Application of gamma delta T cells in treatment of non-tumor intestinal diseases

Abstract

The invention relates to the technical field of biomedicine, in particular to application of gamma delta T cells in treatment of non-tumor intestinal diseases. According to the invention, by increasing the abundance of gamma delta T cells in vivo, promoting the differentiation of epithelial cells of intestinal mucosa and inhibiting the activation of fibroblasts and macrophages, the intestinal barrier function is improved, the intestinal fibrosis is relieved and the intestinal inflammatory response is reduced respectively, and the problem of pain spots existing in clinical practice treatment can be effectively solved.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technology, and in particular to the application of γδT cells in treating non-tumor intestinal diseases. Background Art

[0002] Inflammatory bowel disease (IBD) is a group of chronic, nonspecific intestinal inflammatory diseases with unclear etiology, including ulcerative colitis (UC) and Crohn's disease (CD). Impaired intestinal epithelial cell differentiation, along with fibroblast and macrophage activation, are the primary pathological mechanisms of intestinal mucosal damage, intestinal obstruction, intestinal strictures, and intestinal inflammation in IBD. Common clinical manifestations include abdominal pain, diarrhea, bloody stools, weight loss, fatigue, loss of appetite, and, particularly in children, slowed growth and development. Without timely and effective treatment, the disease progression can worsen, leading to complications including massive gastrointestinal bleeding, intestinal perforation, intestinal stenosis, intestinal obstruction, intestinal fistulas, anal fistulas, abdominal abscesses, and colorectal cancer. Due to the complex pathogenesis, which remains to be fully elucidated, current symptomatic treatment is tailored to the specific clinical presentation and may include enteral nutrition, glucocorticoids, immunosuppressants, and biologic agents to suppress inflammation. However, long-term use of these drugs may cause side effects to the body and increase the risk of drug resistance. At the same time, the disease is prone to relapse, seriously affecting children's growth and development and quality of life.

[0003] As an emerging immunotherapy, γδT cell therapy demonstrates promising therapeutic potential and broad application prospects. γδT cells are primarily distributed in the intestinal mucosa and subcutaneous tissues. They secrete different cytokines, forming an immune regulatory network encompassing immune, inflammatory, and non-immune components to maintain homeostasis. Although studies have shown that healthy intestinal epithelial cells express organ-specific butyric acid-like (BTNL) 3 / 8 proteins that regulate Vγ4 γδ T cell function, and that BTNL3 / 8-deficient mutations are associated with IBD severity, the mechanisms underlying inflammatory bowel disease (IBD), particularly in patients with resistance to remifentanil (anti-TNF) therapy or very early onset, remain largely unresolved. Current clinical treatment strategies remain limited, and targeted therapeutic agents are lacking. Summary of the Invention

[0004] The present invention covers the following technical solutions: The present invention relates to the use of gamma delta T cells in preparing medicines for treating non-tumor intestinal diseases.

[0005] The present invention unexpectedly discovered through testing: In recent years, with the aging of society and the change of people's lifestyle, the incidence and recurrence rates have shown a significant upward trend. At present, clinical treatment is mainly based on anti-inflammatory symptom relief, and there is almost no ability of curative epithelial repair. In clinical practice, it is found that some IBD patients in clinical remission still have varying degrees of mucosal damage and mucosal inflammation, and the extracellular matrix reconstruction and fibrosis at the inflammatory site cause intestinal stenosis and obstruction in some patients. Patients often need to be hospitalized repeatedly or even need surgical treatment, and long-term medication is prone to side effects and drug resistance, which increases the difficulty of treatment. It is especially difficult to treat patients with very early gene mutations, which seriously affects children's growth and development and quality of life. The present invention increases the abundance of γδT cells in the body, promotes the differentiation of intestinal epithelial cells, inhibits fibroblast activation and macrophage activation, thereby improving the intestinal barrier function, alleviating intestinal fibrosis and lowering intestinal inflammatory response, which can effectively solve the pain points in clinical practice treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0006] In order to more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the specific embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0007] Figure 1 The expression and distribution of γδT cells in intestinal mucosal tissues during the active and remission phases of inflammatory bowel disease; A1-A4 show the non-lesioned intestine of a normal patient. B1-B3 show the stained intestinal specimens from a patient with ulcerative colitis in remission, and B4 shows the stained intestinal specimens from a patient with active ulcerative colitis. C1-C3 show the stained intestinal specimens from a patient with Crohn's disease in remission, and C4 shows the stained intestinal specimens from a patient with active Crohn's disease.

[0008] Figure 2 To investigate the effect of γδT cell transfusion on intestinal inflammation in a DSS-induced mouse enteritis model; Figure A is a schematic diagram of the acute model establishment; Figure B is the disease activity score curve for the three groups (disease activity score calculation formula: weight score + stool consistency score + bloody stool score); Figure C is the weight change curve of mice; Figure D is the anatomical diagram of the gross specimen after model establishment; Figure E shows the correlation between changes in intestinal length; Figure G is hematoxylin-eosin staining; Figure H is an 800x scanning electron microscopy of the intestine; Figure I is glycogen staining; Figure J is intestinal immunofluorescence (DAPI is a cell nuclear marker, red is the intestinal differentiation-related protein CDX2) Figure 3 To reverse the intestinal fibrosis of the DSS-induced enteritis mouse model by infusing γδT cells.

[0009] Figure A is a schematic diagram of the chronic model; Figure B is the weight change curve of mice in the three groups; Figure C is the disease activity score curve (disease activity score calculation formula: weight score + stool consistency score + bloody stool score); Figure D is the anatomical diagram of the gross specimen after the model is completed; Figure E is the correlation change of intestinal length; Figure G is hematoxylin-eosin staining; Figure H is an 800x scanning electron microscopy of the intestine; Figure I is glycogen staining; Figure J is intestinal immunofluorescence (DAPI is a cell nucleus marker, red is the intestinal differentiation-related protein CDX2); Figure K is Masson staining. DETAILED DESCRIPTION

[0010] Reference will now be made in detail to embodiments of the present invention, one or more examples of which are described below. Each example is provided to illustrate, not to limit, the present invention. Indeed, it will be apparent to those skilled in the art that various modifications and variations may be made to the present invention without departing from the scope or spirit of the invention. For example, features illustrated or described as part of one embodiment may be used in another embodiment to produce further embodiments.

[0011] Unless otherwise indicated, all terms (including technical and scientific terms) used to disclose the present invention have the same meaning as commonly understood by one of ordinary skill in the art to which the present invention belongs. By way of further guidance, the following definitions are provided to better understand the teachings of the present invention. The terms used herein in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention.

[0012] Unless otherwise indicated, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, terms and laboratory procedures related to protein and nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology, and immunology used herein are those widely used in the respective fields and are standard procedures. To facilitate a better understanding of the present invention, definitions and explanations of relevant terms are provided below.

[0013] The terms "and / or", "or / and", and "and / or" used in the present invention include any one of two or more related listed items, and also include any and all combinations of the related listed items, wherein the arbitrary and all combinations include any combination of two related listed items, any more related listed items, or all related listed items. It should be noted that when at least three items are connected by at least two conjunctions selected from "and / or", "or / and", and "and / or", it should be understood that in the present invention, the technical solution undoubtedly includes technical solutions connected by "logical and" and also undoubtedly includes technical solutions connected by "logical or". For example, "A and / or B" includes three parallel solutions of A, B and A+B. For example, the technical solution of "A, and / or, B, and / or, C, and / or, D" includes any one of A, B, C, and D (that is, the technical solution of all being connected by "logical OR"), and also includes any and all combinations of A, B, C, and D, that is, the combination of any two or any three of A, B, C, and D, and also includes the four-item combination of A, B, C, and D (that is, the technical solution of all being connected by "logical AND").

[0014] As used herein, the terms "comprising," "including," and "comprising" are synonymous and are inclusive or open-ended and do not exclude additional, unrecited members, elements, or method steps.

[0015] The recitation of numerical ranges herein by endpoints includes all numbers and fractions subsumed within the range, as well as the recited endpoints.

[0016] In addition, when describing representative embodiments of the present invention, this specification may present the method and / or process of the present invention as a specific sequence of steps. However, to the extent that the method or process does not rely on the specific order of steps set forth herein, the method or process should not be limited to the specific order of steps described. As one of ordinary skill in the art will appreciate, other sequences of steps are also possible. Therefore, the specific order of steps set forth in the specification should not be interpreted as limiting the claims. In addition, claims to the method and / or process of the present invention should not be limited to the execution of the steps in the order in which they are written, and those skilled in the art will readily recognize that the sequence can be changed and still remain within the spirit and scope of the present invention.

[0017] Concentration values ​​used in this invention include fluctuations within a certain range. For example, fluctuations within a certain precision range are permitted. For example, for a 2% value, fluctuations within a range of ±0.1% are permitted. For larger values ​​or values ​​that do not require overly precise control, greater fluctuations are permitted. For example, for 100 mM, fluctuations within ranges of ±1%, ±2%, ±5%, etc. are permitted. Regarding molecular weight, fluctuations within a range of ±10% are permitted.

[0018] As used herein, the singular articles "a," "an," and "the" include plural referents unless otherwise indicated.

[0019] In the present invention, descriptions such as "plurality" and "multiple" refer to quantities greater than or equal to 2 unless otherwise specified.

[0020] In the present invention, the technical features described in an open manner include closed technical solutions composed of the listed features, and also include open technical solutions containing the listed features.

[0021] In the present invention, "preferably", "better", "more preferably", and "suitably" are merely descriptions of preferred implementation methods or examples, and should be understood to not limit the scope of protection of the present invention. In the present invention, "optionally", "optional", and "optional" refer to being optional, that is, to being selected from either of the two parallel options of "with" or "without". If multiple "options" appear in a technical solution, unless otherwise specified and without contradiction or mutual restriction, each "optional" is independent.

[0022] As used herein, the term "treatment" refers to administering to a patient an internal or external therapeutic agent, such as a cell comprising any modification of the present application or a pharmaceutical composition comprising a modified cell, wherein the patient has one or more symptoms of the disease, and it is known that the therapeutic agent has a therapeutic effect on these symptoms. Typically, the patient is administered an amount (therapeutically effective amount) of the therapeutic agent that effectively improves, alleviates, prevents, delays, controls, reverses or eliminates one or more symptoms of the disease. The desired effect of treatment includes reducing the rate of disease progression, improving or alleviating the disease state, and the prognosis of regression or improvement. For example, if one or more symptoms associated with enteritis are alleviated or eliminated, including but not limited to, repairing intestinal mucosal damage, reducing the symptoms of intestinal fibrosis, improving the quality of life of those individuals with the disease, reducing the dosage of other drugs needed for treating the disease, delaying the progression of the disease, and / or prolonging individual survival, the individual is successfully "treated", and the method of treatment is whether it involves the participation of a medical professional or whether it is implemented in a medical institution.

[0023] As used herein, the term "administer" generally refers to delivering a substance to a subject in need thereof by any route known in the art. Pharmaceutical carriers and formulations or compositions are also well known in the art. Routes of administration may include intravenous, intramuscular, intradermal, subcutaneous, transdermal, mucosal, intratumoral, and / or mucosal.

[0024] For the purposes of this invention, "enteritis" refers to any inflammatory pathological condition affecting intestinal tissues (including but not limited to the small intestine, colon, and rectum). This condition can be caused by a variety of factors, including but not limited to infectious factors (such as bacteria, viruses, fungi, and parasites) and non-infectious factors (such as autoimmune reactions, drug reactions, radiation damage, ischemia, and allergic reactions). Clinical manifestations may include diarrhea, abdominal pain, vomiting, fever, bloody stools, and weight loss. Pathological features may include mucosal congestion, edema, ulceration, and cellular infiltration. This definition is intended to encompass all known and potentially identifiable types of intestinal inflammatory diseases.

[0025] For the purposes of this invention, the term "γδT cells" generally refers to a T cell subset whose T cell receptor (TCR) is composed of a γ (gamma) chain and a δ (delta) chain, and is not limited to a specific TCR Vγ and Vδ chain combination, surface markers, functional status, or tissue distribution. TCRγ chains include, but are not limited to, Vδ1, Vδ2, Vδ3, and Vδ5. TCRδ chains include, but are not limited to, Vγ2, Vγ3, Vγ4, Vγ5, Vγ8, and Vγ9. The term "γδT cells" encompasses different γδT cell types formed by the combination of different TCRδ and TCRγ chains. For example, a γδT cell expressing a TCR containing the delta chain variable region 1 (Vδ1) can be referred to as a Vδ1T cell. Vδ1T cells can be further divided into Vγ2Vδ1T, Vγ3Vδ1T, Vγ4Vδ1T, Vγ5Vδ1T, Vγ8Vδ1T, and Vγ9Vδ1T, depending on the type of γ chain variable region. For example, a γδT cell expressing a TCR containing the variable region 9 of the γ chain (Vγ9) and the variable region 2 of the δ chain (Vδ2) can be referred to as a Vγ9Vδ2T cell. γδT cells can be obtained from peripheral blood, or they can be isolated or derived from lymphoid tissues including the spleen, lymph nodes, bone marrow, thymus, and non-lymphoid tissues including the intestine, skin, lung, and liver. The γδT cells can be mammalian γδT cells, such as rodent γδT cells and primate γδT cells. In a specific embodiment, the γδT cells are human γδT cells. In a specific embodiment, the γδT cells are mouse γδT cells. In a specific embodiment, the γδT cells are monkey γδT cells. The term also includes γδT cells obtained from the body, as well as γδT cells cultured and passaged in vitro or ex vivo. The γδT cells can be engineered γδT cells, such as those with exogenous gene transfer, γδT cells with endogenous gene knockout, or CAR γδT cells or TCR γδT cells. γδT cells express functional TCR γδ and, in addition, possess at least one of the following surface markers: CD3+, CD4+, CD8+, CD4-, CD8-, NKG2D+, CD56+, CD27+, or CD27-. Functionally, these include naturally unactivated γδT cells, activated γδT cells, memory γδT cells, in vitro expanded γδT cells, and engineered γδT cells that have undergone genetic modification (e.g., CAR-γδT, TCR-γδT, etc.). Furthermore, these encompass γδT cells in healthy individuals and disease-associated γδT cells (e.g., tumor-infiltrating γδT cells, γδT cells in the infected microenvironment, and γδT cells in autoimmune diseases).

[0026] All documents mentioned in the present invention are cited as references in the present invention, just as each document is cited as a reference separately. Unless they conflict with the purpose of the invention and / or technical solution of the present invention, the cited documents involved in the present invention are cited with all their contents and all their purposes. When the present invention involves cited documents, the definitions of relevant technical features, terms, nouns, phrases, etc. in the cited documents are also cited. When the present invention involves cited documents, the examples and preferred embodiments of the cited relevant technical features may also be incorporated into the present invention as references, but are limited to the ability to implement the present invention. It should be understood that when the cited content conflicts with the description in the present invention, the present invention shall prevail or be modified adaptively based on the description of the present invention.

[0027] The present invention relates to the use of gamma delta T cells in preparing medicines for treating non-tumor intestinal diseases.

[0028] The present invention uses mice to establish acute and chronic enteritis models, and observes that γδT cells can promote intestinal epithelial cell differentiation and intestinal mucosal repair by reinfusing them through the tail vein, inhibiting macrophage activation to relieve intestinal inflammation, and reducing fibroblast activation to relieve fibroblast activation and relieve intestinal fibrosis in chronic models. Specifically, the present invention uses the strategy of reinfusing DSS-induced acute and chronic enteritis models by reinfusing γδT cells respectively, to illustrate that the γδT cells isolated from the spleen of mice that are reinfused not only promote the increase in CDX2 expression in intestinal epithelial cells, improve microvilli structural damage, and restore barrier function reconstruction, especially the significant increase in the number of goblet cells, but also that the reinfuse of γδT cells can relieve intestinal inflammation. In addition, in the chronic model, the reinfuse of γδT cells can significantly inhibit fibroblast activation and relieve intestinal fibrosis. It can be seen that in patients with inflammatory bowel diseases (such as colitis, UC and CD) with abnormal defects in intestinal γδT cells, the reinfuse of γδT cells can significantly relieve the symptoms of enteritis.

[0029] The γδT cells described herein are generally heterogeneous and can be a collection of γδT cells with different molecular markers. In some embodiments, the proportion of CD39+ γδT cells in the γδT cells is less than 50%, for example, less than 40%, less than 30%, less than 20%, less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2%, or less than 1%. In some embodiments, the γδT cells are substantially free of CD39+ γδT cells.

[0030] In some embodiments, the γδ T cells are γδ T cells isolated from the spleen.

[0031] In some embodiments, the intestinal disease includes at least one of colitis and inflammatory bowel disease, wherein the intestinal disease can be acute or chronic.

[0032] In some embodiments, the inflammatory bowel disease includes at least one of ulcerative colitis (UC), Crohn's disease (CD), indeterminate colitis (IC), microscopic colitis, and segmental colitis associated with diverticulosis (SCAD).

[0033] In some embodiments, the symptoms of the intestinal disease include at least one of intestinal barrier damage, intestinal inflammation, intestinal fibrosis, and intestinal stenosis.

[0034] In some embodiments, the drug further comprises a pharmaceutically acceptable carrier.

[0035] As used herein, "pharmaceutically acceptable carriers" include any material that allows the component to maintain biological activity when combined with the active ingredient and does not react with the subject's immune system. Examples include, but are not limited to, any of standard pharmaceutical carriers (such as phosphate-buffered saline solutions, water, emulsions (such as oil / water emulsions)) and various types of wetting agents. Exemplary diluents for aerosol or parenteral administration are phosphate-buffered saline (PBS) or physiological (0.9%) saline. Compositions comprising such carriers are formulated by well-known conventional methods (see, for example, Remington's Pharmaceutical Sciences, 18th edition, A. Gennaro, ed., Mack Publishing Co., Easton, PA, 1990; and Remington, The Science and Practice of Pharmacy, 21st edition, Mack Publishing, 2005).

[0036] In some embodiments, the drug is in an injectable dosage form, preferably a dosage form suitable for intravenous injection.

[0037] In some embodiments, the subject of the medicament is a mammal.

[0038] In some embodiments, the mammal is a primate.

[0039] In some embodiments, the primate is a human.

[0040] According to yet another aspect of the present invention, it also relates to a method for treating a non-tumor intestinal disease in a patient in need thereof, comprising administering a therapeutically effective amount of the γδ T cells described above to the patient.

[0041] It should be understood that the envisioned treatment methods also preferably include the combined administration of other clinical regimens, such as drug therapy, including aminosalicylic acids (such as mesalazine, sulfasalazine), glucocorticoids (such as prednisone, budesonide), immunomodulators (such as azathioprine, 6-mercaptopurine, methotrexate), monoclonal antibody drugs (such as infliximab, adalimumab, vedemumab, ustekinumab), small molecule targeted drugs (such as tofacitinib); diet and nutritional support, surgical treatment, probiotics, psychological intervention and other methods can also be used in combination.

[0042] A "therapeutically effective amount" refers to an amount effective at the dosage and for the duration required to achieve the desired therapeutic effect. The therapeutically effective amount of γδ T cells may vary depending on factors such as the disease state, age, sex, and weight of the subject to be treated, and the ability of the γδ T cells to elicit the desired response in the subject. The dosing regimen can be adjusted to provide the optimal therapeutic response. A therapeutically effective amount is also generally an amount in which any toxic or deleterious effects of the γδ T cells are outweighed by the beneficial effects of the treatment. The dosage administered depends largely on the condition and size of the subject being treated, as well as the treatment formulation, treatment frequency, and route of administration. The regimen for continued treatment, including dosage, formulation, and frequency, can be guided by the initial response and clinical judgment.

[0043] In some embodiments, the γδ T cells are administered to the subject daily, weekly, biweekly, monthly, every 2 months, every 3 months, every 6 months, annually, every 2 years, every 5 years, or once a lifetime.

[0044] Exemplary routes of administration and delivery include intravenous (IV), intraarticular, intraperitoneal (IP), intraarterial, intramuscular, parenteral, subcutaneous, intrapleural, dermal, transdermal, parenteral, e.g., transmucosal, intracranial, intraspinal, oral (digestive), mucosal, respiratory, intranasal, intubation, intrapulmonary, intrapulmonary instillation, buccal, sublingual, intravascular, intrathecal, intracavitary, iontophoresis, intraocular, intraglandular, intraorgan, intrafallopian tube.

[0045] In some embodiments, γδ T cells are delivered to a mammalian subject by, for example, intravenous injection into the mammalian subject.

[0046] The effect of administering the γδ T cells of the present invention can be to prevent the development of a disease, stop the progression of a disease, reverse the progression of a disease, etc.

[0047] "Patient" or "subject" is used interchangeably in the present invention and refers to mammals, including but not limited to primates (preferably humans), pigs and other farm animals, sports animals, pets, primates, horses, dogs, cats, giant pandas, rodents (including mice, rats, guinea pigs), etc.

[0048] The embodiments of the present invention will be described in detail below with reference to the examples. It should be understood that these examples are intended to illustrate the present invention only and are not intended to limit the scope of the invention. For experimental methods in the following examples where specific conditions are not specified, reference is made to the guidance provided in the present invention, and may also be made to experimental manuals or conventional conditions in the art, other experimental methods known in the art, or conditions recommended by the manufacturer.

[0049] In the following specific examples, the measured parameters of raw material components may have slight deviations within the range of weighing accuracy unless otherwise specified. For temperature and time parameters, acceptable deviations caused by instrument testing accuracy or operational accuracy are allowed.

[0050] The basic experimental methods involved in the following examples are: 1. Immunofluorescence Paraffin-embedded sections of intestinal tissue or organoids were dewaxed and hydrated, followed by antigen retrieval and blocking with goat serum for 1 hour. The sections were then incubated with primary antibodies in a humidified chamber at 4°C overnight in the dark. The sections were then washed with PBS and incubated with Alexa-647- or Alexa-488-conjugated mouse or rabbit secondary antibodies for another 1 hour at room temperature in the dark. The sections were then mounted with a mounting medium containing a fluorescence quencher and DAPI. The sections were imaged and analyzed using a Leica X-ray fluorescence microscope.

[0051] 2. Masson staining The sections were routinely dewaxed into distilled water and, according to the instructions of the Masson trichrome staining solution kit, sequentially stained with Weigert iron hematoxylin, acid differentiation solution, Masson blue solution, Ponceau fuchsin staining solution, weak acid solution, phosphobaric acid solution, and benzidine staining solution, followed by rapid dehydration, xylene clearing, and neutral gum sealing.

[0052] 3. Hematoxylin-eosin staining (HE) The sections were routinely dewaxed into distilled water, stained with hematoxylin, differentiated with differentiation solution, and then stained with eosin. The excess staining solution was discarded and the sections were rapidly dehydrated. After dehydration, the sections were transparentized, mounted, and observed under a microscope.

[0053] 4. AB-PAS staining The sections were dewaxed and hydrated, and according to the instructions of the AB-PAS staining kit, the sections were stained with Alcian blue solution, oxidized by adding or immersing in an oxidant, covered with Schiff staining solution, stained with hematoxylin solution for nuclei, differentiated with acidic differentiation solution, and blued with Scott blue solution. The sections were then dehydrated with conventional graded ethanol, transparentized with xylene, and mounted with neutral gum.

[0054] Example 1 Intestinal mucosal γδT cells are significantly reduced in inflammatory bowel disease, especially in the active stage of the intestinal mucosa Experimental methods: Intestinal mucosal tissue specimens from different stages of remission and active phases and a control group were collected. Immunofluorescence technology was used to detect the intestinal mucosa at different stages using γδTCR antibodies, and the number of γδT cells in the intestinal mucosa of different groups at different stages was statistically analyzed.

[0055] The results showed that compared with the control group, the number of γδT cells in the intestinal mucosa of ulcerative colitis and Crohn's disease decreased significantly, especially in the active stage of inflammatory bowel disease, the number of γδT cells decreased more significantly. It was further found that γδT cells were mainly concentrated around the intestinal epithelial cells ( Figure 1 ).

[0056] Example 2: Reinfusion of γδT cells in animals to alleviate acute and chronic enteritis Experimental Methods: Acute and chronic enteritis models were induced in mice using a γδT cell transfusion strategy combined with dextran sulfate sodium (DSS). In the acute model, mice were fed water containing 2.5% DSS until day 3. Two million γδT cells isolated from the spleens of healthy mice were transfused into the tail vein once. Body weight and disease activity index were monitored during this period. In the chronic model, mice were fed water containing 2.5% DSS until day 7. At this time, mice were fed regular water and received a single tail vein transfusion of mouse γδT cells. This process was repeated two weeks later. This process was repeated twice. Body weight and disease activity index were monitored during this period.

[0057] 1. γδT cell infusion alleviates symptoms of intestinal inflammation and intestinal mucosal damage The results of the present invention show that the experimental group designs in acute enteritis and chronic enteritis models are as follows: Figure 2 A (acute model) and Figure 3 A (chronic model), the infusion of γδT cells effectively alleviated the weight loss and increased disease activity index in the DSS-induced model group ( Figure 2 BC, Figure 3 BC), total intestinal and colorectal length recovery ( Figure 2 DF, Figure 3 DF). HE staining results showed that the reinfusion of γδT cells could alleviate the infiltration of intestinal inflammatory cells ( Figure 2 G, Figure 3G), electron microscopy results showed that γδT cell infusion effectively improved the shortening and loss of intestinal microvilli in the DSS-induced enteritis model ( Figure 2 H, Figure 3 H). Simultaneously, immunofluorescence staining and AB-PAS staining results showed that compared with the DSS-induced model group, the number of CDX2, a transcription factor that regulates intestinal cell differentiation, and goblet cells in the intestinal epithelial cells of the γδT cell reinfusion group increased significantly ( Figure 2 IJ, Figure 3 IJ).

[0058] 2. γδT cell infusion alleviates intestinal fibrosis Furthermore, this study showed through Masson staining that in the control group, after staining the intestinal wall structure, collagen fibers appeared blue and muscle fibers appeared red, with a clear boundary between the two. In the DSS-induced chronic enteritis model, abnormal proliferation of collagen fibers in the intestinal mucosa and submucosa was observed under the microscope. The blue area increased significantly and was distributed disorderly, interwoven into an irregular network structure, and some areas even formed dense fiber bundles. The fibrosis area is not only limited to the lamina propria, but can also extend to the muscularis mucosa and deeper tissues, accompanied by destruction of the muscularis mucosa structure, atrophy and rupture of muscle fibers, and a reduction in the area of ​​the red area. In the γδT re-infusion group, the area of ​​collagen deposition and the area of ​​fiber bundles were significantly reduced ( Figure 3 K).

[0059] The experimental results support that tail vein reinfusion of γδT cells can effectively improve intestinal inflammation, intestinal mucosal damage, barrier function destruction and intestinal fibrosis in gastrointestinal diseases such as colitis, IBD, UC or CD.

[0060] The above-described embodiments merely represent several implementation methods of the present invention, and their descriptions are relatively specific and detailed, but they should not be construed as limiting the scope of the patent. It should be noted that a person skilled in the art may make several modifications and improvements without departing from the concept of the present invention, and these modifications and improvements fall within the scope of protection of the present invention. Therefore, the scope of protection of the patent for this invention shall be based on the appended claims, and the description and drawings may be used to interpret the content of the claims.

Claims

1. Application of γδT cells in the preparation of drugs for the treatment of non-tumor intestinal diseases.

2. The use according to claim 1, characterized in that The intestinal disease includes at least one of colitis and inflammatory bowel disease.

3. The use according to claim 2, characterized in that The inflammatory bowel disease includes at least one of ulcerative colitis, Crohn's disease, indeterminate colitis, microscopic colitis, and segmental colitis associated with diverticular disease.

4. The use according to claim 1, characterized in that The symptoms of the intestinal disease include at least one of intestinal barrier damage, intestinal inflammation, intestinal fibrosis, and intestinal stenosis.

5. The use according to any one of claims 1 to 4, characterized in that The γδ T cells are γδ T cells isolated from the spleen.

6. The use according to any one of claims 1 to 4, characterized in that: The drug further comprises a pharmaceutically acceptable carrier.

7. The use according to claim 6, wherein the medicine is in the form of an injection.

8. The use according to any one of claims 1 to 4 and 7, characterized in that: The subject of the drug is a mammal.

9. The use according to claim 8, characterized in that The mammal is a primate.

10. The use according to claim 9, characterized in that The primate is a human.

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