CN111808800B - In-vitro induced immunosuppressive myeloid suppressor cell and preparation and application thereof - Google Patents

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

In vitro induced immunosuppressive myeloid suppressor cell and its preparation and application

technical field

The invention relates to a preparation and acquisition method of in vitro induced immunosuppressive myeloid suppressor cells, belonging to the field of cell biology and clinical application.

Background technique

Myeloid-Derived Suppressor Cells (MDSCs) are myeloid progenitors with naive myeloid-derived cells and can differentiate into mature granulocytes, macrophages or dendritic cells downstream under certain circumstances. In mice, MDSCs characteristically co-express the surface molecules Myeloid-cell LineageDifferentiation Antigen-1 (Gr-1) and CD11b (ie αM-integrin). MDSCs can be roughly divided into granular MDSCs (CD11b ⁺ Gr-1 ⁺ Ly6G ^high Ly6C ^- , PMN-MDSCs) and mononuclear MDSCs (CD11b ⁺ Gr-1 ⁺ Ly6G) according to the expression of Gr-1 subsets Ly6C and Ly6G in MDSCs ^- Ly6C ^high , M-MDSC) two groups (Veglia F, Perego M, Gabrilovich D. Myeloid-derived suppressor cells coming of age. Nat Immunol. 2018;19(2):108-119.).

MDSC,nMDSC)没有免疫抑制功能，而在病理状态下体内诱导的MDSC，如脓毒血症小鼠及负荷肝癌小鼠骨髓内分选出的CD11b⁺Gr-1⁺MDSC，具有强大的免疫抑制性，可以有效抑制CD4⁺及CD8⁺T细胞增殖，且具有一定的剂量相关性(Yan He,BeibeiWang,Bei Jia,Jieying Guan,Hui Zeng*,Zhiqiang Pan*.Effects of adoptivetransferring different sources of Myeloid Derived Suppressor cells in micecorneal transplant survival.Transplantation 2015；99:2102–2108.)，但体内病理环境诱导具有抑制功能性MDSC有效但存在高风险。Studies have reported that in tumor and inflammatory states, MDSCs can induce the differentiation and proliferation of regulatory T cells (Treg), affect Treg/Th17 balance, promote M2 macrophage polarization, and enhance the expression of Arg-1, Nos2 and Nox2 , through direct cell contact and indirect action of cell secretions, suppressing effector T cells (CD11b ⁺ Gr-1 ⁺ Ly6G ^- Ly6C ^high , M-MDSC) two groups (VegliaF, Perego M, Gabrilovich D.Myeloid-derived suppressorcells coming of age. Nat Immunol. 2018;19(2):108-119.). CD11b ⁺ Gr-1 ⁺ MDSCs isolated from normal mouse bone marrow under physiological conditions (

MDSCs, nMDSCs) have no immunosuppressive function, while MDSCs induced in vivo under pathological conditions, such as CD11b ⁺ Gr-1 ⁺ MDSCs sorted from the bone marrow of septic mice and liver cancer-bearing mice, have strong immunosuppression It can effectively inhibit the proliferation of CD4 ⁺ and CD8 ⁺ T cells, and has a certain dose correlation (Yan He, Beibei Wang, Bei Jia, Jieying Guan, Hui Zeng*, Zhiqiang Pan*. Effects of adoptivetransferring different sources of Myeloid Derived Suppressor cells in micecorneal transplant survival. Transplantation 2015;99:2102–2108.), but in vivo pathological environment induction is effective in inhibiting functional MDSCs but carries a high risk.

MDSCs have been shown to contribute to neovascularization and neolymphangiogenesis in tumor-burdened models and can assist tumor escape and metastasis (Veglia F, Perego M, Gabrilovich D. Myeloid-derived suppressorcells coming ofage. Nat Immunol. 2018; 19 (2): 108-119.), while the effect of MDSCs on pathological neovascularization in transplantation models has not been reported. We observed that after MDSC was cleared with anti-Gr-1 antibody in mice with allogeneic corneal transplantation, the graft bed and graft were rapidly neovascularized, and a large number of new blood vessels and lymphatic vessels grew into the graft in the early postoperative period; Simultaneous adoptive transfer of MDSCs induced in vivo or in vitro during transplantation showed a strong inhibitory effect on corneal graft neovascularization and neovascularization while suppressing rejection (Yan He, Beibei Wang, BeiJia, Jieying Guan, Hui Zeng *, Zhiqiang Pan*. Effects of adoptive transferring different sources of Myeloid Derived Suppressor cells in mice cornealtransplant survival. Transplantation 2015; 99:2102–2108.) (Yan He, Bei Jia, HuiZeng, Zhiqiang Pan*, The Roles of Sepsis-Induced Myeloid Derived SuppressorCells in Mice Corneal Skin and Combinedtransplantation, Transpl Immunol, 2016, 34, 8-13.).

With the in-depth research of MDSCs in tumors, transplantation, and autoimmune diseases, the need for stable acquisition of functional MDSCs is prominent. However, under different induction environments, the immune function and yield of MDSCs vary greatly. How to induce stable and efficient in vitro MDSCs? It is of great significance to develop functional MDSCs for research and future applications, and there is no relevant research for the time being.

SUMMARY OF THE INVENTION

In order to solve the technical problems that the existing immunosuppressive myelosuppressive cells are mainly extracted in vivo, the effect is unstable, and the in vitro induction technology is lacking, the first object of the present invention is to provide a preparation method of in vitro induced immunosuppressive myeloid suppressor cells , aiming to realize the high-yield in vitro directional induction preparation of ex vivo generated MDSC by cytokine-induced differentiation of bone marrow cells (evMDSC) with immunosuppressive function.

The second object of the present invention is to provide an evMDSC induced by the in vitro induction method.

The third object of the present invention is to provide an application of the in vitro induction method for inducing evMDSC.

A preparation method for inducing immunosuppressive myeloid suppressor cells in vitro, which is obtained by culturing myeloid suppressor cells in vitro under the induction of an inducer;

The inducers include cytokines GM-CSF and IL-6.

The invention provides an in vitro directional induction preparation method of evMDSC. It is found in the research of the present invention that GM-CSF and IL-6 have synergy, and evMDSC with high activity and high yield can be obtained at a lower concentration. Not only that, the inventors also found that the evMDSCs prepared by synergistic induction of GM-CSF and IL-6, compared with evMDSCs extracted in vivo and obtained by other methods, inhibited the new blood vessels and new lymphatic vessels of corneal transplantation, as well as prolongation of corneal transplantation. Corneal grafts have better results in terms of life cycle.

The study found that on the basis of the synergistic induction of GM-CSF and IL-6, the further control of the proportion and concentration of the combination will help to further improve the yield of evMDSC and further enhance the evMDSC with better effect in corneal transplantation. Subtype (PMN-evMDSC) selectivity.

Preferably, the mass ratio of GM-CSF and IL-6 is 0.5-1:0.5-1; more preferably 1:1.

In the present invention, the concentration of the inducer is 1-20 ng/mL; preferably, it is 8-12 ng/mL. The inducer of the present invention, based on the synergy of GM-CSF and IL-6, can unexpectedly obtain excellent directed induction effect at a lower content, for example, a higher content of evMDSC can be obtained , to improve the selectivity of active cell subtypes.

In the present invention, the myeloid suppressor cells are bone marrow cells of healthy donors. For example, the myeloid suppressor cells are bone marrow cells of healthy mice.

The method of the present invention is obtained by culturing myeloid suppressor cells in a medium containing the inducer.

In the present invention, the medium can be obtained by mixing the required content of the inducer on the basis of the existing conventional medium suitable for the growth of bone marrow cells.

Preferably, the medium comprises 8-12% fetal bovine serum, 85-95% 1640 medium, 0.5-1.5% penicillin-streptomycin double antibody, and 1-20 ng/mL inducer.

Preferably, the culture conditions are: culture at a temperature of 37±0.2° C., 5±0.1% CO ₂ , and the culture time is greater than or equal to 5 days; preferably 7 to 9 days. The study found that the days of culturing can unexpectedly achieve the synergy between the factors, help to unexpectedly improve the evMDSC content, and significantly improve the expression of PMN-evMDSC.

In the present invention, after in vitro culture, the target evMDSC can be isolated by using existing methods, and the subtype identification and determination of the target evMDSC can be carried out by using the existing methods.

Preferably, after in vitro culture, immunomagnetic bead separation method is used to sort out evMDSCs, and the number and phenotype of evMDSCs, as well as the expression ratio of PMN/M-evMDSCs, are detected by cell counting and flow cytometry, and Giemsa staining is used to identify the two evMDSCs. According to the cell morphology of each subgroup, suitable culture conditions were screened out, and a functional MDSC system for inducing immunosuppression in vitro was preliminarily established.

The present invention also includes in vitro induced immunosuppressive myeloid suppressor cells prepared by the preparation method.

Preferably, the in vitro induced immunosuppressive myeloid suppressor cells are PMN-MDSC subtype cells and/or M-MDSC subtype cells.

The present invention also provides an application of the in vitro induced immunosuppressive myeloid suppressor cells for preparing immunosuppressive biological preparations.

Preferably, it is used for the preparation of immunosuppressive biological preparations that inhibit CD4+ T cells.

Further preferably, it is used in the preparation of immunosuppressive biological preparations for inhibiting immune rejection of organ transplantation.

More preferably, it is used in the preparation of immunosuppressive biological preparations for inhibiting the immune rejection of corneal transplantation.

More preferably, it is used in the preparation of immunosuppressive biological preparations for inhibiting the growth of pathological neovascularization and/or new lymphatic vessels in corneal transplantation; Use in immunosuppressive biologics.

The present invention also provides an immunosuppressive biological preparation, comprising the in vitro induced immunosuppressive myeloid suppressor cells prepared by the preparation method.

Preferably, the in vitro induced immunosuppressive myeloid suppressor cells are PMN-MDSC subtype cells.

Preferably, the immunosuppressive biological preparation is a corneal transplantation immunosuppressive biological preparation.

The research of the present invention unexpectedly found that the evMDSC, especially the PMN-MDSC subtype cells obtained by the method of the present invention can effectively inhibit the growth of new blood vessels and new lymphatic vessels in corneal transplantation, and can effectively prolong the life cycle of the transplanted sheet.

In the present invention, the animal model and biological performance research steps are as follows: first, the effect of depleting MDSCs in vivo on pathological neovascularization after penetrating keratoplasty. Allogeneic penetrating keratoplasty mice were used as a model, and they were divided into 3 groups, namely, the anti-Gr-1 antibody group was intraperitoneally injected twice a week after surgery, the isotype control IgG group was intraperitoneally injected twice a week after the operation, and the mice without In the treatment group, the corneal pathological neovascularization in each group was observed. The corneas of each group were immunofluorescently stained with antibodies that specifically recognize CD31 (endothelial cell marker) and LYVE-1 (lymphatic endothelial cell marker), and the intensity changes of new blood vessels and lymphatic vessels in each group were evaluated. Finally, the effects of in vitro-induced adoptive transfer of MDSCs on immune tolerance and pathological neovascularization of penetrating keratoplasty in mice were observed. Allogeneic penetrating keratoplasty mice were used as models, and they were divided into 5 groups. On the day of surgery, PBS, nMDSC, evMDSC, PMN-evMDSC, and M-evMDSC were injected retrospectively into the non-surgical eyes, and the median corneal grafts in each group were observed. Survival and pathological neovascularization.

beneficial effect

1. The present invention provides a brand-new idea of evMDSC in vitro directed induction synthesis; and it is found that the GM-CSF and IL-6 have a synergistic induction effect, which can reduce the dosage of the inducer, and can also increase the yield. Not only that, but also can improve the selectivity of PMN-evMDSC cell subtypes with better performance in the field of corneal transplantation.

2. The evMDSC obtained by the method of the present invention can effectively inhibit the growth of new blood vessels and new lymphatic vessels in corneal transplantation, and can effectively prolong the service period of the transplanted sheet.

Description of drawings

Fig. 1 is the ratio of MDSCs cultured in vitro under different days and different cytokine concentrations in Example 1;

Fig. 2 is the PMN-MDSC ratio under the in vitro culture of different days and different cytokine concentrations of Example 1;

Fig. 3 is the ratio of M-MDSC under in vitro culture with different days and different cytokine concentrations in Example 1;

Fig. 4 is the PMN/M-MDSC cell morphology after magnetic bead sorting in Example 1;

Figure 5 is a flow chart of the inhibition of proliferation of MDSCs in each group of Example 3 and CD4+ T cells co-cultured

Figure 6 shows the proliferation inhibition ratio of co-culture of MDSCs and CD4+ T cells in each group of Example 3

Figure 7 shows the time of intraperitoneal injection of antibody anti-Gr-1 or control IgG in mice after corneal transplantation in Example 4

Fig. 8 is the direct view under the microscope 7 days and 15 days after corneal transplantation in Example 4

Fig. 9 is corneal immunofluorescence after corneal transplantation in Example 4

Figure 10 shows the proportion of corneal neovascularization perfusion area after corneal transplantation in Example 4

Fig. 11 is the proportion of corneal neoplastic lymphatic vessel perfusion area after corneal transplantation in Example 4

Figure 12 shows the survival rate of adoptively transferred MDSC corneal grafts after corneal transplantation in Example 5

Figure 13 is the corneal pathological section of the adoptively transferred MDSC after corneal transplantation in Example 5

Fig. 14 is the direct view under the microscope after corneal transplantation in Example 5

Fig. 15 shows corneal immunofluorescence after corneal transplantation in Example 5

Figure 16 shows the proportion of corneal neovascularization perfusion area after corneal transplantation in Example 5

Fig. 17 is the proportion of corneal neoplastic lymphatic vessel perfusion area after corneal transplantation in Example 5

Detailed ways

Example 1

Establishment of a functional MDSC system for inducing immunosuppression in vitro

(1) Obtaining mouse bone marrow cells: In a sterile environment, male BALB/c mice at 6-8 weeks were sacrificed and the bilateral tibia and fibula were taken out, the medullary cavity was washed with PBS, and the cells were collected in a centrifuge tube to obtain nMDSC cells.

(2) Preparation of medium: The basal medium was prepared according to 10% fetal bovine serum, 90% 1640 medium, and 1% penicillin-streptomycin double antibody, and different concentrations of cytokines GM-CSF and IL-6 (0 , 5, 10, 20ng/ml; the ratio of GM-CSF and IL-6 is 1:1).

(3) Cell seeding and culture: The cells were seeded in 24-well plates at ^{5×10 5} cells/well, medium containing different concentrations of cytokines was added, and cultured at 37° C., 5% CO ₂ and saturated humidity for different days ( 0, 3, 5, 7, 9, 11 days), and the medium should be changed in half every other day.

(4) Obtaining evMDSCs: The bone marrow cells cultured in vitro were collected into centrifuge tubes, labeled with magnetic beads and combined with biotin anti-Gr1 and anti-CD11b monoclonal antibodies, and evMDSCs were obtained by magnetic column sorting. After sorting, the isolated cells were tested for purity by flow cytometry.

(5) Phenotypic analysis: The evMDSCs in each group were labeled with CD11b, Gr-1, Ly-6g, Ly-6c and then analyzed by flow cytometry. Factor induction peaked on day 7 and increased in a dose-dependent manner (between 0-10 ng/mL), neither extending the incubation time (9-11 days) nor doubling the cytokine concentration (20 ng/mL) significantly improved The yield of evMDSC is shown in Figure 1-3. Giemsa staining was used to identify the cell morphology of the two subpopulations of evMDSC. The results showed that the two cell populations, CD11b ⁺ Ly6G ⁺ Ly6C ^lo and CD11b ⁺ Ly6G ^- ly6C ^hi separated by magnetic beads had polymorphic nuclear morphology and mononuclear morphology, respectively. That is, the corresponding two subgroups of MDSC, PMN-MDSC and M-MDSC, are shown in Figure 4.

According to the analysis of the experimental results, the co-culture of bone marrow cells with the cytokines GM-CSF and IL-6 at a concentration of 10 ng/mL for 7 days in vitro is the best condition for inducing immunosuppressive functional MDSCs in vitro. The morphological features are all as expected.

Example 2

CD4 ⁺ T cell proliferation inhibition assay

(1) Obtain mouse CD4 ⁺ T cells: dissect and dissociate the spleen of 6-8 week old male Balb/c mice in a sterile environment. After grinding, the collected liquid is filtered through a 200-mesh nylon mesh and collected in a centrifuge tube, and added 3 times. The red blood cell lysate in the cell volume was labeled with magnetic beads and combined with CD4 monoclonal antibody, and CD4 ⁺ T was obtained by magnetic column sorting. After sorting, the isolated cells were tested for purity by flow cytometry.

(2) CFSE staining of CD4 ⁺ T cells: Wash the CD4 ⁺ T cell suspension twice with PBS at room temperature, resuspend to 5-10× ^{10 6} cells/mL, add 1 uM CFSE to the final concentration, mix and incubate at room temperature for 10 min in the dark. Add 4-5 times the volume of complete medium to stop the incubation, incubate on ice for 5 min, wash three times with complete medium, and add 0.4 μg/mL anti-mouse CD28 monoclonal antibody.

(3) Co-culture of CD4 ⁺ T cells and evMDSC: Add 1x10 ⁵ (100uL) of the above mouse spleen CD4 ⁺ T cells to a 96-well plate pre-coated with 10 μg/mL anti-mouse CD3e mAb, and add an equal amount The evMDSCs (prepared in Example 1) and their subtypes or nMDSCs were co-cultured at a ratio of 1:1 for 3 days, and a positive control group and a negative control group were set at the same time.

(4) Flow cytometry analysis: The results of proliferation inhibition showed that compared with the positive control group, total evMDSC or PMN-evMDSC inhibited about 51% CD4 ⁺ T cell proliferation, M-evMDSC inhibited about 42%, nMDSC almost did not Inhibit function, as shown in Figure 5-6.

Cytokine-induced and cultured total evMDSC, PMN-evMDSC and M-evMDSC all had inhibitory effects on the proliferation of CD4 ⁺ T cells. The MDSCs induced by the above methods can be used as an effective source for the production of evMDSCs with immunosuppressive functions in vitro.

Example 3

The effect of depleted MDSC in vivo on pathological neovascularization after penetrating keratoplasty

(1) Construction of penetrating keratoplasty mouse model: pure 6-8 week-old male Balb/c mice were used as recipients, and C57BL/6 mice were used as donors for corneal transplantation, which was called allogeneic corneal transplantation (Allograft, referred to as Allo). If the donor and recipient are both BALB/c, it is called allogeneic corneal transplantation (Isograft, Iso for short). The recipient cornea was sutured in advance to induce the neovascularization of the implant bed, and corneal transplantation was performed one week later: a 2.25mm diameter graft was taken, a 2.0mm diameter graft bed, and the graft was watertightly sutured on the implant bed with 11/0 silk continuous suture. The eyelid is sutured with 8/0 silk thread. The eyelid sutures were removed on the third postoperative day, and the corneal sutures were removed on the seventh postoperative day.

(2) Depletion of MDSCs in vivo: The model mice were divided into 3 groups, that is, the group that received intraperitoneal injection of 0.1 ml of 1 mg/ml anti-Gr-1 antibody twice a week after surgery, and the same volume of isotype was injected intraperitoneally twice a week after surgery. For the control IgG group and the untreated group, the specific injection time is shown in Figure 7.

(3) Evaluation of pathological neovascularization: In vivo observation under the microscope showed that compared with the isotype control IgG group and the untreated group, the anti-Gr-1 antibody-treated graft and implant bed could be observed on the 7th day after surgery. Massive neovascularization was formed, as shown in FIG. 8 . 15 days after taking the cornea, immunofluorescence staining with CD31 and LYVE-1 showed that the allogeneic group had almost no new blood vessels and lymphatic vessel growth, and the allogeneic group had a large number of new lymphatic vessels and partial angiogenesis, as shown in Figure 9 , while in the anti-Gr-1 antibody treatment group, both the neovascular perfusion area and the lymphatic perfusion area were significantly increased as shown in Figure 10-11.

MDSCs play an important role in inhibiting neovascularization and maintaining immune tolerance after penetrating keratoplasty. Depletion of MDSCs in vivo with anti-Gr-1 antibody will promote pathological neovascularization and lymphangiogenesis in grafts and implant beds.

Example 4

The effect of adoptive transfer of evMDSC on the growth and survival of pathological neovascularization in mouse penetrating keratoplasty

(1) Construction of a penetrating keratoplasty mouse model (same as Example 3).

(2) Adoptive transfer: evMDSC and its subtypes and nMDSC cells obtained in vitro were injected at a cell concentration of 5×10 ⁶ cells/ml into the non-operated eyeballs of mice on the day of surgery, and 0.1 ml was injected into each mouse. That is, 5×10 ⁵ cells, and the control group is injected with the same amount of PBS after the ball.

(3) Survival observation: Compared with the PBS control group, adoptive transfer of nMDSCs could not prolong the survival time of grafts, while adoptive transfer of evMDSCs or PMN-evMDSCs could prolong the median survival of grafts by nearly two times. Adoption of an equal amount of M-evMDSC resulted in a slight increase (approximately 1.5-fold) in the median survival of the grafts, as shown in FIG. 12 . At the same time H&E staining showed improved immune cell infiltration, epithelial keratinization, collagen fiber disturbance and matrix edema of the grafts, as shown in FIG. 13 .

(4) Evaluation of pathological neovascularization: In vivo observation of corneal neovascularization under microscope showed that adoptive transfer of evMDSC, PMN-evMDSC and M-evMDSC significantly reduced the formation of pathological neovascularization in grafts and implant beds, as shown in Figure 14 . The corneas were harvested 15 days after surgery for immunofluorescence staining with CD31 and LYVE-1, showing that adoptive transfer of nMDSCs did not reduce neovascularization compared with the PBS control group, as shown in Figures 15-17, while evMDSCs and PMN-evMDSCs Adoptive transfer can reduce the area of pathological neovascular perfusion and lymphatic perfusion by about 60%.

In the present invention, the mouse bone marrow cells are cultured in vitro with cytokines GM-CSF and IL-6 to obtain MDSCs with immunosuppressive function. It can effectively and stably obtain MDSCs with immunosuppressive function; adoptive transfer of evMDSC to corneal transplant mice can prolong the survival period of corneal grafts, slow down the occurrence of immune rejection after corneal transplantation, and effectively inhibit the The formation of pathological neovascularization has a therapeutic effect on the rejection after keratoplasty. The unique feature of the present invention lies in that, after in vitro cytokine culture, MDSCs without immunosuppressive function are induced into MDSCs with strong immunosuppressive functions, which can be used to prepare and effectively treat rejection after corneal transplantation and effectively inhibit pathological diseases. Biologics for 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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