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WO2012003810A1 - Foxp3及调节性ｔ细胞的调节因子及其应用 - Google Patents

Foxp3及调节性ｔ细胞的调节因子及其应用 Download PDF

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WO2012003810A1
WO2012003810A1 PCT/CN2011/077032 CN2011077032W WO2012003810A1 WO 2012003810 A1 WO2012003810 A1 WO 2012003810A1 CN 2011077032 W CN2011077032 W CN 2011077032W WO 2012003810 A1 WO2012003810 A1 WO 2012003810A1
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foxp3
cells
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vaccine
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French (fr)
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李斌
林芳
陈祚珈
李志远
潘帆
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Institut Pasteur of Shanghai of CAS
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Institut Pasteur of Shanghai of CAS
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Priority to US13/809,267 priority Critical patent/US20130122046A1/en
Priority to EP11803163.2A priority patent/EP2591797A4/en
Publication of WO2012003810A1 publication Critical patent/WO2012003810A1/zh
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
    • A61K38/53—Ligases (6)
    • A—HUMAN NECESSITIES
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    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
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    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
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    • A61K40/00—Cellular immunotherapy
    • A61K40/40—Cellular immunotherapy characterised by antigens that are targeted or presented by cells of the immune system
    • A61K40/41—Vertebrate antigens
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    • A—HUMAN NECESSITIES
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    • C12Y603/02—Acid—amino-acid ligases (peptide synthases)(6.3.2)
    • C12Y603/02019—Ubiquitin-protein ligase (6.3.2.19), i.e. ubiquitin-conjugating enzyme
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Definitions

  • the invention relates to the field of molecular biology and biomedicine. More specifically, the invention relates to the use of a ubiquitination pathway-related factor, an agonist or antagonist thereof, for modulating FOXP3 activity, IL-2 activity and/or IFN- ⁇ activity.
  • the present invention relates to a negative regulator of regulation of FOXP3 activity, and the use thereof to modulate the immune system by down-regulating FOXP3 activity, thereby treating or preventing a disease or condition associated with hyperactivity of FOXP3 and use as an immunological adjuvant. Background technique
  • the Fox transcription factor family is a large family of transcription factors characterized by a forkhead/winged helix (FKH) with diverse functions. This family member plays a different role in the various processes of cell development (References 1 and 2).
  • FOXP3 is a regulatory T cell (regulatory T cell)-specific transcription factor that plays an important regulatory role in the development and function of Treg and has the function of down-regulating the immune response.
  • the immunoregulatory function of FOXP3-mediated Treg is through the formation of protein complexes by FOXP3 and several transcriptional co-regulatory proteins (such as transcription factors, co-suppressors, coactivators, histones, and chromatin remodeling factors) to dynamically regulate gene specificity. Transcription (References 3 and 4). Under different conditions of stimulation, the different states of the FOXP3 protein complex and its transcriptional activity have different effects on Treg and immune system regulation (References 3 and 4).
  • FOXP3+ regulatory T cells belong to a class of T cell subsets that express CD4, CD25 and transcription factor FOXP3 in T lymphocytes, and their normal functions are essential for the dynamic regulation of human immune homeostasis.
  • FOXP3 binds to multiple transcription factors and an enzymatically active histone acetyltransferase/deacetylase complex, which inhibits T cell stimulating Transcriptional activation of inflammatory cytokines is required.
  • the activity of FOXP3 protein post-translational modification, transcription complex assembly and modified enzymes is dynamically regulated by T cell receptor and inflammatory cytokine receptor signals.
  • FOXP3 is a lysine acetylated protein, and FOXP3 proline-rich N-terminus can directly recruit histidine acetyltransferase TIP60 (Tat interaction protein, 60 kDa), which mediates transcriptional repression of FOXP3. Activity (see Reference 6 and Figure 1).
  • IL-2 is a lymphocyte factor that is normally produced by lectins or antigen-activated T lymphocytes. It can proliferate cytotoxic T cells, natural killer cells and lymphokine activated killer cells, and enhance their killing activity. It can also promote lymphocyte secretion of antibodies and interferons, and has antiviral, antitumor and immune functions. . Human recombinant IL-2 has been used in tumors, inflammatory reactions, acute infectious diseases or chronic infectious diseases.
  • INF-Y is a cytokine of water-soluble dimers, usually produced by natural killer cells, natural killer T cells, and cytotoxic T cells.
  • INF- ⁇ is an important cytokine against pathogens and bacterial infections in the innate and adaptive immune system. It directly inhibits viral replication in vivo and has antiviral, immunomodulatory and antitumor properties.
  • INF-Y binds to its receptor to regulate the JAK-STAT pathway, activates antigen-presenting cells, and promotes the differentiation of type I helper T cells (Thl cells) by up-regulating the transcription factor T-bet; it can induce antiviral protein synthesis, It can treat viral diseases such as sexually transmitted diseases; it can reduce the levels of type I and III collagen mRNA in the liver, prevent and slow the occurrence of liver fibrosis, and show that it is effective for slow-lived liver and cirrhosis.
  • a ubiquitination pathway-related factor for the preparation of a composition for modulating FOXP3 activity, IL-2 activity and/or IFN- ⁇ activity, wherein General
  • the factors associated with the priming pathway are selected from the group consisting of: Toll-like receptors, ubiquitinated ligases, pro-inflammatory cytokine family receptors, and/or their coding sequences.
  • said modulation of FOXP3 activity, IL-2 activity and/or IFN- ⁇ activity is positive or negative regulation, wherein said ubiquitination pathway-related factor or agonist thereof is used Negatively modulating FOXP3 activity and upregulating IL-2 activity and/or IFN- ⁇ activity, the antagonist of the ubiquitination pathway-associated factor is used to positively regulate FOXP3 activity and negatively regulate IL-2 activity and/or IFN- ⁇ activity.
  • the agonist is IL-6.
  • the antagonist is MG-132.
  • the pro-inflammatory cytokine family receptor is selected from the group consisting of: IL-6R, TGFp receptor, IL-2 receptor, TNF-[alpha] receptor or GITR.
  • the use is a use of a ubiquitination pathway-related factor in the preparation of a composition that negatively regulates FOXP3 activity, wherein the ubiquitination pathway-related factor is selected from the group consisting of: Toll-like receptors, Ubiquitinated ligases and/or their coding sequences.
  • the ubiquitinated ligase is one or more selected from the group consisting of STUB TRAF6, Smurf Smurf2, TRAF2, TRAF3, TRAF4, p300, RNF31 or RBCK1.
  • the ubiquitinated ligase is preferably STUB1.
  • the ubiquitinated ligase is a complex that also has molecular chaperone activity.
  • the ubiquitin-linked enzyme activates a downstream protein for the Toll-like receptor signaling pathway, preferably TRAF6.
  • the Toll-like receptor is one or more selected from the group consisting of TLR TLR2, TLR3, TLR4, TLR5, TLR7, TLR8 or TLR9.
  • the Toll-like receptor is selected from the group consisting of: TLR3, TLR7, TLR8 or TLR9.
  • downstream kinase IRAKI of the Toll-like receptor signaling pathway promotes ubiquitination of FOXP3.
  • the composition for modulating FOXP3 activity, IL-2 activity and/or IFN- ⁇ activity is for use in the treatment and/or prophylaxis of FOXP3, IL-2 and/or IFN- ⁇ activity A disorder or disease associated with a disorder.
  • the activity disorder is hyperactivity or activity is too low.
  • the disease or condition associated with dysregulation of FOXP3, IL-2 and/or IFN-[gamma] activity is selected from the group consisting of: a tumor, an inflammatory response, an acute infectious disease, or a chronic infectious disease.
  • the tumor is selected from the group consisting of: prostate cancer, breast cancer, liver cancer, glioma, intestinal cancer, cervical cancer, non-small cell lung cancer, lung cancer, pancreatic cancer, gastric cancer, bladder cancer, Skin cancer, rhabdomyosarcoma, tongue squamous cell carcinoma, nasopharyngeal carcinoma, ovarian cancer, placental villus cancer, glioma, lymphoma, leukemia, rectal adenocarcinoma or melanoma.
  • the inflammatory response is selected from the group consisting of: allergic inflammation, folliculitis, tonsillitis, pneumonia, hepatitis, nephritis, acne, asthma, autoimmune diseases, chronic inflammation, chronic prostatitis, kidney Glomerulonephritis, hypersensitivity, inflammatory bowel disease, pelvic inflammatory disease, reperfusion injury, rheumatoid arthritis, transplant rejection, vasculitis or interstitial cystitis.
  • the infectious disease is selected from the group consisting of: plague, cholera, infectious atypical pneumonia, AIDS, viral hepatitis, polio, human infection with highly pathogenic avian influenza, measles, epidemics Hemorrhagic fever, rabies, epidemic encephalitis, hand, foot and mouth disease, dengue fever, anthrax, bacterial and amoebic dysentery, tuberculosis, typhoid fever and paratyphoid fever, epidemic cerebrospinal meningitis, whooping cough, diphtheria, newborn Tetanus, scarlet fever, brucellosis, gonorrhea, syphilis, leptospirosis, schistosomiasis, malaria, influenza, mumps, rubella, acute hemorrhagic conjunctivitis, leprosy, epidemics and places Typhus typhus, kala-azar, echinococcosis, fil
  • composition for modulating FOXP3, IL-2 activity and/or IFN- ⁇ activity is further used as a vaccine adjuvant or vaccine.
  • the vaccine is selected from the group consisting of: a vaccine for treating or preventing a viral infection, a bacterial infection, an inflammation, a parasitic infection or a tumor, such as a hand, foot and mouth disease vaccine.
  • the vaccine is selected from the group consisting of: tetanus vaccine, diphtheria, tetanus, pertussis vaccine, influenza vaccine, yellow fever vaccine, polio vaccine, tuberculosis vaccine, measles vaccine, rubella vaccine, meningitis A Vaccine, rabies vaccine, hepatitis B vaccine, measles, mumps, rubella triple vaccine, Haemophilus influenzae type b vaccine, typhoid vaccine, hepatitis A vaccine, cervical cancer vaccine, pneumonia vaccine, chickenpox vaccine, meningococcal vaccine, Japanese encephalitis vaccine, dysentery Vaccine, cholera vaccine, AIDS vaccine, hepatitis C vaccine, hepatitis E vaccine, hand, foot and mouth disease vaccine, bronchitis vaccine, group A meningococcal vaccine or human avian influenza vaccine.
  • the vaccine is a DNA vaccine, a recombinant vector vaccine, an inactivated or attenuated pathogen-based vaccine, a subunit vaccine, or a cancer cell vaccine.
  • the composition is a pharmaceutical composition, a nutraceutical composition or a vaccine composition.
  • the content of the regulator in the composition is from 0.05 to 99.5% by weight, preferably 0.1 to 95% by weight, more preferably 1 to 90% by weight, still more preferably 5 to 80% by weight.
  • the composition further comprises a molecular chaperone required for ubiquitination, and the molecular chaperone is preferably: HSP70, HSC70 or HSP90.
  • composition further comprises a pharmaceutically, hygienically or immunologically acceptable carrier.
  • the composition is an injection, a tablet, a granule, a powder or a capsule.
  • a composition for modulating (e.g., negatively regulating) FOXP3 activity, IL-2 activity and/or IFN- ⁇ activity comprising:
  • ubiquitination pathway-related factors selected from the group consisting of ubiquitinated ligases, Toll-like receptors, pro-inflammatory cytokine family receptors, and/or coding sequences thereof; and/or An agonist or antagonist of a ubiquitination pathway-related factor;
  • the composition is for use in treating or preventing a disease or condition associated with a disorder of FOXP3, IL-2 and/or IFN- ⁇ activity (ie, hyperactivity or hypoactivity; or as a vaccine) Agent.
  • a disease or condition associated with a disorder of FOXP3, IL-2 and/or IFN- ⁇ activity ie, hyperactivity or hypoactivity; or as a vaccine
  • composition is further used to prepare a vaccine composition, which further comprises an immunogenic active substance.
  • the composition further comprises one or more other active substances which modulate FOXP3, IL-2 and/or IFN- ⁇ , preferably TIP60.
  • the ubiquitinated ligase is one or more selected from the group consisting of: STUB TRAF6, Smurf Smurf2, TRAF2, TRAF3, TRAF4, p300, RNF31 or RBCKl o
  • the ubiquitinated ligase is preferably STUB 1.
  • the ubiquitinated ligase is a complex that also has molecular chaperone activity.
  • the ubiquitin-linked enzyme activates a downstream protein for the Toll-like receptor signaling pathway, preferably TRAF6.
  • the Toll-like receptor is one or more selected from the group consisting of TLR1, TLR2, TLR3, TLR4, TLR5, TLR7, TLR8 or TLR9.
  • the Toll-like receptor is selected from the group consisting of: TLR3 or TLR9.
  • the downstream kinase IRAKI of the Toll-like receptor signaling pathway promotes ubiquitination of FOXP3.
  • the composition for modulating FOXP3 activity, IL-2 activity and/or IFN- ⁇ activity is for use in the treatment and/or prophylaxis of FOXP3, IL-2 and/or IFN- ⁇ activity A disorder or disease associated with a disorder.
  • the disease or condition associated with the activity disorder is selected from the group consisting of: a tumor, an inflammatory response, an acute infectious disease, or a chronic infectious disease.
  • the tumor, inflammatory response, acute infectious disease or chronic infectious disease can be as described above.
  • the vaccine is a DNA vaccine, a recombinant vector vaccine, an inactivated or attenuated pathogen-based vaccine, a subunit vaccine, or a cancer cell vaccine.
  • the composition is a pharmaceutical composition, a nutraceutical composition or a vaccine composition.
  • a method of modulating FOXP3 activity, IL-2 activity and/or IFN- ⁇ activity comprising administering one or more regulatory factors selected from the group consisting of or Agents or antagonists: ubiquitinated ligases, Toll-like receptors, pro-inflammatory cytokine family receptors and/or their coding sequences.
  • the ubiquitinated ligase is one or more selected from the group consisting of STUB1, TRAF6, Smurf Smurf2, TRAF2, TRAF3, TRAF4, p300, RNF31 or RBCK1, preferably STUB1.
  • the Toll-like receptor is one or more selected from the group consisting of TLRU TLR2, TLR3, TLR9, TLR4, TLR5, TLR7 or TLR8, preferably TLR3 or TLR9.
  • the pro-inflammatory cytokine family receptor is one or more selected from the group consisting of IL-6R, TGFp receptor, IL-2 receptor, TNF- ⁇ receptor or GITR .
  • the method is further for treating or preventing a disease or condition associated with a disorder of FOXP3, IL-2 and/or IFN- ⁇ activity.
  • the method is further for increasing the immunogenicity of the vaccine.
  • Figure 1 Application model of FOXP3 inhibitors known in the prior art.
  • Figure 2 FOXP3 transcriptional activity model. The effect of the inhibitor on FOXP3 transcriptional activity was examined by measuring the luciferase activity under different inhibitors using a cellular level luciferase system.
  • FIG. 3 Effect of TLR signaling and its downstream activator on FOXP3.
  • Figure 4 FOXP3 combines STUB1 with other E3.
  • Figure 4 (A): Left panel: HA-FOXP3a and Myc-STUB 1 were co-transformed into 293T cells, co-immunoprecipitated with HA and Myc antibodies, respectively, and detected by immunoblotting; Right panel: In the reverse validation assay, 293T cells were co-transfected with Flag-FOXP3 and Myc-STUB1, and co-immunoprecipitated by Flag and Myc antibodies, respectively, and detected by immunoblotting.
  • Figure 4(B) Other immune signaling pathways can be detected by co-transforming E3 with Flag-FOXP3a, HA-Ubi into 293T cells. Cell lysates were immunoprecipitated with Flag antibody and detected with Myc antibody.
  • Figure 5 STUB1 can cause FOXP3 ubiquitination.
  • Figure 5 Myc-STUB 1, HA-Ub and Flag-FOXP3a or Flag-FOXPl were transferred to 293T cells.
  • the cell lysate was immunoprecipitated with HA antibody, and the ubiquitinated protein was detected by immunoblotting;
  • Figure 6 STUB1 promotes ubiquitination degradation of FOXP3.
  • FIG. 7 STUB1 inhibits FOXP3 transcriptional activity.
  • Figure 7(D) Lentiviral-mediated transfection was used to express full-length FOXP3 protein and STUB 1 in primary CD4+ T cells to investigate whether STUB1 can attenuate the inhibitory effect of FOXP3 on IL-2 expression.
  • Figure 8 Identification of key regions of FOXP3 interacting with STUB1 (Fig. 8 ( ⁇ ), role of chaperones in interactions (Fig. 8(B), 8(C) and possible mechanisms (Fig. 8(D)) .
  • Figure 9 In vitro negative regulation of STUB 1 expression of FOXP3 protein and function of mouse primary Treg cells. Overexpression of STUB 1 reduced the expression of FOXP3 in cells (Fig. 9(A)), while increasing the expression of inflammatory factors IL-2 (Fig. 9(B)) and IFN- ⁇ (Fig. 9(C)); cell proliferation assay Results (Fig. 9(D)), curve 1 represents the addition of only CD25-, curve 2 represents GFP+CD25-, and curve 3 represents unactivated CD25-.
  • Figure 10 Experimental colitis in a mouse model Overexpression of STUB 1 in Treg cells attenuates the immunosuppressive function of Treg cells.
  • Figure 11 Western blotting experiments demonstrate that the synergistic effects of IL-6 and TGF-beta can promote the degradation of FOXP3.
  • Figure 12 The effect of TGF- ⁇ and IL-6 stimulation on IL-6 receptor expression levels was demonstrated by FACS experiments.
  • Figure 13 The effect of TGF- ⁇ and IL-6/TGF-p stimuli on FOXP3 expression levels was demonstrated by FACS experiments.
  • IP immunoprecipitation
  • IB immuno-blotting
  • FOXP3 is subject to a series of temporal and spatial regulation, including post-transcriptional modification, translation or modification, and protein interactions.
  • One of the factors includes enzymes, small molecule compounds, etc. as negative regulators of FOXP3, in different Signal stimulation can down-regulate the activity of FOXP3, thereby regulating the entire immune system.
  • ubiquitination pathway-related factors down-regulate FOXP3 activity, and these ubiquitination pathway-related factors are FOXP3 negative regulators - Toll-like Receptors, ubiquitinated ligases, pro-inflammatory cytokine family receptors and/or their coding sequences.
  • the inventors found that under the action of natural immune signals, such as different TLR signals (such as TLR3 and TLR9), the FOXP3 protein is degraded, indicating the presence of a negative regulator of FOXP3 protein. Moreover, the inventors have discovered through further research that another important post-translational modification ubiquitination also exists in the FOXP3 protein. After further research, the inventors also discovered for the first time that there is a close relationship between TLR signaling pathway and ubiquitination: TLR signaling downstream ubiquitinating linker such as TRAF6, or protein kinases such as IRAKI, and various cell pattern recognition receptors (such as IL-6, etc. can promote the ubiquitination of FOXP3 protein.
  • TLR signaling downstream ubiquitinating linker such as TRAF6, or protein kinases such as IRAKI
  • various cell pattern recognition receptors Such as IL-6, etc. can promote the ubiquitination of FOXP3 protein.
  • Ubiquitination modification generally occurs in many physiological regulation, leading to changes in the activity and function of the modified substrate protein, and ubiquitination degradation is also a major pathway in protein degradation.
  • K48-polyubiquitination leads to protein passage.
  • E3 can promote the ubiquitination of FOXP3.
  • E3, STUB 1 activated by stress signals (STIP 1 homology and U-box containing protein, STIP 1 homology and U-box containing protein l)/CHIP (C-terminus of Hsc70 interacting protein, C terminus of Hsc70 -interacting protein), which can significantly cause ubiquitination degradation of FOXP3.
  • STUB 1 is capable of binding to the FOXP3 protein mediated by a chaperone.
  • the K30A mutant STUB 1 could not bind to the chaperone protein and could not bind to FOXP3.
  • the H260 mutant STUB 1 lost E3 activity and could still bind to FOXP3, but could not promote its ubiquitination modification.
  • the inventors found in the FOXP3 transcriptional activity model that overexpression of STUB 1 significantly reduced the transcriptional activity of FOXP3. It can be seen that at the post-translational modification level, STUB 1 acts as a negative regulator of FOXP3, which promotes the ubiquitination of FOXP3 through molecular chaperones.
  • STUB 1 also enhances the expression of the inflammatory factors IL-2 and IFN- ⁇ , and reverses the inhibitory effect of FOXP3 on IL2 expression.
  • a ubiquitination pathway-associated factor an agonist or antagonist thereof, can also be used to modulate IL-2 activity and/or IFN- ⁇ activity.
  • the present inventors have provided for the first time to disclose ubiquitination pathway-related factors - ubiquitinated ligase, Toll-like receptor, pro-inflammatory cytokine family receptor and/or their coding sequences and The use of an agonist or antagonist thereof as a modulator of FOXP3 activity, IL-2 activity and/or IFN- ⁇ activity.
  • the identification and application of these regulatory factors is by regulating the activity of FOXP3, IL-2 and/or IFN- ⁇ .
  • Regulating regulatory T cells treating and/or preventing diseases or conditions associated with dysregulation of FOXP3, IL-2 and/or IFN- ⁇ activity (eg, tumors;) provides a new approach while also providing the use of these regulatory factors as vaccines
  • Adjuvant a new method to increase the immunogenicity of viral infections.
  • the inventors have completed the present invention.
  • modulating factor or "activity regulating factor” refers to a ubiquitination pathway-associated factor or an agonist or antagonist thereof that modulates FOXP3, IL-2 and/or IFN by affecting the ubiquitination pathway.
  • the activity of gamma is further used in the prevention and/or treatment of diseases or symptoms associated with dysregulation of FOXP3, IL-2 and/or IFN- ⁇ activity.
  • the term "ubiquitination pathway-associated factor” refers to a factor that reduces the activity of FOXP3 by affecting the ubiquitination pathway.
  • the ubiquitination pathway-related factors include: ubiquitination ligase, a cell pattern recognition receptor (such as a Toll-like receptor;), a pro-inflammatory cytokine family receptor, and/or their coding sequences.
  • TLR Tell-like receptor
  • I-type transmembrane receptors and pathogen pattern recognition receptors in the innate immune system, in acute inflammatory responses, cells.
  • Signal transduction and apoptosis play an important role.
  • TLR1-TLR10 Ten functional family members of TLR (TLR1-TLR10) have been identified in humans. According to the chromosomal distribution, gene structure and the conservation of its amino acid sequence, human TLRs receptors can be divided into five subgroups, S ⁇ TLR2, TLR3, TLR4, TLR5 and TLR9 subpopulations.
  • the TLR2 subpopulation includes TLR1, TLR2, TLR6 and TLR10; the TLR9 subpopulation includes TLR7 and TLR9; TLR3, TLR4 and TLR5 each form a subpopulation.
  • TLRs for use in a preferred embodiment of the invention include, but are not limited to, TLR3, TLR9, TLR1, TLR2, TLR4, TLR5, TLR7 or TLR8, preferably TLRs of TLR2, TLR3 and TLR9 subpopulations, more preferably TLR3, TLR7, TLR8 or T R9.
  • ubiquitinated ligase or "E3” are used interchangeably and refer to the third enzyme required for the binding of ubiquitin to a target protein, which selectively recognizes and binds to a specific target protein. , plays an important role in ubiquitin-mediated degradation of the target protein substrate.
  • E3 can be divided into two types: (1) HECT domain ubiquitin ligase (HECT E3s), including Smurf2, E6, AP, ARF, BPl, etc.;
  • HECT E3s HECT domain ubiquitin ligase
  • RING E3s The RING domain family of ubiquitin ligases (RING E3s), the most typical feature of the RING domain family is the ring finger domain, which is also the family
  • An important factor in the action of ubiquitin ligase, RING E3s includes a large number of members, such as: Mdm2 (Hdm2), APC, SCF, IAP, Skp2 and so on.
  • E3 employed in a preferred embodiment of the invention includes, but is not limited to: S TUB 1, TRAF6, Smurfl, Smurf2, TRAF2, TRAF3, TRAF4, p300 or RBCK1, preferably STUB 1.
  • the E3 employed in the present invention simultaneously has the function or activity of binding to a molecular partner.
  • STUB1 (STIP1 homology and U-Box containing protein 1, STIP 1 homology and U-Box domain contain proteins) has both an auxiliary chaperone function and E3 ubiquitin-protein ligase activity, and is a ubiquitin-protease that degrades abnormal proteins.
  • the molecular switch between the system and the lysosomal system can coordinate the balance and stability of the intracellular protein control system and play a key role in protein quantity control.
  • an "agonist" of a ubiquitination pathway-related factor refers to a substance capable of promoting expression of a factor involved in a ubiquitination pathway or increasing the activity of a factor involved in a ubiquitination pathway, which may also be referred to as "in the present invention”.
  • Agonist Agonists useful in the present invention include, but are not limited to, IL6.
  • an "antagonist" of a ubiquitination pathway-related factor refers to a substance capable of inhibiting the expression of a factor involved in a ubiquitination pathway or reducing the activity of a factor involved in a ubiquitination pathway, which may also be referred to as "in the present invention”.
  • Inhibitor Antagonists useful in the present invention include, but are not limited to, MG-132.
  • proteins or polypeptides of the invention may be naturally purified products, either chemically synthesized or recombinantly derived from prokaryotic or eukaryotic hosts (eg, bacteria, yeast, higher plant, insect and mammalian cells; produce.
  • prokaryotic or eukaryotic hosts eg, bacteria, yeast, higher plant, insect and mammalian cells; produce.
  • the above definition of terms of the invention also includes conservative variant polypeptides of said proteins or polypeptides, or homologous polypeptides thereof.
  • the protein or polypeptide is derived from a human or other eukaryotic organism, such as a mouse, rat, cow or monkey, and the like, and is highly conserved between them.
  • Variants of the protein or polypeptide of the invention include, but are not limited to, one or more (typically 1-50, preferably 1-30, more preferably 1-20, optimal 1-10, For example 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;) amino acid deletions, insertions and/or substitutions, and addition of one or several at the C-terminus and / or N-terminus (usually; Within 20, preferably less than 10, more preferably less than 5; amino acids.
  • the protein or polypeptide when substituted with amino acids of similar or similar properties, the protein or polypeptide is usually not changed.
  • the addition of one or several amino acids at the C-terminus and/or the N-terminus generally does not alter the function of the protein or polypeptide.
  • Those skilled in the art can readily determine these variants based on common knowledge and/or routine experimentation in the art. The way, without affecting the activity of the protein or polypeptide.
  • “conservative variant polypeptide” means compared to a known TLR or E3 amino acid sequence, Up to 20, preferably up to 10, more preferably up to 5, optimally up to 3 amino acids are formed by substitution of similar or similar amino acids, and still have the same or similar functions as the original TLR or E3 .
  • coding sequence refers to a sequence encoding a ubiquitinated ligase or Toll-like receptor of the invention, or a highly homologous sequence thereof or a molecule that hybridizes to the sequence under stringent conditions. Or a family gene molecule that is highly homologous to the above molecule.
  • stringent conditions means: (1) hybridization and elution at lower ionic strength and higher temperatures, such as 0.2 X SSC, 0.1% SDS, 60 °C ; or (2) hybridization Adding a denaturant such as 50% (v/v) formamide, 0.1% calf serum / 0.1% Ficoll, 42 ° C, etc.; or (3) at least 50% identity between the two sequences, Hybridization occurs preferably at 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, or 90% or more, and more preferably 95% or more.
  • the sequence can be the complement of the sequence defined in (a).
  • the full length sequence of the coding sequence of the present invention or a fragment thereof can usually be obtained by a PCR amplification method, a recombinant method or a synthetic method.
  • primers can be designed in accordance with the disclosed nucleotide sequences, particularly open reading frame sequences, and can be prepared using commercially available cDNA libraries or conventional methods known to those skilled in the art.
  • the library is used as a template to amplify the relevant sequences. When the sequence is long, it is often necessary to perform two or more PCR amplifications, and then the amplified fragments are spliced together in the correct order. combination
  • the modulators of the invention may also be used in the preparation of a therapeutic or prophylactic pharmaceutical composition, a nutraceutical composition or a vaccine composition.
  • the invention provides a composition
  • a composition comprising (a) a safe and effective amount of a modulator of the invention; and (b) a pharmaceutically acceptable carrier or excipient.
  • the amount of the regulatory factor of the present invention is usually from 10 ⁇ g to 100 mg / dose, preferably from 100 to 1000 ⁇ g / dose.
  • an effective amount refers to an amount of a therapeutic agent that treats, alleviates or prevents a target disease or condition, or an amount that exhibits a detectable therapeutic or prophylactic effect.
  • the precise and effective amount for a subject depends 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 condition, routine experimentation can be used to determine the effective amount that the clinician can judge.
  • the pharmaceutical composition may also contain a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable carrier refers to a carrier for the administration of a therapeutic agent.
  • pharmaceutical carriers which do not themselves induce the production of antibodies harmful to the individual receiving the composition and which are not excessively toxic after administration. These carriers are used by It is well known to those of ordinary skill in the art. A full discussion of pharmaceutically acceptable excipients can be found in Remington's Pharmaceutical Sciences (Mack Pub. Co., NJ 1991).
  • Such carriers include, but are not limited to:: saline, buffer, dextrose, water, glycerol, ethanol, adjuvants, and combinations thereof.
  • the pharmaceutically acceptable carrier in the therapeutic composition may contain a liquid such as water, saline, glycerol and ethanol.
  • auxiliary substances such as wetting or emulsifying agents, pH buffering substances and the like may also be present in these carriers.
  • the immunological composition may also contain an immunological adjuvant.
  • the therapeutic compositions may be in the form of injectables, such as liquid solutions or suspensions; solid forms such as liquid carriers, which may be formulated in solution or suspension prior to injection.
  • composition of the invention can be administered directly to the subject.
  • the subject to be prevented or treated may be an animal; especially a human.
  • the therapeutic or prophylactic pharmaceutical composition (including vaccine) comprising the regulatory factor of the present invention can be administered orally, subcutaneously, intradermally, intravenously or the like.
  • the therapeutic dosage regimen can be a single dose regimen or a multiple dose regimen.
  • ubiquitination pathway-related factor- ubiquitinated ligase, Toll-like receptor, pro-inflammatory cytokine family receptor and/or their coding sequences are disclosed as FOXP3 activity and IL-2 activity for the first time in the present invention. And/or the use of an IFN- ⁇ activity modulator;
  • a regulatory factor of the present invention which regulates regulatory T cells by regulating the activity of FOXP3, IL-2 and/or IFN- ⁇ , treating and/or preventing with FOXP3, IL-2 and/or IFN - diseases or symptoms associated with Y activity disorders (eg tumors, etc.) provide new pathways;
  • the present invention also provides a novel method for using the regulatory factor of the present invention as a vaccine adjuvant to improve the immunogenicity of a vaccine.
  • the nuclear expression vector; pET28a and pET21-MBP are prokaryotic expression vectors
  • the lentiviral packaging vector plasmids (; FUGW, del8.9 and VSV-G) were constructed by the inventors' laboratory clones (see Reference 4).
  • the El, UbcH5b and HSP70 genes were amplified from human peripheral blood mononuclear cDNA libraries.
  • the primers were designed according to the sequence downloaded by Genbank as follows:
  • TLR signal downstream kinase IRAKI expression plasmid pIPIRAKl was constructed for this laboratory (see Reference 4, IRAKI full gene sequence reference Entrez Gene: 3654); ubiquitinated ligase TRAF6 was provided by Professor Yongwon Choi (see Reference 7, or It can be obtained by commercial (such as Origin), or by conventional means of cloning;).
  • HA antibody F-7
  • Myc antibody 9E10
  • Flag antibody M2
  • F0XP3 antibody hFOXY
  • STUB1 antibody C3B6
  • HRP-conjugated anti-mouse secondary antibody was purchased from Promega.
  • Human HEK293T (purchased from the Chinese Academy of Sciences Cell Bank (Catalog No.: GNHul7)) contains 10% FBS, 100 units /ml of streptomycin in DMEM (Dulbecco, s modified Eagle's medium), cultured at 37 ° C, 5% CO 2 .
  • DMEM Dulbecco, s modified Eagle's medium
  • Jurkat E6. 1 T cells purchased from the Chinese Academy of Sciences Cell Bank (Catalog No.: TCHU123) containing 10% FBS, 100 units/ml chelnanmycin, 100 units/ml non-essential amino acids and 100 units/ml sodium pyruvate The culture was carried out in 1640 medium, 37 ° C, 5% CO 2 .
  • FUGW-TAP-FOXP3, del 8.9 and VSV-G were co-transfected into human embryonic kidney cell line HEK293.
  • the cell culture supernatant was collected 48 and 72 hours after transfection.
  • the virus was purified by ultracentrifugation and infected with Jurkat E6.
  • FOXP3 Jurkat stably expresses cell lines.
  • TLR ligand reagents were purchased from Apotech.
  • MG132 was purchased from Merck.
  • Protein AG-beads was purchased from Shanghai Yueke Biotechnology Co., Ltd.
  • the cells were cytoplasmic extract (10 mM Hepes, pH 7.9, 10 mM KCl, 0.1 mM EDTA, 1 mM DTT, 0.5 mM PMSF, ⁇ complete protease inhibitor mixed test! J (complete protease inhibitor cocktails, Cat. No. 1-697-498; Roche Biochem), 1 mM Na 3 VO 4 ) , placed on ice for 15 minutes, then added to a final concentration of 0.6% NP-40, vortexed (Votex O sec., centrifuged at 12000g for 30 seconds). The supernatant obtained is the cytoplasmic fraction, and the precipitate is the nuclear component.
  • cytoplasmic extract (10 mM Hepes, pH 7.9, 10 mM KCl, 0.1 mM EDTA, 1 mM DTT, 0.5 mM PMSF, ⁇ complete protease inhibitor mixed test! J (complete protease inhibitor cocktails, Cat. No. 1-697-498; Roche Biochem
  • the nuclear fraction is used as a nuclear suspension (20 mM Hepes pH 7.9, 400 mM NaCl, 1 mM EDTA, 1 mM DTT, 1 mM PMSF, chymotrypsin inhibitor).
  • the mixed reagent, 1 mM Na 3 VO 4 was dissolved, gently spun at 4 ° C for 30 minutes, and then centrifuged at 16,060 g, 4 ° C for 15 minutes. The resulting insoluble precipitate was a chromatin subcomponent.
  • Cells with RIPA buffer (20 mM Tris/HCL pH 7.5, 150 mM NaCl, 1% NP-40, 0.5% Na-DOC, 1 mM EDTA and protease inhibitor I mM PMSF, IX Cocktail, phosphatase inhibitor 1 mM Na 3 VO 4 , ImM NaF) cleavage.
  • the cells were incubated with the primary antibody for 1 hour in the cell lysate, and then agarose beads conjugated with Protein A/G were added for 1 hour. After three washes with RIPA buffer, the bound proteins were detected by SDS-PAGE.
  • the protein sample was electrophoresed by SDS-PAGE protein, transferred to a nitrocellulose membrane, blocked with 5% skim milk powder TBST for one hour, added with primary antibody for one hour, and then added with HRP-conjugated secondary antibody. Hours, exposure to color with ECL substrate.
  • the 8 X FK luciferase reporter gene contains 8 repeat forkhead (Forkhead) binding regions in the enhancer region, and the 5 X Gal luciferase reporter gene contains 5 repeat Gal4 expression cassettes in the enhancer region.
  • pBind-FOXP3 expresses the Gal4 binding region fused to FOXP3.
  • the corresponding plasmid was transferred into 293T cells, and luciferase activity and ⁇ -galactosidase activity were measured using a luciferase substrate and a ⁇ -galactosidase substrate, respectively. Each experiment was repeated three times or more independently, and the results were expressed as mean and standard deviation.
  • the enzyme substrate used was purchased from Biyuntian Company.
  • the STUB 1 mutant K30A, H260Q was obtained by mutation PCR (see Reference 5).
  • the site-directed mutagenesis kit was purchased from Stratagene, and verified by DNA sequencing after PCR.
  • the prokaryotic expression vector carrying 6His-El, 6His-UbcH5b, 6His-STUB1, 6His-HSP70 and 6His-FLAG-Ubiqutin was transformed into Rosetta/PlysS competent cells, induced by IPTG, harvested, lysed, and purified by nickel column (Qiagen) Company;), finally purified protein obtained by fast protein liquid chromatography (GE).
  • MBP-FOXP3 and MBP proteins were expressed as described above, harvested, lysed, purified by amylose resin (NEB), dialyzed with standard buffer (25 mM Tris-HCl (pH 7.5), 150 mM N-chloride). Sodium, ⁇ ⁇ ⁇ -mercaptoethanol and 10% glycerol). Finally, all protein concentrations were determined by the Brandford method (Shanghai Biyuntian Biotechnology Co., Ltd.;).
  • the cells were fixed in 4% formaldehyde, 0.5% Triton-X 100 was transfected, blocked and labeled with the corresponding antibodies. DAPI Label the nucleus. Finally, the cells were observed under a confocal microscope (LEICA SP5).
  • mice purchased from Jackson Laboratories, USA
  • Thyl. l mice purchased from the United States
  • regulatory T cells and CD4 + CD25_CD62L hlgh effector T cells were obtained by antibody labeling and magnetic bead sorting.
  • regulatory T cells and effector T cells were cultured for 80 hours after labeling effector T cells with ⁇ CFSE (Invitrogen), and the proliferation of effector T cells was detected by flow cytometry to analyze the proliferation status. .
  • Lymphocytes were isolated from BALB/c mice (purchased from Jackson Laboratory, USA), and natural CD4+CD25-CD62L hlgh effector T cells were obtained by antibody labeling and magnetic bead sorting, and injected into BALB/c via tail vein.
  • RAG2-/- immunodeficient mice purchased from Taconic, USA
  • the experimental mice were divided into two groups, and wild type CD4 + CD25 + Treg cells and STUB 1 -transformed Treg cells (2x10 5 ) were injected through the tail vein, respectively.
  • Weekly observations were used to assess symptoms, and once the symptoms were severe or weight loss was 20%, the experimental mice were sacrificed humanely.
  • TLR signals (such as TLR3, TLR9) can be used as a negative adjustment signal for FOXP3.
  • Test 2 Effect of TLR signal downstream activation protein TRAF 6 X-inch FOXP 3 ubiquitination
  • TLR signal downstream activation protein TRAF6 expression plasmid and FOXP3 expression plasmid Myc-FOXP3a were co-transformed into 293T cells. After 48 hours, the cells were harvested, immunoprecipitated, and then detected by protein electrophoresis and immunoblotting.
  • TLR signals (such as TLR3, TLR9) can pass its downstream ubiquitinating linker
  • TRAF6 promotes ubiquitination of FOXP3.
  • Test 3 Effect of TLR signal downstream activation protein IRAKI on FOXP3 ubiquitination
  • TLR signal downstream kinase expression plasmids IRAKI and FOXP3 expression plasmid Myc-FOXP3a were co-transfected into 293T cells. After 48 hours, the cells were harvested, immunoprecipitated, and then detected by protein electrophoresis and immunoblotting.
  • TLR signals (such as TLR3, TLR9) can be promoted by its downstream kinase IRAKI
  • HA-FOXP3 a and Myc-STUB 1 were co-transfected into 293 T cells, and co-immunoprecipitated with HA and Myc antibodies, respectively, and detected by immunoblotting (results shown in the left panel of Figure 4 (A);).
  • 293T cells were co-transfected with Flag-FOXP3 and Myc-STUB1, co-immunoprecipitated with Flag and Myc antibodies, respectively, and detected by immunoblotting (results shown in Figure 4(A) right ;).
  • FOXP3 can be co-immunoprecipitated with STUB1, and STUB1 can also specifically pull down FOXP3, suggesting that the interaction between STUB1 and FOXP3 is specific and exogenous tag expression is non-dependent.
  • ubiquitin ligase which plays a key role in other signaling pathways, can also interact with FOXP3.
  • the FOXP3 protein can interact with ubiquitin ligase and be modified by ubiquitination.
  • the FOXP3 protein is capable of interacting with STUB1 ubiquitin ligase regulated by stress signaling.
  • STUB 1 can lead to ubiquitination of FOXP3
  • Myc-STUB 1, HA-Ub and Flag-FOXP3a or Flag-FOXPl were co-transferred into 293T cells.
  • the cell lysate was immunoprecipitated with an HA antibody, and the ubiquitinated protein was detected by immunoblotting (the results are shown in Fig. 5(A)).
  • Myc-STUB1, HA-Ub and Flag-FOXP3a were co-transferred into Jurkat T cells, and the cell lysate was immunoprecipitated with HA antibody and detected by immunoblotting (results shown in Fig. 5(B);).
  • Flag-FOXP3, HA-Ub and Myc-STUB 1 were co-transferred into 293T cells.
  • the cells were treated with 5 ⁇ M MG132 for different times before the cells were taken.
  • the lysate was immunoprecipitated with Myc antibody and detected by immunoblotting (the results are shown in Figure 6 (A));
  • the STUB1-H260Q mutant was co-transformed into 293T cells.
  • the cells were treated with 5 ⁇ M MG132 for 4 hours, immunoprecipitated with sputum antibody and detected by immunoblotting (the results are shown in Fig. 6(B);).
  • STUB1 negatively regulates the protein level of FOXP3 by causing ubiquitination degradation of FOXP3.
  • STUB 1 inhibits FOXP3 transcriptional activity
  • the FOXP3 activity was detected by the 8 X FK luciferase reporter system, and the FOXP3, STUB1, 8 X FK luciferase reporter gene and MSV-P-Gal control were co-transfected into 293T cells, and luciferase activity was measured with ⁇ -Gal. After the activity was normalized, the results of three independent experiments were expressed as the mean ⁇ standard deviation (results shown in Figure 7 (A) Shown ;).
  • the pBind system (construction 4) was used to construct pBind-FOXP3, STUB 1 and pGal5-luciferase reporter gene and MSV-P-Gal control into 293T cells, and luciferase activity was normalized by ⁇ -Gal activity. Thereafter, the results of three independent experiments were expressed as mean ⁇ standard deviation (results are shown in Fig. 7(B);).
  • FOXP3 acts as a transcriptional repressor and can reduce the transcriptional activity of the reporter gene.
  • STUB 1 acts as a negative regulator of FOXP3 and has a function of inhibiting FOXP3.
  • the inventors further examined the transcriptional repression of overexpression of luciferase reporter gene expression levels of HSP70 and STUB1 on the FOXP3-mediated IL-2 promoter drive.
  • the corresponding plasmid was transfected into Jurkat T cells, and the luciferase activity expressed by the cells was analyzed by transfecting the cells with PMA and ionomycin before receiving the samples.
  • FOXP3 significantly inhibited the IL-2 promoter-driven luciferase expression level, and HSP70 co-expression had no effect on this inhibition.
  • the inventors also identified a key region of FOXP3 that interacts with STUB1. As shown in Fig. 8(A), the zinc finger and leucine zipper domain of FOXP3 proved to be an essential region for binding to STUB1.
  • HA-FOXP3 binds to the wild-type STUB 1 of the Myc tag, or the chaperone lacks the STUB 1-K30A mutant, or ubiquitin ligase activity.
  • 293T cells were co-transfected with the deleted STUB1-H260Q mutant, and then STUB1 was co-immunoprecipitated with Myc antibody. Next, HA antibody and Myc antibody were used for immunoblot detection.
  • Fig. 10(A) the degree of development of colitis was determined by monitoring the body weight of mice and immunohistochemical analysis.
  • Fig. 10(A) simultaneous transplantation of Teff (utilitative T cells) and STUB 1 overexpression was compared with the control group.
  • the body weight of Treg cells was significantly decreased, and the degree of inflammation in the immunohistochemical sections was significantly increased, which could not effectively inhibit the development of inflammation (Fig. 10(B) and 10(C)).
  • CD4+ T cells can differentiate into four different subtypes under different cytokines and environmental factors: TH1, TH2, TH17 and iTreg. These four T cell subsets have specific cytokines and transcription factors. Previous studies have found that CD4+ T cells differentiate into TH17 under the action of TGF- ⁇ and inflammatory factor IL-6. However, under the action of TGF- ⁇ , CD4+ T cells can differentiate into iTreg. The promotion of TGF- ⁇ in TH17 differentiation and the inhibitory effect of IL-6 in iTreg differentiation are unclear.
  • the cell lines used in Western blotting and FACS experiments were stabilized Jurkat-HA-FOXP3 (as described in Materials and Methods); the amount of IL-6 added was 20 ng/ml; the amount of TGF- ⁇ added was 10 ng/ml.
  • the IL-6I3 ⁇ 4t body (purchased from Santa Cruz) and the Cy3-labeled anti-Rabbit secondary antibody (purchased from Jackson) were stained and detected by flow cytometry PE channel. .
  • the results of Western blotting are shown in Figure 11, and the results of the F AC S experiment are shown in Figures 12 and 13.
  • TGF- ⁇ promoted the expression of IL-6 receptor, but IL-6 had no effect on the expression of its own receptor. It has been reported that TGF- ⁇ can inhibit the negative feedback factor SOCS3 of TH17, and thus can interact with TH17 specific cytokines such as IL-6 and IL-21 to promote the differentiation of TH17. Therefore, TGF- ⁇ promotes the differentiation of TH17 through various mechanisms, such as: promoting the degradation of FOXP3 and up-regulating the IL-6 receptor in the presence of IL-6.
  • CD4+CD25+ regulatory T cells control Leishmania major persistence and immunity (CD4+CD25+ regulatory T cells to Gyula Manetia Survival and Immunity Control). Nature 420, 502-507.
  • composition and method for modulation of suppressor T cell activation composition and method for modulating inhibitory T cell activity.
  • TRAF6 is a critical factor for dendritic cell maturation and development ( TRAF6 is a key factor regulating the maturation and development of dendritic cells). Immunity. 2003 Sep; 19(3): 353-63.

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Abstract

本发明公开了泛素化途径相关因子、其激动剂或拮抗剂在制备调节FOXP3、IL-2和/或IFN-γ活性的组合中的用途，其中泛素化途径相关因子选自：Toll样受体、泛素化连接酶、促炎症细胞因子家族受体和/或其编码序列。本发明调节FOXP3、IL-2和/或活性的组合物还可用作疫苗佐剂或疫苗。

Description

FOXP3及调节性 T细胞的调节因子及其应用 技术领域
本发明涉及分子生物和生物医药领域。 更具体而言， 本发明涉及泛素化途径 相关因子、 其激动剂或拮抗剂在调节 FOXP3活性、 IL-2活性和 /或 IFN-γ活性中的用 途。 本发明尤其涉及调节 FOXP3活性的负调节因子、 及其通过下调 FOXP3活性对 免疫系统进行调节， 从而治疗或预防与 FOXP3活性过高相关的疾病或症状的用途 以及作为免疫佐剂的用途。 背景技术
Fox转录因子家族是一个以具有叉头螺旋结构 (forkhead/winged helix, FKH) 为特征， 功能各异的转录因子大家族。 这个家族成员在细胞发育的各个进程中 起着不同的作用 (参考文献 1和 2)。
FOXP3是调节性 T细胞 (Treg， regulatory T cells)特异性转录因子， 对 Treg 的发育和功能起着重要的调节作用， 具有下调免疫应答的功能。 FOXP3介导的 Treg的免疫调节功能是通过 FOXP3与若干转录共调节蛋白 (例如转录因子、共抑 制因子、共激活因子、 组蛋白以及染色质重建因子)形成蛋白复合体以动态调控 基因的特异性转录 (参考文献 3和 4)。 在不同剌激条件下， FOXP3蛋白复合体的 不同状态及其转录活性对 Treg及免疫系统调节都有不同的效果 (参考文献 3和 4)。
FOXP3+调节性 T细胞 (FOXP3+Tregs)属于 T淋巴细胞中表达 CD4、 CD25及 转录因子 FOXP3的一类 T细胞亚型， 其正常功能对于人体免疫稳态的动态调节 必不可少。 调节性 T细胞的发育及功能失调， 与多种重大免疫相关性疾病， 包 括自身免疫性疾病、 炎症反应、 急性及慢性传染性疾病、 肿瘤免疫耐受、 移植 排斥以及过敏性疾病的生理病变进程密切相关 (；参考文献 1和参考文献 2)。
尽管本研究领域近年来进展很快， 但许多重大及根本性的问题仍然尚待回 答， 例如在细胞水平上调节性 T细胞如何调节其它免疫细胞活性， 以及在分子 水平上 FOXP3采用何种机制使得调节性 T细胞获得免疫抑制活性。
不断积累的实验证据显示， FOXP3基因表达的水平及持续状态对于天然调 节性 T细胞的发育成熟及功能至关重要。 此外， FOXP3结合多重转录因子以及 具有酶学活性的组蛋白乙酰转移酶 /脱乙酰基酶复合体， 这对于抑制 T细胞中促 炎症类细胞因子的转录激活是必需的。 并且， FOXP3蛋白翻译后修饰、 转录复 合体装配及其修饰酶类的活性受到 T细胞受体及炎症细胞因子受体信号的动态 调节。 先前的研究发现 FOXP3是一个赖氨酸乙酰化蛋白， 并且 FOXP3富含脯氨 酸的 N端可以直接招募组氨酸乙酰转移酶 TIP60(Tat interaction protein, 60 kDa)， 从而介导 FOXP3的转录抑制活性 (；参见参考文献 6以及图 1)。
IL-2是一种淋巴细胞因子， 通常由凝集素或抗原激活的 T淋巴细胞产生。 可使 细胞毒性 T细胞， 自然杀伤细胞和淋巴因子活化的杀伤细胞增殖， 并使其杀伤活性 增强， 还可以促进淋巴细胞分泌抗体和干扰素， 具有抗病毒、抗肿瘤和增强机体免 疫功能等作用。 人重组 IL-2在肿瘤、 炎症反应、 急性传染性疾病或慢性传染性疾病 中已有相应运用。
INF- Y是水溶性二聚体的细胞因子， 通常由自然杀伤细胞、 自然杀伤 T细胞及 细胞毒性 T细胞产生。 INF- γ是固有及适应性免疫系统中抗病原体及细菌感染的重 要细胞因子，能直接抑制病毒体内复制，具有抗病毒、免疫调节及抗肿瘤特性。 INF- Y结合激活其受体调节 JAK-STAT通路， 激活抗原提呈细胞， 通过上调转录因子 T-bet而促进 I型辅助 T细胞 (Thl细胞)的分化等功能； 能诱导抗病毒蛋白合成， 治疗 性病等病毒性疾病； 能降低肝内 I、 III型胶原 mRNA水平， 阻止和减慢肝纤维化的 发生， 表明对慢活肝及肝硬化有效。
因此， 深入研究调节性 T细胞及转录因子 FOXP3、 以及 IL-2和 IFN-γ的生化 活性、 生理功能及其调控的分子机理， 对于治疗性控制人体内调节性 T细胞的 免疫活性以及基于调节性 T细胞的免疫治疗皆至关重要。 这为将基础免疫研究 转化为临床研究， 以及理解人类特有的重大传染性疾病提供了新的研究命题与 挑战。 发明内容
本发明的主要目的之一是提供泛素化途径相关因子及其激动剂或拮抗剂在 调节 FOXP3活性、 IL-2活性和 /或 IFN-γ活性中的用途。 本发明的另一目的在于提 供这些调节因子在调节 FOXP3活性、 IL-2活性和 /或 IFN-γ活性、 从而调控免疫系 统中的用途。 本发明的另一目的还在于提供一种采用本发明所述调节因子治疗 调节性 T细胞相关疾病的方法及应用。
在本发明的第一方面中， 提供了泛素化途径相关因子、其激动剂或拮抗剂在 制备调节 FOXP3活性、 IL-2活性和 /或 IFN- Y活性的组合物中的用途， 其中所述泛 素化途径相关因子选自： Toll样受体、 泛素化连接酶、 促炎症细胞因子家族受体和 /或它们的编码序列。
在本发明的一个实施方式中， 对 FOXP3活性、 IL-2活性和 /或 IFN- γ活性的所 述调节是正调节或负调节，其中，所述泛素化途径相关因子或其激动剂用于负调节 FOXP3活性和正调节 IL-2活性和 /或 IFN-γ活性， 所述泛素化途径相关因子的拮抗剂 用于正调节 FOXP3活性和负调节 IL-2活性和 /或 IFN-γ活性。
在一个优选例中， 所述激动剂为 IL-6。
在另一个优选例中， 所述拮抗剂为 MG-132。
在本发明的另一个实施方式中， 所述促炎症细胞因子家族受体选自： IL-6R、 TGFp受体、 IL-2受体、 TNF- α受体或 GITR。
在本发明的一个优选实施方式中， 所述用途是泛素化途径相关因子在制备负 调节 FOXP3活性的组合物中的用途， 其中所述泛素化途径相关因子选自： Toll样受 体、 泛素化连接酶和 /或它们的编码序列。
在本发明的另一个实施方式中， 所述泛素化连接酶是选自下组中的一种或多 种： STUB TRAF6、 Smurf Smurf2、 TRAF2、 TRAF3、 TRAF4、 p300、 RNF31 或 RBCK1。
在一个优选例中， 所述泛素化连接酶优选为 STUB1。
在另一个优选例中， 所述泛素化连接酶是复合体， 其还兼具分子伴侣活性。 在另一个优选例中， 所述泛素链接酶为 Toll样受体信号通路激活下游蛋白， 优 选 TRAF6。
在本发明的另一个实施方式中， 所述 Toll样受体是选自下组中的一种或多种： TLR TLR2, TLR3, TLR4, TLR5, TLR7, TLR8或 TLR9。
在一个优选例中， 所述 Toll样受体选自： TLR3、 TLR7、 TLR8或 TLR9。
在另一个优选例中， 所述 Toll样受体信号通路下游激酶 IRAKI可促进 FOXP3 泛素化。
在本发明的另一个实施方式中， 所述调节 FOXP3活性、 IL-2活性和 /或 IFN-γ活 性的组合物用于治疗和 /或预防与 FOXP3、 IL-2和 /或 IFN-γ活性失调相关的疾病或症 状。
在本发明的一个优选例中， 所述活性失调是活性过高或活性过低。
在本发明的另一个实施方式中， 所述与 FOXP3、 IL-2和 /或 IFN-γ活性失调相关 的疾病或症状选自： 肿瘤、 炎症反应、 急性传染性疾病或慢性传染性疾病。 在本发明的另一个实施方式中， 所述肿瘤选自： 前列腺癌、 乳腺癌、 肝癌、 胶质瘤、 肠癌、 子宫颈癌、 非小细胞肺癌、 肺癌、 胰腺癌、 胃癌、 膀胱癌、 皮 肤癌、 横纹肌癌、 舌鳞癌、 鼻咽癌、 卵巢癌、 胎盘绒毛癌、 神经胶质瘤、 淋巴 瘤、 白血病、 直肠腺癌或黑色素瘤。
在本发明的另一个实施方式中， 所述炎症反应选自： 过敏性炎症、 毛囊炎、 扁桃体炎、 肺炎、 肝炎、 肾炎、 痤疮、 哮喘、 自身免疫性疾病、 慢性炎症、 慢性前 列腺炎、 肾小球肾炎、 超敏反应、 炎性肠道疾病、 盆腔炎、 再灌注损伤、 类风湿关 节炎、 移植排斥反应、 血管炎或间质性膀胱炎。
在本发明的另一个实施方式中， 所述传染性疾病选自： 鼠疫、 霍乱、 传染性 非典型肺炎、 艾滋病、 病毒性肝炎、 脊髓灰质炎、 人感染高致病性禽流感、 麻疹、 流行性出血热、 狂犬病、 流行性乙型脑炎、 手足口病、 登革热、 炭疸、 细菌性和阿 米巴性痢疾、 肺结核、 伤寒和副伤寒、 流行性脑脊髓膜炎、 百日咳、 白喉、 新生儿 破伤风、 猩红热、 布鲁氏菌病、 淋病、 梅毒、 钩端螺旋体病、 血吸虫病、 疟疾、 流 行性感冒、 流行性腮腺炎、 风疹、 急性出血性结膜炎、 麻风病、 流行性和地方性斑 疹伤寒、 黑热病、 包虫病、 丝虫病， 除霍乱、 细菌性和阿米巴性痢疾、 伤寒和副伤 寒以外的感染性腹泻病、 或真菌感染。
在本发明的另一个实施方式中， 所述调节 FOXP3、 IL-2活性和 /或 IFN-γ活性的 组合物进一步用作疫苗佐剂或疫苗。
在本发明的另一个实施方式中， 所述疫苗选自： 治疗或预防病毒感染、 细菌 感染、 炎症、 寄生虫感染或肿瘤的疫苗， 例如手足口病疫苗。
在一个优选例中， 所述疫苗选自： 破伤风疫苗、 白喉、 破伤风、 百日咳疫苗、 流感疫苗、 黄热病疫苗、 脊髓灰质炎疫苗、 结核疫苗、 麻疹疫苗、 风疹疫苗、 甲型 脑膜炎疫苗、 狂犬病疫苗、 乙肝疫苗、 麻疹、 腮腺炎、 风疹三联疫苗、 b型流感嗜 血杆菌疫苗、 伤寒疫苗、 甲肝疫苗、 宫颈癌疫苗、 肺炎疫苗、 水痘疫苗、 流脑疫 苗、 乙脑疫苗、 痢疾疫苗、 霍乱疫苗、 艾滋病疫苗、 丙肝疫苗， 戊肝疫苗、 手 足口病疫苗、 支气管炎疫苗、 A群流脑疫苗或人禽流行性感冒疫苗。
在另一个优选例中， 所述疫苗是 DNA疫苗、 重组载体疫苗、 灭活或减毒的基 于病原体的疫苗、 亚单位疫苗或癌细胞疫苗。
在本发明的另一个实施方式中， 所述组合物是药物组合物、 保健品组合物或 疫苗组合物。
在一个优选例中， 所述组合物中调节因子的含量为 0.05-99.5重量％， 优选 0.1-95重量％， 更优选 1-90重量％， 更优选 5-80重量％。
在另一个优选例中， 所述组合物中还包含泛素化所需的分子伴侣， 所述分子 伴侣优选为： HSP70、 HSC70或 HSP90。
在另一个优选例中， 所述组合物还包含药学上、 保健品学上或免疫学上可接 受的载体。
在另一优选例中， 所述组合物是注射剂、 片剂、 颗粒剂、 粉剂或胶囊。 在本发明的第二方面中， 提供了一种调节 (如负调节) FOXP3活性、 IL-2活性和 /或 IFN-γ活性的组合物， 其包含：
(a)—种或多种选自下组的泛素化途径相关因子： 泛素化连接酶、 Toll样受体、 促炎症细胞因子家族受体和 /或它们的编码序列； 和 /或所述泛素化途径相关因子的 激动剂或拮抗剂；
(b)药学上、 免疫学上或保健品学上可接受的载体。
在一个优选例中， 所述组合物用于治疗或预防与 FOXP3、 IL-2和 /或 IFN-γ活性 失调 (即活性过高或活性过低;)相关的疾病或症状或用作疫苗佐剂。
在另一个优选例中， 所述组合物进一步用于制备疫苗组合物， 所述疫苗组合 物中还包含具有免疫原性的活性物质。
在一个优选例中， 所述组合物还包含一种或多种可调节 FOXP3、 IL-2和 /或 IFN-γ的其它活性物质， 优选 TIP60。
在本发明的一个实施方式中， 所述泛素化连接酶是选自下组中的一种或多种： STUB TRAF6、 Smurf Smurf2、 TRAF2、 TRAF3、 TRAF4、 p300、 RNF31或 RBCKl o
在一个优选例中， 所述泛素化连接酶优选为 STUB 1。
在另一个优选例中， 所述泛素化连接酶是复合体， 其还兼具分子伴侣活性。 在另一个优选例中， 所述泛素链接酶为 Toll样受体信号通路激活下游蛋白， 优 选 TRAF6。
在本发明的另一个实施方式中， 所述 Toll样受体是选自下组中的一种或多种： TLR1、 TLR2、 TLR3、 TLR4、 TLR5、 TLR7、 TLR8或 TLR9。
在一个优选例中， 所述 Toll样受体选自： TLR3或 TLR9。
在另一个优选例中， 所述 Toll样受体信号通路下游激酶 IRAKI可促进 FOXP3 泛素化。 在本发明的另一个实施方式中， 所述调节 FOXP3活性、 IL-2活性和 /或 IFN-γ活 性的组合物用于治疗和 /或预防与 FOXP3、 IL-2和 /或 IFN-γ活性失调相关的疾病或症 状。
在本发明的另一个实施方式中， 所述与活性失调相关的疾病或症状选自： 肿 瘤、 炎症反应、 急性传染性疾病或慢性传染性疾病。 所述肿瘤、 炎症反应、 急性传 染性疾病或慢性传染性疾病可如上文所述。
在另一个优选例中， 所述疫苗是 DNA疫苗、 重组载体疫苗、 灭活或减毒的基 于病原体的疫苗、 亚单位疫苗或癌细胞疫苗。
在本发明的另一个实施方式中， 所述组合物是药物组合物、 保健品组合物或 疫苗组合物。 在本发明的其它方面中， 提供了一种调节 FOXP3活性、 IL-2活性和 /或 IFN-γ活 性的方法， 所述方法包括给予选自下组的一种或多种调节因子或其激动剂或拮抗 剂： 泛素化连接酶、 Toll样受体、 促炎症细胞因子家族受体和 /或它们的编码序列。
在一个优选例中， 所述泛素化连接酶是选自下组中的一种或多种： STUB1、 TRAF6、 Smurf Smurf2、 TRAF2、 TRAF3、 TRAF4、 p300、 RNF31或 RBCK1， 优选 STUB1。 在另一个优选例中， 所述 Toll样受体是选自下组中的一种或多种： TLRU TLR2, TLR3 , TLR9, TLR4, TLR5, TLR7或 TLR8, 优选 TLR3或 TLR9。 在另一个优选例中， 所述促炎症细胞因子家族受体是选自下组中的一种或多种： IL-6R、 TGFp受体、 IL-2 受体、 TNF- α受体或 GITR。
在另一优选例中， 所述方法进一步用于治疗或预防与 FOXP3、 IL-2和 /或 IFN-γ 活性失调相关的疾病或症状。
在另一优选例中， 所述方法进一步用于提高疫苗的免疫原性。
本发明的其它方面由于本文的公开内容， 对本领域的技术人员而言是显而 易见的。 附图说明
图 1: 现有技术中已知的 FOXP3抑制剂的应用模型。
图 1 ( A)： FOXP3抑制剂能够抑制 FOXP3功能复合体的蛋白 (；如 Tip60)的结 合， 从而抑制 Treg的功能；
图 1(B): FOXP3抑制剂能够作为疫苗佐剂， 增强疫苗的免疫原性。 图 2: FOXP3转录活性模型。 运用细胞水平萤光酶系统， 通过检测不同抑 制剂下的萤光酶活性来反映抑制剂对 FOXP3转录活性的影响。
图 2(A): 采用 8XFK萤光素酶报告系统检测 FOXP3转录活性的模型； 图 2(B): 采用 5XGal萤光素酶报告系统检测 FOXP3转录活性的模型。
图 3: TLR信号及其下游激活蛋白对 FOXP3的影响。
图 3(A): TLR信号对 FOXP3表达量的影响。
图 3(B): TLR信号下游激活蛋白 TRAF6对 FOXP3泛素化的影响。
图 3(C): TLR信号下游激活蛋白 IRAKI对 FOXP3泛素化的影响。
图 4: FOXP3结合 STUB1和其它 E3。
图 4(A): 左图： HA-FOXP3a和 Myc-STUB 1共转入 293T细胞， 分别用 HA和 Myc抗体双向免疫共沉淀， 并用免疫印迹法进行检测； 右图： 反向验证试验中， 用Flag-FOXP3与Myc-STUBl共转染293T细胞， 分别用 Flag和 Myc抗体双向免疫 共沉淀， 并用免疫印迹法进行检测。
图 4(B): 其它免疫信号通路可通过将 E3与 Flag-FOXP3a， HA-Ubi共转入 293T细胞进行检测。 细胞裂解液用 Flag抗体免疫沉淀， 用 Myc抗体检测。
图 5: STUB1能够导致 FOXP3泛素化。
图 5(A): Myc-STUB 1、 HA-Ub与 Flag-FOXP3a或 Flag-FOXPl共转入 293T细 胞。 细胞裂解液用 HA抗体免疫沉淀， 通过免疫印迹检测泛素化的蛋白；
图 5(B): Myc-STUB 1, HA-Ub和 Flag-FOXP3a共转入 Jurkat T细胞， 细胞裂 解液用 HA抗体免疫沉淀并用免疫印迹检测。
图 6: STUB1促进 FOXP3泛素化降解。
图 6(A): Flag-FOXP3a， HA-Ub和 Myc-STUB 1共转入 293T细胞。 细胞收样 前， 用 5 MMG132处理不同时间。 裂解液用 Myc抗体免疫沉淀并用免疫印迹检 图 6(B): Flag-FOXP3a, HA-Ub和 Myc-STUBl及其突变体共转入 293T细胞。 细胞用 5 MMG132处理 4小时后， 用 ΗΑ抗体免疫沉淀并用免疫印迹检测。
图 7: STUB1抑制 FOXP3转录活性。
图 7(A): 用 8XFK萤光素酶报告系统检测 FOXP3活性， 将 FOXP3、 STUB1、 8XFK萤光素酶报告基因和 MSV-P-Gal对照共转入 293T细胞， 萤光素酶活性用 β-Gal活性标准化后， 取三次独立实验的结果用平均值士标准偏差表示；
图 7(B): 用 pBind系统， 将 pBind-FOXP3、 STUB1和 pGal5-萤光素酶报告基 因和 MSV-P-Gal对照共转入 293T细胞， 萤光素酶活性用 β-Gal活性标准化后， 取 三次独立实验的结果用平均值士标准偏差表示。
图 7(C): 过表达 HSP70及 STUB1对 FOXP3介导的 IL-2启动子驱动的萤光素 酶报告基因表达水平的转录抑制作用。
图 7(D): 采用慢病毒介导的转染方法在原代 CD4+T细胞中表达全长 FOXP3 蛋白和 STUB 1 , 研究 STUB1是否能够缓解 FOXP3对 IL-2表达的抑制作用。
图 8: 与 STUB1相互作用的 FOXP3关键区域鉴定 (；图 8(Α》、 分子伴侣在相互 作用中的作用（图 8(B)、 8(C》及可能的机理 (图 8(D))。
图 9： STUB 1体外负调控 FOXP3蛋白表达和小鼠原代 Treg细胞功能的研究。 过表达 STUB 1降低细胞内 FOXP3的表达量 (图 9(A))， 同时提高炎症因子 IL-2(图 9(B))和 IFN-γ的表达（图 9(C)) ; 细胞增殖实验结果（图 9(D))， 曲线 1代表仅加入 CD25-, 曲线 2代表 GFP+CD25-, 曲线 3代表未活化 CD25-。
图 10: 实验性结肠炎小鼠模型体内 Treg细胞中过表达 STUB 1能减弱 Treg细 胞的免疫抑制功能。
图 11 : 通过 Western blotting实验证明 IL-6和 TGF-beta的协同作用能够促进 FOXP3的降解。 图 11(A): IL-6剌激下的 FOXP3降解； 图 11(B): IL-6/TGF_p剌激 下的 FOXP3降解。
图 12: 通过 FACS实验证明 TGF-β和 IL-6剌激对 IL-6受体表达水平的影响。 图 12(A) : TGF-β剌激下的 IL-6受体表达水平； 图 12(B) : TGF-β剌激下的 IL-6 受体表达水平。
图 13: 通过 FACS实验证明 TGF-β和 IL-6/TGF-p剌激对 FOXP3表达水平的影 响。 图 13(A): TGF-β剌激下的 FOXP3表达水平； 图 13(B): IL-6/TGF_p剌激下的 FOXP3表达水平。
以上图中， IP是指免疫沉淀（immunoprecipitation) , IB是指免疫印迹 (immuno- blotting)。 具体实施方式
发明人认为 FOXP3会受到一系列时间和空间上的调控， 包括转录后修饰、 翻译或修饰以及蛋白相互作用等， 其中有一类因子包括酶、 小分子化合物等作 为 FOXP3的负调节因子， 在不同的信号剌激下能够下调 FOXP3的活性， 从而调 控整个免疫系统。 通过长期而深入的研究， 发明人发现了泛素化途径相关因子 可下调 FOXP3活性， 这些泛素化途径相关因子为 FOXP3负调节因子—— Toll样 受体、 泛素化连接酶、 促炎症细胞因子家族受体和 /或它们的编码序列。
具体而言， 发明人发现： 在天然免疫信号作用下， 比如不同的 TLR信号 (如 TLR3和 TLR9)，会促使 FOXP3蛋白降解，这说明存在 FOXP3蛋白的负调节因子。 并且， 发明人通过进一步研究还发现另一重要的翻译后修饰 泛素化， 也存 在于 FOXP3蛋白中。 经过进一步研究， 发明人还首次发现了 TLR信号途径与泛 素化之间存在密切的联系： TLR信号下游泛素化链接酶如 TRAF6 , 或蛋白激酶 如 IRAKI、以及多种细胞模式识别受体 (；如 IL-6等）皆可以促进 FOXP3蛋白的泛素 化。
泛素化修饰普遍发生于许多生理调节中， 导致修饰底物蛋白的活性和功能 的改变， 并且泛素化降解也是蛋白降解中一个很主要的途径， 比如 K48-多泛素 化会导致蛋白通过蛋白酶体的降解。 免疫信号通路中， 有许多重要的泛素化连 接酶 (E3)， 比如 TLR通路中的 TRAF6、 TGF-β信号下的 Smurfl。
发明人在对这些 E3的逐一检测中发现， E3能够促使 FOXP3的泛素化修饰。 例如， 受压力信号激活的 E3， STUB 1(STIP 1同源和 U-box域包含蛋白， STIP 1 homology and U-box containing protein l)/CHIP(Hsc70相互作用蛋白的 C末端， C terminus of Hsc70-interacting protein) , 能够明显的导致 FOXP3的泛素化降解。
STUB 1能够在分子伴侣的介导下结合 FOXP3蛋白。 K30A突变的 STUB 1不 能和分子伴侣蛋白结合， 也就无法结合 FOXP3 ; 而 H260突变的 STUB 1失去了 E3 的活性， 仍能结合 FOXP3 , 但不能促进其泛素化修饰。 发明人在 FOXP3转录活 性模型中发现， 过量表达 STUB 1能够显著降低 FOXP3的转录活性。 可见， 在翻 译后修饰水平上， STUB 1能作为 FOXP3的一个负调节分子， 通过分子伴侣的介 导促使 FOXP3的泛素化降解。
并且， 体内外实验均进一步证实了 STUB 1可负调控 FOXP3蛋白表达和小鼠 原代 Treg 细胞的功能。
此外， 发明人还发现 STUB 1的过表达还可提高炎症因子 IL-2和 IFN-γ的表 达， 且可反转 FOXP3对 IL2表达的抑制作用。 由此， 确定了泛素化途径相关因子、 其激动剂或拮抗剂还可用于调节 IL-2活性和 /或 IFN-γ活性。
基于上述研究， 本发明人在本发明中提供了首次揭示了泛素化途径相关因 子一一泛素化连接酶、 Toll样受体、促炎症细胞因子家族受体和 /或它们的编码序 列及其激动剂或拮抗剂作为 FOXP3活性、 IL-2活性和 /或 IFN-γ活性调节因子的用 途。 这些调节因子的确定与应用， 为通过调节 FOXP3、 IL-2和 /或 IFN-γ的活性来 调控调节性 T细胞， 治疗和 /或预防与 FOXP3、 IL-2和 /或 IFN-γ活性失调相关的疾 病或症状 (例如肿瘤;)提供了新途径，同时也提供了利用这些调节因子作为疫苗佐 剂， 提高病毒感染的免疫原性的新方法。 在此基础上， 发明人完成了本发明。 泛素化途径相关因子、 其激动剂或拮抗剂
如本文所用， 术语 "调节因子"或 "活性调节因子" 是指泛素化途径相关 因子或其激动剂或拮抗剂， 它们可通过影响泛素化途径来调节 FOXP3、 IL-2和 / 或 IFN-γ的活性， 从而进一步用于与 FOXP3、 IL-2和 /或 IFN-γ活性失调相关的疾病或 症状的预防和 /或治疗中。
如本文所用， 术语 "泛素化途径相关因子"是指可通过影响泛素化途径来 降低 FOXP3的活性的因子。 在本发明中， 泛素化途径相关因子包括： 泛素化连 接酶、 细胞模式识别受体 (；如 Toll样受体；)、 促炎症细胞因子家族受体和 /或它们的 编码序列。
如本文所用， 术语 " Toll样受体" 或 " TLR" 可互换使用， 均是指是天然 免疫系统中特异的 I-型跨膜受体及病原模式识别受体， 在急性炎症反应、 细胞 信号转导和细胞凋亡中起重要作用。
目前在人体中已鉴定了 10种 TLR的功能型家族成员 (TLR1- TLR10)。 根据 染色体分布、 基因结构及其氨基酸序列的保守性， 人的 TLRs受体可以分为 5个 亚群， S卩 TLR2、 TLR3、 TLR4、 TLR5和 TLR9亚群。 TLR2亚群包括 TLR1、 TLR2、 TLR6和 TLR10; TLR9亚群包括 TLR7和 TLR9; TLR3， TLR4和 TLR5各自形成一 个亚群。
本发明中首次揭示了 TLR对 FOXP3的负调节作用。 在本发明的优选实施方 式中所采用的 TLR包括（但不限于）： TLR3、 TLR9、 TLR1、 TLR2、 TLR4、 TLR5、 TLR7或 TLR8，优选 TLR2、 TLR3及 TLR9亚群的 TLR，更优选 TLR3、 TLR7、 TLR8 或 T R9。
如本文所用， 术语 "泛素化连接酶 "或 " E3 "可互换使用， 均是指泛素与 靶蛋白结合所需要的第三个酶， 其可选择性的识别并结合特异的靶蛋白， 在泛 素介导的降解靶蛋白底物的选择性方面具有重要作用。 根据识别靶蛋白序列中 结构域不同， E3可分为两大类型： （l)HECT结构域家族的泛素连接酶 (HECT E3s)， 包括 Smurf2、 E6、 AP、 ARF、 BPl等； （2)RING结构域家族的泛素连接酶 (RING E3s), RING结构域家族最典型的特点是具有环指结构域， 也是该家族具 有泛素连接酶作用的重要因素， RING E3s包括大量成员， 如： Mdm2(Hdm2), APC、 SCF、 IAP、 Skp2等等。
本发明中首次揭示了 E3对 FOXP3的负调节作用。在本发明的优选实施方式 中所采用的 E3包括 (但不限于）： S TUB 1、 TRAF6、 Smurfl、 Smurf2、 TRAF2、 TRAF3、 TRAF4、 p300或 RBCK1， 优选 STUB 1。 优选本发明所采用的 E3同时兼具结合分 子伴侣的功能或活性。 STUB 1(STIP1 homology and U-Box containing protein 1， STIP 1同源和 U-Box域包含蛋白)兼有辅助分子伴侣功能和 E3泛素 -蛋白连接酶 活性，是异常蛋白降解的泛素-蛋白酶系统和溶酶体系统二者间转换的分子开关, 能够协调细胞内蛋白质量控制系统的平衡和稳定,在蛋白质量控制中起关键作 用。
如本文所用， 泛素化途径相关因子的 "激动剂"是指能够促进泛素化途径相 关因子的表达或提高泛素化途径相关因子的活性的物质， 在本发明中也可称之为 "促效剂" 。 可用于本发明中的激动剂包括但不限于： IL6。
如本文所用， 泛素化途径相关因子的 "拮抗剂"是指能够抑制泛素化途径相 关因子的表达或降低泛素化途径相关因子的活性的物质， 在本发明中也可称之为 "抑制剂" 。 可用于本发明中的拮抗剂包括但不限于： MG-132。
本发明的上述蛋白质或多肽可以是天然纯化的产物， 或是化学合成的产物， 或使用重组技术从原核或真核宿主 (；例如， 细菌、 酵母、 高等植物、 昆虫和哺乳动 物细胞;)中产生。
应理解， 本发明的上述术语定义中也包括所述蛋白质或多肽的保守性变异 多肽、 或其同源多肽。 在本发明的一个实施方式中， 所述蛋白质或多肽来自人 或其它真核生物， 例如小鼠、 大鼠、 牛或猴等， 且它们之间具有高度的保守性。
本发明蛋白质或多肽的变异形式包括 (但并不限于 一个或多个 (通常为 1-50 个， 较佳的 1-30个， 更佳的 1-20个， 最佳的 1-10个， 例如 1、 2、 3、 4、 5、 6、 7、 8、 9或 10个；)氨基酸的缺失、 插入和 /或取代， 以及在 C末端和 /或 N末端添加一个或数个 (；通常为 20个以内， 较佳地为 10个以内， 更佳地为 5个以内；)氨基酸。 例如， 在本领 域中，用性能相近或相似的氨基酸进行取代时，通常不会改变蛋白质或多肽的功能。 又比如， 在 C末端和 /或 N末端添加一个或数个氨基酸通常也不会改变蛋白质或多肽 的功能。 本领域技术人员可根据本领域常识和 /或常规试验很容易的确定这些变异 方式， 而不会影响蛋白或多肽的活性。
在本发明中， "保守性变异多肽" 指与已知的 TLR或 E3氨基酸序列相比， 有至多 20个， 较佳的至多 10个， 更佳的至多 5个， 最佳的至多 3个氨基酸被性质 相似或相近氨基酸所替换而形成， 且仍具有原 TLR或 E3相同或类似功能的多肽。
如本文所用，术语 "编码序列 "是指编码本发明所述的泛素化连接酶或 Toll 样受体的序列， 或是它们的高度同源序列或在严格条件下与所述序列杂交的分 子或与上述分子高度同源的家族基因分子。
如本文所用， 术语 "严格条件"是指： （1)在较低离子强度和较高温度下的杂 交和洗脱， 如 0.2 X SSC,0.1%SDS， 60 °C ; 或 (2)杂交时加有变性剂， 如 50%(v/v)甲 酰胺， 0.1%小牛血清 /0.1%Ficoll， 42°C等； 或 (3)仅在两条序列之间的相同性至少在 50%， 优选 55%以上、 60%以上、 65%以上、 70%以上、 75%以上、 80%以上、 85% 以上或 90%以上， 更优选是 95%以上时才发生杂交。 例如， 所述序列可为 (a)中所限 定序列的互补序列。
本发明的编码序列全长序列或其片段通常可以用 PCR扩增法、 重组法或人工 合成的方法获得。 对于 PCR扩增法， 可根据本发明所公开的有关核苷酸序列， 尤其 是开放阅读框序列来设计引物， 并用市售的 cDNA库或按本领域技术人员已知的常 规方法所制备的 cDNA库作为模板， 扩增而得有关序列。 当序列较长时， 常常需要 进行两次或多次 PCR扩增， 然后再将各次扩增出的片段按正确次序拼接在一起。 组合物
本发明的调节因子还可用于制备治疗性或预防性的药物组合物、 保健品组 合物或疫苗组合物。
因此， 另一方面， 本发明还提供了一种组合物， 它含有 (a)安全有效量的本 发明的调节因子； 以及 (b)药学上可接受的载体或赋形剂。 本发明调节因子的数 量通常为 10微克 -100毫克 /剂， 较佳的为 100-1000微克 /剂。
本文所用的术语 "有效量"指治疗剂治疗、 缓解或预防目标疾病或状况的 量， 或是表现出可检测的治疗或预防效果的量。 对于某一对象的精确有效量取 决于该对象的体型和健康状况、 病症的性质和程度、 以及选择给予的治疗剂和 /或治疗剂的组合。 因此， 预先指定准确的有效量是没用的。 然而， 对于某给定 的状况而言， 可以用常规实验来确定该有效量， 临床医师是能够判断出来的。
药物组合物还可含有药学上可接受的载体。 术语 "药学上可接受的载体" 指用于治疗剂给药的载体。 该术语指这样一些药剂载体： 它们本身不诱导产生 对接受该组合物的个体有害的抗体， 且给药后没有过分的毒性。 这些载体被本 领域普通技术人员所熟知。 在 Remington's Pharmaceutical Sciences(Mack Pub. Co. , N.J. 1991)中可找到关于药学上可接受的赋形剂的充分讨论。 这类载体包 括 (但并不限于；)： 盐水、 缓冲液、 葡萄糖、 水、 甘油、 乙醇、 佐剂及其组合。
治疗性组合物中药学上可接受的载体可含有液体， 如水、 盐水、 甘油和乙 醇。 另外， 这些载体中还可能存在辅助性的物质， 如润湿剂或乳化剂、 pH缓冲 物质等。 此外， 免疫组合物中还可以含有免疫佐剂。
通常， 可将治疗性组合物制成可注射剂， 例如液体溶液或悬液； 还可制成 在注射前适合配入溶液或悬液中、 液体载体的固体形式。
一旦配成本发明的组合物， 可将其直接给予对象。 待预防或治疗的对象可 以是动物； 尤其是人。
本发明的含本发明调节因子的治疗或预防性药物组合物 (包括疫苗；)， 可以 经口服、 皮下、 皮内、 静脉注射等方式应用。 治疗剂量方案可以是单剂方案或 多剂方案。 本发明的主要优点
(1)本发明中首次揭示了泛素化途径相关因子一一泛素化连接酶、 Toll样受 体、 促炎症细胞因子家族受体和 /或它们的编码序列作为 FOXP3活性、 IL-2活性 和 /或 IFN- γ活性调节因子的用途；
(2)本发明调节因子的确定与应用， 为通过调节 FOXP3、 IL-2和 /或 IFN- Y的 活性来调控调节性 T细胞， 治疗和 /或预防与 FOXP3、 IL-2和 /或 IFN- Y活性失调 相关的疾病或症状 (例如肿瘤等;)提供了新途径；
(3)本发明还提供了利用本发明的调节因子作为疫苗佐剂， 以提高疫苗免疫 原性的新方法。 实施例
下面结合具体实施例， 进一步阐述本发明。 应理解， 这些实施例仅用于说 明本发明而不用于限制本发明的范围。 本领域技术人员可对本发明做出适当的 修改、 变动， 这些修改和变动都在本发明的范围之内。
下列实施例中未注明具体条件的实验方法， 可采用本领域中的常规方法， 例如参考《分子克隆实验指南》 (；第三版，纽约，冷泉港实验室出版社， New York: Cold Spring Harbor Laboratory Press, 1989)或按照供应商所建议的条件。 DNA 的测序方法为本领域常规的方法， 也可由商业公司提供测试。
除非另外说明， 否则百分比和份数按重量计算。 除非另行定义， 文中所使 用的所有专业与科学用语与本领域熟练人员所熟悉的意义相同。 此外， 任何与 所记载内容相似或均等的方法及材料皆可应用于本发明方法中。 文中所述的较 佳实施方法与材料仅作示范之用。 材料与方法
1. 质粒和载体：
N端 HA -、 Myc -、 FLAG-标签的 FOXP3、 不同 FOXP3片段真核表达载体、 STUB 1、 Ub (泛素）及 pIPMyc2 -STUB1、 pIPHA2-FOXP3、 其它 E3质粒 (pIPHA2、 pIPMyc2、 pIPFLAG2是真核表达载体； pET28a和 pET21-MBP是原核表达载体) 和慢病毒包装载体质粒 (； FUGW，del8.9和 VSV-G)由发明人实验室克隆构建 (；参照 参考文献 4)。
El、 UbcH5b和 HSP70基因从人类外周血单核细胞 cDNA文库中扩增获得。 根据 Genbank下载的序列设计引物如下：
Figure imgf000015_0001
TLR信号下游激酶 IRAKI表达质粒 pIPIRAKl为本实验室克隆构建 (；参照参 考文献 4， IRAKI全基因序列参照 Entrez Gene: 3654)； 泛素化连接酶 TRAF6由 Yongwon Choi教授提供 (参照参考文献 7、 或可通过市售 (如 Origin公司）获得， 或 通过常规途径克隆；)。
2. 抗体：
HA抗体（F- 7)， Myc抗体（9E10)购自 Santa Cruz公司。 Flag抗体（M2)购自 Sigma 公司。 F0XP3抗体（hFOXY)购自 eBioscience公司。 STUB1抗体（C3B6)购自 Cell Signaling公司。 HRP偶联的抗鼠二抗购自 Promega公司。
3. 细胞：
人 HEK293T (购自中国科学院细胞库（目录号： GNHul7) )在含有 10%FBS， 100单位 /毫升青链霉素的 DMEM (Dulbecco， s modified Eagle' s medium) , 37°C， 5%C02中培 养。
Jurkat E6. 1 T细胞（购自中国科学院细胞库 （目录号： TCHU123 ) )在含有 10%FBS， 100单位 /毫升青链霉素， 100单位 /毫升非必需氨基酸和 100单位 /毫升 丙酮酸钠的 1640培养基， 37°C， 5%CO2中培养。
细胞转染： 利用 Lipofectamin 2000<;invitrogen)进行脂质体转染， 转染 48小 时后收样分析。
FUGW-TAP-FOXP3 , del 8.9 和 VSV-G共同转染人胚肾细胞株 HEK293 , 转 染后 48及 72小时收集细胞培养基上清， 超速离心纯化病毒并感染 Jurkat E6. 1最 终获得 TAP-FOXP3 Jurkat稳定表达细胞系。
4. 试剂及细胞核及染色质亚组分的分离：
各种 TLR配体试剂均购自 Apotech公司。
MG132购自 Merck公司。
Protein AG-beads购自上海悦克生物技术有限公司。
细胞用细胞质提取液（10 mM Hepes, pH7.9， 10 mM KCl， 0.1 mM EDTA, 1 mM DTT , 0.5 mM PMSF , Γ完全蛋白酶抑制剂混合试齐 !J(complete protease inhibitor cocktails , Cat. No. 1-697-498； Roche Biochem), 1 mM Na3VO4) , 放置 冰上 15分钟，然后加入终浓度 0.6%的 NP-40，经涡旋 (Votex O秒裂解后， 12000g 离心 30秒。 所得上清为细胞质部分， 沉淀即为细胞核组分。 将细胞核组分用细 胞核悬浮液 (20 mM Hepes pH7.9, 400 mM NaCl, 1 mM EDTA, 1 mM DTT, 1 mM PMSF, Γ蛋白酶抑制剂混合试剂， 1 mM Na3VO4)溶解后， 置于 4°C匀速缓缓旋 转 30分钟，然后在 16,060 g , 4°C 离心 15分钟。所得不溶沉淀物为染色质亚组分。
5. 免疫沉淀：
细胞用 RIPA缓冲液 （20mM Tris/HCL pH7.5， 150mM NaCl, 1% NP-40, 0.5% Na-DOC， ImM EDTA以及蛋白酶抑制剂 ImM PMSF， I X Cocktail, 磷酸 酶抑制剂 ImM Na3VO4， ImM NaF)裂解。 在细胞裂解液中先加入一抗孵育 1小 时， 再加入偶联 ProteinA/G的琼脂糖珠子作用 1小时。用 RIPA缓冲液裂解洗涤三 次后， 用 SDS-PAGE检测所结合的蛋白质。
6. 免疫印迹：
蛋白样品通过 SDS-PAGE蛋白电泳后， 转膜至硝酸素纤维膜， 再用 5%脱脂 奶粉 TBST中封闭一小时， 加入一抗孵育一小时， 再加入 HRP偶联的二抗孵育一 小时， 用 ECL底物曝光显色。
7. 萤光素酶实验：
8 X FK萤光素酶报告基因在增强子区含有 8个重复叉头 (Forkhead)结合区， 5 X Gal萤光素酶报告基因在增强子区含 5个重复 Gal4表达框。 pBind-FOXP3(参照 参考文献 4)表达与 FOXP3融合的 Gal4结合区。 在 293T细胞中转入相应的质粒， 用萤光素酶底物和 β-半乳糖苷酶底物分别测定萤光素酶活性和 β半乳糖苷酶活 性。 每次实验独立重复三次以上， 所得结果用平均值与标准偏差表示。 所用的 酶底物购自碧云天公司。
8. 基因定点突变
STUB 1突变体 K30A， H260Q是通过突变 PCR得到 (参照参考文献 5)。
Figure imgf000017_0001
定点突变试剂盒购自 Stratagene公司， PCR后 DNA测序验证。
9. 蛋白的表达及纯化
将携带 6His-El、 6His-UbcH5b、 6His-STUB l、 6His-HSP70和 6His-FLAG -Ubiqutin的原核表达载体转化 Rosetta/PlysS感受态细胞， IPTG诱导表达， 收细 胞， 裂解， 镍柱纯化 (Qiagen公司；)， 最后经快速蛋白液相色谱 (GE公司）获得纯 化蛋白。 MBP-FOXP3和 MBP蛋白按上述方法表达， 收细胞， 裂解， 直链淀粉 树脂法纯化 (NEB公司）， 用标准缓冲液透析洗脱蛋白 (25 mM Tris-HCl (pH 7.5)、 150 mM N氯化钠、 ΙΟ ηιΜ β巯基乙醇和 10%甘油）。最后用 Brandford法测定所有 蛋白浓度 (上海碧云天生物技术有限公司；)。
10. 体外泛素化实验
将 O. l g 6His-El、 0.5μg 6His-UbcH5b、 2 g 6His-STUB l、 2 g 6His-HSP70、 10μg 6His-FLAG-Ubiquitin和 2 g MBP-FOXP3或 MBP力卩入 ΙΟΟμΙ 含 50mM Tris/HCl(pH 7.4)、 2mM氯化镁、 ImM DTT、 4mM ATP的反应液中， 混合均匀， 30°C孵育两小时后加入 SDS上样缓冲液终止反应。将以上反应体系进行 Western blotting， 用抗 FLAG标签抗体分析各体系中的泛素化程度。
11. 免疫荧光
细胞经 4%甲醛固定， 0.5% Triton-X 100穿膜， 封闭及相应抗体标记。 DAPI 标记细胞核。 最后细胞于共聚焦显微镜下观察结果 (LEICA SP5)。
12. 细胞增殖检测实验
分别从 Thyl.2小鼠（购自美国 Jackson实验室）及 Thyl. l小鼠（购自美国
Jackson实验室；)体内分离出淋巴细胞， 通过抗体标记、磁珠分选得到调节性 T细胞 及 CD4+CD25_CD62Lhlgh 效应性 T细胞。如前所述，用 ΙμΜ CFSE(Invitrogen公司） 标记效应性 T细胞后将调节性 T细胞及效应性 T细胞培养 80小时， 通过流式细胞 仪检测效应性 T细胞荧光强度变化， 分析其增殖状况。
13. 结肠炎模型构建及免疫组化
从 BALB/c小鼠 (；购自美国 Jackson实验室） 体内分离出淋巴细胞， 通过抗体标 记、 磁珠分选得到天然 CD4+CD25— CD62Lhlgh效应性 T细胞， 通过尾静脉注射至 BALB/c RAG2-/-免疫缺陷型小鼠（购自美国 Taconic 公司）体内（~1χ106个细胞 / 只；)。 实验小鼠分为两组， 分别通过尾静脉注射野生型 CD4+CD25+Treg细胞和 STUB 1转化的 Treg细胞 (2xl05)。 每周观察评估症状， 一旦症状严重或体重减轻 20%则人道牺牲实验小鼠。
T细胞重建 8周后从小鼠体内取出结肠组织， 10%甲醛固定， 石蜡包埋切片 及苏木精 -伊红染色。 评估结肠组织病变情况， 等级 0: 无明显病变； 等级 1 : 最 低程度的免疫细胞浸润， 伴随或非伴随轻微的内皮细胞增殖； 等级 2 : 轻微免 疫细胞浸润， 伴随轻微至中度的内皮细胞增殖和轻微至中度的杆状细胞分泌粘 液减少； 等级 3 : 中度免疫细胞浸润， 中度至严重的内皮细胞增殖和中度至严 重的杆状细胞分泌粘液减少； 等级 4 : 发生于肠壁全程的高度免疫细胞浸润， 高度内皮细胞增殖和高度粘液分泌减少； 等级 5 : 伴随溃疡性炎症的高度肠壁 全程免疫细胞浸润， 管状肠腺缺失。 实施例 1. TLR信号对 FOXP3的影响
测试 1. TLR信号对 FOXP 3表达量的影响
在 Jurkat T细胞中转入 FOXP3表达 Myc-FOXP3a， 48小时后加入不同的 TLR 配体剌激 (；按 Apotech试剂盒步骤操作；)。 细胞收样后， 分离出细胞核与染色质成 分。 分别通过蛋白电泳和免疫印迹法进行检测。
实验结果如图 3a所示。 结果显示： 随着不同 TLR信号剌激， FOXP3蛋白的 表达有所改变。 在 TLR信号 (；尤其是 TLR3、 TLR9)作用下， FOXP3的蛋白水平显 著降低。 该结果表明： TLR信号（比如 TLR3、 TLR9)可作为 FOXP3的负调节信号。 测试 2. TLR信号下游激活蛋白 TRAF 6 X寸 FOXP 3泛素化的影响
在 293T细胞中共转入 TLR信号下游激活蛋白 TRAF6表达质粒及 FOXP3表 达质粒 Myc-FOXP3a， 48小时后细胞收样， 进行免疫沉淀， 然后分别通过蛋白 电泳和免疫印迹法进行检测。
实验结果如图 3b所示。 结果显示： 共表达 TLR信号下游具有泛素化链接酶 活性的接头蛋白 TRAF6可以明显促进 FOXP3泛素化。
该结果表明： TLR信号（比如 TLR3、 TLR9)可通过其下游泛素化链接酶
TRAF6促进 FOXP3泛素化。 测试 3. TLR信号下游激活蛋白 IRAKI对 FOXP3泛素化的影响
在 293T细胞中共转入 TLR信号下游激酶表达质粒 IRAKI及 FOXP3表达质 粒 Myc-FOXP3a， 48小时后细胞收样， 进行免疫沉淀， 然后分别通过蛋白电泳 和免疫印迹法进行检测。
实验结果如图 3c所示。结果显示：共表达 TLR信号下游激酶表达质粒 IRAKI 可以明显促进 FOXP3泛素化。
该结果表明： TLR信号（比如 TLR3、 TLR9)可通过其下游激酶 IRAKI促进
FOXP3泛素化。 实施例 2. FOXP3与 STUB1和其它 E3的结合
将 HA-FOXP3 a和 Myc-STUB 1共转入 293 T细胞， 分别用 HA和 Myc抗体双向 免疫共沉淀， 并用免疫印迹法进行检测 (结果如图 4(A)左图所示；)。 在反向验证 试验中， 用 Flag-FOXP3与 Myc-STUBl共转染 293T细胞， 分别用 Flag和 Myc抗体 双向免疫共沉淀， 并用免疫印迹法进行检测 (结果如图 4(A)右图所示；)。
其它免疫信号通路可通过将 E3与 Flag-FOXP3a， HA-Ubi共转入 293T细胞进 行检测。细胞裂解液用 Flag抗体免疫沉淀，用 Myc抗体检测 (结果如图 4(B)所示；)。
结果显示： FOXP3可以与 STUB1作免疫共沉淀反应， STUB1也可以特异地 拉下 FOXP3 , 暗示 STUB1与 FOXP3的相互作用是特异的和外源标签表达非依赖 性的。 并且， 在其它信号通路中起关键作用的泛素连接酶也能够与 FOXP3有相 互作用。
该结果表明： FOXP3蛋白能够与泛素连接酶相互作用，并受到泛素化修饰。 尤其是， FOXP3蛋白能够和受压力信号调控的 STUB1泛素连接酶相互作用。 实施例 3. STUB 1能够导致 FOXP3泛素化
将 Myc-STUB 1、 HA-Ub与 Flag-FOXP3a或 Flag-FOXPl共转入 293T细胞。 细 胞裂解液用 HA抗体免疫沉淀， 通过免疫印迹检测泛素化的蛋白 (；结果如图 5(A) 所示）。 将 Myc-STUB1、 HA-Ub和 Flag-FOXP3a共转入 Jurkat T细胞， 细胞裂解液 用 HA抗体免疫沉淀并用免疫印迹检测 (结果如图 5(B)所示；)。
结果显示： 泛素连接酶 STUB 1能够特异性作用于 FOXP3 , 促使 FOXP3蛋白 的泛素化修饰。
该结果表明： 泛素化修饰是 FOXP3存在的翻译后修饰之一， FOXP3是 STUB 1泛素连接酶的特异性底物。 实施例 4. STUB 1促进 FOXP3泛素化降解
将 Flag-FOXP3， HA-Ub和 Myc-STUB 1共转入 293T细胞。细胞收样前，用 5μΜ MG132处理不同时间。裂解液用 Myc抗体免疫沉淀并用免疫印迹检测 (；结果如图 6(A)所示)；
将 Flag-FOXP3 a、 HA-Ub和 Myc-STUB 1或分子伴侣结合缺失 S TUB 1-K30A 突变体或泛素连接酶活性缺失 STUB1-H260Q突变体共转入 293T细胞。 细胞用 5μΜ MG132处理 4小时后，用 ΗΑ抗体免疫沉淀并用免疫印迹检测 (；结果如图 6(B) 所示；)。
结果显示： MG132的抑制能明显提高泛素化的 FOXP3蛋白水平， 有结合分 子伴侣和泛素连接酶活性的 STUB 1会导致 FOXP3的泛素化降解。 此外， 尽管泛 素连接酶活性缺失的 H260Q-STUB 1突变体仍然可以与 FOXP3结合，但是 H260Q 和 Κ3 OA两种突变体 STUB 1均不能够促进 FOXP3的泛素化修饰， 这表明 STUB 1 的酶活性对 FOXP3的泛素化修饰起到重要作用，
该结果表明： STUB1通过导致 FOXP3的泛素化降解而负调控 FOXP3的蛋白 水平。 实施例 5. STUB 1抑制 FOXP3转录活性
用 8 X FK萤光素酶报告系统检测 FOXP3活性， 将 FOXP3、 STUB1、 8 X FK 萤光素酶报告基因和 MSV-P-Gal对照共转入 293T细胞， 萤光素酶活性用 β-Gal 活性标准化后， 取三次独立实验的结果用平均值士标准偏差表示 (结果如图 7(A) 所示；)。
用 pBind系统 (；参照参考文献 4构建 将 pBind-FOXP3、 STUB 1和 pGal5-萤光 素酶报告基因和 MSV-P-Gal对照共转入 293T细胞， 萤光素酶活性用 β-Gal活性标 准化后， 取三次独立实验的结果用平均值士标准偏差表示 (结果如图 7(B)所示；)。
结果显示： FOXP3作为转录抑制子能够降低报告基因的转录活性， 而
STUB 1的加入则削弱了 FOXP3的转录抑制能力。
该结果表明： STUB 1作为 FOXP3的负调节因子具有抑制 FOXP3的功能。 发明人进一步检测了过表达 HSP70及 STUB1对 FOXP3介导的 IL-2启动子驱 动的萤光素酶报告基因表达水平的转录抑制作用。 转染相应质粒到 Jurkat T细 胞， 在收样前用 PMA和伊屋诺霉素剌激转染细胞后对细胞表达的萤光素酶活性 进行分析。我们发现， FOXP3显著抑制了 IL-2启动子驱动的萤光素酶表达水平， HSP70共表达对该抑制作用未见影响。 然而， FOXP3同 STUB1的共表达几乎完 全反转了 FOXP3对 IL-2启动子的抑制作用， 并且 HSP70能够明显增强 STUB1对 FOXP3转录抑制的反转作用（图 7(C 。
已报道的研究成果显示， FOXP3对于原代 T细胞中 IL-2的表达起到抑制作 用。因此，我们利用慢病毒介导的转染方法在原代 CD4+T细胞中表达全长 FOXP3 蛋白和 STUB 1 , 以研究 STUB 1是否能够缓解 FOXP3对 IL-2表达的抑制作用。 如 在 Jukart细胞中一致， FOXP3几乎完全封闭了天然 T细胞中 IL-2的表达。 然而， 过表达 STUB 1可以反转 FOXP3对 IL2表达的抑制作用（图 7(D))。 实施例 6. 与 STUB1相互作用的 FOXP3关键区域鉴定、分子伴侣在相互作用中的 作用及可能的机理
发明人还鉴定了与 STUB1相互作用的 FOXP3关键区域。 如图 8(A)所示， FOXP3的锌指与亮氨酸拉链结构域被证明是与 STUB1结合的必需区域。
为鉴定 FOXP3与 STUB1的相互作用是否是分子伴侣 HSC70/HSP70依赖性 的， 将 HA-FOXP3与 Myc标签的野生型 STUB 1 , 或分子伴侣结合缺失 STUB 1-K30A突变体， 或泛素连接酶活性缺失 STUB1-H260Q突变体共转染 293T 细胞， 然后以 Myc抗体对 STUB1作免疫共沉淀。 接下来用 HA抗体和 Myc抗体做 免疫印迹检测。 结果显示， 野生型 STUB1和 H260Q-STUB1突变体均可以与 FOXP3结合， 但是分子伴侣结合缺失的 K30A-STUB1突变体却不可以与 FOXP3 结合 (；图 8(Β))。
为验证 STUB l是否可以促进 FOXP3表达水平的下调， 将 HA-FOXP3、 Myc-STUBK FLAG-HSP70在 293T细胞中共表达， 并以不转染 FLAG-HSP70的 细胞为对照。 结果显示： STUB1与 HSP70可以协同促进 FOXP3的降解（图 8(C；)。
这些结果暗示 HSC70和 HSP70等分子伴侣是 FOXP3与 STUB 1相互作用的必 需的介导蛋白。
免疫荧光分析显示， STUB 1可以大量的表达在稳定表达 TAP-FOXP3的 Jurkat细胞外周。 我们还发现在热激剌激条件下 STUB1会逐渐向细胞核内聚集 (；图 8(D; 。 在热激条件下 STUBl定位的变化反映出其与 FOXP3接触机会的增加， 从而促进了它们的结合， 这一过程可能是应激条件下 FOXP3被降解， 从而调节 免疫的机理。 实施例 7. STUB 1体外负调控 FOXP3蛋白表达和小鼠原代 Treg细胞的功能
为验证体外 STUB1是否可在小鼠原代 Treg细胞中负调控 FOXP3的表达， 进 行了以下实验： 从 C57BL/6小鼠分离出原代 Treg细胞， 分别感染携带空载 (携带 GFP标签)或携带 STUB1基因的慢病毒载体。如图 9(A)所示，与空载对照组相比， 过表达 STUB 1降低细胞内 FOXP3表达量， 同时提高炎症因子 IL-2和 IFN-γ的表达 (；图 9(B)和 9(C))。 以上实验结果说明 Treg细胞缺失部分基因沉默功能。
此外， 发明人还通过细胞增殖实验进一步验证了 STUB 1过表达能极大程度 影响 Treg细胞抑制细胞增殖的能力（图 9(D))。
这些研究结果表明在原代 Treg细胞中表达 STUB l能降低 FOXP3的表达量， 并影响其功能。 实施例 8. 实验性结肠炎小鼠模型体内 Treg 细胞中过表达 STUBl能减弱 Treg细 胞的免疫抑制功能
为验证过表达 STUB1能否在体内影响 Treg 细胞功能， 将天然 CD4+CD25— CD62Lhlgh T细胞移植到 Rag2— Λ小鼠体内， 构建了 Thl介导的小鼠结肠 炎模型 44。 在此模型中共移植 CD4+CD25+Treg细胞能有效抑制炎症， 控制疾病 发展。
本实验中， 通过小鼠体重的监测及免疫组化分析判定结肠炎的发展程度。 如图 10(A)所示， 与对照组相比， 同时移植 Teff (效用性 T细胞)和 STUB 1过表达的 Treg细胞小鼠体重明显下降， 其免疫组化切片中炎症程度明显提高， 不能有效 抑制炎症发展 (图 10(B)和 10(C))。
以上体内实验结果与体外实验实验结果相符， 并再次证明了 STUB 1是 FOXP3的负调控因子， 也是 Treg细胞功能的关键调控因子。 实施例 9. 泛素化途径相关因子 IL-6和 TGF-β促进 FOXP3降解
CD4+T细胞在不同的细胞因子和环境因素下， 可以分化成四种不同的亚 型： TH1、 TH2、 TH17和 iTreg。 这四种 T细胞亚型有其特定的细胞因子和转录 因子。 已有的研究发现， 在 TGF-β和炎症因子 IL-6的作用下， CD4+T细胞分化 为 TH17 , 然而在 TGF-β的作用下， CD4+T细胞能够分化为 iTreg。 TGF-β在 TH17 分化中起的促进作用以及 IL-6在 iTreg分化中起到的抑制作用，其机制尚不清楚。
本实施例中通过 western blotting和 F ACS等实验证明 IL-6和 TGF-β的协同作 用能够促进 FOXP3的降解， 初步设想其机制是通过泛素化依赖的方式降解。
Western blotting和 FACS 实 验 中 所 采 用 的 细 胞 系 为 稳 转 的 Jurkat-HA-FOXP3(如材料和方法中所描述）； 所加 IL-6的量为 20ng/ml； 所加 TGF-β的量为 10ng/ml。 利用 IL-6I¾t体（购自 Santa Cruz公司） ， 以及 Cy3标记的 anti-Rabbit二抗（购自 Jackson公司）染色，通过流式细胞仪的 PE通道进行检测。。 Western blotting的结果如图 11所示， F AC S实验结果如图 12和 13所示。
由图 11中的 western blotting 结果可以看出， 在 IL-6单独作用时， FOXP3的 表达水平没有变化， 而在 IL-6/TGF-P的作用下 FOXP3的表达水平在 12小时开始 下降。
由图 12中的 FACS结果可以看出， TGF-β促进了 IL-6 受体的表达， 但是 IL-6 对本身受体的表达却没有影响。 有报道发现， TGF-β可以抑制 TH17的负反馈因 子 SOCS3 , 从而可以与 IL-6、 IL-21等 TH17特定细胞因子相互作用， 促进 TH17 的分化。 所以 TGF-β是通过多种机制促进 TH17的分化， 比如： 在 IL-6存在下， 促进 FOXP3的降解， 上调 IL-6受体等。
并且， 由图 13中的 FACS结果可以看出， TGF-β能够极大幅度的促进 FOXP3 的表达， 然而在 IL-6/TGF-P的作用下， FOXP3的表达水平在 12小时开始下降， 这与上述 IL-6受体在 12小时时开始上调相符合。 可见 IL-6可以逆转 TGF-β促进 FOXP3表达的作用， 进而通过某种机制促进 FOXP3的降解。 在本发明提及的所有文献都在本申请中引用作为参考， 就如同每一篇文献 被单独引用作为参考那样。此外应理解，在阅读了本发明的上述讲授内容之后， 本领域技术人员可以对本发明作各种改动或修改， 这些等价形式同样落于本申 请所附权利要求书所限定的范围。 参考文献
1. Belkaid, Y" Piccirillo, C.A., Mendez, S" Shevach, E.M., 和 Sacks, D.L. (2002). CD4+CD25+ regulatory T cells control Leishmania major persistence and immunity (CD4+CD25+调节性 T细胞对硕大利什曼原虫存活率和免疫性的控制). Nature 420, 502-507.
2. Belkaid, Y.禾口 Rouse, B.T. (2005). Natural regulatory T cells in infectious disease (传染 病中的天然调节性 T细胞）. Nat Immunol 6, 353-360.
3. Li, B.禾口 Greene, M.I. (2008). Special regulatory T-cell review: FOXP3 biochemistry in regulatory T cells—how diverse signals regulate suppression (特异性调节性 T细胞综述： FOXP3 在调节性 T细胞中的生化性质 _ _不同信号如何进行抑制的调控). Immunology 123, 17-19.
4. Li, B., Samanta, A., Song, X., Iacono, K.T., Bembas, K., Tao, R., Basu, S., Riley, J.L., Hancock, W W., Shen, Y.,等 (2007). FOXP3 interactions with histone acetyltransferase and class II histone deacetylases are required for repression (阻遏所需的 FOXP3与组氨酸乙酰转移酶和 II 型组氨酸脱乙酰基酶的相互作用）. Proc Natl Acad Sci U S A 104, 4571-4576.
5. Qian , S.， McDonough， H., Boellman, F., Cyr, D M., Patterson, C"等（2006). CHIP-mediated stress recovery by sequential ubiquitination of substrates and Hsp70. (STUB 1与 Hsp70作用作为压力信号途径的泛素连接酶）。 Nature 440,551-555.
6. WO 2007/084775， Composition and method for modulation of suppressor T cell activation (调节抑制性 T细胞活性的组合物和方法)。
7. Kobayashi T, Walsh PT, Walsh MC, Speirs KM, Chiffoleau E, King CG, Hancock WW, Caamano JH, Hunter CA, Scott P, Turka LA, Choi Y. TRAF6 is a critical factor for dendritic cell maturation and development (TRAF6是一个调节树突状细胞成熟及发育的关键性因子）. Immunity. 2003 Sep; 19(3):353-63.

Claims

权利要求书
1. 泛素化途径相关因子、 其激动剂或拮抗剂在制备调节 FOXP3活性、 IL-2活性 和 /或 IFN-γ活性的组合物中的用途， 其中所述泛素化途径相关因子选自： Toll样受体、 泛素化连接酶、 促炎症细胞因子家族受体和 /或它们的编码序列。
2. 如权利要求 1所述的用途， 其特征在于， 对 FOXP3活性、 IL-2活性和 /或 IFN-γ 活性的所述调节是正调节或负调节， 其中， 所述泛素化途径相关因子或其激动剂用于 负调节 FOXP3活性和正调节 IL-2活性和 /或 IFN-γ活性， 所述泛素化途径相关因子的拮 抗剂用于正调节 FOXP3活性和负调节 IL-2活性和 /或 IFN-γ活性。
3. 如权利要求 1所述的用途， 其特征在于， 所述促炎症细胞因子家族受体选自： IL-6R、 TGFp受体、 IL-2 受体、 TNF- α受体或 GITR。
4. 如权利要求 1所述的用途， 其特征在于， 所述用途是泛素化途径相关因子在制 备负调节 FOXP3活性的组合物中的用途， 其中所述泛素化途径相关因子选自： Toll样 受体、 泛素化连接酶和 /或它们的编码序列。
5. 如权利要求 1-4中任一项所述的用途， 其特征在于， 所述泛素化连接酶是选自 下组中的一种或多种： STUB 1、 TRAF6、 Smurf Smurf2、 TRAF2、 TRAF3、 TRAF4、 p300、 RNF31或 RBCK1。
6. 如权利要求 1-4中任一项所述的用途， 其特征在于， 所述 Toll样受体是选自下 组中的一种或多种： TLR1、 TLR2、 TLR3、 TLR4、 TLR5、 TLR7、 TLR8或 TLR9。
7. 如权利要求 1-4中任一项所述的用途，其特征在于，所述调节 FOXP3活性、 IL-2 活性和 /或 IFN-γ活性的组合物用于治疗和 /或预防与 FOXP3、 IL-2和 /或 IFN-γ活性失调 相关的疾病或症状。
8. 如权利要求 7所述的用途，其特征在于，所述活性失调是活性过高或活性过低。
9. 如权利要求 7所述的用途， 其特征在于， 所述与 FOXP3、 IL-2和 /或 IFN-γ活性 失调相关的疾病或症状选自： 肿瘤、 炎症反应、 急性传染性疾病或慢性传染性疾病。
10. 如权利要求 9所述的用途， 其特征在于， 所述肿瘤选自： 前列腺癌、 乳腺癌、 肝癌、 胶质瘤、 肠癌、 子宫颈癌、 非小细胞肺癌、 肺癌、 胰腺癌、 胃癌、 膀胱癌、 皮肤癌、 横纹肌癌、 舌鳞癌、 鼻咽癌、 卵巢癌、 胎盘绒毛癌、 神经胶质瘤、 淋巴 瘤、 白血病、 直肠腺癌或黑色素瘤；
所述炎症反应选自： 过敏性炎症、 毛囊炎、 扁桃体炎、肺炎、 肝炎、 肾炎、 痤疮、 哮喘、 自身免疫性疾病、 慢性炎症、 慢性前列腺炎、 肾小球肾炎、 超敏反应、 炎性肠 道疾病、 盆腔炎、 再灌注损伤、 类风湿关节炎、 移植排斥反应、 血管炎或间质性膀胱 炎；
所述传染性疾病选自： 鼠疫、 霍乱、 传染性非典型肺炎、 艾滋病、 病毒性肝炎、 脊髓灰质炎、 人感染高致病性禽流感、 麻疹、 流行性出血热、 狂犬病、 流行性乙型脑 炎、 手足口病、 登革热、 炭疸、 细菌性和阿米巴性痢疾、 肺结核、 伤寒和副伤寒、 流 行性脑脊髓膜炎、 百日咳、 白喉、 新生儿破伤风、 猩红热、 布鲁氏菌病、 淋病、 梅毒、 钩端螺旋体病、 血吸虫病、 疟疾、 流行性感冒、 流行性腮腺炎、 风疹、 急性出血性结 膜炎、 麻风病、 流行性和地方性斑疹伤寒、 黑热病、 包虫病、 丝虫病， 除霍乱、 细菌 性和阿米巴性痢疾、 伤寒和副伤寒以外的感染性腹泻病、 或真菌感染。
11. 如权利要求 1所述的用途， 其特征在于， 所述调节 FOXP3活性、 IL-2活性和 / 或 IFN-γ活性的组合物进一步用作疫苗佐剂或疫苗。
12. 如权利要求 11所述的用途， 其特征在于， 所述疫苗选自： 治疗或预防病毒感 染、 细菌感染、 炎症、 寄生虫感染或肿瘤的疫苗。
13. 如权利要求 11所述的用途， 其特征在于， 所述疫苗是手足口病疫苗。
14. 如权利要求 1所述的用途， 其特征在于， 所述组合物是药物组合物、 保健品 组合物或疫苗组合物。
15. 一种调节 FOXP3活性、 IL-2活性和 /或 IFN-γ活性的组合物， 其包含：
(a)—种或多种选自下组的泛素化途径相关因子： 泛素化连接酶、 Toll样受体、 促 炎症细胞因子家族受体和 /或它们的编码序列； 和 /或所述泛素化途径相关因子的激动 剂或拮抗剂； 和
(b)药学上、 免疫学上或保健品学上可接受的载体。
16. 如权利要求 15所述的组合物， 其特征在于，
所述泛素化连接酶是选自下组中的一种或多种： STUB1、 TRAF6、 SmurfK Smurf2、 TRAF2、 TRAF3、 TRAF4、 p300、 RNF31或 RBCK1 ;
所述 Toll样受体是选自下组中的一种或多种： TLR1、 TLR2、 TLR3、 TLR4、 TLR5、 TLR7, TLR8或 TLR9; 所述促炎症细胞因子家族受体选自： IL-6R、 TGFP受体、 IL-2 受体、 TNF- α受 体或 GITR。
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