WO2021227056A1 - 一种t细胞及其制备方法和应用 - Google Patents

一种t细胞及其制备方法和应用 Download PDF

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WO2021227056A1
WO2021227056A1 PCT/CN2020/090639 CN2020090639W WO2021227056A1 WO 2021227056 A1 WO2021227056 A1 WO 2021227056A1 CN 2020090639 W CN2020090639 W CN 2020090639W WO 2021227056 A1 WO2021227056 A1 WO 2021227056A1
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cells
ryr2
cancer
cell
expression
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PCT/CN2020/090639
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French (fr)
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石彦
蒙俊辰
王晓博
耿爽
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清华大学
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Priority to JP2022570128A priority Critical patent/JP2023526614A/ja
Priority to AU2020447334A priority patent/AU2020447334A1/en
Priority to PCT/CN2020/090639 priority patent/WO2021227056A1/zh
Priority to CA3178951A priority patent/CA3178951A1/en
Priority to US17/998,855 priority patent/US20230220339A1/en
Priority to CN202080100913.4A priority patent/CN115698266A/zh
Priority to EP20935084.2A priority patent/EP4151721A4/en
Publication of WO2021227056A1 publication Critical patent/WO2021227056A1/zh

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Definitions

  • the present invention relates to the field of biotechnology, in particular to a T cell and a preparation method and application thereof, as well as a reagent for regulating Ryr2 expression, a reagent for regulating Ca 2+ basic oscillation, a reagent for regulating m-Calpain activity, and a regulating T cell and DC cell
  • a reagent for regulating Ryr2 expression a reagent for regulating Ca 2+ basic oscillation
  • a reagent for regulating m-Calpain activity a regulating T cell and DC cell
  • Treg Regulatory T cells
  • Natural Treg cells were first reported by Sakaguchi et al. in 1995. They accounted for about 5%-10% of the number of CD4+ T cells in peripheral blood. FoxP3 It is a sign of natural Treg. Regulatory T cells can be divided into two types: natural Treg (n Treg) and acquired Treg (a Treg).
  • Treg cells The inhibitory mechanism of Treg cells is a research hotspot. References: Past, Present, and Future of Regulatory T Cell Therapy in Transplantation and Autoimmunity (Romano et al., Frontiers in Immunology, 2019) The disclosed mechanisms, including T cell lysis, surface protein extraction, local production of adenosine, etc., Both require Treg combination. In recent years, a series of research reports have shifted the focus of research to the direct inhibition of dendritic cells.
  • Ryr2 is a calcium release channel in the endoplasmic reticulum/sarcoplasmic reticulum (ER/SR).
  • ER/SR endoplasmic reticulum/sarcoplasmic reticulum
  • the prior art discloses that the Ryr2 gene is related to sudden cardiac death, arrhythmia, and coronary heart disease, such as:
  • Non-patent literature A family study of sudden cardiac death caused by new mutations in the Ryr2 gene (Shen Tong et al., Journal of Integrated Traditional Chinese and Western Medicine Cardiovascular Diseases, 2019.02) disclosed that the missense mutation c.G4107C in the Ryr2 gene has labor dyspnea. Symptoms such as, chest pain, that is, missense mutations in the Ryr2 gene may be related to sudden cardiac death.
  • Non-patent literature The role of CASQ2 and Ryr2 in the abnormal rhythm of cardiomyocytes induced by diacetylmorphine (Hu Xiayun et al., Journal of Xinjiang Medical University, 2019.03) discloses the relationship between Ryr2 and calcium channels, and clarifies its involvement in cardiomyocyte calcium channels The process of abnormalities and arrhythmias.
  • Non-patent literature A theoretical discussion on the correlation between Ryr2 gene expression and the onset of coronary heart disease with heart-qi deficiency syndrome (Ma Yuexiang et al., Chinese Medicine Information, 2012) discloses that Ryr2 dysfunction can lead to a decline in heart function.
  • the basic motive force of blood circulation, the deficiency of heart qi is consistent with the hypofunction of the heart; the expression of Ryr2 gene can further reveal the essence of the syndrome of heart qi deficiency.
  • Non-patent literature The inhibitory effect of carvedilol on Ryr2-mediated spontaneous calcium oscillations (Xiao Jianmin et al., Chinese Journal of Pathophysiology, 2013) discloses that carvedilol can inhibit Ryr2-mediated spontaneous calcium oscillations, and shows It is superior to other ⁇ -blockers in reducing heart failure mortality.
  • the present invention finds the regulatory switch-Ryr2 of Treg cell function, and obtains the technology that overexpression of FoxP3 can reduce the expression of Ryr2, and specifically determines the target location.
  • a large number of experiments have confirmed that knocking down or knocking out Ryr2 in T cells makes the CD4+ T cell pool become immunosuppressive cells, and the obtained T cells have functions similar to Treg cells.
  • good results have been achieved in the treatment of viral infections, asthma, allergies, colitis and tumors, and the autoimmunity of the related systems of tinea mice has been restored.
  • the first aspect of the present invention provides a T cell in which the Ryr2 gene is deleted or FoxP3 is overexpressed.
  • At least exon 7 of the Ryr2 gene is deleted in the T cell.
  • At least a guanine-rich sequence in the Ryr2 gene is deleted from the T cell.
  • the guanine-rich sequence is located 200 bp before the start codon of the Ryr2 gene.
  • the guanine-rich sequence is a nucleotide sequence containing at least 4 guanines (G). More preferably, it contains at least 4-20 guanine (G) nucleotides.
  • At least 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, etc. are included.
  • the guanine-rich sequence contains GCAGGGG.
  • the T cells are selected from cytotoxic T cells, helper T cells, regulatory/suppressive T cells (Treg) or memory T cells.
  • the T cell is a Tconv cell.
  • the second aspect of the present invention provides a method for preparing the T cell of the present invention, wherein the preparation method is selected from shRNA, siRNA, CRISPR/Cas9, zinc finger nuclease technology, transcription activator-like effector Nuclease technology or homing endonuclease.
  • the shRNA is SEQ ID NO: 5 and 6.
  • the third aspect of the present invention provides a shRNA that knocks down the Ryr2 gene of T cells, so that the T cell Ca 2+ basal oscillation is reduced, the m-Calpain (m-calpain) activity is reduced, and it is compatible with DC cells. Improved binding strength, has the function of immunosuppressive cells or has the function of treating infectious diseases, inflammation or tumors.
  • the shRNA is SEQ ID NO: 5 and 6.
  • the fourth aspect of the present invention provides a T cell in which Ryr2 is overexpressed.
  • the T cells are selected from cytotoxic T cells, helper T cells, regulatory/suppressive T cells (Treg) or memory T cells.
  • the T cells are Treg cells.
  • the fifth aspect of the present invention provides reagents for regulating FoxP3 expression, reagents for regulating Ryr2 expression, reagents for regulating Ca 2+ basic oscillation, reagents for regulating m-Calpain activity, or reagents for regulating the binding strength of T cells and DC cells.
  • the infectious disease is selected from bacterial infections, viral infections or fungal infections; further preferably viral infections, pneumonia with septic shock, peritonitis, bacteremia, sepsis or sepsis; selected viral infections From acute viral infection or chronic viral infection; preferably influenza virus, parainfluenza virus, herpes virus (such as HSV-1, EBV), measles virus, vesicular stomatitis virus, hepatitis B virus, hepatitis C virus, human immunodeficiency Virus, lymphocytic choriomeningitis virus or human papilloma virus.
  • influenza virus parainfluenza virus
  • herpes virus such as HSV-1, EBV
  • measles virus such as HSV-1, EBV
  • measles virus such as HSV-1, EBV
  • measles virus such as HSV-1, EBV
  • measles virus such as HSV-1, EBV
  • measles virus such as HSV-1, E
  • the inflammation may be inflammation of any tissue, including but not limited to adrenal gland, adrenal medulla, anus, appendix, bladder, blood, bone, bone marrow, brain, breast, cecum, central nervous system (Include or exclude the brain), cerebellum, cervix, colon, duodenum, endometrium, epithelial cells (e.g.
  • kidney epithelial cells gallbladder, esophagus, glial cells, heart, ileum, jejunum, kidney, lacrimal gland , Larynx, liver, lung, lymph, lymph nodes, lymphoblasts, maxilla, mediastinum, mesenteric, myometrium, nasopharyngeal, omentum, oral cavity, ovary, pancreas, parotid gland, peripheral nervous system, peritoneum, pleura, prostate, Salivary glands, sigmoid colon, skin, small intestine, soft tissue, spleen, stomach, testes, thymus, thyroid, tongue, tonsils, trachea, uterus, vulva, white blood cells.
  • the inflammation is selected from systemic lupus erythematosus, rheumatoid arthritis, psoriatic arthritis, scleroderma, asthma, atopic dermatitis, organ-specific inflammatory diseases, allergies (such as allergic Rhinitis), folliculitis, tonsillitis, pneumonia, hepatitis, nephritis, acne, autoimmune disease, chronic prostatitis, glomerulonephritis, hypersensitivity, colitis, inflammatory bowel disease, pelvic inflammatory disease, reperfusion injury , Transplant rejection, vasculitis or interstitial cystitis.
  • systemic lupus erythematosus rheumatoid arthritis, psoriatic arthritis, scleroderma, asthma, atopic dermatitis, organ-specific inflammatory diseases, allergies (such as allergic Rhinitis), folliculitis, tonsillitis, pneumonia, hepatitis, nephritis, acne, autoimmune disease
  • the tumor may be any poor cell proliferation (or any disease that manifests itself as poor cell proliferation), neoplasm, or poor cell proliferation, neoplasm, or increased tendency or risk of tumor. It can be benign or malignant, or primary or secondary (metastatic).
  • a neoplasm can be any abnormal growth or proliferation of cells, and can be located in any tissue. Examples of tissues include adrenal gland, adrenal medulla, anus, appendix, bladder, blood, bone, bone marrow, brain, breast, cecum, central nervous system (including or excluding the brain), cerebellum, cervix, colon, duodenum, Endometrium, epithelial cells (e.g.
  • renal epithelial cells gallbladder, esophagus, glial cells, heart, ileum, jejunum, kidney, lacrimal gland, larynx, liver, lung, lymph, lymph nodes, lymphoblasts, maxilla, mediastinum , Mesenteric, myometrium, nasopharyngeal, omentum, oral cavity, ovary, pancreas, parotid gland, peripheral nervous system, peritoneum, pleura, prostate, salivary gland, sigmoid colon, skin, small intestine, soft tissue, spleen, stomach, testis, thymus, thyroid , Tongue, tonsils, trachea, uterus, vulva, white blood cells.
  • the tumor is selected from prostate cancer, breast cancer, liver cancer, glioma (such as glioma), bowel cancer, cervical cancer, non-small cell lung cancer, lung cancer, pancreatic cancer, gastric cancer, bladder cancer, Skin cancer, rhabdomyocarcinoma, tongue squamous cell carcinoma, nasopharyngeal carcinoma, ovarian cancer, placental choriocarcinoma, lymphoma (e.g.
  • non-Hodgkin’s lymphoma Hodgkin’s lymphoma, skin T-cell lymphoma), leukemia, rectal adenocarcinoma , Medulloblastoma, meningioma, neurofibromas (e.g. neurofibrosarcoma), ependymoma, schwannoma, astrocytoma, melanoma, mesothelioma, myeloma, chronic myelogenous leukemia, acute Myeloid leukemia, myelodysplastic syndrome, chronic lymphocytic leukemia, epidermoid carcinoma, colon cancer, thymic cancer, blood cancer, head and neck cancer, or oropharyngeal cancer.
  • neurofibromas e.g. neurofibrosarcoma
  • ependymoma schwannoma
  • astrocytoma melanoma
  • mesothelioma myeloma
  • the sixth aspect of the present invention provides reagents for regulating the expression of FoxP3, reagents for regulating the expression of Ryr2, reagents for regulating Ca 2+ basic oscillation, reagents for regulating the activity of m-Calpain, reagents for regulating the binding strength of T cells and DC cells or The application of the T cells of the present invention in the treatment of infectious diseases, inflammations or tumors.
  • the seventh aspect of the present invention provides reagents for regulating the expression of FoxP3, reagents for regulating the expression of Ryr2, reagents for regulating Ca 2+ basic oscillation, reagents for regulating the activity of m-Calpain, reagents for regulating the binding strength of T cells and DC cells or The application of the T cells of the present invention in the preparation of medicines for treating infectious diseases, inflammations or tumors.
  • the eighth aspect of the present invention provides regulation of FoxP3 expression, regulation of Ryr2 expression, regulation of Ca 2+ basal oscillations, regulation of m-Calpain activity, regulation of the binding strength of T cells and DC cells, or T cells of the present invention in the treatment of infections Diseases, inflammations or tumors, or applications in the preparation of medicines for the treatment of infectious diseases, inflammations or tumors.
  • the infectious disease is selected from bacterial infections, viral infections or fungal infections; further preferably viral infections, pneumonia with septic shock, peritonitis, bacteremia, sepsis or sepsis; selected viral infections From acute viral infection or chronic viral infection; preferably influenza virus, parainfluenza virus, herpes virus (such as HSV-1, EBV), measles virus, vesicular stomatitis virus, hepatitis B virus, hepatitis C virus, human immunodeficiency Virus, lymphocytic choriomeningitis virus or human papilloma virus.
  • influenza virus parainfluenza virus
  • herpes virus such as HSV-1, EBV
  • measles virus such as HSV-1, EBV
  • measles virus such as HSV-1, EBV
  • measles virus such as HSV-1, EBV
  • measles virus such as HSV-1, EBV
  • measles virus such as HSV-1, E
  • the inflammation may be inflammation of any tissue, including but not limited to adrenal gland, adrenal medulla, anus, appendix, bladder, blood, bone, bone marrow, brain, breast, cecum, central nervous system (Include or exclude the brain), cerebellum, cervix, colon, duodenum, endometrium, epithelial cells (e.g.
  • kidney epithelial cells gallbladder, esophagus, glial cells, heart, ileum, jejunum, kidney, lacrimal gland , Larynx, liver, lung, lymph, lymph nodes, lymphoblasts, maxilla, mediastinum, mesenteric, myometrium, nasopharyngeal, omentum, oral cavity, ovary, pancreas, parotid gland, peripheral nervous system, peritoneum, pleura, prostate, Salivary glands, sigmoid colon, skin, small intestine, soft tissue, spleen, stomach, testes, thymus, thyroid, tongue, tonsils, trachea, uterus, vulva, white blood cells.
  • the inflammation is selected from systemic lupus erythematosus, rheumatoid arthritis, psoriatic arthritis, scleroderma, asthma, atopic dermatitis, organ-specific inflammatory diseases, allergies (such as allergic Rhinitis), folliculitis, tonsillitis, pneumonia, hepatitis, nephritis, acne, autoimmune disease, chronic prostatitis, glomerulonephritis, hypersensitivity, colitis, inflammatory bowel disease, pelvic inflammatory disease, reperfusion injury , Transplant rejection, vasculitis or interstitial cystitis.
  • systemic lupus erythematosus rheumatoid arthritis, psoriatic arthritis, scleroderma, asthma, atopic dermatitis, organ-specific inflammatory diseases, allergies (such as allergic Rhinitis), folliculitis, tonsillitis, pneumonia, hepatitis, nephritis, acne, autoimmune disease
  • the tumor may be any poor cell proliferation (or any disease that manifests itself as poor cell proliferation), neoplasm, or poor cell proliferation, neoplasm, or increased tendency or risk of tumor. It can be benign or malignant, or primary or secondary (metastatic).
  • a neoplasm can be any abnormal growth or proliferation of cells, and can be located in any tissue. Examples of tissues include adrenal gland, adrenal medulla, anus, appendix, bladder, blood, bone, bone marrow, brain, breast, cecum, central nervous system (including or excluding the brain), cerebellum, cervix, colon, duodenum, Endometrium, epithelial cells (e.g.
  • renal epithelial cells gallbladder, esophagus, glial cells, heart, ileum, jejunum, kidney, lacrimal gland, larynx, liver, lung, lymph, lymph nodes, lymphoblasts, maxilla, mediastinum , Mesenteric, myometrium, nasopharyngeal, omentum, oral cavity, ovary, pancreas, parotid gland, peripheral nervous system, peritoneum, pleura, prostate, salivary gland, sigmoid colon, skin, small intestine, soft tissue, spleen, stomach, testis, thymus, thyroid , Tongue, tonsils, trachea, uterus, vulva, white blood cells.
  • the tumor is selected from prostate cancer, breast cancer, liver cancer, glioma (such as glioma), bowel cancer, cervical cancer, non-small cell lung cancer, lung cancer, pancreatic cancer, gastric cancer, bladder cancer, Skin cancer, rhabdomyocarcinoma, tongue squamous cell carcinoma, nasopharyngeal carcinoma, ovarian cancer, placental choriocarcinoma, lymphoma (e.g.
  • non-Hodgkin’s lymphoma Hodgkin’s lymphoma, skin T-cell lymphoma), leukemia, rectal adenocarcinoma , Medulloblastoma, meningioma, neurofibromas (e.g. neurofibrosarcoma), ependymoma, schwannoma, astrocytoma, melanoma, mesothelioma, myeloma, chronic myelogenous leukemia, acute Myeloid leukemia, myelodysplastic syndrome, chronic lymphocytic leukemia, epidermoid carcinoma, colon cancer, thymic cancer, blood cancer, head and neck cancer, or oropharyngeal cancer.
  • neurofibromas e.g. neurofibrosarcoma
  • ependymoma schwannoma
  • astrocytoma melanoma
  • mesothelioma myeloma
  • the ninth aspect of the present invention provides reagents for increasing FoxP3 expression, reagents for reducing Ryr2 expression, reagents for reducing Ca 2+ basal oscillation, reagents for reducing m-Calpain activity, reagents for improving the binding strength of T cells and DC cells, or The application of the above-mentioned Ryr2 gene-deficient T cells in the preparation of medicines for the treatment of infectious diseases or inflammations.
  • the tenth aspect of the present invention provides reagents for increasing FoxP3 expression, reagents for reducing Ryr2 expression, reagents for reducing Ca 2+ basal oscillation, reagents for reducing m-Calpain activity, reagents for improving the binding strength of T cells and DC cells, or The application of the above-mentioned T cells lacking Ryr2 gene in the treatment of infectious diseases or inflammation.
  • the eleventh aspect of the present invention provides to increase FoxP3 expression, decrease Ryr2 expression, decrease Ca 2+ basal oscillation, decrease m-Calpain activity or increase the binding strength of T cells and DC cells in the treatment of infectious diseases, inflammation or in preparation Application in medicines for the treatment of infectious diseases and inflammation.
  • the infectious disease is selected from bacterial infections, viral infections or fungal infections; further preferably viral infections, pneumonia with septic shock, peritonitis, bacteremia, sepsis or sepsis; selected viral infections From acute viral infection or chronic viral infection; preferably influenza virus, parainfluenza virus, herpes virus (such as HSV-1, EBV), measles virus, vesicular stomatitis virus, hepatitis B virus, hepatitis C virus, human immunodeficiency Virus, lymphocytic choriomeningitis virus or human papilloma virus.
  • influenza virus parainfluenza virus
  • herpes virus such as HSV-1, EBV
  • measles virus such as HSV-1, EBV
  • measles virus such as HSV-1, EBV
  • measles virus such as HSV-1, EBV
  • measles virus such as HSV-1, EBV
  • measles virus such as HSV-1, E
  • the inflammation may be inflammation of any tissue, including but not limited to adrenal gland, adrenal medulla, anus, appendix, bladder, blood, bone, bone marrow, brain, breast, cecum, central nervous system (Include or exclude the brain), cerebellum, cervix, colon, duodenum, endometrium, epithelial cells (e.g.
  • kidney epithelial cells gallbladder, esophagus, glial cells, heart, ileum, jejunum, kidney, lacrimal gland , Larynx, liver, lung, lymph, lymph nodes, lymphoblasts, maxilla, mediastinum, mesenteric, myometrium, nasopharyngeal, omentum, oral cavity, ovary, pancreas, parotid gland, peripheral nervous system, peritoneum, pleura, prostate, Salivary glands, sigmoid colon, skin, small intestine, soft tissue, spleen, stomach, testes, thymus, thyroid, tongue, tonsils, trachea, uterus, vulva, white blood cells.
  • the inflammation is selected from systemic lupus erythematosus, rheumatoid arthritis, psoriatic arthritis, scleroderma, asthma, atopic dermatitis, organ-specific inflammatory diseases, allergies (such as allergic Rhinitis), folliculitis, tonsillitis, pneumonia, hepatitis, nephritis, acne, autoimmune disease, chronic prostatitis, glomerulonephritis, hypersensitivity, colitis, inflammatory bowel disease, pelvic inflammatory disease, reperfusion injury , Transplant rejection, vasculitis or interstitial cystitis.
  • systemic lupus erythematosus rheumatoid arthritis, psoriatic arthritis, scleroderma, asthma, atopic dermatitis, organ-specific inflammatory diseases, allergies (such as allergic Rhinitis), folliculitis, tonsillitis, pneumonia, hepatitis, nephritis, acne, autoimmune disease
  • the twelfth aspect of the present invention provides reagents for reducing FoxP3 expression, reagents for increasing Ryr2 expression, reagents for increasing Ca 2+ basal oscillation, reagents for increasing m-Calpain activity, and reagents for reducing the binding strength of T cells and DC cells Or the application of the above-mentioned T cells overexpressing Ryr2 in the preparation of drugs for treating tumors.
  • the thirteenth aspect of the present invention provides reagents for reducing FoxP3 expression, reagents for increasing Ryr2 expression, reagents for increasing Ca 2+ basal oscillation, reagents for increasing m-Calpain activity, and reagents for reducing the binding strength of T cells and DC cells Or the application of the above-mentioned Ryr2 overexpressing T cells in the treatment of tumors.
  • the fourteenth aspect of the present invention provides for reducing FoxP3 expression, increasing Ryr2 expression, increasing Ca 2+ basal oscillation, increasing m-Calpain activity, and reducing the binding strength of T cells and DC cells in the treatment of tumors or the preparation of drugs for the treatment of tumors Applications.
  • the tumor may be any poor cell proliferation (or any disease that manifests itself as poor cell proliferation), neoplasm, or poor cell proliferation, neoplasm, or increased tendency or risk of tumor. It can be benign or malignant, or primary or secondary (metastatic).
  • a neoplasm can be any abnormal growth or proliferation of cells, and can be located in any tissue. Examples of tissues include adrenal gland, adrenal medulla, anus, appendix, bladder, blood, bone, bone marrow, brain, breast, cecum, central nervous system (including or excluding the brain), cerebellum, cervix, colon, duodenum, Endometrium, epithelial cells (e.g.
  • renal epithelial cells gallbladder, esophagus, glial cells, heart, ileum, jejunum, kidney, lacrimal gland, larynx, liver, lung, lymph, lymph nodes, lymphoblasts, maxilla, mediastinum , Mesenteric, myometrium, nasopharyngeal, omentum, oral cavity, ovary, pancreas, parotid gland, peripheral nervous system, peritoneum, pleura, prostate, salivary gland, sigmoid colon, skin, small intestine, soft tissue, spleen, stomach, testis, thymus, thyroid , Tongue, tonsils, trachea, uterus, vulva, white blood cells.
  • the tumor is selected from prostate cancer, breast cancer, liver cancer, glioma (such as glioma), bowel cancer, cervical cancer, non-small cell lung cancer, lung cancer, pancreatic cancer, gastric cancer, bladder cancer, Skin cancer, rhabdomyocarcinoma, tongue squamous cell carcinoma, nasopharyngeal carcinoma, ovarian cancer, placental choriocarcinoma, lymphoma (e.g.
  • non-Hodgkin’s lymphoma Hodgkin’s lymphoma, skin T-cell lymphoma), leukemia, rectal adenocarcinoma , Medulloblastoma, meningioma, neurofibromas (e.g. neurofibrosarcoma), ependymoma, schwannoma, astrocytoma, melanoma, mesothelioma, myeloma, chronic myelogenous leukemia, acute Myeloid leukemia, myelodysplastic syndrome, chronic lymphocytic leukemia, epidermoid carcinoma, colon cancer, thymic cancer, blood cancer, head and neck cancer, or oropharyngeal cancer.
  • neurofibromas e.g. neurofibrosarcoma
  • ependymoma schwannoma
  • astrocytoma melanoma
  • mesothelioma myeloma
  • the fifteenth aspect of the present invention provides the application of increasing FoxP3 expression in reducing Ryr2 expression, reducing Ca 2+ basal oscillation, reducing m-Calpain activity, or improving the binding strength of T cells and DC cells.
  • the sixteenth aspect of the present invention provides the application of regulating Ryr2 expression in regulating Ca 2+ basal oscillation, regulating m-Calpain activity, or improving the binding strength of T cells and DC cells.
  • the regulation is to decrease or increase.
  • the seventeenth aspect of the present invention provides the application of regulating Ca 2+ basic oscillations in regulating the activity of m-Calpain or regulating the binding strength of T cells and DC cells.
  • the regulation is to decrease or increase.
  • the eighteenth aspect of the present invention provides the application of regulating the activity of m-Calpain in regulating the binding strength of T cells and DC cells.
  • the regulation is to decrease or increase.
  • the nineteenth aspect of the present invention provides an antagonist of Ryr2, which targets exon 7 of the Ryr2 gene or a guanine-rich sequence in the Ryr2 gene.
  • the guanine-rich sequence is located 200 bp before the start codon of the Ryr2 gene.
  • the guanine-rich sequence contains GCAGGGG.
  • the twentieth aspect of the present invention provides a method for regulating the binding strength of T cells and DC cells, the method comprising regulating the expression of Ryr2.
  • the regulation is to decrease or increase.
  • the method for reducing the binding strength of T cells and DC cells includes increasing the expression of Ryr2, and the method for increasing the binding strength of T cells and DC cells includes reducing the expression of Ryr2.
  • said reducing the expression of Ryr2 includes overexpressing FoxP3 in T cells or adding a Ryr2 inhibitor.
  • the Ryr2 inhibitor is selected from the antagonist of the present invention, rianodine, dantrolene or JTV519.
  • said increasing the expression of Ryr2 includes decreasing the expression of FoxP3 in T cells or adding a Ryr2 driver.
  • the Ryr2 driver is selected from the group consisting of niacinamide adenine dinucleotide phosphate, caffeine, p-chlorom-cresol, ryanodine, chlorantraniliprole, cyantraniliprole, and type B adrenal glands Sulfonate, 4-chloro-3-methylphenol, cyantraniliprole, cyclotriprom, cyclic adenosine diphosphate ribose, suramin sodium, flutraniliprole or trifluoperazine.
  • said reducing the expression of Ryr2 includes deleting exon 7 of the Ryr2 gene in the T cell.
  • the shRNA is used as SEQ ID NO: 5 and 6 to knock out exon 7 of the Ryr2 gene.
  • reducing the expression of Ryr2 includes knocking down or knocking out the Ryr2 gene in T cells. More preferably, a guanine-rich sequence in the Ryr2 gene in the T cell is knocked down or knocked out. More preferably, the guanine-rich sequence is located 200 bp before the start codon of the Ryr2 gene. Most preferably, the guanine-rich sequence contains GCAGGGG.
  • the guanine-rich sequence is a nucleotide sequence containing at least 4 guanines (G). More preferably, it contains at least 4-10 guanine (G) nucleotide sequences.
  • At least 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, etc. are included.
  • the twenty-first aspect of the present invention provides a method for reducing Ryr2 expression in T cells, reducing Ca 2+ basal oscillation, and reducing m-Calpain activity.
  • the method includes overexpressing FoxP3 in T cells, knocking down Lower or knock out the Ryr2 gene in T cells or add Ryr2 inhibitors.
  • At least exon 7 of the Ryr2 gene is knocked out.
  • the shRNA is used as SEQ ID NO: 5 and 6 to knock out exon 7 of the Ryr2 gene.
  • the method includes knocking down or knocking out the Ryr2 gene in T cells. More preferably, a guanine-rich sequence in the Ryr2 gene in the T cell is knocked down or knocked out. More preferably, the guanine-rich sequence is located 200 bp before the start codon of the Ryr2 gene. Most preferably, the guanine-rich sequence contains GCAGGGG.
  • the guanine-rich sequence is a nucleotide sequence containing at least 4 guanines (G). More preferably, it contains at least 4-10 guanine (G) nucleotide sequences.
  • At least 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, etc. are included.
  • the Ryr2 inhibitor is selected from the antagonist of the present invention, rianodine, dantrolene or JTV519.
  • the twenty-second aspect of the present invention provides a method for increasing Ryr2 expression in T cells, increasing Ca 2+ basal oscillation, and increasing m-Calpain activity.
  • the method includes reducing the expression of FoxP3 in T cells or Add Ryr2 driver.
  • the Ryr2 driver is selected from the group consisting of nicotinic acid amide adenine dinucleotide phosphate, caffeine, p-chlorom-cresol, ryanodine, chlorantraniliprole, cyantraniliprole, and type B adrenaline , 4-Chloro-3-methylphenol, cyantraniliprole, cyclotraniliprole, cyclic adenosine diphosphate ribose, suramin sodium, flutraniliprole or trifluoperazine.
  • the twenty-third aspect of the present invention provides a method for transforming Tconv cells into similar functions as Treg cells.
  • the method includes overexpressing FoxP3 of Tconv cells or knocking down or knocking out the Ryr2 gene of Tconv cells.
  • the method includes knocking down or knocking out at least exon 7 of the Ryr2 gene.
  • the shRNA is used as SEQ ID NO: 5 and 6 to knock out exon 7 of the Ryr2 gene.
  • the method includes knocking down or knocking out a guanine-rich sequence in the Ryr2 gene in Tconv cells.
  • the guanine-rich sequence is located 200 bp before the start codon of the Ryr2 gene. More preferably, the guanine-rich sequence contains GCAGGGG.
  • the 1.5 kb or 40 kb nucleotide sequence in the promoter region of the Ryr2 gene is knocked down or knocked out.
  • the functions similar to Treg cells include, but are not limited to, the strength of binding to DC cells, and related surface markers (such as CD25, GITR, CTLA-4, CD39, PD-1, LAG-3, TIM-3)
  • the expression level of Ca 2+ is low, the basal oscillation of Ca 2+ is low, the digestion of CMAC is low, the digestion of talin is reduced, and the immune transition is reduced.
  • the twenty-fourth aspect of the present invention provides a method for treating infectious diseases or inflammation, the method comprising administering to an individual an effective amount of the Ryr2 gene-deficient T cell of the present invention.
  • the twenty-fifth aspect of the present invention provides a method for the treatment of infectious diseases or inflammation, the method is selected from the group consisting of overexpression of FoxP3 in T cells of an individual, reduction of Ryr2 expression in T cells, and reduction of Ca 2 + Basic shaking, reduce m-Calpain activity, increase the binding strength of T cells and DC cells or add Ryr2 inhibitor.
  • the Ryr2 inhibitor is selected from the antagonist of the present invention, rianodine, dantrolene or JTV519.
  • the infectious disease is selected from bacterial infections, viral infections or fungal infections; further preferably viral infections, pneumonia with septic shock, peritonitis, bacteremia, sepsis or sepsis; selected viral infections From acute viral infection or chronic viral infection; preferably influenza virus, parainfluenza virus, herpes virus (such as HSV-1, EBV), measles virus, vesicular stomatitis virus, hepatitis B virus, hepatitis C virus, human immunodeficiency Virus, lymphocytic choriomeningitis virus or human papilloma virus.
  • influenza virus parainfluenza virus
  • herpes virus such as HSV-1, EBV
  • measles virus such as HSV-1, EBV
  • measles virus such as HSV-1, EBV
  • measles virus such as HSV-1, EBV
  • measles virus such as HSV-1, EBV
  • measles virus such as HSV-1, E
  • the inflammation may be inflammation of any tissue, including but not limited to adrenal gland, adrenal medulla, anus, appendix, bladder, blood, bone, bone marrow, brain, breast, cecum, central nervous system (Include or exclude the brain), cerebellum, cervix, colon, duodenum, endometrium, epithelial cells (e.g.
  • kidney epithelial cells gallbladder, esophagus, glial cells, heart, ileum, jejunum, kidney, lacrimal gland , Larynx, liver, lung, lymph, lymph nodes, lymphoblasts, maxilla, mediastinum, mesenteric, myometrium, nasopharyngeal, omentum, oral cavity, ovary, pancreas, parotid gland, peripheral nervous system, peritoneum, pleura, prostate, Salivary glands, sigmoid colon, skin, small intestine, soft tissue, spleen, stomach, testes, thymus, thyroid, tongue, tonsils, trachea, uterus, vulva, white blood cells.
  • the inflammation is selected from systemic lupus erythematosus, rheumatoid arthritis, psoriatic arthritis, scleroderma, asthma, atopic dermatitis, organ-specific inflammatory diseases, allergies (such as allergic Rhinitis), folliculitis, tonsillitis, pneumonia, hepatitis, nephritis, acne, autoimmune disease, chronic prostatitis, glomerulonephritis, hypersensitivity, colitis, inflammatory bowel disease, pelvic inflammatory disease, reperfusion injury , Transplant rejection, vasculitis or interstitial cystitis.
  • systemic lupus erythematosus rheumatoid arthritis, psoriatic arthritis, scleroderma, asthma, atopic dermatitis, organ-specific inflammatory diseases, allergies (such as allergic Rhinitis), folliculitis, tonsillitis, pneumonia, hepatitis, nephritis, acne, autoimmune disease
  • the twenty-sixth aspect of the present invention provides a method for treating tumors, the method comprising administering to an individual an effective amount of the Ryr2 overexpressing T cells of the present invention.
  • the twenty-seventh aspect of the present invention provides a method for treating tumors, the method is selected from the group consisting of reducing the expression of FoxP3 in T cells, increasing the expression of Ryr2 in T cells, increasing Ca 2+ basal oscillation, Improve the activity of m-Calpain, reduce the binding strength of T cells and DC cells or add Ryr2 driver.
  • the Ryr2 driver is selected from the group consisting of nicotinic acid amide adenine dinucleotide phosphate, caffeine, p-chlorom-cresol, ryanodine, chlorantraniliprole, cyantraniliprole, and type B adrenaline , 4-Chloro-3-methylphenol, cyantraniliprole, cyclotronamide, cyclic adenosine diphosphate ribose, suramin sodium, flutraniliprole or trifluoperazine.
  • the tumor may be any poor cell proliferation (or any disease that manifests itself as poor cell proliferation), neoplasm, or poor cell proliferation, neoplasm, or increased tendency or risk of tumor. It can be benign or malignant, or primary or secondary (metastatic).
  • a neoplasm can be any abnormal growth or proliferation of cells, and can be located in any tissue. Examples of tissues include adrenal gland, adrenal medulla, anus, appendix, bladder, blood, bone, bone marrow, brain, breast, cecum, central nervous system (including or excluding the brain), cerebellum, cervix, colon, duodenum, Endometrium, epithelial cells (e.g.
  • renal epithelial cells gallbladder, esophagus, glial cells, heart, ileum, jejunum, kidney, lacrimal gland, larynx, liver, lung, lymph, lymph nodes, lymphoblasts, maxilla, mediastinum , Mesenteric, myometrium, nasopharyngeal, omentum, oral cavity, ovary, pancreas, parotid gland, peripheral nervous system, peritoneum, pleura, prostate, salivary gland, sigmoid colon, skin, small intestine, soft tissue, spleen, stomach, testis, thymus, thyroid , Tongue, tonsils, trachea, uterus, vulva, white blood cells.
  • the tumor is selected from prostate cancer, breast cancer, liver cancer, glioma (such as glioma), bowel cancer, cervical cancer, non-small cell lung cancer, lung cancer, pancreatic cancer, gastric cancer, bladder cancer, Skin cancer, rhabdomyocarcinoma, tongue squamous cell carcinoma, nasopharyngeal carcinoma, ovarian cancer, placental choriocarcinoma, lymphoma (e.g.
  • non-Hodgkin’s lymphoma Hodgkin’s lymphoma, skin T-cell lymphoma), leukemia, rectal adenocarcinoma , Medulloblastoma, meningioma, neurofibromas (e.g. neurofibrosarcoma), ependymoma, schwannoma, astrocytoma, melanoma, mesothelioma, myeloma, chronic myelogenous leukemia, acute Myeloid leukemia, myelodysplastic syndrome, chronic lymphocytic leukemia, epidermoid carcinoma, colon cancer, thymic cancer, blood cancer, head and neck cancer, or oropharyngeal cancer.
  • neurofibromas e.g. neurofibrosarcoma
  • ependymoma schwannoma
  • astrocytoma melanoma
  • mesothelioma myeloma
  • the “regulation” in the present invention includes increasing (increasing) or decreasing (decreasing).
  • the "agent for regulating the expression of FoxP3” includes an agent for increasing the expression of FoxP3 or an agent for reducing the expression of FoxP3;
  • the "agent for regulating the expression of Ryr2” includes an agent for increasing the expression of Ryr2 or an agent for decreasing the expression of Ryr2.
  • treatment in the present invention includes, but is not limited to, slowing, interrupting, preventing, controlling, stopping, alleviating, or reversing the progress or severity of a sign, symptom, disorder, disease or disease, but does not necessarily involve all disease-related Complete elimination of signs, symptoms, symptoms, or disorders.
  • the "reagent” in the present invention represents general reagents, high-purity reagents, analytical reagents, instrumental analysis reagents, clinical diagnostic reagents, biochemical reagents, inorganic ion chromogenic reagents or reagents that can produce corresponding functions or achieve a certain purpose.
  • Device or equipment for example, the "agent for increasing the expression of FoxP3” includes any agent that can increase the expression of FoxP3 in T cells, such as agents that introduce FoxP3 gene into T cells, or agents that increase the transcription or expression of FoxP3 gene in T cells.
  • the "agent that reduces FoxP3 expression” includes any agent that can reduce FoxP3 expression in T cells, such as an agent that knocks out or knocks down the FoxP3 gene in T cells, or an agent that inhibits the transcription or expression of FoxP3 gene, and so on.
  • the "reagents for reducing Ryr2 expression” include, but are not limited to, reagents required to knock down or knock out the Ryr2 gene, or reagents that increase FoxP3 expression, or reagents that reduce Ryr2 transcription or expression; for example, the antagonist of the present invention , Rianodine, dantrolene or JTV519 and so on.
  • the "reagents for increasing Ryr2 expression” include, but are not limited to, reagents for introducing Ryr2 gene into T cells, reagents for increasing the transcription or expression of Ryr2 gene in T cells, or reagents for reducing FoxP3 expression in T cells, and the like.
  • the " agent for reducing Ca 2+ basal oscillation” includes, but is not limited to, an agent for reducing Ryr2 expression.
  • the “ agents that increase Ca 2+ basal oscillations” include, but are not limited to, agents that increase the expression of Ryr2.
  • the "agent that reduces the activity of m-Calpain” includes, but is not limited to, an agent that reduces the expression of Ryr2.
  • agents that increase the activity of m-Calpain include, but are not limited to, agents that increase the expression of Ryr2.
  • agents that increase the binding strength of T cells and DC cells include, but are not limited to, agents that reduce the expression of Ryr2.
  • agents that reduce the binding strength of T cells and DC cells include, but are not limited to, agents that increase the expression of Ryr2.
  • the "individual” in the present invention includes but is not limited to non-human mammals or humans.
  • the non-human mammals include but are not limited to monkeys, dogs, mice or rats.
  • the "effective amount” in the present invention refers to the amount or dose of the agent or drug of the present invention that provides the desired treatment after being administered to an individual or organ in a single or multiple doses.
  • the "CMAC” described in the present invention is a blue fluorescent dye with a chemical name of 7-amino-4-chloromethylcoumarin.
  • Figure 1 Analysis of the protein expression of m-Calpain in Tconv and Treg cells isolated from wild-type mice; Figures A and C are the mRNA levels of m-Calpain, and Figure 1B is the protein level of m-Calpain; all values are Mean value + SEM.
  • Figure 2 The basic oscillation of Ca 2+ in a single Tconv and Treg cell at rest.
  • FIG. 3 Mean fluorescence intensity (MFI) of Ca 2+ oscillations in Tconv and Treg at rest. Tconv and Treg cells were stained with Fluo 4-AM and analyzed for MFI.
  • Figure 4 The standard deviation (SD) of the Ca 2+ oscillation intensity shown in Figure 2.
  • FIG. 5 The GEO database analyzes the levels of calcium regulation-related proteins between Treg and Tconv, and ranks them according to the magnitude of the difference.
  • Figure 7 Determination of calpain activity in Tconv (left) and Treg (right) cells after Ryr activation (4-CMC treatment) by digestion with the calpain substrate CMAC.
  • Figure 8 Using 5mM JTV519 inhibitor to inhibit calcium ion basic oscillation in Tconv cells (middle panel), wild-type Tconv cells (left panel) and Treg cells (right panel) after Ryr2 was used.
  • Figure 9 The standard deviation of calcium ion basal oscillations in Tconv cells, wild-type Tconv cells and Treg cells after inhibiting Ryr2 with 5mM JTV519 inhibitor.
  • Figure 10 Using qPCR to analyze the mRNA transcription levels of Tconv cells isolated from WT mice and Tconv cells after shRNA knockdown of the Ryr2 gene.
  • FIG. 11 Compared with wild-type Tconv cells, Tconv cells after knocking down the Ryr2 gene using shRNA have Ca 2+ basal oscillations.
  • Figure 12 Standard deviation of Ca 2+ basal oscillation between Tconv cells and wild-type Tconv cells after knocking down the Ryr2 gene using shRNA.
  • FIG. 13 JTV519-treated Tconv (left), calpain activity in JTV519-treated Treg (middle) and Ryr2 knockdown Tconv (right).
  • Figure 14 Detection of adhesion based on T cell adhesion to DC2.4.
  • the left picture shows the average adhesion of untreated Treg, Tconv and Tconv treated with JTV519
  • the right picture shows the average adhesion of blank interference control (shCtrl) and Ryr2 knockdown Tconv.
  • Figure 15 Measurement of adhesion between OT-II T cells and DC2.4 cells presenting OVA;
  • Figure A shows the three-cell AFM analysis mode,
  • Figure B shows W/O cells (cell-free group), Blank interference control (shCtrl), Tconv knocked down by Ryr2, and Treg were used to inhibit the average adhesion of cells.
  • Figure 16 Inhibition of OT-II T cell division test results
  • Figure A is the FACS diagram of Tconv cells without Treg, Treg, blank interference control (shCtrl) or Ryr2 knockdown inhibiting the division of OT-II T cells
  • Figure B is The inhibition efficiency of blank interference control (shCtrl) or Ryr2 knockdown Tconv cells standardized by Treg (defined as 100% inhibition) and no Treg (defined as 0% inhibition); where, *, P ⁇ 0.05; **, P ⁇ 0.01; ***, P ⁇ 0.001; ****, P ⁇ 0.0001.
  • Figure 17 Analysis of Itpr1, Cacnb1, Trpm1, Trpm4, Trpv2, Orai1 and Orai3 gene shRNA knockdown efficiency in Tconv cells isolated from WT mice using qPCR.
  • FIG. 18 Untreated Treg cells, blank interference control Tconv (shCtrl), Ryr2 knockdown Tconv, Itpr1 knockdown Tconv, Cacnb1 knockdown Tconv, Trpm1 knockdown Tconv, Trpm4 knockdown Tconv, Trpv2 knockdown The average adhesion of low Tconv, Orai1 knockdown Tconv and Orai3 knockdown Tconv.
  • FIG. 19 FoxP3 overexpression silences Ryr2 gene transcription, specifically, the level of Ryr2 mRNA in Tconv, A20, 3T3 and Renca cells overexpressed by FoxP3.
  • FIG. 20 FoxP3 overexpression silences Ryr2 gene transcription.
  • Foxp3ChIP-seq Tconv transfected with Foxp3 (top) or untransfected with Foxp3 (bottom).
  • the arrow indicates the TSS site of the Ryr2 gene.
  • the triangle is the significantly different binding peak identified by ChIP-seq, and this position mainly overlaps with the promoter region (box) of the gene.
  • FIG. 21 FoxP3 overexpression silences Ryr2 gene transcription.
  • the figure above is a schematic diagram of the construction of Ryr2 promoter-luciferase reporter vector, TSS, transcription start site.
  • the figure below shows the transcription level driven by the Ryr2 promoter in 3T3 or Renca cells overexpressed by FoxP3.
  • Figure 22 FoxP3 overexpression silences Ryr2 gene transcription.
  • Figure A is a schematic diagram of the truncated vector of the Ryr2 promoter-luciferase reporter vector, which is the result of performing sequence segment deletion in the sequence, and
  • Figure B is the transcription level driven by the Ryr2 promoter in 3T3 overexpressed by FoxP3.
  • Figure 23 FoxP3 overexpression silences Ryr2 gene transcription, where Figure A is a schematic diagram of the two truncated vectors of the Ryr2 promoter-luciferase reporter vector and the knockout strategy, Figure B shows the difference in 3T3 cells overexpressed by FoxP3 There was no statistical difference in the level of transcription driven by the truncated vector or the Ryr2 promoter expressed by the FoxP3 binding sequence, NS.
  • Figure 24 FoxP3 overexpression silences Ryr2 gene transcription.
  • FIG 25 T cells that knock down Ryr2 gene (Ryr2-/-Tconv) induce immunosuppression in a mouse tinea dermatophyte (Scurfy) model and restore the immune homeostasis of Scurfy mice.
  • Figure A shows WT and after injection of PBS
  • Figure B shows the survival rate of mice injected with Treg, Ryr2+/+(Foxp3-)Tconv and Ryr2-/-Tconv, respectively.
  • Figure 26 Construction of conditional knockdown mice in which Cre relies on knockdown of exon 7 in the Ryr2 gene.
  • Figure 27 Flow cytometric detection of CD4+ and CD8+ T cells in Ryr2+/+ (wild type without Ryr2 knockout) mice, CD4-Cre/Ryr2fl/+ mice and CD4-Cre/Ryr2-/- mice Distribution in the spleen and thymus.
  • Figure 28 FoxP3+/CD4+ splenocyte ratio in mice.
  • Figure 29 Flow cytometric analysis of surface markers related to Treg function in WTTconv(Ryr2+/+) and Ryr2-/-Tconv (including CD25, GITR, CTLA-4, CD39, PD-1, LAG-3, TIM-3) . And ELISA to detect the levels of IL-10 and TGF- ⁇ in the supernatant after 24h anti-CD3/anti-CD28 stimulation.
  • FIG 30 T cells knocking down Ryr2 have an inhibitory function based on cell contact in vitro.
  • Tconv isolated from WT Figure A
  • CD4-Cre Figure B
  • Ryr2fl/fl(Ryr2-/-) mice were compared with Treg ( Figure C), and calcium ion oscillation was detected in 2mM Ca 2+ HBSS Level and its standard deviation (Figure D).
  • Figure 31 Detection of calpain activity in Treg, Ryr2+/+ and Ryr2-/-Tconv cells using CMAC substrate degradation.
  • Figure 32 Western blot reveals the results of in vivo substrate Talin degradation in the three cell types of Treg, Ryr2+/+ and Ryr2-/-Tconv.
  • Tconv knocking down Ryr2 has an inhibitory function based on cell contact in vitro; all data points from four independent DC-T cell pairs (points in Figures A and B) collected on the same day under each condition Used to generate the bar graph of Figure C, each T-DC pair collected at least 15 adhesion readings; specifically WT Tconv ( Figure A) and Ryr2-/-Tconv cells (Figure B) to DC2.4 Adhesion, and average adhesion ( Figure C).
  • Figure 34 Adhesion experiment between OT-II T cells and DC2.4 cells presenting OVA.
  • the suppressor cells on DC are Treg, WT Tconv or Ryr2-/-Tconv cells; where Figure A shows Three-cell AFM measurement device.
  • Figure B shows the average OT-II-DC adhesion of Treg, WT Tconv or Ryr2-/-Tconv as suppressor cells.
  • Figure 35 Relative inhibition efficiency of Treg-free, Treg, Ryr+/+ and Ryr2-/-Tconv, where Treg-free is 0% and Treg is 100%.
  • FIG. 36 T cells knocking down Ryr2 gene (Ryr2-/-Tconv) induce immunosuppression in a mouse herpes infection model.
  • the figure shows the analysis of HSV-1 titers in plantar tissues after adoptive transfer of cells, HSV-1 , Treg, Ryr2-/-Tconv or Ryr2+/+Tconv are adoptive transfer PBS, Treg or Ryr2-/-Tconv, Ryr2+/+Tconv, respectively.
  • FIG. 37 T cells with knockdown of Ryr2 gene (Ryr2-/-Tconv) induce immunosuppression in a murine herpes infection model.
  • the picture shows the delayed-type hypersensitivity after adoptive transfer of cells and re-attack with UV-inactivated HSV-1 antigen (DTH), where the thickness of the sole without antigen re-attack is set to 100%.
  • DTH UV-inactivated HSV-1 antigen
  • FIG. 38 T cells knocking down the Ryr2 gene (Ryr2-/-Tconv) induce immunosuppression in a murine herpes infection model.
  • the upper picture shows the experimental protocol in the DTH analysis, and the lower picture shows a representative plantar swelling.
  • FIG 39 T cells with knockdown of Ryr2 gene (Ryr2-/-Tconv) induce immunosuppression in a mouse asthma model.
  • Figure A shows the total infiltration of BALF
  • Figure B shows lymphocyte infiltration
  • Figure C shows eosinophils. infiltration.
  • the values of the measurement results are the average of 3 experiments.
  • the blank represents the blank control group.
  • the PBS, Treg, Ryr2-/-Tconv and Ryr2+/+Tconv groups are all asthma models injected with the same amount of PBS, Treg, Ryr2-/-Tconv And Ryr2+/+Tconv.
  • FIG 40 T cells that knock down Ryr2 gene (Ryr2-/-Tconv) induce immunosuppression in a mouse asthma model, where Figure A is the sensitization scheme of the asthma model, and Figure B is a representative lung H&E tissue section.
  • the scale is representative 200 ⁇ m, PBS, Treg, Ryr2-/-Tconv and Ryr2+/+Tconv groups were all injected with the same amount of PBS, Treg, Ryr2-/-Tconv and Ryr2+/+Tconv in the asthma model.
  • FIG 41 T cells knocking down the Ryr2 gene (Ryr2-/-Tconv) induce immunosuppression in a mouse colitis (IBD) model, where blank represents a blank control group, and DSS represents water treatment.
  • IBD mouse colitis
  • FIG 42 T cells knocking down the Ryr2 gene (Ryr2-/-Tconv) induce immunosuppression in a mouse colitis (IBD) model, where Figure A is the induction scheme of a DSS-induced colitis model, and Figure B is a representative Image of the colon. Figure C is a photomicrograph of a section of the colon. The scale bar represents 200 ⁇ m.
  • FIG 43 T cells knocking down the Ryr2 gene (Ryr2-/-Tconv) induce immunosuppression in a mouse colon cancer model.
  • the tumor volume growth curves of each group of mice were measured respectively.
  • Group 1 MC38+PBS;
  • Group 2 MC38+Ryr2+/+Tconv;
  • Group 3 MC38+Treg;
  • Group 4 MC38+Ryr2-/-Tconv.
  • FIG 44 T cells with knockdown of Ryr2 gene (Ryr2-/-Tconv) induce immunosuppression in a mouse dermatophyte (Scurfy) model and restore the immune homeostasis of Scurfy mice.
  • the picture shows the Kaplan-Meier survival curve of mice , Where, Figure A is the survival rate of mice after WT and PBS injection, and Figure B is the survival rate of mice injected with Treg, Ryr2+/+(Foxp3-)Tconv and Ryr2-/-Tconv, respectively.
  • FIG 45 T cells knocking down the Ryr2 gene (Ryr2-/-Tconv) induce immunosuppression in a mouse tinea dermatophyte (Scurfy) model and restore the immune homeostasis of Scurfy mice.
  • Figure A shows the body of Scurfy mice. Symptoms and treatment.
  • Figure B shows representative H&E staining of various organs (thymus, spleen, lung, ear, liver, pancreas, small intestine, colon). Model mice and WT mice injected with PBS. Samples were collected at the third week. The adoptively transferred Treg cells, Foxp3-(Ryr2+/+)Tconv and Ryr2-/-Tconv mice were collected at 8-12 weeks.
  • FIG. 46 Foxp3 overexpression inhibits Ryr2 is the key effect mechanism of T cell immunosuppression.
  • the left picture shows the T cell mechanism after Foxp3 overexpression inhibits Ryr2, and the right picture shows the wild type T cell mechanism.
  • mice All mice are of C57BL/6 background. Ryr2fl/fl mice and CAG-LSL-Ryr2-flag transgenic mice were produced by GemPharmatech Co., Ltd and were identified by genotype.
  • OT-II transgenic mice were purchased from Jackson Laboratory. Wild type, Foxp3-IRES-GFP, Ryr2fl/fl, Ryr2 transgene, OT-II transgene, CD4-Cre and Foxp3-Cre-YFP, female Foxp3+/- mice are raised in the Animal Center of Tsinghua University. All mice were raised under specific pathogen-free (SPF) conditions. All animal experiments were in compliance with animal welfare guidelines and were approved by the IACUC Animal Experiment Ethics Committee of Tsinghua University.
  • DC2.4 cells are a gift from Ken Rock UMass School of Medicine. Vero cells come from Dr. Xu Tan from the School of Pharmacy, Tsinghua University. Renca cells came from Dr. An Guangyu from Beijing Chaoyang Hospital. HEK293FT cells are a gift from Dr. Wei Guo from Tsinghua University School of Medicine.
  • MC38 cells were purchased from the American Type Culture Collection (ATCC). MC38, Renca, NIH-3T3 cells and Vero cells were cultured in DMEM containing 10% FBS, 100 U/mL penicillin, and 100 mg/mL streptomycin. All other cells were grown in RPMI-1640 with the same dose and 50 ⁇ M ⁇ -mercaptoethanol.
  • Mouse CD4+CD25+Treg and CD4+CD25-Tconv were isolated from the spleen using mouse CD4+T cell isolation kit (StemCell, 19852) and mouse CD25Treg cell positive selection kit (Stem cell, 18788). Treg and Tconv cells are sometimes sorted from Foxp3-IERS-GFP mouse CD4+ splenocytes by FACS.
  • Mouse CD11c selection kit II (Stem cell, 28007) was used to isolate mouse DC from the spleen.
  • OT-II T cells were isolated from OT-II spleen cells by the mouse CD4+ T cell isolation kit (Stem cell, 19852), and sometimes sorted by FACS with anti-TCRV ⁇ 2 antibody.
  • Antibodies and reagents All primary cell isolation kits are from StemCell. Recombinant human IL-2 comes from R&D systems. Anti-mouse CD3e monoclonal antibodies, anti-mouse CD28 and anti-mouse TCRV ⁇ 2-PE antibodies were from eBioscience. Flow cytometry antibodies are from eBioscience, including anti-CD4, anti-CD8, anti-GITR, anti-CD25, anti-PD-1, anti-CTLA-4, anti-Tim-3 and anti-LAG3 antibodies. Anti-FoxP3 antibody was from Invitrogen.
  • the following antibodies were used for Western blot analysis: m-Calpain large subunit (type M) antibody and anti-mouse IgG-HRP linked antibody were from CST.
  • Anti-talin antibody (8D4) was purchased from Sigma-Aldrich.
  • the pLVX-IRES-mcherry vector is a gift from Dr. Xiaohua Shen, School of Medicine, Tsinghua University.
  • Example 1 Preparation of T cells knocked down Ryr2 gene or FoxP3 overexpression
  • the shRNA based on lentivirus is used to knock down the Ryr2 gene. Specifically, the pre-synthesized shRNA sequence is inserted into the pLKO.1 vector.
  • the EndoFree Plasmid Midi Kit (CWBIO, CW2105S) was used to purify plasmids including shRNA and packaging constructs (pMD2.G and psPAX2) from transformed E. coli.
  • pMD2.G and psPAX2 The EndoFree Plasmid Midi Kit
  • 293FT cells were cultured in a 10cm dish and cultured to 60-80% coverage. Change the medium 2 hours before DNA transfection.
  • Neofect Using Neofect (Neofect), 2.5 ⁇ g packaging vector pMD2.G and 2.5 ⁇ gpsPAX2, 5 ⁇ g pLKO.1 vector (with inserted shRNA) were transfected into 293FT cells. After 72 hours, the lentivirus was harvested and T cells were infected with Polybrene (final concentration 8 ⁇ g/mL). 48 hours after virus infection, the cells were sorted by Aria cytometer (BD Biosciences). The knockdown efficiency was verified by real-time quantitative PCR.
  • the shRNA sequence is as follows:
  • Control-shRNA 5'-CCGGcaacaagatgaagagcaccaaCTCGAGttggtgctcttcatcttgttgTTTTTG-3' (SEQ ID NO: 3) and 5'-AATTCAAAAAcaacaagatgaagagcaccaaCTCGAGttggtgctgtcttcatcttgttgg-3' (SEQ ID NO: 3) and 5'-AATTCAAAAAcaacaagatgaagagcaccaaCTCGAGttggtgctgtcttcatcttgttgg-3' (SEQ ID TGTACCTAGCATTACCTTCATTACCTAGTTCAGTCCTAGTTCAGTCCTTCATTAGTACCTAGTTCAGTCCTACCTACCTAGTTCAGTCCTAGTTGCTACCTAGTTGCTACCGTTGCGTCCTACCTAGCCTTGCGTCCTAGTTCCTAGCATTACCTAGTTCCGT 5) and 5'-AATTCAAAAAAccgctaat
  • Orai1-shRNA 5'-CCGGcacaaccaccaactcggtcaaaCTCGAGtttgaccgagttgaggttgtgTTTTTG-3' (SEQ ID NO: 9) and
  • Trpm1-shRNA Trpm1-shRNA
  • the primer sequence is as follows:
  • Mouse GAPDH 5'-CATCACTGCCACCCAGAAGACTG-3' (SEQ ID NO: 21) and 5'-ATGCCAGTGAGCTTCCCGTTCAG-3' (SEQ ID NO: 22);
  • Mouse 18S RNA 5'-CGGACAGGATTGACAGATTG-3' (SEQ ID NO: 23) and 5'-CGGACAGGATTGACAGATTG-3' (SEQ ID NO: 24);
  • Mouse FoxP3, 5'-CCCATCCCCAGGAGTCTTG-3' SEQ ID NO: 27) and 5'-ACCATGACTAGGGGCACTGTA-3' (SEQ ID NO: 28);
  • Mouse m-Calpain 5'-3' and 5'-3' ; Rat Itpr1, 5'-CGTTTTGAGTTTGAAGGCGTTT-3'
  • lentiviruses produced by cell lines carrying pLVX-FoxP3-IRES-mcherry and FoxP3-overexpression was carried out according to the above-mentioned knockdown scheme.
  • the expression of FoxP3 was verified by RT-PCR.
  • Fc blocking agent CD16/32 antibody; 2.4G2
  • surface antibodies CD4, CD8, GITR, CD25, PD-1, CTLA-4, TIM-3, LAG3
  • Fc blocking agent CD16/32 antibody; 2.4G2
  • surface antibodies CD4, CD8, GITR, CD25, PD-1, CTLA-4, TIM-3, LAG3
  • FoxP3/transcription factor fixation/permeabilization buffer Thermo Fisher
  • PFA paraformaldehyde
  • Tregs 10 6 purified Tregs, Ryr2+/+ and Ryr2-/-Tconv were stimulated with anti-CD3/anti-CD28 antibody. After 72 hours, the supernatant was collected.
  • a high-binding 96-well ELISA plate (Nunc) was coated with anti-mouse IL-10 and anti-mouse TGF- ⁇ capture antibodies at 4°C overnight. After drying, the plate was blocked with 2% BSA in PBS for 1 hour at room temperature. After washing, add 100 uL of the diluted cell supernatant to triplicate wells, and then incubate for 1 hour at room temperature.
  • the plate was then washed with PBST (0.05% Tween20, Sigma-Aldrich, in PBS) and incubated with HRP-labeled goat anti-mouse IL-10 and TGF- ⁇ detection antibodies for 0.5 hours at room temperature.
  • TMB 100 mL/well was added, and the plate was incubated in the dark at room temperature for 10 minutes, and then H 2 SO 4 (50 uL, 1 M) was added to each well to stop the reaction.
  • ELISA microplate reader Bio-Rad
  • OD optical density
  • Treg, Tconv and A20 cells were stained with 2mM fluo-4AM (Thermo Fisher) in HBSS-HEPS (10mM) buffer at 37°C for 1 hour. After washing, the cells were adhered to a poly-L-lysine coating (0.1 mg/mL; Sigma-Aldrich) round glass slide installed in a sandwich self-made chamber at room temperature. After 15 minutes, rinse with buffer to remove excess non-adherent cells. For adherent cells, NIH-3T3 and Renca cells attached to glass slides were stained with 2mM fluo-4AM (Thermo Fisher) in HBSS-HEPS (10mM) buffer at 37°C for 1 hour.
  • 2mM fluo-4AM Thermo Fisher
  • the measurement chamber is then placed on an Olympus IX-73 microscope equipped with a 20x (numerical aperture: 0.8) or 40x (numerical aperture: 1.2) Olympus objective lens.
  • Ca 2+ oscillations were recorded for 20 minutes at 6 s intervals.
  • cells labeled with Fluo-4 were treated with JTV519 (Sigma) in the dark at room temperature for 30 minutes, and then images were acquired.
  • JTV519 Sigma
  • 5 minutes of Ca2+ oscillations were recorded at 1s intervals. 50 to 80 seconds after starting to acquire images, 4-CmC (Sigma) buffer was added to induce the release of intracellular calcium.
  • 4-CmC (Sigma) buffer was added to induce the release of intracellular calcium. Experiments were carried out in phenol red-free HBSS medium with or without Ca2+.
  • a charge-coupled device camera (ORCA-AG, Hamamatsu) was used to record the emission signal at 468-550nm excited by a 488nm laser. Data collection is controlled by NIS-Elements 3.0 software (Nikon). Use ImageJ to analyze the average fluorescence intensity change of a single cell over time and normalize it according to the fluorescence intensity of the first frame (Fluo-4F/F0). The Ca 2+ oscillation peak value of a single cell is displayed, and the standard deviation of the Ca 2+ fluctuation intensity is calculated as Mean ⁇ SD.
  • the cells were collected and lysed with RIPA buffer (Beyotime, P0013B). Centrifuge the cell lysate and collect the supernatant. The total protein was quantified with BCA protein assay kit (Beyotime, P0012). After mixing with 3XSDS loading buffer and boiling for 5 minutes, the protein was loaded on a 7.5% PAGE gel (EpiZyme, PG111). The protein was then transferred to the NC membrane and immunoblotted with the indicated primary and secondary antibodies. Finally, Super ECL detection reagent (Yeasen, 36208ES76) was used to detect the immunostained bands.
  • Ryr2 can be knocked down in Tconv of CD4-cre ( Figure 26). At the same time, knockdown of Ryr2 Tconv cells did not affect their basic functions, including the body weight and development speed of mice. And the distribution of CD4+ and CD8+ markers in thymocytes and peripheral blood is almost the same as that of WT mice (see Figure 27 for flow cytometry results). The proportion of FoxP3+CD4+ T cells also remained unchanged ( Figure 28). Compared with WT Tconv, the surface markers that may be related to Treg function also remain unchanged (see Figure 29 for flow cytometry and ELISA test results).
  • Example 2 Verify the effect of FoxP3 expression on Ryr2 gene, and determine the location of the target
  • this example established a relationship between it and FoxP3 expression and determined the location of the target.
  • FoxP3 was overexpressed in T cells, B cells (A20), 3T3 and Renca tumor lines (using the method of Example 1). In these four cases, FoxP3 overexpression significantly blocked Ryr2 gene transcription, indicating that FoxP3 spontaneously targets Ryr2 (Figure 19).
  • the ChIP-seq data is downloaded from the GEO data set.
  • the GEO ID is: Foxp3 in Tconv GSM989036, and FoxP3 in Tconv cells is transduced as an expression marker- Foxp3GSM989034.
  • IGV v2.4.14 uses the mouse reference genome mm8 to facilitate the visualization of ChIP-seq data. As shown in the screenshot at a specific gene locus. When using the IGV program to concentrate the data set in a 40 kb region around the Ryr2 gene, a strong signal from a 1.5 kb promoter region was detected on the untransfected control group ( Figure 20).
  • the FoxP3 binding site is: the GCAGGGG sequence repeated twice.
  • FoxP3 overexpression loses the ability to inhibit luciferase, indicating that it is the binding site of FoxP3 in the promoter ( Figure 23).
  • this example triggers the Ryr2-mediated Ca 2+ flux of 3T3 and A20 cells, including the FoxP3 overexpression group and the FoxP3 non-overexpression group.
  • Figure 24 shows that under the action of FoxP3, the Ca 2+ signal driven by 4-CmC in FoxP3 transfected cells is much smaller. It shows that Ryr2 is indeed directly controlled by FoxP3 and is a key channel for T cell inhibitory activity.
  • the dual luciferase report determination steps involved in this embodiment refer to the literature Identification and characterization of MAVS, a mitochondrial antiviral signaling protein that activates NF-kappaB and IRF 3 (Seth et al., Cell 122, 669-682, 2005) and LFA-1-mediated adhesion is regulated by cytoskeletal restraint and by a Ca2+-dependent protease, calpain (Stewart et al., J Cell Biol 140, 699-707, 1998).
  • the mouse Ryr2 reporter plasmid was constructed by subcloning the 1500bp Ryr2 promoter into the luciferase expression pGL3 vector.
  • Neofect (KS2000) was used to co-transfect 1.25 ⁇ 105 FoxP3 overexpressed -3T3, FoxP3 overexpressed Renca or FoxP3 overexpressed A20 cells with 300ng Ryr2 reporter gene and Renilla luciferase reporter gene plasmid. 36 hours after transfection, cell lysates were prepared and analyzed using the dual luciferase reporter assay system (Promega).
  • Example 3 The effect of inhibiting Ryr2 on the level of Ca 2+ in T cells
  • m-Calpain Due to the low activity of m-Calpain in Treg, this example predetermines whether its expression level is low. At the same time, m-Calpain is regulated by the availability of intracellular Ca 2+. Therefore, the Ca 2+ signals of Tconv and Treg in the resting state were detected.
  • GEO database analysis According to three RNA-Seq studies published in NCBI GEO database (GSE71162), proteins are classified according to their expression levels. Sort calcium-related proteins by GO analysis. The functions of these proteins are annotated based on UniProt (http://www.uniprot.org/). The proteins are ranked by the degree of expression difference between Treg and Tconv.
  • Ryr receptors are located on the er membrane (Ryr1, 2 and 3). In human PBMC, Ryr1 is expressed in the CD19+ population, and Ryr2 is expressed in the CD3+ part. In order to confirm the low expression of Ryr2 in Treg, stimulating factor 4-CMC was used to observe the digestibility of CMAC (calpain substrate). The results are shown in Figure 7. 4-CMC can completely induce Calpain activity, while Treg is not sensitive to this treatment, confirming that the expression of Ryr2 in Treg is reduced.
  • This example further suppress variations verification signal after Ryr2 Ca 2+, the results shown in FIG. 8 and 9, either standard or Ca 2+ peak difference signal, after suppressing Ryr2 Tconv Treg cells are similar effect, and the relatively In Tconv cells that did not inhibit Ryr2, the calcium level was significantly reduced.
  • the lower Ca 2+ levels of T cell subsets are basically controlled by the transcriptional inhibition of Ryr2.
  • calpain activity is measured.
  • 200 ⁇ L 10 5 T cells in medium containing 20 ⁇ M calpain substrate CMAC t-BOC-Leu- Met, Thermo Fisher
  • CMAC t-BOC-Leu- Met, Thermo Fisher
  • the fluorescent signal emitted from the digested substrate was measured as calpain activity by a Fortech flow cytometer through the Hoechst blue channel.
  • the inhibitor JTV519 was added 30 minutes before the experiment, and the activators 4-CmC and CMAC were added at the same time.
  • the in vitro inhibitory function The purified OTII T cells were stained with CellTrace CFSE (Thermo Fisher Scientifc). Spleen cells from the purified 10 4 DC, 2 ⁇ 10 4 OTII T cells, 2 ⁇ 10-Treg or Ryr2 - / - knock Ryr2 or low Tconv Tconv 2 ⁇ M OVA323-339 peptide and mixed in a 96-well U bottom plate and Cultured in each well, and assessed the proliferation of OTII T cells by CFSE dilution by a Fortessa flow cytometer. The inhibition percentage was calculated from (1-proliferation%), and then the data was normalized by setting the Treg-free group as 0% inhibition and the Treg group as 100% inhibition.
  • the single-cell force spectroscopy procedure based on the atomic force microscope was as described above, and the experiment was carried out using the JPK CellHesion device (see reference: Alum interaction with dendritic cell membrane lipids is essential for its adjuvanticity (Flach et al., Nat Med 17, 479- 487, 2011) and reference: Strong adhesion by regulatory T cells induces dendritic cell cytoskeletal polarization and contact-dependent lethargy (Chen et al., The Journal of experimental medicine, 2017)).
  • DC2.4 cells were cultured on untreated glass dishes. T cells were treated with 200U/mL human IL-2 overnight.
  • the disk is moved into an AFM compatible chamber and installed on the machine table. Coat the clean cantilever with CellTak (BD) and paste it onto a single T cell on the disk. Lower the AFM cantilever carrying a single T cell, make it contact and interact with a single DC for 15s, and then move upward until the two cells are completely separated. Obtain the force curve. Then repeat the process.
  • the DC2.4 cells cultured on the glass dish were treated with 100 ⁇ g/mL soluble OVA protein for 4h before the experiment.
  • Treg/Tconv cells treated with IL-2 were stained with 10 ⁇ M CFSE, and then DC2.4 cells were incubated with these fluorescently labeled Treg or Tconv cells for about 20 minutes, and then unlabeled OT-II T cells were added.
  • the Treg/Tconv cell-DC pair identified by the ultraviolet flash lamp is then approached through the tip of the cantilever with OT-II T cells.
  • the adhesion inhibition of OT-II-DC mediated by Treg/T transformation was analyzed. In each cycle, the AFM cantilever with a single T cell is lowered in 0.5–2 ⁇ m increments until the first force curve is generated.
  • the T cell on the cantilever interacts with the DC for 15s, and then moves upward until the two cells are completely separated. Place the incubator equipped with the machine at 37°C and 5% CO 2 . In all experiments, at least 14 force curves were collected for further analysis. Use JPK image processing software to process the force curve. Only round and strong cells are selected for AFM gluing. For each SCFS experiment, within a few minutes, a pair of T-DCs are used to generate force readings for each upper and lower cycle. Use at least three pairs of data for each condition.
  • the right sole was re-excited with UV-inactivated HSV-1 (10 6 pfu/20 ⁇ L PBS) at 6 dpi, and then the left sole was used as a control to measure plantar swelling at 7 dpi. 16-21 mice per group.
  • Example 6 The role of Ryr2-/-T cells in a mouse asthma model
  • Ovalbumin induces airway inflammation.
  • OVA Ovalbumin
  • alum adjuvant 100 ⁇ g+4mg
  • intratracheal OVA shocks were repeated at the back of the tongue.
  • Iv by adoptive transfer of Treg 10 6, Ryr2 - / - Tconv, Ryr2 + / + Tconv or PBS.
  • BALF infiltration and histology were analyzed on the 32nd day.
  • the skin, ears, liver and other tissues were all fixed in 4% nerve buffered formalin at a temperature of 4°C. Then embed the sample in paraffin. Cut into five to six micron thick glass slides. All slides were stained with hematoxylin and eosin.
  • Example 7 The role of Ryr2-/-T cells in a mouse colitis model
  • DSS-induced mouse experimental colitis model can refer to the literature Exhibition of Dectin-1 Signaling Ameliorates Colitis by Inducing Lactobacillus-Mediated Regulatory T Cell Expansion in the Intestine (Tang et al., Cell Host Microbe 18, 183-197, 2015) and Myeloid- Derived Suppressor Cells Are Controlled by Regulatory T Cells via TGF-beta during Murine Colitis (Lee et al., Cell Rep 17, 3219-3232, 2016).
  • iv adoptive transfer of 3 ⁇ 10 6 Ryr2-/-Tconv wild-type Tconv or Treg was transferred to 6-week-old male C57BL/6 mice.
  • the skin, ears, liver and other tissues were all fixed in 4% nerve buffered formalin at a temperature of 4°C.
  • the sample was then embedded in paraffin wax. Cut into five to six micron thick glass slides. All slides were stained with hematoxylin and eosin.
  • Example 8 The role of Ryr2-/-T cells in a mouse colon cancer model
  • the transplanted tumor mouse model can be established by referring to the literature Oxidative stress controls regulatory T cell apoptosis and suppressor activity and PD-L1-blockade resistance in tumor (Maj et al., Nat Immunol18, 1332-1341, 2017).
  • MC38 colon cancer cells were washed twice in PBS, and then 5 ⁇ 10 5 MC38 cells and 10 6 T cells in 200 ⁇ L of PBS were subcutaneously injected into the abdomen subcutaneously of 6-week-old male C57BL/6 mice. From the 7th day after tumor injection Use vernier calipers to monitor tumor growth. The volume is calculated as (length ⁇ width ⁇ width)/2.
  • Group 1 MC38+PBS
  • Group 2 MC38+Ryr2+/+Tconv
  • Group 3 MC38+Treg
  • Group 4 MC38+Ryr2-/-Tconv.
  • Example 9 Restoring immune homeostasis effect of Ryr2-/-T cells in mouse skin tinea model
  • Ryr2-/-Tconv cells play the most obvious role in general inflammation caused by multi-organ autoimmunity. For this reason, this example constructs a Foxp3-deficient mouse model to verify that Foxp3-deficient immunodeficient mice are in Ryr2-/-Tconv Restoration of immune homeostasis under cell regulation.
  • adoptive transfer of T cells is performed every three or four days, and then every two weeks.
  • the body weight of the tinea rash model and male WT litters were recorded, and the survival rate was monitored. All mice were sampled on the day of birth, the sf mutant gene was genotyped by PCR, and the genotype was verified by sequencing.
  • the primers of FoxP3PCR are 5'-CATCCCACTGTGACGAGATG-3' (SEQ ID NO: 1) and 5'-ACTTGGAGCACAGGGGTCT-3' (SEQ ID NO: 2).
  • PBS-infused mice were analyzed at week 3, and mice with adoptive transfer of Treg and Ryr2-/-Tconv were analyzed at week 8-12.
  • the skin, ears, liver and other tissues were all fixed in 4% nerve buffered formalin at a temperature of 4°C.
  • the sample was then embedded in paraffin wax. Cut into five to six micron thick glass slides. All slides were stained with hematoxylin and eosin.
  • tinea dermatoses mice (FoxP3-/-, C57BL/6) were injected with PBS, Treg, Ryr2+/+Tconv or Ryr2-/-Tconv, respectively.
  • PBS PBS
  • Treg PBS
  • Ryr2+/+Tconv a mice injected with PBS
  • Treg PBS
  • Ryr2-/-Tconv survived for at least 20 weeks ( Figures 44, 25).
  • mice injected with PBS or Ryr2+/+Tconv showed severe thyroiditis, splenitis, pneumonia, dermatitis, hepatitis, pancreatitis, gastritis, and colitis.
  • the mice injected with Treg or Ryr2-/-Tconv were completely treated for these pathologies, that is, the loss of the combined effect of FoxP3-deficient mice was restored, indicating that Ryr2 regulates the immune function of T cells.
  • Ryr2-/-T and Treg cells have shown functional equivalence in mouse herpes infection models, asthma models, colitis models and colon cancer models, and can also reduce the immune deficiency The mouse regained its immune function.
  • Ryr2-/-T can all play the same role.

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Abstract

提供了一种缺失Ryr2基因的T细胞及制备方法。还提供了Ryr2的拮抗剂，过表达Ryr2的T细胞，调控Ryr2表达、调控Ca2+基础振荡、调控m-Calpain活性、提高T细胞与DC细胞的结合强度的方法及应用，实现该功能的试剂在制备治疗感染性疾病、炎症或肿瘤的药物中的应用，以及将Tconv细胞转化为与Treg细胞类似功能的方法。

Description

一种T细胞及其制备方法和应用 技术领域
本发明涉及生物技术领域，具体涉及一种T细胞及其制备方法和应用，以及调控Ryr2表达的试剂、调控Ca 2+基础振荡的试剂、调控m-Calpain活性的试剂、调控T细胞与DC细胞的结合强度的试剂在制备治疗感染性疾病、炎症或肿瘤的药物中的应用。
背景技术
调节性T细胞(Treg)是抑制性T细胞的一种功能亚群，天然的Treg细胞于1995年由Sakaguchi等首次报道，它们约占外周血CD4+T细胞数的5％-10％，FoxP3是天然的Treg的标志。调节性T细胞可分为两类：天然Treg(n Treg)和获得性Treg(a Treg)。
Treg细胞的抑制机制是一个研究热点。参考文献：Past,Present,and Future of Regulatory T Cell Therapy in Transplantation and Autoimmunity(Romano et al.，Frontiers in Immunology，2019)公开的机制，包括T细胞溶解、表面蛋白提取、腺苷的局部生成等，都需要Treg结合。近年来，一系列的研究报告将研究重点转移到直接抑制树突细胞上。主要理由在于：一方面，在CD4+T细胞的数量上，Treg细胞远远超过了树突状细胞，但与树突状细胞相比，Treg细胞在数量上仍然占有优势，在体内的比例大约为2:1，这就推断了抑制能力的更合理和更公正的使用。其次，DC/Treg直接结合在体内和体外都很常见。虽然这种约束被认为是对DC的压制，但在一种确切的操作方式方面尚未达成一致意见。参考文献：Trans-endocytosis of CD80 and CD86:a molecular basis for the cell-extrinsic function of CTLA-4(Qureshi et al.，Science 332，2011)中Sakaguchi的团队提出，这主要是通过CTLA-4在Treg上表达的胞吞作用实现的。Shevach在最近的一篇论文中提出，MHC II类提取在抑制抗原递呈中的DC中发挥了重要作用。
然而，本申请发明人认为Treg与DC之间的结合是通过LFA-1和ICAM-1介导的，结合本身异常强烈，以致于DC细胞骨架被严重扭曲，无法支持抗原呈递给其他T细胞(参 考文献：Strong adhesion by regulatory T cells induces dendritic cell cytoskeletal polarization and contact-dependent lethargy(Chen et al.，The Journal of experimental medicine，2017))。随着时间的推移，这些差异可能会得到解决，从而产生一个关于Treg如何通过接触抑制DC的更精确和全面的图像。然而，为了继续讨论，有一个基本的问题需要回答：为什么Treg将DC以如此强大的力量绑定在一起，以及这种过度的力量如何影响DC。
Ryr2是内质网/肌浆网上(ER/SR)的一种钙释放通道，现有技术公开了Ryr2基因与心源性猝死、心律失常、冠心病相关，例如：
非专利文献：Ryr2基因新突变致心源性猝死一家系研究(申童等，中西医结合心血管病杂志，2019.02)公开了携带Ryr2基因上的错义突变c.G4107C均存在劳动性呼吸困难、胸痛等症状，即Ryr2基因上的错义突变可能与心源性猝死相关。
非专利文献：CASQ2和Ryr2在二乙酰吗啡致心肌细胞节律异常中的作用(胡夏韵等，新疆医科大学学报，2019.03)公开了Ryr2与钙通道之间的关系，以及明确其参与心肌细胞钙离子通道异常及心律失常的过程。
非专利文献：Ryr2基因表达与冠心病心气虚证发病相关性的理论探讨(马月香等，中医药信息，2012)公开了Ryr2功能失调可导致心功能下降，与中医学心主血脉，心气是推动血液运行的基本动力，心气亏虚导致心功能减退相吻合；从Ryr2基因表达可进一步揭示心气虚证的证候实质。
非专利文献：卡维地洛对Ryr2介导的自发性钙振荡的抑制作用(肖建民等，中国病理生理杂志，2013)公开了卡维地洛可以抑制Ryr2介导的自发性钙振荡，并表明其在降低心衰死亡率方面优于其他β受体阻滞剂。
但是上述现有技术中并没有公开Ryr2与T细胞及感染性疾病、炎症或肿瘤的相关性。
发明内容
本发明为弥补现有技术的空白，找到了Treg细胞功能的调控开关-Ryr2，并获得FoxP3 过表达可以降低Ryr2的表达的技术，而且具体确定了靶向位置。同时，经大量试验证实，敲低或敲除T细胞中的Ryr2使CD4+T细胞池成为免疫抑制细胞，获得的T细胞具有类似于Treg细胞的功能。进一步的，通过对Ryr2的调控，治疗病毒感染、哮喘、过敏、结肠炎和肿瘤均取得了很好的效果，并恢复皮癣小鼠相关系统的自身免疫。
具体的，本发明的第一方面，提供了一种T细胞，所述的T细胞中缺失Ryr2基因或过表达FoxP3。
优选的，所述的T细胞中至少缺失Ryr2基因的7号外显子。
优选的，所述的T细胞中至少缺失Ryr2基因中的一段富含鸟嘌呤的序列。
优选的，所述的一段富含鸟嘌呤的序列位于Ryr2基因的起始密码子前200bp中。
优选的，所述的一段富含鸟嘌呤的序列为至少包含4个鸟嘌呤(G)的核苷酸序列。进一步优选的，至少包含4-20个鸟嘌呤(G)的核苷酸。
在本发明的一个具体实施方式中，至少包含4、5、6、7、8、9、10、11、12、13、14、15等等。
优选的，所述的一段富含鸟嘌呤的序列包含GCAGGGG。
优选的，所述的T细胞选自细胞毒T细胞、辅助T细胞、调节/抑制T细胞(Treg)或记忆T细胞。
在本发明的一个具体实施方式中，所述的T细胞为Tconv细胞。
本发明的第二方面，提供了一种本发明所述的T细胞的制备方法，所述的制备方法选自采用shRNA、siRNA、CRISPR/Cas9、锌指核酸酶技术、转录激活子样效应因子核酸酶技术或归巢核酸内切酶。
优选的，所述的shRNA为SEQ ID NO：5、6。
本发明的第三方面，提供了一种shRNA，所述的shRNA敲低T细胞的Ryr2基因，使得T细胞Ca 2+基础振荡降低、m-Calpain(m-钙蛋白酶)活性降低、与DC细胞的结合强度提高、具有免疫抑制细胞的功能或具有治疗感染性疾病、炎症或肿瘤的功能。
优选的，所述的shRNA为SEQ ID NO：5、6。
本发明的第四方面，提供了一种T细胞，所述的T细胞中过表达Ryr2。
优选的，所述的T细胞选自细胞毒T细胞、辅助T细胞、调节/抑制T细胞(Treg)或记忆T细胞。
在本发明的一个具体实施方式中，所述的T细胞为Treg细胞。
本发明的第五方面，提供了调控FoxP3表达的试剂、调控Ryr2表达的试剂、调控Ca 2+基础振荡的试剂、调控m-Calpain活性的试剂或调控T细胞与DC细胞的结合强度的试剂在制备治疗感染性疾病、炎症或肿瘤的药物中的应用。
优选的，所述的感染性疾病选自细菌感染、病毒感染或真菌感染；进一步优选为病毒感染、肺炎伴感染性休克、腹膜炎、菌血症、脓毒症或败血症；所述的病毒感染选自急性病毒感染或慢性病毒感染；优选为流感病毒、副流感病毒、疱疹病毒(例如HSV-1、EBV)、麻疹病毒、水泡口炎病毒、乙型肝炎病毒、丙型肝炎病毒、人免疫缺陷病毒、淋巴细胞脉络丛脑膜炎病毒或人乳头瘤病毒。
优选的，所述的炎症可以是任何组织出现的炎症，所述的组织包括但不限于肾上腺、肾上腺髓质、肛门、阑尾、膀胱、血液、骨、骨髓、脑、乳腺、盲肠、中枢神经系统(包括或排除大脑)、小脑、子宫颈、结肠、十二指肠、子宫内膜、上皮细胞(例如肾上皮细胞)、胆囊、食道、神经胶质细胞、心脏、回肠、空肠、肾、泪腺、喉、肝、肺、淋巴、淋巴结、淋巴母细胞、上颌骨、纵隔、肠系膜、子宫肌层、鼻咽、网膜、口腔、卵巢、胰腺、腮腺、周围神经系统、腹膜、胸膜、前列腺、唾液腺、乙状结肠、皮肤、小肠、软组织、脾、胃、睾丸、胸腺、甲状腺、舌、扁桃体、气管、子宫、外阴、白细胞。进一步优选的，所述的炎症选自系统性红斑狼疮、类风湿性关节炎、银屑病关节炎、硬皮病、哮喘、特应性皮炎、器官特异性炎性疾病、过敏(例如过敏性鼻炎)、毛囊炎、扁桃体炎、肺炎、肝炎、肾炎、痤疮、自身免疫性疾病、慢性前列腺炎、肾小球肾炎、超敏反应、结肠炎、炎性肠道疾病、盆腔炎、再灌注损伤、移植排斥反应、血管炎或间质性膀胱炎。
优选的，所述的肿瘤可以是任何不良的细胞增殖(或本身表现为不良细胞增殖的任何疾病)、赘生物，或不良细胞增殖、赘生物或肿瘤的倾向性或风险增加。其可以是良性或恶性的，也可以是原发性或继发性(转移性)。赘生物可以是细胞的任何异常生长或增殖，并且可以位于任何组织中。组织的实例包括肾上腺、肾上腺髓质、肛门、阑尾、膀胱、血液、骨、骨髓、脑、乳腺、盲肠、中枢神经系统(包括或排除大脑)、小脑、子宫颈、结肠、十二指肠、子宫内膜、上皮细胞(例如肾上皮细胞)、胆囊、食道、神经胶质细胞、心脏、回肠、空肠、肾、泪腺、喉、肝、肺、淋巴、淋巴结、淋巴母细胞、上颌骨、纵隔、肠系膜、子宫肌层、鼻咽、网膜、口腔、卵巢、胰腺、腮腺、周围神经系统、腹膜、胸膜、前列腺、唾液腺、乙状结肠、皮肤、小肠、软组织、脾、胃、睾丸、胸腺、甲状腺、舌、扁桃体、气管、子宫、外阴、白细胞。进一步优 选的，所述的肿瘤选自前列腺癌、乳腺癌、肝癌、胶质瘤(例如神经胶质瘤)、肠癌、宫颈癌、非小细胞肺癌、肺癌、胰腺癌、胃癌、膀胱癌、皮肤癌、横纹肌癌、舌鳞癌、鼻咽癌、卵巢癌、胎盘绒毛癌、淋巴瘤(例如非霍奇金淋巴瘤、霍奇金淋巴瘤、皮肤T细胞淋巴瘤)、白血病、直肠腺癌、成神经管细胞瘤、脑膜瘤、神经纤维瘤(例如神经纤维肉瘤)、室管膜瘤、神经鞘瘤、星形细胞瘤、黑色素瘤、间皮瘤、骨髓瘤、慢性粒细胞白血病、急性髓性白血病、骨髓增生异常综合征、慢性淋巴细胞白血病、表皮样癌、结肠癌、胸腺癌、血液癌、头颈癌或口咽癌。
本发明的第六方面，提供了调控FoxP3表达的试剂、调控Ryr2表达的试剂、调控Ca 2+基础振荡的试剂、调控m-Calpain活性的试剂、调控T细胞与DC细胞的结合强度的试剂或本发明所述的T细胞在治疗感染性疾病、炎症或肿瘤中的应用。
本发明的第七方面，提供了调控FoxP3表达的试剂、调控Ryr2表达的试剂、调控Ca 2+基础振荡的试剂、调控m-Calpain活性的试剂、调控T细胞与DC细胞的结合强度的试剂或本发明所述的T细胞在制备治疗感染性疾病、炎症或肿瘤的药物中的应用。
本发明的第八方面，提供了调控FoxP3表达、调控Ryr2表达、调控Ca 2+基础振荡、调控m-Calpain活性、调控T细胞与DC细胞的结合强度或本发明所述的T细胞在治疗感染性疾病、炎症或肿瘤或者在制备治疗感染性疾病、炎症或肿瘤的药物中的应用。
优选的，所述的感染性疾病选自细菌感染、病毒感染或真菌感染；进一步优选为病毒感染、肺炎伴感染性休克、腹膜炎、菌血症、脓毒症或败血症；所述的病毒感染选自急性病毒感染或慢性病毒感染；优选为流感病毒、副流感病毒、疱疹病毒(例如HSV-1、EBV)、麻疹病毒、水泡口炎病毒、乙型肝炎病毒、丙型肝炎病毒、人免疫缺陷病毒、淋巴细胞脉络丛脑膜炎病毒或人乳头瘤病毒。
优选的，所述的炎症可以是任何组织出现的炎症，所述的组织包括但不限于肾上腺、肾上腺髓质、肛门、阑尾、膀胱、血液、骨、骨髓、脑、乳腺、盲肠、中枢神经系统(包括或排除大脑)、小脑、子宫颈、结肠、十二指肠、子宫内膜、上皮细胞(例如肾上皮细胞)、胆囊、食道、神经胶质细胞、心脏、回肠、空肠、肾、泪腺、喉、肝、肺、淋巴、淋巴结、淋巴母细胞、上颌骨、纵隔、肠系膜、子宫肌层、鼻咽、网膜、口腔、卵巢、胰腺、腮腺、周围神经系统、腹膜、胸膜、前列腺、唾液腺、乙状结肠、皮肤、小肠、软组织、脾、胃、睾丸、胸腺、甲状腺、舌、扁桃体、气管、子 宫、外阴、白细胞。进一步优选的，所述的炎症选自系统性红斑狼疮、类风湿性关节炎、银屑病关节炎、硬皮病、哮喘、特应性皮炎、器官特异性炎性疾病、过敏(例如过敏性鼻炎)、毛囊炎、扁桃体炎、肺炎、肝炎、肾炎、痤疮、自身免疫性疾病、慢性前列腺炎、肾小球肾炎、超敏反应、结肠炎、炎性肠道疾病、盆腔炎、再灌注损伤、移植排斥反应、血管炎或间质性膀胱炎。
优选的，所述的肿瘤可以是任何不良的细胞增殖(或本身表现为不良细胞增殖的任何疾病)、赘生物，或不良细胞增殖、赘生物或肿瘤的倾向性或风险增加。其可以是良性或恶性的，也可以是原发性或继发性(转移性)。赘生物可以是细胞的任何异常生长或增殖，并且可以位于任何组织中。组织的实例包括肾上腺、肾上腺髓质、肛门、阑尾、膀胱、血液、骨、骨髓、脑、乳腺、盲肠、中枢神经系统(包括或排除大脑)、小脑、子宫颈、结肠、十二指肠、子宫内膜、上皮细胞(例如肾上皮细胞)、胆囊、食道、神经胶质细胞、心脏、回肠、空肠、肾、泪腺、喉、肝、肺、淋巴、淋巴结、淋巴母细胞、上颌骨、纵隔、肠系膜、子宫肌层、鼻咽、网膜、口腔、卵巢、胰腺、腮腺、周围神经系统、腹膜、胸膜、前列腺、唾液腺、乙状结肠、皮肤、小肠、软组织、脾、胃、睾丸、胸腺、甲状腺、舌、扁桃体、气管、子宫、外阴、白细胞。进一步优选的，所述的肿瘤选自前列腺癌、乳腺癌、肝癌、胶质瘤(例如神经胶质瘤)、肠癌、宫颈癌、非小细胞肺癌、肺癌、胰腺癌、胃癌、膀胱癌、皮肤癌、横纹肌癌、舌鳞癌、鼻咽癌、卵巢癌、胎盘绒毛癌、淋巴瘤(例如非霍奇金淋巴瘤、霍奇金淋巴瘤、皮肤T细胞淋巴瘤)、白血病、直肠腺癌、成神经管细胞瘤、脑膜瘤、神经纤维瘤(例如神经纤维肉瘤)、室管膜瘤、神经鞘瘤、星形细胞瘤、黑色素瘤、间皮瘤、骨髓瘤、慢性粒细胞白血病、急性髓性白血病、骨髓增生异常综合征、慢性淋巴细胞白血病、表皮样癌、结肠癌、胸腺癌、血液癌、头颈癌或口咽癌。
本发明的第九方面，提供了提高FoxP3表达的试剂、降低Ryr2表达的试剂、降低Ca 2+基础振荡的试剂、降低m-Calpain活性的试剂、提高T细胞与DC细胞的结合强度的试剂或上述缺失Ryr2基因的T细胞在制备治疗感染性疾病或炎症的药物中的应用。
本发明的第十方面，提供了提高FoxP3表达的试剂、降低Ryr2表达的试剂、降低Ca 2+基础振荡的试剂、降低m-Calpain活性的试剂、提高T细胞与DC细胞的结合强度的试剂或上述缺失Ryr2基因的T细胞在治疗感染性疾病或炎症中的应用。
本发明的第十一方面，提供了提高FoxP3表达、降低Ryr2表达、降低Ca 2+基础 振荡、降低m-Calpain活性或提高T细胞与DC细胞的结合强度在治疗感染性疾病、炎症或在制备治疗感染性疾病、炎症的药物中的应用。
优选的，所述的感染性疾病选自细菌感染、病毒感染或真菌感染；进一步优选为病毒感染、肺炎伴感染性休克、腹膜炎、菌血症、脓毒症或败血症；所述的病毒感染选自急性病毒感染或慢性病毒感染；优选为流感病毒、副流感病毒、疱疹病毒(例如HSV-1、EBV)、麻疹病毒、水泡口炎病毒、乙型肝炎病毒、丙型肝炎病毒、人免疫缺陷病毒、淋巴细胞脉络丛脑膜炎病毒或人乳头瘤病毒。
优选的，所述的炎症可以是任何组织出现的炎症，所述的组织包括但不限于肾上腺、肾上腺髓质、肛门、阑尾、膀胱、血液、骨、骨髓、脑、乳腺、盲肠、中枢神经系统(包括或排除大脑)、小脑、子宫颈、结肠、十二指肠、子宫内膜、上皮细胞(例如肾上皮细胞)、胆囊、食道、神经胶质细胞、心脏、回肠、空肠、肾、泪腺、喉、肝、肺、淋巴、淋巴结、淋巴母细胞、上颌骨、纵隔、肠系膜、子宫肌层、鼻咽、网膜、口腔、卵巢、胰腺、腮腺、周围神经系统、腹膜、胸膜、前列腺、唾液腺、乙状结肠、皮肤、小肠、软组织、脾、胃、睾丸、胸腺、甲状腺、舌、扁桃体、气管、子宫、外阴、白细胞。进一步优选的，所述的炎症选自系统性红斑狼疮、类风湿性关节炎、银屑病关节炎、硬皮病、哮喘、特应性皮炎、器官特异性炎性疾病、过敏(例如过敏性鼻炎)、毛囊炎、扁桃体炎、肺炎、肝炎、肾炎、痤疮、自身免疫性疾病、慢性前列腺炎、肾小球肾炎、超敏反应、结肠炎、炎性肠道疾病、盆腔炎、再灌注损伤、移植排斥反应、血管炎或间质性膀胱炎。
本发明的第十二方面，提供了降低FoxP3表达的试剂、提高Ryr2表达的试剂、提高Ca 2+基础振荡的试剂、提高m-Calpain活性的试剂、降低T细胞与DC细胞的结合强度的试剂或上述过表达Ryr2的T细胞在制备治疗肿瘤的药物中的应用。
本发明的第十三方面，提供了降低FoxP3表达的试剂、提高Ryr2表达的试剂、提高Ca 2+基础振荡的试剂、提高m-Calpain活性的试剂、降低T细胞与DC细胞的结合强度的试剂或上述过表达Ryr2的T细胞在治疗肿瘤中的应用。
本发明的第十四方面，提供了降低FoxP3表达、提高Ryr2表达、提高Ca 2+基础振荡、提高m-Calpain活性、降低T细胞与DC细胞的结合强度在治疗肿瘤或制备治疗肿瘤的药物中的应用。
优选的，所述的肿瘤可以是任何不良的细胞增殖(或本身表现为不良细胞增殖的任何疾病)、赘生物，或不良细胞增殖、赘生物或肿瘤的倾向性或风险增加。其可以 是良性或恶性的，也可以是原发性或继发性(转移性)。赘生物可以是细胞的任何异常生长或增殖，并且可以位于任何组织中。组织的实例包括肾上腺、肾上腺髓质、肛门、阑尾、膀胱、血液、骨、骨髓、脑、乳腺、盲肠、中枢神经系统(包括或排除大脑)、小脑、子宫颈、结肠、十二指肠、子宫内膜、上皮细胞(例如肾上皮细胞)、胆囊、食道、神经胶质细胞、心脏、回肠、空肠、肾、泪腺、喉、肝、肺、淋巴、淋巴结、淋巴母细胞、上颌骨、纵隔、肠系膜、子宫肌层、鼻咽、网膜、口腔、卵巢、胰腺、腮腺、周围神经系统、腹膜、胸膜、前列腺、唾液腺、乙状结肠、皮肤、小肠、软组织、脾、胃、睾丸、胸腺、甲状腺、舌、扁桃体、气管、子宫、外阴、白细胞。进一步优选的，所述的肿瘤选自前列腺癌、乳腺癌、肝癌、胶质瘤(例如神经胶质瘤)、肠癌、宫颈癌、非小细胞肺癌、肺癌、胰腺癌、胃癌、膀胱癌、皮肤癌、横纹肌癌、舌鳞癌、鼻咽癌、卵巢癌、胎盘绒毛癌、淋巴瘤(例如非霍奇金淋巴瘤、霍奇金淋巴瘤、皮肤T细胞淋巴瘤)、白血病、直肠腺癌、成神经管细胞瘤、脑膜瘤、神经纤维瘤(例如神经纤维肉瘤)、室管膜瘤、神经鞘瘤、星形细胞瘤、黑色素瘤、间皮瘤、骨髓瘤、慢性粒细胞白血病、急性髓性白血病、骨髓增生异常综合征、慢性淋巴细胞白血病、表皮样癌、结肠癌、胸腺癌、血液癌、头颈癌或口咽癌。
本发明的第十五方面，提供了提高FoxP3表达在降低Ryr2表达、降低Ca 2+基础振荡、降低m-Calpain活性或提高T细胞与DC细胞的结合强度中的应用。
本发明的第十六方面，提供了调控Ryr2表达在调控Ca 2+基础振荡、调控m-Calpain活性或者提高T细胞与DC细胞的结合强度中的应用。优选的，所述的调控为降低或提高。
本发明的第十七方面，提供了调控Ca 2+基础振荡在调控m-Calpain活性或调控T细胞与DC细胞的结合强度中的应用。优选的，所述的调控为降低或提高。
本发明的第十八方面，提供了调控m-Calpain活性在调控T细胞与DC细胞的结合强度中的应用。优选的，所述的调控为降低或提高。
本发明的第十九方面，提供了一种Ryr2的拮抗剂，所述的拮抗剂靶向Ryr2基因7号外显子或Ryr2基因中的一段富含鸟嘌呤的序列。优选的，所述的一段富含鸟嘌呤的序列位于Ryr2基因的起始密码子前200bp中。进一步优选的，所述的一段富含鸟嘌呤的序列包含GCAGGGG。
本发明的第二十方面，提供了一种调控T细胞与DC细胞的结合强度的方法，所述的方法包括调控Ryr2表达。
优选的，所述的调控为降低或提高。其中，所述的降低T细胞与DC细胞的结合强度的方法包括提高Ryr2表达，提高T细胞与DC细胞的结合强度的方法包括降低Ryr2表达。
优选的，所述的降低Ryr2表达包括过表达T细胞中的FoxP3或加入Ryr2抑制剂。进一步优选的，所述的Ryr2抑制剂选自本发明所述的拮抗剂、利阿诺定、硝苯呋海因或JTV519。
优选的，所述的提高Ryr2表达包括降低T细胞中的FoxP3的表达或加入Ryr2驱动剂。进一步优选的，所述的Ryr2驱动剂选自烟酸酰胺腺嘌呤二核苷酸磷酸、咖啡因、对氯代间甲酚、兰尼碱、氯虫苯甲酰胺、氰虫酰胺、B型肾上腺素、4-氯-3-甲基苯酚、溴氰虫酰胺、环溴虫酰胺、环腺苷二磷酸核糖、苏拉明钠、氟氰虫酰胺或三氟拉嗪。
优选的，所述的降低Ryr2表达包括使所述T细胞中缺失Ryr2基因的7号外显子。
在本发明的一个具体实施方式中，采用shRNA为SEQ ID NO：5、6敲除Ryr2基因的7号外显子。
优选的，降低Ryr2表达包括敲低或敲除T细胞中的Ryr2基因。进一步优选的，敲低或敲除T细胞中的Ryr2基因中的一段富含鸟嘌呤的序列。更进一步优选的，所述的一段富含鸟嘌呤的序列位于Ryr2基因的起始密码子前200bp中。最为优选的，所述的一段富含鸟嘌呤的序列包含GCAGGGG。
优选的，所述的一段富含鸟嘌呤的序列为至少包含4个鸟嘌呤(G)的核苷酸序列。进一步优选的，至少包含4-10个鸟嘌呤(G)的核苷酸序列。
在本发明的一个具体实施方式中，至少包含4、5、6、7、8、9、10、11、12、13、14、15等等。
本发明的第二十一方面，提供了一种降低T细胞中的Ryr2表达、降低Ca 2+基础振荡、降低m-Calpain活性的方法，所述的方法包括过表达T细胞中的FoxP3、敲低或敲除T细胞中的Ryr2基因或加入Ryr2抑制剂。
优选的，至少敲除Ryr2基因的7号外显子。
在本发明的一个具体实施方式中，采用shRNA为SEQ ID NO：5、6敲除Ryr2基因的7号外显子。
优选的，所述的方法包括敲低或敲除T细胞中的Ryr2基因。进一步优选的，敲低或敲除T细胞中的Ryr2基因中一段富含鸟嘌呤的序列。更进一步优选的，所述的 一段富含鸟嘌呤的序列位于Ryr2基因的起始密码子前200bp中。最为优选的，所述的一段富含鸟嘌呤的序列包含GCAGGGG。
优选的，所述的一段富含鸟嘌呤的序列为至少包含4个鸟嘌呤(G)的核苷酸序列。进一步优选的，至少包含4-10个鸟嘌呤(G)的核苷酸序列。
在本发明的一个具体实施方式中，至少包含4、5、6、7、8、9、10、11、12、13、14、15等等。
优选的，所述的Ryr2抑制剂选自本发明所述的拮抗剂、利阿诺定、硝苯呋海因或JTV519。
本发明的第二十二方面，提供了一种提高T细胞中的Ryr2表达、提高Ca 2+基础振荡、提高m-Calpain活性的方法，所述的方法包括降低T细胞中的FoxP3的表达或加入Ryr2驱动剂。
优选的，所述的Ryr2驱动剂选自烟酸酰胺腺嘌呤二核苷酸磷酸、咖啡因、对氯代间甲酚、兰尼碱、氯虫苯甲酰胺、氰虫酰胺、B型肾上腺素、4-氯-3-甲基苯酚、溴氰虫酰胺、环溴虫酰胺、环腺苷二磷酸核糖、苏拉明钠、氟氰虫酰胺或三氟拉嗪。
本发明的第二十三方面，提供了一种将Tconv细胞转化为与Treg细胞类似功能的方法，所述的方法包括过表达Tconv细胞的FoxP3或者敲低或敲除Tconv细胞的Ryr2基因。
优选的，所述的方法包括至少敲低或敲除Ryr2基因的7号外显子。
在本发明的一个具体实施方式中，采用shRNA为SEQ ID NO：5、6敲除Ryr2基因的7号外显子。
优选的，所述的方法包括敲低或敲除Tconv细胞中的Ryr2基因中的一段富含鸟嘌呤的序列。进一步优选的，所述的一段富含鸟嘌呤的序列位于Ryr2基因的起始密码子前200bp中。更进一步优选的，所述的一段富含鸟嘌呤的序列包含GCAGGGG。
在本发明的一个具体实施方式中，敲低或敲除Ryr2基因的启动子区域的1.5kb或40kb长度的核苷酸序列。
优选的，所述的与Treg细胞类似功能包括但不限于与DC细胞结合的强度、相关表面标志物(例如CD25、GITR、CTLA-4、CD39、PD-1、LAG-3、TIM-3)的表达量、Ca 2+水平较低、Ca 2+基础振荡低、CMAC消化低、talin消化降低以及降低免疫过渡等。
本发明的第二十四方面，提供了一种治疗感染性疾病或炎症的方法，所述的方法包括向个体施加有效量的本发明所述的缺失Ryr2基因的T细胞。
本发明的第二十五方面，提供了一种治疗感染性疾病或炎症的方法，所述的方法选自使个体的T细胞中的FoxP3过表达、降低T细胞中的Ryr2表达、降低Ca 2+基础振荡、降低m-Calpain活性、提高T细胞与DC细胞的结合强度或加入Ryr2抑制剂。
优选的，所述的Ryr2抑制剂选自本发明所述的拮抗剂、利阿诺定、硝苯呋海因或JTV519。
优选的，所述的感染性疾病选自细菌感染、病毒感染或真菌感染；进一步优选为病毒感染、肺炎伴感染性休克、腹膜炎、菌血症、脓毒症或败血症；所述的病毒感染选自急性病毒感染或慢性病毒感染；优选为流感病毒、副流感病毒、疱疹病毒(例如HSV-1、EBV)、麻疹病毒、水泡口炎病毒、乙型肝炎病毒、丙型肝炎病毒、人免疫缺陷病毒、淋巴细胞脉络丛脑膜炎病毒或人乳头瘤病毒。
优选的，所述的炎症可以是任何组织出现的炎症，所述的组织包括但不限于肾上腺、肾上腺髓质、肛门、阑尾、膀胱、血液、骨、骨髓、脑、乳腺、盲肠、中枢神经系统(包括或排除大脑)、小脑、子宫颈、结肠、十二指肠、子宫内膜、上皮细胞(例如肾上皮细胞)、胆囊、食道、神经胶质细胞、心脏、回肠、空肠、肾、泪腺、喉、肝、肺、淋巴、淋巴结、淋巴母细胞、上颌骨、纵隔、肠系膜、子宫肌层、鼻咽、网膜、口腔、卵巢、胰腺、腮腺、周围神经系统、腹膜、胸膜、前列腺、唾液腺、乙状结肠、皮肤、小肠、软组织、脾、胃、睾丸、胸腺、甲状腺、舌、扁桃体、气管、子宫、外阴、白细胞。进一步优选的，所述的炎症选自系统性红斑狼疮、类风湿性关节炎、银屑病关节炎、硬皮病、哮喘、特应性皮炎、器官特异性炎性疾病、过敏(例如过敏性鼻炎)、毛囊炎、扁桃体炎、肺炎、肝炎、肾炎、痤疮、自身免疫性疾病、慢性前列腺炎、肾小球肾炎、超敏反应、结肠炎、炎性肠道疾病、盆腔炎、再灌注损伤、移植排斥反应、血管炎或间质性膀胱炎。
本发明的第二十六方面，提供了一种治疗肿瘤的方法，所述的方法包括向个体施加有效量的本发明所述的过表达Ryr2的T细胞。
本发明的第二十七方面，提供了一种治疗肿瘤的方法，所述的方法选自使个体降低T细胞中的FoxP3的表达、提高T细胞中的Ryr2表达、提高Ca 2+基础振荡、提高m-Calpain活性、降低T细胞与DC细胞的结合强度或加入Ryr2驱动剂。
优选的，所述的Ryr2驱动剂选自烟酸酰胺腺嘌呤二核苷酸磷酸、咖啡因、对氯代间甲酚、兰尼碱、氯虫苯甲酰胺、氰虫酰胺、B型肾上腺素、4-氯-3-甲基苯酚、溴氰虫酰胺、环溴虫酰胺、环腺苷二磷酸核糖、苏拉明钠、氟氰虫酰胺或三氟拉嗪。
优选的，所述的肿瘤可以是任何不良的细胞增殖(或本身表现为不良细胞增殖的任何疾病)、赘生物，或不良细胞增殖、赘生物或肿瘤的倾向性或风险增加。其可以是良性或恶性的，也可以是原发性或继发性(转移性)。赘生物可以是细胞的任何异常生长或增殖，并且可以位于任何组织中。组织的实例包括肾上腺、肾上腺髓质、肛门、阑尾、膀胱、血液、骨、骨髓、脑、乳腺、盲肠、中枢神经系统(包括或排除大脑)、小脑、子宫颈、结肠、十二指肠、子宫内膜、上皮细胞(例如肾上皮细胞)、胆囊、食道、神经胶质细胞、心脏、回肠、空肠、肾、泪腺、喉、肝、肺、淋巴、淋巴结、淋巴母细胞、上颌骨、纵隔、肠系膜、子宫肌层、鼻咽、网膜、口腔、卵巢、胰腺、腮腺、周围神经系统、腹膜、胸膜、前列腺、唾液腺、乙状结肠、皮肤、小肠、软组织、脾、胃、睾丸、胸腺、甲状腺、舌、扁桃体、气管、子宫、外阴、白细胞。进一步优选的，所述的肿瘤选自前列腺癌、乳腺癌、肝癌、胶质瘤(例如神经胶质瘤)、肠癌、宫颈癌、非小细胞肺癌、肺癌、胰腺癌、胃癌、膀胱癌、皮肤癌、横纹肌癌、舌鳞癌、鼻咽癌、卵巢癌、胎盘绒毛癌、淋巴瘤(例如非霍奇金淋巴瘤、霍奇金淋巴瘤、皮肤T细胞淋巴瘤)、白血病、直肠腺癌、成神经管细胞瘤、脑膜瘤、神经纤维瘤(例如神经纤维肉瘤)、室管膜瘤、神经鞘瘤、星形细胞瘤、黑色素瘤、间皮瘤、骨髓瘤、慢性粒细胞白血病、急性髓性白血病、骨髓增生异常综合征、慢性淋巴细胞白血病、表皮样癌、结肠癌、胸腺癌、血液癌、头颈癌或口咽癌。
本发明所述的“调控”包括调高(提高)或调低(降低)。例如：所述的“调控FoxP3表达的试剂”包括提高FoxP3表达的试剂或降低FoxP3表达的试剂；所述的“调控Ryr2表达的试剂”包括提高Ryr2表达的试剂或降低Ryr2表达的试剂。
本发明所述的“治疗”包括但不限于减缓、中断、阻止、控制、停止、减轻、或逆转一种体征、症状、失调、病症或疾病的进展或严重性，但不一定涉及所有疾病相关体征、症状、病症、或失调的完全消除。
本发明所述的“试剂”代表可以产生相应功能或为实现某一目的所使用的通用试剂、高纯试剂、分析试剂、仪器分析试剂、临床诊断试剂、生化试剂、无机离子显色剂试剂或装置或设备。例如：所述的“提高FoxP3表达的试剂”包括任何可以提高T细胞中FoxP3表达的试剂，例如向T细胞中导入FoxP3基因的试剂，或者提高T细胞中FoxP3基因转录或表达的试剂等等。所述的“降低FoxP3表达的试剂”包括任何可以降低T细胞中FoxP3表达的试剂，例如敲除或敲低T细胞中FoxP3基因的试剂，或者抑制FoxP3基因转录或表达的试剂等等。所述的“降低Ryr2表达的试剂”包括但 不限于敲低或敲除Ryr2基因所需要的试剂，或者提高FoxP3表达的试剂，或者降低Ryr2转录或表达的试剂；例如本发明所述的拮抗剂、利阿诺定、硝苯呋海因或JTV519等。所述的“提高Ryr2表达的试剂”包括但不限于向T细胞中导入Ryr2基因的试剂、提高T细胞中Ryr2基因转录或表达的试剂或者降低T细胞中FoxP3表达的试剂等等。所述的“降低Ca 2+基础振荡的试剂”包括但不限于降低Ryr2表达的试剂。所述的“提高Ca 2+基础振荡的试剂”包括但不限于提高Ryr2表达的试剂。所述的“降低m-Calpain活性的试剂”包括但不限于降低Ryr2表达的试剂。所述的“提高m-Calpain活性的试剂”包括但不限于提高Ryr2表达的试剂。所述的“提高T细胞与DC细胞的结合强度的试剂”包括但不限于降低Ryr2表达的试剂。所述的“降低T细胞与DC细胞的结合强度的试剂”包括但不限于提高Ryr2表达的试剂。
本发明所述的“个体”包括但不限于非人哺乳动物或人。优选的，所述的非人哺乳动物包括但不限于是猴子、狗、小鼠或大鼠等。
本发明所述的“有效量”是指在以单个或多个剂量给予至个体或器官之后提供所希望的治疗的本发明所述试剂或药物的量或剂量。
本发明所述的“CMAC”为蓝色荧光染料，化学名称为7-氨基-4-氯甲基香豆素。
附图说明
以下，结合附图来详细说明本发明的实施例，其中：
图1：从野生型小鼠分离的Tconv和Treg细胞中m-Calpain的蛋白表达分析；其中，图A、C为m-Calpain的mRNA水平，图1B为m-Calpain的蛋白水平；所有值均为平均值+SEM。
图2：静息状态下Ca 2+在单个Tconv和Treg细胞中的基础振荡，检测系统为2mM Ca 2+的HBSS中加入Ca 2+探针Fluo 4-AM；其中，每根线代表一个细胞，n：一组数据点中的生物重复次数，n＝20。
图3：静息状态下Tconv和Treg中Ca 2+振荡的平均荧光强度(MFI)。Tconv和Treg细胞用Fluo 4-AM染色并分析MFI。
图4：图2中所示的Ca 2+振荡强度的标准差(SD)。
图5：GEO数据库分析Treg和Tconv之间钙离子调节相关蛋白的水平，并按差异大小排序。
图6：纯化的Tconv和Treg中Ryr2mRNA表达的qPCR分析。
图7：通过钙蛋白酶底物CMAC消化，测定Ryr激活(4-CMC处理)后的Tconv(左)和Treg(右)细胞中的钙蛋白酶活性。
图8：采用5mM JTV519抑制剂抑制Ryr2后的Tconv细胞(中图)、野生型Tconv细胞(左图)与Treg细胞(右图)中的钙离子基础振荡。
图9：采用5mM JTV519抑制剂抑制Ryr2后的Tconv细胞、野生型Tconv细胞与Treg细胞中的钙离子基础振荡的标准差。
图10：使用qPCR分析从WT小鼠分离的Tconv细胞以及shRNA敲低Ryr2基因后的Tconv细胞的mRNA转录水平。
图11：使用shRNA敲低Ryr2基因后的Tconv细胞与野生型Tconv细胞相比Ca 2+基础振荡。
图12：使用shRNA敲低Ryr2基因后的Tconv细胞与野生型Tconv细胞相比Ca 2+基础振荡的标准差。
图13：JTV519处理的Tconv(左)，在JTV519处理的Treg(中)和Ryr2敲低的Tconv(右)中的钙蛋白酶活性。
图14：检测基于T细胞粘附于DC2.4的粘附力。其中，左图为未经处理的Treg，Tconv与经JTV519处理的Tconv的平均粘附力，右图为空白干扰对照(shCtrl)与Ryr2敲低的Tconv的平均粘附力。
图15：OT-II T细胞与递呈OVA的DC2.4细胞之间的粘附力测定；图A展示了三细胞AFM分析模式图，图B展示了W/O细胞(无细胞组)、空白干扰对照(shCtrl)、Ryr2敲低的Tconv、Treg作为抑制细胞的平均粘附力。
图16：抑制OT-II T细胞的分裂测试结果，图A为无Treg、Treg、空白干扰对照(shCtrl) 或Ryr2敲低的Tconv细胞抑制OT-II T细胞的分裂的FACS图；图B为经过Treg(定义为100％抑制)和无Treg(定义为0％抑制)标准化后的空白干扰对照(shCtrl)或Ryr2敲低的Tconv细胞的抑制效率；其中，*，P<0.05；**，P<0.01；***，P<0.001；****，P<0.0001。
图17：使用qPCR分析从WT小鼠分离的Tconv细胞中的Itpr1，Cacnb1，Trpm1，Trpm4，Trpv2，Orai1和Orai3基因shRNA敲低效率。
图18：未经处理的Treg细胞，空白干扰对照Tconv(shCtrl)，Ryr2敲低的Tconv，Itpr1敲低的Tconv，Cacnb1敲低的Tconv，Trpm1敲低的Tconv，Trpm4敲低的Tconv，Trpv2敲低的Tconv，Orai1敲低的Tconv和Orai3敲低的Tconv的平均粘附力。
图19：FoxP3过表达沉默Ryr2基因转录，具体的，为FoxP3过表达的Tconv、A20、3T3和Renca细胞中的Ryr2mRNA水平。
图20：FoxP3过表达沉默Ryr2基因转录。Foxp3ChIP-seq，转染Foxp3(上)或未转染Foxp3(下)的Tconv。箭头所示Ryr2基因的TSS位点。三角是ChIP-seq识别出的差异显著的结合峰，该位置主要与该基因的启动子区域(方框)重叠。
图21：FoxP3过表达沉默Ryr2基因转录。上图为Ryr2启动子-荧光素酶报告载体的构建示意图，TSS，转录起始位点。下图为FoxP3过表达的3T3或Renca细胞中Ryr2启动子驱动的转录水平。
图22：FoxP3过表达沉默Ryr2基因转录。图A为在序列中执行顺序段删除结果，即Ryr2启动子-荧光素酶报告载体的截短载体的示意图，图B为在FoxP3过表达的3T3中Ryr2启动子驱动的转录水平。
图23：FoxP3过表达沉默Ryr2基因转录，其中，图A为Ryr2启动子-荧光素酶报告载体的2个截短载体及敲除策略的示意图，图B为FoxP3过表达的3T3细胞中不同的截短载体或FoxP3结合序列表达的Ryr2启动子驱动的转录水平，N.S.，无统计学差异。
图24：FoxP3过表达沉默Ryr2基因转录。在不含钙离子的HBSS中，4-CmC刺激FoxP3 过表达的A20或3T3细胞，钙离子随时间变化的平均轨迹；N＝3，在每个独立实验中，每个单次测量至少收集30个细胞用于分析。
图25：敲低Ryr2基因的T细胞(Ryr2-/-Tconv)诱导小鼠皮癣(Scurfy)模型的免疫抑制，并恢复Scurfy小鼠的免疫稳态，其中，图A为WT和注射PBS后的小鼠存活率，图B为分别注射Treg、Ryr2+/+(Foxp3-)Tconv和Ryr2-/-Tconv的小鼠存活率。
图26：在Ryr2基因中Cre依赖敲低第7号外显子的条件敲低小鼠构建。
图27：通过流式检测CD4+和CD8+T细胞在Ryr2+/+(未敲除Ryr2的野生型)小鼠，CD4-Cre/Ryr2fl/+小鼠和CD4-Cre/Ryr2-/-小鼠的脾和胸腺中的分布。
图28：小鼠中FoxP3+/CD4+脾细胞的比例。
图29：流式分析WTTconv(Ryr2+/+)、Ryr2-/-Tconv中的Treg功能相关表面标志物(包括CD25、GITR、CTLA-4、CD39、PD-1、LAG-3、TIM-3)。以及ELISA检测24h抗CD3/抗CD28刺激后，上清液中的IL-10和TGF-β水平。
图30：敲低Ryr2的T细胞在体外具有基于细胞接触的抑制功能。从WT(图A)或CD4-Cre、Ryr2fl/fl(Ryr2-/-)(图B)小鼠中分离的Tconv与Treg(图C)对比，在2mM Ca 2+的HBSS中检测钙离子振荡水平及其标准差(图D)。
图31：使用CMAC底物降解在Treg，Ryr2+/+和Ryr2-/-Tconv细胞中检测钙蛋白酶活性。
图32：通过蛋白质印迹揭示在Treg，Ryr2+/+和Ryr2-/-Tconv的三种细胞类型中的体内底物Talin降解结果。
图33：敲低Ryr2的Tconv在体外具有基于细胞接触的抑制功能；在同一天收集的每种条件下来自四个独立DC-T细胞对的所有数据点(图A、B中的点)均用于生成图C的条形图，每个T-DC对至少收集了15个粘附力读数；具体为WT Tconv(图A)和Ryr2-/-Tconv细胞(图B)对DC2.4的粘附力，以及平均粘附力(图C)。
图34：OT-II T细胞与递呈OVA的DC2.4细胞之间的粘附实验，DC上的抑制细胞为 Treg、WT Tconv或Ryr2-/-Tconv细胞；其中，图A为展示的是三细胞AFM测定装置，图B为Treg、WT Tconv或Ryr2-/-Tconv作为抑制细胞的平均OT-II-DC粘附力。
图35：无Treg、Treg、Ryr+/+和Ryr2-/-Tconv的相对抑制效率，其中，设无Treg为0％，Treg为100％。
图36：敲低Ryr2基因的T细胞(Ryr2-/-Tconv)诱导小鼠疱疹感染模型的免疫抑制，图为细胞过继转移后在足底组织中的HSV-1滴度的分析，HSV-1、Treg、Ryr2-/-Tconv或Ryr2+/+Tconv分别为过继转移PBS、Treg或Ryr2-/-Tconv、Ryr2+/+Tconv。
图37：敲低Ryr2基因的T细胞(Ryr2-/-Tconv)诱导小鼠疱疹感染模型的免疫抑制，图为细胞过继转移和紫外线灭活的HSV-1抗原再攻击后的迟发型超敏反应(DTH)，其中，不进行抗原再攻击的足底厚度设为100％。
图38：敲低Ryr2基因的T细胞(Ryr2-/-Tconv)诱导小鼠疱疹感染模型的免疫抑制，其中，上图为DTH分析中的实验方案，下图为代表性的足底肿胀。
图39：敲低Ryr2基因的T细胞(Ryr2-/-Tconv)诱导小鼠哮喘模型的免疫抑制，其中，图A为BALF的总浸润，图B为淋巴细胞浸润，图C为嗜酸性粒细胞浸润。测定结果的数值均为3次试验的平均值，空白代表空白对照组，PBS、Treg、Ryr2-/-Tconv及Ryr2+/+Tconv组均为哮喘模型注射等量PBS、Treg、Ryr2-/-Tconv及Ryr2+/+Tconv。
图40：敲低Ryr2基因的T细胞(Ryr2-/-Tconv)诱导小鼠哮喘模型的免疫抑制，其中图A为哮喘模型致敏方案，图B为代表性的肺部H&E组织切片，比例尺代表200μm，PBS、Treg、Ryr2-/-Tconv及Ryr2+/+Tconv组均为哮喘模型注射等量PBS、Treg、Ryr2-/-Tconv及Ryr2+/+Tconv。
图41：敲低Ryr2基因的T细胞(Ryr2-/-Tconv)诱导小鼠结肠炎(IBD)模型的免疫抑制，其中，空白代表空白对照组，DSS代表水处理。
图42：敲低Ryr2基因的T细胞(Ryr2-/-Tconv)诱导小鼠结肠炎(IBD)模型的免疫抑制，其中，图A为DSS诱导的结肠炎模型的诱导方案，图B为代表性结肠图像，图C 为结肠切片的显微照片，比例尺代表200μm。
图43：敲低Ryr2基因的T细胞(Ryr2-/-Tconv)诱导小鼠结肠癌模型的免疫抑制，分别测量各组小鼠肿瘤体积生长曲线，第1组：MC38+PBS；第2组：MC38+Ryr2+/+Tconv；第3组：MC38+Treg；第4组：MC38+Ryr2-/-Tconv。
图44：敲低Ryr2基因的T细胞(Ryr2-/-Tconv)诱导小鼠皮癣(Scurfy)模型的免疫抑制，并恢复Scurfy小鼠的免疫稳态，图为小鼠的Kaplan-Meier生存曲线，其中，图A为WT和注射PBS后的小鼠存活率，图B为分别注射Treg、Ryr2+/+(Foxp3-)Tconv和Ryr2-/-Tconv的小鼠存活率。
图45：敲低Ryr2基因的T细胞(Ryr2-/-Tconv)诱导小鼠皮癣(Scurfy)模型的免疫抑制，并恢复Scurfy小鼠的免疫稳态，其中，图A为Scurfy小鼠的身体症状和治疗，图B为各器官(胸腺、脾、肺、耳朵、肝、胰腺、小肠、结肠)的代表性H&E染色，注射PBS的模型小鼠、WT小鼠，在第3周采集样品，过继转移Treg细胞、Foxp3-(Ryr2+/+)Tconv和Ryr2-/-Tconv的小鼠在第8-12周采集。
图46：Foxp3过表达抑制Ryr2是T细胞免疫抑制作用的关键效应机制，左图为Foxp3过表达抑制Ryr2后T细胞的作用机制，右图为野生型T细胞的作用机制。
具体实施方式
下面将结合本发明实施例中的附图，对本发明实施例中的技术方案进行清楚、完整地描述，显然，所描述的实施例仅是本发明的部分实施例，而不是全部。基于本发明中的实施例，本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例，都属于本发明保护的范围。
实施例中所用试验动物、试剂及装置的来源：
小鼠。所有小鼠均为C57BL/6背景。Ryr2fl/fl小鼠和CAG-LSL-Ryr2-flag转基因小鼠由GemPharmatech Co.，Ltd生产，并通过基因型鉴定。OT-II转基因小鼠购自Jackson实验室。野生型，Foxp3-IRES-GFP，Ryr2fl/fl，Ryr2转基因，OT-II转基因，CD4-Cre 和Foxp3-Cre-YFP，雌性Foxp3+/-小鼠在清华大学动物中心饲养。所有小鼠均在无特定病原体(SPF)条件下饲养。所有动物实验均符合动物福利准则，并获得清华大学动物实验伦理委员会IACUC的批准。
细胞系和原代细胞培养。DC2.4细胞是Ken Rock UMass医学院的馈赠。Vero细胞来自清华大学药学院的谭旭博士。Renca细胞来自北京朝阳医院安广宇医生。HEK293FT细胞是清华大学医学院的郭伟博士的馈赠。MC38细胞购自美国典型培养物保藏中心(ATCC)。在含有10％FBS，100U/mL青霉素，100mg/mL链霉素的DMEM中培养MC38，Renca，NIH-3T3细胞和Vero细胞。所有其他细胞均在具有相同剂量和50μMβ-巯基乙醇的RPMI-1640中生长。通过PCR分析测试所有细胞系的支原体污染。使用小鼠CD4+T细胞分离试剂盒(StemCell，19852)和小鼠CD25Treg细胞阳性选择试剂盒(Stem cell，18782)从脾脏中分离出小鼠CD4+CD25+Treg和CD4+CD25-Tconv。Treg和Tconv细胞有时通过FACS从Foxp3-IERS-GFP小鼠CD4+脾细胞中分选出来。用小鼠CD11c选择试剂盒II(Stem cell，28007)从脾脏中分离出小鼠DC。通过小鼠CD4+T细胞分离试剂盒(Stem cell，19852)从OT-II脾细胞中分离出OT-II T细胞，有时通过FACS用抗TCRVα2抗体进行分选。
抗体和试剂。所有原代细胞分离试剂盒均来自StemCell。重组人IL-2来自R&D systems。抗小鼠CD3e单克隆抗体，抗小鼠CD28和抗小鼠TCRVα2-PE抗体来自eBioscience。流式抗体来自eBioscience，包括抗CD4，抗CD8，抗GITR，抗CD25，抗PD-1，抗CTLA-4，抗Tim-3和抗LAG3抗体。抗FoxP3抗体来自Invitrogen。使用了以下克隆：抗小鼠CD4，抗小鼠CD8，抗小鼠GITR，抗小鼠CD25，抗小鼠PD-1，抗小鼠/大鼠CTLA-4，抗小鼠/大鼠FoxP3(FJK-16s)，抗小鼠/人Tim-3，抗小鼠LAG3，抗小鼠PD-1(J43.1)，抗小鼠CTLA4(UC10-4B9)，抗小鼠GITR(DTA-1)。以下抗体用于蛋白质印迹分析：m-Calpain大亚基(M型)抗体和抗小鼠IgG-HRP连接的抗体来自CST。抗talin抗体(8D4)购自Sigma-Aldrich。
从清华大学生物医学分析中心的shRNA文库平台购买的pLKO.1载体用于我们的所有 基因敲低实验。所有预先设计的shRNA序列均由Ruiboxingke Biotech合成。
pLVX-IRES-mcherry载体是清华大学医学院沈晓华博士的馈赠。
实施例1：敲低Ryr2基因或FoxP3过表达的T细胞的制备
1、敲低Ryr2基因试验步骤
基于慢病毒的shRNA用于敲低Ryr2基因，具体的，将预先合成的shRNA序列插入pLKO.1载体。使用EndoFree质粒Midi试剂盒(CWBIO，CW2105S)从转化的大肠杆菌中纯化包括shRNA的质粒和包装构建体(pMD2.G和psPAX2)。按照制造商的说明进行慢病毒包装。简而言之，将293FT细胞培养在10cm皿中，培养到60-80％覆盖。DNA转染前2小时更换培养基。使用Neofect(Neofect)，将2.5μg包装载体pMD2.G和2.5μgpsPAX2、5μgpLKO.1载体(带有插入的shRNA)转染到293FT细胞中。72小时后，收获慢病毒并用Polybrene(终浓度为8μg/mL)感染T细胞。病毒感染后48小时，通过Aria细胞仪(BD Biosciences)分选细胞。通过实时定量PCR验证了敲低效率。shRNA序列如下：
对照-shRNA：5'-CCGGcaacaagatgaagagcaccaaCTCGAGttggtgctcttcatcttgttgTTTTTG-3'(SEQ ID NO：3)和5’-AATTCAAAAAcaacaagatgaagagcaccaaCTCGAGttggtgctcttcatcttgttgg-3’(SEQ ID NO：4)；Ryr2-shRNA：5'-CCGGccgctaatgaagccatataaaCTCGAGTTTATATGGCTTCATTAGCGGTTTTTG-3'(SEQ ID NO：5)和5’-AATTCAAAAAccgctaatgaagccatataaaCTCGAGTTTATATGGCTTCATTAGCGG-3’(SEQ ID NO：6)；Itpr1—shRNA：5'-CCGGgcagtaggtaagaagttattaCTCGAGtaataacttcttacctactgcTTTTTG-3'(SEQ ID NO：7)和5'-AATTCAAAAAgcagtaggtaagaagttattaCTCGAGtaataacttcttacctactgc-3'(SEQ ID NO：8)；
Orai1-shRNA：5'-CCGGcacaaccaccaactcggtcaaaCTCGAGtttgaccgagttgaggttgtgTTTTTG-3'(SEQ ID NO：9)和
'-AATTCAAAAAcacaacctcaactcggtcaaaCTCGAGtttgaccgagttgaggttgtg-3'(SEQ ID NO：10)；Orai3-shRNA：
5'-CCGGgcccttgcttatctgtataatCTCGAGattatacagataagcaagggcTTTTTTGG-3'(SEQ ID NO：11)和5'-AATTCAAAAAAgcccttgcttatctgtataatCTCGAGattatacagataagcaagggc-3'(SEQ ID NO：12)；Trpm1-shRNA：
5'-CCGGcggagtgaacatgcagcatttCTCGAGaaatgctgcatgttcactccgTTTTTG-3'(SEQ ID NO：13)和5'-AATTCAAAAAcggagtgaacatgcagcatttCTCGAGaaatgctgcatgttcactccg-3'(SEQ ID NO：14)；Trpm4—shRNA：
5'-CCGGgcacatcttcacggtgaacaaCTCGAGttgttcaccgtgaagatgtggttTTTG-3'(SEQ ID NO：15)和5'-AATTCAAAAAgcacatcttcacggtgaacaaCTCGAGttgttcaccgtgaagatgtgc-3'(SEQ ID NO：16)；Trpv2—shRNA：
5'-CCGGccaaggaacttgtttctatttCTCGAGaaatagaaacaagttccttggTTTTTG-3'(SEQ ID NO：17)和5'-AATTCAAAAAccaaggaacttgtttctatttCTCGAGaaatagaaacaaagagctccttgg-3'(SEQ ID NO：18)；Cacnb1–shRNA：
5'-CCGGtaggaacgcaatggatattaaCTCGAGttaatatccattgcgttcctaTTTTTG-3'(SEQ ID NO：19)和5'-AATTCAAAAAtaggaacgcaatggatattaaCTCGAGttaatattccattgcgttccta-3'(SEQ ID NO：20)。
2、通过实时定量PCR验证了敲低效率。具体步骤如下：
使用TRIzol试剂(Invitrogen)从指示的细胞中提取总RNA，并用逆转录酶M-MLV(TaKaRa)合成第一链cDNA。使用SYBR Green Master Mix(No Rox)(Yeasen)进行实时定量PCR。GAPDH或18S RNA用于标准化对照。引物序列如下：
鼠GAPDH，5'-CATCACTGCCACCCAGAAGACTG-3'(SEQ ID NO：21)和5'-ATGCCAGTGAGCTTCCCGTTCAG-3’(SEQ ID NO：22)；小鼠18S RNA，5'-CGGACAGGATTGACAGATTG-3'(SEQ ID NO：23)和5’-CGGACAGGATTGACAGATTG-3’(SEQ  ID NO：24)；鼠Ryr2，5’-ATGGCTTTAAGGCACAGCG-3’(SEQ ID NO：25)和5’-CAGAGCCCGAATCATCCAGC-3’(SEQ ID NO：26)；鼠FoxP3，5’-CCCATCCCCAGGAGTCTTG-3'(SEQ ID NO：27)和5'-ACCATGACTAGGGGCACTGTA-3'(SEQ ID NO：28)；鼠m-Calpain，5'-3'和5'-3'；鼠Itpr1，5'-CGTTTTGAGTTTGAAGGCGTTT-3'(SEQ ID NO：29)和5'-CATCTTGCGCCAATTCCCG-3'(SEQ ID NO：30)；鼠Orai1，5'-GATCGGCCAGAGTTACTCCG-3'(SEQ ID NO：31)和5'-TGGGTAGTCATGGTCTGTGTC-3'(SEQ ID NO：32)；鼠Orai3，5’-GGCTACCTGGACCTTATGGG-3'(SEQ ID NO：33)和5'-GCAGGCACTAAATGTGACC-3'(SEQ ID NO：34)；鼠Trpm1、5'-ATCCGAGTCTCCTACGACACC-3'(SEQ ID NO：35)和5'-CAGTTTGGACTGCATCTCGAA-3’(SEQ ID NO：36)；鼠Trpm4、5'-GGACTGCACACAGGCATTG-3'(SEQ ID NO：37)和5'-GTACCTTGCGGGGAATGAGC-3’(SEQ ID NO：38)；鼠Trpv2，5'-TGCTGAGGTGAACAAAGGAAAG-3'(SEQ ID NO：39)和5'-TCAAACCGATTTGGGTCCTGT-3'(SEQ ID NO：40)；小鼠Cacnb1，5′-GGCAGCAAGTTATCTCCCAG-3'(SEQ ID NO：41)和5'-CCACAGGATGATTGGCGTCTT-3'(SEQ ID NO：42)；HSV-1gB，5’-AACGCGACGCACATCAAG-3'(SEQ ID NO：43)和5'-CTGGTACGCGATCAGAAAGC-3'(SEQ ID NO：44)；HSV-1LAT，5'-GGGTGGGCTCGTGTTACAG-3'(SEQ ID NO：45)和5'-GGACGGGTAAGTAACAGAGTCTCTA-3’(SEQ ID NO：46)。
3、FoxP3过表达试验步骤
构建载体，使用Treg总RNA的cDNA作为模板，使用以下引物通过PCR扩增FoxP3：正向引物5'-ATCGCTCGAGATGTGCACACCTAGGCCA-3'(SEQ ID NO：47)和反向引物5'-ATCGGAATTCTCAAGGGCAGGGATTGGA-3'(SEQ ID NO：48)。凝胶纯化扩增的片段，消化(XhoI和EcoRI)并克隆到pLVX-IRES-mcherry质粒中，得到pLVX-FoxP3-IRES-mcherry构建体。根据上述敲低方案进行了携带pLVX-FoxP3-IRES-mcherry和FoxP3-过表达的细胞系生成的慢病毒生产。通过RT-PCR验证FoxP3表达。
4、流式方法检测步骤
将细胞与Fc阻断剂(CD16/32抗体；2.4G2)孵育5分钟，然后与表面抗体(CD4，CD8，GITR，CD25，PD-1，CTLA-4，TIM-3，LAG3)在室温下避光放置15分钟。FoxP3细胞染色，将表面染色的细胞与FoxP3/转录因子固定/通透缓冲液(Thermo Fisher)孵育30分钟，然后与抗体孵育2小时。直接分析染色的细胞，或偶尔使用FACS Diva软件使用Fortessa细胞仪(BD Biosciences)用1％多聚甲醛(PFA)固定后进行分析。用Flowjo软件分析数据。
5、ELISA检测步骤
为了检测IL-10和TGF-β，用抗CD3/抗CD28抗体刺激了10 6个纯化的Treg，Ryr2+/+和Ryr2-/-Tconv。72小时后，收集上清液。将高结合力的96孔ELISA板(Nunc)在过夜于4℃涂上抗小鼠IL-10和抗小鼠TGF-β捕获抗体。干燥后，在室温下将板用PBS中的2％BSA封闭1小时。洗涤后，在一式三份的孔中加入100uL稀释的细胞上清液，然后在室温下孵育1小时。然后将板用PBST(0.05％Tween20，Sigma-Aldrich，在PBS中)洗涤，并在室温下与HRP标记的山羊抗小鼠IL-10和TGF-β检测抗体孵育0.5小时。加入TMB(100mL/孔)，并将板在室温下在黑暗中温育10分钟，然后每孔加入H 2SO 4(50uL，1M)以终止反应。立即使用ELISA酶标仪(Bio-Rad)在450nm下读取光密度(OD)。
6、钙成像。
对于非贴壁细胞，将Treg，Tconv和A20细胞用HBSS-HEPS(10mM)缓冲液中的2mM fluo-4AM(Thermo Fisher)在37℃染色1小时。洗涤后，使细胞粘附于室温下安装在夹心的自制室中的聚-L-赖氨酸涂层(0.1mg/mL；Sigma-Aldrich)圆形玻璃片上。15分钟后，用缓冲液冲洗除去多余的非贴壁细胞。对于贴壁细胞，将贴在玻璃片上的NIH-3T3和Renca细胞用HBSS-HEPS(10mM)缓冲液中的2mM fluo-4AM(Thermo Fisher)在37℃染色1小时。然后将测量室放置在配备有20倍(数值孔径：0.8)或40倍(数值孔径：1.2)Olympus物镜的Olympus IX-73显微镜上。除非另有说明，否则以6s的间隔记录20分钟的Ca 2+振荡。在抑制实验中，将Fluo-4标记后的细胞在室温下于黑暗处用JTV519 (Sigma)处理30分钟，然后获取图像。对于刺激性实验，以1s的间隔记录5分钟的Ca2+振荡。开始获取图像50到80s后，添加4-CmC(Sigma)缓冲液以诱导细胞内钙的释放。在含或不含Ca2+的无酚红HBSS培养基中进行实验。用电荷耦合器件照相机(ORCA-AG，滨松)记录由488nm激光激发的468–550nm处的发射信号。数据收集由NIS-Elements 3.0软件(Nikon)控制。使用ImageJ分析单个细胞随时间的平均荧光强度变化，并根据第一帧的荧光强度标准化(Fluo-4F/F0)。显示单个细胞的Ca 2+振荡峰值，计算Ca 2+波动强度的标准偏差为Mean±SD。
7、蛋白质印迹(western blot)测步骤
收集细胞并用RIPA缓冲液(Beyotime，P0013B)裂解。离心细胞裂解液并收集上清液。用BCA蛋白质测定试剂盒(Beyotime，P0012)定量总蛋白质。与3XSDS上样缓冲液混合并煮沸5分钟后，将蛋白质上样到7.5％PAGE凝胶(EpiZyme，PG111)上。然后将蛋白质转移到NC膜上，并用所示的一抗和二抗进行免疫印迹。最后，用Super ECL检测试剂(Yeasen，36208ES76)检测免疫染色的条带。
8、试验结果
在CD4-cre的Tconv中可以敲低Ryr2(图26)。同时，敲低Ryr2的Tconv细胞并没有影响其基础功能，包括小鼠的体重与发育速度。以及胸腺细胞和外周血CD4+、CD8+标记物的分布与WT小鼠几乎相同(流式检测结果见图27)。FoxP3+CD4+T细胞的比例也保持不变(图28)。与WT Tconv相比，可能与Treg功能相关的表面标记也保持不变(流式及ELISA检测结果见图29)。敲低Ryr2的Tconv伴随着基础Ca 2+水平的降低以及振荡强度的降低(钙成像结果见图30)。与未处理的Tconv相比，Ryr2-/-Tconv中的CMAC消化降低到与Treg类似的水平(图31)。与Treg一样，Ryr2-/-Tconv中大量缺失talin消化(western blot检测结果见图32)。
实施例2：验证FoxP3表达对Ryr2基因的影响，以及确定靶点位置
为确定Ryr2基因调节的特异性，本实施例将其与FoxP3表达建立联系，并确定靶点 位置。
首先在T细胞、B细胞(A20)、3T3和Renca肿瘤系中都过表达了FoxP3(采用实施例1的方法)。在这四种情况中，FoxP3的过表达显著阻断了Ryr2基因转录，这表明FoxP3自发靶向Ryr2(图19)。
然后，制备一个用于基因表达分析的全基因组Chip-seq数据集，ChIP-seq数据是从GEO数据集下载，GEO ID为：Tconv GSM989036中的Foxp3，Tconv细胞中的FoxP3转导为表达标志-Foxp3GSM989034。IGV(v2.4.14)使用小鼠参考基因组mm8促进了ChIP-seq数据的可视化。如在特定基因位点的屏幕截图所示。采用IGV程序将该数据集集中在Ryr2基因周围的一个40kb区域时，在未转染的对照组上检测到一个1.5kb启动子区域的强信号(图20)。
进一步的，克隆上述区域，并在此启动子控制下构建了荧光素酶报告基因的表达载体，用于表达3T3和Renca细胞，包括过表达FoxP3组和未过表达FoxP3组。从图21可以看出，过表达FoxP3降低了荧光素酶的活性。然后，在序列中执行顺序段删除(参见图22A)。在一系列的实验中，逐步将抑制活性缩小到TSS位点后～300-500bp的一个限定区域，使其在Ryr2起始密码子(图22B)之前200bp。
最后确定FoxP3结合位点为：重复两次出现的GCAGGGG序列。当这两个位点发生突变时，FoxP3过表达失去了抑制荧光素酶的能力，表明其是该启动子中FoxP3的结合位点(图23)。同时，为了进一步证实荧光素酶活性抑制与Ryr2活性相关，本实施例触发了Ryr2介导的3T3和A20细胞的Ca 2+通量，包括FoxP3过表达组和FoxP3未过表达组。图24显示，在FoxP3的作用下，4-CmC在FoxP3转染的细胞中驱动的Ca 2+信号要小得多。表明Ryr2确实受FoxP3的直接控制，是T细胞抑制活性的关键通道。
其中，本实施例中涉及的双荧光素酶报告测定步骤参见文献Identification and characterization of MAVS,a mitochondrial antiviral signaling protein that activates NF-kappaB and IRF 3(Seth等，Cell 122，669-682，2005)和LFA-1-mediated  adhesion is regulated by cytoskeletal restraint and by a Ca2+-dependent protease,calpain(Stewart等，J Cell Biol 140，699-707，1998)。通过将1500bp的Ryr2启动子亚克隆到荧光素酶表达pGL3载体中来构建鼠Ryr2报道质粒。合成完整的启动子序列和截短的启动子，并通过测序确认质粒正确。使用Neofect(KS2000)将1.25×105FoxP3过表达的-3T3，FoxP3过表达的Renca或FoxP3过表达的A20细胞与300ng Ryr2报告基因和海肾荧光素酶报告基因质粒共转染。转染后36小时，制备细胞裂解物并使用双重荧光素酶报告测定系统(Promega)进行分析。
实施例3：抑制Ryr2对T细胞中Ca 2+水平的影响
1、Tconv和Treg中的m-Calpain差异，及与DC结合的能力
介于Treg中m-Calpain活性低，本实施例预确定其表达水平是否低。同时由于m-Calpain，是由细胞内Ca 2+的可用性调节。因此，对静息状态下Tconv和Treg的Ca 2+信号进行了检测。
结果如图1A、B、C所示，Tconv和Treg中的m-Calpain在蛋白或mRNA水平上均无差异。而且虽然基础Ca 2+波动是包括Tconv在内的许多免疫细胞共有的一个共同特征，但这种活性在Treg中基本不存在。即，单个Treg没有显示Ca 2+振荡的峰值(图2和图3)。Ca 2+波动强度的标准差在Treg中明显小于Tconv(图4)。即m-Calpain活性在Treg中被阻断，使得其具有基于LFA-1/icam-1的异常强的与DC结合的能力。
2、确定影响Ca 2+水平的因素
当T细胞激活时，Ca 2+信号来自于IP3R通道，这是基于免疫酪氨酸的信号级联扩大到继发性Ca 2+扩增事件的结果，而这种激活诱导信号在静息细胞中是不存在的。本实施例采用数据库GEO DataSets(参见文献：A mechanism for expansion of regulatory T-cell repertoire and its role in self-tolerance(Feng et al.，Nature，2015))中涉及的几种影响Ca 2+信号调节的因素数据，具体见图5，验证了Treg中基础Ca 2+水平较低的原因，并根据Treg和Tconv表达的差异程度进行排序。结果见图5所示，Ryr2差异最大， Tconv的表达水平比Treg高5倍log2以上(32倍线性)，p值为2.57×10 -14。采用qPCR进一步分析证实该结论(参见图6)。
其中，GEO数据库分析：根据已发表在NCBI GEO数据库(GSE71162)中的三项RNA-Seq研究，按照表达水平对蛋白质进行分类。通过GO分析对钙相关蛋白进行排序。这些蛋白质的功能基于UniProt(http://www.uniprot.org/)进行注释。通过Treg和Tconv之间表达差异的程度对蛋白质进行排名。
Ryr受体位于er膜上(Ryr1、2和3)，在人PBMC中，Ryr1在CD19+群体中表达，Ryr2在CD3+部分中表达。为证实Ryr2在Treg中的表达量低，采用刺激因子4-CMC，观察CMAC(calpain底物)的消化率。结果如图7所示，4-CMC完全能够诱导Calpain活性，而Treg对这种处理不敏感，证实了Treg中Ryr2的表达降低。
3、抑制Tconv细胞的Ryr2可以降低Ca 2+水平及基础振荡
本实施例进一步验证抑制Ryr2后Ca 2+信号的变化，结果如图8、9所示，无论是峰值还是Ca 2+信号的标准差，抑制Ryr2后的Tconv细胞均为Treg效果相近，且相对于未抑制Ryr2的Tconv细胞，钙离子水平显著降低。
综上所述，T细胞亚群的Ca 2+水平较低基本是由于Ryr2的转录抑制所控制。
实施例4：阻断Ryr2可以使T细胞与DC结合紧
1、敲低Ryr2的Tconv可以降低Ca 2+水平
为从遗传学上证实Ryr2抑制是Tconv中降低Ca 2+水平的原因，本实施例进行了shRNA干扰敲低Ryr2。Ryr2特异性shRNA敲低与未敲低Ryr2转录mRNA水平(图10)比较。敲低Ryr2后的Tconv与对照组相比细胞基础Ca 2+水平显著降低(图11和12)。敲低后，Ca 2+峰值降低，且Ca 2+变化的标准差也降低。与低m-Calpain底物消化处理结果一致，例如JTV519处理的抑制Ryr2(图13)。
其中，钙蛋白酶活性测量。为了测量钙蛋白酶活性，将10 5T细胞在200μL含有20μM钙蛋白酶底物CMAC(t-BOC-Leu-Met，Thermo Fisher)的培养基中于暗处在室温下孵育。 孵育5分钟后，以1％PFA(Biosharp)终止反应，并将细胞立即置于冰上。通过Fortech流式细胞仪通过Hoechst蓝通道测定从消化的底物发出的荧光信号作为钙蛋白酶活性。在所有实验中，在实验前30分钟添加抑制剂JTV519，同时将活化剂4-CmC与CMAC添加。
2、Tconv细胞中Ryr2的缺失对T细胞与DC的结合的影响
为了验证Ryr2的减少和m-Calpain活性的丧失对T细胞与DC的结合的影响。本实施例使用shRNA和JTV519在Tconv中阻断Ryr2。结果显示，两种处理都诱导了与树突状细胞的强烈接触，强度甚至高于Treg(图14)。
进一步的，证实经处理的Tconv与DC的结合增加导致抗原特异性Tconv与相同的DC形成稳定的结合，进行了三重细胞结合力分析(参见文献：Strong adhesion by regulatory T cells induces dendritic cell cytoskeletal polarization and contact-dependent lethargy(Chen et al.，The Journal of experimental medicine，2017))。结果显示，Treg介导的阻断，Ryr2降低的Tconv可以显著抑制OTII和负载OVA的DC之间的结合强度(图15)。因此，在单细胞水平上证实，Ryr2活性降低的Tconv在阻止Tconv于DC相互作用的能力上与Treg功能相当。然后，在一个典型的DC介导的抗原呈递试验中测试了shRNA处理的Tconv抑制OTII激活的能力。结果如图16所示，这些细胞在减少OTII分裂方面表现出类似于Treg的方式。在一项关于其他可能以与Ryr2相似的方式起作用的潜在Ca 2+通道活动的调查中，产生了一组shRNA来降低它们在CD4Tconv细胞中的活性，并通过SCFS测试它们与DC的结合。结果如图17、18所示，除Ryr2外，其他敲低均未增加处理T细胞与DC的结合，证实了Ryr2的独特参与。
其中，体外抑制功能。纯化的OTII T细胞用CellTrace CFSE(Thermo Fisher Scientifc)染色。将来自脾细胞的10 4个纯化的DC，2×10 4OTII T细胞，2×10-Treg或Ryr2-/-Tconv或Ryr2敲低的Tconv和2μM OVA323-339肽混合并在96孔U底板的每个孔中培养，并通过Fortessa流式细胞仪通过CFSE稀释评估OTII T细胞的增殖。由(1-增殖％)计算抑制百分比，然后通过将无Treg组作为0％抑制、将Treg组作为100％抑制来进行数据归 一化。
3、采用力谱证实Ryr2缺失时，与DC的结合情况
同样的，力谱也表明Ryr2缺失时，与DC的结合增加(图33)。与此同时，在三细胞力分析中，干扰DC与T细胞接触的能力也增强了(图34)。更重要的是，Ryr2-/-Tconv在抑制抗原阳性树突状细胞刺激的OTII扩增的能力上与Treg类似(图35)。因此，通过简单地限制Tconv细胞中的Ryr2在体外就可以获得了抑制DC介导的T细胞活化的能力。图46显示了抑制Ryr2的T细胞的免疫抑制作用。
其中，基于原子力显微镜的单细胞力谱步骤如前所述，使用JPK CellHesion装置进行了实验(参见参考文献：Alum interaction with dendritic cell membrane lipids is essential for its adjuvanticity(Flach等，Nat Med 17，479-487，2011)和参考文献：Strong adhesion by regulatory T cells induces dendritic cell cytoskeletal polarization and contact-dependent lethargy(Chen等，The Journal of experimental medicine，2017))。简而言之，为了测量双细胞系统中的T-DC粘附力，将DC2.4细胞培养在未处理的玻璃盘上。用200U/mL的人IL-2处理T细胞过夜。磁盘被移入AFM兼容的腔室并安装到机器台上。将干净的悬臂涂上CellTak(BD)，然后将其粘贴到磁盘上的单个T细胞上。降低携带单个T细胞的AFM悬臂，使其与单个DC接触并相互作用15s，然后向上移动，直到两个细胞完全分离。获得力曲线。然后重复该过程。对于三细胞系统，在实验前，将玻璃盘上培养的DC2.4细胞用100μg/mL可溶性OVA蛋白处理4h。用10μM CFSE对经IL-2处理的Treg/Tconv细胞进行染色，然后将DC2.4细胞与这些荧光标记的Treg或Tconv细胞孵育约20分钟，然后添加未标记的OT-II T细胞。然后通过带有OT-II T细胞的悬臂尖端接近用紫外线闪光灯识别的Treg/Tconv细胞-DC对。分析了Treg/T转化介导的OT-II-DC粘附抑制。在每个循环中，带有单个T细胞的AFM悬臂以0.5–2μm的增量降低，直到生成第一条力曲线。然后将悬臂上的T细胞与DC相互作用15s，然后向上移动，直到两个细胞完全分离。将装有机器的培养箱置于37℃和5％CO 2的条件下。在所 有实验中，至少收集了14条力曲线用于进一步分析。使用JPK图像处理软件处理力曲线。仅选择圆形和坚固的细胞进行AFM胶合。对于每个SCFS实验，在几分钟的时间内，使用一对T-DC生成每个上下周期的力读数。每种条件至少使用三对数据。
实施例5：HSV-1感染中Ryr2-/-T细胞的作用
1、疱疹感染模型的制备及试验步骤
由10 6pfu HSV-1(F株)在20μL PBS中的后足底引起的。在3dpi时，将细胞与2×10 5个细胞/20μLPBS、Treg或Ryr2-/-Tconv、Ryr2+/+Tconv过继转移到足底中。在7dpi用匀浆的足底组织测试病毒效价。每组4-6只小鼠，试验重复3次。
对于DTH，右足底在6dpi时用紫外线灭活的HSV-1(10 6pfu/20μLPBS)重新激发，然后在7dpi时以左足底作为对照测量足底肿胀。每组16-21只小鼠。
2、试验结果
在疱疹感染模型中，大约注射Treg或Ryr2-/-Tconv 1log时，HSV-1pfu计数开始增加。Ryr2+/+Tconv的输注与单独的病毒(PBS)接种相比没有增加(参见图36)。其次，将同样的模型用于检测DTH响应。2次接种HSV-1后，致敏的足底肿胀，但注射Treg和Ryr2-/-Tconv的足底增厚均减小，而对照组Ryr2+/+Tconv和单纯接种病毒(PBS)均未减小增厚(图37，图38)。
实施例6：小鼠哮喘模型中Ryr2-/-T细胞的作用
1、哮喘模型的制备及试验步骤
卵清蛋白(OVA)诱发气道炎症。在第0天和第14天，两次i.p注射OVA加明矾佐剂(100μg+4mg)以致敏小鼠。在第21、23和25天，在舌后部反复进行气管内OVA冲击。通过i.v过继转移10 6的Treg、Ryr2-/-Tconv、Ryr2+/+Tconv或PBS。在第32天分析BALF浸润和组织学。
2、H&E组织学步骤
皮肤，耳朵，肝脏和其他组织全部固定在4％神经缓冲的福尔马林中，温度为4℃。然 后将样品包埋在石蜡中。切成五到六微米厚的载玻片。所有玻片均用苏木精和曙红染色。
3、试验结果
按照图40A的致敏时间表产生的OVA致敏哮喘模型中，输注Treg和Ryr2-/-Tconv在限制BALF细胞数量上具有同等效果，淋巴细胞和嗜酸性粒细胞的减少程度也相似(图39)。组织学检测见图40B。
实施例7：小鼠结肠炎模型中Ryr2-/-T细胞的作用
1、DSS诱导的结肠炎(IBD)模型的制备及试验步骤
DSS诱导的小鼠实验性结肠炎模型可以参照文献Inhibition of Dectin-1Signaling Ameliorates Colitis by Inducing Lactobacillus-Mediated Regulatory T Cell Expansion in the Intestine(Tang等，Cell Host Microbe 18，183-197，2015)和Myeloid-Derived Suppressor Cells Are Controlled by Regulatory T Cells via TGF-beta during Murine Colitis(Lee等，Cell Rep 17，3219-3232，2016)。简而言之，通过i.v过继转移3×10 6Ryr2-/-Tconv，野生型Tconv或Treg转移到6周龄的雄性C57BL/6小鼠中。第二天，口服4％DSS(w/v)诱导结肠炎(Yeason，MW＝36,000-50,000Da)，持续7天，然后再饲喂普通的饮用水。正常对照小鼠用PBS治疗并给予正常饮用水。在第10天处死小鼠，解剖结肠并测量结肠长度。然后将结肠在4％PFA中于48小时内于4℃固定，用于随后的H&E染色。通过3DHISTECH Pannoramic SCAN(3DHISTECH)观察结肠切片。每组11-18只小鼠。
2、H&E组织学步骤
皮肤，耳朵，肝脏和其他组织全部固定在4％神经缓冲的福尔马林中，温度为4℃。然后将样品包埋在石蜡中。切成五到六微米厚的载玻片。所有玻片均用苏木精和曙红染色。
3、试验结果
按照图42A的诱导时间表产生的结肠炎模型中，注射Treg和Ryr2-/-Tconv组的结肠长度缩短。与Ryr2+/+Tconv相比，Treg和Ryr2-/-Tconv组均能减轻结肠损伤(图41、 42)。
实施例8：小鼠结肠癌模型中Ryr2-/-T细胞的作用
1、结肠癌肿瘤模型的制备及试验步骤
可以参照文献Oxidative stress controls regulatory T cell apoptosis and suppressor activity and PD-L1-blockade resistance in tumor(Maj等，Nat Immunol18，1332-1341，2017)建立移植的肿瘤小鼠模型。简而言之，将MC38结肠癌细胞在PBS中洗涤两次，然后将200μL PBS中的5×10 5MC38细胞与10 6T细胞皮下注射进6周龄雄性C57BL/6小鼠的腹部皮下。从肿瘤注射后第7天起每
Figure PCTCN2020090639-appb-000001
天使用游标卡尺监测肿瘤生长。体积计算为(长×宽×宽)/2。第1组：MC38+PBS；第2组：MC38+Ryr2+/+Tconv；第3组：MC38+Treg；第4组：MC38+Ryr2-/-Tconv。每组十只小鼠。
2、试验结果
在MC38肿瘤模型中，Treg和Ryr2-/-Tconv的注入促进了肿瘤的生长，而PBS和Ryr2+/+Tconv组未见明显作用(图43)。
实施例9：小鼠皮癣模型中Ryr2-/-T细胞的恢复免疫稳态作用
Ryr2-/-Tconv细胞在多器官自身免疫导致的普遍炎症中的作用最为明显，为此，本实施例构建Foxp3缺陷的小鼠模型，验证在Foxp3缺陷的免疫缺陷小鼠在Ryr2-/-Tconv细胞调节下的免疫稳态恢复情况。
1、皮癣小鼠(Scurfy)模型
参照文献A requisite role for induced regulatory T cells in tolerance based on expanding antigen receptor diversity(Haribhai等，Immunity 35，109-122，2011)和TGF-beta-induced Foxp3+ regulatory T cells rescue scurfy mice(Huter等，Eur J Immunol 38，1814-1821，2008)建立了Foxp3缺陷型小鼠的皮癣模型。纯化5×10 6个T细胞，在PBS中洗涤两次，并重悬于50μL PBS中进行腹膜内注射。在新生的同源皮癣小鼠出生后第2天或第3天注射。在前两周，每三或四天进行一次T细胞过继转移，然后 每两周进行一次。过继转移时，记录皮癣模型和雄性WT同窝仔的体重，并监测存活率。所有的小鼠在出生当天采样，通过PCR对sf突变基因进行基因分型，并通过测序验证基因型。FoxP3PCR的引物是5'-CATCCCACTGTGACGAGATG-3'(SEQ ID NO：1)和5'-ACTTGGAGCACAGGGGTCT-3'(SEQ ID NO：2)。对于组织学，在第3周分析注入PBS的小鼠，在第8-12周分析过继转移Treg和Ryr2-/-Tconv的小鼠。
2、H&E组织学步骤
皮肤，耳朵，肝脏和其他组织全部固定在4％神经缓冲的福尔马林中，温度为4℃。然后将样品包埋在石蜡中。切成五到六微米厚的载玻片。所有玻片均用苏木精和曙红染色。
3、试验结果
在出生后2-3天，对皮癣小鼠(FoxP3-/-、C57BL/6)分别注射PBS、Treg、Ryr2+/+Tconv或Ryr2-/-Tconv。不出所料，注射PBS或Ryr2+/+Tconv的小鼠在出生后2-4周内全部死亡。相比之下，所有注射Treg或Ryr2-/-Tconv的小鼠至少存活了20周(图44、25)。而且，观察6个月后，依然未见注射Ryr2-/-Tconv的皮癣小鼠死亡或出现任何明显异常。
组织学结果如图45所示。与预期一样，注射PBS或Ryr2+/+Tconv的小鼠表现出严重的甲状腺炎、脾炎、肺炎、皮炎、肝炎、胰腺炎、胃炎和结肠炎。而注射Treg或者Ryr2-/-Tconv的小鼠均完全治疗了这些病理，即FoxP3缺陷小鼠的综合效应功能缺失得以恢复，表明Ryr2调节T细胞的免疫功能。
综上所述，Ryr2-/-T和Treg细胞在小鼠疱疹感染模型、哮喘模型、结肠炎模型及结肠癌模型中，都显示出了功能的等效性，而且也可以将免疫缺陷的小鼠恢复免疫功能。也就是说，在一些已知Treg具有免疫调节作用的疾病中，Ryr2-/-T均可以发挥同等的作用。
以上详细描述了本发明的优选实施方式，但是，本发明并不限于上述实施方式中的具体细节，在本发明的技术构思范围内，可以对本发明的技术方案进行多种简单变型，这些简单变型均属于本发明的保护范围。
另外需要说明的是，在上述具体实施方式中所描述的各个具体技术特征，在不矛盾的情况下，可以通过任何合适的方式进行组合，为了避免不必要的重复，本发明对各种可能的组合方式不再另行说明。

Claims (41)

  1. 一种T细胞，其特征在于，所述的T细胞中缺失Ryr2基因。
  2. 根据权利要求1所述的T细胞，其特征在于，所述的T细胞中缺失Ryr2基因的7号外显子。
  3. 根据权利要求1所述的T细胞，其特征在于，所述的T细胞中至少缺失Ryr2基因中的一段富含鸟嘌呤的序列；优选的，所述的一段富含鸟嘌呤的序列位于Ryr2基因的起始密码子前200bp中。
  4. 根据权利要求3所述的T细胞，其特征在于，所述的一段富含鸟嘌呤的序列包含GCAGGGG。
  5. 根据权利要求1-4任一所述的T细胞，其特征在于，所述的T细胞为Tconv细胞。
  6. 一种权利要求1-5任一所述的T细胞的制备方法，其特征在于，所述的制备方法选自采用shRNA、siRNA、CRISPR/Cas9、锌指核酸酶技术、转录激活子样效应因子核酸酶技术或归巢核酸内切酶。
  7. 根据权利要求6所述的制备方法，其特征在于，所述的shRNA如SEQ ID NO：5、6所示。
  8. 一种shRNA，其特征在于，所述的shRNA敲低T细胞的Ryr2基因，使得T细胞Ca 2+基础振荡降低、m-Calpain活性降低、与DC细胞的结合强度提高、具有免疫抑制细胞的功能或具有治疗感染性疾病、炎症或肿瘤的功能。
  9. 根据权利要求8所述的shRNA，其特征在于，所述的shRNA为SEQ ID NO：5、6。
  10. 一种T细胞，其特征在于，所述的T细胞中过表达Ryr2。
  11. 根据权利要求10所述的T细胞，其特征在于，所述的T细胞为Treg细胞。
  12. 调控Ryr2表达的试剂、调控Ca 2+基础振荡的试剂、调控m-Calpain活性的试剂、调控T细胞与DC细胞的结合强度的试剂或权利要求1-5和10-11任一所述的T细胞在制备治疗感染性疾病、炎症或肿瘤的药物中的应用。
  13. 调控Ryr2表达的试剂、调控Ca 2+基础振荡的试剂、调控m-Calpain活性的试剂、调控T细胞与DC细胞的结合强度的试剂或权利要求1-5和10-11任一所述的T细胞在治疗感染性疾病、炎症或肿瘤中的应用。
  14. 降低Ryr2表达的试剂、降低Ca 2+基础振荡的试剂、降低m-Calpain活性的试 剂、提高T细胞与DC细胞的结合强度的试剂或权利要求1-5任一所述的T细胞在制备治疗感染性疾病或炎症的药物中的应用。
  15. 降低Ryr2表达的试剂、降低Ca 2+基础振荡的试剂、降低m-Calpain活性的试剂、提高T细胞与DC细胞的结合强度的试剂或权利要求1-5任一所述的T细胞在治疗感染性疾病或炎症中的应用。
  16. 提高Ryr2表达的试剂、提高Ca 2+基础振荡的试剂、提高m-Calpain活性的试剂、降低T细胞与DC细胞的结合强度的试剂或权利要求10-11任一所述的T细胞在制备治疗肿瘤的药物中的应用。
  17. 提高Ryr2表达的试剂、提高Ca 2+基础振荡的试剂、提高m-Calpain活性的试剂、降低T细胞与DC细胞的结合强度的试剂或权利要求10-11任一所述的T细胞在治疗肿瘤中的应用。
  18. 根据权利要求12-15任一所述的应用，其特征在于，所述的感染性疾病选自细菌感染、病毒感染或真菌感染；进一步优选为病毒感染、肺炎伴感染性休克、腹膜炎、菌血症、脓毒症或败血症；所述的病毒感染选自急性病毒感染或慢性病毒感染；优选为流感病毒、副流感病毒、疱疹病毒、麻疹病毒、水泡口炎病毒、乙型肝炎病毒、丙型肝炎病毒、人免疫缺陷病毒、淋巴细胞脉络丛脑膜炎病毒或人乳头瘤病毒。
  19. 根据权利要求12-15任一所述的应用，其特征在于，所述的炎症选自系统性红斑狼疮、类风湿性关节炎、银屑病关节炎、硬皮病、哮喘、特应性皮炎、器官特异性炎性疾病、过敏、毛囊炎、扁桃体炎、肺炎、肝炎、肾炎、痤疮、自身免疫性疾病、慢性前列腺炎、肾小球肾炎、超敏反应、结肠炎、炎性肠道疾病、盆腔炎、再灌注损伤、移植排斥反应、血管炎或间质性膀胱炎。
  20. 根据权利要求12-13和16-17任一所述的应用，其特征在于，所述的肿瘤选自前列腺癌、乳腺癌、肝癌、胶质瘤、肠癌、宫颈癌、非小细胞肺癌、肺癌、胰腺癌、胃癌、膀胱癌、皮肤癌、横纹肌癌、舌鳞癌、鼻咽癌、卵巢癌、胎盘绒毛癌、淋巴瘤、白血病、直肠腺癌、成神经管细胞瘤、脑膜瘤、神经纤维瘤、室管膜瘤、神经鞘瘤、星形细胞瘤、黑色素瘤、间皮瘤、骨髓瘤、慢性粒细胞白血病、急性髓性白血病、骨髓增生异常综合征、慢性淋巴细胞白血病、表皮样癌、结肠癌、胸腺癌、血液癌、头颈癌或口咽癌。
  21. 调控Ryr2表达在调控Ca 2+基础振荡、调控m-Calpain活性或者提高T细胞与 DC细胞的结合强度中的应用。
  22. 调控Ca 2+基础振荡在调控m-Calpain活性或调控T细胞与DC细胞的结合强度中的应用。
  23. 调控m-Calpain活性在调控T细胞与DC细胞的结合强度中的应用。
  24. 根据权利要求21-23任一所述的应用，其特征在于，所述的调控为降低或提高。
  25. 一种Ryr2的拮抗剂，其特征在于，所述的拮抗剂靶向Ryr2基因7号外显子或Ryr2基因中的一段富含鸟嘌呤的序列；优选的，所述的一段富含鸟嘌呤的序列位于Ryr2基因的起始密码子前200bp中；进一步优选的，所述的一段富含鸟嘌呤的序列包含GCAGGGG。
  26. 一种调控T细胞与DC细胞的结合强度的方法，其特征在于，所述的方法包括调控Ryr2表达。
  27. 根据权利要求25所述的方法，其特征在于，所述的调控为降低或提高；优选的，所述的降低T细胞与DC细胞的结合强度的方法包括提高Ryr2表达，提高T细胞与DC细胞的结合强度的方法包括降低Ryr2表达。
  28. 根据权利要求26所述的方法，其特征在于，降低Ryr2表达包括过表达T细胞中的FoxP3或加入Ryr2抑制剂，提高Ryr2表达包括降低T细胞中的FoxP3的表达或加入Ryr2驱动剂；其中，所述的Ryr2抑制剂选自权利要求25所述的Ryr2的拮抗剂、利阿诺定、硝苯呋海因或JTV519；所述的Ryr2驱动剂选自烟酸酰胺腺嘌呤二核苷酸磷酸、咖啡因、对氯代间甲酚、兰尼碱、氯虫苯甲酰胺、氰虫酰胺、B型肾上腺素、4-氯-3-甲基苯酚、溴氰虫酰胺、环溴虫酰胺、环腺苷二磷酸核糖、苏拉明钠、氟氰虫酰胺或三氟拉嗪。
  29. 根据权利要求26或27所述的方法，其特征在于，降低Ryr2表达包括敲低或敲除T细胞中的Ryr2基因；优选的，敲低或敲除T细胞中的Ryr2基因中一段富含鸟嘌呤的序列；进一步优选的，所述的一段富含鸟嘌呤的序列位于Ryr2基因的起始密码子前200bp中；最为优选的，所述的一段富含鸟嘌呤的序列包含GCAGGGG。
  30. 根据权利要求26或27所述的方法，其特征在于，所述的降低Ryr2表达包括使T细胞中缺失Ryr2基因的7号外显子。
  31. 一种降低T细胞中的Ryr2表达、降低Ca 2+基础振荡、降低m-Calpain活性的方法，其特征在于，所述的方法包括过表达T细胞中的FoxP3、敲低或敲除T细胞中的 Ryr2基因或加入Ryr2抑制剂。
  32. 一种提高T细胞中的Ryr2表达、提高Ca 2+基础振荡、提高m-Calpain活性的方法，其特征在于，所述的方法包括降低T细胞中的FoxP3的表达或加入Ryr2驱动剂。
  33. 一种将Tconv细胞转化为与Treg细胞类似功能的方法，其特征在于，所述的方法包括过表达Tconv细胞的FoxP3或者敲低或敲除Tconv细胞的Ryr2基因。
  34. 根据权利要求32所述的方法，其特征在于，所述的方法包括敲低或敲除Tconv细胞中的Ryr2基因中的一段富含鸟嘌呤的序列；优选的，所述的一段富含鸟嘌呤的序列位于Ryr2基因的起始密码子前200bp中；进一步优选的，所述的一段富含鸟嘌呤的序列包含GCAGGGG。
  35. 根据权利要求32所述的方法，其特征在于，所述的方法包括敲除Ryr2基因的7号外显子。
  36. 一种治疗感染性疾病或炎症的方法，其特征在于，所述的方法包括向个体施加有效量的权利要求1-5任一所述的T细胞。
  37. 一种治疗感染性疾病或炎症的方法，其特征在于，所述的方法选自使个体的T细胞中的FoxP3过表达、降低T细胞中的Ryr2表达、降低Ca 2+基础振荡、降低m-Calpain活性、提高T细胞与DC细胞的结合强度或加入Ryr2抑制剂。
  38. 根据权利要求35或36所述的方法，其特征在于，所述的感染性疾病选自细菌感染、病毒感染或真菌感染；进一步优选为病毒感染、肺炎伴感染性休克、腹膜炎、菌血症、脓毒症或败血症；所述的病毒感染选自急性病毒感染或慢性病毒感染；优选为流感病毒、副流感病毒、疱疹病毒、麻疹病毒、水泡口炎病毒、乙型肝炎病毒、丙型肝炎病毒、人免疫缺陷病毒、淋巴细胞脉络丛脑膜炎病毒或人乳头瘤病毒；所述的炎症选自系统性红斑狼疮、类风湿性关节炎、银屑病关节炎、硬皮病、哮喘、特应性皮炎、器官特异性炎性疾病、过敏、毛囊炎、扁桃体炎、肺炎、肝炎、肾炎、痤疮、自身免疫性疾病、慢性前列腺炎、肾小球肾炎、超敏反应、结肠炎、炎性肠道疾病、盆腔炎、再灌注损伤、移植排斥反应、血管炎或间质性膀胱炎。
  39. 一种治疗肿瘤的方法，其特征在于，所述的方法包括向个体施加有效量的权利要求10-11任一所述的T细胞。
  40. 一种治疗肿瘤的方法，其特征在于，所述的方法选自使个体降低T细胞中的 FoxP3的表达、提高T细胞中的Ryr2表达、提高Ca 2+基础振荡、提高m-Calpain活性、降低T细胞与DC细胞的结合强度或加入Ryr2驱动剂。
  41. 根据权利要求38或39所述的方法，其特征在于，所述的肿瘤选自前列腺癌、乳腺癌、肝癌、胶质瘤、肠癌、宫颈癌、非小细胞肺癌、肺癌、胰腺癌、胃癌、膀胱癌、皮肤癌、横纹肌癌、舌鳞癌、鼻咽癌、卵巢癌、胎盘绒毛癌、淋巴瘤、白血病、直肠腺癌、成神经管细胞瘤、脑膜瘤、神经纤维瘤、室管膜瘤、神经鞘瘤、星形细胞瘤、黑色素瘤、间皮瘤、骨髓瘤、慢性粒细胞白血病、急性髓性白血病、骨髓增生异常综合征、慢性淋巴细胞白血病、表皮样癌、结肠癌、胸腺癌、血液癌、头颈癌或口咽癌。
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