Method for reducing immune function of dendritic cells by 27-site single ubiquitin molecule

Abstract

The invention discloses a method for reducing the immune function of dendritic cells by using 27-site single ubiquitin molecules, and relates to the technical field of gene engineering. The 27-site single ubiquitin molecule is a ubiquitin molecule only retaining 27-site lysine, dendritic cells are separated from mouse bone marrow, the bone marrow-derived dendritic cells are pretreated with the 27-site single ubiquitin molecule, then in-vitro adoptive immunization is carried out, and finally an enzyme-linked immunosorbent assay spot experiment and a flow cytometry experiment are carried out. Experiments prove that the antigen presenting capability of the treated dendritic cells is obviously reduced, and the antigen-specific CTL inducing capability of the treated dendritic cells is also reduced; the 27-site single ubiquitin molecule can reduce the capability of generating IFN-gamma, perforin and granzyme B by lymphocytes in a dendritic cell transfer receptor. The 27-site single ubiquitin molecule inhibits splenocytes of a transfer receptor to generate perforin and granzyme B through a lysosome way.

Description

Technical Field

[0001] The present invention relates to the technical field of genetic engineering, and particularly relates to a method for reducing the immunological function of dendritic cells by single ubiquitin molecules at 27 sites. Background Art

[0002] Dendritic cells (DCs) are professional antigen-presenting cells that play a role in innate immunity and adaptive immunity. The cytotoxic T cells (CTLs) induced by them play an important role in killing tumors and clearing intracellular pathogenic microorganisms. The activation and proliferation of CTLs mainly depend on the MHC class I molecule presentation and cross-presentation pathways of DCs for antigens. The MHC class I molecule presentation pathway refers to the process in which endogenous antigens are degraded into antigen peptides and loaded onto MHC class I molecules, and then presented to CD8+ T cells; cross-presentation refers to the process in which exogenous antigens are taken up, processed and then combined with MHC class I molecules by DCs to form an antigen peptide-MHC class I molecule complex, and then presented to CTLs. The killing of tumor cells and virus-infected host cells by CTLs involves the release of effects mediated by perforin and granzyme B. Perforin is a Ca2+-dependent hydrophobic molecule that can form channels on the target cell membrane and further induce granzyme to enter the cytoplasm of the target cell, promoting apoptosis of the target cell. Since the relative molecular mass of exogenous antigens is relatively large, it is restricted to process antigens through the endogenous pathway and play a role in killing target cells. Therefore, the clearance of tumors and viruses by CTLs induced by DC antigen presentation is very important (Fu C, Jiang A. Dendritic cells and CD8T cell immunity in tumor microenvironment [J]. Front Immunol, 2018, 9: 3059. DOI: 10.3389 / fimmu.2018.03059).

[0003] The ubiquitin molecule (Ub) consists of 76 amino acids and is widely expressed in nucleated cells and the cytoplasm. The complete ubiquitination process requires the cooperation of ubiquitin-activating enzyme E1, conjugating enzyme E2, and ligase E3. Ubiquitination affects CD86 expression and MHC-II molecule trafficking via MARCH1 (membrane-associated RING-CH type finger 1), suggesting that the ubiquitination-regulated pattern recognition receptor signaling pathway plays a role in regulating innate immune responses and dendritic cell-mediated adaptive immunity. In a Listeria infection model of UCH-L1 knockout mice, it was found that the deubiquitinating enzyme UCH-L1 can reduce MHC-I molecule recycling, decrease its co-localization with late endosomes / lysosomes, and negatively regulate cross-presentation, thereby affecting CTL induction mediated by DCs, suggesting that the ubiquitination process is involved in the adaptive immune response of the body.

[0004] During the ubiquitination process, proteolysis targets the 26S proteasome, and the specificity of ubiquitinated substrates is determined by E3 ligases, which can be divided into RING (really interesting new gene) type E3 ligases and HECT (homologous to the E6AP carboxyl terminus) type E3 ligases. When polyubiquitin chains are formed, the carboxyl-terminal glycine residue of the ubiquitin molecule binds to the lysine residue or methionine residue of another ubiquitin molecule, and is classified into ubiquitination in ways such as K6, K11, K27, K29, K33, K48, and K63 according to the lysine position where ubiquitination occurs. (Embgenbroich M, Burdorfs. Current concepts of antigen cross-presentation[J].Front Immunol. 2018;9:1643.)

[0005] The processes of ubiquitination and deubiquitination are of great significance in the maturation, migration, adaptive immunity, and maintenance of homeostasis of DCs. You X et al. inhibited the ubiquitination of the NF-κB pathway with the ubiquitination inhibitor PYR-41 or thalidomide, and observed a decrease in the recruitment of p97 and Sec61 to endosomes and a reduction in the cytoplasmic translocation of internalized antigens, thereby impairing the cross-presentation ability of DCs. When DCs undergo endocytosis, the ubiquitination of the MHC-II molecule B chain can regulate the expression of MHC-II molecules. (Jancic C, Savina A, Wasmeier C, et al. Rab27a regulates phagosomal pH and NADPH oxidase recruitment to dendritic cell phagosomes[J]. Nat Cell Biol. 2007;9(4): 367-378). Both MARCH1 and MARCH8 can ubiquitinate MHC-II molecules. MARCH1 negatively regulates the ubiquitination of CD86, inhibiting the autocrine of IL-10 and the induction of CTLs. Hrd1, a transmembrane protein in the endoplasmic reticulum, is responsible for targeting misfolded proteins to the proteasome for degradation. During inflammation, Hrd1 promotes the ubiquitination and subsequent autodegradation of BIMP-1 by interacting with BIMP-1 (B lymphocyte-induced maturation protein 1), mediating the activation of CD4+ T cells. Rhbdd3 promotes the K27 ubiquitination of lysine 302 of NEMO (IKKr) through its ubiquitin-binding-association (UBA) domain, recruits the deubiquitinase A20 to ubiquitinate lysine 268 with K27, thereby inhibiting the K63 ubiquitination of NEMO and suppressing the activation of the NF-κB pathway (Samie M, Cresswell P. The transcription factor TFEB acts as a molecular switch that regulates exogenous antigen-presentation pathways[J]. Nat Immunol. 2015;16(7):729-736.). Summary of the Invention

[0006] The object of the present invention is to provide a method for reducing the immune function of dendritic cells by 27-site monoubiquitin molecules in view of the problems such as the lack of controllable immunosuppressive means in the prior art; the inhibition of perforin and granzyme B production by transfusing receptor splenocytes by 27-site monoubiquitin molecules is achieved through the lysosomal pathway. Precise immune regulation is realized by specifically down-regulating the antigen presentation function and CTL activity.

[0007] The present invention includes the following steps:

[0008] 1) Isolate dendritic cells from mouse bone marrow;

[0009] 2) Pretreat bone marrow-derived dendritic cells with 27-site monoubiquitin molecules and exogenous antigens, and then perform adoptive transfer in vitro. Transfer the treated DCs to recipient mice by adoptive transfer;

[0010] 3) Detect the expression of effector molecules of CTL in the spleen of recipient mice.

[0011] In step 1), the specific method for isolating dendritic cells from mouse bone marrow can be as follows: First, euthanize the mouse by dislocation. After alcohol disinfection, aseptically isolate the femur and tibia, remove the redundant muscle tissue, and flush out the bone marrow until the bone turns from red to grayish white. Grind the bone marrow into a single-cell suspension with the bottom of a sterile syringe; After centrifugation and discarding the supernatant, add erythrocyte lysate to lyse erythrocytes. After the solution turns from red to white, add an equal volume of complete culture medium to terminate; Centrifuge again and discard the supernatant, and adjust the cell concentration to 1×106 cells / mL with the culture medium; Add cytokines GM-CSF and IL4 to make their final concentrations 30 ng / mL and 1 ng / mL; Incubate in an incubator; On the 4th day, discard the suspended cells in the supernatant, wash the bottom of the dish with PBS, and continue to culture the cells adherent to the bottom of the dish for 3-4 days, which are the bone marrow-derived dendritic cells.

[0012] In step 2), the specific steps of the adoptive immunization experiment in vitro can be as follows: Add 27-site monoubiquitin molecules (5-30 μM) and exogenous antigens (ovalbumin, OVA 50-100 μg / mL) to induced DCs on Day 6-7, and after acting for 4-6 h, collect the cells into a centrifuge tube, resuspend the cells with PBS and centrifuge to remove the influence of excess 27-site monoubiquitin molecules and chicken ovalbumin; Resuspend the cells with an appropriate amount of normal saline again and adjust the cell concentration, and transfer the same number of dendritic cells into C57BL / 6 female mice with the same genetic background through the abdominal cavity; To find that the inhibition of the immune function of dendritic cells by 27-site monoubiquitin molecules is achieved through the lysosomal pathway, the induced dendritic cells are blocked with Bafilomycin A1 10 nM in advance to block the lysosomal function, and then the steps of 27-site monoubiquitin molecule and antigen stimulation and dendritic cell transfer are carried out. Take the spleen and lymph nodes after 5-7 days to make cell suspensions for subsequent experiments.

[0013] In step 3), to detect the effector molecule expression of CTL in the spleen of the recipient mice for verifying the inhibitory effect of monoubiquitin molecule at site 27 on the immune function of bone marrow progenitor cells, enzyme-linked immunosorbent spot assay (ELISPOT) and flow cytometry can be performed.

[0014] The specific steps of the ELISPOT assay can be as follows: First, prepare the concentration of the mouse spleen suspension. Dislocate and sacrifice the adoptive immunization experimental mice and aseptically isolate the spleens. After lysing the red blood cells with red blood cell lysate, adjust the suspension concentration to 1×10 7 / mL; Second, activate the pre-coated IFN-γ antibody detection plate with complete culture medium, add the suspension, that is, 1×10 6 / well, add SIINFEKL at 2 - 4 μg / mL, and culture for 16 - 24 h; After hypotonic lysis of the cells with pre-cooled double-distilled water, successively add biotin-IFN-γ and avidin-HRP antibodies and wash the plate with washing buffer during incubation; Finally, freshly prepare the AEC chromogenic solution for chromogenic reaction, and rinse with water to terminate the experiment. After the detection plate is naturally air-dried, take pictures with an ELISPOT image analysis system and count the number of spots.

[0015] The specific steps of the flow cytometry can be as follows:

[0016] Intracellular staining: To detect the expression of IFN-γ, perforin, and granzyme B in CD8+ T lymphocytes in mouse spleen cells after the action of DCs, first grind the mouse spleen cells to make a spleen cell suspension, then stain the spleen cells with CD8 antibody at the recommended concentration in the manual in the dark at 4°C, and then fix and permeabilize the cells with Fix / Permeabilization Solution in the dark at 4°C for 20 min. Then wash with PBS, and combine each antibody with the appropriate volume of staining buffer according to the recommended amount in the manual, stain in the dark at 4°C for 30 min, and wash the cells with flow cytometry staining buffer. Finally, perform upper machine detection with a FACSCalibur flow cytometer, and analyze the obtained data using Cytexpert software.

[0017] The bone marrow progenitor cells treated by the described method can be used in the preparation of research tools related to reducing the immune function of dendritic cells.

[0018] The bone marrow progenitor cells treated by the described method can be used in the preparation of research tools related to inhibiting the antigen-specific CTL induction ability mediated by dendritic cells.

[0019] The bone marrow progenitor cells treated by the described method can be used in the preparation of research tools related to reducing the ability of lymphocytes in dendritic cell transfer recipients to produce IFN-γ, perforin, and granzyme B.

[0020] Compared with the prior art, the present invention has the following outstanding advantages:

[0021] The present invention uses monoubiquitin molecule at site 27 to treat dendritic cells. Experiments have shown that the treated dendritic cells can significantly reduce the antigen presentation ability of dendritic cells and inhibit the ability to induce antigen-specific CTL mediated by dendritic cells; monoubiquitin molecule at site 27 can reduce the ability of lymphocytes in dendritic cell transfer receptors to produce IFN-γ, perforin, and granzyme B. The inhibition of perforin and granzyme B production by monoubiquitin molecule at site 27 in transfer receptor spleen cells is achieved through the lysosomal pathway. The treated bone marrow progenitor cells have potential application value in the preparation of immune-related research tools, can be used to develop research models for regulating the immune function of dendritic cells, contribute to exploring the pathogenesis of immune-related diseases, provide new ideas and targets for disease treatment and drug research and development, and are of great significance for promoting basic research and clinical applications in the field of immunology. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 Microscopic photographs of mouse dendritic cells induced by GM-CSF and IL-4 for 4 days and 6 days.

[0023] Figure 2 Diagram showing the inhibition of antigen-specific CTL production in transfer receptor spleen cells by monoubiquitin molecule at site 27.

[0024] Figure 3 Diagram showing the inhibition of perforin production in transfer receptor spleen cells by monoubiquitin molecule at site 27.

[0025] Figure 4 Diagram showing the reduction of enhanced granzyme B production in transfer receptor spleen cells by monoubiquitin molecule at site 27.

[0026] Figure 5 Diagram showing the reduction of IFN-γ production in transfer receptor spleen cells by monoubiquitin molecule at site 27.

[0027] Figure 6 Diagram showing the inhibition of perforin production in transfer receptor spleen cells by monoubiquitin molecule at site 27 through the lysosomal pathway.

[0028] Figure 7 Diagram showing the inhibition of granzyme B production in transfer receptor spleen cells by monoubiquitin molecule at site 27 through the lysosomal pathway. DETAILED DESCRIPTION OF THE INVENTION

[0029] The following examples will further illustrate the present invention in conjunction with the accompanying drawings. Those not specifically noted adopt the prior art.

[0030] In the embodiment of the present invention, dendritic cells were treated with 27-site monoubiquitin molecules, and the induction ability of dendritic cells to mediate antigen-specific CTL was detected by enzyme-linked immunosorbent assay (Elispot) and flow cytometry.

[0031] The embodiment of the present invention includes the following steps:

[0032] 1) Induction of bone marrow-derived dendritic cells

[0033] First, the mice were killed by dislocation. After disinfection with alcohol, the femur and tibia were aseptically separated with ophthalmic scissors and ophthalmic forceps, and the excess muscle tissue was removed. Secondly, the bone marrow was flushed out with RMPI 1640 complete culture medium until the bone changed from red to grayish white. The bone marrow was ground into a single cell suspension with the bottom of a sterile syringe. After centrifugation and discarding the supernatant, red blood cell lysis solution was added to lyse the red blood cells. After the solution changed from red to white, an equal volume of complete culture medium was added to terminate the suspension. Centrifugation was performed again, the supernatant was discarded, and the cell concentration was adjusted to 1×10 6 / mL; add cytokines GM-CSF and IL-4 to make the final concentrations of 30 ng / mL and 1 ng / mL; culture in a 37°C, 5% CO2 incubator; finally, on the 4th day, discard the suspended cells in the supernatant, rinse the bottom of the dish with PBS, and continue to culture the cells attached to the bottom of the dish for 3 to 4 days, which are bone marrow-derived dendritic cells.

[0034] 2) Pretreatment of bone marrow-derived dendritic cells.

[0035] (1) In vitro adoptive immunization experiments

[0036] On the 6th to 7th day of dendritic cell induction, 27-site monoubiquitin molecules (5-30 μM) and exogenous antigens (ovalbumin, OVA 50-100 μg / mL) were added. After 4-6 hours of action, the cells were collected into centrifuge tubes. The cells were resuspended in PBS and centrifuged to remove the effects of excess 27-site monoubiquitin molecules and chicken ovalbumin. This operation can be repeated 2 to 3 times to remove excess 27-site monoubiquitin molecules and chicken ovalbumin as much as possible, while avoiding excessive operation to affect cell activity; the cells were resuspended again with an appropriate amount of saline and the cell concentration was adjusted, and the same number of DC cells were transfused into C57BL / 6 female mice with the same genetic background via the peritoneal cavity. In order to find that the 27-site monoubiquitin molecule inhibited the immune function of dendritic cells through the lysosomal pathway, the induced dendritic cells were pre-treated with BafilomycinA1 10 nM to block the lysosomal function, and then the 27-site monoubiquitin molecule and antigen stimulation and dendritic cell transfusion steps were performed. After 5 to 7 days, the spleen and lymph nodes were taken out to make cell suspension for subsequent experiments.

[0037] (2) Enzyme-linked immunospot assay (Elispot)

[0038] First, prepare the concentration of mouse spleen suspension. Dislocate and sacrifice the adoptive immunization experimental mice, and aseptically isolate the spleens. After lysing the red blood cells with red blood cell lysate, adjust the suspension concentration to 1×10 7 / mL; Second, activate the pre-coated IFN-γ antibody detection plate with complete culture medium for 20 min, add 100 μL of the suspension, that is, 1×10 6 / well, add SIINFEKL (2 - 4 μg / mL), and culture for 16 - 24 h; Then, hypotonic-lyse the cells with pre-cooled double-distilled water, and successively add biotin-IFN-γ and avidin-HRP antibodies. Wash the plate with washing buffer during incubation; Finally, freshly prepare the AEC chromogenic solution for chromogenic reaction, and rinse with water to terminate the experiment. After the detection plate is naturally dried, take pictures with an ELISPOT image analysis system and count the number of spots.

[0039] The statistical method is as follows:

[0040] The measurement data of each group are expressed as mean ± standard deviation (Sd). GraphPad Prism 5 is used for statistical analysis, and the difference is analyzed by the student t test (Student's t test). P < 0.05 indicates that the difference is statistically significant.

[0041] The morphology of DCs induced by GM-CSF and IL4 for 4 - 6 days is shown in Figure 1 , the figure of the single ubiquitin molecule at site 27 enhancing the transfer receptor spleen antigen-specific CTL is shown in Figure 2 , for DCs induced by GM-CSF and IL4, simultaneously administer the single ubiquitin molecule at site 27 (5 - 30 μM) and exogenous OVA (50 - 100 μg / mL), and transfer an equal number of DCs into syngeneic C57BL / 6 mice via intraperitoneal injection. 5 - 7 days after the DCs are transferred into the mice, take the mouse spleen cells (spleen, SP) for the Elispot experiment. The data are shown as mean ± standard deviation, and the student t test (Student's t test) is used between groups, ***P < 0.001, n = 3. OVA: ovalbumin.

[0042] Experimental results: Microscopic photographs show that dendritic cells can be successfully induced from mouse bone marrow with GM-CSF and IL4. The Elispot experimental results show that the ability of dendritic cells treated with the single ubiquitin molecule at site 27 to induce the generation of spleen cell-specific CTL after loading exogenous antigen and transferring it into mice is reduced by 55%.

[0043] Conclusion: The immune function of DCs treated with single ubiquitin molecule at site 27 combined with OVA antigen is reduced, and the ability to induce the generation of antigen-specific CTLs in the spleen is further decreased.

[0044] Figure showing the enhanced expression of perforin in splenocytes of transfer receptors by single ubiquitin molecule at site 27 is shown in Figure 3 , for DCs induced by GM-CSF and IL-4, after treatment with single ubiquitin molecule at site 27 (5 - 30 μM) and exogenous OVA (50 - 100 μg / mL), an equal number of DCs were transferred intraperitoneally into syngeneic C57BL / 6 mice. 5 - 7 days after the transfer of DCs into mice, proteins were extracted from the small spleens, and the expression of perforin was detected by flow cytometry. The results were presented as histograms analyzed by Prism. OVA: ovalbumin.

[0045] Results: Flow cytometry results showed that with the RID value of the control group set to 1, the production of perforin by CTLs mediated by DCs after treatment with single ubiquitin molecule at site 27 decreased by 49%.

[0046] Conclusion: The perforin secreted by spleen CTLs mediated by DCs treated with single ubiquitin molecule at site 27 combined with OVA antigen is decreased.

[0047] Figure showing the enhanced expression of granzyme B in splenocytes of transfer receptors by single ubiquitin molecule at site 27 is shown in Figure 4 . DCs induced by GM-CSF and IL-4 were treated with single ubiquitin molecule at site 27 (5 - 30 μM) and exogenous OVA (50 - 100 μg / mL), and an equal number of DCs were transferred intraperitoneally into syngeneic C57BL / 6 mice. 5 - 7 days later, after extracting proteins from the mouse spleens, the expression of granzyme B was detected by flow cytometry, and the results were presented as histograms analyzed by Prism. OVA: ovalbumin.

[0048] Experimental results: Flow cytometry results showed that with the control group value set to 1, the production of granzyme B by CTLs mediated by DCs after treatment with single ubiquitin molecule at site 27 decreased by 25%.

[0049] Conclusion: The secretion of granzyme B by CTLs mediated by DCs loaded with OVA antigen by single ubiquitin molecule at site 27 is reduced.

[0050] Figure showing the enhanced expression of IFN-γ in splenocytes of transfer receptors by single ubiquitin molecule at site 27 is shown in Figure 5DCs induced by GM-CSF and IL-4 were treated with monoubiquitin molecules at site 27 (5 - 30 μM) and exogenous OVA (50 - 100 μg / mL), and an equal number of DCs were transferred into syngeneic C57BL / 6 mice via intraperitoneal injection. After 5 - 7 days, the spleen of the mice was taken to extract proteins, and the expression of IFN-γ was detected by flow cytometry and presented as a histogram analyzed by Prism. OVA: ovalbumin

[0051] Results: Experimental results: Flow cytometry results showed that if the value of the control group was set as 1, the production of IFN-γ mediated by CTL by DCs treated with monoubiquitin molecules at site 27 decreased by 64%.

[0052] Conclusion: The secretion of IFN-γ by spleen CTL mediated by DCs treated with monoubiquitin molecules at site 27 combined with OVA antigen decreased.

[0053] The inhibition of perforin production by transporter receptor splenocytes by monoubiquitin molecules at site 27 via the lysosomal pathway is shown in Figure 6 For DCs induced by GM-CSF and IL-4, the lysosomal function was blocked in advance with 10 nM Bafilomycin A1, and then they were treated with monoubiquitin molecules at site 27 (5 - 30 μM) and exogenous OVA (50 - 100 μg / mL), and an equal number of DCs were transferred into syngeneic C57BL / 6 mice via intraperitoneal injection. 5 - 7 days after the transfer of DCs into the mice, the spleen of the mice was taken to extract proteins, and the expression of perforin was detected by flow cytometry and presented as a histogram analyzed by Prism. OVA: ovalbumin.

[0054] Results: Flow cytometry results showed that when the RID value of the control group was set as 1, the production of perforin mediated by CTL by DCs treated with monoubiquitin molecules at site 27 decreased by 79%. After blocking the lysosomal function with 10 nM Bafilomycin A1, the production of perforin mediated by CTL by DCs treated with monoubiquitin molecules at site 27 returned to 93%.

[0055] Conclusion: The inhibition of perforin production by transporter receptor splenocytes by monoubiquitin molecules at site 27 is achieved via the lysosomal pathway.

[0056] The inhibition of granzyme B production by transporter receptor splenocytes by monoubiquitin molecules at site 27 via the lysosomal pathway is shown in Figure 7DCs induced by GM-CSF and IL-4 were pre-blocked with 10 nM Bafilomycin A1 to inhibit lysosomal function, and then treated with monoubiquitin molecules at site 27 (5 - 30 μM) and exogenous OVA (50 - 100 μg / mL). An equal number of DCs were transferred into syngeneic C57BL / 6 mice via intraperitoneal injection. After 5 - 7 days, the spleens of the mice were collected to extract proteins, and the expression of granzyme B was detected by flow cytometry. The bar graph obtained by Prism analysis is presented. OVA: ovalbumin.

[0057] Experimental results: The flow cytometry results showed that if the control group value was set to 1, the production of granzyme B mediated by CTL by DCs treated with monoubiquitin molecules at site 27 decreased to 65%. After blocking lysosomal function with 10 nM Bafilomycin A1, the production of perforin mediated by CTL by DCs treated with monoubiquitin molecules at site 27 recovered to 78%.

[0058] Conclusion: The inhibition of granzyme B production by transfer receptor splenocytes by monoubiquitin molecules at site 27 is achieved through the lysosomal pathway.

[0059] The above embodiments are only preferred embodiments of the present invention and should not be considered as limiting the scope of implementation of the present invention. All equivalent changes and improvements made within the scope of the application of the present invention should still fall within the scope covered by the patent of the present invention.

Claims

1. A method for reducing the immune function of dendritic cells by using a monoubiquitin molecule at site 27, characterized in that The following steps are involved: 1) Dendritic cells were isolated from mouse bone marrow; 2) pre-treating bone marrow-derived dendritic cells with 27-site monoubiquitin molecules and exogenous antigens, and then adoptively transferring the treated DCs into recipient mice in vitro; 3) Detect the expression of effector molecules of CTL in the spleen of recipient mice.

2. The method for reducing the immune function of dendritic cells by using a 27-site monoubiquitin molecule as claimed in claim 1, characterized in that In step 1), the specific method for isolating dendritic cells from mouse bone marrow is as follows: first, the mouse is killed by cervical dislocation, and after disinfection with 75% alcohol, the femur and tibia are aseptically separated, excess muscle tissue is removed, and the bone marrow is flushed out as completely as possible until the bone changes from red to grayish white, and the bone marrow is fully ground into a single cell suspension with the bottom of a sterile syringe; after centrifugation and discarding the supernatant, red blood cell lysis solution is added to lyse the red blood cells, and after the solution changes from red to white, an equal volume of complete culture medium is added to terminate; centrifugation is performed again, the supernatant is discarded, and the cell concentration is adjusted to 1×10 6 / mL; add cytokines IL4 and GM-CSF to make the final concentrations of 1 ng / mL and 30 ng / mL, and culture them in an incubator; on the 4th day, discard the suspended cells in the supernatant, rinse the bottom of the dish with PBS, and continue to culture the cells attached to the bottom of the dish for 2 to 4 days, which are bone marrow-derived dendritic cells.

3. The method for reducing the immune function of dendritic cells by using the 27-site monoubiquitin molecule as claimed in claim 1, characterized in that In step 2), the specific steps of the in vitro adoptive immunization experiment are as follows: adding 27-site monoubiquitin molecules and exogenous antigen chicken ovalbumin OVA to the dendritic cells obtained in step 1), after the action, collecting the cells into a centrifuge tube, resuspending the cells with PBS and centrifuging to remove the influence of excess 27-site monoubiquitin molecules and chicken ovalbumin; resuspending the cells again with an appropriate amount of physiological saline and adjusting the cell concentration, and transferring the same number of dendritic cells into C57BL / 6 female mice with the same genetic background through the peritoneal cavity; taking the spleen after 5 to 7 days to prepare a cell suspension for subsequent experiments.

4. The method for reducing the immune function of dendritic cells by using a 27-site monoubiquitin molecule as claimed in claim 3, characterized in that Add 27-site monoubiquitin molecule 5-30 μM and exogenous antigen chicken ovalbumin OVA 50-100 μg / mL and let act for 4-6 hours.

5. Use of bone marrow precursor cells treated by the method according to any one of claims 1 to 4 in preparing research tools related to reducing the immune function of dendritic cells.

6. Use of bone marrow precursor cells treated by the method of any one of claims 1 to 4 in preparing research tools for inhibiting the ability of dendritic cells to induce antigen-specific CTLs.

7. Use of bone marrow precursor cells treated by the method according to any one of claims 1 to 4 in the preparation of a research tool for reducing the ability of lymphocytes in recipients of dendritic cells to produce IFN-γ, perforin and granzyme B.

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