CN111808800B - In-vitro induced immunosuppressive myeloid suppressor cell and preparation and application thereof - Google Patents
In-vitro induced immunosuppressive myeloid suppressor cell and preparation and application thereof Download PDFInfo
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Abstract
The invention relates to a preparation and acquisition method of in vitro induced immunosuppressive myeloid suppressor cells, belonging to the fields of cytobiology and clinical application; the invention particularly discloses immunosuppressive Myeloid suppressor cells (MDSCs), which are derived from healthy donated individuals and generate immunosuppressive property through in vitro drug induced stimulation, and a preparation and purification method thereof; the invention discovers that the in vitro induction of immunosuppressive myeloid suppressor cells can effectively inhibit CD4 in vitro + The application of the invention can prepare a biological preparation for effectively treating rejection after corneal transplantation and effectively inhibiting pathological neovascularization.
Description
Technical Field
The invention relates to a preparation and acquisition method of in vitro induced immunosuppressive myeloid suppressor cells, belonging to the fields of cytobiology and clinical application.
Background
Myeloid Suppressor Cells (MDSCs) are Myeloid progenitor Cells that are immature in Myeloid-Derived cell morphology and can differentiate downstream into mature granulocytes, macrophages or dendritic Cells under certain circumstances. In mice, MDSCs characteristically co-express surface molecule Myeloid Differentiation Antigen (Myeloid-cell Differentiation Antigen-1, Gr-1) and CD11b (i.e., α M-integrin). A rough classification into granular MDSCs was made on the basis of the expression of the subgroups Ly6C and Ly6G of Gr-1 in MDSCs (CD11 b) + Gr-1 + Ly6G high Ly6C - PMN-MDSC) and mononuclear MDSC (CD11 b) + Gr-1 + Ly6G - Ly6C high M-MDSC) two groups (VegliaF, Perego M, gabriovich d.myeloid-derived supressor cells communication of age.nat immunol.2018; 19(2):108-119.).
Research reports that under the tumor and inflammation states, MDSC can induce the differentiation and proliferation of regulatory T cells (Treg), influence the Treg/Th17 balance, promote the polarization of M2 macrophages, enhance the expression of Arg-1, Nos2 and Nox2, and inhibit the effector T cells (CD11 b) through the direct contact of cells and the indirect action of cell secretion + Gr-1 + Ly6G - Ly6C high M-MDSC) two groups (VegliaF, Perego M, Gabrilovich d.myoloid-derived supressor cells communing of age.nat immunol.2018; 19(2):108-119.). Physiological state CD11b isolated from normal mouse bone marrow + Gr-1 + MDSC(
MDSC, nMDSC) has no immunosuppressive function, and MDSC induced in vivo in pathological state, such as CD11b selected in marrow of sepsis mouse and liver cancer-bearing mouse + Gr-1 + MDSC has strong immunosuppressive property and can effectively inhibit CD4 + And CD8 + T cells proliferate and have a dose-dependence (Yan He, Beibei Wang, Bei Jia, Jieying Guan, Hui Zeng, Zhiqiang Pan. Effects of adaptive transduction sources of fungal depleted supra cell in micro cornual transfer Survival. transplantation 2015; 99: 2102-2108.), but in vivo pathological environment induction is effective in inhibiting functional MDSC but at high risk.
MDSCs have been shown to contribute to neovascularization and lymphangiogenesis in tumor-burdened models and to aid in tumor escape and metastasis (Veglia F, Perego M, Gabriovich D.Myeloid-derived donor cells comorbing of Nat Immunol.2018; 19(2): 108-. We observed that after MDSC was cleared in allogeneic corneal transplantation mice by anti-Gr-1 antibody, the bed and the graft were rapidly vascularized, and a large amount of new blood vessels and lymphatic vessels were grown into the graft in the early stage after the operation; while The simultaneous adoptive transfer of in vivo or in vitro Induced MDSCs in Corneal transplantation surgery also showed strong inhibitory Effects on Corneal graft neovascularization and neonatal lymphangiogenesis while inhibiting rejection (Yan He, Beibei Wang, Bei Jia, joining Guan, Hui Zeng, Zhijiang Pen. Effect of adaptive transfer differential sources of Myel Derived superior Cells in microcornual transfer Surviability 2015; 99: 2102.) (Yan He, Bei Jia, Hui Zeng, Zhijiang, The Roles of Sepsis-Induced superior Cells in vitro transfer tissue culture, culture 2016, 13).
With the intensive research of MDSC in tumor, transplantation and autoimmune disease, the requirement for stable acquisition of functional MDSC is outstanding, but the MDSC has great variation in immune function and yield under different induction environments, so that how to stably and efficiently induce functional MDSC in vitro for research and future application is significant and has no relevant research for a while.
Disclosure of Invention
In order to solve the technical problems of main in vivo extraction, unstable effect and lack of in vitro induction technology of the existing immunosuppressive myeloid suppressor cells, a first objective of the invention is to provide a preparation method of in vitro induced immunosuppressive myeloid suppressor cells, and to realize high-yield in vitro directed induction preparation of immunosuppressive function MDSCs (ex vivo generated MDSC by cytokine-induced differentiation of bone marrow cells, evmdscs).
The second objective of the invention is to provide evMDSC induced by the in vitro induction method.
The third purpose of the invention is to provide the application of the in vitro induction method to obtain the evMDSC.
A method for preparing in vitro induced immunosuppressive myeloid suppressor cell comprises culturing myeloid suppressor cell in vitro under induction of inducer;
the inducer comprises cytokines GM-CSF and IL-6.
The invention provides a preparation method of evMDSC by in vitro directional induction. The research of the invention finds that GM-CSF and IL-6 have cooperativity, and evMDSC with high activity and high yield can be obtained at lower concentration. Moreover, the research of the inventor also finds that the evMDSC prepared by the synergistic induction of GM-CSF and IL-6 has better effects in inhibiting the neovascular and lymphatic vessels of corneal transplantation and prolonging the life cycle of corneal transplantation compared with the evMDSC obtained by in vivo extraction and other ways.
Research finds that on the basis of the synergistic induction of GM-CSF and IL-6, the further control of the matching proportion and concentration is helpful for further improving the yield of evMDSC and further promoting the selectivity of evMDSC subtype (PMN-evMDSC) with better effect on corneal transplantation.
Preferably, the mass ratio of the GM-CSF to the IL-6 is 0.5-1: 0.5-1; more preferably 1: 1.
in the invention, the concentration of the inducer is 1-20 ng/mL; preferably 8-12 ng/mL. The inducer of the invention can unexpectedly obtain excellent directional induction effect at a lower content based on the synergy of GM-CSF and IL-6, for example, higher content of evMDSC can be obtained, and the selectivity of active cell subtype is improved.
In the invention, the myeloid suppressor cell is a bone marrow cell of a healthy donor. For example, the myeloid-suppressor cells are bone marrow cells of healthy mice.
The method of the invention is obtained by culturing myeloid suppressor cells in a culture medium containing the inducer.
In the invention, the culture medium can be obtained by matching the inducer with the required content on the basis of the existing conventional culture medium suitable for the growth of bone marrow cells.
Preferably, the culture medium comprises 8-12% of fetal calf serum, 85-95% of 1640 culture medium, 0.5-1.5% of penicillin-streptomycin double antibody and 1-20 ng/mL of inducer.
Preferably, the culture conditions are: at 37 + -0.2 deg.C and 5 + -0.1% CO 2 Culturing for 5 days or longer; preferably 7 to 9 days. It was found that the days of culture, unexpectedly, enabled synergy between the factors, contributed to the unexpected improvement of evMDSC content, and significantly improved PMN-evMDSC expression.
In the invention, after in vitro culture, target evMDSC can be separated by adopting the existing means, and subtype identification and determination are carried out by adopting the existing method.
Preferably, after in vitro culture, an immunomagnetic bead separation method is adopted to sort out evMDSC, the number and phenotype of evMDSC are detected through cell counting and flow cytometry, the change of PMN/M-evMDSC expression ratio is detected, Giemsa staining is carried out to identify the cell morphology of two subgroups of evMDSC, more appropriate culture conditions are screened out, and an in vitro induced immunosuppression functional MDSC system is initially established.
The invention also comprises the in-vitro induced immunosuppressive myeloid suppressor cell prepared by the preparation method.
Preferably, the in vitro induced immunosuppressive myeloid-lineage suppressor cell is a PMN-MDSC subtype cell and/or an M-MDSC subtype cell.
The invention also provides application of the in vitro induced immunosuppressive myeloid suppressor cell in preparing immunosuppressive biological agents.
Preferably, it is used for the preparation of immunosuppressive biologics that inhibit CD4+ T cells.
Further preferably, it is used for preparing an immunosuppressive biological agent for suppressing an immune rejection reaction in organ transplantation.
Further preferably, the application of the compound is used for preparing an immunosuppressive biological agent for inhibiting the immune rejection reaction of corneal transplantation.
Still more preferably, it is used for the preparation of immunosuppressive biological agents that inhibit the growth of pathological neovessels and/or neolymphatics of corneal transplants; or the application of the derivative in preparing an immunosuppressive biological agent for prolonging the survival time of a corneal graft for corneal transplantation.
The invention also provides an immunosuppressive biological agent, which comprises the in-vitro induced immunosuppressive myeloid suppressor cell prepared by the preparation method.
Preferably, the in vitro induced immunosuppressive myeloid-lineage suppressor cell is a PMN-MDSC subtype cell.
Preferably, the immunosuppressive biological agent is a corneal transplantation immunosuppressive biological agent.
The research of the invention unexpectedly discovers that the evMDSC, particularly the PMN-MDSC subtype cells obtained by the method can effectively inhibit the growth of new blood vessels and new lymphatic vessels in the aspect of corneal transplantation, and can effectively prolong the life cycle of the graft.
In the invention, the research steps of the animal model and the biological performance are as follows: first, the effect of depleting in vivo MDSCs on pathological neovascularization after penetrating corneal transplantation. Using allogeneic permeable cornea transplantation mice as a model, dividing the model into 3 groups, namely, a group which is subjected to intraperitoneal injection twice per week after operation and anti-Gr-1 antibody, a group which is subjected to intraperitoneal injection twice per week after operation and is subjected to isotype control IgG and an untreated group, and observing pathological angiogenesis conditions of the cornea of each group. The intensity changes in the growth of neovasculature and lymphatic vessels in each group were assessed by immunofluorescent staining of each group of the cornea with antibodies that specifically recognize CD31 (endothelial cell marker) and LYVE-1 (lymphatic endothelial marker). Finally, the effect of in vitro induced adoptive transfer of MDSCs on mouse penetrating corneal transplantation immune tolerance and pathological neovascularization growth was observed. Using allogeneic penetrable cornea transplantation mice as a model, dividing the model into 5 groups, injecting PBS, nMDDSC, evMDSC, PMN-evMDSC and M-evMDSC after non-operative eyeball on the day of operation, and observing median survival period and pathological new blood vessel growth condition of each group of corneal graft.
Advantageous effects
1. The invention provides a brand new thought for in vitro directional induction synthesis of evMDSC; and the discovery shows that the GM-CSF and IL-6 have synergistic induction effect, and can obtain evMDSC with high yield on the premise of reducing the dosage of an inducer, and can improve the selectivity of PMN-evMDSC cell subtype with better performance in the field of corneal transplantation.
2. The evMDSC obtained by the method can effectively inhibit the growth of new blood vessels and new lymphatic vessels in the aspect of corneal transplantation, and can effectively prolong the service cycle of the transplant.
Drawings
FIG. 1 is the ratio of MDSC in vitro culture at different days and different cytokine concentrations in example 1;
FIG. 2 is the ratio of PMN-MDSC in vitro culture on different days and at different cytokine concentrations in example 1;
FIG. 3 is the ratio of M-MDSC in vitro culture on different days and at different cytokine concentrations in example 1;
FIG. 4 shows the morphology of PMN/M-MDSC cells after magnetic bead sorting according to example 1;
FIG. 5 is a flow chart of co-culture proliferation inhibition of MDSC and CD4+ T cells in each group in example 3
FIG. 6 shows the ratio of suppression of proliferation in co-culture of MDSC and CD4+ T cells in each group in example 3
FIG. 7 shows the timing of intraperitoneal injection of anti-Gr-1 or control IgG antibody to mice after corneal transplantation in example 4
FIG. 8 is a visual image of the cornea transplanted in example 4 under a microscope at 7 days and 15 days after the transplantation
FIG. 9 shows corneal immunofluorescence after corneal transplantation in example 4
FIG. 10 shows the ratio of the neovascular perfusion area of cornea after corneal transplantation in example 4
FIG. 11 is the ratio of the perfusion area of corneal neolymphatics after corneal transplantation in example 4
FIG. 12 shows the survival rate of adoptive transfer of MDSC corneal graft after corneal transplantation in example 5
FIG. 13 shows the corneal pathological section of adoptive transfer MDSC after corneal transplantation in example 5
FIG. 14 is a visual image of the cornea after the corneal transplantation as in example 5
FIG. 15 shows corneal immunofluorescence after corneal transplantation in example 5
FIG. 16 shows the ratio of the neovascular perfusion area of cornea after corneal transplantation in example 5
FIG. 17 shows the perfusion area ratio of the corneal neolymphatic vessels after corneal transplantation in example 5
Detailed Description
Example 1
Establishment of in vitro induced immunosuppression functional MDSC system
(1) Obtaining mouse bone marrow cells: and (3) taking bilateral tibiofibulas after 6-8 weeks of male BALB/c mice die in a sterile environment, flushing medullary cavities with PBS, and collecting cells in a centrifuge tube to obtain nMDSC cells.
(2) Preparation of a culture medium: a basic culture medium is prepared according to 10% of fetal bovine serum, 90% of 1640 culture medium and 1% of penicillin-streptomycin double antibody, and different concentrations of cell factors GM-CSF and IL-6(0, 5, 10 and 20 ng/ml; the ratio of GM-CSF to IL-6 is 1: 1) are added.
(3) Cell inoculation and culture: cells were plated at 5X10 5 Inoculating each cell/well into 24-well plate, adding culture medium containing different concentrations of cytokine, and culturing at 37 deg.C with 5% CO 2 And culturing under saturated humidity for different days (0, 3, 5, 7, 9, 11 days), and changing the culture solution half every other day strictly.
(4) Acquiring evMDSC: collecting the bone marrow cells cultured in vitro into a centrifuge tube, labeling the bone marrow cells by using magnetic beads, combining the bone marrow cells with biotin anti-Gr 1 and anti-CD 11b monoclonal antibodies, and carrying out magnetic column sorting to obtain the evMDSC. After sorting, the cells were checked for separation purity with a flow cytometer.
(5) And (3) phenotypic analysis: flow cytometric analysis of each group of evMDSCs after labeling with CD11b, Gr-1, Ly-6g, Ly-6c revealed that the ratio of evMDSCs and their subpopulations PMN/M-evMDSCs peaked at 7 days of cytokine induction and increased in a dose-dependent manner (between 0-10 ng/mL), neither prolonged culture time (9-11 days) nor doubled cytokine concentration (20ng/mL) could significantly improve the production of evMDSCs, as shown in FIGS. 1-3. Giemsa staining to identify the cell morphology of two subsets of evMDSC, results show CD11b isolated by magnetic beads + Ly6G + Ly6C lo And CD11b + Ly6G - ly6C hi The two cell populations had polymorphic and mononuclear morphology, respectively, i.e., corresponding to two subpopulations of MDSCs, PMN-MDSC and M-MDSC, as shown in fig. 4.
According to the analysis of experimental results, the in vitro co-culture of bone marrow cells with the cytokines GM-CSF and IL-6 at the concentration of 10ng/mL for 7 days is the best condition for generating the MDSC with the function of inducing the immunosuppression in vitro, and the phenotype and morphological characteristics of the obtained cells are in line with the expectation.
Example 2
CD4 + T cell proliferation inhibition assay
(1) Obtaining mouse CD4 + T cell: dissecting and dissociating spleen of male Balb/c mouse of 6-8 weeks under sterile environment, grinding, collecting liquid, filtering with 200 mesh nylon net, collecting in centrifuge tube, adding erythrocyte lysate of 3 times of cell volume, labeling with magnetic bead, combining with CD4 monoclonal antibody, and separating with magnetic column to obtain CD4 + And T. After sorting, the separation purity of the cells was checked by a flow cytometer.
(2)CD4 + T cell CFSE staining: will CD4 + The T cell suspension was washed twice with room temperature PBS and resuspended to 5-10 × 10 6 Adding 1uM CFSE to the final concentration, mixing, incubating for 10min at room temperature in the dark, adding 4-5 times of complete culture medium to stop incubation, incubating for 5min on ice, washing for 3 times by using complete culture solution, and adding 0.4 mu g/mL anti-mouse CD28 monoclonal antibody.
(3)CD4 + Co-culture of T cells with evMDSC: add 1x10 5 A (100uL) ofThe mouse spleen CD4 + T cells were transferred to a 96-well plate precoated with 10. mu.g/mL anti-mouse CD3e monoclonal antibody, and the same amount of evMDSC and its subtype or nMDSC (prepared in example 1) as described above was added thereto, and the mixture was cultured at a ratio of 1:1 for 3 days while setting a positive control group and a negative control group.
(4) Flow cytometry analysis: the proliferation inhibition results showed that total evMDSC or PMN-evMDSC inhibited CD4 by about 51% compared to the positive control group + T cell proliferation, M-evMDSC inhibited by about 42%, nMDSC had little inhibitory function, as shown in FIGS. 5-6.
Total evMDSC, PMN-evMDSC and M-evMDSC in vitro cytokine induction culture on CD4 + The T cell proliferation has the inhibiting effect, and the total inhibiting effect of the evMDSC and the PMN-evMDSC is equivalent to that of the M-evMDSC, and the inhibiting effect of the PMN-evMDSC is slightly higher than that of the M-evMDSC. MDSC produced by the method can be used as an effective source for producing evMDSC with immunosuppressive function in vitro.
Example 3
Effect of depleted in vivo MDSC on pathological neovascularization after penetrating corneal transplantation
(1) Constructing a penetrating cornea transplantation mouse model: pure line 6-8 week male Balb/C mice were used as recipients and C57BL/6 mice as donors for corneal transplantation, called allogenic corneal transplantation (Allogrft, Allo for short). Donor recipients are BALB/c and are called allogeneic corneal transplantation (Isograft, Iso). The recipient cornea adopts a suture method to induce the neovascular state of the graft in advance, and the cornea transplantation operation is carried out after one week: the diameter of the implant is 2.25mm, the diameter of the implant bed is 2.0mm, 11/0 silk threads are continuously sewed, the implant is sewed on the implant bed in a watertight manner, and 8/0 silk threads are sewed on the eyelid. The eyelid sutures were removed on the third day after surgery and the corneal sutures were removed on the seventh day.
(2) Depletion of MDSCs in vivo: the model-making mice were divided into 3 groups, i.e., a group of 0.1ml of 1mg/ml anti-Gr-1 antibody injected intraperitoneally twice a week after surgery, a group of isotype control IgG injected intraperitoneally twice a week after surgery, and an untreated group, and the specific injection time is shown in fig. 7.
(3) Assessment of pathological neovascularization: microscopic in vivo observation showed that a large amount of neovascularization could be observed on day 7 after the operation in the anti-Gr-1 antibody-treated group and the graft bed, as compared with the isotype control IgG group and the untreated group, as shown in FIG. 8. After 15 days of surgery, the cornea was subjected to immunofluorescence staining with CD31 and LYVE-1, and it was revealed that the allogeneic group had almost no neovascularization and lymphangiogenesis, the allogeneic group had a large number of neovascularization and a partial angiogenesis, as shown in FIG. 9, while the neovascularization perfusion area and the lymphatic perfusion area were both greatly increased in the anti-Gr-1 antibody-treated group, as shown in FIGS. 10 to 11.
MDSC plays an important role in inhibiting angiogenesis and immune tolerance maintenance after penetrating corneal transplantation, and the MDSC exhausted in vivo by using anti-Gr-1 antibody can promote pathological angiogenesis and lymphangiogenesis of the implant and the implanted bed.
Example 4
Effect of adoptive transfer of evMDSC on pathological neovascularization growth and survival time of mouse penetrating corneal transplantation
(1) A penetrating corneal transplantation mouse model was constructed (same as in example 3).
(2) Adoptive transfer: the evMDSC obtained in vitro and the subtype and nMDSC cells are adopted to be 5 multiplied by 10 6 The cell concentration of each cell is 0.1ml, 5 × 10 ml, after injection into non-operative eyeball of mouse on the day of operation 5 One cell, the control group, i.e., the retrobulbar injection, was injected with an equal amount of PBS.
(3) And (3) survival time observation: adoptive transfer of nMDSC did not prolong survival of the grafts compared to the PBS control group, whereas adoptive transfer of evMDSC or PMN-evMDSC extended median survival of the grafts by nearly two-fold. Adoptive with equal amount of M-evMDSC resulted in a slight increase (approximately 1.5 fold) in median survival of the grafts, as shown in fig. 12. While H & E staining showed improvement in immune cell infiltration, epithelial keratinization, collagen fiber disorders and stromal edema of the grafts, as shown in figure 13.
(4) Assessment of pathological neovascularization: corneal neovascularization was observed in vivo under a microscope, and the results showed that adoptive transfer of evMDSC, PMN-evMDSC and M-evMDSC significantly reduced the formation of pathological neovascularization in the graft and the graft bed, as shown in fig. 14. Immunofluorescence staining of the cornea with CD31 and LYVE-1 at 15 days post-surgery showed that adoptive transfer of nMDSC did not reduce neovascularization as compared to the PBS control group, as shown in fig. 15-17, while adoptive transfer of evMDSC and PMN-evMDSC reduced pathological neovascular and lymphatic perfusion areas by about 60%.
The invention adopts the technical scheme that the mouse bone marrow cells are cultured in vitro by using the cell factors GM-CSF and IL-6 to obtain the MDSC with the immunosuppressive function, the culture scheme is simple and convenient to operate and low in cost, and compared with the high mortality rate of a tumor-bearing and sepsis model, the MDSC with the immunosuppressive function can be efficiently and stably obtained; after the evMDSC is adoptively transferred to a cornea transplantation mouse, the survival period of the cornea implant can be prolonged, the occurrence of immunological rejection reaction after the cornea transplantation is slowed down, the formation of pathological neovascularization is effectively inhibited, and the anti-rejection reaction after the cornea transplantation has a treatment effect. The invention is characterized in that MDSC which does not have immunosuppressive function originally is induced into MDSC with strong immunosuppressive function after in vitro cell factor culture, and the MDSC can be used for preparing biological agents for effectively treating rejection after corneal transplantation and effectively inhibiting pathological neovascularization.
Claims (2)
1. The application of in vitro induced immunosuppressive myeloid suppressor cell in preparing immunosuppressive biological preparation for inhibiting pathological neovascularization and/or neovascularization lymphatic vessel growth of corneal transplantation or preparing immunosuppressive biological preparation for prolonging corneal graft survival time of corneal transplantation; wherein the in vitro induced immunosuppressive myeloid-derived suppressor cells are obtained by culturing myeloid-derived suppressor cells in a medium comprising an inducing agent; the myeloid suppressor cell is a bone marrow cell of a healthy donor;
the inducer is cell factors GM-CSF and IL-6; wherein the mass ratio of GM-CSF to IL-6 is 0.5-1: 0.5-1;
the culture medium comprises 8-12% of fetal calf serum, 85-95% of 1640 culture medium and 0.5-1.5% of penicillin-streptomycin double antibody, and the concentration of the inducer is 1-20 ng/mL;
the culture conditions were: culturing at 37 + -0.2 deg.C and 5 + -0.1% CO2 for 7-9 days.
2. The use of claim 1, wherein said myeloid suppressor cells are bone marrow cells of BALB/c healthy mice.
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