Application of rTh1 cell in preparation of medicine for preventing or treating allergic diseases or inflammatory diseases

Abstract

The invention relates to the field of immunology and cell therapy, in particular to application of rTh1 cells in preparation of drugs for preventing or treating allergic diseases or inflammatory diseases, the phenotypes of the rTh1 cells are CD3 (+), CD4 (+), CD25 (-), Foxp3 (-), CXCR5 (-), CCR6 (-), CXCR3 (+), CD73 (+) and cMaf (+), and FR4, PD1 and ICOS are highly expressed. The novel T cell subset rTh1 cell has an immunoregulation function, and has an important value in prevention and treatment of allergic diseases such as food allergy and dust mite allergy, atopic dermatitis, allergic asthma, chronic obstructive pulmonary disease and other Th2-type inflammation related skin diseases such as pemphigus bullosa and urticaria.

Description

Technical Field

[0001] The present invention relates to the fields of immunology and cell therapy, and in particular to the use of rTh1 cells in preparing medicines for preventing or treating allergic diseases or inflammatory diseases. Background Art

[0002] Food allergy is caused by an abnormal immune system response to specific food proteins. Common allergens include peanuts, milk, and eggs. Globally, peanut allergy prevalence is approximately 1%-2%, with higher rates in North America and Europe. Milk allergy prevalence is approximately 2%-3% in infants and young children, though some children gradually develop tolerance with age. Egg allergy prevalence is approximately 1%-2% in children, though most children naturally outgrow the allergy by preschool. The pathogenesis of food allergies primarily involves an IgE-mediated immune response. When allergens enter the body, they stimulate B cells to produce specific IgE antibodies, which bind to FcεRI receptors on mast cells and basophils, leading to cell degranulation and the release of inflammatory mediators such as histamine and leukotrienes, triggering symptoms such as itching, urticaria, dyspnea, and even anaphylactic shock. Non-IgE-mediated or mixed reactions may also be involved, manifesting as gastrointestinal symptoms or eczema. Genetic susceptibility, environmental factors (such as early exposure and intestinal flora imbalance), and the properties of food proteins (such as heat resistance and digestive stability) are all closely related to the development of food allergies. In terms of clinical treatment, the current main strategy is to strictly avoid the intake of allergens and carry an epinephrine auto-injector to deal with acute allergic reactions. In recent years, desensitization treatments such as oral immunotherapy (OIT) and sublingual immunotherapy (SLIT) have made certain progress. By gradually increasing the dose of allergen exposure, immune tolerance is induced, thereby reducing the severity of allergic reactions or achieving desensitization. In addition, biologics (such as the anti-IgE monoclonal antibody omalizumab) are also used as adjuvant therapy to reduce the frequency and severity of allergic reactions. Despite continuous improvements in treatment methods, the management of food allergies still faces challenges, and new diagnostic, efficacy evaluation, and treatment measures are urgently needed in the future.

[0003] Dust mite allergy is a widespread allergic disease globally, with varying prevalence rates in different regions, but overall, the prevalence is high. Studies have shown that about 10%-20% of the global population is allergic to dust mites, with children and adolescents being the main affected groups. Dust mite allergy is an immune response triggered by proteins in dust mite (mainly Dermatophagoides pteronyssus and Dermatophagoides farinae) feces and corpses. The pathogenesis mainly involves IgE-mediated immune response. After dust mite allergens enter the human body through respiratory tract or skin contact, they stimulate B cells to produce specific IgE antibodies, which bind to the surface of FcεRI receptors on mast cells and basophils, leading to cell degranulation and release of histamine, leukotrienes and other inflammatory mediators, causing symptoms such as allergic rhinitis, asthma, eczema, etc. Genetic susceptibility, environmental exposure (such as high humidity and high density of dust mites in living environment), and immune imbalance are important factors affecting the occurrence of dust mite allergy. Clinically, the treatment of dust mite allergy mainly includes avoiding allergen exposure, drug therapy and immunotherapy. Avoidance measures include the use of anti-mite bedding, regular cleaning of the environment, and reducing indoor humidity. Drug therapy mainly includes antihistamines, nasal corticosteroids, leukotriene receptor antagonists, etc., which are used to relieve symptoms. Specific immunotherapy (such as subcutaneous immunotherapy SCIT and sublingual immunotherapy SLIT) can induce immune tolerance by gradually increasing the exposure dose of dust mite allergens, thereby reducing the frequency and severity of symptoms. In recent years, biological agents (such as anti-IgE monoclonal antibody omalizumab) have also shown good efficacy in some refractory dust mite allergic patients. However, the treatment of dust mite allergy still faces challenges such as large individual differences, long treatment course, and limited efficacy in some patients.

[0004] Atopic dermatitis (AD) is a chronic inflammatory skin disease characterized by intense itching and recurrent eczematous lesions. It often first develops in infancy and early childhood, but can affect people of all ages and is one of the most common inflammatory diseases. In developed Western countries, AD affects 20-30% of children and 10-20% of adults, and its prevalence continues to rise. In my country, according to a nationwide study led by our department, the prevalence of AD in infants aged 0-1 years and preschool children aged 1-7 years is as high as 30.48% and 12.94%, respectively. Furthermore, AD often acts as an initiating factor, leading to the development of other allergic diseases, including food allergies, asthma, and allergic rhinitis, a process known as the "atopic progression." Due to its high prevalence, difficulty in achieving a complete cure, recurrent or chronic, and even lifelong course, the clinical manifestations of severe itching and eczematous lesions, the profound physiological and psychological impact on patients, and the risk of inducing atopic processes, AD is becoming a major public health and socioeconomic issue, necessitating an urgent need for primary prevention to reduce its incidence. Classical regulatory T cells (Tregs) play an important role in immune regulation, but their therapeutic efficacy in specific inflammatory diseases is limited. Recent studies have revealed that subpopulations of Th1 cells may possess immunomodulatory functions, but their specific characteristics and functions remain unclear.

[0005] Asthma is a common chronic respiratory disease with an increasing incidence worldwide, particularly in industrialized and urbanized regions. According to statistics, approximately 300 million people worldwide suffer from asthma, with children and adolescents being the primary group affected. The pathogenesis of asthma is complex, with the Th2 immune response playing a crucial role in the development and progression of the disease. The Th2 inflammatory response is primarily driven by cytokines secreted by Th2 cells (such as IL-4, IL-5, and IL-13), leading to airway inflammation, mucus hypersecretion, eosinophil infiltration, and airway hyperresponsiveness. These pathological changes collectively cause the typical symptoms of asthma, such as wheezing, shortness of breath, chest tightness, and cough. Furthermore, environmental factors (such as allergens and air pollution) and genetic susceptibility also play a key role in the pathogenesis of asthma. Understanding the mechanisms of Th2 inflammation will facilitate the development of targeted biologics, such as anti-IL-5 and anti-IL-4 / IL-13 therapies, providing more effective treatment options for asthma patients.

[0006] Common inflammatory skin diseases such as bullous pemphigoid and urticaria involve abnormal reactions of the immune system. Bullous pemphigoid is an autoimmune disease characterized by the appearance of tense blisters on the skin and mucous membranes, often accompanied by severe itching and pain. Its etiology is related to the production of autoantibodies against components of the basement membrane of the skin, leading to inflammatory reactions and blister formation. Urticaria is a more common allergic skin disease characterized by the sudden appearance of wheals (red or pale raised skin lesions) and severe itching, usually triggered by allergens, infections, drugs or physical stimuli, and its pathological mechanism involves the release of histamine and other inflammatory mediators by mast cells. Both require treatment based on the specific etiology and symptoms to alleviate inflammation and improve the quality of life of patients.

[0007] Chronic obstructive pulmonary disease (COPD) is a chronic respiratory disease characterized by persistent airflow limitation. The disease usually progresses, with its core feature being chronic inflammation of the airways and lung tissue. Globally, COPD is one of the main causes of increased morbidity and mortality, posing a significant challenge to individual health and the social medical system. Currently, the treatment goals of COPD are to alleviate symptoms, prevent acute exacerbations, improve quality of life, and slow disease progression. Main treatment methods include inhaled drug therapy, smoking cessation intervention, rehabilitation therapy, oxygen therapy, and non-invasive ventilation. However, due to factors such as difficulty in early identification, strong heterogeneity, and multiple complications, the efficacy is limited. In particular, the development of new drugs for COPD has been slow, and although some new targeted drugs have been introduced, the overall efficacy has been limited, and they have not been able to significantly reverse or stop disease progression.

[0008] Cell therapy, as a cutting-edge medical technology, has shown great potential in the treatment of inflammatory diseases. Its core principle is to use the characteristics and functions of cells to regulate immune responses, repair tissue damage, and inhibit inflammatory processes. Currently, cell therapy in inflammatory diseases is mainly applied in the treatment of rheumatoid arthritis (RA), inflammatory bowel disease (IBD), systemic lupus erythematosus (SLE), and other autoimmune diseases, as well as chronic obstructive pulmonary disease (COPD) and asthma, etc. Regulatory T cells (Tregs) have shown good prospects in the treatment of transplant rejection, type I diabetes, multiple sclerosis (MS), and inflammatory bowel disease (IBD).

[0009] In summary, there is an urgent need for a preventive and therapeutic product that can simultaneously target inflammatory diseases such as food allergies, dust mite allergies, AD, AA, COPD, and other inflammatory skin diseases such as bullous pemphigoid, urticaria, etc. Summary of the Invention

[0010] In view of the above-mentioned shortcomings of the prior art, the object of the present invention is to provide the use of rTh1 cells in the preparation of drugs for preventing or treating allergic diseases or inflammatory diseases, so as to solve the problems in the prior art.

[0011] To achieve the above objectives and other related objectives, the present invention provides the use of rTh1 cells or substances that induce upregulation of rTh1 cells in the preparation of drugs for preventing, treating or diagnosing allergic diseases or inflammatory diseases.

[0012] Preferably, the substance that induces upregulation of rTh1 cells is selected from allergens or allergen component proteins.

[0013] Preferably, the allergic disease is selected from diseases caused by food allergens or inhalant allergens.

[0014] Preferably, the inflammatory disease is selected from atopic dermatitis, allergic asthma, bullous pemphigoid, urticaria or chronic obstructive pulmonary disease.

[0015] The present invention also provides the use of rTh1 cells or substances that induce upregulation of rTh1 cells in combination with glucocorticoids or anti-inflammatory drugs in the preparation of drugs for preventing, treating or diagnosing allergic diseases or inflammatory diseases.

[0016] As described above, the use of rTh1 cells of the present invention in the preparation of a medicament for preventing or treating allergic diseases or inflammatory diseases has the following beneficial effects:

[0017] 1. rTh1 cells significantly inhibited T cells express Th2-type inflammatory factors IL4 and IL13, and their inhibitory ability is superior to that of classical Tregs.

[0018] 2. In vivo application in animals: In mouse AD and AA models, after in vivo injection of rTh1 cells, they were significantly upregulated in the lesion-draining lymph nodes (dLN) and significantly inhibited the AD inflammatory response.

[0019] 3. Human Relevance: rTh1 cells are also present in human peripheral blood mononuclear cells (PBMCs), and their molecular characteristics are essentially the same as those of mouse rTh1. The proportion of rTh1 cells in PBMCs of human AD patients is significantly negatively correlated with AD severity (EASI score, IGA score) and the number of allergens sensitized.

[0020] 4. Application in the treatment of human diseases: The main drugs currently used in clinical practice for the treatment of AD are glucocorticoids and biological agents, both of which have obvious disadvantages. Specifically, long-term or large-area use of glucocorticoids can easily lead to local adverse reactions such as skin atrophy, thinning, pigmentation, and capillary dilation, and may also cause hormone dependence and rebound, with symptoms recurring or worsening after discontinuation of the drug. Long-term medication in children may also affect growth and development. The disadvantages of biological agents include high cost, large individual differences in efficacy, limited efficacy in some patients, and the presence of drug resistance or no response. According to the results of in vitro experiments and animal experiments of the present invention, rTh1 cells sorted from human PBMCs may have significant therapeutic effects on the prevention and treatment of inflammatory diseases such as AD, AA, and COPD. It may even be possible to prevent or treat diseases by inducing the production of rTh1 cells in the patient's body without extracting and isolating rTh1 cells, which greatly simplifies the treatment cycle and cost, and has significant advantages over existing treatment methods. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 Shown are flow cytometry results of rTh1 cells in mice.

[0022] A: Flow cytometry gating strategy for rTh1 (regulatory helper T cell type 1) in mouse mesenteric lymph nodes (MLN), defined as a group of CD3+CD4+ non-Treg, non-Th CXCR3+, CCR6-, CD73+, cMaf+ cells, and their main molecular characteristics are displayed by flow cytometric rainbow plots.

[0023] B: MFI bar chart of the main characteristic molecules of rTh1, confirming its high expression of CD73, FR4, PD1, ICOS, CXCR3, cMa and other molecules.

[0024] Figure 2 These results show that rTh1 cells inhibit Th2 inflammatory factors in vitro.

[0025] A: Purity verification of rTh1, Treg and remaining CD4+ T cells (otherT, OT) after rTh1 and Treg depletion in mouse MLN by flow cytometry sorting.

[0026] BC: Flow-cytometrically sorted rTh1, Treg, and OT cells were co-cultured with CTY (Cell Trace Yellow)-labeled CD4+ naive T cells from mouse kidneys. Flow cytometric analysis was performed to assess the effects of different co-cultures on the production of Th2 inflammatory factors, such as IL4 and IL13, by naive T cells. B is a representative flow cytometric plot of IL4 and IL13 expression by naive T cells, and C is a bar graph.

[0027] Figure 3 Shown is the therapeutic effect of rTh1 cells in a mouse AD model.

[0028] A: Flow-sorted rTh1, Treg, OT, or PBS (Con) were injected into recipient mice, and then the atopic dermatitis model was induced using house dust mites (HDM). From top to bottom, the following are representative images of mouse ear lesions, HE staining, rTh1 flow cytometry images in the lesion-draining lymph nodes (dLN), and Treg images.

[0029] B: Bar graph of mouse ear thickness, epidermal thickness, dermal thickness, mouse ear IL4 level, rTh1 ratio, and Treg ratio in dLN in A.

[0030] Figure 4 It shows that rTh1 cells mediate oral tolerance to various allergens such as dust mites, eggs, peanuts, etc. in mice and the window period of their induction.

[0031] A: Representative photos of ear lesions in mice of different weeks of age who were orally administered with HDM (OR) during neonatal period and PBS (Con) during adulthood, in which HDM was used to induce AD ​​model.

[0032] B: Representative images of HE staining of mice in A.

[0033] C: Bar graph of ear thickness, epidermal thickness, dermal thickness, and IL4 level in the ears of the mice in A.

[0034] D: Representative flow cytometry image of the proportion of rTh1 in dLN of mice in A.

[0035] E: Representative flow cytometry images of the proportion of Tregs in dLN of mice in A.

[0036] F: Bar chart showing the proportion of rTh1 and Treg in dLN of type A mice.

[0037] G: Mice were orally administered with OVA (OR) during the neonatal period, and the control group was orally administered with PBS (Con). OVA was used to induce AD ​​model in adulthood. From left to right are representative photos of mouse ear lesions, representative photos of HE staining, representative photos of rTh1 flow cytometry in dLN, and representative photos of Treg.

[0038] H: Bar graph of mouse ear thickness, epidermal thickness, dermal thickness, mouse ear IL4 level, rTh1 ratio and Treg ratio in dLN in G.

[0039] I: Mice were orally administered with Peanut (OR) during the neonatal period, while the control group was orally administered with PBS (Con). Peanut was used to induce an AD model in adulthood. From left to right are representative images of mouse ear lesions, HE staining, rTh1 flow cytometry, and Treg in dLN.

[0040] J: Bar graph of mouse ear thickness, epidermal thickness, dermal thickness, mouse ear IL4 level, rTh1 ratio and Treg ratio in dLN in I.

[0041] Figure 5 It was shown that rTh1 cells play a key role in oral tolerance to allergens in mice.

[0042] A: Mice were orally administered with HDM (OR) during neonatal life, while the control group was orally administered with PBS (Con). AD models were induced with HDM in adulthood. At the same time, some mice in the oral and control groups were injected with anti-CXCR3 antibodies or anti-CD25 antibodies to antagonize rTh1 and Treg, respectively. The upper row is a representative mouse ear photograph, and the lower row is a representative HE staining image.

[0043] B: Representative rTh1 and Treg flow cytometry images of dLN of mice in each group in A.

[0044] C: Bar graph of ear thickness, epidermal thickness, dermal thickness and IL4 level in ear tissue of mice in A.

[0045] D: Bar graph showing the proportion of rTh1 and Treg in dLN of mice in A.

[0046] Figure 6 The therapeutic effect of rTh1 cells in a mouse asthma model

[0047] A: Mice were orally administered with HDM (Oral, OR) during neonatal life, while the control group was orally administered with PBS (Con). HDM was used to induce an asthma model in adulthood. From left to right are representative images of HE staining, lung function, eosinophil flow cytometry in BALF, rTh1 flow cytometry in lung tissue, and Treg.

[0048] B: Bar graph of lung function, BALF eosinophil percentage and count, lung tissue IL4 level, serum IgE level, lung tissue rTh1 percentage and Treg percentage of mice in A.

[0049] Figure 7 Shown are the results of rTh1 isolation from PBMCs of human AD patients.

[0050] A: The flow cytometry gating strategy for rTh1 in human blood PBMCs is consistent with the definition of mouse rTh1, and its main molecular characteristics are displayed through the flow cytometry rainbow plot.

[0051] B: MFI bar graph of the main characteristic molecules of human rTh1.

[0052] Figure 8Shown is the analysis of the content of human rTh1 cells in the pathogenesis of AD and COPD.

[0053] A: Regression analysis of the proportion of rTh1 and Treg in PBMC of AD patients and the EASI score of AD severity;

[0054] B: Bar chart showing the proportion of rTh1 and Treg in PBMC of AD patients with different AD severity IGA scores;

[0055] C: Regression analysis of the proportion of rTh1 and Treg in PBMC of AD patients and the number of allergens;

[0056] D: Bar graph showing the proportion of rTh1 and Treg in PBMCs of healthy controls (HC), asthma (AS) patients and chronic obstructive pulmonary disease (COPD) patients. DETAILED DESCRIPTION

[0057] The present invention experimentally discovered and identified a new type of immunoregulatory Th1 cell (rTh1), which has the characteristics of clear immune phenotype and significant inflammation inhibition effect. It exhibits significant immunoregulatory function both in vitro and in vivo, especially has unique advantages in inhibiting Th2 type inflammation. The combination of this cell with current clinical anti-inflammatory drugs including glucocorticoids and biologics is expected to reduce side effects and enhance efficacy, and has good translational prospects and clinical significance.

[0058] The present invention provides an immunoregulatory Th1 cell (rTh1). The phenotype of the rTh1 cell is CD3(+), CD4(+), CD25(-), Foxp3(-), CXCR5(-), CCR6(-), CXCR3(+), CD73(+), cMaf(+), and the rTh1 cell highly expresses the immunoregulatory molecules FR4 and PD1 and the activation molecule ICOS.

[0059] The high expression refers to that the expression levels of FR4, PD1 and ICOS are higher than those of traditional immunoregulatory T cells (Treg).

[0060] rTh1 cells are different from traditional immunoregulatory T cells and have unique molecular characteristics. These cells highly express Th1-type chemokine receptor CXCR3 and immunoregulatory transcription factor cMaf, as well as immunoregulatory molecules CD73, FR4, PD1 and activation molecule ICOS.

[0061] The present invention also provides the use of rTh1 cells or substances that induce upregulation of rTh1 cells in the preparation of drugs for preventing, treating or diagnosing allergic diseases or inflammatory diseases.

[0062] The substance that induces upregulation of rTh1 cells includes a substance that can induce upregulation of the number of rTh1 cells in vivo. The upregulation means that the number of cells after induction is increased relative to the number of cells before induction.

[0063] In certain embodiments of the present invention, the substance that induces upregulation of rTh1 cells is selected from an allergen or an allergen component protein. The dosage and timing of administration of the substance that induces upregulation of rTh1 cells must be strictly controlled to achieve the desired effect without triggering a severe allergic reaction or inflammatory response.

[0064] The allergic disease is a disease caused by allergens or allergen component proteins.

[0065] Allergens are substances that can trigger allergic reactions. Allergen component proteins are specific protein molecules present in allergens. They are widely found in nature, including plants, animals, and microorganisms. The allergens described in this invention are not specifically limited and are selected from food allergens or inhalant allergens. The following is a classification and specific composition of common allergens and their component proteins, but the present invention is not limited to these allergens.

[0066] The food allergens are selected from allergens in eggs, milk, peanuts, soybeans, wheat, nuts, seafood, etc., and the allergen component proteins include but are not limited to the following protein components, such as Gal d 1 (ovomucoid), Gal d 2 (ovalbumin), Gal d 3 (conalbumin), Gal d 4 (lysozyme) in eggs; Bos d 8 (casein), Bos d (lactoferrin), Bos d 4, Bos d 5, Bos d 6 in milk; Ara h 1, Ara h 2, Ara h 3, Ara h 9 in peanuts; Gly m 4, Gly m 5, Gly m 6, Gly m 8 in soybeans; Tri a 14, Tri a 18, Tri a 19, Tri a 20, Tri a 25, Tri a 26, Tri a 36-Tri a 45, ω-5 alcohol-soluble protein (Tri a 19), high molecular weight glutenin (Tri a 26), wheat lipid transfer protein (Tri a 14); prolamin superfamily, Cupin superfamily, pathogenesis-related protein (PR) or profilins family in nuts; Pen a 1 in shrimp; Gad c 1 (fish albumin) in fish.

[0067] The inhaled allergens are selected from allergens in dust mites (such as house dust mites or dust mites), pollen, animal dander, fungi, etc., and the allergen component proteins are, for example, Der p 1, Der p 2, Der p 10 in dust mites; Phl p 1, Phl p 5, Phl p 4, Phl p 7, Phl p 12, Bet v 1, Art v 1, Art v 3, Amb a 1, Cyn d 1 in pollen; Fel d 1, Can f 1 in animal dander; Asp f 1, Asp f 15, Alt a 1 in fungi.

[0068] The inflammatory disease is a Th2 type inflammation.

[0069] The Th2 type inflammation is an immune response driven by type 2 helper T cells (Th2) and the cytokines they secrete (such as IL-4, IL-5, IL-13, etc.).

[0070] The inflammatory disease is selected from atopic dermatitis, allergic asthma, bullous pemphigoid, urticaria or chronic obstructive pulmonary disease.

[0071] Atopic dermatitis is a common chronic, recurrent, inflammatory skin disease with a clear familial genetic predisposition. It often occurs in infancy and can continue into adulthood. Patients often suffer from other atopic diseases such as asthma and allergic rhinitis. Its main characteristics are dry skin, eczema-like rashes and severe itching. The itching usually worsens at night and can seriously affect the patient's sleep, daily life, work and study. The cause of the disease is not yet clear, and it may be related to genetics, immune abnormalities, environmental factors, and abnormal skin barrier function. On the basis of genetic factors, the entry of allergens into the human body and the colonization of microorganisms in the skin can lead to abnormal skin immune reactions and inflammation, causing rashes and itching. Adverse stimuli such as scratching and excessive washing can also aggravate skin inflammation.

[0072] Allergic asthma is a type of asthma triggered by allergens. It is the most easily recognizable type of asthma and often occurs in children. Patients often have a family history of allergic diseases or previous allergic diseases such as eczema, allergic rhinitis, and food or drug allergies. When a patient is exposed to an allergen, the allergen enters the body through the respiratory system and reacts with the immune system, triggering allergic asthma. This leads to airway inflammation and hyperresponsiveness, causing recurrent symptoms such as wheezing, shortness of breath, chest tightness, or coughing, which often occur or worsen at night and in the early morning.

[0073] Bullous pemphigoid is an autoimmune skin and mucosal disease that is predominantly seen in the elderly, regardless of gender or ethnicity. The disease is linked to an autoimmune response, in which the immune system produces antibodies that attack healthy cells. These antibodies attack the membrane between the outer layer of the skin and the subepidermal layer, leading to fluid accumulation and formation of blisters. The primary manifestation is tension blisters on the skin. The blister fluid is usually clear or turbid, and may also contain a small amount of blood. Blisters can appear anywhere on the skin, but are most common in flexural areas such as the arms, axillae, legs, lower abdomen, and groin. When the blisters rupture, they form extensive wounds. While the disease course is long, the prognosis is good. Approximately 10%-20% of patients may develop mucosal damage.

[0074] Urticaria is a common skin and mucous membrane disease characterized by wheals and transient edematous papules accompanied by severe itching. Its pathogenesis is complex and is associated with multiple factors, including genetics, autoimmunity, infection, inhaled or ingested allergens, psychiatric factors, and endocrine changes. Allergens trigger an immune response, dilating blood vessels in the skin and increasing vascular permeability, leading to fluid accumulation in the subcutaneous tissue, forming wheals and edematous papules. Urticaria can be categorized as acute or chronic based on its course, with chronic urticaria lasting for months or even years.

[0075] Chronic obstructive pulmonary disease (COPD) is a common chronic lung disease characterized by lung overinflation, accompanied by airway inflammation and airflow limitation, which is progressive. The underlying lesion is destruction of the lung parenchyma. The disease is often caused by factors such as smoking, air pollution, and infection, which can lead to inflammation, narrowing, and obstruction of the small airways in the lungs, preventing air from flowing in and out of the lungs. This in turn leads to alveolar overinflation and rupture, forming bullae, which affect normal lung function. Patients often experience symptoms such as dyspnea, coughing, and expectoration. Dyspnea worsens with activity and can lead to respiratory and heart failure in severe cases.

[0076] In the present invention, the term "prevention" includes preventive treatment that can lead to the desired pharmaceutical and / or physiological effects. Preferably, the effect is to medically block or delay the occurrence of the disease and / or reduce the risk of disease development or worsening.

[0077] For allergic diseases, the prevention can be achieved by taking allergens to induce upregulation of rTh1 cells in the body, thereby improving the symptoms of allergic diseases, such as reduced severity and area of ​​skin lesions; improved lung function, reduced eosinophil infiltration, decreased IgE, etc.

[0078] For inflammatory diseases, the prevention can be achieved by inducing upregulation of rTh1 cells in the body through administration of allergens, thereby improving the symptoms of inflammatory diseases, including inhibiting the expression of Th2 inflammatory factors such as IL4 and / or IL13.

[0079] As used herein, the term "treatment" includes curative or palliative treatment that results in the desired pharmaceutical and / or physiological effect. Preferably, the effect is a medical reduction in one or more symptoms of the disease or a complete elimination of the disease.

[0080] In the present invention, the term "diagnosis" may refer to determining the severity of a disease or the effectiveness of treatment based on the number of rTh1 cells.

[0081] The present invention also provides the use of rTh1 cells or substances that induce upregulation of rTh1 cells in combination with glucocorticoids or anti-inflammatory drugs in the preparation of drugs for preventing, treating or diagnosing allergic diseases or inflammatory diseases.

[0082] The glucocorticoid or anti-inflammatory drug is not specifically limited, and can be, for example, any one or more of the following: prednisone, prednisolone, methylprednisolone, dexamethasone, betamethasone, beclomethasone dipropionate, budesonide, fluticasone propionate, beclomethasone dipropionate, mometasone nasal spray, fluticasone furoate nasal spray, beclomethasone dipropionate nasal spray, mometasone furoate nasal spray, fluticasone propionate nasal spray, beclomethasone dipropionate nasal spray, loteprednol eye drops, prednisolone eye drops, oxymetazoline eye drops, chondroitin sulfate eye drops, hydrocortisone cream, dexamethasone cream, mometasone furoate cream, dexamethasone acetate cream, triamcinolone acetonide econazole cream, and compound betamethasone acetate cream.

[0083] The present invention also provides a pharmaceutical composition comprising the rTh1 cells.

[0084] The present invention also provides a method for treating or preventing a disease, comprising administering a therapeutically effective amount of rTh1 cells to a subject or inducing upregulation of rTh1 cells in the subject.

[0085] The disease is selected from an allergic disease or an inflammatory disease.

[0086] "Subjects" include, but are not limited to, animals, preferably mammals; the mammals are preferably rodents, artiodactyls, perissodactyls, lagomorphs, primates, etc. The mammals include, for example, humans, non-human primates (e.g., monkeys), mice, pigs, cows, goats, rabbits, rats, guinea pigs, hamsters, horses, monkeys, sheep, or other non-human mammals; non-mammals include, for example, non-mammalian vertebrates, such as birds (e.g., chickens or ducks) or fish, and non-mammalian invertebrates. The subject can be a human, such as a patient suffering from a cancer, allergic disease, or inflammatory disease.

[0087] "Treatment" or "treatment" of a condition includes preventing or alleviating the condition, reducing the rate of occurrence or development of a condition, reducing the risk of developing a condition, preventing or delaying the development of symptoms associated with a condition, reducing or stopping symptoms associated with a condition, causing complete or partial reversal of a condition, curing a condition, or a combination of these.

[0088] The term "therapeutically effective amount" or "effective dose" in the present invention refers to the dose or concentration of a drug that is effective in treating a disease or condition associated with an antigen.

[0089] Specifically, when administered to a subject, the dosage varies depending on the patient's age and weight, disease characteristics and severity, and route of administration. The results of animal experiments and various circumstances may be referenced, and the total dosage cannot exceed a certain range.

[0090] Specifically, when administering to a subject, the specific administration method is not limited, and includes, for example, intravenous injection, subcutaneous injection, and aerosol inhalation.

[0091] In some embodiments, the methods described herein can further include co-administration with other compounds or other treatment regimens known in the art, such as glucocorticoids or anti-inflammatory drugs.

[0092] The following describes the embodiments of the present invention through specific examples. Those skilled in the art will readily understand the other advantages and benefits of the present invention from the disclosure herein. The present invention may also be implemented or applied through various other specific embodiments, and the details in this specification may be modified or altered based on different viewpoints and applications without departing from the spirit of the present invention.

[0093] Before further describing the specific embodiments of the present invention, it should be understood that the scope of protection of the present invention is not limited to the specific specific embodiments described below; it should also be understood that the terms used in the examples of the present invention are for describing specific specific embodiments rather than for limiting the scope of protection of the present invention; in the present specification and claims, unless otherwise expressly stated herein, the singular forms "a", "an" and "the" include plural forms.

[0094] When the embodiments provide numerical ranges, it should be understood that, unless otherwise specified in the present invention, both endpoints of each numerical range and any numerical value between the two endpoints may be selected. Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as those generally understood by those skilled in the art. In addition to the specific methods, equipment, and materials used in the embodiments, according to the understanding of the prior art by those skilled in the art and the description of the present invention, any methods, equipment, and materials of the prior art similar or equivalent to the methods, equipment, and materials described in the embodiments of the present invention may also be used to implement the present invention.

[0095] The in vitro experiments of the present invention confirmed that compared with conventional regulatory T cells (Treg), rTh1 cells exhibited a stronger ability to inhibit the expression of Th2-type inflammatory factors (IL4 and IL13). In a mouse model of food and dust mite sensitization, rTh1 cells mediated the oral tolerance of mice to food allergens such as eggs, peanuts, and milk, and dust mite allergens. In an animal model of atopic dermatitis (AD), by in vivo injection of rTh1 cells, a significant increase in the number of rTh1 cells in the lymph nodes draining the lesions was observed, and the AD inflammatory response was effectively inhibited. In a mouse allergic asthma (AA) model, it was found that oral allergens can also effectively reduce asthma inflammation by inducing rTh1. More importantly, the study found that rTh1 cells with similar molecular characteristics also exist in human peripheral blood mononuclear cells (PBMC). Clinical observations have shown that the proportion of rTh1 cells in PBMCs of AD patients is significantly negatively correlated with the severity of the disease, and the proportion of rTh1 cells in the peripheral blood of patients with asthma and chronic obstructive pulmonary disease (COPD) is significantly reduced. Specific examples are as follows.

[0096] Example 1 Isolation and identification of mouse rTh1 cells

[0097] Experimental steps:

[0098] Mouse mesenteric lymph nodes were obtained, ground with a grinding rod, passed through a 70 μm cell strainer, and rinsed with Staining Buffer (DPBS + 2% FBS). The cells were then centrifuged at 500 g for 5 min at 4°C, the supernatant discarded, and the cells were resuspended in 100 μl of Staining Buffer. The cell culture medium was then diluted with the anti-vital dye FVD-Efluor / 455UV and flow cytometry surface antibodies CD3-BV510, CD4-BV786, CD25-SB600, CXCR5-BV650, PD1-Percp / Efluor710, CXCR3-BV421, CCR6-PE / Fire640, CD73-PE / Cy7, FR4-FITC, and ICOS-AF700 at a dilution of 1:100 and incubated at 4°C in the dark for 30 min. Wash with 500 μl of Staining Buffer, centrifuge at 500 g for 5 min at 4°C, discard the supernatant, resuspend in 200 μl of Permeabilization Fixative, and incubate at 4°C in the dark for 12 h. Wash with 500 μl of Permeabilization Buffer, centrifuge at 1000 g for 5 min at 4°C, discard the supernatant, resuspend in 100 μl of Permeabilization Buffer, and add flow cytometry intracellular staining antibodies Foxp3-APC and cMaf-PE at a 1:100 ratio. Incubate at 4°C in the dark for 60 min. Wash with 500 μl of Permeabilization Buffer, centrifuge at 1000 g for 5 min at 4°C, discard the supernatant, resuspend in 500 μl of Staining Buffer, transfer through a 300-mesh flow cytometer into a flow cytometer, and analyze on a BD LSR Fortessa X-20 flow cytometer.

[0099] The results are as follows Figure 1 As shown, rTh1 cells isolated from mouse mesenteric lymph nodes (MLN) had the phenotype of CD3(+)CD4(+)CD25(-)Foxp3(-)CXCR5(-)CCR6(-)CXCR3(+)CD73(+)cMaf(+) and highly expressed FR4, PD1 and ICOS.

[0100] Example 2 Verification of rTh1 cell function in vitro

[0101] The sorted mouse rTh1, Treg and remaining CD4+ T cells (OT) were respectively T cells were co-cultured and treated with anti-CD3, anti-CD28, PMA, ionomycin, monensin, and Brefeldin A. The experimental steps are as follows:

[0102] Mouse MLN single cell suspension preparation and live / dead dye and flow surface staining steps were the same as before. Flow sorting was performed using a BD FACSymphony S6 flow sorter, and the sorted rThl, Treg and other T subpopulations were counted using a cell counting plate. Mouse spleen was passed through a 70 pm Cell Strainer and rinsed with Staining Buffer (DPBS + 2% FBS). Then centrifuged at 500g, 4°C for 5 min, discard the supernatant, and use EasySep TM Mouse CD4+T Cell Isolation Kit (Catalog # 19765) to sort CD4+ T cells, counted using a cell counting plate, and labeled with CellTrace TM Yellow Cell Proliferation Kit (Cat. No. C34573). rThl, Treg and Other T were labeled with 30,000 CTY-labeled CD4+ T cells at a ratio of 1:3, respectively. rThl, Treg and Other T cells were co-cultured in round-bottom 96-well plates and ImmunoCult TM Mouse T Cell Activator Kit (Catalog # 100-1572) for in vitro expansion. The remaining mesenteric lymph node cells and sorted rThl, Treg and Other T were fixed, intracellular marker flow cytometry staining and flow cytometry were the same as before, and the purity of flow cytometry sorting was verified. The remaining sorted CD4+ T cells were stained with live / dead dye FVD-Efluor / 455UV and flow surface staining anti-CD3-BV510, CD4-FITC, CD44-Efluor450, CD62L-APC flow cytometry and flow cytometry were the same as before, and the purity of flow cytometry sorting was verified. After 4 days of in vitro co-culture, eBioscience TM Cell Stimulation Cocktail (with protein transport inhibitor) (500X) (Cat. No. 00-4975-03) for flow cytometry cytokine detection pretreatment, then flow cytometry staining with live / dead dye FVD-Efluor455UV and flow surface antibody CD3-BV510, CD4-BV786 was the same as before, fixed with membrane, intracellular flow cytometry staining with intracellular flow cytometry staining antibody IL4-PE / Cy7, IL13-APC / Efluor780, IFNg-BV605 was the same as before, and flow cytometry was the same as before.

[0103] The results are shown in Figure 2 Figure 6, flow cytometry showed that rThl significantly inhibited the expression of IL4 and IL13 in co-cultured T cells in vitro, confirming that rThl cells significantly inhibited Th2-type inflammatory factor expression.

[0104] Example 3. In vivo application of rTh1 cells

[0105] 3.1 rTh1 cells can significantly improve the symptoms of dust mite-induced AD mouse model

[0106] Flow cytometry isolation of rTh1, Treg, and other T cells from mouse MLNs was performed using the same procedures as previously described. Six-week-old male wild-type C57BL6 / J mice were housed in a SPF environment at the Experimental Animal Center of Xinhua Hospital and acclimated from one to seven weeks of age. A dust mite-induced atopic dermatitis (AD) model was induced by applying MC903 solution (0.075 nmol / μl) and house dust mite (HDM) solution (1 μg / μl) to the ears of each group. The mice were then treated with a 20 μl / ear application of MC903 solution (0.075 nmol / μl) and house dust mite (HDM) solution (1 μg / μl) once daily for 14 days. On day 7 of modeling, 100,000 rTh1, Treg, and other T cells or an equal volume of PBS were injected into the tail vein of each group. Mice were anesthetized intraperitoneally, and ear lesions were photographed. Ear thickness was measured using a vernier caliper, and the mice were then sacrificed by cervical dislocation. Skin from the mid-ear section of the mice was fixed with 4% paraformaldehyde, stained with hematoxylin and eosin, and scanned under a bright-field microscope. The skin on both sides of the mouse ear was harvested by RIPA method to obtain protein, and protein was quantified by BCA method. TM Deluxe Set Mouse IL-4 (Cat#431104) measures IL-4 levels in mouse ears. Lesion-draining lymph nodes were obtained from mice, and flow cytometry staining and flow cytometry were performed as described above.

[0107] The results are as follows Figure 3 As shown in the mouse AD model, in the rTh1 in vivo injection group of mice, rTh1 cells were significantly upregulated in the lesion-draining lymph nodes (dLN), and the mouse ear IL-4 level was significantly reduced, indicating that rTh1 cells have an inhibitory effect on AD inflammation and can significantly improve AD symptoms, and its effect is not inferior to Treg.

[0108] 3.2 Role of rTh1 cells in HDM-, OVA-, or peanut-induced AD mouse models

[0109] In this study, allergens were administered to mice during their neonatal period, especially during the peri-weaning period (3 weeks of age), to induce the production of endogenous rTh1 cells to prevent or treat AD induced by HDM, OVA, or peanut. Early or delayed oral administration of allergens may lack the preventive effect. The experimental steps are as follows:

[0110] Mice were gavaged with HDM (20 μg), ovalbumin (OVA, 2 mg), or peanut (15 mg) allergens on days 15-21 after birth. A control group received an equal dose of PBS for 7 consecutive days. Some mice were orally administered an equal dose of HDM or PBS on days 1-7 or 29-35 after birth. Following gavage, mice were reared for 4 weeks until adulthood. Subsequently, the ears of all mice in each group were smeared with MC903 solution (0.075 nmol / μl) at 20 μl / ear. Furthermore, according to the group, HDM (1 μg / μl), OVA (5 μg / μl), or peanut (10 μg / μl) were smeared at 20 μl / ear once daily for 14 consecutive days to induce AD ​​models in mice. Subsequent skin lesion photography, ear thickness measurement, HE pathology, mouse ear protein IL-4 ELISA, and flow cytometry of lesion-draining lymph nodes were performed as described above.

[0111] The results are as follows Figure 4 As shown in the data, oral administration of dust mites, eggs, and peanuts during the neonatal period can prevent AD inflammation by inducing upregulation of rTh1.

[0112] The AD model was induced in mice by oral gavage with HDM or PBS in early life and in adulthood using MC903 combined with HDM, as described in 3.2. Some mice were intraperitoneally injected with anti-CXCR3 (150 μg), anti-CD25 (150 μg), or control IgG (150 μg) every 3 days throughout the modeling process. Subsequent skin lesion photography, ear thickness measurement, HE pathology, mouse ear protein IL-4 ELISA, and lesion-draining lymph node flow cytometry were performed as described above.

[0113] The results are as follows Figure 5 As shown in the results, AD inflammation could not be improved after antagonizing rTh1 cells with anti-CXCR3 antibodies, demonstrating that rTh1 cells play a key role in preventing AD symptoms in mice.

[0114] 3.3 Preventive or therapeutic effects of rTh1 cells in mouse asthma models

[0115] In this experiment, allergens were administered to mice during the peri-weaning period to induce the production of endogenous rTh1 cells, thereby preventing or treating the symptoms of asthma in mice. The experimental steps are as follows:

[0116] Mice were gavaged with HDM or PBS as before in the early stages of life. Four weeks after gavage, mice were first sensitized by intraperitoneal injection of 60 μg HDM and 2 mg aluminum dissolved in 200 μl PBS. Subsequently, inhalation anesthesia and 20 μl of HDM solution (3 μg / μl) were dripped into the nose once a day for 30 consecutive days to induce a mouse asthma model. Mice were first anesthetized by inhalation and 20 μl of HDM solution (3 μg / μl) were dripped into the nose for respiratory tract provocation, and then lung function was tested using a whole-body plethysmograph. After intraperitoneal anesthesia, the mice were enucleated and blood was collected. Then, they were killed by cervical dislocation. After the blood was allowed to stand at room temperature for more than 1.5 hours, it was centrifuged at 2000g for 20 minutes at room temperature to obtain serum. A vertical midline incision was made in the mouse neck, and the neck muscles were stripped to expose the trachea. A small transverse incision was made on the trachea with ophthalmic scissors. A gavage needle was inserted and ligated with surgical sutures. 500 μl of pre-cooled DPBS (containing 1% FBS) was drawn into the lungs with a 1 ml syringe without the needle. The lungs were slowly injected into the lungs through the gavage needle. The chest cavity was gently pressed for 20 seconds. The lavage fluid was slowly withdrawn while the chest was rhythmically pressed. The alveolar lavage fluid was collected through the gavage needle into a 1.5 mm EP tube. The alveolar lavage fluid was collected again and centrifuged at 4°C, 200 g for 10 minutes. The supernatant was discarded and the cell pellet was precipitated with 100 μl of Staining. Resuspend in buffer and add CD16 / 32Blocker at a 1:100 dilution. After incubation at 4°C for 5 minutes, add the live / dead dye FVD-EF455UV and flow cytometry surface staining antibodies CD45-APC / Fire750, CD3-PE / Cy7, CD11b-BV650, Ly6g-FITC, CD11c-PE / Dazzle594, MHCII-BV510, and Siglec F-APC at a 1:100 dilution. Flow cytometry staining and loading were performed as before, with 100 μl of Counting Beads added to each tube of sample before loading. Mouse lungs were harvested, and the right lower lobe was fixed with 4% paraformaldehyde, stained with HE, and scanned under a brightfield microscope. Protein was obtained from the right upper lobe using RIPA, quantified using the BCA assay, and analyzed using ELISAMAX. TM Deluxe Set Mouse IL-4 (Cat#431104) measures mouse lung IL-4 levels; mince the left lung and digest with 3 ml of tissue digestion buffer (CM1640, Collagenase I 3 mg / ml, DNase I 0.3 mg / ml) at 37°C, 125 rpm for 45 min. Add 3 ml of pre-cooled stop buffer (DPBS, containing 2% FBS and 1 mM EDTA) to terminate the mouse, pass through a 70 μm Cell Strainer and rinse with 3 ml of pre-cooled stop buffer. Centrifuge at 500 g, 4°C for 8 min, discard the supernatant, resuspend in 2 ml of red blood cell lysis buffer, let stand at room temperature for 3 min, add 3 ml of pre-cooled FACS to terminate red blood cell lysis, centrifuge at 500 g, 4°C for 6 min, discard the supernatant, and then perform flow staining and flow cytometry as before.

[0117] The results are as follows Figure 6 As shown in the figure, oral administration of house dust mites to newborns can prevent or treat asthma inflammation by upregulating endogenous rTh1.

[0118] Example 4 Isolation, purification, identification and activity determination of human rTh1 cells

[0119] Peripheral blood (approximately 3 ml / person) was collected from patients with AD, asthma, COPD, and healthy controls using EDTA-anticoagulant tubes. PBMCs were then isolated using Human Peripheral Blood Lymphocyte Separation Medium (Cat: P8610) and cryopreserved in Serum-Free Cell Freezing Medium (Cat: C40100) at -80°C. Frozen PBMCs were rapidly thawed in a 37°C water bath and subsequently stained and loaded onto the flow cytometer as described above.

[0120] The results are as follows Figure 7 As shown, the sorted cells highly expressed molecules such as CD73, TIGIT, PD1, ICOS, CXCR3, and cMaf, indicating that rTh1 cells similar to those in mice also exist in humans.

[0121] Example 5 Correlation Analysis between Human rTh1 Cells and the Severity of AD and COPD

[0122] Methods: rTh1 cells were isolated from PBMCs of human AD, asthma (AS), chronic obstructive pulmonary disease (COPD) patients and healthy controls, and their proportions were correlated with the severity of AD, AS, COPD (EASI score, IGA score) and the number of sensitizing allergens.

[0123] The results are as follows Figure 8 As shown in the results, it can be seen that Th1 decreases with the increasing severity of AD patients and the increasing number of allergens in AD patients, and its proportion in AS and COPD patients is significantly lower than that in healthy controls, further suggesting that rTh1 may have a protective effect in humans against diseases such as AD, AS and COPD similar to that in mice.

[0124] In summary, the present invention provides a novel immunoregulatory function of rTh1 cells, a T cell subset, which provides a new potential target for the prevention and treatment of inflammatory diseases including AD, AA, COPD, etc., and also lays the foundation for the development of rTh1 cell-based immunotherapy strategies. It has important value for the prevention and treatment of inflammatory diseases such as food allergy, dust mite allergy, AD, AA, COPD and other Th2-type inflammatory-related skin diseases such as bullous pemphigoid, urticaria, etc.

[0125] The above examples are intended to illustrate the embodiments disclosed herein and are not to be construed as limiting the present invention. In addition, the various modifications listed herein and variations of the methods in the invention will be apparent to those skilled in the art without departing from the scope and spirit of the present invention. Although the present invention has been specifically described in conjunction with various specific preferred embodiments of the present invention, it should be understood that the present invention should not be limited to these specific embodiments. In fact, various modifications apparent to those skilled in the art as described above to obtain the invention should be included within the scope of the present invention.

Claims

1. Use of rTh1 cells or substances that induce upregulation of rTh1 cells in the preparation of drugs for preventing, treating or diagnosing allergic diseases or inflammatory diseases.

2. The use according to claim 1, characterized in that The phenotype of the rTh1 cells is CD3(+), CD4(+), CD25(-), Foxp3(-), CXCR5(-), CCR6(-), CXCR3(+), CD73(+), cMaf(+), and they highly express FR4, PD1 and ICOS.

3. The use according to claim 1, characterized in that The substance that induces upregulation of rTh1 cells is selected from allergens or allergen component proteins.

4. The use according to claim 1, characterized in that The allergic disease is selected from diseases caused by food allergens or inhalant allergens.

5. The use according to claim 4, characterized in that The food allergen is selected from eggs, milk, peanuts, soybeans, wheat, nuts, seafood or allergen component proteins of the aforementioned substances.

6. The use according to claim 4, characterized in that The inhaled allergen is selected from dust mites, pollen, animal dander, fungi or allergen component proteins of the aforementioned substances.

7. The use according to claim 6, characterized in that The dust mite is selected from house dust mite or dust mite.

8. The use according to claim 1, characterized in that The inflammatory disease is a Th2 type inflammation.

9. The use according to claim 1, characterized in that The inflammatory disease is selected from atopic dermatitis, allergic asthma, bullous pemphigoid, urticaria or chronic obstructive pulmonary disease.

10. Use of rTh1 cells or substances that induce upregulation of rTh1 cells in combination with glucocorticoids or anti-inflammatory drugs in the preparation of drugs for preventing, treating or diagnosing allergic diseases or inflammatory diseases.

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