WO2008148349A1 - Use of beta-arrestin 1 for modulating t cell survival and autoimmunity - Google Patents

Abstract

Use of a beta-arrestin 1 inhibitor for the manufacture of a medicament for the prophylaxis or treatment of a disorder in mammals associated with CD4+ T cell apoptosis is provided. The beta-arrestin 1 inhibitor is selected from (i) compounds inhibiting the activity of beta-arrestin 1; (ii) compounds inhibiting the transcription and/or translation of beta-arrestin 1 gene.

Description

 Application of β-arrestin 1 in regulating tau cell survival and autoimmunity

 The present invention relates to the field of biomedicine, and in particular to the use of an inhibitor of beta inhibitory protein 1 for the preparation of a disease associated with abnormal apoptosis of CD4+ T cells for the treatment or prevention of an object.

 Background technique

In living organisms, sputum cells and sputum cells constitute the acquired immune system to protect the body from pathogens. In order to ensure the normal immune response of the body, the apoptosis of these cells is precisely regulated. In the thymus, depending on the strength of the TCR signal, most of the sputum cells undergo apoptosis through negative selection and positive selection. The surviving sputum cells enter the peripheral blood system to form a peripheral blood CD4 ⁺ and CD8 ⁺ T cell bank. CD4 ⁺ T cells in peripheral blood maintain their homeostasis due to new cell input in the thymus and apoptosis of CD4 ⁺ T in peripheral blood (1, 2). When CD4 ⁺ T cells are exposed to their specific antigen, the cells begin to divide and differentiate into functional CD4 ⁺ T cells. These functional CD4 ⁺ T cells need to be apoptotic in time after the foreign antigen is cleared, otherwise it will cause damage to the normal body. The signaling pathways involved in the apoptosis of these functional CD4 ⁺ T cells are: activation-induced apoptosis (AICD) and activated cell apoptosis (ACAD). In all these processes CD4 ⁺ T cell development, controlling the expression of regulatory molecules CD4 ⁺ T cells and destroy abnormal CD4 ⁺ T cells may be balanced against the autoantigen that the CD4 ⁺ T cells to survive prolonged or CD4 ⁺ T cells The immune response leads to many immune diseases such as autoimmune diseases and lymphocytosis (3-6).

In mammals, apoptosis of CD4 ⁺ T cells is mainly mediated by two pathways (1, 6). One is through cell surface receptors such as TNF and Fas. They primarily mediate apoptosis in activated CD4 ⁺ T cells. Activation of these receptors can induce apoptosis directly by recruiting and activating caspase (7_11). The pathway that mediates apoptosis in CD4 ⁺ T cells corresponding to this pathway is the Bcl-2 family protein-mediated mitochondrial pathway. This family of proteins includes two major classes of anti-apoptotic and pro-apoptotic molecules that synergistically regulate the integrity of the mitochondrial membrane and the release of pro-apoptotic proteins present in mitochondria (12, 13). Experiments have shown that Bcl-2 family proteins mainly mediate spontaneous apoptosis of CD4 ⁺ T cells due to cytokine deficiency, stress or activation (1, 14-17). Bcl-2 is the prototype protein of this protein family. In fc-knockout mice, T cells are prone to apoptosis and leukopenia occurs when the mouse ages (18). In 3⁄4 -overexpressing mice, T cells are not sensitive to many apoptotic stimuli (15, 16), the body's immune response time is longer and the mice spontaneously develop symptoms of autoimmune disease (19).

鼠在哮喘模型中的发病及病情显著低于其 野生型 （30， 31 )。 但是到现在为止， 人们仍然没有发现 parrl在免疫细胞中的功能。 发明内容 The beta inhibitor protein family (Pairs) comprises beta inhibitory protein 1 (PaiTl), beta inhibitory protein 2 (p _a ir2) and the like. They are signal molecules with multiple functions (20) that, on the one hand, mediate endocytosis of multiple receptors (21-24). On the other hand, they regulate these signaling pathways by binding to many signaling molecules, such as: Src family kinase activity, some MAPK signaling pathways (20, 25, 26). --arrestin 1 (gene number: NM-004041) is an important regulatory protein of GPCRs and is distributed in both cytoplasm and nucleus. Recently, it has been found that Parrl not only regulates the signaling pathway in the cytoplasm, but also directly regulates gene transcription in the nucleus (27). It shows that some functions of parrl are realized by affecting the expression of genes. Pairs are a class of ubiquitously expressed proteins, but their protein levels are relatively higher in nerve cells and immune cells, as reported in (28, 29) and in Figure 2A. Recent studies have found that arr2 negatively regulates Toll-like receptor signaling in primary immune cells. And another lab found that

The incidence and condition of the rat in the asthma model was significantly lower than in the wild type (30, 31). But until now, people still have not found the function of parrl in immune cells. Summary of the invention

Upon investigation, the inventors have found that parrl is regulating the survival of CD4 ⁺ T cells and their homeostasis in vivo. This function of arrl is likely to be achieved through its intranuclear function, which promotes the level of acetylation of histone H4 and the expression of this gene in the nucleus. The important physiological significance of parrl regulating CD4 ⁺ T cell survival is further elucidated in the examples of the present application: 1) In the experiment of EAE in mouse model of autoimmune demyelinating disease, ^rr knockout mice are not sensitive to the induction of EAE. The incidence of ^rr transgenic mice was significantly higher than that of wild type; 2) The expression of parrl and Bcl_2 in peripheral blood CD4 ⁺ T cells of patients with autoimmune demyelinating disease MS was significantly higher than that of normal controls. This high expression of ^arr and _C - is important for the survival of CD4 ⁺ T cells. Thus, the present invention provides a novel molecular mechanism for regulating the physiological functions of CD4 ⁺ T cells and provides a basis for utilizing this molecular mechanism to treat autoimmune diseases.

Specifically, the present invention relates to the use of an inhibitor of β-inhibitor 1 in the preparation of a medicament, characterized in that the inhibitor of β-inhibitor 1 is selected from the group consisting of (i) a substance which inhibits the activity of β-inhibitor 1; (ii) A substance which inhibits transcription, translation or both of a gene encoding β inhibitory protein 1 for treating or preventing a disease associated with abnormal apoptosis of CD4 ⁺ T cells in a mammalian subject.

Another aspect of the present invention provides a pharmaceutical composition for treating or preventing a disease associated with abnormal apoptosis of CD4 ⁺ T cells in a subject, characterized in that the pharmaceutical composition comprises an effective amount of an inhibitor of β inhibitory protein 1 As an active ingredient, and a pharmaceutically acceptable carrier, wherein the inhibitor of β inhibitory protein 1 is selected from the group consisting of (i) a substance that inhibits β-inhibitory protein 1 activity; (ii) an inhibition of transcription of a gene encoding β inhibitory protein 1, Translation or the substance of both.

Still another aspect of the present invention provides a method of screening a candidate for a drug for treating or preventing a disease associated with abnormal apoptosis of CD4 ⁺ T cells, the method comprising: a) allowing the candidate substance to Cell contact expressing β-arrestin 1; b) identifying the expression level of β-arrestin 1 in the nucleus of the cell, and expressing the expression level with the β-arrestin in the nucleus of the cell not contacting the candidate substance 1 Comparison of expression levels; c) Selection of a substance that reduces the expression level of β-arrestin 1 in the nucleus as a disease drug for treating or preventing apoptosis associated with abnormal apoptosis of CD4+ T cells.

 Other objects and advantages of the present invention will be apparent from the following detailed description and embodiments. DRAWINGS

和野生型 （WT)小鼠脾细胞（上） 以及淋巴细胞（下），流式细胞分析 CD4 和 CD8分子在这些细胞上的表达。 FACS图是每组 10只小鼠中的代表。 数字显示的 是每个区域的细胞百分比。 (B,C)从

ferrHg和野生型小鼠中分离 CD4⁺和 CD8+ T 细 胞， 这些细胞用 CD3 和 CD28的抗体激活 （C ) 或不激活 （B)。 随后， 细胞培养在单纯的 培养液里。 活细胞的数量是通过排除 PI和膜联蛋白 V阳性的细胞得到。 三次平行实验， * P<0.05, ** P<0.01 和相应对照比较。 (D,E) βα Γ^ fiarrltg和野生型小鼠每组 5只腹腔注射 100 μg SEB。 尾静脉取血， 外周血中 TCR νβ^{8 1/2+} 或 νβ⁶⁺ 阳性的 CD4⁺禾 P CD8⁺ T量用流 式细胞分析得到。 数据从三次平行实验得到， * P<0.05, ** P<0.01 和相应对照比较。 Figure 1 shows that β-arrestin 1 positively regulates the homeostasis and survival of CD4 ⁺ T cells. In the figure: (A) Separation of βωτΐ-ζ-

And wild-type (WT) mouse spleen cells (top) and lymphocytes (bottom), flow cytometry analysis of CD4 and CD8 molecules expression on these cells. The FACS map is representative of 10 mice per group. The numbers show the percentage of cells in each region. (B, C) from

CD4 ⁺ and CD8+ T cells were isolated from ferrHg and wild-type mice, and these cells were activated (C) with or without activation of antibodies to CD3 and CD28 (B). Subsequently, the cells are cultured in a simple medium. The number of viable cells is obtained by excluding PI and Annexin V positive cells. Three parallel experiments, *P<0.05, **P<0.01 compared with the corresponding controls. (D, E) βα Γ^ fiarrltg and wild-type mice were intraperitoneally injected with 100 μg of SEB in each group. Blood was taken from the tail vein, and the amount of TCR νβ ^{8 1/2+} or νβ ⁶⁺ positive CD4 ^{+ and} P CD8 ⁺ T in peripheral blood was obtained by flow cytometry. Data were obtained from three parallel experiments, * P < 0.05, ** P < 0.01 compared to the corresponding controls.

Figure 2 shows the expression of β-arrestin 1 in different tissues of mice and the development of lymphocytes in ferr j and mice. (A) Western blotting method was used to detect the expression of Parrl and Pair2 in different tissues of mice, and actin as an internal reference for loading. (B) Expression of CD3 and B220 in spleen cells and lymphocytes derived from ferr" and W mice (13-18 weeks) by flow cytometry, and the FACS map is representative of 5 mice per group. Percentage of cells in each region. The number of cells in the specific cell population in spleen cells (top) and lymphocytes (bottom) is also shown. (C) Flow cytometry analysis of ferr" and W mice (13-18 weeks) derived from Expression of CD4 and CD8 in thymocytes. The FACS map is representative of more than 5 mice per group. The numbers show the percentage of cells in each region. The number of cells of the specific cell population is also shown. DP indicates double positive, DN indicates double negative, and SP indicates single positive. ** P < 0.05 compared to the corresponding controls.

Figure 3 shows the effect of β-arsenin 1 on IL-2 secretion, Akt and Erk activation in CD4 ⁺ T cells. (A) Spleen CD4 ⁺ T cells were activated with appropriate amounts of antibodies to CD3 and CD28, and IL-2 levels in the culture medium were measured by ELISA 24 hours later. The data was derived from three parallel experiments. (B) βαττϊ'- and wt mouse-derived spleen CD4 ⁺ T cells were activated or not activated with the corresponding amounts of antibodies to CD3 and CD28, and immunoblot assays detected phosphorylated (P-) Akt, (P-) Erk and All Akt, Erk levels. The figure is representative of two parallel experiments.

 Figure 4 shows the expression of β-arrestin and ρ300 and the transcriptional effects of β-arrestin 1 on the Bcl-2 family gene after different treatments. (A) After transfecting the plasmid as shown in the Jurkat cells, the effect of these proteins on the expression of these proteins was examined by Western blotting. (B) After transfecting the plasmid as shown in the Jurkat cells, the RT-qPCR assay detects the transcription levels of /-2, Bax, Bim., Sad and 3^/^/ to normalize the loading. Data were derived from three parallel experiments, ** P < 0.01 compared to the corresponding controls.

ferr (D)和野生型小鼠脾脏中 分离 CD4+ T细胞， 并按上所述方法激活相应的时间。 细胞取样后， 用 RT-qPCR和免疫印 迹分别检测 Bcl-2禾 P Bax的 mRNA水平和 Bcl-2， Bax及 Parrl 的蛋白水平。 mHPRT是 RT-qPCR实验的内参， 而肌动蛋白是免疫印迹实验的内参。所有的免疫印迹和 RT-qPCR实 验平行做三次以上， ** P<0.01 和相应对照比较。 Figure 5 shows that β-arrestin 1 promotes the expression of Bcl-2 in CD4 ⁺ T cells. (A) Spleen CD4 ⁺ T cells were activated with antibodies to CD3 and CD28 for the corresponding time, and the expression of Parrl, Bcl-2 and Bax was analyzed by Western blotting. Actin is used as an internal reference for loading. (B, C, IT)}A βωτΓ (σ>,

CD4+ T cells were isolated from the spleens of ferr (D) and wild type mice and activated for the corresponding time as described above. After the cells were sampled, the mRNA levels of Bcl-2 and P Bax and the protein levels of Bcl-2, Bax and Parrl were detected by RT-qPCR and immunoblotting, respectively. mHPRT is an internal reference for RT-qPCR experiments, and actin is an internal reference for immunoblot experiments. All immunoblots and RT-qPCR experiments were performed in parallel for more than three times, **P<0.01 compared with the corresponding controls.

 Figure 6 shows that β-arrestin 1 promotes the expression of Bd-2 by transcription and the effect of β-arrestin 1 on the CREB and F-kappa B reporter genes. (A) After transfecting the plasmid as shown in the Jurkat cells, RT-qPCR and immunoblotting were used to detect the expression levels of So and So. (B, C) After 33 hours of transfection of ^a/ or ferr in Jurkat cells, use 0, 5, 10 μg of cycloheximide (CHX) (B) or 0, 2, 4 μΜ actinomycin D (Act (C) Cells were treated for 15 hours, and RT-qPCR and Western blotting assays were used to detect and express Sax levels. Data were derived from three parallel experiments, ** P < 0.01 compared to the corresponding controls. CD) HEK293 cells were individually transfected with CREB-luciferase reporter plasmid or co-transfected with different amounts of the ^rr plasmid. Data are expressed in multiples relative to the control. 10 μΜ of forskolin (FK) is a positive control. The data was derived from three parallel experiments. (Ε) In ΗΕΚ293 cells, the NF-kappa Β-luciferase reporter plasmid was transfected with a different amount of βωτΐ plasmid. Data are expressed in multiples relative to the control. T F-α is a positive control. The data was derived from three parallel experiments. *P<0.05, **P<0.01 compared to the corresponding controls.

Figure 7 shows that β-arrestin 1 promotes the level of Bd-2 locus histone H4 acetylation in CD4 ⁺ T cells. Spleen CD4 ⁺ T cells were activated with antibodies to CD3 and CD28, and cells were sampled at different times and analyzed by CHIP assay. Histone H3 acetylation and H4 acetylation antibodies were used in the CHIP assay. In the neutralization of antibody chromatin precipitation The sequences of the 3^-2 and 3^ gene promoter regions were analyzed by qPCR. The data is normalized by the amount in the sample. (A) CD4 ⁺ T cells of wt mice were used, and levels of histone H4 (left) acetylation and H3 (right) acetylation were analyzed. (B) CD4 ⁺ T cells using parrltg and W mice. (C) CD4 ⁺ T cells using ferr and W mice. (D) The histone H3 acetylation and H4 acetylation levels of the Bcl-2 gene region in CD4 ⁺ T cells of βαπ^- and W mice were analyzed by CHIP-qPCR assay. The ratio of W and ferr histone acetylation levels at the same site is shown in the figure. "0" represents the transcription start site, and "u" represents the upstream of the transcription start site. Data were derived from three parallel experiments, ** P < 0.01 compared to the corresponding controls.

 Figure 8 shows the role of p300 in the up-regulation of β-arrestin-1 transcription.

 (A-D) Jurkat cells transfected with the plasmid shown in the figure were analyzed for acetylation levels of histones H4 (A, C) and H3 (B, D) by CHIP assay. The DNA sequences of the promoter regions of Bc!-2 (A, B, C, D) and Βαχ (Α, Β) in the loading and immunoprecipitation complexes were detected by qPCR. (E, F) Jurkat cells transfected with the plasmid shown in the figure were analyzed for transcription levels of Bd-2 (E) and Sax (F) by RT-qPCR. Data were derived from three parallel experiments, ** P < 0.01 compared to the corresponding controls.

Figure 9 shows that β-arrestin 1 promotes experimental autoimmune encephalomyelitis in mice. (Α) Induction of sputum in βωτΓ-, ferrHg and ^ mice at 6-8 weeks, and the onset of the mice was observed daily after induction. The data is the sum of 4 experiments including 10 βωτ -, 10 arrltg and 14 W mice. (B) The spinal cord was removed from EAE mice, fixed and observed for inflammatory infection with hematoxylin blush and Luxol fast blue for the degree of demyelination of the spinal cord. The figure is typical of 3-4 mice. (C) After 8 days of induction of EAE in mice, cell proliferation was detected by stimulating mouse spleen cells with MOG peptides. Data were derived from three parallel experiments, ** P < 0.01 compared to the corresponding controls. (D) After 4 days of induction of EAE in mice, CD4 ⁺ and CD8+ T cells were isolated from spleen cells. Serum-free cells induced apoptosis in these cells, and viable cells were obtained by excluding PI and Annexin V-positive cells. Data were derived from three parallel experiments, ** P < 0.01 compared to the corresponding controls.

Figure 10 shows that β-arrestin 1 is associated with multiple sclerosis disease. (Α, Β) CD4+ T cells were isolated from peripheral blood of 14 patients with multiple sclerosis and 14 healthy subjects, and the transcription levels of ferr (A) and Sc/(B) were detected by RT-qPCR. *P<0.05, **P<0.01 compared to the corresponding controls. (C) Expression of Parrl was interfered with a lentivirus containing βωτΐ siRNA from MBP-specific CD4 ⁺ T cell clones derived from multiple sclerosis patients. Western blotting assays were used to detect the expression levels of ^ and 3^-2. The figure is typical of three experiments. (D) MBP-specific CD4 ⁺ T cell clones were infected with lentiviruses with ferr siRNA. Serum-free cultures induce apoptosis in these cells, and cells that survive and carry GFP are obtained by excluding PI-positive cells. Data were derived from three parallel experiments, ** P < 0.01 compared to the corresponding controls.

Figure 11 shows the expression of β-arrestin 1 in CD4 ⁺ T cells after EAE induction, as well as CD4 ⁺ and

Subcellular localization of β-arrestin 1 in CD8+ T cells. (Α) Western blotting assays were used to detect Parrl levels in mouse spleen CD4 ⁺ T cells induced for eight days. The figure is typical of two parallel experiments. (B) Western blotting assays for β-arrestin 1 levels in CD4 ⁺ and CD8+ T cells. The figure is typical of three parallel experiments. (C, D) CD4 ⁺ and CD8+ T cells were activated for 0 (C) or 72 (D) hours as previously described, and Parrl levels in the nucleus and cytoplasm were detected by immunoblot assay. The figure is typical of three parallel experiments. detailed description CD4 ⁺ T plays an important role in acquired immunity, and abnormal regulation of such apoptosis may lead to autoimmunity. Parrl is a discovered protein that functions in the G protein-coupled receptor signaling pathway, and it also functions to directly regulate gene transcription in the nucleus. In the present invention, the inventors found that parrl is regulating the survival of CD4 ⁺ T cells. In yferr knockout mice, naive and activated CD4 ⁺ T are prone to apoptosis in both in vivo and in vitro experimental conditions compared to their wild type. Parrl regulates the level of acetylation of histone H4 in c-gene and affects its gene expression in CD4 ⁺ T cells. In the mouse model EAE with multiple sclerosis, the pathological severity of the ^rr knockout mice was significantly lower than that of the wild type, while the pathological severity of the yferr transgenic mice was significantly increased. The expression of ^arr in peripheral blood CD4 ⁺ T cells was significantly higher in patients with multiple sclerosis than in normal subjects. The expression of fiarrl siRNAi in CD4 ⁺ T cells from patient sources and reactive with autoantigens decreased its expression, and this type of CD4 ⁺ T cells showed a significant increase in apoptosis under cytokine stress, revealing that parrl regulates CD4 ⁺ T cells. A new function in survival and autoimmunity, and provides a new target for the treatment of autoimmune diseases.

 Therefore, the first aspect of the invention relates to the use of an inhibitor of β-inhibitor 1 in the preparation of a medicament, characterized in that the inhibitor of β-inhibitor 1 is selected from the group consisting of (i) a substance which inhibits the activity of β-inhibitor 1; Ii) A substance which inhibits transcription, translation or both of a gene encoding β inhibitory protein 1 for use in the treatment or prevention of a disease associated with abnormal apoptosis of CD4+ T cells in a mammalian subject.

(Ι) inhibitor of beta inhibitor 1

 The term "inhibitor of beta inhibitory protein 1" herein has a broad meaning including direct or indirect (eg, by reactive intermediates, metabolites, etc.) acting on beta inhibitory protein 1 to inhibit or reduce beta inhibitory protein 1 The biologically active substance specifically includes: (i) a substance that directly inhibits the biological activity of β inhibitory protein 1 by interaction at the protein level; (ii) a substance that inhibits transcription and/or translation of a gene encoding β inhibitory protein 1. The "(biological) activity of beta-inhibitor 1" as used herein refers to the activity of beta-inhibitor 1 to promote survival of CD4+ T cells, the activity of promoting Bcl-2 expression, or the promotion of histone acetylation in the Bcl-2 locus. Horizontal activity.

 It will be understood by those skilled in the art that the scope of β inhibitory protein 1 herein includes not only the full-length sequence of β-arrestin 1 but also various variant forms, fragments, derivatives or analogs of the protein, as long as The variant forms, fragments, derivatives or analogs have the same or similar activities as described above. These variant forms include, but are not limited to;: a plurality of (preferably 1-10, more preferably 1-5, most preferably 1-3) amino acids relative to the amino acid sequence of the polypeptide. a protein that is deleted, inserted, and/or substituted, or a polypeptide that is identical (homologous;) or substantially identical (homologous;) and has the same or similar biological activity or function, for example, at least 60%, more preferably 70% Also preferred are 80%, 90%, or even 95% homologous or identical polypeptides. The difference between these analogs or derivatives and the native protein may be a difference in amino acid sequence and/or a difference in the modified form that does not affect the sequence.

In a preferred embodiment, the substance which inhibits the activity of β inhibitory protein 1 is a substance which specifically binds to β inhibitory protein 1 and thereby inhibits its activity, for example, polyclonal antibodies and monoclonal antibodies specific for β inhibitory protein 1. Antibodies, especially monoclonal antibodies. Here, "specificity" refers to an antibody that binds to β-inhibitor 1 but does not recognize and bind to other unrelated antigen molecules. Also included within the scope of the antibody are various modified forms of the antibody, and fragments thereof such as Fab' or (Fab) ₂ fragments and the like. The antibodies can be prepared by a variety of techniques known to those skilled in the art, such as immunizing animals to produce polyclonal antibodies or hybridoma producing monoclonal antibodies. In addition, other known agents other than antibodies are specific to beta inhibitor 1 The receptors and ligands which bind sexually are also within the scope of the "substance which inhibits the activity of β-inhibitor 1". In another embodiment, the substance which inhibits the activity of β inhibitory protein 1 is a compound having such an inhibitory effect known to those skilled in the art, for example, actinomycin, cycloheximide or a pharmaceutically acceptable salt thereof , derivatives or analogues. Those skilled in the art are also well able to select other substances which inhibit the activity of beta inhibitory protein 1 from the candidate according to the screening method of the present invention. In addition, some agonists of G protein-coupled receptors are known to indirectly affect the activity of parrl by affecting the distribution of β ιτΐ in the nucleus and cytoplasm. Such agonists of G protein-coupled receptors include, for example, but are not limited to, agonists such as δ opioid receptors and kappa opioid receptors, and the like. Therefore, agonists of these G protein coupled receptors are also included in the scope of the present invention.

 "Those substances which inhibit the transcription and/or translation of a gene encoding β-suppressor 1" mainly refer to affecting the expression of β-inhibitor 1 by indirectly acting on a gene of β-inhibitor 1 at a gene level, thereby inhibiting its activity. Those substances, usually nucleic acids.

 In one embodiment, these are antisense sequences of all or part of the sequence encoding the gene for beta inhibitorin 1 (gene number: M-004041). The antisense sequence may typically be between 5 and 200, preferably between 10 and 100, more preferably between 20 and 50 bases in length. Such antisense nucleic acid molecules can be synthesized chemically using natural nucleotides or physics designed to increase molecular biological stability or increase duplex formation with beta inhibitor 1 mRNA or beta inhibitor 1 gene. Various modified nucleotides for stability. The antisense sequence can also be produced by biological methods, and the expression vector is introduced into the cell in the form of a recombinant plasmid, a phagemid or an attenuated virus, in which the antisense sequence is produced under the control of a highly effective regulatory region, Activity can be determined by the type of cell into which the vector is introduced.

In another embodiment, those substances that inhibit transcription and/or translation of a gene encoding beta inhibitory protein 1 are small interfering RNA (siRNA). ₀ RNA interference is a newly discovered in vivo gene silencing phenomenon. Small interfering RNA is a processed product of exogenous double-stranded RNA, which mediates RNA interference effects, recognizes specific mRNA, and silences homologous gene expression in cells. When designing siRNA for mammalian cells, a more effective siRNA has a size of 20-30 bases, more preferably 21-25 bases, and preferably has two prominent bases at the 3' end. The sequence specificity of siRNA is very stringent, and a base mismatch with the target mRNA significantly impairs the effect of gene silencing. One of skill in the art can generally design siRNAs using the following steps: (1) selection of siRNA target sites; (2) sequence homology analysis; (3) design of negative controls. The designed siRNA can also be synthesized by chemical synthesis or in vitro transcription. For a specific example of the construction of β-inhibitor 1 siRNA, see Parruti, G. et al., J Biol Chem 268, 9753-61 (1993).

 In still another embodiment, the substance that inhibits transcription and/or translation of a gene encoding beta inhibitory protein 1 is a ribozyme. A ribozyme is a ribonucleic acid molecule that is capable of specifically pairing with a target RNA molecule, and then cleavage of the latter at a specific site to stop the production of the corresponding protein. There are currently seven major classes of naturally occurring ribozymes, namely a class of introns, a class II intron, an RNA subunit of RNase P, a hammerhead ribozyme, a hairpin ribozyme, a hepatitis delta virus ribozyme. , and VS ribozymes. In the ribozyme family, the hammerhead ribozyme is the smallest (about 40-50 nucleotides in length), which has the advantages of simple structure and simple design. For example, the DNA sequence can be directly chemically synthesized, and then ligated by recombinant DNA into a recombinant plasmid to produce the ribozyme, and then the specificity and efficiency of the designed ribozyme to the RNA substrate molecule can be determined in a test tube. Verification. In addition, PCR can be used to amplify RNA (Saiki, et al. Science 1985; 230: 1350-1354). The ribozyme gene is cloned into a plasmid, adenovirus, or retroviral vector.

Obtaining the above-mentioned nucleic acid sequence (such as antisense sequence, ribozyme) for inhibiting transcription and/or translation of a gene encoding β inhibitory protein 1 Alternatively, small interfering RNAs can be introduced into the subject cells using conventional techniques such as transformation, transfection, infection, and physical techniques such as electroporation and microinjection. Alternatively, it can be delivered by chemical methods such as DNA co-precipitation and incorporation into liposomes or in aerosol or lavage format. Suitable viral vectors, plasmids or cloning vectors for transferring nucleic acid molecules are known in the art. Examples of suitable viral vectors include retroviral vectors, lentiviral vectors, adenoviral vectors, and DNA viral vectors, and the like. These techniques are well known to those skilled in the art.

 Further, in the present invention, it is also conceivable to interfere with transcription of a gene encoding β inhibitory protein 1 by a gene transformation method (for example, using allelic substitution, insertional inactivation, and deletion formation for targeted gene mutagenesis), thereby selectively Inhibition of beta inhibitory protein 1 activity. (II) Use

The inhibitor of β-inhibitor 1 of the present invention can be used for treating a disease associated with abnormal apoptosis of CD4 ⁺ T cells in a subject. Mammalian subjects to which the present invention is applicable include humans, domestic and farm animals, non-human primates, and zoos, playground animals, or pets such as dogs, horses, cats, cows, and the like. In a preferred embodiment, the mammal is a human.

The term "abnormal apoptosis of CD4 ⁺ T cells" as used herein means that compared with CD4 ⁺ T cells in normal subjects,

The level of apoptosis of CD4 ⁺ T cells is decreased and the viability is improved. Apoptosis of CD4 ⁺ T cells refers specifically to the occurrence of CD4+ T cell apoptosis in peripheral blood.

 Diseases associated with abnormal apoptosis of CD4+ T cells include autoimmune diseases and lymphocytosis. Autoimmune diseases refer to diseases caused by autoantibodies or sensitized lymphocytes produced by the body that damage and damage their own tissues and cellular components, leading to tissue damage and organ dysfunction. The underlying mechanism leading to autoimmune diseases is the termination and destruction of immune tolerance. Autoimmune diseases often have the following characteristics: 1 The sensitized lymphocytes in which the high-valent self-antibody and/or self-organological components react can be measured in the blood of the patient. 2 autoantibodies and/or autosensitized lymphocytes act on tissues and cells in which the target antigen is located, causing pathological damage and dysfunction of the corresponding tissues and organs. 3 A similar pathological model can be replicated in animal experiments and the disease can be passively transferred by the patient's serum or lymphocytes. 4 The outcome of the disease is closely related to the intensity of autoimmune response. 5 In addition to some of the secondary autoimmune diseases that are clear of the cause can be resolved with the cure of the primary disease, most of the unexplained autoimmunity is often recurrent and chronic.

 Autoimmune diseases suitable for treatment or prevention with the present invention include, but are not limited to, rheumatoid arthritis, systemic lupus erythematosus, dermatomyositis, scleroderma, multiple sclerosis, myasthenia gravis, demyelination Disease, primary adrenal atrophy, chronic thyroiditis, type I diabetes, chronic non-specific ulcerative colitis, chronic active hepatitis, avian anemia and atrophic gastritis, autoimmune glomerulonephritis, pulmonary and renal hemorrhagic Syndrome, autoimmune hemolytic anemia, idiopathic thrombocytopenic purpura, idiopathic leukopenia, etc.

 In a preferred embodiment, autoimmune diseases suitable for treatment or prevention with the present invention include demyelinating diseases, multiple sclerosis or type I diabetes. (III) Pharmaceutical composition

The present invention also provides a pharmaceutical composition for treating or preventing a disease associated with abnormal apoptosis of CD4 ⁺ T cells in a mammalian subject, the pharmaceutical composition comprising an effective amount of an inhibitor of β inhibitory protein 1 as an activity Component to And a pharmaceutically acceptable carrier, wherein the inhibitor of β inhibitory protein 1 is selected from the group consisting of (i) a substance that inhibits β inhibitory protein 1 activity; (ii) a gene that inhibits β-inhibitor protein 1 transcription, translation, or both Substance.

 The pharmaceutical compositions of the present invention comprise a substance which inhibits β inhibitory protein 1 as described in detail herein or which is identified by the method of the present invention. The active substance may be administered alone, but usually together with a pharmaceutical carrier or the like, which may be selected according to the chosen route of administration and standard pharmaceutical practice. The dose administered should be an "effective amount", that is, an amount that treats, alleviates or prevents the target disease or condition, or an amount that exhibits a detectable therapeutic or prophylactic effect. Dosage dosage according to the use and known factors (such as the pharmacodynamic properties of the specific substance and its administration and route; the age, health and weight of the recipient; the nature and extent of the disease; the type of treatment, the frequency of treatment and the expectation The effect varies). The precise effective amount for a subject will depend on the size and health of the subject, the nature and extent of the condition, and the combination of therapeutic and/or therapeutic agents selected for administration. Therefore, it is useless to specify an accurate effective amount in advance. However, for a given symptom, routine experimentation can be used to determine the effective amount that the clinician can judge.

 The term "pharmaceutically acceptable carrier" refers to a carrier or excipient for the administration of a therapeutic agent which is compatible with the inhibitor of the beta inhibitory protein 1 of the present invention, i.e., can be blended therewith without the usual circumstances. The effect of the pharmaceutical composition is greatly reduced. Specific examples of some substances which can be used as pharmaceutically acceptable carriers or excipients or components thereof are sugars such as lactose, glucose and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives, such as Sodium carboxymethylcellulose, ethylcellulose and methylcellulose; scutellaria powder; malt; gelatin; talc; solid lubricants such as stearic acid and magnesium stearate; calcium sulphate; vegetable oils, such as peanut oil , cottonseed oil, sesame oil, olive oil, corn oil and cocoa butter; polyols such as propylene glycol, glycerin, sorbitol, mannitol and polyethylene glycol; alginic acid; emulsifiers such as Tween; wetting agents, such as laurel Sodium sulfate; coloring agent; flavoring agent; tablet, stabilizer; antioxidant; preservative; pyrogen-free water; isotonic saline solution; and phosphate buffer. In addition, a thorough discussion of pharmaceutically acceptable excipients can be found in Remington's Pharmaceutical Sciences (Mack Pub. Co., N. J. 1991).

 More than one substance described in detail herein or identified by the methods of the invention may also be used in combination with other agents. They may be administered simultaneously or in parallel as separate dosage forms, or as a physical composition of the therapeutic agents of the various components, either alone or in combination. The substance of the present invention can be administrated by mouth, buccal, rectal, parenteral, topical, inhalation. The substance can also be administered by controlled release or sustained release capsule systems and other drug delivery techniques.

For example, for oral administration in the form of a tablet or capsule, the active substance may be combined with a pharmaceutically acceptable non-toxic, orally, inert carrier (such as lactose, starch, sucrose, glucose, methylcellulose, stearic acid) Magnesium, dicalcium phosphate, calcium sulfate, mannitol, sorbitol, etc.; for oral administration of liquid dosage forms, the oral active substance can be combined with any pharmaceutically acceptable oral, non-toxic inert carrier (eg Ethanol, glycerin, water, etc. are combined. If desired, suitable binders, lubricants, disintegrants, and colorants can also be incorporated into the dosage form. Suitable binders include starch, gelatin, natural sugars such as glucosamine or beta-lactose, corn sweeteners, natural and synthetic gums such as acacia, tragacanth or sodium alginate, carboxymethylcellulose. , polyethylene glycol, wax, etc. Suitable lubricants for use in these dosage forms include sodium oleate, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate, sodium chloride, and the like. Examples of the disintegrant include starch, methyl cellulose, qiongyue, bentonite, xanthan gum and the like. The pharmaceutical compositions of the present invention may also be administered in the form of liposome drug delivery systems, such as small unilamellar vesicles, large unilamellar vesicles, and multilamellar vesicles. Liposomes can be formed from a variety of phospholipids, such as cholesterol, stearylamine or phosphatidylcholine. Substances which are described in detail in the present invention and which are identified by the methods of the invention may also be coupled to soluble polymers which are orientable pharmaceutical carriers. Examples of such polymers include polyvinylpyrrolidone, pyran copolymer, polyhydroxypropyl A methacrylamide-phenol, a polyhydroxyethylaspartamide phenol, or a polyethylene oxide-polylysine substituted with a palmitoyl group. These materials can also be coupled to biodegradable polymers used to control drug release. Suitable polymers include polylactic acid, polyglycolic acid, copolymers of polylactic acid and polyglycolic acid, polyε-caprolactone, polyhydroxybutyrate, polyorthoesters, polyacetals, polydihydropyrans Classes, polycyanoacrylates, hydrogel crosslinked or amphiphilic block copolymers. Suitable pharmaceutical carriers and methods of preparation are described in the Remington Medical Sciences Mack Publishing Company, which is a standard reference in the art.

(IV) Identification method

Another aspect of the present invention also provides a method of screening a candidate for a drug for treating or preventing a disease associated with abnormal apoptosis of CD4 ⁺ T cells, the method comprising:

 a) contacting the candidate substance with a cell expressing β-arrestin 1;

 b) identifying the expression level of β-arrestin 1 in the nucleus of the cell, and comparing the expression level to the expression level of β-arrestin 1 in the nucleus of cells not contacted with the candidate substance;

c) A substance which lowers the expression level of β-arrestin 1 in the nucleus is selected as a disease drug for treating or preventing abnormal apoptosis associated with CD4 ⁺ T cells. The invention is further illustrated below in conjunction with specific embodiments. It is to be understood that the examples are merely illustrative of the invention and are not intended to limit the scope of the invention. The practice of the present invention will employ, unless otherwise indicated, conventional techniques of molecular biology, microbiology, recombinant DNA, and immunology, all of which are known to those skilled in the art. These techniques are fully described in the following literature: For example, Sambrook, Guide to Molecular Cloning, 2nd Edition (1989); DNA Cloning, Volumes I and II (DN Glover, 1985); Oligonucleotide Synthesis (Editing by MJ Gait, 1984); Nucleic Acid Hybridization (BD Hames and P SJ Higgins, ed. 1984); Protein Purification: Principles and Practices, 2nd Edition (Springer-Verlag, NY), and the Manual of Experimental Immunology I-IV volume (edited by DC Weir and CC Blackwell 1986). Alternatively, follow the instructions provided by the reagent manufacturer. Example

 Materials and Method

 Patient sample

 In this study, patients who were diagnosed with relapsing-remitting multiple sclerosis were from the Huashan Hospital of Fudan University and the Department of Neurology of Ruijin Hospital of Jiaotong University. After the patient's informed consent was obtained according to local hospital regulations, the patient's peripheral blood samples were taken. The healthy volunteers in this study were from employees or students of the Biochemical Cell Institute of the Shanghai Branch of the Chinese Academy of Sciences, and some were from the Shanghai Blood Center. They also received informed consent as previously mentioned.

2. Human T cell sample preparation

T cells were labeled with a murine anti-human anti-CD4 antibody, and then isolated and purified using a goat anti-mouse IgG magnetic bead, a separation column, and an AutoMACS separation device, and CD4 ⁺ T cells having a purity greater than 90% were obtained by FACS. 3. Mice

C57BL/6 mice were purchased from the Shanghai Experimental Animal Center of the Chinese Academy of Sciences. With C57BL/6

^ar ^^—The mice were presented by Dr. Robert J. Lefkowitz, Duke University Medical Center. ^a mice such as: Rapid xenograft tumor growth in beta-arrestinl transgenic mice due to the elevation of tumor angiogenesis Lin Zoul, 2, Rongxi Yangl, 3 and Gang Pei l described production, and backcrossed with C57BL/6 for 9 generations or more. Animal feeding and operation are carried out in accordance with the regulations of the Experimental Animal Committee of the Shanghai Academy of Life Sciences of the Chinese Academy of Sciences. The animals used were housed in a pathogen free environment and the genome was identified prior to the experiment.

4. Antibodies, reagents, plasmids and interference siRNA

Mouse anti-Bcl-2 and Bax antibodies, PE-conjugated antibodies against murine CD4, CD8, B220, and νβ ⁶ and FITC-conjugated antibodies against murine CD4, CD8, CD3 and νβ ⁸ were purchased from BD Biosciences the company. Rabbit polyclonal antibodies against parrs (Al CT) were kindly provided by Dr. Robert J. Lefkowitz. Antibodies against acetylated histone H4 and histone H3 were purchased from Upstate Biotechnology. IRDyeTM 800CW conjugated anti-mouse IgG and anti-rabbit IgG purified antibodies were purchased from Rockland Corporation. Anti-actin antibodies and reagents Actinomycin D and dexamethasone were purchased from Sigma. Antibodies against Erk and anti-ser217/221 phosphorylated Erk are products of Cell Signaling, Inc., while anti-Akt and anti-ser473 phosphorylated Akt antibodies were purchased from Shanghai KANGCHEN Company. SEB is produced by Beijing Biotinge Biomedicine. Plasmids encoding ^ga/, HA-parrl (described above) (29), HA-/rr2, arrlQ394L were constructed as previously described (32). /^^6/^ΓΓ siRNA, pBS/U6^rr2 siRNA and pBS/U6l non-specific siRNA plasmids were constructed as previously described (29). The p300 wild type and its active region mutant (C/H1 deletion) plasmid was purchased from Upstate. The human MBP small peptide was synthesized and purified by the Anderson Cancer Center Small Peptide Center Laboratory. The purity of the small peptide is greater than 90%.

5. Flow cytometry

The cells were resuspended in PBS buffer containing 1% BSA. Fluorescent antibodies such as CD4, CD8, CD3, B220, TCR Vp ⁸ and Vp ⁶ with the corresponding cell surface antigen are incubated in the recommended buffer for 4 hours at 4 degrees. The labeled cells were washed and analyzed by BD FACSAria instrument.

6. Cell purification, activation and culture

The spleen cells of the mouse were labeled with purified rabbit anti-mouse CD4 antibody or rabbit anti-mouse CD8 antibody, and then isolated and purified by goat anti-rabbit IgG magnetic beads, separation column and AutoMACS separation device, and obtained by FACS test. The purity of T cells is greater than 95%. The isolated and purified T cells were activated in a CD3 (CD3 ε chain) monoclonal antibody culture dish coated with a large buccal mouse anti-mouse and a CD28 monoclonal antibody. Cultures were RPMI 1640 medium plus 10% fetal bovine serum, 2 mM L-glutamine, 5 mM 2-ME, and 100 units/ml penicillin. Myelin matrix protein (MBP)-specific CD4 ⁺ T cells were cultured in RPMI1640 medium containing 20 g/ml MBP small peptide (33) and 100 U/ml human recombinant IL-2.

7. Reverse transcription and real-time quantitative PCR All total RNA in the experiment was extracted with TRIzol according to the Invitrogen guidelines. In the reverse transcription experiments, oligoCdT) primers and the superscript II system were used. All quantitative gene transcription experiments were performed by qPCR. The qPCR system used mainly includes: Brilliant SYBR Green QPCR Master MIX and a Light Cycler detector (Stratagene). The primers used were: murine Bc/-2-sense, 5'-TTC TCC TTC CAG CCT GAG AGC AA-3 ' (SEQ ID NO: 1), antisense, 5'-ATG ACC CCA CCG AAC TCA AAG-3 ( SEQ ID NO: 2)'; murine to-sense, 5'-AGG ATG CGT CCA CCA AG-3 ' (SEQ ID NO: 3), antisense, 5'-AAG TAG AAG AGG GCA ACC AC-3 ' (SEQ ID NO: 4); Mouse HPRT-se e, 5'-CCT GCT GGA TTA CAT TAA AGC ACT G-3 ' (SEQ ID NO: 5), antisense, 5'-TTC AAC ACT TCG AGA GGT CCT-3 ' (SEQ ID NO: 6); Murine HPRT cDNA input control; human HPRT-诵e, 5'-CCT GCT GGA TTA CAT CAA AGC ACT G-3 ' (SEQ ID NO: 7), antisense, 5' -TCC AAC ACT TCG TGG GGT CCT-3 ' (SEQ ID NO: 8); human ^ x-sense 5'- ATC CAG GAT CGA GC A GGG CG-3 ' (SEQ ID NO: 9), antisense, 5 '-ACT CGC TCA GCT TCT TGG TG-3 ' (SEQ ID NO: 10); A Bim-sense, 5' -CAA TGG CTA ACT GGG ACT A-3 ' (SEQ ID NO: 11), antisense, 5' -TCT TCG GCT GCT TGG TAA -3 ' (SEQ ID NO: 12); Human Bad-seme, 5'-CCA GAG TTT GAG CCG AGT GAG-3 ' (SEQ ID NO: 13), antisense, 5' -GCT GTG CTG CCC AGA GGT T-3 ' (SEQ ID NO: 14); Human Bcl-xl-sense, 5' -CAA CCC ATC CTG GCA CCT-3 '(SEQ ID NO: 15), antisense, 5'-CGA TCC GAC TCA CCA ATA CC-3 ' (SEQ ID NO: 16); human βωτΐ-se, 5'-GGG ACG CGA GTG TTC AAG AA-3 '(SEQ ID NO: 17), antisense, 5'-AC A AAC AGG TCC TTG CGA AAG-3 ' (SEQ ID NO: 18). The transcriptional level of X Bcl-2 was done using the Tagman probe: Bcl-2-smse, 5'-CCT GTG GAT GAC TGA GTA CCT GAA-3 ' (SEQ ID NO: 19), antisense, 5 ' -CAG CCA GGA GAA ATC AAA CAG A-3 ' (SEQ ID NO: 20), Taqman probe, 5'-£AGG ATA ACG GAG GCT GGG ATG CCT TTP-3 ' (SEQ ID NO: 21).

8. EAE induction and evaluation

 The amino acid residue sequence at position 35-55 of the MOG antigen used in EAE induction is:

Met-Glu-Val-Gly-Trp-Tyr-Arg-Ser-Pro-Phe-Ser-Arg-Val-Val-His-Leu-Tyr-Arg-Asn-Gly-Lys. This peptide was purchased from BioAsia Biotechnology and its purity was greater than 95%. Acute EAE induction was performed as follows: On the first day, subcutaneous injection of CFA containing 300 μ _§ MOG peptide and 5 mg/ml heat-inactivated Mycobacterium tuberculosis H37Ra peptide chain (produced by BD), and tail vein injection 200 ng/ Pertussis toxin in mice. On the third day, 200 ng/mouse of pertussis toxin was injected into the tail vein again. Mice that induced EAE were weighed and observed daily. According to the severity of the disease, the mice were scored, specifically: 0, without any symptoms of the disease; 1, the tail is weak; 2, - or two hind limbs squat; 3, two hind limb paralysis; 4, fore limb paralysis; 5, dying or dying.

9. Cell survival analysis

 T cells were maintained in serum-free RPMI 1640 medium. At the time point shown on the graph, using annexin

The -V-FLUOS Labeling Kit (Roche Molecular Biochemicals) obtained the percentage of viable cells by excluding PI and annexin V positive cells. In the experiment to detect the survival of GFP-positive cells, the PI alone was excluded. Positive cells gave a percentage of viable cells.

10. Histochemistry observation

 Tissues were removed from EAE mice that were induced for 20 days, fixed in 4% formalin, and embedded in paraffin. Then dye with Luxol fast blue or H&E. Finally, observation photographs were taken with an optical microscope. A total of 3-4 mice were spinal cords, and each mouse took 3 samples for quantitative demyelination and infection (34).

11. Chromatin immunoprecipitation

Chromatin immunoprecipitation (CHIP) was performed according to Upstate Biotech's CHIP kit. The sequence of the specific gene promoter region in the loading and neutralizing immunoprecipitation complex was detected by qPCR. The primers for the specific gene promoter region are designed to be in the base region of 1000 upstream to 500 downstream of the transcription start site. The results obtained were obtained by loading the amount in the precipitate using a loading gauge. The promoters of the gene promoter region and the ^3⁄4/-2 gene locus are: mouse promoter: sense, 5'-GGC AAA CCC TCC CCC ACC ACC TC-3 ' (SEQ ID NO: 22), antisense, 5 '-CCA CCG GAC CGC TTC AGA CCT C-3 ' (SEQ ID NO: 23); murine αχ promoter, sense, 5'-GGG GAA ACA ACC AAC TCT GG-3 ' (SEQ ID NO: 24), antisense 5 '-CAT CAC TGC CGC TGC CTC T-3 ' (SEQ ID NO: 25); murine Bd-2 locus u25,000 sense, 5'-GCT GTT TAT CAG TTA GTG GGT C-3 ' (SEQ ID NO: 26), antisense 5'-GGG TCA GAA GTG GGA GTG-3 ' (SEQ ID NO: 27); u20,000 sense, 5' -TGC CAA GGT TAG CAG GAC-3 ' (SEQ ID NO: 28) , antisense, 5'-CCA GAG GAC AAA TGA GGG-3 ' (SEQ ID NO: 29); ul 5,000 sense, 5 '-CAT TTG TCT GCC CTA TCT-3 ' (SEQ ID NO: 30), antisense, 5 '-TTA ACT GGG AAC ACC TCA-3 ' (SEQ ID NO: 31); ul0,000 sense, 5'-AGT GCT TAT CGC TCT TCC-3 ' (SEQ ID NO: 32), antisense, 5'-CTC CAA ACC TGA CCC TCT-3 ' (SEQ ID NO: 33); u5,000 sense, 5'-GCA GCA AAC AAT CTT CAT-3 ' (SEQ ID NO: 34), antisense, 5 '-AC A CCA ATA CCA ACC CTA-3 ' (SEQ ID NO: 35); 50,000 sense, 5'-CCT TCT CAA TCA GCC AGC AT-3 ' (SEQ ID NO: 36), antisense, 5'-AAG CAA GCC TCC TCA CCC-3 ' (SEQ ID NO: 37); 100,000 sense, 5' -CAC TCA GAG AAG AAT TCA CAC AGA A-3 ' (SEQ ID NO: 38), antisense 5'-TTC TGT GTG AAT TCT TCT CTG AGT G-3 ' (SEQ ID NO: 39); 150,000 sense, 5'-CCC CAG AAC TCA TGT CTC-3 ' (SEQ ID NO: 40), antisense, 5'-TTC CAA TGC TCT CCC AAA-3 ' (SEQ ID NO: 41); 175,000 sense, 5' -CAG AGT GAG TAT TGG AGG AG-3 ' (SEQ ID NO: 42), antisense, 5 '-AAA GAC AGT GGT GGG AAA-3 ' (SEQ ID NO: 43); 176,000 sense, 5' -TGA CCC TGA GGA GAT GGA-3 ' ( SEQ ID NO: 44), antisense, 5'-GGT ATG ACC TGG GCT TCG-3 ' (SEQ ID NO: 45); 181,000 sense, 5'- AGG AGC AAA CTC AGG AAT-3 ' (SEQ ID NO: 46 ), antisense, 5'-AGT CAC AAA GAT GGC AGA-3 ' (SEQ ID NO: 47); 186,000 sense, 5' -CAG ACG CAC CAC TGA TTT-3 ' (SEQ ID NO: 48), antisense, 5 '-AGC AGC CAG CAG ACT TAC-3 ' (SEQ ID NO: 49); 191,000 sense, 5' -TAC GAG CGA TAC ATA CAA C-3 ' (SEQ ID NO: 50), antisense, 5' -CTA GAG CCA GTC CTT CTT-3 ' (SEQ ID NO: 51). People Promoter region: Bcl-2 sense, 5'-GCG ACT CCT GAT TCA TTG-3 ' (SEQ ID NO: 52), antisense 5'- AGG TGC GTT TCC CTG TA-3 ' (SEQ ID NO: 53); Bax Sense, 5'-TAT CGG GAG ATG CTC ATT GGA-3 ' (SEQ ID NO: 54), antisense, 5'-CCC TCG GGA GGT TTG GTC-3 ' (SEQ ID NO: 55).

12. Immunoblotting

 In immunoblot experiments, protein bands were detected by enhanced chemiluminescence methods. In some experiments, IRDye800CW coupled secondary antibody fluorescence quantification was used. The images in these experiments were taken by Odyssey Far Infrared Image System (Li-Cor Bioscience).

13. Cell proliferation experiment

In the cell proliferation assay, 5×10 ⁵ mouse spleen cells were cultured in DMEM complete medium containing MOG or no MOG for 72 hours. [3Η] thymidine of Ιμα was added 16-18 hours before cell analysis.

The [3Η] thymidine incorporated in the DNA was detected by a β plate detector.

14. Cell line

 Jurkat and HEK293 cells were purchased from the American Type Culture Collection and were

Culture in RPMI1640 or MEM (Gibco-BRL) culture medium. Jurkat cells were transfected with Amaxa transfection kit, while HEK293 cells were transfected with calcium phosphate.

15. Detection of cytokines

2 X 10 ⁵ CD4 ⁺ T cells were activated with CD3 and CD28 antibodies for 24 hours as described above, and the supernatants were collected and assayed using the IL-2 ELISA kit according to the PIERCE guidelines. At the same time, purified and known concentrations of recombinant murine IL-2 were used as standard lines for quantification.

16. Extraction of nuclear components

 The nuclear fraction was extracted with some modifications as previously described (27). Specifically, the cells treated by various methods were washed and resuspended in 400 μl of hypotonic buffer for 10 minutes in an ice bath. Add 3μ1 and 10% Ρ-40 and mix and continue to ice bath for 5 minutes. After a brief centrifugation, the resulting precipitate is a crude extract of the nuclear component. This fraction was washed, resuspended in hypotonic buffer and shaken at 4 degrees for 1 hour. After centrifugation, the resulting supernatant is the nuclear component. 17. Reporter gene experiment

 Clontech's pNF-kappa-B-Luc or pCREB-TA-Luc, and pRL-TK, and some of the plasmids shown in the figure were co-transfected into ΗΕΚ293 cells. After 36 hours of transfection, the activity of luciferase was detected using a dual luciferase reporter assay system. Cells treated with 10 ng/ml forskolin (FK) and 10 ng/ml rhTNF-α were used as positive controls for the experiments.

18. Data processing and statistics

Quantitative experimental data is expressed as an average of 3⁄4 ± sem. Use one-way ANOVA to check if there is any statistical difference between the data Different, then use Bonferroni post-hoc to detect significant differences between the two sets of data for multiple sets of data or tow-tailed Student's t. Experimental result

 1. Parrl regulates the homeostasis and survival of peripheral blood CD4+ T cells

鼠和野生型（_wt) 小鼠中胸腺细胞 CD4+ T细胞和 CD8+ T细胞的数量， 发现并没有改变 (图 2C)。 随后， 发明 人检测了外周血 CD4⁺ T细胞的凋亡。 从/ hrrr 小鼠， βαττΐ转基因 （^rrHg) 小鼠和 wt 小鼠脾脏分离得到 CD4⁺ T细胞， 用抗 -CD3 和 抗 -CD28抗体激活或者不激活， 然后在无 血清也无细胞因子的培养基中培养。 图 1B和 1C显示， 激活了的^ rrHg CD4⁺ T细胞生存 能力比其野生型明显增加， 而^ ^ -的 CD4⁺ T细胞无论是在其幼稚还是激活状态下都明 显比其野生型对凋亡更敏感。 这些数据表明： Parrl促进 CD4+ T细胞体外的生存能力。 When studying the ferr knockout/rrl-mouse immune system, it was found that the number of CD4 ⁺ T cells in the peripheral lymphatic system was significantly reduced after 13 weeks (Figs. 1A and 2B). CD4 ⁺ T cells is apoptosis ⁺ T cells and thymus from entering the outer periphery of CD4 to CD4 ⁺ T cells maintained.

And the number of murine + T cells and wild type _(w t) mouse thymocytes CD4 CD8 + T cells, was found not changed (FIG. 2C). Subsequently, the inventors examined apoptosis of peripheral blood CD4 ⁺ T cells. CD4 ⁺ T cells were isolated from / hrrr mice, βαττΐ transgenic (^rrHg) mice and wt mouse spleens, activated or not activated with anti-CD3 and anti-CD28 antibodies, and then cultured in serum-free and cytokine-free Culture in the base. Figures 1B and 1C show that the viability of activated rrHg CD4 ⁺ T cells is significantly higher than that of wild type, while CD4 ⁺ T cells of ^ ^ - are significantly more likely than wild type in their naive or activated state. Death is more sensitive. These data indicate that: Parrl promotes the viability of CD4+ T cells in vitro.

 To further demonstrate the situation in vivo, the inventors used SEB to inject mice. SEB can be specifically activated in the body

鼠的 CD4⁺ νβ8.1/2 Τ细胞凋亡比 W增多。 这些结果表明： 在体内 Parrl也能促 进 CD4⁺ T细胞的存活。 IL-2是促进幼稚和激活的 CD4⁺ T细胞的一个重要细胞因子（55-57)。 当 CD4⁺ T细胞在体外激活后，它们产生大量的 IL-2 ( 1 ) ₀然而，如图 3A所示：

CD4⁺ T细胞产生 IL-2的能力比其 wt要略强。 因此， Parrl不可能是通过影响 CD4⁺ T细胞 IL-2 的产生来调节这些细胞存活的。 2.parrl促进 Bcl-2的表达 CD4 ⁺ νβ8.1/2 Τ cells have no effect on CD4 ⁺ νβ6.1 T cells (16). As shown in Figures 1D and 1E: The number of CD4 ⁺ Vp8.1/2 T cells in each genotype of mice increased significantly after three days of SEB injection, and decreased significantly after seven days of injection. During the next few days, it was observed that: ^rrHg CD4 ⁺ Vp8.1/2 T cell apoptosis was significantly less than W,

The CD4 ⁺ νβ8.1/2 Τ cell apoptosis was increased in mice. These results indicate that Parrl also promotes the survival of CD4 ⁺ T cells in vivo. IL-2 is an important cytokine (55-57) that promotes naive and activated CD4 ⁺ T cells. When CD4 ⁺ T cells are activated in vitro, they produce large amounts of IL-2 ( 1 ) _{0 ,} however, as shown in Figure 3A:

The ability of CD4 ⁺ T cells to produce IL-2 is slightly stronger than its wt. Therefore, Parrl is unlikely to regulate the survival of these cells by affecting the production of IL-4 by CD4 ⁺ T cells. 2.parrl promotes the expression of Bcl-2

Erk的 磷酸化水平； 而 Akt的磷酸化水平在激活状态下的 CD4⁺ T细胞中有所下降。 因为， parrl 能同时促进幼稚和激活了的 CD4⁺ T细胞的存活， 而 parrl并没有影响幼稚 CD4⁺ T细胞中 Akt和 Erk 的磷酸化水平， 所以， 推测 parrl 促进 CD4⁺ T细胞的存活还有其他的机制。 Affymetrix基因芯片数据表明： 通过 siRNA抑制 parrl的表达能降低抗凋亡分子 Bd-2的表 达， 但并不影响 Bcl-2家族其他蛋白的表达， 如： Bim, Bax和 Bcl-xl等。 在 Jurkat T细胞中 进一步验证了这些结果（图 4)，并且发现是核内的 β ιτΐ调控着 的表达， 因为 β ιτ2 和 arrlQ394L( arrl不进核的突变体） （32， 40) 并没有此功能。 Bcl-2是调节幼稚和激活 T细胞存活的一个重要分子 （1， 16)， 因此进一步检测了在 CD4⁺ T细胞中 parrl和 Bcl-2的表达情况。 CD4⁺ T细胞如上述方法激活以后， βαιτΐ和 Bcl-2 的蛋白水平在前两天中逐步下降， 而在激活后第三天升高并在随后保持该水平 （图 5A)。 当进一步在激活 40到 64 小时这段时间内检测 parrl和 Bcl-2的表达情况， 发现 parrl蛋白 水平的升高早于 Bcl-2 (图 5A)。是不是在 CD4⁺ T细胞中， parrl调节着 Bcl-2的表达呢？ 发明人同时激活 y&rrHg小鼠和 W的 CD4⁺ T细胞， 并检测 parrl和 Bc!-2的 mRNA和蛋白 水平。 结果发现， ^rrHg小鼠来源的 CD4⁺ T细胞在激活 48小时后 parrl的水平明显比其 野生型高， 和此一致的是 的 mRNA和蛋白水平也在该时间有显著升高 （图 5B)。 当 和 W小鼠来源的 CD4⁺ T细胞在体外激活后， Bc!-2的 mRNA和蛋白水平在激活后 没有变化并保持在野生型里最低的水平 （图 5C)。 在实验中发现， β ιτ2 的蛋白水平也在 CD4⁺ T细胞激活过程中有跟 parrl类似的变化。发明人在^ rr2-的 CD4⁺ T细胞检测了 Bd-2 的 mRNA和蛋白水平， 发现 表达正常 （图 5D)。 这些结果表明： parrl而不是 parr2 调节着 CD4⁺ T细胞中 Bd-2的表达， 这可能是导致 ^rr ^ P^rrHg的 CD4⁺ T细胞存活能 力异常的原因， 因为这跟^¾/-2敲除和转基因的 CD4+ T细胞存活能力很类似 （18)。 The molecular mechanism of Parrl promoting the survival of CD4 ⁺ T cells was further studied. Considering that activation of Akt (38) and Erk (39) promotes CD4 ⁺ T cell survival, and Parrl itself positively regulates Akt and Erk activity (20), therefore, whether it is due to Parrl upregulation of Akt or Erk Activity to promote the viability of CD4 ⁺ T cells. The parl-'- and wt CD4 ⁺ T cells were activated in vitro as previously described, and their activity was detected using phosphorylated antibodies of Akt and Erk. As shown in Figure 3B, in naive and activated CD4+ T cells,

The phosphorylation level of Erk; while the phosphorylation level of Akt is decreased in activated CD4 ⁺ T cells. Because parrl can simultaneously promote the survival of naive and activated CD4 ⁺ T cells, and parrl does not affect the phosphorylation levels of Akt and Erk in naive CD4 ⁺ T cells, so it is speculated that parrl promotes the survival of CD4 ⁺ T cells. Other mechanisms. Affymetrix gene chip data showed that inhibition of parrl expression by siRNA can reduce the expression of anti-apoptotic molecule Bd-2, but does not affect the expression of other proteins in Bcl-2 family, such as Bim, Bax and Bcl-xl. These results were further validated in Jurkat T cells (Fig. 4) and found to be regulated by β ιτΐ in the nucleus, since β ιτ2 and arrlQ394L (arrls that do not enter the nucleus) (32, 40) do not have this Features. Bcl-2 is an important molecule regulating the survival of naive and activated T cells (1, 16), thus further detecting the expression of parrl and Bcl-2 in CD4 ⁺ T cells. After CD4 ⁺ T cells were activated as described above, the protein levels of βαιτΐ and Bcl-2 gradually decreased in the first two days, and increased on the third day after activation and subsequently maintained (Fig. 5A). When the expression of parrl and Bcl-2 was further detected during the period of 40 to 64 hours of activation, it was found that the level of parrl protein was earlier than that of Bcl-2 (Fig. 5A). Is it that parrl regulates the expression of Bcl-2 in CD4 ⁺ T cells? The inventors simultaneously activated CD4 ⁺ T cells of y&rrHg mice and W, and detected mRNA and protein levels of parrl and Bc!-2. It was found that the level of parrl in the CD4 ⁺ T cells derived from ^ rrHg mice was significantly higher than that in the wild type after 48 hours of activation, and the mRNA and protein levels were also significantly increased at this time (Fig. 5B). . When activated with W mouse-derived CD4 ⁺ T cells in vitro, the mRNA and protein levels of Bc!-2 did not change after activation and remained at the lowest level in the wild type (Fig. 5C). It was found in the experiment that the protein level of β ιτ2 also had a similar change to parrl during CD4 ⁺ T cell activation. The inventors detected mRNA and protein levels of Bd-2 in CD4 ⁺ T cells of ^rr2- and found that expression was normal (Fig. 5D). These results indicate that parrl, but not parr2, regulates the expression of Bd-2 in CD4 ⁺ T cells, which may be responsible for the abnormal viability of CD4 ⁺ T cells that cause ^rr ^ P^rrHg, as this is followed by ^3⁄4/-2 The viability of knockout and transgenic CD4+ T cells is very similar (18).

Because the mRNA and protein levels of 3^-2 change simultaneously during CD4 ⁺ T cell activation, the inventors used

Jurkat T cells to prove whether parrl regulates its expression by affecting the transcription of Sc/. Consistent with Figure 4 (Figure 6A), overexpression of Jurkat T cells or downregulation of parrl with siRNA promoted or inhibited Bcl-2 mRNA and protein levels. Further, the inventors have found that they are effective in inhibiting the up-regulation of Bcl-2 expression by parrl using transcription inhibitors and protein synthesis inhibitors (Figs. 6B and 6C). Thus, parrl regulates its expression by promoting transcription. It has been reported that CREB (41) and F-kappa B (42) are transcription factors that affect Sc/J expression. However, the inventors found that parrl did not affect the activity of the CREB and F-kappa B reporter genes in the reporter gene system (Figures 6D and 6E;). Thus, parrl may be regulated by other means.

3. arrl promotes histone acetylation in the locus

CD4⁺ T细胞激活后， 基因启动 子区的组蛋白 H4乙酰化水平一直保持在相当于野生型中最低的水平（图 7C)。进一步， 发 明人发现 parrl影响着 基因座区从转录起始点上游 10,000bp到下游 186,000bp染色体 区的组蛋白 H4乙酰化水平 （图 7D)。 这些实验表明： parrl很可能是通过促进 基因 座区组蛋白 H4乙酰化水平来上调 基因表达的。 4. p300在 parrl上调 Bcl-2表达中的是必需的 Another way parrl regulates gene expression is by up-regulating the level of acetylation of histone H4 in the promoter region of a specific gene (27). During CD4+ T cell activation, the inventors found that the pattern of changes in the level of histone H4 acetylation in the promoter region of the gene was very similar to that of Bd-2 (Fig. 7A). The level of histone H4 acetylation in the promoter region of the Bcl-2 gene gradually decreased after activation of CD4 ⁺ T cells, reached a minimum at 48 hours, and then increased again at 72 hours. The level of H3 acetylation as a control protein for histone H4 acetylation levels did not change. Further, the inventors found that the effect of parrl on Bd-2 expression is consistent, and parrl also affects the level of histone H4 acetylation in the promoter region of Bd-2 gene. At 0 rrHg CD4 ⁺ T cell activation 48, the histone H4 acetylation level of the Bd-2 gene promoter region was significantly increased relative to the wild type (Fig. 7B).

After activation of CD4 ⁺ T cells, the level of histone H4 acetylation in the promoter region of the gene remained at the lowest level equivalent to that in the wild type (Fig. 7C). Further, the inventors found that parrl affects the level of histone H4 acetylation in the locus region from 10,000 bp upstream to 186,000 bp downstream of the transcription initiation site (Fig. 7D). These experiments show that parrl is likely to upregulate gene expression by promoting histone H4 acetylation in the locus. 4. p300 is required for upregulating Bcl-2 expression in parrl

 The level of acetylation of intracellular histones is regulated by histone deacetylase and histone acetylase. Previous studies have found that parrl promotes acetylation of histone H4 by binding to the histone acetylase p300 (43) and recruiting it to the promoter region of a specific gene (27). Therefore, the inventors examined the role of p300 in up-regulating Sc/expression in β ιτΐ. Consistent with the previous results: In Jurkat cells, parrl, but not parr2 or parrlQ394L, promoted histone H4 acetylation levels in the promoter region of the gene (Fig. 8A). Further, the inventors found that p300 also promotes histone H4 acetylation level and mRNA level in the promoter region of Bcl-2 gene, and this promotion can be further enhanced by co-expression parrl and inhibited by co-expression of rr siRNA (Fig. 8C). And 8E). p300DN (inactive mutant of p300) significantly reduced histone H4 acetylation and Sc/^ mRNA levels in the promoter region of the gene, and also blocked the promotion of parrl (Figures 8C and 8E). These results indicate that p300 plays an important role in the increase in the level of H4 acetylation and the increase in transcription in the parrl-mediated Bd-2 gene promoter region.

5. The role of arrl in multiple sclerosis in autoimmune diseases

Fine regulation of CD4 ⁺ T cell apoptosis by signal-regulating molecules prevents CD4 ⁺ T cells from producing autoimmunity. Considering the important role of parrl in the regulation of apoptosis in CD4 ⁺ T cells, the inventors further investigated whether it would affect autoimmunity. The EAE model of mice is a good animal model of multiple sclerosis (MS) (44). In this model, CD4+ T cells are thought to play a leading role and its pathogenesis is clear. The inventors used MOG35-55 to immunize mice of three different genotypes and induced them to produce EAE. Subsequent observation for 20 days is shown in Fig. 9A, and it is found that the onset of the onset of the mouse has a significant delay Ot: 9.7 ± 2.9 days vs. βωτΐ-ζ '. 16.3 ± 3.0 days) and the severity of the disease is also significant. Decline (; maximum mean disease score t: 3.2 ± 0.27 vs. βαττΓ^: 1.4 ± 0.4). In / hrr tg mice, although the onset of onset was not significantly different from W mice t: 9.7 ± 2.9 days vs. ^ rrHg: 8.7 ± 0.8 days), but the severity of the disease was significantly increased (maximum mean Condition score ferrHg: 4.2 ± 0.3 vs. wt: 3.2 ± 0.2). Consistent with this result, when the inventors detected the degree of spinal cord lesions in mice,

鼠的则较低 （图 9C)。 进一步，发明人用 MOG35-55重剌激 EAE小鼠脾淋巴细胞中纯化得到的 MOG特异的 CD4⁺ T细胞， 并观察在细胞因子胁迫下这些 CD4⁺ T细胞的存活。 和图 1B和 1C一致的是： 从 arrltg小鼠来的 MOG特异的 CD4⁺ T细胞明显比野生型的存活能力强， 而从 小鼠 中来的 CD4⁺ T细胞存活能力则较差（图 9D)。 这些结果表明 parrl在 EAE模型中起着正调 节的作用， 这可能是由于促进 CD4⁺ T细胞的存活能力来实现的。 The degree of myelin is milder; whereas y&rrHg mice are just the opposite (Fig. 9B). These data indicate that parr 1 plays a positive regulatory role in this EAE model. Because parrl can promote the survival of CD4+ T cells, is this mechanism involved in the positive regulation of parrl on autoimmunity? The inventors first used MOG35-55 to re-excite the spleen lymphocytes of sputum mice, and found that the proliferation of spleen lymphocytes in rrHg mice was significantly higher than that in wild type.

The mouse is lower (Fig. 9C). Further, the inventors used MOG35-55 to mobilize MOG-specific CD4 ⁺ T cells purified from spleen lymphocytes of EAE mice, and observed the survival of these CD4 ⁺ T cells under cytokine stress. Consistent with Figures 1B and 1C: MOG-specific CD4 ⁺ T cells from arrltg mice were significantly more viable than wild-type, whereas CD4 ⁺ T cells from mice were less viable (Figure 9D). . These results indicate that parrl plays a positive regulatory role in the EAE model, which may be due to the ability to promote the viability of CD4 ⁺ T cells.

A few years ago, Anne Vroon and his colleagues found that parrl was significantly elevated in peripheral blood after induction of EAE in mice (45). The inventors further found that the protein level of parrl in CD4+ T cells from EAE mice was significantly increased (Fig. 11A;). In view of these findings, the inventors examined the expression of β ιτΐ in CD4 ⁺ T cells of patients with multiple sclerosis, as shown in Figures 10A and 10B: Transcription levels of ^rr and Bc!-2 in CD4 ⁺ T cells from patients with multiple sclerosis Significantly higher than healthy people. Further, infection with a retrovirus with ^rr siRNA from patients with multiple sclerosis From the source of MBP-specific CD4 ⁺ T cells (46), Bcl-2 expression was found to be significantly downregulated (Fig. 10C). When these MBP-specific CD4 ⁺ T cells were subjected to cytokine stress after infection with a retrovirus containing rr siRNA, these cells were significantly more susceptible to apoptosis than retroviruses harboring an empty vector (Fig. 10D). These results indicate that in patients with multiple sclerosis, CD4 ⁺ T cells targeting autoantigens have higher levels of rr expression, which may contribute to the viability of these cells, thereby allowing their regulatory mechanisms to escape multiple sclerosis. development of. discuss

In mammals, the immune system is one of the most dynamic systems in the body. The apoptosis of immune cells in the immune system is regulated by many extracellular and intracellular signals to maintain their normal physiological functions. Parrl is a signal molecule with multiple functions and is involved in the regulation of many signal paths. In addition, it has the ability to directly regulate nuclear-specific gene transcription. The inventors have found in the present invention that parrl specifically regulates the survival and homeostasis of CD4+ T cells, thereby affecting the acquired immune response of the body. Although the activity of Akt is inhibited in activated CD4 ⁺ T cells, the ability of parrl to affect the survival of CD4 ⁺ T cells is mainly achieved by its intranuclear function. In either the naive or activated CD4+ T cell nucleus, parrl upregulates the level of histone H4 acetylation in the promoter region of the gene, thereby promoting gene expression. Further studies have found that parrl plays an important role in demyelinating diseases caused by autoimmunity and type I diabetes caused by streptozotocin. In demyelinating diseases caused by autoimmunity, the high expression of ^rr in CD4+ T cells that specifically cause encephalitis promotes the viability of these cells in vivo. Therefore, the inventors' results indicate that parrl plays an important regulatory role in the survival of CD4+ T cells and autoimmune diseases in humans and mice.

A detailed mechanism analysis found that the regulation of parrl expression in CD4+ T cells was achieved by affecting epigenetic modifications. Bcl-2 regulates cell survival by regulating the integrity of the mitochondrial membrane and the release of pro-apoptotic proteins present in mitochondria (12). In T cells, Bcl-2 must be maintained at an appropriate level, otherwise it will cause the breaking of T cell homeostasis and the confusion of immune response. However, to date, the regulation mechanism of Bcl-2 in T cells has not been well understood. The inventors found that parrl promotes the level of histone acetylation in the gene region and the transcription level of the ^3⁄4/-2 gene, whether in naive or activated CD4+ T cells. This function of parrl in CD4 ⁺ T cells was also found to be conserved in humans and mice. Therefore, these experiments have revealed an epigenetic mechanism that regulates gene expression in addition to revealing a novel mechanism for regulating the survival of CD4 ⁺ T cells. This mechanism has not been reported before.

In this study, parrl promoted T cell survival mainly in peripheral blood CD4 ⁺ but not CD8 ⁺ T cells or thymic T cells. In thymic T cells, as shown in Figure 2A: the expression level of parrl is significantly lower than its expression level in spleen cells or lymph node cells. This difference in expression levels may be responsible for the small role of parrl in thymic T cells. However, as can be seen from Figure 11B, the difference in parrl expression levels in CD4 ⁺ and CD8 ⁺ T cells is not significant. However, the effect of parrl on the survival of CD4 ⁺ and CD8 ⁺ T cells was significantly different. Compared with wild-type cells, the viability of y&rr "CD4 ⁺ T cells was significantly reduced, while there was no significant difference between ^rr CD8 ⁺ T cells. The detailed mechanism showed that the ribs in the nucleus up-regulated the acetylation level of histones. And its transcriptional level, because the expression of parrl does not enter the nuclear mutant parrlQ394L does not affect the expression of Bd-2. Therefore, the inventors suspected whether parrl differs in the subcellular localization of CD4 ⁺ and CD8 ⁺ T cells. As shown in 11C and 11D, parrl is expressed in the nucleus at a higher level than CD8 ⁺ T cells in both naive and activated CD4+ T cells. height of. Therefore, it may be that the distribution of parrl in subcellular levels in CD4 ⁺ and CD8 ⁺ T cells results in a different effect of parrl on the survival of CD4 ⁺ and CD8 ⁺ T cells.

Previous studies have shown that parrl and parr2 are recruited to the cell membrane when GPCR is activated; where they bind to phosphorylated GPCRs causing receptor endocytosis and signal termination (47). However, there is now increasing evidence that the two parrs differ in function and receptor specificity. In this study by the inventors, parrl, but not arr2, promotes the level of acetylation of the Sc/gene region and its transcriptional level. This result of the inventors is consistent with previous conclusions (27), which may be due to the different distribution of the two parr at the subcellular level. Although the two parrs are different in this function, they can regulate the immune response from the animal level. In this study, the inventors found that parrl promoted CD4 ⁺ T cell survival and autoimmunity; another earlier study showed that β ιτ2 knockout mice had significantly lower symptoms than wild-type mice in asthma model experiments. , and this may be due to the influence of β ιτ2 on the migration of immune cells. It can be seen from these results that although the cellular mechanisms affected by pairs are different, they all play a role in promoting immune response in the immune system.

Recent studies by the inventors' laboratory have shown that parrl has the function of directly regulating gene transcription in the nucleus; this function is affected by some GPCRs (27). In addition, in in vitro activated CD4+ T cells, Nguyen K. and Miller BC were found to have expression of delta opioid receptor (DOR) (48). Interestingly, the inventors found in vitro experiments that activation of DOR can upregulate the expression of Bd-2 in a parrl dependent manner. These data suggest that parrl may act as an important signaling regulator molecule to mediate external signals that promote CD4 ⁺ T cell survival and thereby affect the adaptive immune response. Considering the intrinsic physiological function of Ijparrl in CD4 ⁺ T cells, these results not only reveal a mechanism for regulating the immune system under normal physiological conditions, but also provide a possible therapeutic target for autoimmune diseases such as multiple sclerosis. .

 While the invention has been described with respect to the specific embodiments of the present invention, it will be apparent to those skilled in the art Therefore, the appended claims cover all such variations that are within the scope of the invention. All publications, patents and patent applications cited herein are hereby incorporated by reference. references

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Claims

Rights request 1 . Use of an inhibitor of β inhibitory protein 1 in the preparation of a medicament, characterized in that the inhibitor of β inhibitory protein 1 is selected from the group consisting of (i) a substance that inhibits β inhibitory protein 1 activity; (ii) an inhibitory expression of β inhibition A gene transcription, translation, or both of the protein 1 for treating or preventing a disease associated with abnormal apoptosis of CD4 ⁺ T cells in a mammalian subject.  The use according to claim 1, wherein the substance which inhibits the activity of β inhibitory protein 1 is selected from the group consisting of an antibody specific for actinomycin, cycloheximide or β inhibitory protein 1.  The use according to claim 1, wherein the substance which inhibits transcription, translation or both of the gene encoding β inhibitory protein 1 is a small interfering RNA, a coding gene encoding all or part of β inhibitorin 1 Antisense sequence or ribozyme.  The use according to claim 1, wherein the substance which inhibits transcription, translation or both of a gene encoding β inhibitory protein 1 is loaded in a carrier.  5. Use according to claim 4, wherein the vector is selected from the group consisting of a retroviral vector, a lentiviral vector, an adenoviral vector or a DNA viral vector.  6. Use according to claim 5, wherein the mammal is a human.  The use according to claim 1, wherein the disease associated with abnormal apoptosis of CD4+ T cells is selected from an autoimmune disease or a lymphocytosis.  The use according to claim 7, wherein the autoimmune disease is selected from the group consisting of rheumatoid arthritis, systemic lupus erythematosus, dermatomyositis, scleroderma, multiple sclerosis, myasthenia gravis, and detachment. Myelin sheath disease, primary adrenal atrophy, chronic thyroiditis, type I diabetes, chronic non-specific ulcerative colitis, chronic active hepatitis, avian anemia and atrophic gastritis, autoimmune glomerulonephritis, lung and kidney Hemorrhagic syndrome, autoimmune hemolytic anemia, idiopathic thrombocytopenic purpura, idiopathic leukopenia. A pharmaceutical composition for treating or preventing a disease associated with abnormal apoptosis of CD4 ⁺ T cells in a subject, wherein the pharmaceutical composition comprises an effective amount of an inhibitor of β inhibitory protein 1 as an active group And a pharmaceutically acceptable carrier, wherein the inhibitor of β inhibitory protein 1 is selected from the group consisting of (i) a substance that inhibits β inhibitory protein 1 activity; (ii) a gene that inhibits β-inhibitor protein 1 transcription, translation or this The substance of both.  10. A method of screening a candidate for a drug for treating or preventing a disease associated with abnormal apoptosis of CD4+ T cells, the method comprising: a) contacting the candidate substance with a cell expressing β-arrestin 1; b) identifying the expression level of β-arrestin 1 in the nucleus of the cell, and comparing the expression level to the expression level of β-arrestin 1 in the nucleus of cells not in contact with the candidate substance; c) A substance which reduces the expression level of β_inhibitor 1 in the nucleus is selected as a disease drug for treating or preventing abnormal apoptosis associated with CD4 ⁺ T cells.