered a Crohn's disease therapy, and / or administering a Crohn's disease therapy to the subject, e.g., administering to the subject any one or more of the Crohn's disease therapies described elsewhere herein.

[0030] In certain embodiments, the method further comprises subjecting the results of the evaluation step to further analysis, such as subjecting the results of the evaluation step to a model. For example, the method may further comprise subjecting the results of the evaluation step to a model to classify the subject as having or not having Crohn's disease, and / or to classify the subject as having Crohn's disease and not having non-Crohn's disease IBD (e.g., ulcerative colitis). Those skilled in the art will appreciate that, with the benefit of the TCRβ CDR3 sequences set forth in SEQ ID NOs: 1-1996 (e.g., SEQ ID NOs: 1-1281) described herein, a variety of useful models may be applied to the results of the evaluation. In one non-limiting example, the method may further comprise subjecting the results of the evaluation step to a two-feature logistic regression, with features representing the number of Crohn's disease-associated TCRβ CDR3 sequences determined from the sample and the total number of unique TCRβ CDR3 sequences determined from the sample. As shown in the experimental section below, such models show high specificity and sensitivity for Crohn's disease patients.

[0031] In certain embodiments, where the method further comprises subjecting the results of the evaluation step to a model for classification purposes (e.g., as described above), the model may take into account the number of unique Crohn's disease-associated TCRβ CDR3 sequences present in the TCRβ CDR3 sequences determined from the sample (e.g., the greater the number of unique Crohn's disease-associated TCRβ CDR3 sequences, the more likely the model will classify the subject as having Crohn's disease). According to some embodiments, the number of unique Crohn's disease-associated TCRβ CDR3 sequences is not a feature utilized by the model to classify the subject. In certain embodiments, the presence and / or frequency of one or more specific unique Crohn's disease-associated TCRβ CDR3 sequences is the feature(s) used by the model to classify the subject. For example, the presence and / or frequency of one or more specific unique Crohn's disease-associated TCRβ CDR3 sequences may be given relatively greater weight in classifying the subject compared to the presence and / or frequency of one or more other unique Crohn's disease-associated TCRβ CDR3 sequences.

[0032] According to some embodiments, if the classification model weights a particular unique Crohn's disease associated TCRβ CDR3 sequence differently than other unique Crohn's disease associated TCRβ CDR3 sequences, the model may weight the sequence differently using convergent recombination. Different T cells may exhibit convergent recombination, where unique DNA sequences formed in recombination to a first T cell, a second T cell, a third T cell, etc., each resulting in the same protein (CDR3 + V gene + J gene), which is diagnostic of a high likelihood of Crohn's disease. This convergent recombination may be more likely for certain Crohn's disease associated TCRβ CDR3 sequences than others, and the model may take into account these aspects of the signal that reflect the explainable biology of the immune response. Thus, in some embodiments, sequences may be given differential weights based on convergent recombination.

[0033] In certain embodiments, prior to the evaluation step, the method may further comprise one or more steps for determining TCRβ CDR3 sequence from a sample obtained from the subject. For example, the determination may include immunosequencing and evaluation of the T cell repertoire in the biological sample obtained from the subject, for example by high-throughput sequencing (HTS) as described elsewhere herein. The determination may be performed in part using a computer. For example, the analysis of raw sequencing data may be performed by a computer. Extraction, amplification, and sequencing of DNA or RNA from the biological sample may be performed manually, by machine, or a combination thereof. In certain embodiments, the method may further comprise an initial step of obtaining a biological sample from the subject.

[0034] Biological samples (e.g., peripheral blood, intestinal tissue, and / or the like) may be obtained from a variety of subjects. Such subjects may be "mammals" or "mammals," where these terms are used broadly to describe organisms within the class Mammalia, including Carnivora (e.g., dogs and cats), Rodentia (e.g., mice, guinea pigs, and rats), and Primates (e.g., non-human primates such as humans, chimpanzees, and monkeys). In some embodiments, the subject is a human subject.

[0035] Biological samples of interest include those that contain T cells, including, but not limited to, whole blood samples, fractions of whole blood that contain peripheral blood mononuclear cells (e.g., plasma), serum, peripheral blood mononuclear cell (PBMC) samples, gut tissue samples, urine, buffy coat, synovial fluid, bone marrow, cerebrospinal fluid, saliva, lymph, semen, vaginal secretions, urethral secretions, exudates, percutaneous exudates, pharyngeal exudates, nasal secretions, sputum, sweat, bronchoalveolar lavage, tracheal aspirate, fluid from joints, or vitreous humor. T cells may also be obtained from biological samples that may be derived, for example, from solid tissue samples. T cells may be helper T cells (effector T cells or Th cells), cytotoxic T cells (CTLs), memory T cells, and regulatory T cells. In some embodiments, peripheral blood mononuclear cells (PBMCs) are isolated by techniques known to those skilled in the art, for example, Ficoll-Hypaque® density gradient separation.

[0036] Nucleic acids, such as genomic DNA or RNA, may be extracted from lymphocytes by methods known to those of skill in the art. Examples include extracting genomic DNA using the QIAamp® DNA Blood Mini Kit or the Qiagen DNeasy Blood Extraction Kit (both commercially available from Qiagen, Gaithersburg, MD, USA). In some embodiments, 100,000-200,000 cells may be used for analysis of diversity (i.e., about 0.6-1.2 μg of DNA from diploid T cells). Using PBMCs as a source, the number of T cells can be estimated at about 30% of the total cells. Alternatively, total nucleic acids can be isolated from cells containing both genomic DNA and mRNA. In other embodiments, cDNA is transcribed from the mRNA and then used as a template for amplification. RNA molecules can be transcribed into cDNA using known reverse transcription kits, such as, for example, the SMARTer™ Ultra Low RNA Kit for Illumina Sequencing (Clontech, Mountain View, CA), essentially following the supplier's instructions.

[0037] Immune Repertoire Sequencing (Multiplex PCR and High-Throughput Sequencing) According to some embodiments, the TCRβ CDR3 sequence is determined from a sample obtained from a subject by immune cell receptor sequencing (e.g., immune repertoire sequencing).

[0038] "T cell receptor" or "TCR" refers to a disulfide-linked membrane-bound heterodimeric protein consisting of highly variable α and β chains, usually expressed as part of a complex with an invariant CD3 chain molecule. T cells expressing these two chains are called α:β (or αβ) T cells, while a small number of T cells express an alternative receptor, formed by variable γ and σ chains, and are called γσ T cells. TCR development occurs through a lymphocyte-specific process of genetic recombination, which assembles the final sequence from multiple potential segments. This genetic recombination of TCR gene segments in somatic T cells occurs during the early stages of thymic development. The TCRα locus contains variable (V) and joining (J) gene segments (Vα and Jα), whereas the TCRβ locus contains a D gene segment in addition to the Vβ and Jβ segments. Thus, the α chain is generated from VJ recombination, and the β chain is involved in VDJ recombination. This is similar to the development of γδTCR, where the TCRγ chain is involved in VJ recombination and the TCRδ gene is generated from VDJ recombination. The TCRα chain locus consists of 46 variable segments, 8 joining segments, and a constant region. The TCRβ chain locus consists of 48 variable segments followed by two diversity segments, 12 joining segments, and two constant regions. The D and J segments are located within a relatively short 50 kb region, while the variable genes are spread over a large region of 1.5 megabases (TCRα) or 0.67 megabases (TCRβ).

[0039] TCRβ CDR3 sequencing can involve quantitative detection of the sequences of virtually all possible TCR gene rearrangements that may be present in a sample containing lymphocyte DNA.

[0040] The amplified nucleic acid molecule comprising the rearranged TCR region obtained from the biological sample is sequenced using high-throughput sequencing. In one embodiment, a multiplex PCR system is used to amplify the rearranged TCR locus from genomic DNA, as described in U.S. Patent Application Publication No. 2010 / 0330571, filed June 4, 2010, U.S. Patent Application Publication No. 2012 / 0058902, filed August 24, 2011, and International Application No. PCT / US2013 / 062925, filed October 1, 2013 (each of which is incorporated by reference in its entirety).

[0041] To that end, multiplex PCR is performed using a set of forward primers that specifically hybridize to the V segments and a set of reverse primers that specifically hybridize to the J segments of the TCR locus, where the multiplex PCR reaction using the primers allows the amplification of all possible VJ (and VDJ) combinations within a given T cell population.

[0042] Exemplary V segment and J segment primers are described in US2012 / 0058902, US2010 / 033057, WO2010 / 151416, WO2011 / 106738, US2015 / 0299785, WO2012 / 027503, US2013 / 0288237, U.S. Patent No. 9,181,590, U.S. Patent No. 9,181,591, US2013 / 0253842, WO2013 / 188831, each of which is incorporated by reference in its entirety.

[0043] A multiplex PCR system can be used to amplify the rearranged immune cell receptor locus. In certain embodiments, the CDR3 region is amplified from the TCRB CDR3 region locus. Multiple V segment and J segment primers are used to amplify substantially all (e.g., more than 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) of the rearranged immune cell receptor CDR3 coding region to generate a multiplicity of amplified rearranged DNA molecules. In certain embodiments, the primers are designed such that each amplified rearranged DNA molecule is less than 600 nucleotides in length, thereby excluding the amplification products from non-rearranged immune cell receptor locus.

[0044] In some embodiments, two pools of primers are used in a single highly multiplexed PCR reaction. The "forward" pool of primers can include multiple V segment oligonucleotide primers, and the reverse pool can include multiple J segment oligonucleotide primers. In some embodiments, there is a primer that is specific (e.g., has a nucleotide sequence complementary to a unique sequence region thereof) for each V region segment and each J region segment in each TCR or Ig locus. In other embodiments, the primers can hybridize to one or more V or J segments, thereby reducing the number of primers required for multiplex PCR. In certain embodiments, the J segment primers anneal to sequences conserved in the joining ("J") segments.

[0045] Each primer can be designed so that each amplified DNA segment is obtained that contains a sequence portion of sufficient length to unambiguously identify each J segment based on the sequence difference between known J region-encoding gene segments in the human genome database, and also contains a sequence portion to which the J segment-specific primer can anneal for resequencing.This design of V segment-specific primer and J segment-specific primer allows direct observation of the majority of somatic rearrangements present in the immune cell receptor gene repertoire in a subject.

[0046] The multiplex PCR system may comprise at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25, and in certain embodiments at least 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, or 39, and in other embodiments At least 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 65, 70, 75, 80, 85, or more forward primers can be used, each of which specifically hybridizes (i.e., is complementary to) a sequence corresponding to a V region segment. The multiplex PCR system also uses at least 2, 3, 4, 5, 6, or 7, and in certain embodiments at least 8, 9, 10, 11, 12, or 13 reverse primers, or at least 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 or more reverse primers, each of which specifically hybridizes or is complementary to a sequence corresponding to a J region segment. Various combinations of V segment and J segment primers can be used to amplify the full diversity of TCR sequences in the immune cell receptor gene repertoire in a subject.

[0047] Details regarding the multiplex PCR system, including primer oligonucleotide sequences for amplifying TCR sequences, can be found in Robins et al., 2009 Blood 114, 4099; Robins et al., 2010 Sci. Translat. Med. 2:47ra64; Robins et al., 2011 J. Immunol. Meth. doi:10.1016 / j.jim.2011.09.001; Sherwood et al. 2011 Sci. Translat. Med. 3:90ra61; US2012 / 0058902, US2010 / 033057, WO / 2010 / 151416, WO / 2011 / 106738, US No. 2015 / 0299785, WO2012 / 027503, US2013 / 0288237, U.S. Patent No. 9,181,590, U.S. Patent No. 9,181,591, US2013 / 0253842, and WO2013 / 188831, each of which is incorporated by reference in its entirety herein.

[0048] Oligonucleotides or polynucleotides capable of specifically hybridizing or annealing to a target nucleic acid sequence due to the complementarity of the nucleotide bases can do so under moderate to high stringency conditions. In one embodiment, moderate to high stringency conditions suitable for specific PCR amplification of a target nucleic acid sequence can be 25-80 PCR cycles, each cycle including a denaturation step (e.g., above about 95°C for about 10-30 seconds), an annealing step (e.g., at about 60-68°C for about 10-30 seconds), and an extension step (e.g., at about 60-72°C for about 10-60 seconds), optionally, according to certain embodiments, the annealing and extension steps are combined to provide a two-step PCR. As will be appreciated by those skilled in the art, other PCR reagents can be added or modified in the PCR reaction to increase the specificity of primer annealing and amplification, such as altering the magnesium concentration, optionally adding DMSO, and / or using blocked primers, modified nucleotides, peptide-nucleic acids, etc.

[0049] The primer may be a single-stranded DNA. The appropriate length of the primer depends on the intended use of the primer, but typically ranges from 6 to 50 nucleotides, or in certain embodiments, 15 to 35 nucleotides in length. Short primer molecules generally require lower temperatures to form a sufficiently stable hybrid complex with the template. The primer need not reflect the exact sequence of the template nucleic acid, but must be sufficiently complementary to hybridize with the template. The design of suitable primers for amplification of a given target sequence is well known in the art and described in the literature cited herein.

[0050] V segment primers and J segment primers are used to generate multiple amplicons from a multiplex PCR reaction. In certain embodiments, the size of the amplicons ranges from 10, 20, 30, 40, 50, 75, 100, 200, 300, 400, 500, 600, 700, 800 or more nucleotides in length. In certain embodiments, the amplicons have a size of 20-600, 50-600, 20-400, or 50-400 nucleotides in length.

[0051] According to a non-limiting theory, these embodiments take advantage of the current understanding in the art (also described above) that once a T lymphocyte has rearranged its TCR-encoding genes, its progeny cells will have the same immune cell receptor-encoding gene rearrangement, thus resulting in a clonal population (clone) that can be uniquely identified by the presence therein of rearranged (e.g., CDR3-encoding) V and J gene segments, which can be amplified by specific pairwise combinations of V-specific and J-specific oligonucleotide primers disclosed herein.

[0052] The V segment primer and the J segment primer will preferably each include a second sequence at the 5' end of the primer that is not complementary to the target V segment or J segment. The second sequence can include an oligonucleotide having a sequence selected from (i) a universal adaptor oligonucleotide sequence, and (ii) a sequencing platform specific oligonucleotide sequence attached to and positioned 5' to the first universal adaptor oligonucleotide sequence. Examples of universal adaptor oligonucleotide sequences can be pGEX forward and pGEX reverse adaptor sequences.

[0053] The resulting amplicons using the V segment and J segment primers described above include the amplified V and J segments, as well as a universal adaptor oligonucleotide sequence. The universal adaptor sequence can be complementary to the oligonucleotide sequence found in the tailing primer. The tailing primer can be used in a second PCR reaction to generate a second set of amplicons. In some embodiments, the tailing primer can have the general formula (I):

[0054] 5'-P--S--B--U-3' (I),

[0055] where P comprises a sequencing platform-specific oligonucleotide, S comprises a sequencing platform tag-containing oligonucleotide sequence, B comprises an oligonucleotide barcode sequence that can be used to identify the sample source, and U comprises a sequence that is complementary to or the same as the universal adapter oligonucleotide sequence.

[0056] Additional description of universal adapter oligonucleotide sequences, barcodes, and tailing primers can be found in WO2013 / 188831, which is incorporated by reference in its entirety.

[0057] Sequencing may be performed using any of a variety of available high-throughput nucleic acid sequencing machines and systems.Exemplary sequencing systems include Illumina iSeq 100, Miniseq, MiSeq series, NextSeq series (e.g., NextSeq 500 series, NextSeq 1000, NextSeq 2000) and NovaSeq sequencing system (Illumina, Inc., San Diego, CA), Pacific Biosciences Sequel (e.g., Sequel II) sequencing system (Pacific Biosciences, Menlo Park, CA), Oxford Nanopore Technologies MinION™, GridIONx5™, PromethION™, or SmidgION™ nanopore-based sequencing system (Oxford Nanopore Technologies, Oxford, UK), and other systems with similar capabilities.

[0058] In certain embodiments, sequencing is achieved by using a set of sequencing platform-specific oligonucleotides that hybridize to defined regions in the amplified DNA molecule.The sequencing platform-specific oligonucleotides are designed to sequence amplicons so that V-coding gene segments and J-coding gene segments can be uniquely identified by the sequence generated.See, for example, US2012 / 0058902, US2010 / 033057, WO2011 / 106738, US2015 / 0299785, or WO2012 / 027503 (each of which is incorporated by reference in its entirety).

[0059] In some embodiments, raw sequence data is preprocessed to remove errors in the primary sequence of each read and compress data.Nearest neighbor algorithm can be used to collapse data into unique sequence by merging closely related sequences, and remove both PCR error and sequencing error.See, for example, US2012 / 0058902, US2010 / 033057, WO2011 / 106738, US2015 / 0299785, or WO2012 / 027503 (each of which is incorporated by reference in its entirety).

[0060] Sequencing multiplexes of amplified rearranged TCR β CDR3 coding region DNA molecules by high-throughput sequencing (HTS) can be used to generate TCR clonotype profiles containing at least 10,000 TCR clonotype sequences ranging from 20 to 400 nucleotides in length.

[0061] Amplification Bias Control Given that certain primer sets may have higher amplification efficiency than others, multiplex PCR assays may introduce bias in the total number of amplicons generated from a sample. To overcome the problem of such biased utilization of a subpopulation of amplification primers, a method can be used that provides a template composition for standardizing the amplification efficiency of members of an oligonucleotide primer set, the primer set being capable of amplifying rearranged DNAs encoding multiple TCRs in a biological sample that includes DNA from lymphocytes.

[0062] To this end, the template composition is used to normalize the different amplification efficiencies of the primer sets. The template composition can comprise a plurality of different template oligonucleotides of general formula (II).

[0063] 5'-U1-B1-V-B2-RJ-B3-U2-3' (II)

[0064] The component template oligonucleotides are diverse with respect to the nucleotide sequence of each template oligonucleotide. Each template oligonucleotide can differ from each other considerably in nucleotide sequence as a function of the significant sequence variability between the many possible TCR variable (V) and joining (J) region polynucleotides. The sequence of each template oligonucleotide species can also differ from each other as a function of the sequence differences of the U1, U2, B (B1, B2 and B3) and R oligonucleotides contained in a particular template within the diverse plurality of templates.

[0065] V is a polynucleotide comprising at least 20, 30, 60, 90, 120, 150, 180, or 210, and no more than 1000, 900, 800, 700, 600, or 500 contiguous nucleotides of an adaptive immune receptor variable (V) region encoding gene sequence, or its complement, and in each of the plurality of template oligonucleotide sequences, V comprises a unique oligonucleotide sequence.

[0066] J is a polynucleotide comprising at least 15-30, 31-60, 61-90, 91-120, or 120-150, and not more than 600, 500, 400, 300, or 200 contiguous nucleotides of an adaptive immune receptor binding (J) region-encoding gene sequence, or its complement, and in each of the plurality of template oligonucleotide sequences, J comprises a unique oligonucleotide sequence.

[0067] U1 and U2 can each comprise nothing or an oligonucleotide independently having a sequence selected from (i) a universal adapter oligonucleotide sequence, and (ii) a sequencing platform-specific oligonucleotide sequence bound to and positioned 5' to the universal adapter oligonucleotide sequence.

[0068] B1, B2 and B3 are each either nothing or a first and second nucleotide of 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900 or 1000 (including all integer values ​​therebetween) contiguous nucleotides. and an oligonucleotide B comprising a second oligonucleotide barcode sequence, where in each of the plurality of template oligonucleotide sequences, B comprises a unique oligonucleotide sequence, where (i) the first barcode sequence uniquely identifies a unique V oligonucleotide sequence of the template oligonucleotide, and (ii) the second barcode sequence uniquely identifies a unique J oligonucleotide sequence of the template oligonucleotide.

[0069] R can contain none or a restriction enzyme recognition site that comprises an oligonucleotide sequence that is not present in V, J, U1, U2, B1, B2, and B3.

[0070] The method is used with a template composition to determine heterogeneous nucleic acid amplification capabilities among members of a set of oligonucleotide amplification primers capable of amplifying productively rearranged DNA encoding one or more TCRs in a biological sample comprising DNA from lymphocytes of a subject. The method can include (a) amplifying DNA of a template composition to standardize the amplification efficiency of the oligonucleotide primer set in a multiplex polymerase chain reaction (PCR), the template composition including (i) the above-mentioned template composition (II), in which each template oligonucleotide in the plurality of template oligonucleotides is present in substantially equimolar amounts, and (ii) an oligonucleotide amplification primer set capable of amplifying productively rearranged DNA encoding one or more TCRs in a biological sample comprising DNA from lymphocytes of the subject.

[0071] The primer set can include: (1) a plurality of V segment oligonucleotide primers, each independently capable of specifically hybridizing to at least one polynucleotide encoding a TCR V region polypeptide or its complement, in substantially equimolar amounts, where each V segment primer comprises a nucleotide sequence of at least 15 contiguous nucleotides complementary to at least one functional TCR V region encoding gene segment, and where the plurality of V segment primers specifically hybridize to substantially all functional TCR V region encoding gene segments present in the template composition; and (2) a plurality of J segment oligonucleotide primers, each independently capable of specifically hybridizing to at least one polynucleotide encoding a TCR J region polypeptide or its complement, in substantially equimolar amounts, where each J segment primer comprises a nucleotide sequence of at least 15 contiguous nucleotides complementary to at least one functional TCR J region encoding gene segment, and where the plurality of J segment primers specifically hybridize to substantially all functional TCR J region encoding gene segments present in the template composition.

[0072] The V-segment and J-segment oligonucleotide primers are capable of facilitating amplification in said multiplex polymerase chain reaction (PCR) of substantially all of the template oligonucleotides in the template composition to generate a plurality of amplified template DNA molecules, said plurality of amplified template DNA molecules being sufficient to quantify the diversity of the template oligonucleotides in the template composition, and each amplified template DNA molecule in the plurality of amplified template DNA molecules being less than 1000, less than 900, less than 800, less than 700, less than 600, less than 500, less than 400, less than 300, less than 200, less than 100, less than 90, less than 80, or less than 70 nucleotides in length.

[0073] The method for determining heterogeneous nucleic acid amplification capacity may further include (b) sequencing all or a sufficient portion of each of the plurality of amplified template DNA molecules to determine, for each unique template DNA molecule in the plurality of amplified template DNA molecules, the relative frequency of occurrence of (i) the template-specific oligonucleotide DNA sequence, and (ii) the template oligonucleotide; and (c) comparing the relative frequency of occurrence for each unique template DNA sequence from the template composition, wherein heterogeneous frequencies of occurrence for one or more template DNA sequences indicate heterogeneous nucleic acid amplification capacity among members of a set of oligonucleotide amplification primers.

[0074] Further details regarding the aforementioned bias control methods are provided in US 2013 / 0253842, U.S. Pat. No. 9,150,905, US 2015 / 0203897, and WO 2013 / 169957, which are incorporated by reference in their entireties.

[0075] Estimation of PCR template abundance To estimate the average read coverage per input template in multiplex PCR and sequencing approach, a set of synthetic TCR templates (as described above) can be used that includes each combination of V. beta and J. beta gene segments.These synthetic molecules can be as described in the above general formula (II) and in US2013 / 0253842, US Patent No. 9,150,905, US2015 / 0203897, and WO2013 / 169957, which are incorporated by reference in their entirety.

[0076] These synthetic molecules can be included in each PCR reaction at very low concentrations so that only a fraction of the synthetic templates are observed. Using known concentrations of the synthetic template pool, one can simulate the relationship between the number of unique synthetic molecules observed and the total number of synthetic molecules added to the reaction (which is very close to one to one at the low concentrations used). The synthetic molecules allow the calculation of the average number of sequencing reads obtained per molecule of PCR template for each PCR reaction, and an estimate of the number of T cells or B cells in the input material with each unique TCR or Ig rearrangement, respectively.

[0077] Table 1 - Crohn's disease-associated TCRβ CDR3 amino acid sequences [Table 1] TIFF2024515435000003.tif250161TIFF2024515435000004.tif250161TIFF2024515435000005.tif249165TIFF2024515435000006.tif250165TIFF2024515435000007.tif250162TIFF2024515435000008.tif250165TIFF2024515435000009.tif250165TIFF2024515435000010.tif251164TIFF2024515435000011.tif251162TIFF2024515435000012.tif250165TIFF2024515435000013.tif250165TIFF2024515435000014.tif251161TIFF2024515435000015.tif250165TIFF2024515435000016.tif250165TIFF2024515435000017.tif249164TIFF2024515435000018.tif249164TIFF2024515435000019.tif249164TIFF2024515435000020.tif249164TIFF2024515435000021.tif252166TIFF2024515435000022.tif250165TIFF2024515435000023.tif251165TIFF2024515435000024.tif251161TIFF2024515435000025.tif251164TIFF2024515435000026.tif251165TIFF2024515435000027.tif251162TIFF2024515435000028.tif250165TIFF2024515435000029.tif249165TIFF2024515435000030.tif250165TIFF2024515435000031.tif251165TIFF2024515435000032.tif250166TIFF2024515435000033.tif250165TIFF2024515435000034.tif250164TIFF2024515435000035.tif250166TIFF2024515435000036.tif250166TIFF2024515435000037.tif85169.

[0078] Table 2 - Additional Crohn's disease-associated TCRβ CDR3 amino acid sequences [Table 2] TIFF2024515435000039.tif251153TIFF2024515435000040.tif251151TIFF2024515435 000041.tif251156TIFF2024515435000042.tif251156TIFF2024515435000043.tif25015 6TIFF2024515435000044.tif251152TIFF2024515435000045.tif251155TIFF2024515435 000046.tif250155TIFF2024515435000047.tif250153TIFF2024515435000048.tif25015 2TIFF2024515435000049.tif251157TIFF2024515435000050.tif249154TIFF2024515435 000051.tif250153TIFF2024515435000052.tif251156TIFF2024515435000053.tif25115 6TIFF2024515435000054.tif251151TIFF2024515435000055.tif251153TIFF2024515435 000056.tif251153TIFF2024515435000057.tif251156TIFF2024515435000058.tif59159

[0079] In Tables 1 and 2 herein, the amino acid sequence represents the TCRβ CDR3 segment of a TCR, while V##-## or J##-## refer to the standard two-tiered coding system [family]-[gene] for specific portions of the human genome that may be used as part of a TCR rearrangement formed in response to antigen exposure. The first two digits reflect the member of the family, and the second two digits reflect the specific gene, if any, from within that family. Thus, by way of example, TCRBV06 indicates a sequence match to a specific family of variable (V) chain sequences, while TCRBV06-05 indicates a more precise discrimination to a specific gene from within the family of variable chain sequences.

[0080] The identities of these V-gene and J-gene sequences can be found in the international ImMunoGeneTics information system (http: / / www.imgt.org), including www.imgt.org / download / V-QUEST / IMGT_V-QUEST_reference_directory / Homo_sapiens / TR / TRBV.fasta.

[0081] Treatment method Also provided by the present disclosure are methods of treatment. According to some embodiments, methods are provided that include administering Crohn's disease therapy to a subject identified as comprising T cells expressing a T cell receptor β chain (TCRβ) comprising a TCRβ CDR3 sequence set forth in SEQ ID NOs: 1-1996 (e.g., SEQ ID NOs: 1-1281). In certain embodiments, the method includes administering Crohn's disease therapy to a subject identified as comprising T cells expressing two or more (e.g., two or more unique) TCRβs comprising a TCRβ CDR3 sequence set forth in SEQ ID NOs: 1-1996 (e.g., SEQ ID NOs: 1-1281). According to some embodiments, the method includes administering Crohn's disease therapy to a subject identified as having Crohn's disease using a model / classifier as described elsewhere herein. Such models include, but are not limited to, those that use bi-feature logistic regression with features representing the number of Crohn's disease-associated TCRβ CDR3 sequences determined from the sample and the total number of unique TCRβ CDR3 sequences determined from the sample. As shown in the experimental section below, such models show high specificity and sensitivity for Crohn's disease patients. In certain embodiments, the model may take into account the number of unique Crohn's disease-associated TCRβ CDR3 sequences present in the TCRβ CDR3 sequences determined from the sample (e.g., the greater the number of unique Crohn's disease-associated TCRβ CDR3 sequences, the more likely the model will classify the subject as having Crohn's disease). According to some embodiments, the number of unique Crohn's disease-associated TCRβ CDR3 sequences is not a feature utilized by the model to classify the subject. In certain embodiments, the presence and / or frequency of one or more particular unique Crohn's disease-associated TCRβ CDR3 sequences is the feature(s) used by the model to classify the subject. For example, the presence and / or frequency of one or more particular unique Crohn's disease-associated TCRβ CDR3 sequences may be given relatively greater weight in classifying the subject compared to the presence and / or frequency of one or more other unique Crohn's disease-associated TCRβ CDR3 sequences.According to some embodiments, where the classification model weights a particular unique Crohn's disease associated TCR β CDR3 sequence differently than other unique Crohn's disease associated TCR β CDR3 sequences, the model may use convergent recombination to weight that sequence differently, as described elsewhere herein.

[0082] Any suitable Crohn's disease therapy may be administered to the subject identified as above. Crohn's disease therapies are known and may vary depending on the patient's age, stage of disease, and / or the like. In certain embodiments, the Crohn's disease therapy includes administering to the subject a therapeutically effective amount of an anti-inflammatory drug. Non-limiting examples of anti-inflammatory drugs that find use in treating Crohn's disease include corticosteroids (e.g., prednisone, budesonide, or combinations thereof) and 5-aminosalicylates, such as sulfasalazine, mesalamine, or combinations thereof. According to some embodiments, the Crohn's disease therapy includes administering to the subject a therapeutically effective amount of an immunosuppressant. Immunosuppressants that find use in treating Crohn's disease include, but are not limited to, azathioprine, mercaptopurine, methotrexate, or any combination thereof. In certain embodiments, the Crohn's disease therapy includes administering to the subject a therapeutically effective amount of a monoclonal antibody. Non-limiting examples of monoclonal antibodies that find use in the treatment of Crohn's disease include natalizumab, vedolizumab, infliximab, adalimumab, certolizumab pegol, ustekinumab, or any combination thereof. According to some embodiments, the Crohn's disease therapy includes administering to the subject a therapeutically effective amount of an antibiotic (e.g., ciprofloxacin, metronidazole, or a combination thereof). In certain embodiments, the Crohn's disease therapy includes surgery. According to some embodiments, the surgery is suitable for Crohn's disease and not ulcerative colitis. In certain embodiments, the surgery is suitable for Crohn's disease and not irritable bowel syndrome. According to some embodiments, the surgery is suitable for Crohn's disease and not celiac disease. In certain embodiments, the surgery is suitable for Crohn's disease with fistulization or structuring and not Crohn's disease without fistulization or structuring. According to some embodiments, the surgery is suitable for ileal / ileocolonic Crohn's disease and not colonic Crohn's disease.

[0083] In certain embodiments, prior to administering a Crohn's disease therapy to the subject, the method includes identifying the subject as having Crohn's disease, but not ulcerative colitis, based on identifying the subject as comprising T cells expressing one or more TCRβ comprising one or more TCRβ CDR3 sequences set forth in SEQ ID NOs: 1-1996 or 1-1281. According to some embodiments, the Crohn's disease therapy (e.g., drugs and / or surgery) is a treatment appropriate for Crohn's disease, but not ulcerative colitis.

[0084] According to some embodiments, prior to administering a Crohn's disease therapy to the subject, the method includes identifying the subject as having Crohn's disease, but not irritable bowel syndrome, based on identifying the subject as comprising T cells expressing one or more TCRβ that comprise one or more TCRβ CDR3 sequences set forth in SEQ ID NOs: 1-1996 or 1-1281. In certain embodiments, the Crohn's disease therapy (e.g., drugs and / or surgery) is a therapy appropriate for Crohn's disease, but not irritable bowel syndrome.

[0085] In certain embodiments, prior to administering a Crohn's disease therapy to the subject, the method includes identifying the subject as having Crohn's disease, but not celiac disease, based on identifying the subject as comprising T cells expressing one or more TCRβ that comprise one or more TCRβ CDR3 sequences set forth in SEQ ID NOs: 1-1996 or 1-1281. According to some embodiments, the Crohn's disease therapy (e.g., drugs and / or surgery) is a treatment appropriate for Crohn's disease, but not celiac disease.

[0086] According to some embodiments, prior to administering a Crohn's disease therapy to the subject, the method includes identifying the subject as having Crohn's disease with fistulization or structuring, as opposed to Crohn's disease without fistulization or structuring, based on identifying the subject as comprising T cells expressing one or more TCRβ comprising one or more TCRβ CDR3 sequences set forth in SEQ ID NOs: 1-1996 or SEQ ID NOs: 1-1281. In certain embodiments, the Crohn's disease therapy (e.g., drugs and / or surgery) is a therapy appropriate for Crohn's disease with fistulization or structuring, as opposed to Crohn's disease without fistulization or structuring.

[0087] In certain embodiments, prior to administering Crohn's disease therapy to the subject, the method includes identifying the subject as having ileal / ileocolonic Crohn's disease, but not colonic Crohn's disease, based on identifying the subject as comprising T cells expressing one or more TCRβ comprising one or more TCRβ CDR3 sequences set forth in SEQ ID NOs: 1-1996 or 1-1281. According to some embodiments, the Crohn's disease therapy (e.g., drugs and / or surgery) is a treatment appropriate for ileal / ileocolonic Crohn's disease, but not colonic Crohn's disease.

[0088] Various therapies for the treatment of Crohn's disease (including specific treatments of Crohn's disease or subtypes thereof, but not including ulcerative colitis, irritable bowel syndrome, or celiac disease, including specific treatments of specific Crohn's disease subtypes, e.g., Crohn's disease with fistulization or structuring but not Crohn's disease without fistulization or ileal / ileocolonic Crohn's disease but not colonic Crohn's disease) are known, e.g., as described in Gade et al. (2020) Cureus 12(5):e8351; Cushing & Higgins (2021) JAMA 325(1):69-80; Shi & Ng (2018) J Gastroenterol. 53(9): 989-998; and Sulz et al. (2020) Digestion 101 Suppl 1:43-57 (DOI: No. 10.1159 / 000506364, the disclosures of which are incorporated herein by reference in their entireties for all purposes.

[0089] According to some embodiments, the method is effective for treating Crohn's disease in an individual. "Treating" or "treatment" refers to at least improving symptoms associated with Crohn's disease (e.g., diarrhea, fatigue, abdominal pain, abdominal cramps, rectal bleeding, unintentional weight loss, or any combination thereof), where improvement is used broadly to refer to at least reducing the magnitude of the parameters (e.g., symptoms) associated with the Crohn's disease being treated. Thus, treatment also includes the situation where Crohn's disease, or at least symptoms associated with Crohn's disease, are completely inhibited (e.g., prevented from occurring or stopped, e.g., ended), so that an individual no longer suffers from Crohn's disease, or at least symptoms that characterize Crohn's disease.

[0090] Dosage may depend on the severity and responsiveness of the disease state to be treated. Optimal dosing schedules can be calculated from measurements of drug accumulation in the body of an individual. The administering physician can determine the optimal dosage, dosing method, and repetition period. Optimal dosages may vary depending on the relative potency of individual therapeutic agents, and are generally determined by ECs found to be effective in in vitro and in vivo animal models, etc. 50 The dosage can be estimated based on the following. Generally, the dosage is about 0.01 μg to about 100 g per kg of body weight and may be given one or more times daily, weekly, monthly, or yearly. In certain embodiments, the dosage is about 1 μg / kg to 100 mg / kg or more, depending on the factors mentioned above. The treating physician can estimate the repetition period of administration based on the measured residence time and concentration of the therapeutic agent in bodily fluids or tissues. Following successful treatment, it may be desirable to subject the subject to maintenance therapy to prevent recurrence of the disease state, in which case the therapeutic agent is administered at a maintenance dose ranging from about 0.01 μg to about 100 g per kg of body weight, from once or more times daily to once every few months, once every six months, once a year, or any other suitable frequency.

[0091] The therapeutic methods of the present disclosure may include administering a single type of therapeutic agent to a subject, or may include administering two or more types of therapeutic agents to a subject, either separately or by administration of a cocktail of different therapeutic agents. For example, in certain embodiments, two or more therapeutic agents (e.g., anti-inflammatory agents, immunosuppressants, monoclonal antibodies, and / or antibiotics) that find use in treating Crohn's disease as described elsewhere herein may be administered to a subject, e.g., two or more, three or more, four or more, or five or more of such therapeutic agents.

[0092] One or more therapeutic agents may be administered to a subject using any available method and route suitable for drug delivery, including in vivo and ex vivo methods, and systemic and local administration routes. Conventional and pharmacologic acceptable routes of administration include oral and parenteral routes of administration. Parenteral routes of administration of interest include, but are not limited to, injection (e.g., intravenous, intraarterial, topical, subcutaneous, or intramuscular injection), intranasal, intratracheal, intradermal, topical application, ocular, nasal, and other parenteral routes of administration. Routes of administration may be combined as needed, or adjusted depending on the therapeutic agent and / or the desired effect. The therapeutic agent may be administered in a single dose or in multiple doses. In some embodiments, the therapeutic agent is administered intravenously. In some embodiments, the therapeutic agent is administered by injection, for example, for systemic delivery (e.g., intravenous infusion) or to a localized site.

[0093] A "therapeutically effective amount" or "effective amount" refers to the amount of a therapeutic agent that, when administered to a mammal or other subject for treating a disease, is sufficient to effect such treatment for the disease. A "therapeutically effective amount" will vary depending on the therapeutic agent, the disease and its severity, and the age, weight, etc., of the subject being treated.

[0094] In some embodiments, the Crohn's disease therapy is adoptive cell therapy.Non-limiting examples of adoptive cell therapy include those involving administering to a subject an effective amount of recombinant cells (e.g., recombinant immune cells such as T cells) that express a T cell receptor that contains the Crohn's disease-related TCRβ CDR3 sequence identified to be present in the TCR expressed by the subject's T cells.Similar to CAR therapy, TCR therapy modifies the patient's T lymphocytes ex vivo before being administered back into the patient's body.TCR-T cell therapy has a broader range of targets, since the target antigens identified by CAR-T cell therapy are all cell surface proteins, while TCR-T cell therapy can recognize intracellular antigen fragments presented by MHC molecules. TCR therapy approaches are known and are described, for example, in Zhang et al. (2019) Technol Cancer Res Treat. 18:1533033819831068; Govers et al. (2010) Trends in Molecular Medicine 16(2):77-87; Zhao et al. (2019) Front. Immunol. 10:2250.

[0095] Nucleic acids encoding a T cell receptor β chain comprising a TCRβ CDR3 sequence as set forth in SEQ ID NOs: 1-1996 (e.g., SEQ ID NOs: 1-1281) are also provided. For example, in certain embodiments, an expression vector is provided comprising a nucleic acid sequence encoding a T cell receptor β chain comprising a TCRβ CDR3 sequence as set forth in SEQ ID NOs: 1-1996 (e.g., SEQ ID NOs: 1-1281), operably linked to a nucleic acid expression control sequence. A "vector" has the ability to introduce a nucleic acid sequence into a target cell (e.g., viral vectors, non-viral vectors, particle carriers, and liposomes). Typically, a "vector construct", "expression vector", or "gene transfer vector" refers to any nucleic acid construct capable of directing the expression of a nucleic acid of interest and capable of introducing a nucleic acid sequence into a target cell. Thus, the term includes cloning and expression vehicles, as well as viral vectors.

[0096] To express a desired T cell receptor β chain comprising a TCR β CDR3 sequence as set forth in SEQ ID NOs: 1-1996 (e.g., SEQ ID NOs: 1-1281), a nucleotide sequence encoding the T cell receptor β chain can be inserted into an appropriate vector, for example, using recombinant DNA techniques known in the art. Exemplary viral vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (e.g., herpes simplex viruses), pox viruses, papilloma viruses, and papova viruses (e.g., SV40). Illustrative examples of expression vectors include, but are not limited to, pClneo vector (Promega) for expression in mammalian cells, pLenti4 / V 5-DEST™, pLenti6 / V 5-DEST™, murine stem cell virus (MSCV), MSGV, Moloney murine leukemia virus (MMLV), and pLenti6.2 / V5-GW / lacZ (Invitrogen) for lentivirus-mediated gene transfer and expression in mammalian cells. In certain embodiments, the nucleic acid sequence encoding the T cell receptor β chain may be ligated into any such expression vector for expression of the T cell receptor β chain in mammalian cells.

[0097] The expression control sequences, control elements, or regulatory sequences present in the expression vector are the origin of replication, selection cassettes, promoters, enhancers, translation initiation signals (Shine Dalgarno or Kozak sequences), introns, polyadenylation sequences, 5' and 3' untranslated regions, and / or the like, which are the untranslated regions of the vector that interact with host cell proteins to carry out transcription and translation. Such elements can vary in their strength and specificity. Depending on the vector system and host utilized, any number of suitable transcription and translation elements may be used, including ubiquitous promoters and inducible promoters.

[0098] The components of an expression vector are operably linked such that they are in a relationship permitting them to function in their intended manner. In some embodiments, the term refers to the functional linkage between a nucleic acid expression control sequence (such as a promoter and / or enhancer) and a second polynucleotide sequence, e.g., a nucleic acid encoding a T cell receptor beta chain, where the expression control sequence directs the transcription of the nucleic acid encoding the T cell receptor beta chain.

[0099] In some embodiments, the expression vector is an episomal vector, or a vector that is maintained extrachromosomally. As used herein, the term "episomal" refers to a vector that is capable of replicating without integration into the chromosomal DNA of the host cell and without gradual loss from dividing host cells, and also means that the vector replicates extrachromosomally or episomally. Such vectors may be engineered to carry sequences coding for origins of DNA replication or "ori" from alpha-, beta-, or gamma-herpesviruses, adenovirus, SV40, bovine papillomavirus, yeast, and the like. The host cell may contain viral replication transactivator proteins that activate replication. Alphaherpesviruses have a relatively short growth cycle, a variable host range, and efficiently destroy infected cells, establishing latent infections primarily in sensory ganglia. Illustrative examples of alphaherpesviruses include HSV1, HSV2, and VZV. Betaherpesviruses have a long growth cycle and a limited host range. Infected cells often become enlarged. Non-limiting examples of beta-herpesviruses include CMV, HHV-6, and HHV-7. Gamma-herpesviruses are specific for either T-lymphocytes or B-lymphocytes, and often show latency in lymphoid tissues. Illustrative examples of gamma-herpesviruses include EBV and HHV-8.

[0100] Also provided is a recombinant cell comprising any of the expression vectors of the present disclosure, comprising a nucleic acid encoding a T cell receptor β chain comprising a TCR β CDR3 sequence set forth in SEQ ID NOs: 1 to 1996, for example, SEQ ID NOs: 1 to 1281. In a specific embodiment, a cell is provided that expresses a TCR comprising a T cell receptor β chain comprising a TCR β CDR3 sequence set forth in SEQ ID NOs: 1 to 1996 (for example, SEQ ID NOs: 1 to 1281) on the cell surface.

[0101] In some embodiments, the cell of the present disclosure is a eukaryotic cell. Eukaryotic cells of interest include, but are not limited to, yeast cells, insect cells, mammalian cells, etc. Mammalian cells of interest include, for example, mouse cells, non-human primate cells, human cells, etc.

[0102] "Recombinant host cell", "host cell", "cell", "cell line", "cell culture", and other such terms referring to microorganisms or higher eukaryotic cell lines refer to cells that can be or have been used as recipients of recombinant vectors or other transferred DNA, including the progeny of transfected cells. Host cells may be cultured as single cells or multicellular entities (e.g., tissues, organs, or organoids) that contain the expression vectors of the present disclosure.

[0103] In one aspect, the cells provided herein comprise immune cells. Non-limiting examples of recombinant immune cells that may comprise any of the expression vectors of the present disclosure include T cells, B cells, natural killer (NK) cells, macrophages, monocytes, neutrophils, dendritic cells, mast cells, basophils, and eosinophils. In some embodiments, the immune cells are T cells. Examples of T cells include naive T cells (T N ), cytotoxic T cells (T CTL ), memory T cells (T MEM ), T memory stem cells (T SCM ), central memory T cells (T CM ), effector memory T cells (T EM ), tissue-resident memory T cells (T RM ), effector T cells (TEFF ), regulatory T cells (T REG ), helper T cells (T H , T H 1. T H 2. T H 17) CD4+ T cells, CD8+ T cells, virus-specific T cells, alpha-beta T cells (T αβ ), and gamma delta T cells (T γδ In another embodiment, the cells provided herein include stem cells, e.g., embryonic stem cells or adult stem cells.

[0104] Also provided are methods of making the cells of the present disclosure. In some embodiments, such methods include transfecting or transducing a cell with a nucleic acid or expression vector of the present disclosure (e.g., an expression vector comprising a nucleic acid encoding a T cell receptor β chain comprising a TCRβ CDR3 sequence set forth in SEQ ID NOs: 1-1996 (e.g., SEQ ID NOs: 1-1281). The terms "transfection" or "transduction" are used to refer to the introduction of foreign DNA into a cell. A cell is "transfected" when the foreign DNA is introduced inside the cell membrane. Many transfection techniques are commonly known in the art. See, for example, Sambrook et al., (2001) Molecular Cloning, a Laboratory manual, 3rd ed., Cold Spring Harbor Laboratories, New York; Davis et al., (1995) Basic Methods in Molecular Biology, 2nd ed., McGraw-Hill; and Chu et al., (1981) Gene 13:197. Using such techniques, one or more foreign DNA moieties can be introduced into a suitable host cell. The term refers to both stable and transient uptake of genetic material.

[0105] In some embodiments, the cells of the disclosure are generated by transfecting cells with a viral vector encoding a T cell receptor β chain comprising a TCR β CDR3 sequence set forth in SEQ ID NO: 1-1996 (e.g., SEQ ID NO: 1-1281). In some embodiments, such methods include activating a T cell population (e.g., T cells obtained from an individual to whom TCR T cell therapy is to be administered), stimulating the T cell population to proliferate, and transducing the T cells with a viral vector encoding a T cell receptor β chain comprising a TCR β CDR3 sequence set forth in SEQ ID NO: 1-1996 (e.g., SEQ ID NO: 1-1281). In some embodiments, the T cells are transduced with a retroviral vector (e.g., a gamma retroviral vector or a lentiviral vector) encoding a T cell receptor β chain comprising a TCR β CDR3 sequence set forth in SEQ ID NO: 1-1996 (e.g., SEQ ID NO: 1-1281). In some embodiments, T cells are transduced with a lentiviral vector encoding a T cell receptor β chain comprising a TCR β CDR3 sequence set forth in SEQ ID NOs: 1-1996 (eg, SEQ ID NOs: 1-1281).

[0106] The cells of the present disclosure may be autologous / autologous ("autologous") or non-autologous ("non-autologous", e.g., allogeneic, syngeneic, or xenogeneic). As used herein, "autologous" refers to cells from the same individual. As used herein, "allogeneic" refers to cells of the same species that are genetically different from the cell being compared. As used herein, "syngeneic" refers to cells of a different individual that are genetically identical to the cell being compared. In some embodiments, the cells are T cells obtained from a mammal. In some embodiments, the mammal is a primate. In some embodiments, the primate is a human.

[0107] T cells may be obtained from a number of sources, including, but not limited to, peripheral blood, peripheral blood mononuclear cells, bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue from a site of infection, ascites, pleural effusion, spleen tissue, and tumors. In certain embodiments, T cells may be obtained from a unit of blood drawn from an individual using any number of known techniques, such as sedimentation (e.g., FICOLL™ separation).

[0108] In some embodiments, isolated or purified T cell populations are used. CTL and T H The lymphocytes are purified from PBMCs. CTL and T H Lymphocytes may be divided into naive (T N ), Memory (T MEM ), and effector (T EFF ) T cell subpopulations. Suitable approaches for such selection are known and include, for example, magnetic activated cell sorting (MACS), in which TNs are CD45RA + CD62L + CD95 ‐ and TSCM is CD45RA + CD62L + CD95 + and TCM is CD45RO + CD62L + CD95 + and TEM is CD45RO + CD62L ‐ CD95 + An example of such a selection approach is described in Wang et al. (2016) Blood 127(24):2980-90.

[0109] Specific subpopulations of T cells expressing one or more of the following markers: CD3, CD4, CD8, CD28, CD45RA, CD45RO, CD62, CD127, and HLA-DR may be further isolated by positive or negative selection techniques. In some embodiments, specific subpopulations of T cells expressing one or more of the markers selected from the group consisting of CD62L, CCR7, CD28, CD27, CD122, CD127, CD197, or CD38 or CD62L, CD127, CD197, and CD38 are further isolated by positive or negative selection techniques. In some embodiments, the produced T cell composition does not express one or more of the following markers: CD57, CD244, CD 160, PD-1, CTLA4, TIM3, and LAG3. In some embodiments, the produced T cell compositions do not substantially express one or more of the following markers: CD57, CD244, CD 160, PD-1, CTLA4, TIM3, and LAG3.

[0110] To achieve a therapeutically effective dose of the T cell composition, the T cells may be subjected to one or more rounds of stimulation, activation and / or expansion. T cells can be activated and expanded generally using the methods described, for example, in U.S. Patent Nos. 6,352,694, 6,534,055, 6,905,680, 6,692,964, 5,858,358, 6,887,466, 6,905,681, 7,144,575, 7,067,318, 7,172,869, 7,232,566, 7,175,843, 5,883,223, 6,905,874, 6,797,514, and 6,867,041, each of which is incorporated by reference in its entirety for all purposes. In some embodiments, the T cells are activated and expanded for about 1 to 21 days, e.g., about 5 to 21 days. In some embodiments, the T cells are activated and expanded for about 1 to about 4 days, about 1 to about 3 days, about 1 to about 2 days, about 2 to about 3 days, about 2 to about 4 days, about 3 to about 4 days, or about 1 day, about 2 days, about 3 days, or about 4 days before a nucleic acid (e.g., an expression vector) encoding a polypeptide is introduced into the T cells.

[0111] In some embodiments, the T cells are activated and expanded for about 6 hours, about 12 hours, about 18 hours, or about 24 hours before a nucleic acid (e.g., an expression vector) encoding a T cell receptor β chain comprising a TCR β CDR3 sequence set forth in SEQ ID NOs: 1-1996 (e.g., SEQ ID NOs: 1-1281) is introduced into the T cells. In some embodiments, the T cells are activated simultaneously with the introduction of a nucleic acid (e.g., an expression vector) encoding a T cell receptor β chain into the T cells.

[0112] In some embodiments, suitable conditions for T cell culture include an appropriate medium (e.g., Minimum Essential Medium or RPMI Medium 1640 or X-vivo 15 (Lonza)) and one or more factors necessary for proliferation and viability, including, but not limited to, serum (e.g., fetal bovine or human serum), interleukin-2 (IL-2), insulin, IFN-γ, IL-4, IL-7, IL-21, GM-CSF, IL-10, IL-12, IL-15, TGFβ, and TNF-α, or any other additives suitable for the growth of cells known to one of skill in the art. Further illustrative examples of cell culture media include, but are not limited to, RPMI 1640, Click, AEVI-V, DMEM, MEM, a-MEM, F-12, X-Vivo 15, and X-Vivo 20, Optimizer, supplemented with amino acids, sodium pyruvate, and vitamins, and either serum-free or supplemented with an appropriate amount of serum (or plasma) or a defined set of hormones, and / or cytokine(s) in an amount sufficient for T cell growth and proliferation.

[0113] In some embodiments, the nucleic acid (e.g., expression vector) encoding the T cell receptor β chain is introduced into the cell (e.g., T cell) by microinjection, transfection, lipofection, heat shock, electroporation, transduction, gene gun, microinjection, DEAE-dextran mediated transfer, etc. In some embodiments, the nucleic acid (e.g., expression vector) encoding the T cell receptor β chain is introduced into the cell (e.g., T cell) by AAV transduction. The AAV vector may include an ITR from AAV2 and a serotype from any one of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, or AAV 10. In some embodiments, the AAV vector includes an ITR from AAV2 and a serotype from AAV6. In some embodiments, the nucleic acid (e.g., expression vector) encoding the T cell receptor β chain is introduced into the cell (e.g., T cell) by lentiviral transduction. Lentiviral vector backbones can be derived from HIV-1, HIV-2, Visna-Maedi virus (VMV), Caprine Arthritis-Encephalomyelitis Virus (CAEV), Equine Infectious Anemia Virus (EIAV), Feline Immunodeficiency Virus (FIV), Bovine Immunodeficiency Virus (BIV), or Simian Immunodeficiency Virus (SIV). Lentiviral vectors can be integration-competent or integrase-deficient lentiviral vectors (TDLV). In one embodiment, an IDLV vector containing an HIV-based vector backbone (i.e., HIV cis-acting sequence elements) is used.

[0114] Computer-Readable Medium and System A computer readable medium and system are also provided by the present disclosure.

[0115] In certain aspects, one or more computer readable media are provided having stored thereon one or more TCR β CDR3 sequences set forth in SEQ ID NOs: 1-1996 (e.g., SEQ ID NOs: 1-1281). The number of TCR β CDR3 sequences set forth in SEQ ID NOs: 1-1996 (e.g., SEQ ID NOs: 1-1281) stored on the one or more computer readable media can vary. For example, the one or more computer readable media can include one or more TCR β CDR3 sequences set forth in SEQ ID NOs: 1-1996 (e.g., SEQ ID NOs: 1-1281). One or more, two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, fifteen or more, twenty or more, twenty-five or more, thirty or more, thirty-five or more, forty or more, forty-five or more, fifty or more, seventy-five or more, one ... Where less than all of the TCR β CDR3 sequences set forth in SEQ ID NOs: 1-1996 (e.g., SEQ ID NOs: 1-1281) are stored on one or more computer readable media, the one or more computer readable media may have stored thereon any desired (e.g., as described above) number and combination of the TCR β CDR3 sequences set forth in SEQ ID NOs: 1-1996 (e.g., SEQ ID NOs: 1-1281).In some embodiments, the one or more computer readable media comprises a TCRβ polypeptide as set forth in SEQ ID NOs: 1-1996 (e.g., SEQ ID NOs: 1-1281). Up to 1996, up to 1281, up to 1000, up to 950, up to 900, up to 850, up to 800, up to 750, up to 700, up to 650, up to 600, up to 550, up to 500, up to 450, up to 400, up to 350, up to 300, up to 250, up to 200, up to 190, up to 180, up to 170, up to 160, up to 150, up to 140, up to 130, up to 120, up to 110, up to 100, up to 90, up to 80, up to 70, up to 60, up to 50, up to 40, up to 30, up to 20, or up to 10 of the CDR3 sequences may be stored thereon in any desired combination.

[0116] Systems for evaluating TCRβ CDR3 sequences are also provided. According to some embodiments, systems for evaluating TCRβ CDR3 sequences are provided, such systems comprising one or more processors and one or more computer readable media. The one or more computer readable media comprises instructions stored thereon, which when executed by the one or more processors, cause the one or more processors to evaluate TCRβ CDR3 sequences determined from a sample obtained from a subject (e.g., a subject identified as having IB or suspected of having IBD, including, but not limited to, a subject presenting with one or more non-specific symptoms consistent with Crohn's disease) for the presence or absence of one or more TCRβ CDR3 sequences set forth in SEQ ID NOs: 1-1996, e.g., SEQ ID NOs: 1-1281. According to some embodiments, the number of TCRβ CDR3 sequences determined from a sample obtained from a subject is between 1,000 and 2,000,000. For example, in certain embodiments, the number of TCRβ CDR3 sequences determined from a sample obtained from a subject is between 1,000 and 2,000,000. The number of CDR3 sequences is 2,000,000 or less (e.g., 1,500,000 or less, 1,250,000 or less, 1,000,000 or less, 750,000 or less, or 500,000 or less), but can be 1,000 or more, 5,000 or more, 10,000 or more, 15,000 or more, 20,000 or more, 25,000 or more, 30,000 or more, 35,000 or more, 40,000 or more, 45,000 or more, 50,000 or more, 55,000 or more, 60,000 or more, 65,000 or more, 70,000 or more, 75,000 or more, 80,000 or more, 85,000 or more, 90,000 or more, 95,000 or more, or 100,000 or more. The number of TCR β CDR3 sequences set forth in SEQ ID NOs: 1 to 1996 (eg, SEQ ID NOs: 1 to 1281) to which the determined TCR β CDR3 sequence is compared may vary.For example, the determined TCRβ CDR3 sequence is a TCRβ CDR3 sequence set forth in SEQ ID NOs: 1 to 1996 (e.g., SEQ ID NOs: 1 to 1281). or each of the CDR3 sequences. When the determined TCR β CDR3 sequence is compared to fewer than all of the TCR β CDR3 sequences set forth in SEQ ID NOs: 1-1996 (e.g., SEQ ID NOs: 1-1281), the determined TCR β CDR3 sequence may be compared to any desired (e.g., as described above) number and combination of the TCR β CDR3 sequences set forth in SEQ ID NOs: 1-1996, e.g., SEQ ID NOs: 1-1281.

[0117] The one or more computer readable media may further include instructions stored thereon that, when executed by the one or more processors, cause the one or more processors to perform one or more additional steps based on the results of the evaluating step. For example, if it is determined from the evaluating step that none of the TCRβ CDR3 sequences set forth in SEQ ID NOs: 1-1996 (e.g., SEQ ID NOs: 1-1281) are present in the TCRβ CDR3 sequences determined from the sample obtained from the subject, the instructions may further cause the one or more processors to, for example, identify the subject as not having Crohn's disease, identify the subject as not being administered a Crohn's disease therapy, and / or the like. Also, by way of example, if it is determined from the evaluating step that one or more (e.g., two or more, three or more, four or more, five or more, or ten or more) of the TCR β CDR3 sequences set forth in SEQ ID NOs: 1-1996 (e.g., SEQ ID NOs: 1-1281) are present in the TCR β CDR3 sequences determined from a sample obtained from a subject (e.g., a subject identified as having IB or suspected of having IBD, including, but not limited to, a subject exhibiting one or more non-specific symptoms consistent with Crohn's disease), the instructions may further cause the one or more processors to, for example, predict that the subject has Crohn's disease, diagnose the subject as having Crohn's disease, identify the subject as one who should be administered Crohn's disease therapy, and / or the like.

[0118] In certain embodiments, the one or more computer-readable media may further include instructions stored thereon, which when executed by the one or more processors, cause the one or more processors to subject the results of the evaluation step to further analysis, such as subjecting the results of the evaluation step to a model. For example, the instructions may cause the one or more processors to subject the results of the evaluation step to a model to classify the subject as having or not having Crohn's disease, and / or to classify the subject as having Crohn's disease and not having non-Crohn's disease IBD (e.g., ulcerative colitis). Those skilled in the art will appreciate that, with the benefit of the TCRβ CDR3 sequences set forth in SEQ ID NOs: 1-1996 (e.g., SEQ ID NOs: 1-1281) described herein, a variety of useful models may be applied to the results of the evaluation. In one non-limiting example, the instructions may cause the one or more processors to subject the results of the evaluation step to a two-feature logistic regression, with features representing the number of Crohn's disease-associated TCRβ CDR3 sequences determined from the sample and the total number of unique TCRβ CDR3 sequences determined from the sample. As shown in the experimental section below, such models demonstrate high specificity and sensitivity for Crohn's disease.

[0119] In certain embodiments, when the one or more computer-readable media further comprises instructions stored thereon, which when executed by the one or more processors cause the one or more processors to subject the results of the evaluation step to a model for purposes of classification (e.g., as described above), the model may take into account the number of unique Crohn's disease-associated TCRβ CDR3 sequences present in the TCRβ CDR3 sequences determined from the sample (e.g., the greater the number of unique Crohn's disease-associated TCRβ CDR3 sequences, the more likely the model will classify the subject as having Crohn's disease). According to some embodiments, the number of unique Crohn's disease-associated TCRβ CDR3 sequences is not a feature utilized by the model to classify the subject. In certain embodiments, the presence and / or frequency of one or more specific unique Crohn's disease-associated TCRβ CDR3 sequences is the feature(s) used by the model to classify the subject. For example, the presence and / or frequency of one or more specific unique Crohn's disease-associated TCRβ CDR3 sequences may be given relatively greater weight in classifying the subject compared to the presence and / or frequency of one or more other unique Crohn's disease-associated TCRβ CDR3 sequences.

[0120] Various processor-based systems may be used to implement embodiments of the present disclosure. Such systems may include a system architecture in which components of the system are in electrical communication with each other using a bus. The system architecture may include a processing unit (CPU or processor) and a cache, variously coupled to a system bus. The bus couples various system components, including system memory (e.g., read-only memory (ROM) and random access memory (RAM)), to the processor.

[0121] The system architecture may include a cache of high-speed memory directly connected to the processor, in close proximity to the processor, or integrated as part of the processor. The system architecture may copy data from memory and / or storage to the cache for quick access by the processor. In this way, the cache may provide a performance boost that avoids delays in the processor while waiting for the data. These and other modules may control or be configured to control the processor to perform various operations. Other system memories may also be available for use. The memory may include multiple different types of memory with different performance characteristics. The processor may include any general-purpose processor, as well as hardware or software modules, such as a first module, a second module, and a third module stored in a storage device, configured to control the processor, as well as special-purpose processors where the software instructions are built into the actual processor design. The processor may be essentially a fully self-contained computing system, including multiple cores or processors, buses, memory controllers, caches, etc. Multi-core processors may be symmetrical or asymmetrical.

[0122] To enable user interaction with the computing system architecture, the input device may represent any number of input mechanisms, such as, for example, a microphone for speech, a touch-sensitive screen for gesture or graphical input, a keyboard, a mouse, motion input, speech, etc. The output device may also be one or more of a number of output mechanisms. In some instances, a multimodal system may enable a user to provide multiple types of input for communicating with the computing system architecture. The communication interface may generally control and manage user input and system output. There is no restriction to operation with any particular hardware arrangement, so the basic functions herein may be easily replaced with improved hardware or firmware arrangements as they are developed.

[0123] The storage device is typically a non-volatile memory and may be a hard disk or other type of computer-readable medium capable of storing data accessible by a computer, such as a magnetic cassette, a flash memory card, a solid-state memory device, a digital versatile disk, a cartridge, random access memory (RAM), read-only memory (ROM), and hybrids thereof.

[0124] The storage device may include a software module for controlling the processor. Other hardware or software modules are contemplated. The storage device may be connected to a system bus. In one aspect, a hardware module performing a specific function may include a software component stored in a computer-readable medium in association with the necessary hardware elements, such as a processor, a bus, an output device, etc., to perform various functions of the disclosed technology.

[0125] Embodiments within the scope of the present disclosure may also include tangible and / or non-transitory computer-readable storage media, or devices for carrying or having computer-executable instructions or data structures stored thereon. Such tangible computer-readable storage devices may be any available device accessible by a general purpose computer or a special purpose computer, including the functional design of any special purpose processor as described above. By way of example and not limitation, such tangible computer-readable devices may include RAM, ROM, EEPROM, CD-ROM or other optical disk storage, magnetic disk storage or other magnetic storage, or any other device that can be used to carry or store desired program code in the form of computer-executable instructions, data structures, or processor chip designs. When information or instructions are provided to a computer over a network or another communications connection (either wired, wireless, or a combination thereof), the computer properly views the connection as a computer-readable medium. Thus, any such connection is properly referred to as a computer-readable medium. Combinations of the above should also be included within the scope of computer-readable storage devices.

[0126] Computer-executable instructions include, for example, instructions and data that cause a general purpose computer, special purpose computer, or special purpose processing device to perform a certain function or group of functions. Computer-executable instructions also include program modules that are executed by a computer in a stand-alone or network environment. Generally, program modules include routines, programs, components, data structures, objects, and design-specific functions such as special purpose processors that perform tasks or implement abstract data types. Computer-executable instructions, associated data structures, and program modules represent examples of the program code means for executing steps of the methods disclosed herein. A particular arrangement of such executable instructions or associated data structures represents examples of corresponding acts for implementing the functions described in such steps.

[0127] Other embodiments of the present disclosure may be practiced in networked computing environments having many types of computer system configurations including personal computers, handheld devices, multiprocessor systems, microprocessor-based or programmable consumer electronics, network PCs, minicomputers, mainframe computers, etc. Embodiments may also be practiced in distributed computing environments where tasks are performed by local and remote processing devices that are coupled (either by hardwired links, wireless links, or a combination thereof) through a communications network. In a distributed computing environment, program modules may be located in both local and remote memory storage devices.

[0128] Notwithstanding the appended claims, the present disclosure is also defined by the following embodiments.

[0129] 1. A computer-implemented method for evaluating T cell receptor beta chain complementarity determining region 3 (TCRβ CDR3) sequences, comprising: A computer-implemented method comprising evaluating TCRβ CDR3 sequences determined from a sample obtained from a subject for the presence or absence of one or more TCRβ CDR3 sequences set forth in SEQ ID NOs: 1-1996.

[0130] 2. The computer-implemented method of embodiment 1, comprising evaluating TCRβ CDR3 sequences determined from a sample obtained from the subject for the presence or absence of one or more TCRβ CDR3 sequences set forth in SEQ ID NOs: 1-1281.

[0131] 3. The computer-implemented method of embodiment 1 or embodiment 2, wherein prior to the evaluating, the subject is identified as having or suspected of having inflammatory bowel disease (IBD).

[0132] 4. A computer-implemented method according to any one of embodiments 1-3, wherein the subject, at the time of evaluation, has one or more non-specific symptoms consistent with Crohn's disease.

[0133] 5. The computer-implemented method of embodiment 4, wherein the one or more non-specific symptoms are selected from the group consisting of diarrhea, fatigue, abdominal pain, abdominal cramps, rectal bleeding, unintentional weight loss, and any combination thereof.

[0134] 6. The computer-implemented method of any one of embodiments 1-5, wherein the TCRβ CDR3 sequences determined from a sample obtained from the subject comprise 10,000 or more TCRβ CDR3 sequences.

[0135] 7. The computer-implemented method of any one of embodiments 1-6, wherein the TCR β CDR3 sequence determined from the sample obtained from the subject is determined by performing amplification and high-throughput sequencing of genomic DNA present in the sample obtained from the subject.

[0136] 8. A computer-implemented method according to any one of embodiments 1 to 7, wherein the sample obtained from the subject is a peripheral blood sample.

[0137] 9. A computer-implemented method according to any one of embodiments 1 to 7, wherein the sample obtained from the subject is an intestinal tissue sample.

[0138] 10. A method comprising administering Crohn's disease therapy to a subject identified as containing T cells expressing a T cell receptor β chain (TCRβ) containing a TCRβ CDR3 sequence set forth in SEQ ID NOs: 1-1996.

[0139] 11. The method of embodiment 10, comprising administering Crohn's disease therapy to a subject identified as comprising T cells expressing TCRβ comprising a TCRβ CDR3 sequence set forth in SEQ ID NOs: 1-1281.

[0140] 12. The method of embodiment 10 or embodiment 11, wherein the Crohn's disease therapy comprises administering to the subject a therapeutically effective amount of an anti-inflammatory drug.

[0141] 13. The method of embodiment 12, wherein the Crohn's disease therapy comprises administering to the subject a therapeutically effective amount of a corticosteroid.

[0142] 14. The method of embodiment 13, wherein the corticosteroid is prednisone, budesonide, or a combination thereof.

[0143] 15. The method of any one of embodiments 10-14, wherein the Crohn's disease therapy comprises administering to the subject a therapeutically effective amount of a 5-aminosalicylate.

[0144] 16. The method of embodiment 15, wherein the 5-aminosalicylate is sulfasalazine, mesalamine, or a combination thereof.

[0145] 17. The method of any one of embodiments 10-16, wherein the Crohn's disease therapy comprises administering to the subject a therapeutically effective amount of an immunosuppressant.

[0146] 18. The method of embodiment 17, wherein the immunosuppressant is azathioprine, mercaptopurine, methotrexate, or any combination thereof.

[0147] 19. The method of any one of embodiments 10-18, wherein the Crohn's disease therapy comprises administering to the subject a therapeutically effective amount of a monoclonal antibody.

[0148] 20. The method of embodiment 19, wherein the monoclonal antibody is natalizumab, vedolizumab, infliximab, adalimumab, certolizumab pegol, ustekinumab, or any combination thereof.

[0149] 21. The method of any one of embodiments 10-20, wherein the Crohn's disease therapy comprises administering to the subject a therapeutically effective amount of an antibiotic.

[0150] 22. The method of embodiment 10 or embodiment 11, further comprising identifying the subject as having Crohn's disease, but not ulcerative colitis, based on identifying the subject as comprising T cells expressing one or more TCRβ comprising one or more TCRβ CDR3 sequences set forth in SEQ ID NOs: 1-1996 or 1-1281, prior to administering Crohn's disease therapy to the subject.

[0151] 23. The method of embodiment 10 or embodiment 11, further comprising identifying the subject as having Crohn's disease, rather than irritable bowel syndrome, based on identifying the subject as comprising T cells expressing one or more TCRβ comprising one or more TCRβ CDR3 sequences set forth in SEQ ID NOs: 1-1996 or 1-1281, prior to administering Crohn's disease therapy to the subject.

[0152] 24. The method of embodiment 10 or embodiment 11, further comprising identifying the subject as having Crohn's disease, rather than celiac disease, based on identifying the subject as comprising T cells expressing one or more TCRβ comprising one or more TCRβ CDR3 sequences set forth in SEQ ID NOs: 1-1996 or 1-1281, prior to administering Crohn's disease therapy to the subject.

[0153] 25. The method of any one of embodiments 22-24, wherein the Crohn's disease therapy is a therapy suitable for Crohn's disease and not ulcerative colitis, irritable bowel syndrome, and / or celiac disease.

[0154] 26. The method of embodiment 10 or embodiment 11, further comprising identifying the subject as having Crohn's disease with fistulization or structuring, as opposed to Crohn's disease without fistulization or structuring, based on identifying the subject as comprising T cells expressing one or more TCRβ comprising one or more TCRβ CDR3 sequences set forth in SEQ ID NOs: 1-1996 or SEQ ID NOs: 1-1281, prior to administering Crohn's disease therapy to the subject.

[0155] 27. The method of embodiment 26, wherein the Crohn's disease therapy is a therapy suitable for Crohn's disease with fistulization or structuring, rather than Crohn's disease without fistulization or structuring.

[0156] 28. The method of embodiment 10 or embodiment 11, further comprising identifying the subject as having ileal / ileocolonic Crohn's disease rather than colonic Crohn's disease based on identifying the subject as comprising T cells expressing one or more TCRβ comprising one or more TCRβ CDR3 sequences set forth in SEQ ID NOs: 1-1996 or 1-1281 prior to administering Crohn's disease therapy to the subject.

[0157] 29. The method of embodiment 28, wherein the Crohn's disease therapy is a therapy suitable for ileal / ileocolonic Crohn's disease, and not colonic Crohn's disease.

[0158] 30. A non-transitory computer-readable medium having stored thereon one or more TCRβ CDR3 sequences set forth in SEQ ID NOs: 1-1996 or 1-1281.

[0159] 31. The non-transitory computer-readable medium of embodiment 30, having five or more TCRβ CDR3 sequences stored thereon, as set forth in SEQ ID NOs: 1-1996 or 1-1281.

[0160] 32. The non-transitory computer-readable medium of embodiment 30, having stored thereon 10 or more TCRβ CDR3 sequences set forth in SEQ ID NOs: 1-1996 or 1-1281.

[0161] 33. The non-transitory computer-readable medium of embodiment 30, having stored thereon 100 or more TCRβ CDR3 sequences set forth in SEQ ID NOs: 1-1996 or 1-1281.

[0162] 34. A system for evaluating TCRβ CDR3 sequences, comprising: one or more processors; and one or more non-transitory computer readable media comprising instructions stored thereon that, when executed by one or more processors, cause the one or more processors to evaluate a TCR β CDR3 sequence determined from a sample obtained from the subject for the presence or absence of one or more TCR β CDR3 sequences set forth in SEQ ID NOs: 1-1996 or SEQ ID NOs: 1-1281.

[0163] 35. The system of embodiment 34, wherein the TCRβ CDR3 sequences determined from a sample obtained from the subject include 10,000 or more TCRβ CDR3 sequences.

[0164] 36. The system of embodiment 34 or embodiment 35, wherein the instructions, when executed by the one or more processors, cause the one or more processors to evaluate the determined TCR β sequence for the presence or absence of five or more TCR β CDR3 sequences set forth in SEQ ID NOs: 1-1996 or SEQ ID NOs: 1-1281.

[0165] 37. The system of embodiment 34 or embodiment 35, wherein the instructions, when executed by the one or more processors, cause the one or more processors to evaluate the determined TCR β sequence for the presence or absence of ten or more TCR β CDR3 sequences set forth in SEQ ID NOs: 1-1996 or SEQ ID NOs: 1-1281.

[0166] 38. The system of embodiment 34 or embodiment 35, wherein the instructions, when executed by the one or more processors, cause the one or more processors to evaluate the determined TCR β sequence for the presence or absence of 100 or more TCR β CDR3 sequences set forth in SEQ ID NOs: 1-1996 or SEQ ID NOs: 1-1281.

[0167] The following examples are offered by way of illustration and not by way of limitation. EXAMPLES

[0168] experiment Example 1 - Prediction of Crohn's disease from TCR repertoire data Described in the Examples is the identification of Crohn's disease associated TCRβ sequences, sometimes referred to herein as "Crohn's disease enhancer sequences", "enhancement sequences", and the like.

[0169] T cell receptor repertoires were generated by immunosequencing of whole blood samples, peripheral blood mononuclear cells (PBMCs), or buffy coat preparations of blood. Briefly, genomic DNA was extracted from blood or cell samples using standard extraction kits. ∼18 μg of genomic DNA was then input into a multiplex PCR reaction to amplify the CDR3 region of the TCR β chain, followed by high-throughput sequencing (immunoSEQ assay as described above).

[0170] An early diagnostic model predicting Crohn's disease from TCR repertoire data was run on blood samples from patients undergoing Crohn's disease-related surgery from a cohort from Saint-Louis Hospital (Paris). Cases during training included 362 Crohn's disease patients before surgical intervention. Controls during training included over 1000 healthy adults from other datasets (healthy blood donors collected via contract research organizations and other studies). The model first identifies unique TCR sequences that are elevated in Crohn's disease case samples compared to controls using a one-tailed Fisher's exact test. Unique sequences are identified by their V gene, J gene, and TCRβ CDR3 amino acid sequences. A bi-characteristic logistic regression was then performed with dependent variables E and N, where E is the number of unique TCRβ DNA sequences encoding enhanced sequences and N is the total number of unique TCRβ DNA sequences in the subject. As illustrated diagrammatically in Figure 1 , hundreds of Crohn's disease-associated TCRβ sequences that distinguished Crohn's disease patients from controls were identified and used in logistic regression (model AUC = 0.945, 71.6% sensitivity at 99.5% specificity).

[0171] Model performance was then tested on a hold-out dataset not used in training. As shown in Figure 2, high model scores (strong signal) were observed only for Crohn's disease patients, but not for other healthy / disease groups tested.

[0172] As shown in FIG. 3, Crohn's disease-associated TCRβ sequences were found to cluster in association with class II MHC and exhibit high convergent recombination in Crohn's patients but not controls.

[0173] As an additional biological validation of these TCR sequences, tissue samples from Crohn's disease patients at Saint-Louis Hospital were also sequenced using the immunoSEQ assay. These tissue-derived repertoires revealed that Crohn's disease-associated TCRβ sequences identified in blood were also present and enriched in intestinal tissue samples compared to blood samples from the same patients. As shown in Figure 4, a similar number of Crohn's disease-associated TCRβ sequences were observed in blood and intestinal tissue. Because the total sequences in the tissue samples are small, a significantly larger proportion of sequences in the tissue are enhanced sequences. The signals in the blood and intestinal tissue of the matched specimens were found to be strongly correlated.

[0174] A number of additional clinical samples were then collected and immunosequenced using the ImmunoSEQ assay to build an improved model. These samples included additional samples from Saint-Louis Hospital, as well as from multiple studies conducted by the University of Kiel (Germany), and from the Inflammatory Bowel Disease Association. A total of 3,890 Crohn's disease subjects were collected, and 80% of these subject samples were used for training to develop a new classifier for Crohn's disease (20% were left as holdouts). Over 4,000 negative controls from other studies of adults (without inflammatory bowel disease) were also used for training. 1,281 unique TCRβ CDR3 sequences significantly associated with Crohn's disease were identified (Table 1 herein). This list showed a high degree of overlap from sequences identified in the initial model, but added hundreds of additional TCRβ sequences. Other models were trained with Crohn's disease, including different selection cohorts and location-based models for ileal versus colonic disease that identified other Crohn's disease-associated sequences (see Table 2 herein).

[0175] The holdout data from this larger model was tested for performance as previously described. As shown in Figure 5, the sensitivity observed in the holdout data is high across the three major Crohn's disease cohorts, with high specificity for other diseases including irritable bowel syndrome and celiac disease, conditions that also lead to underlying problems in the gut (Figure 5, top right). The model shows significantly higher signal in individuals with more complex disease with fistula formation or strictures (Figure 5, bottom left), and is also elevated in ileal / ileocolonic disease over colonic disease (Figure 5, bottom right). It is believed that different TCRβ sequences contribute to the signal in the ileum versus colon.

[0176] Therefore, the foregoing merely illustrates the principles of the present disclosure. It will be appreciated that those skilled in the art will be able to devise various arrangements that embody the principles of the present invention and are within the spirit and scope thereof, although not expressly described or shown herein. Furthermore, all examples and conditional language described herein are intended primarily to aid the reader in understanding the principles of the present invention and the concepts contributed by the inventor to advance the art, and should not be interpreted as being limited to such specifically described examples and conditions. Furthermore, all statements herein that describe the principles, aspects, and embodiments of the present invention and their specific examples are intended to encompass both structural and functional equivalents thereof. In addition, such equivalents are intended to include both currently known equivalents and equivalents developed in the future, i.e., any developed elements that perform the same function, regardless of structure. Thus, the scope of the present invention is not intended to be limited to the exemplary embodiments shown and described herein.

Claims

1. 1. A computer-implemented method for evaluating T cell receptor beta chain complementarity determining region 3 (TCRβ CDR3) sequences, comprising: A computer-implemented method comprising evaluating TCRβ CDR3 sequences determined from a sample obtained from a subject for the presence or absence of one or more TCRβ CDR3 sequences set forth in SEQ ID NOs: 1-1996.

2. 2. The computer-implemented method of claim 1, comprising evaluating the TCRβ CDR3 sequences determined from the sample obtained from the subject for the presence or absence of one or more TCRβ CDR3 sequences set forth in SEQ ID NOs: 1-1281.

3. 3. The computer-implemented method of claim 1 or claim 2, wherein prior to said evaluating, said subject is identified as having or suspected of having inflammatory bowel disease (IBD).

4. The computer-implemented method of any one of claims 1 to 3, wherein the subject, at the time of said evaluating, has one or more non-specific symptoms consistent with Crohn's disease.

5. 5. The computer-implemented method of claim 1, wherein the TCRβ CDR3 sequences determined from the sample obtained from the subject comprise 10,000 or more TCRβ CDR3 sequences.

6. 6. The computer-implemented method of any one of claims 1 to 5, wherein the TCR β CDR3 sequence determined from the sample obtained from the subject is determined by performing amplification and high-throughput sequencing of genomic DNA present in the sample obtained from the subject.

7. The computer-implemented method of any one of claims 1 to 6, wherein the sample obtained from the subject is a peripheral blood sample or an intestinal tissue sample.

8. A method for determining whether a subject is a patient for whom Crohn's disease therapy should be administered, said method comprising evaluating a TCRβ CDR3 sequence determined from a sample obtained from said subject, wherein if said subject is identified as comprising T cells expressing a T cell receptor β chain (TCRβ) comprising a TCRβ CDR3 sequence set forth in SEQ ID NOs: 1-1996, said subject is determined to be a patient for whom Crohn's disease therapy should be administered.

9. The method according to claim 8, wherein the subject is determined to be a patient for whom Crohn's disease therapy should be performed if the subject is identified as containing T cells expressing TCRβ comprising a TCRβ CDR3 sequence set forth in SEQ ID NOs: 1 to 1281.

10. 10. The method of claim 8 or 9, wherein the Crohn's disease therapy comprises administering to the subject a therapeutically effective amount of an anti-inflammatory drug, a corticosteroid, a 5-aminosalicylate, an immunosuppressant, an antibiotic, or any combination thereof.

11. 10. The method of claim 8 or 9, further comprising identifying the subject as having Crohn's disease and not ulcerative colitis based on identifying that the subject comprises T cells expressing one or more TCRβ comprising one or more TCRβ CDR3 sequences set forth in SEQ ID NOs: 1-1996 or 1-1281 prior to administering the Crohn's disease therapy to the subject.

12. 10. The method of claim 8 or 9, further comprising identifying the subject as having Crohn's disease and not irritable bowel syndrome based on identifying the subject as comprising T cells expressing one or more TCRβ comprising one or more TCRβ CDR3 sequences set forth in SEQ ID NOs: 1-1996 or 1-1281 prior to administering the Crohn's disease therapy to the subject.

13. 10. The method of claim 8 or 9, further comprising identifying the subject as having Crohn's disease and not celiac disease based on identifying the subject as comprising T cells expressing one or more TCRβ comprising one or more TCRβ CDR3 sequences set forth in SEQ ID NOs: 1-1996 or 1-1281 prior to administering the Crohn's disease therapy to the subject.

14. 10. The method of claim 8 or 9, further comprising identifying the subject as having Crohn's disease with fistulizing or structuring as opposed to Crohn's disease without fistulizing or structuring based on identifying the subject as comprising T cells expressing one or more TCRβ comprising one or more TCRβ CDR3 sequences set forth in SEQ ID NOs: 1-1996 or SEQ ID NOs: 1-1281 prior to administering the Crohn's disease therapy to the subject.

15. A non-transitory computer readable medium storing one or more TCRβ CDR3 sequences set forth in SEQ ID NOs: 1-1996 or 1-1281.

16. 16. The non-transitory computer readable medium of claim 15, storing five or more TCRβ CDR3 sequences set forth in SEQ ID NOs: 1-1996 or 1-1281.

17. 16. The non-transitory computer readable medium of claim 15, storing 10 or more TCRβ CDR3 sequences set forth in SEQ ID NOs: 1-1996 or 1-1281.

18. A system for evaluating TCRβ CDR3 sequences, comprising: one or more processors; and one or more non-transitory computer readable media comprising instructions stored thereon that, when executed by the one or more processors, cause the one or more processors to evaluate a TCRβ CDR3 sequence determined from a sample obtained from a subject for the presence or absence of one or more TCRβ CDR3 sequences set forth in SEQ ID NOs: 1-1996 or 1-1281.

19. The system of claim 18, wherein the TCRβ CDR3 sequences determined from the sample obtained from the subject comprise 10,000 or more TCRβ CDR3 sequences.

20. 20. The system of claim 18 or 19, wherein the instructions, when executed by the one or more processors, cause the one or more processors to evaluate the determined TCR β sequence for the presence or absence of ten or more TCR β CDR3 sequences set forth in SEQ ID NOs: 1-1996 or 1-1281.

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