Biomarker or target for diagnosing or treating immune system diseases

Abstract

A biomarker or target for diagnosing or treating immune system diseases, comprising Dock2, AP-1 and / or Sub1. The Dock2, AP-1 and / or Sub1 are highly expressed in a plurality of immune system diseases, such that down-regulating the expression of the Dock2, AP-1 and / or Sub1 or functions thereof can be used for treating a plurality of immune system diseases.

Description

A biomarker or target for diagnosing or treating immune system diseases Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to a biomarker or target for diagnosing or treating immune system diseases. Background Art

[0002] A fundamental characteristic of the immune system is its unique ability to fully distinguish between self and non-self to prevent abnormal infection and colonization. Maintaining a state of immune homeostasis requires precise control of the activity and function of immune cells to avoid unnecessary responses against the self. Central and peripheral lymphoid organs maintain immune tolerance, allowing the body's self-reactive T and B lymphocytes to be eliminated or silenced under normal circumstances. The immune system has evolved multiple mechanisms to control self-reactivity. Defects in one or more of these mechanisms may lead to a breakdown of tolerance, allowing self-reactive lymphocytes to evade immune surveillance and cause autoimmune diseases. A wide range of autoimmune diseases have been described, with variable age of onset, tissue distribution, and clinical and functional impact. Most of these diseases are incurable and require lifelong treatment. Autoimmune diseases affect approximately one in ten individuals and have complex etiologies, variable symptoms and signs, and incompletely understood pathogenesis.

[0003] Inflammatory bowel disease (IBD) is a chronic, relapsing intestinal disorder. The etiology and pathophysiology of IBD are complex and multifactorial, including genetic, environmental, and microbial components. Currently, IBD is difficult to diagnose and has no cure, necessitating an urgent need for effective strategies to address this issue.

[0004] Multiple sclerosis (MS) is a typical autoimmune disease characterized by demyelination of the white matter of the central nervous system (CNS). The disorder of immune tolerance mediated by T cells leads to inflammatory damage to the myelin sheath and axons. Because of its high incidence, chronic course and tendency to occur in young people, it has become one of the most important neurological diseases. Although the exact cause of MS is still unclear, it is generally believed that myelin-specific CD4 + The activation of T cells is a central step in the initiation of neuroinflammation. The strongest genetic risk factors for multiple sclerosis are HLA-DRA, interleukin (IL)-2R, and IL-7R genes, which emphasizes the role of CD4 + The importance of T cells. The fact that adoptive transfer of bone marrow-specific T cells can induce EAE further strengthens the view that MS is a T cell-mediated autoimmune disease. +The observation that T cell transfer is sufficient to initiate EAE has led most studies of the pathogenic mechanism to focus on CD4 + The pathogenic role of T cells.

[0005] The central nervous system has several unique barriers to maintain homeostasis and limit the infiltration of leukocytes. To initiate CNS autoimmunity, self-antigen-specific T cells are activated in the periphery and enter the CNS, where they are reactivated by APCs presenting self-antigens, which triggers the release of cytokines that activate and recruit other inflammatory cells, thereby causing inflammation. However, the CNS uses various barriers such as the blood-brain barrier (BBB) ​​and the blood-cerebrospinal fluid barrier (BCSFB) to restrict the entry of T cells. To overcome these barriers, the coordinated use of selectins, integrins, and chemokines is required for efficient migration into the CNS. In the context of immune dysregulation, blocking the migration of immune cells into the CNS has been shown to be an effective approach for treating MS. A recent study found that DICAM (dual immunoglobulin domain containing cell adhesion molecule) promotes neuroinflammation by facilitating the migration of Th17 lymphocytes across the blood-brain barrier endothelium. Pharmacological neutralization of DICAM reduced the trafficking of Th17 cells across the blood-brain barrier in both mice and humans and alleviated disease symptoms in a mouse model of autoimmune encephalomyelitis. These results suggest that blocking DICAM with monoclonal antibodies may be a promising therapeutic approach. Natalizumab is an approved monoclonal antibody designed to specifically impede this process by targeting the integrin VLA4, which is expressed on virtually all immune cell subsets. Although natalizumab has demonstrated some efficacy in treating MS patients, it has been associated with severe rebound MS activity after discontinuation and life-threatening central nervous system infections. This is due to the impaired immune surveillance of the central nervous system caused by the ubiquitous expression of VLA4 by both protective and pathogenic leukocytes. Therefore, the search for novel targets with both efficacy and minimal side effects is crucial.

[0006] The transcriptional activator SUB1 (also known as PC4) was identified as a coactivator of RNA polymerase II-dependent transcription. It contains a unique, conserved nonspecific DNA-binding domain and is involved in diverse DNA-dependent processes, including replication, DNA repair, and transcription. Complete knockout of SUB1 in mice results in embryonic lethality, reflecting its diverse functions. As a transcriptional regulator, multiple studies have demonstrated that SUB1 can directly initiate transcription of multiple genes, such as PLK1, BUB1B, and C-MYC. Furthermore, SUB1 interacts with distinct domains of activators, including VP16, GAL4, AP2, HIV-TAT, P53, and SMYD3, to regulate their functions. At present, most of the research on the function of SUB1 is still focused on tumor cells. For example, the non-patent literature: MicroRNA-101 regulated transcriptional modulator SUB1 plays a role in prostate cancer (Chakravarthi BV, Goswami MT, et al., Oncogene. 2016 Dec 8; 35(49): 6330-6340) disclosed that SUB1 expression is elevated in prostate cancer cells; another non-patent literature: The prognostic value of SUB1 in liver cancer patients (Huang Yongping, Tang Dejun, Liu Dong, et al., Lingnan Modern Clinical Surgery, 2020, 20(04) disclosed that SUB1 is highly expressed in liver cancer tissues, and SUB1 can be used as a prognostic factor for overall survival and disease-free survival of liver cancer patients. However, the relationship between SUB1 and immune system diseases has not been disclosed in the prior art. Summary of the Invention

[0007] This application found that Sub1 is expressed in CD4 + Highly expressed in T cells. Sub1 deletion downregulates Dock2 expression, inhibiting GTPase activity and F-actin polymerization, thereby inhibiting CD4 + T cells migrate to the central nervous system and block the occurrence of immune system diseases (especially multiple sclerosis or colitis). Furthermore, the mechanism was determined to be that Sub1 regulates Dock2 by promoting the transcriptional expression of Junb gene, and that Sub1 and JunB bind to form Sub1 / JunB complex, which promotes the opening of transcription of Dock2 gene site and further regulates the expression of Dock2. + The key regulator of T cell migration is to block CD4 +The potential therapeutic target of T cell migration to the central nervous system is of great significance for curbing the occurrence and development of immune system diseases (especially multiple sclerosis or colitis).

[0008] In a first aspect, the present invention provides a use of a biomarker or target in the preparation of a product for diagnosis, monitoring, severity assessment, efficacy assessment or prognosis assessment, and / or prevention or treatment of an immune system disease, wherein the biomarker or target comprises one or more of Dock2, AP-1 or Sub1.

[0009] Preferably, the biomarkers or targets are genes and / or proteins.

[0010] Among them, Dock2 is cytoplasmic division protein 2 or its encoding gene; Sub1 is an auxiliary activator of RNA polymerase II-dependent transcription or its encoding gene.

[0011] Sub1 is a 127-amino acid DNA-binding protein. Its N-terminus contains two serine- and acidic-rich (SEAC) regions separated by a lysine-rich (K-rich) region, encompassing amino acids 1-63. The C-terminus of Sub1 (spanning amino acids 64-127) contains a high-affinity single-stranded DNA (ssDNA) binding domain that has a structural function in ssDNA.

[0012] AP-1 is a transcription factor (activator protein-1), a dimeric complex that forms highly conserved homodimers or heterodimers from one or more proteins of the basic leucine zipper structure Jun protein family, Fos protein family, activating transcription factor ATF (activating transcription factor) protein family, or musculoaponeurotic fibrosarcoma (MAF) protein family, which can activate the transcription of downstream target genes.

[0013] In a specific embodiment of the present invention, the AP-1 includes one or more proteins from the Jun protein family, the Fos protein family, the ATF protein family or the MAF protein family.

[0014] Preferably, the Jun protein family includes one or more of Jun (eg, v-Jun or c-Jun), JunB or JunD.

[0015] Preferably, the Fos protein family includes one or more of Fos (v-Fos or c-Fos), FosB, Fra-1, and Fra-2.

[0016] Preferably, the ATF protein family includes one or more of ATF2, ATF3 / LRF1, B-ATF, JDP1 or JDP2.

[0017] Preferably, the MAF protein family includes one or more of c-Maf, MafB, MafA, MafG, MafF, MafK or Nrl.

[0018] In a specific embodiment of the present invention, the AP-1 is a dimer composed of Jun (one or more of c-Jun, JunB or JunD) and / or Fos (one or more of c-Fos, FosB, Fra-1 or Fra-2) multigene family members.

[0019] In a specific embodiment of the present invention, the AP-1 is a protein of the Jun protein family, preferably JunB.

[0020] Specifically, JunB (JunB Proto-Oncogene, AP-1 Transcription Factor Subunit) belongs to the basic zipper (bZIP) protein family, which contains a conserved leucine zipper (LZ) domain and a basic motif (BM) domain rich in basic amino acids.

[0021] Preferably, the diagnosis, monitoring, severity assessment, efficacy assessment and / or prognostic assessment of immune system diseases includes detecting the presence or expression level of biomarkers.

[0022] Preferably, the expression level of the biomarker is the expression level of the mRNA and / or protein of the biomarker.

[0023] Preferably, the detecting the presence or expression level of the biomarker comprises using a reagent.

[0024] Further preferably, the reagents may include but are not limited to reagents required for PCR, reagents required for RPA, reagents required for LAMP, reagents required for ERA, reagents required for RCA, reagents required for Western blot, reagents required for immunohistochemistry, reagents required for sequencing, reagents required for liquid chromatography or reagents required for mass spectrometry, etc.

[0025] Preferably, when the expression level of the biomarker mRNA and / or protein is significantly higher than a threshold, it indicates that the individual suffers from an immune system disease, wherein the threshold is obtained in an early stage through experiments.

[0026] Preferably, the prevention and / or treatment of immune system diseases includes regulating the expression level or function of the target.

[0027] Preferably, the expression level of the target is the expression level of the target mRNA and / or protein.

[0028] Preferably, the regulation includes up-regulation or down-regulation.

[0029] Preferably, regulating the function of Sub1 includes regulating its DNA binding function, or its binding function with JunB.

[0030] Preferably, the DNA binding function of Sub1 includes ssDNA binding function.

[0031] Preferably, the DNA binding function of Sub1 includes binding to the promoter site of JunB.

[0032] In one embodiment of the present invention, the regulation of Sub1 function comprises mutating Sub1. Preferably, the mutation comprises one or more of F77A, K78G, or K80G.

[0033] Preferably, regulating the function of JunB includes regulating the function of its BM domain, such as the function of binding to Sub1 or the function of binding to Dock2. Preferably, the site binding to Dock2 includes a promoter or an enhancer.

[0034] Preferably, said downregulation comprises knockout or knockdown.

[0035] Preferably, the knockout or knockdown includes but is not limited to the use of CRISPR / Cas system, tissue-specific knockout system or introduction of interfering RNA targeting Sub1.

[0036] Preferably, the down-regulating the expression of Sub1 comprises using a reagent.

[0037] Preferably, the reagents include but are not limited to reagents required for knockout or knockdown (such as materials required for tissue-specific knockout systems, materials required for CRISPR / Cas systems or interfering RNA, etc.), or reagents required for downregulating gene transcription and / or translation, etc.

[0038] Preferably, the interfering RNA includes but is not limited to one or more of siRNA, dsRNA, shRNA, aiRNA or microRNA (miRNA, such as microRNA-101).

[0039] Preferably, the Cas protein used in the CRISPR / Cas system is selected from Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas5d, Cas5t, Cas5h, Cas5a, Cas6, Cas7, Cas8, Cas9, Cas10, Csy1, Csy2, Csy3, Csy4, Cse1, Cse2, Cse3, Cse4, Cse5e, Cscl, Csc2, Csa5, Csn1, Csn2, Csml, Csm2, Csm3, Csm4, Csm5, Csm6, Cmrl, Cmr3, Cmr4, Cmr5, Cmr6, Csbl, Csb2, Csb3, Csx17, Csx14, CsxlO, Csx16, CsaX, Csx3, Csxl, CsxlS, Csfl, Csf2, CsO, Csf4, Csdl, Csd2, Cstl, Cst2, Cshl, Csh2, Csal, Csa2, Csa3, Csa4, Csa5, C2cl, C2c2, C2c3, Cpfl, CARF, DinG, homologs thereof, or modified forms thereof.

[0040] The tissue-specific knockout technology includes but is not limited to the Cre / loxp recombinase system. The Cre / loxp recombinase system is a site-specific recombinase technology that can perform deletions, insertions, translocations, and inversions at specific sites on DNA. This system can be used to modify DNA in cells for specific cell types or using specific external stimuli. The Cre recombinase recognizes the inverted repeat sequences at both ends of the loxP site and combines to form a dimer. This dimer then combines with the dimer on another loxP site to form a tetramer. The loxP site is directional, and the two sites connected by the tetramer are parallel in direction. The DNA sequence between the two loxP sites is then cut by the Cre recombinase. Then, DNA ligase quickly and efficiently connects these chains. If the two loxP sites are located on the same DNA chain and in the same direction, Cre recombinase mediates the excision of the sequence between the loxP sites; if the two loxP sites are located on the same DNA chain and in opposite directions, Cre recombinase mediates the inversion of the sequence between the loxP sites; if the two loxP sites are located on different DNA chains or chromosomes, Cre recombinase mediates the exchange of the two DNA chains or chromosomal translocation.

[0041] In a specific embodiment of the present invention, the two loxP sites in the Cre / loxP recombinase system are set at both ends of the target gene and arranged in the same direction (flox / flox), and then the Cre recombinase is introduced to mediate the excision of the sequence between the loxP.

[0042] Preferably, the downregulation of the expression level of Sub1 means that Sub1 cannot be transcribed or the transcribed protein is not expressed or the activity is reduced, preferably including knocking out all or part of exons 1 to 5 of the Sub1 gene, such as any one or a combination of two or more of exons 1, 2, 3, 4 or 5.

[0043] In one embodiment of the present invention, downregulating the expression of Sub1 comprises knocking out exon 3 and exon 4 of the Sub1 gene, preferably introns 3-4. Preferably, the knockout comprises tissue-specific knockout, such as inserting LoxP sites at the 5' end of exon 3 and the 3' end of exon 4, followed by the introduction of Cre recombinase.

[0044] Preferably, the down-regulation of Sub1 expression level comprises knocking out the Sub1 gene in T cells. The T cells may be CD4 + T cells or CD8 + T cells, more preferably CD4 + CD8 + T cells. Preferably, the knockout or knockdown comprises the combined use of a tissue-specific knockout system and a CRISPR / Cas system. Further preferably, the knockout or knockdown of Sub1 comprises inserting LoxP sites at the 5' end of exon 3 and the 3' end of exon 4 of the Sub1 gene using the CRISPR / Cas system, and then introducing Cre recombinase (e.g., T cell-specific Cre recombinase, including but not limited to CD4-Cre and / or CD8-Cre).

[0045] Preferably, downregulating the expression level of JunB comprises knocking out or knocking down JunB or its BM domain.

[0046] It should be understood that Sub1 enhances histone modifications of chromatin (such as methylation modification or acetylation modification, histone methylation modification includes H3K4me1: indicating monomethylation of lysine 4 on histone 3; histone acetylation modification includes H3K27ac: indicating acetylation of lysine 27 on histone 3) to increase the openness of chromatin, thereby recruiting AP-1 transcription factors to bind to the Dock2 promoter to drive transcription. At the same time, Sub1 further stabilizes the occupancy of AP-1 (especially JunB) on the Dock2 promoter through interaction with AP-1 (especially JunB), thereby promoting AP-1 (especially JunB)-mediated transcription activation.

[0047] Preferably, downregulating Sub1 prevents and / or treats immune system diseases by reducing JunB expression or function.

[0048] Preferably, downregulating Sub1 prevents and / or treats immune system diseases by reducing the expression or function of Dock2.

[0049] Preferably, downregulating Sub1 prevents and / or treats immune system diseases by blocking or reducing the binding of Sub1 to JunB.

[0050] Preferably, the biomarker or target is one or more biomarkers or targets selected from the central nervous system, body fluids, cells, tissues or organs.

[0051] Preferably, the body fluid comprises blood or cerebrospinal fluid.

[0052] Preferably, the tissue includes one or more of brain tissue, spinal cord, spleen or lymphatic tissue.

[0053] Preferably, the cells include immune cells. More preferably, the immune cells include one or more of lymphocytes, dendritic cells, monocytes / macrophages, granulocytes or mast cells.

[0054] Preferably, the lymphocytes include T cells.

[0055] In a specific embodiment of the present invention, the biomarker or target is a biomarker or target in an immune cell, such as a T cell.

[0056] Preferably, the T cells include Tconv cells or Treg cells;

[0057] Preferably, the T cells include CD8 + T cells, CD4 + T cells, CD25 + T cells, CD25 - T cells, Foxp3 + T cells, CD127 + T cells or CD45RB + One or more types of T cells.

[0058] Preferably, the product includes a reagent for detecting the presence or expression level of a biomarker (the expression level of the biomarker is preferably the expression level of the mRNA and / or protein of the biomarker), or the product includes a reagent for regulating the expression level of a target or its function (the expression level of the target is preferably the expression level of the mRNA and / or protein of the target).

[0059] In a specific embodiment of the present invention, the product includes a drug, or a kit, test paper, mass spectrometer or biochip for diagnosis, monitoring, severity assessment, efficacy assessment and / or prognosis assessment of immune system diseases.

[0060] Preferably, the drug includes an agent that regulates the expression level of the target or its function. Further preferably, the regulation includes up-regulation and / or down-regulation.

[0061] In a specific embodiment of the present invention, the drug downregulates the expression level of target mRNA and / or protein or its function. The drug can be an antibody or a small molecule inhibitor, etc. For example, the drug can be one or more of anti-Dock2 antibody, anti-Sub1 antibody, interfering RNA (preferably microRNA, such as microRNA-101) or IRF4 transcription factor antibody.

[0062] Downregulating the expression of Sub1 can downregulate the expression or function of JunB.

[0063] Downregulating the function of Sub1 (particularly the ssDNA binding function) can downregulate the expression or function of JunB.

[0064] Downregulating the expression of Sub1 and / or downregulating the function of Sub1 (particularly the ssDNA binding function) can downregulate the binding of Sub1 to JunB.

[0065] Downregulating Sub1 expression further downregulates Dock2 expression by downregulating JunB expression or its function.

[0066] Downregulating the function of Sub1 (particularly the ssDNA binding function) further downregulates the expression of Dock2 by downregulating the expression or function of JunB.

[0067] Downregulating the expression of Sub1 and / or downregulating the function of Sub1 (particularly the ssDNA binding function) further downregulates the expression of Dock2 by downregulating the binding of Sub1 to JunB.

[0068] Downregulation of Sub1 can downregulate the expression of Dock2.

[0069] Downregulating the expression or function of JunB or its BM domain can downregulate the expression of Dock2.

[0070] Downregulating the expression or function of Sub1, downregulating the expression or function of JunB or its BM domain, downregulating the binding of Sub1 to JunB and / or downregulating the expression of Dock2 can block or slow down the T cell (including CD4 T cells) mediated by Dock2. + T cells, CD8 +T cells or CD4 + CD8 + T cells) migration, thereby preventing or treating immune system diseases.

[0071] Preferably, the T cell migration includes CD4 + T cell migration into the central nervous system.

[0072] Downregulating the expression or function of Sub1 can further downregulate the expression of Dock2, inhibit GTPase activity and F-actin polymerization, and thus inhibit CD4 + T cells migrate to the central nervous system and block the occurrence of immune system diseases.

[0073] Preferably, the drug further comprises pharmaceutically acceptable excipients.

[0074] Preferably, the pharmaceutically acceptable excipient is selected from one or a combination of two or more of a diluent, an absorbent, a wetting agent, a binder, a disintegrant, a lubricant, a solvent, a pH regulator, a buffer, an antioxidant, a metal ion chelator, an antibacterial agent or an isotonicity regulator.

[0075] Preferably, the drug can be in the form of a suspension, powder, granule, tablet, aqueous solution, cream, gel or emulsion. The various dosage forms of the drug can be prepared according to conventional production methods in the pharmaceutical field.

[0076] Preferably, the pharmaceutical preparation is a unit dose preparation.

[0077] Preferably, the active ingredient (agent for regulating the expression of the target or its function) contained in the drug is calculated by volume or mass ratio to include any value between 0.001% and 99.9%, for example, 0.001, 0.01, 0.1, 0.2, 0.3, 0.4, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98, 99, 99.1, 99.5, 99.9%, etc.

[0078] According to the requirements of the specific embodiment, the medicine may also contain other suitable therapeutic agents.

[0079] The medicine is suitable for parenteral administration such as, for example, preparations for administration by intravenous, intramuscular, intradermal and subcutaneous routes include aqueous and non-aqueous isotonic sterile injections, which may contain antioxidants, buffers, antibacterials, and solutes that render the preparation isotonic with the recipient's blood, and aqueous and non-aqueous sterile suspensions, which may contain suspending agents, solubilizers, thickeners, stabilizers or preservatives. The preparations may be present in unit dose or multi-dose sealed containers such as ampoules and bottles. Injectable solutions and suspensions can be prepared from sterile powders, granules and tablets of the types previously described.

[0080] Preferably, the administration of the drug includes but is not limited to intravenous infusion, local administration, intraperitoneal administration or intrathecal administration, etc.

[0081] Preferably, the kit, test paper, mass spectrometer or biochip specifically detects the presence or expression level of a biomarker, and preferably, the kit, test paper, mass spectrometer or biochip contains reagents for detecting the presence or expression level of a biomarker.

[0082] Preferably, the expression level of the biomarker is the expression level of the biomarker mRNA and / or protein.

[0083] Preferably, the kit is selected from an immunomagnetic bead detection kit, an agglutination detection kit, a liquid phase chip detection kit, an enzyme-linked immunosorbent assay kit, a fluorescent immunoassay kit or a mass spectrometry detection kit.

[0084] The kit further comprises a diluent, a cleaning solution, a buffer solution, a substrate and / or a stop solution.

[0085] Preferably, the immune system disease is suppression of T cells (such as CD4 + T cells, CD8 + T cells or CD4 + CD8 + T cell) migration is beneficial for the prevention or treatment of immune system diseases.

[0086] More preferably, the T cell migration is Dock2-mediated T cell migration, and the T cell migration includes T cell migration to the central nervous system.

[0087] Preferably, the immune system disease includes one or more of autoimmune diseases, immunoproliferative diseases, inflammatory diseases or immunodeficiency diseases.

[0088] Preferably, the immune system disease is an autoimmune disease of central nervous system white matter demyelinating lesions.

[0089] Preferably, the inflammation includes but is not limited to infectious diseases or hypersensitivity diseases.

[0090] Preferably, the inflammatory disease comprises one or more of asthma, rheumatoid arthritis, chronic obstructive pulmonary disease, psoriasis, colitis, chronic inflammatory demyelinating polyneuropathy, giant cell arteritis, glomerulonephritis, juvenile idiopathic arthritis, synovitis, osteitis, polymyositis, Wegener's granulomatosis, psoriasis, Behçet's syndrome, polyarteritis nodosa, Takayasu's arteritis, graft-versus-host disease, adult-onset Still's disease, hidradenitis suppurativa, antiphospholipid syndrome, cryoglobulinemic vasculitis, hidradenitis suppurativa, idiopathic membranitis, autoimmune hepatitis, erythema nodosum, myelodysplastic syndrome or amyloidosis.

[0091] Preferably, the autoimmune disease comprises one or more of rheumatoid arthritis, multiple sclerosis, autoimmune hemolytic anemia, chronic inflammatory demyelinating polyneuropathy, glomerulonephritis, juvenile idiopathic arthritis, polymyalgia rheumatica, systemic lupus erythematosus, thrombotic / idiopathic thrombocytopenic purpura, Sjögren's syndrome, polymyositis, Wegener's granulomatosis, Takayasu arteritis, sarcoidosis, pyoderma gangrenosum, Kawasaki's disease, relapsing polychondritis, celiac disease, autoimmune hepatitis, myasthenia gravis, SAPHO syndrome, Graves' disease, myelodysplastic syndrome, pemphigus or amyloidosis.

[0092] Preferably, the immunoproliferative disease includes one or more of myeloma (including multiple myeloma), leukemia (acute lymphocytic leukemia, B-cell chronic lymphocytic leukemia, naked cell acute lymphocytic leukemia, histo-monocyte acute monocytic leukemia, hairy cell leukemia), lymphoma (including lymphoblastoma, non-Hodgkin's lymphoma, Hodgkin's lymphoma, Burkitt's lymphoma), Sézary syndrome, mycosis fungoides, essential macroglobulinemia, infectious mononucleosis or acute histiocytosis.

[0093] Preferably, the immune system disease includes one or more of systemic lupus erythematosus, rheumatoid arthritis, antineutrophil cytoplasmic antibody-associated vasculitis, Stevens-Johnson syndrome, Sjögren's syndrome, systemic vasculitis, scleroderma, dermatomyositis, mixed connective tissue disease, Hashimoto's thyroiditis, primary myxedema, ankylosing spondylitis, adult-onset Still's disease, Behçet's syndrome, systemic sclerosis, multiple sclerosis, autoimmune hepatitis, polyarteritis nodosa or colitis.

[0094] Preferably, the colitis includes one or more of inflammatory bowel disease (such as ulcerative colitis or Crohn's disease), infectious colitis, ischemic colitis or pseudomembranous colitis.

[0095] In a specific embodiment of the present invention, the immune system disease is multiple sclerosis or colitis.

[0096] In a second aspect, the present invention provides a biomarker or target for diagnosing and / or treating immune system diseases, wherein the biomarker or target comprises one or more of Dock2, AP-1 or Sub1.

[0097] In a third aspect of the present invention, an immune cell is provided, wherein the target expression level in the immune cell is downregulated, and / or the immune cell comprises an agent for downregulating the target expression level or an agent for downregulating the target function; the target comprises one or more of Dock2, AP-1 or Sub1.

[0098] Preferably, the target expression level is the expression level of target mRNA and / or protein.

[0099] Preferably, downregulation of target expression includes knockout or knockdown, preferably using a CRISPR / Cas system, tissue-specific knockout, or introduction of interfering RNA targeting the target.

[0100] Preferably, downregulating the expression of Sub1 includes knocking out all or part of exons 1 to 5 of the Sub1 gene, further preferably knocking out exon 3 and exon 4 of the Sub1 gene, and preferably introns 3-4 are knocked out.

[0101] Preferably, down-regulating the expression level of JunB includes knocking out the BM domain of JunB.

[0102] Preferably, downregulating the function of Sub1 includes downregulating the DNA binding function of Sub1, or the function of binding to JunB.

[0103] Preferably, the DNA binding function of Sub1 includes ssDNA binding function.

[0104] Preferably, the DNA binding function of Sub1 includes binding to the promoter site of JunB.

[0105] Preferably, downregulating the function of JunB includes downregulating the function of its BM domain, preferably the function of binding to Sub1 or the function of binding to Dock2. Preferably, the site binding to Dock2 includes a promoter or an enhancer.

[0106] Preferably, the immune cells include one or more of lymphocytes, dendritic cells, monocytes / macrophages, granulocytes or mast cells.

[0107] Preferably, the lymphocytes include T cells.

[0108] Preferably, the T cells include Tconv cells or Treg cells.

[0109] Preferably, the T cells include one or more of CD8+T cells, CD4+T cells, CD25+T cells, CD25-T cells, Foxp3+T cells, CD127+T cells or CD45RB+T cells.

[0110] A fourth aspect of the present invention provides a method for blocking or reducing the binding of Sub1 to JunB in the preparation of a product for preventing and / or treating immune system diseases.

[0111] Preferably, said blocking or reducing the binding between Sub1 and JunB comprises:

[0112] A) downregulating the expression or function of Sub1; or

[0113] B) downregulating JunB expression or its function;

[0114] Preferably, blocking or reducing the binding of Sub1 to JunB achieves prevention and / or treatment of immune system diseases by reducing the expression of Dock2. Preferably, the definition of immune system diseases is the same as that of the first aspect of the present invention.

[0115] In a fifth aspect, the present invention provides a method for screening drugs for preventing or treating immune system diseases, wherein the method comprises adding the drug to be tested to a sample and detecting the expression level or function of the target, wherein the target comprises one or more of Dock2, AP-1 or Sub1.

[0116] Preferably, the expression level of the target is the expression level of the target mRNA and / or protein.

[0117] Preferably, the sample includes but is not limited to one or more of the central nervous system, body fluids, cells, tissues or organs.

[0118] Preferably, the body fluid comprises blood or cerebrospinal fluid.

[0119] Preferably, the tissue includes one or more of brain tissue, spinal cord, spleen or lymphatic tissue.

[0120] Preferably, the cells include immune cells. More preferably, the immune cells include one or more of lymphocytes, dendritic cells, monocytes / macrophages, granulocytes or mast cells.

[0121] Preferably, the lymphocytes include T cells.

[0122] Preferably, the T cells include Tconv cells or Treg cells.

[0123] Preferably, the T cells include CD8 + T cells, CD4 + T cells, CD25 + T cells, CD25 - T cells, Foxp3 + T cells, CD127 + T cells or CD45RB + One or more types of T cells.

[0124] In one embodiment of the present invention, the cells are CD4 + T cells, CD8 + T cells or CD4 + CD8 + T cells.

[0125] Preferably, the sample is extracted from a subject suffering from an immune system disease.

[0126] Preferably, the drug to be tested that can downregulate the expression level of the target in the sample or its function has a preventive or therapeutic effect.

[0127] Preferably, the relevant definitions of targets and immune system diseases are the same as those in the first aspect of the present invention.

[0128] In a sixth aspect, the present invention provides a method for diagnosing, monitoring, severity assessment, efficacy assessment and / or prognostic assessment of immune system diseases.

[0129] Preferably, the method comprises detecting the presence or expression level of a biomarker, and the biomarker comprises one or more of Dock2, AP-1 or Sub1.

[0130] Preferably, the expression level of the biomarker is the expression level of the mRNA and / or protein of the biomarker.

[0131] Preferably, the biomarker is one or more biomarkers selected from the central nervous system, body fluids, cells, tissues or organs.

[0132] Preferably, the body fluid comprises blood or cerebrospinal fluid.

[0133] Preferably, the biomarker is a biomarker in an immune cell, such as a T cell. Preferably, the T cell includes a Tconv cell or a Treg cell.

[0134] Preferably, the T cells include CD8 + T cells, CD4 + T cells, CD25+ T cells, CD25 - T cells, Foxp3 + T cells, CD127 + T cells or CD45RB + One or more types of T cells.

[0135] Preferably, when the expression level of the biomarker is higher than a threshold, it indicates that the disease is present or the risk is higher.

[0136] Preferably, the biomarkers, diagnosis, monitoring, severity assessment, efficacy assessment and / or prognosis assessment of immune system diseases, and the definitions of immune system diseases are the same as those of the first aspect of the present invention.

[0137] In a seventh aspect, the present invention provides a method for preventing and / or treating immune system diseases.

[0138] Preferably, the method comprises using the above-mentioned immune cells, and / or regulating the expression level or function of a target, wherein the target comprises one or more of Dock2, AP-1 or Sub1;

[0139] Preferably, the expression level of the target is the expression level of the target mRNA and / or protein.

[0140] Preferably, the regulation includes up-regulation or down-regulation.

[0141] Preferably, regulating the function of Sub1 includes regulating its DNA binding function, or its binding function with JunB.

[0142] Preferably, the DNA binding function of Sub1 includes ssDNA binding function.

[0143] Preferably, the DNA binding function of Sub1 includes binding to the promoter site of JunB.

[0144] In one embodiment of the present invention, the regulation of Sub1 function comprises mutating Sub1. Preferably, the mutation comprises one or more of F77A, K78G, or K80G.

[0145] Preferably, regulating the function of JunB includes regulating the function of its BM domain, such as the function of binding to Sub1 or the function of binding to Dock2. Preferably, the site binding to Dock2 includes a promoter or an enhancer.

[0146] Preferably, the method comprises administering to the subject an effective amount of the above-mentioned immune cells, or an agent that downregulates the expression or function of the target.

[0147] Preferably, the target is one or more targets selected from the central nervous system, body fluids, cells, tissues or organs.

[0148] Preferably, the body fluid comprises blood or cerebrospinal fluid.

[0149] Preferably, the target is a target in an immune cell, such as a T cell. Preferably, the T cell includes a Tconv cell or a Treg cell. Preferably, the T cell includes a CD8 + T cells, CD4 + T cells, CD25 + T cells, CD25 - T cells, Foxp3 + T cells, CD127 + T cells or CD45RB + One or more types of T cells.

[0150] Preferably, the immune cells are autologous or allogeneic.

[0151] Preferably, downregulating Sub1 prevents and / or treats immune system diseases by reducing JunB expression or function.

[0152] Preferably, downregulating Sub1 prevents and / or treats immune system diseases by reducing the expression or function of Dock2.

[0153] Preferably, downregulating Sub1 prevents and / or treats immune system diseases by blocking or reducing the binding of Sub1 to JunB.

[0154] Preferably, the relevant limitations on the expression level of the regulatory target or its function and immune system diseases are the same as those in the first aspect of the present invention.

[0155] In an eighth aspect, the present invention provides a method for inhibiting immune cell migration, which comprises using the above-mentioned immune cells, and / or regulating the expression level or function of the target, wherein the target comprises one or more of Dock2, AP-1 or Sub1.

[0156] Preferably, the expression level of the target is the expression level of the target mRNA and / or protein.

[0157] Preferably, the regulation includes up-regulation or down-regulation.

[0158] Preferably, regulating the function of Sub1 includes regulating its DNA binding function, or its binding function with JunB.

[0159] Preferably, the DNA binding function of Sub1 includes ssDNA binding function.

[0160] Preferably, the DNA binding function of Sub1 includes binding to the promoter site of JunB.

[0161] In one embodiment of the present invention, the regulation of Sub1 function comprises mutating Sub1. Preferably, the mutation comprises one or more of F77A, K78G, or K80G.

[0162] Preferably, regulating the function of JunB includes regulating the function of its BM domain, such as its binding to Sub1 or its binding to Dock2. Preferably, the site binding to Dock2 includes a promoter or enhancer. Preferably, the immune cells include one or more of lymphocytes, dendritic cells, monocytes / macrophages, granulocytes, or mast cells. Preferably, the lymphocytes include T cells.

[0163] Preferably, the T cells include Tconv cells or Treg cells.

[0164] Preferably, the T cells include CD8 + T cells, CD4 + T cells, CD25 + T cells, CD25 - T cells, Foxp3 + T cells, CD127 + T cells or CD45RB + One or more types of T cells.

[0165] Preferably, said migration comprises migration to the central nervous system.

[0166] Preferably, the immune cell migration includes Dock2-mediated T cell migration.

[0167] Preferably, downregulating Sub1 inhibits immune cell migration by reducing JunB expression or function.

[0168] Preferably, downregulating Sub1 inhibits immune cell migration by reducing Dock2 expression or function.

[0169] Preferably, downregulating Sub1 inhibits immune cell migration by blocking or reducing the binding of Sub1 to JunB.

[0170] Preferably, immune system diseases can be prevented and / or treated by inhibiting Dock2-mediated T cell migration.

[0171] Preferably, the relevant limitations on the expression level of the regulatory target or its function and immune system diseases are the same as those in the first aspect of the present invention.

[0172] In a ninth aspect, the present invention provides a method for constructing an animal model of an immune system disease, the method comprising regulating the expression level or function of a target, wherein the target comprises one or more of Dock2, AP-1 or Sub1.

[0173] Preferably, the construction method includes regulating the expression level of the target in the immune cell or its function. The immune cell includes one or more of lymphocytes, dendritic cells, monocytes / macrophages, granulocytes or mast cells, preferably T cells. Preferably, the T cell includes Tconv cells or Treg cells. Preferably, the T cell includes CD8 + T cells, CD4 + T cells, CD25 + T cells, CD25 - T cells, Foxp3 + T cells, CD127 + T cells or CD45RB + One or more types of T cells.

[0174] Preferably, the regulation is upregulation. Preferably, the expression level of the target is the expression level of the target mRNA and / or protein.

[0175] Preferably, the construction method includes upregulating the expression of Dock2.

[0176] Preferably, the upregulation of Dock2 expression includes overexpression of Dock2 or its DHR-2 domain; or upregulation of the binding of Sub1 to JunB; or upregulation of the expression level or function of Sub1 and / or JunB.

[0177] In one embodiment of the present invention, the construction method includes overexpressing Dock2 or its DHR-2 domain in an animal model to promote the GTPase activity and F-actin polymerization ability of T cells, and promote Dock2-mediated T cell (including CD4 + T cells, CD8 + T cells, CD4 + CD8 + T cell) migration.

[0178] In a specific embodiment of the present invention, the construction method includes upregulating the binding of Sub1 and JunB in an animal model, promoting Dock2 gene transcription, thereby promoting the GTPase activity and F-actin polymerization ability of T cells, and promoting Dock2-mediated T cell (including CD4 + T cells, CD8 + T cells, CD4 + CD8+ T cell) migration.

[0179] Upregulating the expression of Sub1 can upregulate the expression or function of JunB.

[0180] Upregulating the function of Sub1 (particularly the ssDNA binding function) can upregulate the expression or function of JunB.

[0181] Upregulating the expression of Sub1 and / or upregulating the function of Sub1 (particularly the ssDNA binding function) can upregulate the binding of Sub1 to JunB.

[0182] Upregulating Sub1 expression further upregulates Dock2 expression by upregulating JunB expression or its function.

[0183] Upregulating the function of Sub1 (particularly the ssDNA binding function) further upregulates the expression of Dock2 by upregulating the expression or function of JunB.

[0184] Upregulating the expression of Sub1 and / or upregulating the function of Sub1 (particularly the ssDNA binding function) further upregulates the expression of Dock2 by upregulating the binding of Sub1 to JunB.

[0185] Upregulating the expression or function of JunB or its BM domain can upregulate the expression of Dock2.

[0186] Upregulation of Sub1 can upregulate the expression of Dock2.

[0187] Upregulating the expression or function of JunB or its BM domain can upregulate the expression of Dock2.

[0188] Upregulating the expression or function of Sub1, upregulating the expression or function of JunB or its BM domain, upregulating the binding of Sub1 to JunB and / or upregulating the expression of Dock2 can promote the activation of Dock2-mediated T cells (including CD4 T cells) + T cells, CD8 + T cells or CD4 + CD8 + T cells) migration, promoting the occurrence or development of immune system diseases.

[0189] The animal model is a non-human mammal or a human, such as rats, mice, monkeys, pigs, cows, horses, sheep, dogs, cats, etc.

[0190] The tenth aspect of the present invention provides an animal model of immune system disease obtained by the construction method described in the ninth aspect.

[0191] The animal model is a non-human mammal or a human, such as rats, mice, monkeys, pigs, cows, horses, sheep, dogs, cats, etc.

[0192] The eleventh aspect of the present invention provides a use of the immune system disease animal model described in the tenth aspect in screening drugs for preventing and / or treating immune system diseases.

[0193] Preferably, the relevant limitations on immune system diseases and drugs are the same as those in the first aspect of the present invention.

[0194] The twelfth aspect of the present invention provides a method for inhibiting the secretion of inflammatory cytokines, wherein the method comprises using the above-mentioned immune cells, and / or regulating the expression level or function of the target, wherein the target comprises one or more of Dock2, AP-1 or Sub1.

[0195] Preferably, the expression level of the target is the expression level of the target mRNA and / or protein.

[0196] Preferably, the regulation includes up-regulation or down-regulation.

[0197] Preferably, regulating the function of Sub1 includes regulating its DNA binding function, or its binding function with JunB.

[0198] Preferably, the DNA binding function of Sub1 includes ssDNA binding function.

[0199] Preferably, the DNA binding function of Sub1 includes binding to the promoter site of JunB.

[0200] In one embodiment of the present invention, the regulation of Sub1 function comprises mutating Sub1. Preferably, the mutation comprises one or more of F77A, K78G, or K80G.

[0201] Preferably, regulating the function of JunB includes regulating the function of its BM domain, such as its binding to Sub1 or its binding to Dock2. Preferably, the site of Dock2 binding includes a promoter or enhancer. Preferably, downregulating Sub1 inhibits inflammatory cytokine secretion by reducing JunB expression or function.

[0202] Preferably, down-regulating Sub1 inhibits the secretion of inflammatory cytokines by reducing the expression or function of Dock2.

[0203] Preferably, downregulating Sub1 inhibits the secretion of inflammatory cytokines by blocking or reducing the binding of Sub1 to JunB.

[0204] Preferably, the relevant limitations on the expression level of the regulatory target or its function are the same as those in the first aspect of the present invention.

[0205] Preferably, the inflammatory cytokines include one or both of IL-17A and IFN-γ.

[0206] The term "include" or "comprising" in the present invention is an open description containing the specified components or steps described, as well as other specified components or steps that do not substantially affect them.

[0207] The term "and / or" in this invention includes all combinations of the items connected by the term, and each combination should be considered to have been listed separately herein. For example, "A and / or B" includes "A," "A and B," and "B." For another example, "A, B and / or C" includes "A," "B," "C," "A and B," "A and C," "B and C," and "A and B and C."

[0208] As used herein, "treating" means slowing, interrupting, preventing, controlling, stopping, alleviating, or reversing the progression or severity of a sign, symptom, disorder, condition, or disease after the disease has begun to develop, but does not necessarily involve the complete elimination of all disease-associated signs, symptoms, conditions, or disorders.

[0209] The "prognosis assessment" mentioned in the present invention refers to predicting the possible course and outcome of a disease, including determining the specific consequences of the disease (such as recovery, the appearance or disappearance of certain symptoms, signs, complications and other abnormalities, and death).

[0210] The "evaluation of therapeutic efficacy" mentioned in the present invention refers to the evaluation of a patient's response to treatment.

[0211] The "monitoring" mentioned in the present invention refers to observing the occurrence and development of a disease.

[0212] The term "prevention" as used in the present invention refers to an individual taking specific measures to prevent the occurrence of a disease before the disease is diagnosed or develops.

[0213] The "subject" of the present invention can be any animal, including humans and non-human animals. The non-human animals include all vertebrates, for example, mammals, such as non-human primates (particularly higher primates), sheep, dogs, rodents (such as mice or rats), guinea pigs, goats, pigs, cats, rabbits, cattle, and any livestock or pets; as well as non-mammals, such as chickens, amphibians, reptiles, etc.

[0214] The "effective amount" of the present invention refers to the amount or dose of the product of the present invention that provides the desired effect after being administered to a subject, cell or organ in a single or multiple doses.

[0215] The term "diagnosis" in the present invention refers to finding out whether a patient has had a disease or condition in the past, at the time of diagnosis, or in the future, or to finding out the progression or possible future progression of a disease.

[0216] The term "pharmaceutically acceptable" as used herein means that the pharmaceutical composition neither significantly stimulates the organism nor inhibits the biological activity and properties of the active substance of the administered product. BRIEF DESCRIPTION OF THE DRAWINGS

[0217] Hereinafter, embodiments of the present invention will be described in detail with reference to the accompanying drawings, in which:

[0218] Figure 1: Sub1 expression in CD4 T cells of patients with autoimmune diseases + Expression analysis in T cells;

[0219] Figure 2: Sub1 expression in CD4 T cells of MS patients + Expression analysis in T cells;

[0220] Figure 3: WT and Sub1 -KO Mouse Sub1 mRNA expression;

[0221] Figure 4: WT and Sub1 -KO Flow cytometry and quantitative detection of mouse T cell development, where DN represents double negative cells CD4 - CD8 - DP indicates double positive cells CD4 + CD8 + ; In each group of bar graphs, from left to right, it represents the WT group and Sub1 -KO Group;

[0222] Figure 5: Using myelin oligodendrocyte glycoprotein (MOG 35-55 ) Flowchart of peptide immunization-induced autoimmune encephalomyelitis EAE;

[0223] Figure 6: WT and Sub1 -KO EAE clinical scores of mice at different days after immunization;

[0224] Figure 7: HE staining results;

[0225] Figure 8: LFB staining results;

[0226] Figure 9: WT and Sub1 -KO Mouse central nervous system and peripheral CD4 + T cell analysis;

[0227] Figure 10: CD4 + Experimental process of T cell adoptive transfer to induce EAE;

[0228] Figure 11: CD4+ EAE clinical scores after different days of T cell adoptive infusion;

[0229] Figure 12: Flow cytometry analysis results of mouse brain;

[0230] Figure 13: Flow cytometry analysis of mouse spinal cord;

[0231] Figure 14: RNA-seq differentially expressed genes volcano plot and KEGG pathway analysis. In Figure 14a, the left side of the double vertical dashed line indicates downregulation, the right side indicates upregulation, and the area between the double vertical dashed lines and below the single horizontal dashed line indicates no significant difference.

[0232] Figure 15: Sub1 deletion inhibits CD4 + T cell chemotaxis in vitro and homing ability in vivo;

[0233] Figure 16: Sub1 deficiency downregulates Dock2 expression, inhibiting Rac activation and F-actin polymerization;

[0234] Figure 17: Overexpression of Dock2 rescues Sub1-deficient CD4 + T cell function, in Figure 17d, the bar graphs of each group represent the WT-vector group, Sub1 -KO -vector group, Sub1 -KO -Dock2DHR2 group and Sub1 - KO -Dock2DHR2 V1538A Group;

[0235] Figure 18: Sub1 enhances chromatin accessibility by increasing histone H3K27ac and H3K4me1 modifications. In Figures 18c and 18d, TSS indicates the transcription start site, and TES indicates the transcription end site. In Figures 18e and 18f, P indicates promoter, E1 indicates enhancer 1, and E2 indicates enhancer 2.

[0236] Figure 19: Sub1 directly upregulates Junb transcriptional expression;

[0237] Figure 20: Sub1 interacts with JunB protein;

[0238] Figure 21: The Sub1 / JunB complex promotes transcription of the open Dock2 gene locus;

[0239] Figure 22: Flowchart of the Rag mouse enteritis model induced by T cell transplantation, where MACS represents magnetic bead sorting, FACS represents flow cytometry sorting, and iv represents intravenous injection;

[0240] Figure 23: Mouse weight record;

[0241] Figure 24: Diagram of the mouse colorectal structure;

[0242] Figure 25: HE staining results of colon;

[0243] Figure 26: Colorectal and peripheral CD4 in enteritis mice + T cell analysis.

[0244] In each figure, * indicates P < 0.05, ** indicates P < 0.01, *** indicates P < 0.001, **** indicates P < 0.0001, and NS indicates no significant difference. DETAILED DESCRIPTION

[0245] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, not all. All other embodiments obtained by ordinary technicians in this field based on the embodiments of the present invention without making any creative efforts shall fall within the scope of protection of the present invention.

[0246] Some of the material sources and experimental methods involved in this application are as follows:

[0247] 1. Mouse Source

[0248] Sub1 fl / fl (Sub1 is located on mouse chromosome 15, NCBI reference sequence: NM_011294. Exons 3-4 serve as conditional knockout regions, and deletion of this region will lead to loss of mouse Sub1 gene function. Sub1 fl / fl Mice were induced using CRISPR / Cas-mediated genome engineering technology, with loxP sites inserted at both ends of exons 3-4) and CD4-Cre mice were purchased from Saiye Biotechnology Co., Ltd. fl / fl Sub1 mice were crossed with CD4-Cre mice to generate -KO Mice. All mice were housed in a specific pathogen-free (SPF) facility.

[0249] 2. EAE model induction

[0250] Myelin oligodendrocyte glycoprotein (MOG) peptide 35-55 8-10 week old mice (WT and Sub1) were immunized with an emulsion of 100 μg / mouse and 5 mg / mL of Mycobacterium tuberculosis H37Ra extract (complete Freund's adjuvant, 200 μl / mouse) in equal proportions in their limbs. -KOOn days 0 and 2 after immunization, 200 ng of pertussis toxin (PTX) was injected intraperitoneally. Clinical symptoms of EAE were assessed daily according to the following scores: 0, no clinical symptoms; 1, limp tail; 2, hind limb weakness; 3, hind limb paralysis; 4, quadriplegia; 5, moribund state.

[0251] 3. Adoptive transfer of T effector cells to induce EAE

[0252] WT,Sub1 -KO On the seventh day of EAE modeling, spleen cells were isolated and prepared at a concentration of 10 7 Cells were suspended at a concentration of 10 cells / mL and placed in a T75 culture flask. The cells were cultured for 3 days at 37°C, 5% CO2, and 50 μg / mL MOG. Th17 polarization conditions included 20 ng / mL mIL-23, 20 ng / mL mIL6, and 2 ng / mL mTGFβ. Re-stimulated cells were collected and washed with PBS. 1×10 cells / mL of Th17 were injected intravenously into C57BL / 6 recipient mice. 7 Recipient mice were intraperitoneally injected with 200 ng of pertussis toxin (PTX) on days 0 and 2 after T cell transfer, and the animals were monitored daily for the development of EAE.

[0253] 4. Isolation of T cells from the central nervous system (CNS)

[0254] Mice were perfused cardiacally with cold PBS. Under hydrostatic pressure, the forebrain and cerebellum were flushed with PBS, and the spinal cord was flushed out of the spinal canal. The tissue was then ground through a 70 μm filter, and the trituration fluid was collected and centrifuged over a 70% / 30% Percoll gradient to isolate mononuclear cells. Mononuclear cells were aspirated from the interstitial layer, washed, and resuspended in PBS for subsequent flow cytometric analysis.

[0255] 5. Streaming analysis

[0256] For surface markers, cells were stained with the indicated antibodies for 30 minutes at 4°C. To detect T lymphocyte cytokine expression, cells were activated with 500 ng / ml ionomycin and 50 ng / ml PMA in the presence of Brefeldin A and Monenin solution at 37°C for 4 hours. Live cells were then identified using Violet LIVE / DEAD fixation and staining reagents, and cells were fixed and permeabilized using a fixation and permeabilization kit. Cells were acquired on a BD LSR Fortessa flow cytometer and analyzed using FlowJo version 10 software.

[0257] 6. RNA extraction and real-time quantitative PCR (RT-PCR)

[0258] Total RNA was extracted from cultured cells or tissues using an RNA extraction kit (RNAfast200, Shanghai Feijie) according to the manufacturer's protocol. RNA was reverse transcribed using an RT kit (Yeasen). cDNA was subsequently analyzed by SYBR-based real-time PCR. Results were normalized using GAPDH, and data are presented as mean ± standard deviation. P values ​​were calculated using the Student's t-test.

[0259] 7. Western blot

[0260] Cell samples were lysed in RIPA buffer (Beyotime, P0013B) and supplemented with protease and phosphatase inhibitors (MCE). Protein concentration was determined using the BCA protein assay (Thermo Fisher Scientific). Proteins were separated using a 12.5% ​​SDS-PAGE gel (Yakoshi) and then transferred to a 0.22 μm polyvinylidene fluoride (PVDF) membrane. Blocking was performed with 5% BSA at room temperature for 1.5 hours, followed by incubation with primary antibodies at 4°C overnight and then with secondary antibodies for 1 hour.

[0261] 8. Chemotaxis Assay

[0262] Chemotaxis assays were performed using a 24-well Transwell plate (pore size 5 μm; CoStar). Cells were starved for 4–6 h in serum-free medium. Cells were resuspended in complete medium (at a concentration of 1 × 10 6 100 μl of cell suspension was added to the upper chamber, and 500 μl of complete culture medium containing chemokines was added to the lower chamber. After incubation at 37°C, 5% CO₂ for 2 hours, all cells in the lower chamber were collected and counted by flow cytometry to calculate the percentage of migrated cells. CCL21, CCL19, and CXCL12 chemokines were purchased from R&D Company.

[0263] 9. T cell homing detection

[0264] Extraction of WT and Sub1 -KO Mouse spleen cells were labeled with different colors of Cell Tracker dyes and mixed in a 1:1 ratio and injected intravenously into C57 / B6 recipient mice (1×10 per mouse). 7 4 and 24 hours later, lymphocytes were isolated from the blood and secondary lymphoid organs of recipient mice and analyzed by flow cytometry.

[0265] 10. Rac activity detection

[0266] After the cells were stimulated with CCL21 (1 μg / ml), 1× Mg 2+Lysis buffer (MLB; 25 mM Hepes (pH 7.5), 150 mM NaCl, 1% Igepal CA-630, 10 mM MgCl2, 1 mM EDTA, 10% glycerol; Millipore) was used, followed by centrifugation at 20,000 g for 1 minute at 4°C. An equal amount of total cell lysate was retained as a control, and the remaining lysate was incubated with agarose beads containing the GST-fused Rac-binding domain of PAK1 for 1 hour at 4°C. The beads were washed twice with 1× MLB buffer and suspended in SDS-PAGE sample buffer (62.5 mM Tris-HCl (pH 6.8), 2% SDS, 10% glycerol, 0.005% bromophenol blue, 2.5% 2-mercaptoethanol). Bound proteins and total cell lysate were separated by SDS-PAGE on a 12.5% ​​polyacrylamide gel and probed with a monoclonal antibody against Rac1.

[0267] 11. F-actin polymerization detection

[0268] The cells were resuspended in RPMI / 1% FCS / 10 mM HEPES (pH 7.5) to a volume of 5 × 10 6 Cells were plated at 400 cells / ml and stored at 37°C. Before adding chemokines, an aliquot was removed from each sample to determine baseline F-actin levels. CCL21 was added to the cell suspension and stimulated at 37°C. Aliquots were removed at the prescribed stimulation time and immediately fixed with 4% paraformaldehyde for 10 minutes. After washing with PBS, samples were stained with FITC-Phalloidin (Molecular Probes) as a probe for F-actin and analyzed by flow cytometry.

[0269] 12. Extraction and culture of primary T cells

[0270] Mouse lymph nodes were removed, ground using a 70 μm filter, and resuspended in T cell primary culture medium at 37°C with 5% CO. 2 T cell primary culture medium formula: RPMI 1640 (500 ml) + 10% FBS + 1% NEAA + 1% sodium pyruvate + 1% P / S + 1.74 μl β-mercaptoethanol.

[0271] 13. Dual luciferase reporter assay

[0272] The promoter construct used in the experiment was generated by cloning the full-length -2000 to +200 region of the mouse Dock2 promoter and inserting it between the NheI and XhoI restriction sites of the pGL3-Basic vector. 293T cells were co-transfected with PEI transfection reagent at a ratio of 9:1:0.1: transcription factor:luc reporter plasmid:pRL-TK (renilla internal reference plasmid). A total of 3 μg of plasmid and 12 μl of PEI were transfected per well of a six-well plate. Forty-eight hours after transfection, cells were washed and lysed, and firefly and Renilla luciferase activities were assessed using a multi-function microplate reader.

[0273] 14. Co-immunoprecipitation (Co-IP)

[0274] Sub1 and AP-1 cDNA sequences were cloned into the lentiviral vectors pcDNA3.1-FLAG and pcDNA6.0-HIS, respectively. Flag-tagged Sub1 and His-tagged AP-1 proteins were expressed in 293T cells, and whole-cell lysates were prepared using lysis buffer (50 mM Tris-HCl pH 7.4, 150 mM NaCl, 5 mM EDTA, 10% glycerol, 1% Triton X-100, 1 mM PMSF). The cell lysate was mixed with Sepharose beads (Sigma) conjugated to an anti-Flag antibody and incubated overnight at 4°C with peripheral rotation. The supernatant was removed, and immunoblotting analysis was performed using anti-Flag and anti-His antibodies.

[0275] 15. RNA-seq analysis

[0276] WT and Sub -KO CD4 T cells were isolated from spleen cells of EAE mice + T cells were lysed with Trizol. Sequencing was performed by Lianchuan Biotechnology Co., Ltd. Differential gene expression was analyzed using the DESeq2 package. Significance was determined by setting a false discovery rate (FDR) threshold of less than 0.05 and a |log2FC| greater than 1. All differentially expressed genes were then subjected to GO function and KEGG pathway analysis.

[0277] 16. ATAC-seq analysis

[0278] Sorting of WT and Sub1 -KO Mouse spleen CD4 + T cells were cryopreserved. Sequencing was performed by Panosun Biotechnology Co., Ltd. ATAC-seq sequencing yielded raw reads, which were filtered to remove adapters, cleaned for contamination, and aligned to the reference genome (GRCm38). Unique mapped reads were used for subsequent information analysis.

[0279] 17. ChIP-seq analysis

[0280] Mouse primary CD4 + T cells were cross-linked with 1% formaldehyde for 10 minutes at room temperature, and cross-linking was terminated with 0.125 mol / L glycine. Library construction and sequencing were performed by Lianchuan Biotechnology Co., Ltd. Clean reads were mapped to the mouse genome (GRCm38) using Bowtie2 (v2.2.6) software. Peak detection was performed using the MACS (v2.1.1) peak-finding algorithm, and peak sites of gene signatures were annotated using the ChIPseeker R package.

[0281] 18. Intestinal inflammation mouse model

[0282] WT,Sub1 -KO The spleen and lymph nodes of mice were taken and CD4 + T cells were sorted and stained with CD25 and CD45RB, and then CD25 T cells were sorted using fluorescence activated cytometry. – CD45RB + CD4 + T cells were collected and injected intravenously into Rag mice (i.e., B6-Rag1 KO mice), with 5×10 T cells per mouse. 5 The body weight of mice was measured every five days.

[0283] 19. Isolation of T cells in the colon

[0284] On day 30 of inducing enteritis, mice were harvested, their colons removed, and feces and other contents removed. The intestines were then cut longitudinally and washed with PBS. The intestines were cut into approximately 2 cm long segments and washed thoroughly in PBS with vigorous shaking. The intestines were transferred to solution A (DTT+EDTA+HEPES) and incubated at 37°C at 200 rpm for 10 minutes, followed by vigorous shaking for 1 minute. The intestines were then transferred to solution B (EDTA+HEPES) and incubated at 37°C at 200 rpm for 10 minutes, followed by vigorous shaking for 1 minute. The intestines were washed with 1640 medium, minced, and incubated in digestion solution (Col VIII+DNase I+1640) at 37°C for 55 minutes. After digestion, PBS was added and shaken vigorously for 1 minute. The digested tissue fluid was filtered through a 70 μm filter and centrifuged at 1800 rpm for 5 minutes to collect the cell pellet. The collected cells were subjected to 80% / 40% Percoll gradient centrifugation to isolate mononuclear cells. Mononuclear cells were aspirated from the middle layer, washed, and resuspended in PBS for subsequent flow cytometry analysis.

[0285] 20. Histological analysis

[0286] Histological analysis of mouse colons was performed. Mice were sacrificed on day 30 after induction of enteritis, and colons were fixed in 4% (w / v) paraformaldehyde at 4°C overnight. Paraffin-embedded sections were prepared. H&E staining was performed on 5 μm-thick transverse paraffin sections.

[0287] Example 1 Sub1 in CD4 T cells of patients with various autoimmune diseases + Highly expressed in T cells

[0288] Through large-scale immune cell gene expression analysis and whole genome sequence analysis from 337 patients diagnosed with 10 immune system diseases and 79 healthy volunteers, it was found that Sub1 is expressed in CD4 T cells of patients with multiple autoimmune diseases (such as Steven-Johnson syndrome (SjS), rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), systemic sclerosis (SSc), adult-onset Still's disease (AOSD), Behçet's syndrome (BD)). + As shown in Figure 1, Sub1 is highly expressed in T cells, indicating that it may be a key pathogenic factor leading to autoimmune diseases and is also a CD4 + An important regulator of T cell function.

[0289] Furthermore, single-cell data analysis was performed on the blood and cerebrospinal fluid of multiple sclerosis (MS) patients and healthy volunteers. + There is also obvious high expression in T cells, as shown in Figure 2.

[0290] Example 2 Sub1 deletion can significantly inhibit CD4 + T cell infiltration prevents EAE from occurring

[0291] To explore the function of Sub1 in MS, a T cell-specific knockout of Sub1 (Sub1 -KO ) mouse model. First, the knockout efficiency of Sub1 was detected by q-PCR to determine -KO Sub1 was knocked out in the mouse model (see Figure 3 ), and the T cell development of the knockout mice was then tested. It was found that the loss of Sub1 did not affect the normal development of T cells, as shown in Figure 4 .

[0292] Next, myelin oligodendrocyte glycoprotein (MOG 35-55 ) peptide immunization WT and Sub1 -KO Mice were induced to develop experimental allergic encephalomyelitis (EAE). The induction process is shown in Figure 5. EAE is a disease characterized by specific sensitization of CD4+ It is an autoimmune disease mediated mainly by T cells, characterized by the infiltration of mononuclear cells around small blood vessels in the central nervous system and demyelination. Its pathological changes are similar to those of multiple sclerosis. Therefore, the EAE animal model is an important way to study the pathological process and pathogenesis of MS, and is of great significance in the study of clinical neuroimmunology. The study found that the deletion of the Sub1 gene completely inhibited the onset of EAE, and no EAE symptoms appeared during the experiment (see Figure 6 for clinical scores). The onset of EAE mice is accompanied by inflammatory cell infiltration and myelin damage in the central nervous system, especially in the white matter of the spinal cord. WT and Sub1 mice were taken. -KO Pathological analysis of the spinal cord of EAE mice at the peak stage showed that the purple-blue granules in the white matter of the spinal cord of WT EAE mice were dense and clustered. The infiltration of inflammatory cells in the spinal cord of Sub1-deficient EAE mice was significantly reduced, indicating that Sub1 deficiency can reduce the infiltration of inflammatory cells into the CNS (Figure 7). Luxol fast blue (LFB) can bind to myelin and stain it blue. LFB is not only used to detect spinal cord demyelination, but also to show whether the myelin is intact, degenerated, and the degree of necrosis and repair in pathological conditions. The experimental results showed that compared with WT mice, Sub1-deficient EAE mice had a significantly reduced infiltration of inflammatory cells in the spinal cord, indicating that Sub1 deficiency can reduce the infiltration of inflammatory cells into the CNS (Figure 7). -KO LFB staining of the mouse spinal cord showed a significant increase in the blue part, and the spinal cord had good structural integrity without vacuoles ( Figure 8 ).

[0293] We further examined the T cell status in the central nervous system (CNS) and periphery. -KO Flow cytometry analysis was performed on the brain, spinal cord, spleen and lymph nodes of mice. -KO Mouse CD4 + The total number of infiltrating T cells was significantly reduced, and the number of CD4 T cells producing IL-17A and IFN-γ was significantly reduced. + T cells were significantly reduced in the spleen and lymph nodes. + The number of T cells and the production of IL-17A and IFN-γ were significantly different between WT and Sub1 -KO There was no significant difference in the expression of Sub1 in mice (see Figure 9), indicating that the loss of Sub1 did not impair the expression of CD4 + T cell activity.

[0294] To further verify that Sub1 deficiency can inhibit the pathogenesis of EAE, adoptive transfer of T cells was used to induce EAE. The experimental process is shown in Figure 10. -KO CD4 +The recipient mice that received T cell transfusion never showed EAE symptoms (Figure 11). At the peak of the disease, the brain and spinal cord of the mice were taken for flow cytometry analysis. + T cells hardly infiltrate into the CNS (Figures 12 and 13). + T cell infiltration into the CNS prevents the occurrence of EAE.

[0295] Example 3 Sub1 deletion inhibits CD4 + T cell migration.

[0296] To further explore the regulation of Sub1 on CD4 + Mechanism of T cell function. On the 7th day after EAE model establishment, WT and Sub1 -KO Mouse spleen CD4 + T cells were analyzed by RNA-seq. The volcano plot showed that the difference between the two groups was not significant. + Compared with T cells, Sub1 -KO CD4 + T cells upregulated 52 genes and downregulated 84 genes (Figure 14a). KEGG results showed that the signaling pathways related to the downregulated genes caused by Sub1 deficiency were mainly cytokine-cytokine receptor interaction and chemokine signaling pathway (Figure 14b).

[0297] Next, a transwell experiment was performed in vitro to verify whether Sub1 would affect CD4 + The migration ability of T cells. Three different chemokines, CCL21, CCL19 and CXCL12, were used to attract CD4 + The results showed that Sub1 deletion could significantly inhibit the migration of CD4 + The in vitro migration ability of T cells (Fig. 15a) is consistent with the previous EAE model in which CD4 + The results of the study were consistent with the results of the study on the reduction of T cell infiltration. The primitive lymphocytes continuously migrate or home from the blood to the secondary lymphoid organs (SLOs), such as the spleen, peripheral lymph nodes (pLN) and mesenteric lymph nodes (mLN), as well as the gut-associated lymphoid tissues. To further prove that Sub1 affects CD4 + The migration ability of T cells was tested in vivo (the schematic diagram is shown in Figure 15b). The results showed that Sub1 deficiency weakened the CD4 + The ability of T cells to home to secondary lymphoid organs (see Figure 15c).

[0298] Example 4 Sub1 deficiency inhibits Rac activity and F-actin polymerization by downregulating Dock2 expression

[0299] Further analysis of the genes most significantly affected by Sub1 deletion, as shown in the heat map (Figure 16a), revealed that Sub1, Dock2, IL-17 (IL17a and / or IL17f), IFN-γ (Ifng) and other genes were significantly affected by Sub1 deletion. -KO The expression of Dock2 was significantly downregulated in the 1447 cells of the 247 cells. Existing studies have shown that Dock2 can affect cell membrane polarization and cytoskeleton dynamics by activating Rac, thereby regulating the migration, proliferation and activation of lymphocytes and innate immune cells. First, the expression of Dock2 was detected at the RNA and protein levels by RT-PCR and western blot, respectively. Consistent with the results of RNA-seq, the expression of Dock2 was significantly downregulated after the loss of Sub1 (Figure 16b and c).

[0300] The small GTPase Rac is a key regulator of F-actin polymerization and a prerequisite for cell migration. In lymphocytes, chemokine-mediated Rac activation is strongly dependent on Dock2, a member of the CDM protein family. Therefore, we further examined whether downregulation of Dock2 would lead to impaired Rac activation. Western blot results showed that Sub1 deficiency significantly decreased the expression of active Rac (Rac-GTP) (Figure 16d). Since phalloidin can selectively bind to filamentous actin (F-actin) rather than actin monomers (G-actin), the degree of F-actin polymerization is measured by the intensity of phalloidin staining. The results showed that Sub1 deficiency significantly weakened the polymerization ability of F-actin (Figure 16e).

[0301] Example 5 Overexpression of Dock2 restores Sub1-deficient CD4 + T cell migration ability

[0302] To verify the Sub1-regulated CD4 + T cell migration is mediated by Dock2, and a Dock2 complementation experiment was performed. It is known that Dock2 contains four domains: SH3, DHR-1, DHR-2 and PAA (Figure 17a). Among them, the V1538 site in the DHR-2 domain has been shown to directly bind to and activate Rac. When this site mutates (V1538A), the Rac GEF activity is almost completely lost. Therefore, Dock2-DHR2 functional truncation and Dock2-DHR2 were constructed respectively. V1538A The results showed that overexpression of Dock2-DHR2 could significantly restore the survival of Sub1 -KO CD4 +Rac activation and F-actin polymerization ability of T cells (Figure 17b and c). Furthermore, in vitro transwell experiments also showed that overexpression of Dock2-DHR2 could restore Sub1 -KO CD4 + In vitro migration ability of T cells overexpressing mutant Dock2-DHR2 V1538A Unable to recover CD4 + The migration ability of T cells ( FIG. 17 d ).

[0303] Example 6 Sub1 promotes chromatin openness by increasing histone modification

[0304] Next, we further explored how Sub1 regulates the expression of Dock2. -KO Mouse spleen CD4 + T cells underwent ATAC-seq to describe the accessibility of chromatin. The results in Figure 18a show that the overall chromatin openness was significantly weakened after Sub1 loss, which indicates that Sub1 can enhance chromatin accessibility and thus regulate gene transcription. Further gene ontology (GO) enrichment analysis of genes with differential chromatin accessibility revealed that these genes were mainly associated with GTPase regulator activity and actin binding function (Figure 18b). Histone modification is a key marker reflecting the transcriptional state of chromatin. Specifically, monomethylation of lysine 4 on histone 3 (H3K4me1) and acetylation of lysine 27 on histone 3 (H3K27ac) are markers of transcriptionally active regions. In order to elucidate the epigenetic landscape, ChIP-seq was used to depict the expression of H3K4me1 and H3K27ac in WT and Sub1. -KO CD4 + The distribution in T cells. The results in Figure 18 c and d show that Sub1 -KO CD4 + The overall levels of H3K4me1 and H3K27ac were significantly reduced in T cells.

[0305] Furthermore, the annotation of ChIP-seq combined with ATAC-seq peak graphs showed that the positions of H3K27ac and H3K4me1 modifications on Dock2 were basically the same, and the degree of chromatin openness at the modified site was significantly increased, and this region was predicted to be a super enhancer (e in Figure 18). Considering the function of Sub1 as a transcriptional activator of multiple genes, it is speculated that Sub1 may directly regulate the transcription of Dock2. A dual luciferase reporter gene experiment was then performed to evaluate the effect of Sub1 on promoter and enhancer activity. The results in Figure 18f show that (where Luc is the luciferase fluorescence signal and RTLK is the fluorescence signal of the internal reference sea renilla) Sub1 cannot directly regulate Dock2 transcription. This observation indicates that intermediate factors are involved in the transcriptional regulation of Dock2 by Sub1, highlighting the complexity of gene regulation.

[0306] Example 7 Sub1 directly upregulates the transcriptional expression of Junb

[0307] To further elucidate the mechanism by which Sub1 regulates Dock2 expression, we used the Find Individual Motif Occurrences (FIMO) software tool to predict potential transcription factors at specific binding sites within the Dock2 promoter and enhancer regions. FIMO predictions indicated that several components of the activator protein 1 (AP-1) complex, such as Fos, Jun, and Junb, may directly bind to the regulatory region of the Dock2 gene (Table 1). The AP-1 family is a ubiquitous dimeric transcriptional complex involved in numerous cellular and physiological functions.

[0308] Table 1 FIMO transcription factor prediction analysis of Dock2 promoter and enhancer regions

[0309] Subsequently, Flag ChIP-seq was performed on the overexpression of Flag-Sub1 in OT-II cells. Flag ChIP-seq analysis showed that there was a large amount of Sub1 binding at the Junb gene site, and the direct modification sites of H3K27ac and H3K4me1 were highly consistent with the binding sites of Sub1 (Figure 19a). The occupancy of Sub1 on the Junb promoter was further verified by ChIP-qPCR analysis (Figure 19b, where Retro-SUB1 (flag-tag) indicates that the cells were infected with retrovirus and overexpressed flag-sub1, and the IgG group was the control group to exclude the non-specific binding of the antibody itself; in Figure 19b, the signal of the Flag group was significantly higher than that of the IgG group, proving that there was direct binding, and "% of input" indicates that the result is a standardized result). Further, by comparing WT and Sub1-KO qPCR and WB analysis of OT-II cells confirmed that the expression level of Junb was significantly decreased after knocking out or knocking down Sub1 (c and d in Figure 19). Sub1 is a DNA-binding protein with a length of 127 amino acids. It contains two regions rich in serine and acidic residues (SEAC) separated by a lysine-rich (K-rich) region at the N-terminus, and the N-terminal domain contains amino acids 1-63. In addition, the C-terminus of the protein contains a high-affinity single-stranded DNA (ssDNA) binding domain (e in Figure 19). The C-terminal domain of Sub1, spanning amino acids 64-127, was found to form a dimer fold, providing an unusual binding surface for two antiparallel ssDNA chains. The F77A / K78G / K80G mutation resulted in a significant loss of ssDNA binding ability. Dual-luciferase reporter assays revealed that the C-terminal ssDNA-binding domain of Sub1 is responsible for promoting Junb expression, while the presence of a mutant DNA-binding domain (mDB) abolished this transcriptional-promoting effect ( Figure 19 , f). These results suggest that Sub1 directly promotes Junb gene transcription.

[0310] Example 8: The Sub1 / JunB complex promotes transcription of the open Dock2 gene locus

[0311] JunB belongs to the basic zipper (bZIP) protein family, whose members contain a conserved leucine zipper (LZ) domain and a basic motif (BM) domain rich in basic amino acids ( Figure 21 a).

[0312] The physical interaction between Sub1 and Junb proteins was depicted by in vitro Co-IP experiments (Figure 20, where WB detection was performed on the Input group and IP group, IB indicates WB incubation with antibodies to detect FLAG and HIS tags. The presence of bands in the Input group indicates successful protein expression, and the presence of bands in the IP group indicates interaction between Sub1 and Junb proteins). This interaction may help Junb to accurately recruit to the Dock2 gene site opened by Sub1, thereby improving the expression of Dock2 in CD4 + Transcriptional activation effects in T cells.

[0313] In order to confirm the direct regulatory role of Junb in Dock2 transcription, the full-length promoter and predicted enhancer of Dock2 were cloned and their sequences were inserted into luciferase reporter plasmids. Further plasmids of JunB full-length (Full-length), JunB LZ-deficient truncated (dLZ), JunB BM-deficient truncated (dBM) and JunB mutant LZ domain (mLZ) were constructed. Dual luciferase reporter gene analysis showed that the BM domain of JunB is necessary for directly binding to the promoter and enhancer to promote Dock2 transcriptional expression (Figure 21b). Since Sub1 is responsible for the accessibility of the Dock2 site and interacts with JunB, the following is the expression of JunB in WT and Sub1. -KO CD4 + Flag-Junb was overexpressed in T cells, followed by Flag ChIP-seq to characterize the expression of JunB in WT and Sub1 -KO CD4 + The peak graph annotation shows that JunB has a clear binding signal at the Dock2 site, and this signal is not expressed in Sub1-deficient CD4 + ChIP-qPCR further verified the enrichment of JunB binding signals on the enhancer and promoter of Dock2, and the binding signals were significantly reduced in Sub1-deficient CD4 T cells (Figure 21c). + It was strongly reduced in T cells (Fig. 21d). Taken together, these results indicate that the Sub1 / JunB complex promotes transcription of the open Dock2 gene locus.

[0314] Example 9 Treatment of enteritis by inhibiting Sub1

[0315] To further investigate the role of Sub1 in autoimmune diseases, we used a mouse model of enteritis to verify the function of Sub1. -KO CD4 T cells were purified from the spleen and lymph nodes of mice + T cells were then sorted using fluorescence-activated cell sorting (FACS) to identify CD25 – CD45RB + CD4 + T cells were then injected intravenously into recipient Rag mice to achieve a T cell-induced colitis model (Figure 22). The body weight of mice was measured every five days. The experimental results showed that the WT CD4 + The body weight of the recipient mice after T cell infusion decreased significantly (Figure 23). On the 30th day, the colon of the mice was taken for morphological observation and tissue section staining (for methods, see 20, Histological Analysis). The morphological observation results showed that the recipient mice received WT CD4 +The colon of the recipient mice after T cell infusion was significantly shortened and thickened (Figure 24). HE staining results showed that the colon of the recipient mice after WT CD4 + The submucosal layer of the colon of the recipient mice that received T cell infusion was significantly thickened, and the infiltration of inflammatory cells increased significantly (Figure 25). -KO CD4 + CD4 + The infiltration of T cells was significantly reduced, and the production of IL-17A and IFN-γ by CD4 + In the spleen and mesenteric lymph nodes, Sub1 -KO CD4 + The number of T cells and the production of IL-17A and IFN-γ also decreased significantly (Figure 26). Overall, these results indicate that Sub1 reduction or deletion can significantly inhibit the inflammatory phenotype of intestinal inflammation and reduce CD4 + Infiltration of T in colonic tissue.

[0316] The preferred embodiments of the present invention are described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the technical concept of the present invention, various simple modifications can be made to the technical solution of the present invention, and these simple modifications all fall within the scope of protection of the present invention.

[0317] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any appropriate manner without contradiction. In order to avoid unnecessary repetition, the present invention will not further describe various possible combinations.

Claims

1. Use of biomarkers or targets in the preparation of products for diagnosis, monitoring, severity assessment, efficacy assessment, prognosis assessment, prevention and / or treatment of immune system diseases, characterized in that: The biomarkers or targets include one or more of Dock2, AP-1 or Sub1.

2. The use according to claim 1, characterized in that: The AP-1 includes one or more proteins from the Jun protein family, the Fos protein family, the ATF protein family or the MAF protein family; Preferably, the Jun protein family includes one or more of Jun (such as v-Jun or c-Jun), JunB or JunD; Preferably, the Fos protein family includes one or more of Fos (such as v-Fos or c-Fos), FosB, FRA-1, and FRA-2; Preferably, the ATF protein family includes one or more of ATF2, ATF3 / LRF1, B-ATF, JDP1 or JDP2; Preferably, the MAF protein family includes one or more of c-Maf, MafB, MafA, MafG, MafF, MafK or Nrl.

3. The use according to claim 1 or 2, characterized in that: The biomarkers or targets are genes and / or proteins.

4. The use according to any one of claims 1 to 3, characterized in that: The diagnosis, monitoring, severity assessment, efficacy assessment and / or prognosis assessment of immune system diseases includes detecting the presence or expression of biomarkers; Preferably, the expression level of the biomarker is the expression level of the mRNA and / or protein of the biomarker.

5. The use according to any one of claims 1 to 3, characterized in that: The prevention and / or treatment of immune system diseases includes regulating the expression level of the target or its function; Preferably, the expression level of the target is the expression level of the mRNA and / or protein of the target; Preferably, the regulation includes up-regulation or down-regulation; Preferably, regulating the function of Sub1 includes regulating its DNA binding function, or its function of binding to JunB; Preferably, regulating the function of JunB includes regulating the function of its BM domain, such as the function of binding to Sub1 or the function of binding to Dock2. Preferably, the site binding to Dock2 includes a promoter or an enhancer.

6. The use according to claim 5, characterized in that: Said down-regulation includes knocking out or knocking down; Preferably, down-regulating the expression of Sub1 includes knocking out all or part of exons 1 to 5 of the Sub1 gene, further preferably knocking out exon 3 and exon 4 of the Sub1 gene, preferably introns 3-4 are knocked out; Preferably, down-regulating the expression level of JunB comprises knocking out or knocking down JunB or its BM domain.

7. The use according to claim 5, characterized in that: Downregulating Sub1 can prevent and / or treat immune system diseases by reducing JunB expression or function.

8. The use according to claim 5, characterized in that: Downregulating Sub1 can prevent and / or treat immune system diseases by reducing the expression or function of Dock2.

9. The use according to claim 5, characterized in that: Downregulating Sub1 prevents and / or treats immune system diseases by blocking or reducing the binding of Sub1 to JunB.

10. The use according to any one of claims 1 to 9, characterized in that: The biomarker or target is one or more biomarkers or targets selected from the central nervous system, body fluids, cells, tissues or organs; Preferably, the body fluid comprises blood or cerebrospinal fluid; Preferably, the biomarker or target is a biomarker or target in an immune cell, such as a T cell. Preferably, the T cells include Tconv cells or Treg cells; Preferably, the T cells include CD8 + T cells, CD4 + T cells, CD25 + T cells, CD25 - T cells, Foxp3 + T cells, CD127 + T cells or CD45RB + One or more types of T cells.

11. The use according to any one of claims 1 to 10, characterized in that: The product includes a reagent for detecting the presence or expression of a biomarker (the expression of the biomarker is preferably the expression of the mRNA and / or protein of the biomarker), or the product includes a reagent for regulating the expression of a target or its function (the expression of the target is preferably the expression of the mRNA and / or protein of the target).

12. The use according to any one of claims 1 to 11, characterized in that: Immune system diseases include one or more of autoimmune diseases, immunoproliferative diseases or inflammatory diseases; Preferably, the inflammatory disease includes an infectious disease or a hypersensitivity disease; Preferably, the inflammatory disease comprises one or more of asthma, rheumatoid arthritis, chronic obstructive pulmonary disease, psoriasis, colitis, chronic inflammatory demyelinating polyneuropathy, giant cell arteritis, glomerulonephritis, juvenile idiopathic arthritis, synovitis, osteitis, polymyositis, Wegener's granulomatosis, psoriasis, Behcet's syndrome, polyarteritis nodosa, Takayasu arteritis, graft-versus-host disease, adult-onset Still's disease, hidradenitis suppurativa, antiphospholipid syndrome, cryoglobulinemia vasculitis, hidradenitis suppurativa, idiopathic membranitis, autoimmune hepatitis, erythema nodosum, myelodysplastic syndrome or amyloidosis; Preferably, the autoimmune disease comprises one or more of rheumatoid arthritis, multiple sclerosis, autoimmune hemolytic anemia, chronic inflammatory demyelinating polyneuropathy, glomerulonephritis, juvenile idiopathic arthritis, polymyalgia rheumatica, systemic lupus erythematosus, thrombotic / idiopathic thrombocytopenic purpura, Sjögren's syndrome, polymyositis, Wegener's granulomatosis, Takayasu arteritis, sarcoidosis, pyoderma gangrenosum, Kawasaki's disease, relapsing polychondritis, relapsing polychondritis, celiac disease, autoimmune hepatitis, myasthenia gravis, SAPHO syndrome, Graves' disease, myelodysplastic syndrome, pemphigus or amyloidosis; Preferably, the immunoproliferative disease includes one or more of myeloma (including multiple myeloma), leukemia (acute lymphocytic leukemia, B-cell chronic lymphocytic leukemia, naked cell acute lymphocytic leukemia, histo-monocyte acute monocytic leukemia, hairy cell leukemia), lymphoma (including lymphoblastoma, non-Hodgkin's lymphoma, Hodgkin's lymphoma, Burkitt's lymphoma), Sezary syndrome, mycosis fungoides, essential macroglobulinemia, infectious mononucleosis or acute histiocytosis.

13. An immune cell, characterized in that: The target expression level in the immune cells is downregulated, and / or the immune cells include an agent for downregulating the target expression level or an agent for downregulating the target function; the targets include one or more of Dock2, AP-1 or Sub1; Preferably, the target expression level is the expression level of the target mRNA and / or protein.

14. The immune cell according to claim 13, characterized in that Downregulation of target expression includes knockout or knockdown, preferably using CRISPR / Cas system, tissue-specific knockout or introduction of interfering RNA targeting the target; Preferably, down-regulating the expression of Sub1 includes knocking out all or part of exons 1 to 5 of the Sub1 gene, further preferably knocking out exon 3 and exon 4 of the Sub1 gene, preferably introns 3-4 are knocked out; Preferably, down-regulating the expression level of JunB includes knocking out JunB or its BM domain.

15. The immune cell according to claim 13 or 14, characterized in that Down-regulating the function of Sub1 includes down-regulating the DNA binding function of Sub1, or the function of binding to JunB.

16. The immune cell according to any one of claims 13 to 15, characterized in that: Down-regulating the function of JunB includes down-regulating the function of its BM domain, preferably the function of binding to Sub1 or the function of binding to Dock2, and preferably the site binding to Dock2 includes a promoter or an enhancer.

17. The immune cell according to any one of claims 13 to 16, characterized in that: The immune cells include one or more of lymphocytes, dendritic cells, monocytes / macrophages, granulocytes or mast cells; Preferably, the lymphocytes include T cells; Preferably, the T cells include Tconv cells or Treg cells; Preferably, the T cells include CD8 + T cells, CD4 + T cells, CD25 + T cells, CD25 - T cells, Foxp3 + T cells, CD127 + T cells or CD45RB + One or more types of T cells.

18. Use of blocking or reducing the binding of Sub1 to JunB in preparing products for preventing and / or treating immune system diseases.

19. The use according to claim 18, characterized in that The blocking or reducing the binding between Sub1 and JunB includes: A) downregulating the expression or function of Sub1; or, B) Down-regulating JunB expression or its function.

20. The use according to claim 18 or 19, characterized in that Blocking or reducing the binding of Sub1 to JunB can prevent and / or treat immune system diseases by reducing the expression of Dock2.

21. The use according to any one of claims 18 to 20, characterized in that: The immune system diseases include one or more of autoimmune diseases, immunoproliferative diseases or inflammatory diseases; Preferably, the inflammatory disease includes an infectious disease or a hypersensitivity disease; Preferably, the inflammatory disease comprises one or more of asthma, rheumatoid arthritis, chronic obstructive pulmonary disease, psoriasis, colitis, chronic inflammatory demyelinating polyneuropathy, giant cell arteritis, glomerulonephritis, juvenile idiopathic arthritis, synovitis, osteitis, polymyositis, Wegener's granulomatosis, psoriasis, Behcet's syndrome, polyarteritis nodosa, Takayasu arteritis, graft-versus-host disease, adult-onset Still's disease, hidradenitis suppurativa, antiphospholipid syndrome, cryoglobulinemia vasculitis, hidradenitis suppurativa, idiopathic membranitis, autoimmune hepatitis, erythema nodosum, myelodysplastic syndrome or amyloidosis; Preferably, the autoimmune disease comprises one or more of rheumatoid arthritis, multiple sclerosis, autoimmune hemolytic anemia, chronic inflammatory demyelinating polyneuropathy, glomerulonephritis, juvenile idiopathic arthritis, polymyalgia rheumatica, systemic lupus erythematosus, thrombotic / idiopathic thrombocytopenic purpura, Sjögren's syndrome, polymyositis, Wegener's granulomatosis, Takayasu arteritis, sarcoidosis, pyoderma gangrenosum, Kawasaki's disease, relapsing polychondritis, relapsing polychondritis, celiac disease, autoimmune hepatitis, myasthenia gravis, SAPHO syndrome, Graves' disease, myelodysplastic syndrome, pemphigus or amyloidosis; Preferably, the immunoproliferative disease includes one or more of myeloma (including multiple myeloma), leukemia (acute lymphocytic leukemia, B-cell chronic lymphocytic leukemia, naked cell acute lymphocytic leukemia, histo-monocyte acute monocytic leukemia, hairy cell leukemia), lymphoma (including lymphoblastoma, non-Hodgkin's lymphoma, Hodgkin's lymphoma, Burkitt's lymphoma), Sezary syndrome, mycosis fungoides, essential macroglobulinemia, infectious mononucleosis or acute histiocytosis.

22. A method for constructing an animal model of immune system disease, characterized in that: The construction method includes regulating the expression level or function of the target, and the target includes one or more of Dock2, AP-1 or Sub1; Preferably, the regulation is upregulation; Preferably, the expression level of the target is the expression level of the target mRNA and / or protein.

23. The construction method according to claim 22, characterized in that: The construction method includes up-regulating the expression of Dock2; Preferably, the up-regulation of Dock2 expression includes over-expression of Dock2 or its DHR-2 domain; or, up-regulation of the binding of Sub1 and JunB; or, up-regulation of the expression level or function of Sub1 and / or JunB.

24. An animal model of immune system disease obtained by the construction method according to claim 22 or 23.

25. Use of the immune system disease animal model according to claim 24 in screening drugs for preventing and / or treating immune system diseases.

26. A method for diagnosing, monitoring, severity assessment, efficacy assessment and / or prognosis assessment of an immune system disease, characterized in that: The method includes detecting the presence or expression of a biomarker, wherein the biomarker includes one or more of Dock2, AP-1 or Sub1; Preferably, the expression level of the biomarker is the expression level of the mRNA and / or protein of the biomarker.

27. The method according to claim 23, characterized in that The biomarkers are one or more biomarkers selected from the central nervous system, body fluids, cells, tissues or organs; Preferably, the body fluid comprises blood or cerebrospinal fluid; Preferably, the biomarker is a biomarker in an immune cell, such as a T cell; Preferably, the T cells include Tconv cells or Treg cells; Preferably, the T cells include CD8 + T cells, CD4 + T cells, CD25 + T cells, CD25 - T cells, Foxp3 + T cells, CD127 + T cells or CD45RB + One or more types of T cells.

28. The method according to any one of claims 26-27, characterized in that: Immune system diseases include one or more of autoimmune diseases, immunoproliferative diseases or inflammatory diseases; Preferably, the inflammatory disease includes an infectious disease or a hypersensitivity disease; Preferably, the inflammatory disease comprises one or more of asthma, rheumatoid arthritis, chronic obstructive pulmonary disease, psoriasis, colitis, chronic inflammatory demyelinating polyneuropathy, giant cell arteritis, glomerulonephritis, juvenile idiopathic arthritis, synovitis, osteitis, polymyositis, Wegener's granulomatosis, psoriasis, Behcet's syndrome, polyarteritis nodosa, Takayasu arteritis, graft-versus-host disease, adult-onset Still's disease, hidradenitis suppurativa, antiphospholipid syndrome, cryoglobulinemia vasculitis, hidradenitis suppurativa, idiopathic membranitis, autoimmune hepatitis, erythema nodosum, myelodysplastic syndrome or amyloidosis; Preferably, the autoimmune disease comprises one or more of rheumatoid arthritis, multiple sclerosis, autoimmune hemolytic anemia, chronic inflammatory demyelinating polyneuropathy, glomerulonephritis, juvenile idiopathic arthritis, polymyalgia rheumatica, systemic lupus erythematosus, thrombotic / idiopathic thrombocytopenic purpura, Sjögren's syndrome, polymyositis, Wegener's granulomatosis, Takayasu arteritis, sarcoidosis, pyoderma gangrenosum, Kawasaki's disease, relapsing polychondritis, relapsing polychondritis, celiac disease, autoimmune hepatitis, myasthenia gravis, SAPHO syndrome, Graves' disease, myelodysplastic syndrome, pemphigus or amyloidosis; Preferably, the immunoproliferative disease includes one or more of myeloma (including multiple myeloma), leukemia (acute lymphocytic leukemia, B-cell chronic lymphocytic leukemia, naked cell acute lymphocytic leukemia, histo-monocyte acute monocytic leukemia, hairy cell leukemia), lymphoma (including lymphoblastoma, non-Hodgkin's lymphoma, Hodgkin's lymphoma, Burkitt's lymphoma), Sezary syndrome, mycosis fungoides, essential macroglobulinemia, infectious mononucleosis or acute histiocytosis.

29. A method for preventing and / or treating immune system diseases, characterized in that: The method comprises using the immune cell of any one of claims 13 to 17, and / or regulating the expression level or function of a target, wherein the target comprises one or more of Dock2, AP-1 or Sub1; Preferably, the expression level of the target is the expression level of the mRNA and / or protein of the target; Preferably, the regulation includes up-regulation or down-regulation; Preferably, regulating the function of Sub1 includes regulating its DNA binding function, or its function of binding to JunB; Preferably, regulating the function of JunB includes regulating the function of its BM domain, such as the function of binding to Sub1 or the function of binding to Dock2. Preferably, the site binding to Dock2 includes a promoter or an enhancer.

30. The method according to claim 29, characterized in that The method comprises administering to a subject an effective amount of the immune cell according to any one of claims 13 to 17, or an agent that downregulates the expression level or function of a target.

31. The method according to claim 29 or 30, characterized in that The target is one or more targets selected from the central nervous system, body fluids, cells, tissues or organs; Preferably, the body fluid comprises blood or cerebrospinal fluid; Preferably, the target is a target in an immune cell, such as a T cell; Preferably, the T cells include Tconv cells or Treg cells; Preferably, the T cells include CD8 + T cells, CD4 + T cells, CD25 + T cells, CD25 - T cells, Foxp3 + T cells, CD127 + T cells or CD45RB + One or more types of T cells.

32. The method according to any one of claims 29 to 31, characterized in that: The immune cells are autologous or allogeneic.

33. The method according to any one of claims 29 to 32, characterized in that: Said down-regulation includes knocking out or knocking down; Preferably, down-regulating the expression of Sub1 includes knocking out all or part of exons 1 to 5 of the Sub1 gene, further preferably knocking out exon 3 and exon 4 of the Sub1 gene, preferably introns 3-4 are knocked out; Preferably, down-regulating the expression level of JunB comprises knocking out or knocking down JunB or its BM domain.

34. The method according to claim 29, characterized in that Downregulating Sub1 can prevent and / or treat immune system diseases by reducing JunB expression or function.

35. The method according to claim 29, characterized in that Downregulating Sub1 can prevent and / or treat immune system diseases by reducing the expression or function of Dock2.

36. The method according to claim 29, characterized in that Downregulating Sub1 prevents and / or treats immune system diseases by blocking or reducing the binding of Sub1 to JunB.

37. The method according to any one of claims 29 to 36, characterized in that: Immune system diseases include one or more of autoimmune diseases, immunoproliferative diseases or inflammatory diseases; Preferably, the inflammatory disease includes an infectious disease or a hypersensitivity disease; Preferably, the inflammatory disease comprises one or more of asthma, rheumatoid arthritis, chronic obstructive pulmonary disease, psoriasis, colitis, chronic inflammatory demyelinating polyneuropathy, giant cell arteritis, glomerulonephritis, juvenile idiopathic arthritis, synovitis, osteitis, polymyositis, Wegener's granulomatosis, psoriasis, Behcet's syndrome, polyarteritis nodosa, Takayasu arteritis, graft-versus-host disease, adult-onset Still's disease, hidradenitis suppurativa, antiphospholipid syndrome, cryoglobulinemia vasculitis, hidradenitis suppurativa, idiopathic membranitis, autoimmune hepatitis, erythema nodosum, myelodysplastic syndrome or amyloidosis; Preferably, the autoimmune disease comprises one or more of rheumatoid arthritis, multiple sclerosis, autoimmune hemolytic anemia, chronic inflammatory demyelinating polyneuropathy, glomerulonephritis, juvenile idiopathic arthritis, polymyalgia rheumatica, systemic lupus erythematosus, thrombotic / idiopathic thrombocytopenic purpura, Sjögren's syndrome, polymyositis, Wegener's granulomatosis, Takayasu arteritis, sarcoidosis, pyoderma gangrenosum, Kawasaki's disease, relapsing polychondritis, relapsing polychondritis, celiac disease, autoimmune hepatitis, myasthenia gravis, SAPHO syndrome, Graves' disease, myelodysplastic syndrome, pemphigus or amyloidosis; Preferably, the immunoproliferative disease includes one or more of myeloma (including multiple myeloma), leukemia (acute lymphocytic leukemia, B-cell chronic lymphocytic leukemia, naked cell acute lymphocytic leukemia, histo-monocyte acute monocytic leukemia, hairy cell leukemia), lymphoma (including lymphoblastoma, non-Hodgkin's lymphoma, Hodgkin's lymphoma, Burkitt's lymphoma), Sezary syndrome, mycosis fungoides, essential macroglobulinemia, infectious mononucleosis or acute histiocytosis.

38. A method for screening drugs for preventing or treating immune system diseases, characterized in that: The method comprises adding a drug to be tested to a sample and detecting the expression level or function of a target, wherein the target comprises one or more of Dock2, AP-1 or Sub1; Preferably, the expression level of the target is the expression level of the target mRNA and / or protein.

39. The method according to claim 38, characterized in that The sample includes one or more of the central nervous system, body fluids, cells, tissues or organs; Preferably, the body fluid comprises blood or cerebrospinal fluid; Preferably, the cells include immune cells, such as T cells; Preferably, the T cells include Tconv cells or Treg cells; Preferably, the T cells include CD8 + T cells, CD4 + T cells, CD25 + T cells, CD25 - T cells, Foxp3 + T cells, CD127 + T cells or CD45RB + One or more types of T cells.

40. The method according to any one of claims 38-39, characterized in that: The sample is extracted from a subject suffering from an immune system disease.

41. The method according to any one of claims 38 to 40, characterized in that: Immune system diseases include one or more of autoimmune diseases, immunoproliferative diseases or inflammatory diseases; Preferably, the inflammatory disease includes an infectious disease or a hypersensitivity disease; Preferably, the inflammatory disease comprises one or more of asthma, rheumatoid arthritis, chronic obstructive pulmonary disease, psoriasis, colitis, chronic inflammatory demyelinating polyneuropathy, giant cell arteritis, glomerulonephritis, juvenile idiopathic arthritis, synovitis, osteitis, polymyositis, Wegener's granulomatosis, psoriasis, Behcet's syndrome, polyarteritis nodosa, Takayasu arteritis, graft-versus-host disease, adult-onset Still's disease, hidradenitis suppurativa, antiphospholipid syndrome, cryoglobulinemia vasculitis, hidradenitis suppurativa, idiopathic membranitis, autoimmune hepatitis, erythema nodosum, myelodysplastic syndrome or amyloidosis; Preferably, the autoimmune disease comprises one or more of rheumatoid arthritis, multiple sclerosis, autoimmune hemolytic anemia, chronic inflammatory demyelinating polyneuropathy, glomerulonephritis, juvenile idiopathic arthritis, polymyalgia rheumatica, systemic lupus erythematosus, thrombotic / idiopathic thrombocytopenic purpura, Sjögren's syndrome, polymyositis, Wegener's granulomatosis, Takayasu arteritis, sarcoidosis, pyoderma gangrenosum, Kawasaki's disease, relapsing polychondritis, relapsing polychondritis, celiac disease, autoimmune hepatitis, myasthenia gravis, SAPHO syndrome, Graves' disease, myelodysplastic syndrome, pemphigus or amyloidosis; Preferably, the immunoproliferative disease includes one or more of myeloma (including multiple myeloma), leukemia (acute lymphocytic leukemia, B-cell chronic lymphocytic leukemia, naked cell acute lymphocytic leukemia, histo-monocyte acute monocytic leukemia, hairy cell leukemia), lymphoma (including lymphoblastoma, non-Hodgkin's lymphoma, Hodgkin's lymphoma, Burkitt's lymphoma), Sezary syndrome, mycosis fungoides, essential macroglobulinemia, infectious mononucleosis or acute histiocytosis.

42. A method for inhibiting immune cell migration, characterized in that: The method comprises using the immune cell of any one of claims 13 to 17, and / or regulating the expression level or function of a target; the target comprises one or more of Dock2, AP-1 or Sub1; Preferably, the expression level of the target is the expression level of the mRNA and / or protein of the target; Preferably, the regulation includes up-regulation or down-regulation; Preferably, regulating the function of Sub1 includes regulating its DNA binding function, or its function of binding to JunB; Preferably, regulating the function of JunB includes regulating the function of its BM domain, such as the function of binding to Sub1 or the function of binding to Dock2. Preferably, the site binding to Dock2 includes a promoter or an enhancer.

43. The method according to claim 42, characterized in that The immune cells include one or more of lymphocytes, dendritic cells, monocytes / macrophages, granulocytes or mast cells; Preferably, the lymphocytes include T cells; Preferably, the T cells include Tconv cells or Treg cells; Preferably, the T cells include CD8 + T cells, CD4 + T cells, CD25 + T cells, CD25 - T cells, Foxp3 + T cells, CD127 + T cells or CD45RB + One or more types of T cells.

44. The method according to claim 42 or 43, characterized in that The migration includes migration to the central nervous system; Preferably, the immune cell migration includes Dock2-mediated T cell migration.

45. The method according to any one of claims 42-44, characterized in that: Said down-regulation includes knocking out or knocking down; Preferably, the down-regulation of the expression of Sub1 includes knocking out all or part of exons 1 to 5 of the Sub1 gene, and further preferably knocking out exon 3 and exon 4 of the Sub1 gene, preferably introns 3-4 are knocked out; Preferably, down-regulating the expression level of JunB comprises knocking out or knocking down JunB or its BM domain.

46. ​​The method according to claim 42, characterized in that Downregulation of Sub1 inhibits immune cell migration by reducing JunB expression or function.

47. The method according to claim 42, characterized in that Downregulation of Sub1 inhibits immune cell migration by reducing Dock2 expression or function.

48. The method according to claim 42, characterized in that Downregulation of Sub1 inhibits immune cell migration by blocking or reducing the binding of Sub1 to JunB.

49. A method for inhibiting the secretion of inflammatory cytokines, characterized in that: The method comprises using the immune cell of any one of claims 13 to 17, and / or regulating the expression level or function of a target; the target comprises one or more of Dock2, AP-1 or Sub1; Preferably, the expression level of the target is the expression level of the mRNA and / or protein of the target; Preferably, the regulation includes up-regulation or down-regulation; Preferably, regulating the function of Sub1 includes regulating its DNA binding function, or its function of binding to JunB; Preferably, regulating the function of JunB includes regulating the function of its BM domain, such as the function of binding to Sub1 or the function of binding to Dock2. Preferably, the site binding to Dock2 includes a promoter or an enhancer.

50. The method according to claim 49, characterized in that Said down-regulation includes knocking out or knocking down; Preferably, the down-regulation of the expression of Sub1 includes knocking out all or part of exons 1 to 5 of the Sub1 gene, and further preferably knocking out exon 3 and exon 4 of the Sub1 gene, preferably introns 3-4 are knocked out; Preferably, down-regulating the expression level of JunB comprises knocking out or knocking down JunB or its BM domain.

51. The method according to claim 47, characterized in that Downregulation of Sub1 inhibits the secretion of inflammatory cytokines by reducing JunB expression or function.

52. The method of claim 47, wherein: Downregulation of Sub1 inhibits the secretion of inflammatory cytokines by reducing the expression or function of Dock2.

53. The method according to claim 47, characterized in that Downregulation of Sub1 inhibits the secretion of inflammatory cytokines by blocking or reducing the binding of Sub1 to JunB.

54. The method according to any one of claims 49 to 53, characterized in that: The inflammatory cytokines include one or both of IL-17A and IFN-γ.

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