CN117683877A - Biomarkers or targets for diagnosing or treating immune system diseases - Google Patents

Abstract

The invention relates to the technical field of biological medicine, in particular to a biomarker or target spot for diagnosing or treating immune system diseases. The biomarker or target comprises Dock2 and/or Sub1. The Dock2 and/or Sub1 is highly expressed in various immune system diseases, and down-regulating Dock2 and/or Sub1 can be used for treating various immune system diseases (especially multiple sclerosis).

Description

Biomarkers or targets for diagnosing or treating immune system diseases

Technical Field

The invention relates to the technical field of biological medicine, in particular to a biomarker or target spot for diagnosing or treating immune system diseases.

Background

One of the fundamental features of the immune system is its unique ability to distinguish between self and non-self sufficiently to prevent abnormal infection and colonization. Maintenance of the immune balance requires precise control of the activity and function of immune cells to avoid unnecessary responses to itself. The central and peripheral lymphoid organs maintain the body's immune tolerance so that autoreactive T, B lymphocytes produced by the body can be cleared or silenced under normal conditions. The immune system evolved a variety of mechanisms that control the autoreaction, wherein defects in one or more mechanisms may lead to disruption of tolerance, enabling autoreactive lymphocytes to evade immune system monitoring, leading to autoimmune disease. A wide range of autoimmune diseases are described as having variable onset age, tissue distribution, clinical and functional effects. Most of these diseases cannot be cured and require life-long treatment. Autoimmune diseases affect about one tenth of individuals, and have complex etiology, variable symptoms and signs, and an unclear pathogenesis.

Multiple sclerosis (multiple sclerosis, MS) is an autoimmune disease characterized by typical central nervous system (central nervous system, CNS) white matter demyelinating lesions, T cell mediated immune tolerance disorders that in turn result in inflammatory lesions of the myelin sheath and axons. Because of its high incidence, chronic disease processes and susceptibility to young people, it has become one of the most important neurological diseases. Although the exact cause of multiple sclerosis is not known, myelin-specific CD4 is widely recognized ⁺ Activation of T cells is a central step in the onset of neuroinflammation. The strongest genetic risk factors for multiple sclerosis are HLA-DRA, interleukin (IL) -2R and IL-7R genes, which underscores CD4 ⁺ Importance of T cells. Bone marrow-specific T cell adoptive transfer can induce the occurrence of EAE further potentiate the view that MS is a T cell mediated autoimmune disease. Furthermore, only CD4 ⁺ T cell transfer is sufficient to trigger EAE, an observation that has focused on CD4 for the majority of pathogenic mechanism studies ⁺ Pathogenic effects of T cells.

The central nervous system has several unique barriers to maintain homeostasis and limit leukocyte infiltration. To initiate central nervous system autoimmunity, autoantigen-specific T cells are activated peripherally and enter the central nervous system where they are reactivated by APCs presenting autoantigens, which trigger the release of cytokines, thereby activating and recruiting other inflammatory cells, causing inflammation. However, the central nervous system uses various disorders, such as the Blood Brain Barrier (BBB) and the blood cerebrospinal fluid barrier (BCSFB), to limit T cell entry. To break through these disorders, coordinated use of selectins, integrins and chemokines is required to migrate effectively to the central nervous system. In the case of immune disorders, blocking migration of immune cells to the central nervous system has proven to be an effective method of treating MS. A recent study found that DICAM (dual immunoglobulin domain containing cell adhesion molecule) promotes neuroinflammation by promoting migration of TH17 lymphocytes onto the blood brain barrier endothelium, reduces transport of mouse and human TH17 cells across the blood brain barrier by drug neutralization of DICAM, and alleviates disease symptoms in the mouse autoimmune encephalomyelitis model, suggesting that blocking DICAM with monoclonal antibodies may be a promising treatment. Natalizumab is an approved monoclonal antibody intended to specifically block this process by targeting integrin VLA4 expressed on nearly all immune cell subsets. Although natalizumab has a therapeutic effect on treating MS patients, natalizumab has been found to be associated with severe rebound of MS activity following withdrawal, as well as with life-threatening central nervous system infections, due to impaired central nervous system immune monitoring caused by the ubiquity of VLA4 expressed by protective and pathogenic leukocytes. It is therefore important to find new targets for effectiveness and low side effects.

Chemokine signals are important components of lymphocyte trafficking, activation and survival, with dynamic cytoskeletal changes occurring throughout T cell transendothelial migration (TEM). Rac is a member of the GTPase Rho family and is the primary driver of the cytoskeletal dynamics of actin downstream of most chemokine receptors. Rac circulates between inactive (GDP-bound) and active (GTP-bound) states, this switching being mainly due to the action of the guanine nucleotide exchange factor (GEF). GEFs consist of two family members, the classical Dbl-homolog (DH) domain-containing protein family and the non-classical Dock protein family. The Dock family of proteins includes highly conserved Dock homology domains DHR-1 and DHR-2, which play a key role in cell proliferation, migration and activation. Based on sequence identityThe family of Dock proteins includes 4 types, dock-a, dock-b, dock-c, and Dock-d. The Dock-A subfamily includes Dock1, dock2 and Dock5, which all activate the small G protein Rac, but not Cdc42. Among these subfamily members, dock2 is specifically expressed on immune cells, including bone marrow cells and T/B lymphocytes, while the expression of other Dock proteins is not limited to immune cells. Dock2 regulates the migration, proliferation and activation of lymphocytes and innate immune cells by activating Rac to affect cell membrane polarization and cytoskeletal dynamics. By using Dock2 ^-/- In mice, dock2 has been determined to be critical for Rac activation and chemotaxis of a variety of chemokine receptor downstream lymphocytes, including CCR7 and CXCR 4. There is growing evidence that Dock2 regulates the development of various inflammatory diseases, such as leishmania major infection, human Immunodeficiency Virus (HIV) infection, allergic diseases caused by T and B cell combined immunodeficiency, and the like.

The transcriptional activator (SUB 1, also known as PC 4) was identified as a cofactor for RNA polymerase ii dependent transcription, which comprises a unique conserved non-specific DNA binding domain and is involved in different DNA dependent processes including replication, DNA repair and transcription. In mice, complete knockout of SUB1 results in embryonic lethality, which reflects its multiple functions. As transcription regulators, several studies have demonstrated that SUB1 can directly initiate transcription initiation of multiple genes, such as PLK1, BUB1B, and C-MYC, among others. In addition, SUB1 interacts with different domains of activating factors such as VP16, GAL4, AP2, HIV-TAT, P53, and SMYD3 to regulate their function. Currently, research on SUB1 function is mostly focused on tumor cells, such as non-patent literature: microRNA-101regulated transcriptional modulator SUB1 plays a role in prostate cancer (Chakravarthi BV, goswami MT, et al; oncogene.2016Dec 8;35 (49): 6330-6340) discloses that SUB1 expression is elevated in prostate cancer cells; further, non-patent literature: the prognostic value of SUB1 in liver cancer patients (Huang Yongping, tang Dejun, liu Dong, etc., in modern clinical surgery in the south of the Ling, 2020,20 (04) discloses that SUB1 is highly expressed in liver cancer tissues, SUB1 can be used as a prognostic factor for overall survival and disease-free survival of liver cancer patients, but the relationship between SUB1 and immune system diseases is not disclosed in the prior art.

Disclosure of Invention

The present application found that Sub1 was CD4 in patients with various immune system diseases ⁺ High expression in T cells. Down-regulating Dock2 expression by deletion of Sub1, inhibiting GTPase activity and F actin polymerization, thereby inhibiting CD4 ⁺ Migration of T cells to the central nervous system blocks the occurrence of immune system diseases, particularly multiple sclerosis. Define Sub1 as CD4 ⁺ The key regulator of T cell migration is the blockade of CD4 ⁺ The potential therapeutic targets for T cell migration to the central nervous system are of great significance in suppressing the development and progression of immune system diseases, particularly multiple sclerosis. Specific:

in a first aspect of the invention, there is provided the use of Dock2 and/or Sub1 as a biomarker or target in the manufacture of a product for diagnosis, monitoring, severity assessment, efficacy assessment or prognosis assessment, and/or for the prevention or treatment of a disease of the immune system.

Preferably, said Dock2 and/or Sub1 is a gene and/or protein.

Wherein Dock2 is cytokinin 2 or a gene encoding the same; sub1 is a cofactor for RNA polymerase II-dependent transcription or a gene encoding the same.

Preferably, said diagnosing, monitoring, severity assessing, efficacy assessing and/or prognosis assessing an immune system disorder comprises detecting the presence or absence of Dock2 and/or Sub1 or the amount of mRNA and/or protein expressed thereof.

Preferably, the detection of the expression level of mRNA and/or protein of Dock2 and/or Sub1 comprises the use of a reagent.

Further preferably, the reagents may include, but are not limited to, PCR reagents, RPA reagents, LAMP reagents, ERA reagents, RCA reagents, western blot reagents, immunohistochemical reagents, sequencing reagents, liquid phase reagents, mass spectrometry reagents, and the like.

Preferably, when the expression level of mRNA and/or protein of Dock2 and/or Sub1 is significantly higher than a threshold value, which is obtained earlier by experiments, it indicates that the individual suffers from an immune system disorder.

Preferably, the prevention or treatment of immune system diseases comprises modulation of the expression of mRNA and/or protein of Dock2 and/or Sub 1.

Preferably, the modulation comprises up-modulation or down-modulation.

Further preferred, the prevention or treatment of immune system disorders comprises knocking out or knocking down Sub1, more preferred, the knocking out or knocking down Sub1 comprises but is not limited to using a CRISPR/Cas system, a tissue specific knocking out system or introducing interfering RNA or microRNA (e.g. microRNA-101) targeting Sub 1.

Preferably, the knocking out or knocking down Sub1 comprises using an agent.

Preferably, the agents include, but are not limited to, agents required for knockout or knockdown (e.g., materials required for tissue-specific knockout systems, materials required for CRISPR/Cas systems, interfering RNAs or micrornas, etc.), or agents required to inhibit gene transcription and/or translation, etc.

Preferably, the interfering RNA includes, but is not limited to, one or more than two of siRNA, dsRNA, shRNA, aiRNA or miRNA.

Preferably, the method comprises the steps of, the Cas protein used in the CRISPR/Cas system is selected from Casl, caslB, cas, cas3, cas4, cas5d, cas5t, cas5h, cas5a, cas6, cas7, cas8, cas9, caslO, csyl, csy2, csy3, csy4, csel, cse2, cse3, cse4, cse5e, cscl, csc2, csa5, csnl, 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 2, cshl 2, al, a2, csa3, cscl 4, csc2, C84, C2, C, or a modification thereof.

The tissue-specific knockout techniques include, but are not limited to, the Cre/loxp recombinase system, which is a site-specific recombinase technique that performs deletions, insertions, translocations and inversions at specific sites on DNA, with which the DNA in the cell can be modified for a specific cell type or with specific external stimuli. The Cre recombinase recognizes the inverted repeats at both ends of the loxP site and binds to form a dimer, which then binds to the dimer at the other loxP site to form a tetramer. The LoxP site is directional and the two sites of tetramer ligation are parallel in direction. The DNA sequence between the two loxP sites is then cleaved by Cre recombinase. Next, DNA ligase rapidly and efficiently ligates these strands. If two loxP sites are positioned on the same DNA chain and have the same direction, cre recombinase mediates sequence excision between loxP sites; if two loxP sites are located on the same DNA strand and are opposite in direction, cre recombinase mediates sequence inversion between loxP; if the two loxP sites are located on different DNA strands or chromosomes, cre recombinase mediates the exchange or chromosomal translocation of the two DNA strands.

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

Preferably, the knockout or knocking down of Sub1 is such that Sub1 is not transcribed or the protein after transcription is not expressed or activity is reduced, preferably comprising knocking out all or part of exons 1 to 5 of the Sub1 gene, e.g. any one or a combination of two or more of exons 1, 2, 3, 4 or 5.

In one embodiment of the invention, the knockout or knockdown of Sub1 comprises knocking out exon 3 and exon 4 of the Sub1 gene, preferably 3-4 introns are knocked out. Preferably, the knockout comprises the use of tissue specific knockout, e.g., insertion of the 5 'end of exon 3 and the 3' end of exon 4 into the LoxP site followed by reintroduction of Cre recombinase.

Preferably, the knocking out or knocking down Sub1 comprises knocking out a Sub1 gene in a T cell. The T cells may be CD4 ⁺ T cells or CD8 ⁺ T cells, further preferably CD4 ⁺ CD8 ⁺ T cells. Preferably, the knockout or knockdown comprises the combined use of tissue-specificThe knockdown system and CRISPR/Cas system, further preferably, the knockdown or knockdown of Sub1 comprises inserting LoxP sites at the 5 'end of exon 3 and 3' end of exon 4 of the Sub1 gene using the CRISPR/Cas system, followed by the introduction of Cre recombinases (e.g., T cell specific Cre recombinases including but not limited to CD4-Cre and/or CD 8-Cre). Preferably, said Dock2 and/or Sub1 is derived from 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 tissue comprises one or more of brain tissue, spinal cord, spleen or lymph tissue;

preferably, the cells include immune cells, and more preferably, the immune cells include one or more of lymphocytes, dendritic cells, monocytes/macrophages, granulocytes or mast cells.

Preferably, the lymphocytes comprise T cells, preferably, the T cells comprise CD8 ⁺ T cells, CD4 ⁺ T cells, CD4 ⁺ CD8 ⁺ T cells, treg cells (CD 4 ⁺ 、CD25 ⁺ 、Foxp3 ⁺ 、CD127 ⁺ )。

In one embodiment of the present invention, the cell is CD4 ⁺ T cells, CD8 ⁺ T cells or CD4 ⁺ CD8 ⁺ T cells.

Preferably, the product comprises an agent for detecting the presence or absence of Dock2 and/or Sub1 or the amount of mRNA and/or protein expressed therein, or the product comprises an agent for modulating the amount of Dock2 and/or Sub1 mRNA and/or protein expressed therein (preferably up-or down-regulated, more preferably down-regulated).

In one embodiment of the invention, sub1 enhances histone modification (e.g., methylation modification or acetylation modification) of chromatin to increase the degree of opening of chromatin to recruit the binding of an AP-1 transcription factor to the promoter of Dock2 to drive transcription, while Sub1 further stabilizes the occupancy of AP-1 on the promoter of Dock2 by interaction with AP-1 to promote AP-1 mediated transcriptional activation.

Wherein, AP-1 is a transcription factor, is a dimer complex, and forms a highly conserved homodimer or heterodimer by Jun protein, fos protein, activating transcription factor ATF (activating transcription factor) or muscle aponeurosis fiber sarcoma MAF (musculoaponeurotic fibrosarcoma) family of basic leucine zipper structure, and can activate transcription of downstream target genes.

Preferably, the Jun protein family comprises one or more of v-Jun, c-Jun, junB or JunD;

preferably, the Fos protein family comprises one or more than two of v-Fos, c-Fos, fosB, fra-1 and Fra-2;

preferably, the ATF protein family comprises one or more than two of ATF2, ATF3/LRF1, B-ATF, JDP1 or JDP 2;

preferably, the MAF protein family comprises one or more of c-Maf, mafB, mafA, mafG, mafF, mafK or Nrl.

In one 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) polygenic family members.

The histone methylation modification comprises H3K4me1 (representing the single methylation of lysine at position 4 of H3 histone).

The histone acetylation modification includes H3K27ac (indicating acetylation of lysine at position 14 of H3 histone).

In one embodiment of the invention, the product comprises a drug, or a kit, test paper, mass spectrometry or biochip for diagnosis, monitoring, severity assessment, efficacy assessment and/or prognosis of an immune system disorder.

Preferably, the medicament comprises an agent that modulates Dock2 and/or Sub1, further preferably, the modulation comprises up-regulation and/or down-regulation.

In one embodiment of the present invention, the drug down regulates Dock2 and/or Sub1, the drug may be an antibody or a small molecule inhibitor, etc., for example, the drug may be one or more than two of an anti-Dock 2 antibody, an anti-Sub 1 antibody, a microRNA (e.g., microRNA-101) or an IRF4 transcription factor antibody.

Down-regulation of Sub1 down-regulates the expression of Dock 2.

Down-regulating Sub1 blocks or slows down Dock2 mediated CD4 ⁺ T cells (comprising CD 4) ⁺ CD8 ⁺ T cells) to prevent or treat immune system disorders.

Said CD4 ⁺ T cell migration including CD4 ⁺ Migration of T cells to the central nervous system.

Down-regulating Sub1 inhibits GTPase Activity and polymerization of F-actin by down-regulating the expression of Dock2, thereby inhibiting CD4 ⁺ Migration of T cells to the central nervous system blocks the onset of immune system disorders.

Preferably, the medicament further comprises pharmaceutically acceptable auxiliary materials.

Preferably, the pharmaceutically acceptable auxiliary materials are selected from one or more than two of diluents, absorbents, wetting agents, adhesives, disintegrants, lubricants, solvents, pH regulators, buffers, antioxidants, metal ion chelating agents, bacteriostats or isotonicity regulators.

Preferably, the pharmaceutical formulation may be in the form of a suspension, powder, granule, tablet, aqueous solution, cream, gel or emulsion. The various dosage forms of the medicament can be prepared according to the conventional production method in the pharmaceutical field.

Preferably, the pharmaceutical formulation is preferably a unit dose formulation.

Preferably, the active ingredient (agent for modulating Dock2 and/or Sub 1) contained in the medicament comprises any one of 0.001% -99.9% by volume or mass ratio, 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.

Other suitable therapeutic agents may also be included in the medicament, as desired in particular embodiments.

Formulations of the medicament suitable for parenteral administration such as, for example, by intravenous, intramuscular, intradermal and subcutaneous routes include aqueous and nonaqueous isotonic sterile injection solutions which may contain antioxidants, buffers, bacteriostats, and solutes which render the formulation isotonic with the blood of the recipient, and aqueous and nonaqueous sterile suspensions which may contain suspending agents, solubilizers, thickening agents, stabilizers or preservatives. The formulations may be presented in unit-dose or multi-dose sealed containers, such as ampules and vials. Injectable solutions and suspensions may be prepared from sterile powders, granules and tablets of the type previously described.

Preferably, the drug is administered by intravenous infusion, topical administration, intraperitoneal administration, intrathecal administration, or the like.

Preferably, the kit, test paper, mass spectrum or biochip specifically detects the expression level of mRNA and/or protein of Dock2 and/or Sub1, preferably comprises a reagent for detecting mRNA and/or protein of Dock2 and/or Sub 1.

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

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

Preferably, the immune system disorder is T cell inhibition (e.g., CD4 ⁺ T cells) migration is beneficial in preventing or treating immune system disorders.

Further preferably, the T cell migration is Dock2 mediated T cell migration. The T cell migration includes migration of T cells to the central nervous system.

Preferably, the immune system disease comprises one or more of an infectious disease, a hypersensitivity disease, an autoimmune disease, an immunoproliferation disease or an immunodeficiency disease.

Preferably, the immune system disorder is an autoimmune disorder of the central nervous system white matter demyelinating lesions.

Preferably, the autoimmune disease includes but is not limited to immune-mediated inflammatory diseases.

Further preferred, the immune system disorder comprises one or more of systemic lupus erythematosus, rheumatoid arthritis, anti-neutrophil cytoplasmic antibody associated vasculitis, shi Difen predominance syndrome, sjogren's syndrome, systemic vasculitis, scleroderma, dermatomyositis, mixed connective tissue disease, hashimoto's thyroiditis, primary myelitis, compulsive spondylitis, adult stell's disease, behcet's syndrome, systemic sclerosis, multiple sclerosis, autoimmune hepatitis, polyarteritis nodosa, or ulcerative colitis.

In one embodiment of the invention, the immune system disorder is multiple sclerosis.

In a second aspect of the invention there is provided a biomarker for an immune system disorder, said biomarker comprising Dock2 and/or Sub1.

In a third aspect of the present invention, there is provided a method for screening a drug for preventing or treating an immune system disorder, the method comprising treating a sample with a drug to be tested and detecting the amount of mRNA and/or protein expression of Dock2 and/or Sub1.

Preferably, the sample includes, but is not limited to, a body fluid, a cell, a tissue, or an organ.

Preferably, the body fluid comprises blood or cerebrospinal fluid.

Preferably, the tissue comprises one or more of brain tissue, spinal cord, spleen or lymph tissue;

preferably, the cells include immune cells, and more preferably, the immune cells include one or more of lymphocytes, dendritic cells, monocytes/macrophages, granulocytes or mast cells.

Preferably, the lymphocytes comprise T cells, preferably, the T cells comprise CD8 ⁺ T cells, CD4 ⁺ T cell,CD4 ⁺ CD8 ⁺ T cells, treg cells (CD 4 ⁺ 、CD25 ⁺ 、Foxp3 ⁺ 、CD127 ⁺ )。

In one embodiment of the present invention, the cell is CD4 ⁺ T cells, CD8 ⁺ T cells or CD4 ⁺ CD8 ⁺ T cells.

Preferably, the drug to be tested which can down-regulate Dock2 and/or Sub1 in the sample has a preventive or therapeutic effect.

In a fourth aspect of the invention, a method of diagnosing, monitoring, severity assessment, efficacy assessment and/or prognosis of an immune system disorder is provided.

Preferably, the method comprises detecting the presence or absence of Dock2 and/or Sub1 or the expression level of its mRNA and/or protein.

When the expression level of Dock2 and/or Sub1 is above the threshold, a higher risk or disease is indicated.

In a fifth aspect of the invention, a method of preventing and/or treating an immune system disorder is provided.

Preferably, the method comprises regulating the amount of mRNA and/or protein expression of Dock2 and/or Sub 1.

Preferably, the adjusting comprises down-adjusting.

In one embodiment of the invention, the method comprises administering to a subject in need thereof a therapeutically effective amount of an agent that down-regulates the amount of mRNA and/or protein of Dock2 and/or Sub 1.

Preferably, the agent that down regulates the amount of mRNA and/or protein of Dock2 and/or Sub1 may be the agent required for knocking out or knocking down Sub 1.

In a sixth aspect of the invention, there is provided a method of inhibiting immune cell migration, said method comprising modulating the amount of mRNA or protein expression of Dock2 and/or Sub 1.

Preferably, the adjusting comprises down-adjusting.

Preferably, the immune cells include one or more of lymphocytes, dendritic cells, monocytes/macrophages, granulocytes or mast cells. Preferably, the lymphocytes comprise T cells.

Further preferably, the T cells comprise CD8 ⁺ T cells, CD4 ⁺ T cells, treg cells (CD 4 ⁺ 、CD25 ⁺ 、Foxp3 ⁺ 、CD127 ⁺ )。

Preferably, the migration includes migration to the central nervous system.

Preferably, the immune cell migration is Dock2 mediated T cell migration.

Preferably, the immune system disorder is prevented or treated by inhibiting Dock 2-mediated T cell migration.

In a specific embodiment of the invention, the method comprises down-regulating the expression of Sub1, preferably by the Cre/loxp recombinase system.

In a seventh aspect of the invention, there is provided a method of constructing an animal model of an immune system disorder, said method comprising modulating the expression of mRNA and/or protein of Dock2 and/or Sub 1.

Preferably, the construction method comprises modulating the expression of Dock2 and/or Sub1 in immune cells. The immune cells include one or more of lymphocyte, dendritic cell, mononuclear/macrophage, granulocyte or mast cell, preferably T cell, such as CD8 ⁺ T cells, CD4 ⁺ T cells, CD4 ⁺ CD8 ⁺ T cells, treg cells (CD 4 ⁺ 、CD25 ⁺ 、Foxp3 ⁺ 、CD127 ⁺ )。

Preferably, the adjustment is an up-adjustment.

Preferably, the up-regulation Sub1 is over-expression Sub1 and/or over-expression Dock2.

Preferably, the up-regulating Dock2 is over-expressing Dock2 or a DHR-2 domain thereof.

Upregulation of Sub1 can upregulate the expression of Dock2.

Upregulation of Sub1 may promote Dock 2-mediated CD4 ⁺ T cells (comprising CD 4) ⁺ CD8 ⁺ T cells) migration promotes the development or progression of immune system diseases.

In one embodiment of the inventionIn a mode, the construction method comprises over-expressing Dock2 or DHR-2 domain thereof in animal model to promote CD4 ⁺ GTPase Activity and F-actin polymerization capability of T cells, promoting Dock 2-mediated CD4 ⁺ T cells (comprising CD 4) ⁺ CD8 ⁺ T cells) migrate.

The animal model is a non-human mammal or human, such as a rat, mouse, monkey, pig, cow, horse, sheep, dog, cat, etc.

In an eighth aspect of the present invention, there is provided a method for inhibiting cytokine secretion, the method comprising regulating the expression level of Sub 1. Preferably includes regulating the expression level of mRNA or protein of Sub 1.

Preferably, the modulation comprises up-modulation or down-modulation.

Preferably, the cytokine comprises one or both of IL-17A or IFN-gamma.

The terms "comprising" or "includes" are used in this specification to be open-ended, having the specified components or steps described, and other specified components or steps not materially affected.

The term "and/or" in this disclosure encompasses all combinations of items to which the term is attached, and should be taken as the individual combinations have been individually listed herein. For example, "a and/or B" includes "a", "a and B", and "B". Also for example, "A, B and/or C" include "a", "B", "C", "a and B", "a and C", "B and C" and "a and B and C".

The term "treatment" as used herein means slowing, interrupting, arresting, controlling, stopping, alleviating, or reversing the progression or severity of one sign, symptom, disorder, condition, or disease after the disease has begun to develop, but does not necessarily involve the complete elimination of all disease-related signs, symptoms, conditions, or disorders.

As used herein, "prognostic evaluation" refers to predicting the likely course and outcome of a disease, including determining the specific outcome of the disease (e.g., recovery, appearance or disappearance of certain symptoms, signs and complications, and death) and other abnormalities.

As used herein, "efficacy assessment" refers to assessing a patient's response to treatment.

The term "monitoring" as used herein refers to observing the occurrence and development of a disease.

The term "prevention" as used herein means that an individual takes specific measures to prevent the occurrence of a disease before the disease is diagnosed or has not developed.

The term "diagnosis" in the present invention refers to ascertaining whether a patient has a disease or condition in the past, at the time of diagnosis, or in the future, or to ascertaining the progression of a disease or the likely progression in the future.

By "pharmaceutically acceptable" is meant that the biological activity and properties of the active substance of the administered product are neither significantly stimulated nor inhibited by the organism.

Drawings

Embodiments of the present invention are described in detail below with reference to the attached drawing figures, wherein:

fig. 1: sub1 CD4 in autoimmune disease patients ⁺ Analysis of the expression level in T cells;

fig. 2: sub1 CD4 in MS patients ⁺ Analysis of the expression level in T cells;

fig. 3: WT and Sub1 ^-KO Expression level of mouse Sub1 mRNA;

fig. 4: WT and Sub1 ^-KO Flow cytometry and quantitative detection of mouse T cell development, wherein DN represents double negative cell CD4 ^- CD8 ^- DP represents double positive cell CD4 ⁺ CD8 ⁺ ；

Fig. 5: using Myelin Oligodendrocyte Glycoprotein (MOG) _35-55 ) A flow chart of peptide immunity eliciting autoimmune encephalomyelitis EAE;

Fig. 6: WT and Sub1 ^-KO EAE clinical scores of mice after different days of immunization;

fig. 7: HE staining results;

fig. 8: LFB staining results;

fig. 9: WT and Sub1 ^-KO Mouse central nervous system and peripheral CD4 ⁺ T cell analysis;

fig. 10: CD4 ⁺ Experimental flow for T cell adoptive feedback to induce EAE to occurA program;

FIG. 11CD4 ⁺ EAE clinical scores after T cell adoptive reinfusion for different days;

fig. 12: flow analysis results of mouse brain;

fig. 13: results of flow analysis of mouse spinal cord;

fig. 14: RNA-seq differential gene volcanic mapping and KEGG pathway analysis;

fig. 15: sub1 deletion inhibition of CD4 ⁺ T cell chemotaxis in vitro and homing ability in vivo;

fig. 16: sub1 deletion down regulates Dock2 expression, inhibits Rac activation and F-actin polymerization;

fig. 17: overexpression of Dock2 rescue of Sub1 deleted CD4 ⁺ T cell function;

fig. 18: sub1 enhances chromatin accessibility, and cooperates with JUNB to promote expression of Dock 2;

fig. 19: sub1 increases histone H3K27ac and H3K4me1 modifications, promoting chromatin opening.

In the drawings, P < 0.05, P < 0.01, P < 0.001, P < 0.0001, and ns no significant difference.

Detailed Description

The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is apparent that the described embodiments are only some embodiments of the present invention, not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.

The sources of some materials and experimental methods involved in this application are as follows:

1. mouse origin

Sub1 ^fl/fl (Sub 1 is located on mouse chromosome 15, NCBI reference sequence: NM-011294. Exon 3-4 is used as a conditional knockout region, deletion of this region will result in loss of mouse Sub1 Gene function. Sub1 ^fl/fl The mice are mice with LoxP sites inserted at both ends of exons 3-4) and CD4-Cre by using CRISPR/Cas mediated genome engineering technologyPurchased from racing biotechnology limited. Sub1 ^fl/fl Hybridization of mice with CD4-Cre mice resulted in Sub1 ^-KO And (3) a mouse. All mice were housed in Specific Pathogen Free (SPF) facilities.

2. EAE model induction

With a peptide MOG comprising Myelin Oligodendrocyte Glycoprotein (MOG) _35-55 Emulsion of (100. Mu.g/min) and Mycobacterium tuberculosis H37Ra extract (5 mg/mL) (complete Freund's adjuvant, 200 ul/min) was immunized in balanced proportions on limbs in 8-10 weeks old mice (WT and Sub 1) ^-KO ). 200ng Pertussis Toxin (PTX) was intraperitoneally injected on day 0 and day 2 post immunization. Clinical symptoms of EAE were assessed daily according to score 0, no clinical symptoms; 1 min, soft tail; 2 minutes, weakness of hind limbs; 3 minutes, hind limb paralysis; 4 minutes, quadriplegia; 5 minutes, dying state.

3. T effector cell adoptive feedback induced EAE

WT,Sub1 ^-KO Spleen cells were isolated on day seven of mice EAE molding to prepare a concentration of 10 ⁷ Cell suspensions were placed in T75 flasks per cell/mL. Cells at 37℃with 5% CO ₂ The cells were cultured for 3 days at 50. Mu.g/mL MOG under Th17 polarization conditions of 20ng/mL mIL-23, 20ng/mL mIL6, mTGFβ2ng/mL. The re-stimulated cells were collected and washed with PBS. Intravenous injection of 1X 10 into C57BL/6 receptor mice ⁷ Individual cells. Recipient mice were intraperitoneally injected with 200ng Pertussis Toxin (PTX) on day 0 and day 2 post T cell transfer, and animals were monitored daily for EAE development.

4. T cell isolation in the Central Nervous System (CNS)

Mice hearts were perfused with cold PBS. The forebrain and cerebellum were washed with PBS under hydrostatic pressure, and spinal cord was washed out of the spinal canal. The tissue was ground through a 70 μm filter, the grinding fluid was collected and then subjected to 70%/30% percoll gradient centrifugation to isolate mononuclear cells. Mononuclear cells were aspirated from the medium, washed and resuspended in PBS for subsequent flow analysis.

5. Flow analysis

For surface markers, cells were stained with the indicated antibodies for 30 minutes at 4 ℃. To detect the expression of T lymphocyte cytokines, cells were activated with 500ng/ml ionomycin and 50ng/ml PMA for 4h at 37℃in the presence of Brefeldin A and Monenin solutions. Viable cells were then identified using Violet LIVE/DEAD fixation staining reagents and the cells were fixed and permeabilized using a fixation and permeabilization kit. Cells were collected on a BD LSR Fortessa flow machine and analyzed using Flowjo version 10 software.

6. RNA extraction and real-time fluorescent quantitative PCR (RT-PCR)

Total RNA was extracted from cultured cells or tissues using an RNA extraction kit (RNAfast 200, shanghai Fei Jie) according to the manufacturer's protocol. Reverse transcription of RNA uses the RT kit (Yeasen). The cDNA was then subjected to SYBR-based real-time PCR analysis. The results were normalized using GAPDH and the data were expressed as mean ± standard deviation. The p-value was calculated using the Student's test.

7. Protein immunoblotting (western blot)

Cell samples were lysed in RIPA buffer (Beyotime, P0013B) and protease and phosphatase inhibitors (MCE) were added. Protein concentration was determined using BCA protein assay (Thermo Fisher Scientific). Proteins were separated on a 12.5% SDS-PAGE gel (elegance enzyme) and then transferred to a 0.22 μm polyvinylidene fluoride (PVDF) membrane. Blocking with 5% bsa for 1.5 hours at room temperature followed by incubation with primary antibody overnight at 4 ℃ followed by incubation with secondary antibody for 1 hour.

8. Chemotaxis assay

Chemotaxis experiments were performed using a Transwell 24-well plate (pore size 5 μm; coStar). Cells were starved in serum-free medium for 4-6h. Cells were resuspended in complete medium (1X 10 concentration ⁶ Individual cells/ml), 100. Mu.l of the cell suspension was added to the upper chamber and 500. Mu.l of complete medium containing chemokines was added to the lower chamber. After 2 hours of resting at 37 ℃,5% CO2, all cells in the lower chamber were harvested, counted by flow cytometry and the percentage of migrating cells calculated. CCL21, CCL19 and CXCL12 chemokines are purchased from R &Company D.

9. T cell homing detection

Extraction of WT and Sub1 ^-KO Mouse spleen cells were labeled with Cell Tracker dyes of different colors, respectively, 1:1 Mixed intravenous injectionIs injected into C57/B6 receptor mice (1X 10 each) ⁷ Individual cells). After 4 and 24 hours, lymphocytes were isolated from the recipient mouse blood and secondary lymphoid organs for flow detection.

10. Rac Activity assay

Immediately after stimulation of the cells with CCL21 (1. Mu.g/ml), 1 XMg was added ²⁺ Lysis buffer [ MLB;25mM Hepes (pH 7.5), 150mM NaCl,1% Igepal CA-630,10mM MgCl2,1mM EDTA,10% glycerol; millipore]Then centrifuged at 20000g for 1 min at 4 ℃. An equal amount of total cell lysate was kept as a control, and the remaining lysate was incubated with agarose beads containing the GST-fused Rac binding domain of PAK1 at 4℃for 1 hour. Washing with 1 XMLB buffer 2 times, suspending in SDS-PAGE sample buffer [62.5mM trichloride (pH 6.8), 2% SDS,10% glycerol, 0.005% bromophenol blue, 2.5% 2-mercaptoethanol ]]. Binding proteins and total cell lysates were separated on a 12.5% polyacrylamide gel by SDS-PAGE and detected with monoclonal antibody Rac 1.

11. F-actin polymerization assay

Cells were resuspended to 5X 10 in RPMI/1% FCS/10mM HEPES (pH 7.5) ⁶ Individual cells/ml and stored at 37 ℃. Aliquots were removed from each sample to determine baseline F-actin levels prior to chemokine addition. CCL21 was added to the cell suspension and stimulated at 37 ℃, aliquots were taken at the prescribed stimulation times and immediately fixed with 4% paraformaldehyde for 10 minutes. After washing with PBS, the sample was stained with FITC-Phalloidin (Molecular Probes) as a probe for F-actin and analyzed by flow cytometry.

12. Primary T cell extraction, culture

The lymph nodes of mice were taken, ground with a 70 μm filter, resuspended in T cell primary medium, 37℃and 5% CO ² Culturing under the condition. T cell primary medium formulation: RPMI 1640 (500 ml) +10%FBS+1%NEAA+1%sodium pyruvate+1%P/S+1.74 μl β -mercaptoethanol.

13. Double luciferase reporter assay

Promoter constructs for experiments were generated by cloning the full length-2000- +200 region of the murine Dock2 promoter and inserting it between the nhei and XhoI cleavage sites of pGL3-Basic vectors. PEI transfection reagent is adopted, and the following transcription factors are adopted: luc reporter plasmid: pRL-TK (renilla reference plasmid) =9: 1:0.1 of the proportion of cotransfected 293T cells. Six well plates were transfected with 3. Mu.g total amount of plasmid per well, PEI 12. Mu.l. After 48 hours of transfection, cells were washed and lysed and firefly luciferase and Renilla luciferase activities were evaluated sequentially using a multifunctional microplate reader.

14. RNA-seq analysis

WT and Sub ^-KO Sorting CD4 from spleen cells of EAE mice ⁺ T cells, cell pellet were lysed in Trizol. Sequencing was performed by the company Lithospermum limited. Differential gene expression was analyzed using the DESeq2 package. The significance list was determined by setting the False Discovery Rate (FDR) threshold to less than 0.05 and |log2FC| > 1. All differentially expressed genes were subsequently subjected to GO function and KEGG pathway analysis.

15. ATAC-seq analysis

Sorting WT and Sub1 ^-KO Mouse spleen CD4 ⁺ T cells, cryopreserved. Sequencing was done by penoxen biosciences, inc. The original data (raw reads) were obtained after ATAC-seq sequencing, filtered to remove adaptors, decontaminated, aligned to the reference genome (GRCm 38), and subjected to subsequent information analysis using Unique mapped reads.

16. ChIP-seq analysis

Primary CD4 of mouse ⁺ T cells were crosslinked with 1% formaldehyde at room temperature for 10 minutes and the crosslinking was terminated with 0.125mol/L glycine. Library construction and sequencing was done by the company Lichuan Biolimited. Clean reads were mapped to mouse genome (GRCm 38) using Bowtie2 (v 2.2.6) software. Peak detection was performed using MACS (v2.1.1) peak finding algorithm, and peak positions of gene signatures were annotated using the ChIPseeker R package.

Example 1SUB1 CD4 in patients with various autoimmune diseases ⁺ High expression in T cells

By large-scale immune cell gene expression analysis, as well as whole genome sequence analysis, of patients from 337 diagnosed with 10 immune system diseases and 79 healthy volunteers, SUB1 was found to be in a variety of autoimmune diseasesCD4 of patients with (e.g., shi Difen Crohn's syndrome (SjS), rheumatoid Arthritis (RA), systemic Lupus Erythematosus (SLE), systemic sclerosis (SSc), adult Steve disease (AOSD), behcet's syndrome (BD)) ⁺ T is highly expressed, as shown in FIG. 1, indicating that SUB1 may be a key causative agent of autoimmune disease, as well as CD4 ⁺ An important regulatory factor for T cell function.

Further, single cell data analysis was performed on blood and cerebrospinal fluid of Multiple Sclerosis (MS) patients and healthy volunteers. CD4 of SUB1 in patients with multiple sclerosis was also found ⁺ There is also significantly higher expression in T cells, as shown in figure 2.

Example 2Sub1 deletion significantly inhibits CD4 in the CNS ⁺ Infiltration of T cells to prevent EAE from occurring

To explore the function of Sub1 in MS, T cell specific knockout Sub1 was constructed (Sub 1 ^-KO ) Is a mouse model of (2). First, the knockout efficiency of Sub1 was examined by q-PCR, and Sub1 was determined ^-KO Sub1 was knocked out (see fig. 3), and then the T cell development of the knocked-out mice was examined, and it was found that Sub1 deletion did not affect the normal development of T cells, as shown in fig. 4.

Next, myelin Oligodendrocyte Glycoprotein (MOG) _35-55 ) Peptide immunization of WT and Sub1 ^-KO Mice, thereby eliciting experimental allergic encephalomyelitis EAE (experimental allergic encephalomyelitis, scheme of priming procedure see FIG. 5, EAE is a CD4 specifically sensitized ⁺ T-cell mediated, predominantly autoimmune diseases characterized by the appearance of mononuclear cell infiltration and demyelination around small blood vessels in the central nervous system, have pathological changes similar to multiple sclerosis. Therefore, the EAE animal model is an important path for researching the pathological process and pathogenesis of MS and has important significance in the research of clinical neuroimmunology. The deletion of Sub1 gene was found to completely suppress the onset of EAE, and no EAE symptoms occurred at all during the experiment (see fig. 6 for clinical scores). WT and Sub1 were taken during the onset of EAE mice with concomitant infiltration of inflammatory cells and myelin damage in the central nervous system, particularly spinal cord white matter ^-KO Pathological section analysis is carried out on spinal cord of EAE mice in peak period, HE results show that the WT group EAE mice have dense purple blue particles in spinal cord white matter areas, the purple blue particles are clustered, and the infiltration degree of myelitis cells of the EAE mice with Sub1 deletion is obviously reduced, which indicates that the Sub1 deletion can reduce infiltration of inflammatory cells to CNS (figure 7). The Luxol Fast Blue (LFB) can be dyed blue in combination with myelin sheath (myelin), the LFB is not only used for detecting spinal cord demyelination, but also can show whether myelin sheath is complete, denatured, necrotic degree and repaired under pathological conditions, and experimental results show that compared with WT mice, sub1 ^-KO Mouse spinal LFB staining showed a significant increase in blue portion, better spinal structural integrity, and no cavitation (fig. 8).

T cell conditions in the Central Nervous System (CNS) and in the periphery were further examined. WT or Sub1 was collected during peak disease period after EAE modeling ^-KO The mice brain, spinal cord, spleen and lymph nodes were subjected to flow assay. Sub1 was found in brain and spinal cord ^-KO Mouse CD4 ⁺ The total infiltration number of T cells is significantly reduced, producing CD4 of IL-17A, IFN-gamma ⁺ T cells were significantly reduced. CD4 in spleen and lymph nodes ⁺ Number of T cells and IL-17A and IFN-gamma production in WT and Sub1 ^-KO There were no significant differences in mice (see fig. 9), indicating that the deletion of Sub1 did not damage CD4 ⁺ T cell viability.

To further verify that Sub1 deletion inhibited EAE onset, EAE was induced by T cell adoptive transfer, the experimental procedure is shown in schematic 10. Experimental results show that Sub1 is accepted ^-KO CD4 ⁺ Recipient mice with T cell reinfusion consistently did not develop EAE symptoms (fig. 11). During peak onset period, mouse brain and spinal cord were taken for flow analysis, and Sub1 deleted CD4 ⁺ T cells hardly infiltrate into the CNS (fig. 12 and 13). The above results indicate that Sub1 deletion inhibits CD4 ⁺ Infiltration of T cells into the CNS prevents EAE from occurring.

EXAMPLE 3Sub1 deletion inhibition of CD4 ⁺ Migration of T cells.

To further explore Sub1 modulation of CD4 ⁺ T cell function mechanism, at EAE post-molding stageFor 7 days, WT and Sub1 were taken ^-KO Spleen CD4 of mice ⁺ T cells were subjected to RNA-seq analysis. Volcanic plot results show that with WT CD4 ⁺ Compared with T cells, sub1 ^-KO CD4 ⁺ T cells up-regulate 52 genes and down-regulate 84 genes (FIG. 14 a), and KEGG results show that the deletion of Sub1 results in down-regulating gene-related signaling pathways mainly cytokine-cytokine receptor interactions, chemokine signaling pathways, etc. (FIG. 14 b).

Next, a transwell experiment was performed in vitro to verify whether Sub1 would affect CD4 ⁺ Migration ability of T cells. Chemotactic for CD4 using three different chemokines of CCL21, CCL19 and CXCL12, respectively ⁺ T migration, the result shows that the Sub1 deletion can obviously inhibit CD4 ⁺ The in vitro migration capacity of T cells (FIG. 15, a), which is comparable to CD4 in the CNS of the previous EAE model ⁺ The results for reduced T cell infiltration were consistent. Primary lymphocytes continually migrate or home from the blood to Secondary Lymphoid Organs (SLOs), such as the spleen, peripheral lymph nodes (pLN), and mesenteric lymph nodes (mLN), as well as gut-associated lymphoid tissues. To further demonstrate that Sub1 affects CD4 ⁺ Migration ability of T cells in vivo T cell homing experiments were performed (schematic representation is shown in fig. 15 b). The results indicate that the Sub1 deletion reduces CD4 ⁺ Ability of T cells to home to secondary lymphoid organs (see c in fig. 15).

Example 4Sub1 deletion inhibits Rac Activity and F-actin polymerization by downregulating the expression of Dock2

Further analysis of the genes most significant in the effects of Sub1 deletion, as shown in the heat map (FIG. 16 a), found that Sub1, dock2, IL-17, IFN-gamma, etc. genes were found in Sub1 ^-KO The group was significantly down-regulated. Previous studies have shown that Dock2 can regulate the ability of lymphocytes and innate immune cells to migrate, proliferate and activate by activating Rac to affect cell membrane polarization and cytoskeletal dynamics. First, the expression of Dock2 was detected by RT-PCR and western blot at the RNA and protein levels, respectively, consistent with the results of RNA-seq, with significantly down-regulated Dcok2 expression following Sub1 deletion (b and c in fig. 16).

Small G protein (small GTPase) Rac is a key regulator of F-actin polymerization, a prerequisite for cell migration. In lymphocytes, chemokine-mediated Rac activation is strongly dependent on CDM protein family member Dock2. Thus, it was further examined whether down-regulation of Dock2 would result in impairment of Rac activation. Western blot results showed that the Sub1 deletion significantly reduced the expression of active Rac (Rac-GTP) (d in FIG. 16). Since phalloidin can selectively bind filiform actin (F-actin) rather than actin monomer (G-actin), the degree of polymerization of F-actin is measured by the intensity of phalloidin staining. The results indicate that the deletion of Sub1 significantly reduces the polymerization capacity of F-actin (e in FIG. 16).

EXAMPLE 5 overexpression of Dock2 recovery Sub1 deletion of CD4 ⁺ Migration ability of T cells

To verify Sub1 regulated CD4 ⁺ T cell migration was mediated by Dock2 and a Dock2 back-fill experiment was performed. DocK2 is known to contain four domains, SH3, DHR-1, DHR-2 and PAA (a in FIG. 17). Wherein the V1538 site in the DHR-2 domain has been shown to bind directly to Rac and activate Rac, the Rac GEF activity is almost completely lost when this site is mutated (V1538A). Because the full length of the Dock2 cannot be expressed, a Dock2-DHR2 functional truncated body and the Dock2-DHR2 are respectively constructed ^V1538A Mutants were tested. The result shows that the overexpression of Dock2-DHR2 can obviously recover Sub1 ^-KO CD4 ⁺ Rac activation of T cells and F-actin polymerization capacity (b and c in FIG. 17). Furthermore, in vitro transwell experiments also showed that overexpression of Dock2-DHR2 restored Sub1 ^-KO CD4 ⁺ T cells in vitro migratory capacity, while overexpressing the mutant Dock2-DHR2 ^V1538A Failure to restore CD4 ⁺ Migration ability of T cells (d in fig. 17).

Example 6Sub1 in conjunction with JUNB to promote Dock2 expression

Next, it was further investigated how Sub1 regulates the expression of Dock 2. On day 7 after EAE molding, WT and Sub1 were taken ^-KO Spleen CD4 of mice ⁺ T cells underwent ATAC-seq to describe chromatin accessibility. The results in FIG. 18 a show that the overall chromatin opening degree is significantly reduced after the deletion of Sub1, indicating that Sub1 can enhance chromatin accessibility and thus regulate gene transcription. ATAC (ATAC-seq: chromatin patency assay) Sequence technology) is generally upstream and downstream of the portion of the DNA sequence being transcribed, and the sequences thus obtained can be analyzed in combination with motif to identify which transcription factor is involved in gene expression control. The use of HOMER software for the motif analysis of WT group-specific open chromatin regions found that AP-1 could bind to these open chromatin regions (b in FIG. 18), indicating that Sub1 might regulate expression of these genes in concert with AP-1. The transcription factor AP-1 (activin 1) is a ubiquitous family of dimeric transcriptional complexes involved in a number of cellular and physiological functions. AP-1 dimers are composed of a variety of polygenic protein families that possess highly conserved basic leucine zipper domains (bZip), where leucine zipper domains (LZ) are used for dimerization and Basic Regions (BR) are used for binding to specific DNA motifs. AP-1 is generally defined as a dimeric set consisting of Jun (c-Jun, junB, junD) and Fos (c-Fos, fosB, fra-1, fra-2) polygenic family members.

To verify whether Sub1 synergistically regulates the expression of Dock2 with AP-1, a co-immunoprecipitation (co-IP) experiment was first performed to examine whether Sub1 has an interaction with AP-1. The results in FIG. 18 c show that Sub1 interacts to some extent with all of the Jun family proteins c-Jun, junB, junD, with junB being the most pronounced. To verify whether Sub1 and JunB can directly bind to the promoter of Dock2 to regulate expression, a luciferase reporter plasmid containing the full length of Dock2 promoter was constructed, and a dual luciferase reporter system was used to detect whether Sub1 and JunB bound to the Dock2 promoter sequence. The results show that Sub1 does not bind directly to the Dock2 promoter, whereas JunB binds significantly to the Dock2 promoter and enhances transcription of Dock2 (d in fig. 18)

Example 7Sub1 promotion of chromatin opening by enhanced histone modification

The property of open chromatin to allow for the binding of regulatory factors is called accessibility of chromatin, and chromosomal histone modification is a key factor in regulating accessibility of chromatin. Histone H3K27 acetylation (H3K 27 ac) and H3K4 monomethylation (H3K 4me 1) are known to be markers of gene transcriptional activation, thus for WT and Sub1 ^-KO CD4 ⁺ T cell egg groupChIP-seq analysis of white H3K27ac and H3K4me 1. The results in FIG. 19 a show that the deletion of Sub1 significantly reduced the modified regions of histones H3K27ac and H3K4me 1. The ChIP-seq combined ATAC-seq peak pattern notes showed that the positions of H3K27ac and H3K4me1 modifications on Dock2 were substantially identical and that the chromatin opening at the modified positions was significantly increased, indicating that Sub1 could increase the level of histone H3K27ac and H3K4me1 modifications near Dock2 and thus increase the chromatin opening at its position, thereby recruiting AP-1 transcription factor binding to drive Dock2 transcriptional expression (b in fig. 19).

The preferred embodiments of the present invention have been described in detail above, but the present invention is not limited to the specific details of the above embodiments, and various simple modifications can be made to the technical solution of the present invention within the scope of the technical concept of the present invention, and all the simple modifications belong to the protection scope of the present invention.

In addition, the specific features described in the above embodiments may be combined in any suitable manner, and in order to avoid unnecessary repetition, various possible combinations are not described further.

Claims (10)

1. Use of dock2 and/or Sub1 as biomarkers or targets for the preparation of a product for diagnosis, monitoring, severity assessment, efficacy assessment, prognosis assessment, prevention and/or treatment of a disease of the immune system.
2. 2. The use according to claim 1, wherein Dock2 and/or Sub1 is a gene and/or protein.
3. 3. The use according to any one of claims 1-2, wherein said diagnosing, monitoring, severity assessing, efficacy assessing or prognosis assessing an immune system disorder comprises detecting the presence or absence of Dock2 and/or Sub1 or the amount of mRNA and/or protein expressed thereof.
4. 4. The use according to any one of claims 1 to 3, wherein said preventing or treating of an immune system disorder comprises modulating the expression of mRNA and/or protein of Dock2 and/or Sub1;

   preferably, the modulation comprises up-modulation or down-modulation.
5. 5. The use according to claim 4, wherein said preventing or treating an immune system disorder comprises knocking out or knocking down Sub1;

   Preferably, the knockdown or knockdown of Sub1 includes the use of a CRISPR/Cas system, tissue specific knockdown, introduction of interfering RNA or micrornas (e.g., microRNA-101) targeting Sub 1.
6. 6. The use according to claim 1, wherein said Dock2 and/or Sub1 is derived from one or more of the central nervous system, body fluids, cells, tissues or organs;

   preferably, the body fluid comprises blood or cerebrospinal fluid;

   preferably, said Dock2 and/or Sub1 is derived from an immune cell, such as a T cell, preferably CD4 ⁺ T cells or CD8 ⁺ T cells, further preferably CD4 ⁺ CD8 ⁺ T cells.
7. 7. The use according to any one of claims 1 to 6, wherein said product comprises an agent for detecting the presence or absence of Dock2 and/or Sub1 or the expression level of mRNA and/or protein thereof, or wherein said product comprises an agent for regulating the expression level of Dock2 and/or Sub1 mRNA and/or protein.
8. 8. The use according to any one of claims 1 to 7, wherein the immune system disorder comprises one or more of an infectious disease, a hypersensitivity disease, an autoimmune disease, an immunoproliferation disease or an immunodeficiency disease;

   preferably, the autoimmune disease comprises an immune-mediated inflammatory disease;

   Further preferred, the immune system disorder comprises one or more of systemic lupus erythematosus, rheumatoid arthritis, anti-neutrophil cytoplasmic antibody associated vasculitis, shi Difen predominance syndrome, sjogren's syndrome, systemic vasculitis, scleroderma, dermatomyositis, mixed connective tissue disease, hashimoto's thyroiditis, primary myelitis, compulsive spondylitis, adult stell's disease, behcet's syndrome, systemic sclerosis, multiple sclerosis, autoimmune hepatitis, polyarteritis nodosa, or ulcerative colitis.
9. 9. A method of constructing an animal model of an immune system disorder, comprising modulating the expression of mRNA and/or protein of Dock2 and/or Sub 1;

   preferably, the adjustment is an up-adjustment.
10. 10. The method of claim 9, wherein said method of construction comprises overexpressing Dock2 or its DHR-2 domain.