TWI486449B - Method for generating immunomodulatory cells, the cells prepared therefrom, and use thereof - Google Patents

Description

Method for preparing immunoregulatory cells, cells prepared by the method and application thereof

The present invention relates to a method of preparing cells having a specific function, and more particularly to a method of preparing immunomodulatory cells.

Autoimmune diseases are caused by abnormal immune responses that antagonize substances or tissues normally present in the human body; in other words, the immune system mistakes certain parts of the body for pathogens and thus attacks their own cells. Transplant rejection is another problem caused by an unwanted immune response. The so-called transplantation is the act of moving a cell, tissue or organ from one place to another. Although transplantation has become a routine medical treatment, the immune system remains the most difficult obstacle to overcome.

Immunosuppressive drugs can reduce the immune response and are usually used to treat/prevent autoimmune diseases and transplant rejection. Immunosuppressive drugs are associated with various side effects, including inhibition of the overall immune system, and vulnerability to infection. Biologics can also be used in such treatments, including, for example, anti-cytokine therapy, T cell clearance therapy, B cell clearance therapy, and tolerance evoked therapy. Recently, cell therapies (such as passive administration of adoptive T-cells or stem cells) have become new options for treating or preventing autoimmune diseases and transplant rejection, but this therapy is still evolving.

Mesenchymal stem cells (hereinafter sometimes referred to as MSCs or MSCs) are a population of multilineage progenitor cells that have the ability to differentiate into a variety of mesenchymal cell lines. Mesenchymal stem cells can be isolated from bone marrow (BM) and several other organs, such as adipose tissue, placenta, amniotic fluid, and fetal tissues, such as the lungs and blood of the fetus. Since mesenchymal stem cells are easily isolated and have been reported to differentiate into extra-mesodermal cell types, such post-natal progenitors have become a popular choice for cell therapy for a variety of diseases.

In addition to the ability to differentiate, leaf stem cells between the bone marrow and the fetus have been reported to have immunomodulatory effects. This function involves the regulation of the proliferation and effector functions of T cells, B cells, monocyte-derived dendritic cells (DCs), and natural killer cells (NKs). The effect of human mesenchymal stem cells on immune cells is mainly regulated by non-cell contact processes, but the exact mechanism remains unknown.

Increasing research shows the importance of immune regulatory immune cells. It has only recently been known that many white blood cell subpopulations produce immunosuppressive effects, including regulatory T lymphocytes, type II macrophages, and immature dendritic cells. However, there is little information on regulatory monocytes.

Hepatocyte growth factor (hereinafter sometimes referred to as HGF) is known as a mitotic promoter and is a developmentally important molecule. Also known as a cell detachment factor, HGF not only provides a strong growth signal, but also induces cell migration. Therefore, the role of HGF in the growth and spread of cancer cells has been well studied; however, the effect of HGF on non-mitotic effects is not known. many. HGF is secreted by middle-cell cells and acts mainly on epithelial cells and endothelial cells, but also on hematopoietic precursor cells. HGF has been shown to play an important role in embryonic organ development, adult organ regeneration, and wound healing. HGF and TGF-β have been reported to act as regulators of T cell proliferation in mixed lymphocyte responses, and by antagonizing HGF and TGF-β neutralizing antibodies, T cell proliferation will be restored. Proof (Di Nicola et al. , Blood 2002; 99: 3838-3843). However, there are also reports showing different conclusions that are independent of the inhibitory effect of mesenchymal stem cells on T cell stimulation by mitotic promoters, thus suggesting a different stimulus-dependent mechanism (Le Blanc et al. , Scand J) Immunol 2004 ; 60 : 307-315; Rasmusson I et al. , Exp Cell Res 2005 ; 305 : 33-41).

In summary, the function and interaction between HGF and the immune system remains unknown.

The present invention provides a method for producing immunoregulatory cells, comprising: treating peripheral mononuclear cells with hepatocyte growth factor (HGF) to induce differentiation of the peripheral monocytes into immunomodulatory leukocytes (immunomodulatory leukocytes) .

The present invention also provides an immunomodulatory cell prepared by the aforementioned method.

The present invention further provides a method for treating a disease caused by an abnormal immune reaction, comprising: administering hepatocyte growth factor (HGF) to a patient suffering from the disease; and inducing differentiation of peripheral monocytes into immunomodulatory properties of the patient White blood cells; and regulate the abnormal immune response.

The present invention provides a method for producing immunoregulatory cells comprising: treating peripheral monocytes with hepatocyte growth factor (HGF) to induce differentiation of the peripheral monocytes into immunoregulatory leukocytes.

The hepatocyte growth factor (HGF) may be a recombinant protein or a natural protein, such as produced by mesenchymal stem cells or other cell lines. In one embodiment, the HGF is derived from mesenchymal stem cells and the HGF is a secreted protein from mesenchymal stem cells. In one embodiment, the mesenchymal stem cell line is derived from a mammal, by way of example and not limitation, for example, human, monkey, rat, mouse, pig, rabbit, dog, cat, and the like.

In the methods of the invention, HGF therapy can be achieved by providing HGF or providing mesenchymal stem cells in contact with peripheral monocytes. In one embodiment, the peripheral monocytes can be co-cultured with mesenchymal stem cells, which produce HGF and secrete it to the environment.

The concentration of HGF used for induction can be, for example but not limited to, for example, 3-40 nanograms (ng) per ml of medium. In one embodiment, the concentration of HGF can be 5 ng/ml, 10 ng/ml, 15 ng/ml, 20 ng/ml, 25 ng/ml, 30 ng/ml, 40 ng/ml or between any two of the above values. In one embodiment, it is preferably 5-30 ng/ml, more preferably 10-30 ng/ml.

In the present invention, the peripheral monocyte means any blood cell having a round cell nucleus. The peripheral monocyte cell line includes lymphocytes, monocytes, macrophages, basophils, and dendritic cells. The lymphocyte line is composed of T cells, B cells, and natural killer cells. In one embodiment, the peripheral monocyte cell line is from a mammal.

HGF can induce differentiation of the peripheral monocytes into immunoregulatory leukocytes, such as myeloid-derived suppressor cells (hereinafter sometimes referred to as MDSCs or MDSCs), monocytes, and the like. In one embodiment, the immunoregulatory leukocyte inhibits proliferation of activated allogeneic lymphocytes.

The bone marrow-derived suppressor cells have various immunomodulatory functions including, for example, production of arginase, production of nitric oxide synthase, induction of regulatory T cells, and the like. In one embodiment, the MDSCs can express the cell markers CD11b and CD33 and do not exhibit CD14; that is, the cells expressed by the bone marrow-derived suppressor cells are labeled CD14 ^- CD11b ⁺ CD33 ⁺ . In one embodiment, the amount of MDSC can be increased by about 1.5 to 5 times by applying the method of the present invention. In one embodiment, a trace amount of MDSC is present in untreated peripheral monocytes, for example, about 1%, and can be expanded to about 3% or more after treatment with HGF. In one embodiment, the MDSC can be amplified to about 3-10% of the peripheral monocytes, preferably about 3-5%.

The monocytes have various immunoregulatory functions including, for example, production of IL-10, production of anti-inflammatory cytokines, regulation of an immune response, and tendency to a Th2-dominant response. In particular, by applying the method of the present invention, the monocyte can be induced to produce IL-10 (i.e., IL10 ⁺ ). In one embodiment, the induced monocytes can also exhibit CD14, but not CD16; that is, the induced monocytes exhibit a cell marker of CD14 ⁺ CD16 ^- IL10 ⁺ . In one embodiment, the CD14 ⁺ CD16 ^- IL10 ⁺ monocytes produced by the method of the present invention may comprise more than about 3% of peripheral monocytes, while the untreated CD14 is absent in untreated peripheral monocytes. ⁺ CD16 ^- IL10 ⁺ , only CD14 ⁺ CD16 ^- IL10 ^- monocytes are present. In a preferred embodiment, the CD14 ⁺ CD16 ^- IL10 ⁺ monocytes are about 5% based on the total amount of peripheral monocytes.

The invention also provides an immunomodulatory cell prepared according to the methods described above. The immunoregulatory cell line is derived from peripheral monocytes, and the cell differentiation can be induced by co-culturing the peripheral monocytes with mesenchymal stem cells and/or treating with HGF secreted by mesenchymal stem cells. In one embodiment, the immunomodulatory cell is a CD14 ^- CD11b ⁺ CD33 ⁺ bone marrow-derived suppressor cell. In another embodiment, the immunomodulatory cell is a CD14 ⁺ CD16 ^- IL10 ⁺ monocyte.

The present invention provides a method for producing immunoregulatory cells from peripheral monocytes in a simple, direct and rapid manner. The method can be further applied to produce immunoregulatory cells that can be clinically applied to autoimmune diseases and transplant rejection of organ transplants.

The invention further provides a method of treating a disease caused by an abnormal immune response. The disease caused by abnormal immune reaction, for example, transplant rejection of organ transplantation, or autoimmune diseases, including: systemic lupus erythematosus, multiple sclerosis, rheumatoid arthritis ( Rheumatoid arthritis), type 1 diabetes mellitus, coeliac disease, Sjögren's syndrome, Hashimoto's thyroiditis, Graves' disease ), immune platelet deficiency iridosis (idiopathic thrombocytopenic purpura) and the like.

In this method of treatment, HGF is administered to a patient suffering from the disease. The patient can be a mammal. In one embodiment, the HGF administered is a protein type, preferably HGF secreted by mesenchymal stem cells, and HGF secreted by leaf stem cells between human sources is more preferred. In another embodiment, a dry leaflet can also be provided. A cell that produces HGF to supply the patient. After administration of HGF, peripheral monocytes of the patient can be induced to differentiate into immunoregulatory white blood cells (eg, MDSC, monocytes, etc.). The immunoregulatory leukocytes derived from the differentiation of the cells have different immune functions, can regulate the immune system of the patient, and can regulate the abnormal immune response accordingly.

Example Example 1: Preparation of immunoregulatory bone marrow-derived suppressor cells (MDSCs) Materials and Methods Cell culture

For example, Yen BL et al., Stem Cells. 2005; 23(1): 3-9 ; Chang CJ et al., Stem Cells. 2006; 24(11): 2466-77 ; Liu KJ et al., Cell Transplant. 2011; 20:1721-30 ) methods for isolating and expanding human placenta-derived leaf stem cells (MSCs) (from stem cell populations between term placenta). A full-term placenta (38-40 weeks of gestation) from a healthy provider's mother is obtained with the consent of the body's review committee. Briefly, the mesenchymal stem cell line was cultured in low glucose-DMEM medium (purchased from Gibco-Invitrogen) containing 10% fetal calf serum (FBS, purchased from HyClone). Mesenchymal stem cells were grown to 70% densified conditioned medium, collected and stored at -80 °C until testing. Human peripheral blood lymphocytes from the optional white blood cell layer of healthy volunteers are obtained with the agreement of the body's review committee. Peripheral blood leukocytes were separated by Ficoll-Paque (1.077 g/ml; Gibco-Invitrogen) density gradient centrifugation as previously described. CD14 ⁺ and CD4 ⁺ T cells were purified from peripheral blood leukocytes using MACS (magnetic beads cell separation) CD14 and CD4 single-set kits (purchased from Miltenyi Biotec) and according to the manufacturer's instructions. Briefly, peripheral blood leukocytes and appropriate magnetic beads were placed at 4 ° C for 20 minutes, followed by washing the complex of peripheral blood leukocytes and magnetic beads with phosphate buffered saline (PBS) containing 1% FBS, and borrowed Divided into a positive fraction and a negative fraction by AutoMACS (Miltenyi Biotec). The positive fraction was collected, and the purified purified cells of CD14 ⁺ and CD4 ⁺ were confirmed to have a purity higher than 98% by FACS analysis, and cultured in RPMI-1640 medium.

Immunophenotype

Cell surface marker analysis was performed using a BD FACSCalibur flow cytometry system (available from BD Biosciences, Mississauga, Canada). Anti-CD14, CD33 and CD11b anti-systems were purchased from BioLegend, San Diego, CA, USA, anti-ARG1 antibodies were purchased from R&D Systems, Minneapolis, MN, USA, anti-iNOS antibodies were purchased from Abcam, Cambridge, UK, and anti-c- The met antibody was purchased from eBiosciences, San Diego, CA, USA, and the remaining antibodies were purchased from BD Biosciences.

Amplification of bone marrow-derived suppressor cells

Peripheral blood leukocytes (PBL) were co-cultured with mesenchymal stem cells or cancer cell lines (10:1 ratio) for 3 days, followed by CD14 ^- CD11b ⁺ CD33 ^- evaluation, which is also a FACS cell separation method for bone marrow-derived suppressor cells. Cell surface markers (using BD Aria Cell Sorter (purchased from BD Biosciences)). According to the manufacturer's instructions (purchased from Miltenyi Biotec, Bergisch Gladbach, Germany), part of the experiment was carried out by replacing the PBL with CD14 ^- cells screened out of the negative fraction of the magnetic beads. A predetermined dose of recombinant human HGF (rhHGF, purchased from R&D Systems) was added to peripheral blood leukocytes for analysis and evaluation of MDSC inhibition of PBL cell division described below. Briefly, allogeneic PBLs were labeled with 2.5 μmol/L fluorescent dye CFSE (carboxyfluorescein succinimidyl ester; purchased from Molecular Probes/Gibco-Invitrogen, Grand Island, NY, USA) for 10 minutes, followed by anti-CD3/CD28 The beads (purchased from Gibco-Invitrogen) were stimulated. Homogenous MSC-dilated bone marrow-derived suppressor cells were isolated by FACS cell isolation and co-cultured with stimulated allogeneic PBL at different cell-target cell (E:T) ratios for 3 days. PBL cell division was assessed by flow cytometry by CFSE stain intensity.

Quantification of HGF secretion

The supernatant was collected in a cell culture dish and assayed for HGF using a commercially available ELISA kit (purchased from R&D Systems) according to the manufacturer's instructions.

RNA interference test

The siRNA (specific small interfering RNA for IL-6 and HGF, purchased from Gibco-Invitrogen) was used and subjected to a knockdown test according to the manufacturer's instructions. The inhibitory effect of siRNA on mesenchymal stem cell-secreting factor was verified by ELISA.

In vivo test

All animal experiments are performed in accordance with the specifications approved by the agency's Animal Care and Use Committee. C57BL/6 mice (4 to 8 weeks old) were from the National Laboratory Animal Center of Taiwan (Taiwan, Taipei). Recombinant mouse HGF (100 ng, purchased from R&D Systems) was injected from the tail vein and sacrificed 3 days after injection. Peripheral blood, bone marrow, and spleen were collected for flow cytometric analysis of Gr1 ⁺ CD11b ⁺ cells.

Statistical Analysis

In the above test, statistical analysis was performed with SPSS 18.0 software (purchased from SPSS Inc., Chicago, IL, USA), and statistical significance was defined as p < 0.05. Student’s t-test is used for comparison between the two groups, and ANOVA is used for comparison between the plural groups.

Example 1-(1) Mesenchymal stem cells can make functional CD14 from peripheral blood leukocytes ^- CD11b ⁺ CD33 ^- Bone marrow-derived suppressor cells increase in size

None of the prior art reports that strong immunosuppressive properties of leaf stem cells (MSCs) between different sources are associated with expansion of bone marrow-derived suppressor cells (MDSCs). Leaf stem cells between bone marrow and placenta have positive CD73, CD105 and CD90 The type of surface marker is expressed and is a negative expression of the hematopoietic marker (including co-stimulatory molecules CD80 and 86) (see Figure 1A). Both mesenchymal stem cell populations can differentiate into osteogenic lineage, chondrogenic lineage, and adipogenic lineage, in accordance with the principle of multivariate mesenchymal stem cells (see Figure 1B).

Mesenchymal stem cells are co-cultured with human allogeneic peripheral blood leukocytes, and subjected to bone marrow-derived suppressor cell analysis. The characteristics of the human system are CD33 and CD11b, but not CD14. As shown in Figures 2A and 2B, both bone marrow and placental mesenchymal stem cells can increase the number of CD14 ^- CD11b ⁺ CD33 ^- cells in peripheral blood leukocytes. Detected between stem cells - CD14 amplification of ^- CD11b ⁺ CD33 ^- cells (hereinafter sometimes referred to as "MSC--induced suppression of bone marrow-derived cells") of the characteristics and biological functions, of which results are shown in FIGS. 2C to 2F. By co-culture, MSC-induced bone marrow-derived suppressor cells have a function of inhibiting lymphocyte proliferation and are dose-dependent (see Figure 2C). MSC-induced bone marrow-derived suppressor cells can express ARG1 and iNOS (see Figures 2D & 2E) and induce a large amount of CD4 ⁺ CD25 ^high T _regs in stimulated peripheral blood leukocytes (see Figure 2F). Accordingly, MSC-induced bone marrow-derived suppressor cells do have a variety of immunomodulatory functions.

Example 1-(2) MSC-induced expansion of bone marrow-derived suppressor cells is regulated by secretory HGF

As shown in Figure 3A, expansion of bone marrow-derived suppressor cells induced by mesenchymal stem cells is not affected by cell-separated culture, suggesting that cell-cell contact is not required and may be related to secretion during this process. factor. As shown in Figure 3B, between the supernatants of leaf stem cells, mesenchymal stem cells secrete a variety of matrix-related factors such as RANTES/CCL5, HGF, and IL-6. IL-6 is known to be involved in bone marrow-derived inhibition of cell proliferation, however, antagonistic antibodies and siRNA inhibition experiments Both showed that IL-6 was not associated with mesenchymal stem cell-induced bone marrow-derived inhibition of cell proliferation (experimental data not shown in the specification).

Since mesenchymal stem cells secrete a large amount of HGF, it is tested and evaluated whether this molecule is involved in mesenchymal stem cell-induced bone marrow-derived inhibition of cell proliferation. As shown in Fig. 3C, the addition of recombinant HGF can amplify bone marrow-derived suppressor cells, and A dose-dependent effect was exhibited, up to a concentration of HGF of about 30 ng/ml, which is similar to the upper limit measured in conditioned medium of mesenchymal stem cells (Fig. 3B). HGF-amplified bone marrow-derived suppressor cells also exhibit iNOS and ARG1 and have inhibitory effector functions (see Figures 3D & 3E, respectively). To confirm the relevance of HGF in mesenchymal stem cell-induced bone marrow-derived suppressor cell expansion, RNA interference was performed on mesenchymal stem cells using siRNA to inhibit HGF secretion. When the HGF secretion of mesenchymal stem cells is effectively inhibited, the mesenchymal stem cell-induced bone marrow-derived suppressor cell expansion phenomenon also disappears (Fig. 3F). Therefore, the experimental data confirmed that the HGF system secreted by mesenchymal stem cells is involved in the proliferation of bone marrow-derived suppressor cells.

HGF from other cell lines was also tested. As shown in Figures 4A and 4B, the amount of HGF secreted by different cell lines is related to the amount of increase in bone marrow-derived suppressor cells.

To verify the above findings in vivo , recombinant HGF was injected intravenously into wild-type C57BL/6 mice. After 3 days, an increase in CD11b ⁺ Gr1 ⁺ bone marrow-derived suppressor cells in the bone marrow of these mice was observed (Fig. 4C), indicating that HGF can amplify bone marrow-derived suppressor cells in an in vivo environment.

Example 1-(3) HGF-induced bone marrow-derived suppressor cell expansion, regulated by c-met, its receptor, and increased STAT3 phosphorylation

Further studies were conducted on the mechanism by which HGF-regulated bone marrow-derived suppressor cells are expanded. Detecting inhibition of bone marrow-derived c-met (HGF cognate receptors) on the cell's performance, and found that CD14 ^- exhibits a low amount of white blood cells continue c-met (FIG. 5A), and In terms of neutralizing antibodies blocked CD14 ^- In the c-met of cells, the HGF-induced bone marrow-derived suppressor cell expansion phenomenon disappears (Fig. 5B). HGF-induced STAT3 phosphorylation by bone marrow-derived suppressor cells was examined to confirm that the major utility of HGF/c-met was regulated by STAT3. As shown in Figure 5C, the addition of exogenous HGF induced phosphorylated STAT3 (pSTAT3) above the baseline, suggesting that the pathway is activated. The addition of a STAT3 inhibitor (cpd188) in the presence of exogenous HGF, the phenomenon of increased pSTAT3 in the bone marrow-derived suppressor cells disappeared and showed a dose-dependent (Fig. 5D). Thus, HGF, by binding to its receptor c-met, results in an increase in STAT3 phosphorylation, which in turn regulates the proliferation of bone marrow-derived suppressor cells.

The mechanism by which HGF-regulated bone marrow-derived suppressor cells are expanded is shown in Fig. 6, and HGF (especially secreted by mesenchymal stem cells) can increase the number of bone marrow-derived suppressor cells. Mechanistically, mesenchymal stem cells induced by bone marrow-derived suppressor cell proliferation via the HGF/c-met pathway, downstream of which is associated with STAT3. The bone marrow-derived suppressor cells expanded by co-culture with mesenchymal stem cells or HGF are functional, exhibit iNOS and ARG1, and have an inhibitory function to induce T _regs . Therefore, HGF-induced bone marrow-derived suppressor cells achieve immunomodulatory functions. Such immunomodulatory functions can be used to treat autoimmune diseases and other immune related diseases such as graft-host diseases.

Example 2: Preparation of immunoregulatory monocytes

In the present invention, it has been found that mesenchymal stem cells can induce an immunomodulatory production of a CD14 ⁺ monocyte population of IL-10 via secretion of hepatocyte growth factor (HGF). The presence of such immunoregulatory monocytes inhibits T cell proliferation and alters the effector function of T cells from Th1 to Th2. The interaction between HGF and CD14 ⁺ monocytes was also tested and this signaling pathway is associated with the production of IL-10 by this immunoregulatory monocyte.

Materials and Methods Cell culture

Human placenta-derived leaf stem cells were isolated and cultured as described in Example 1.

Human histiocytic lymphoma cell line U937 and human T cell acute leukemia cell line Jurkat, obtained from the American Type Culture Center (ATCC), were cultured according to the disclosed procedure. In some experiments, prior to treatment with recombinant human HGF (rhHGF, purchased from Peprotech), ERK1/2 inhibitor U0126 (cell signaling), p38 MAPK inhibitor SB203580 (purchased from Merck), and STAT3 inhibitor cpd188 (purchased) U937 was processed from Merck for 30 minutes.

Cell proliferation test

The cells were stained with 2.5 μM CFSE (carboxyfluorescein diacetate, succinimidyl ester, purchased from Gibco-Invitrogen) for 10 minutes at 25 ° C and 0.1% calf serum albumin (BSA). After washing twice with 0.1% BSA, the cells were redissolved in RPMI-1640 medium and incubated at 25 ° C for 10 minutes. Peripheral blood leukocytes, or peripheral blood leukocytes removed from CD14, purchased as human T cell activators, ie, anti-CD3/CD28 Dynabeads (purchased from Gibco-Invitrogen), or mitotic agent PHA (phytohemagglutinin, 100 μg/ml) From Sigma-Aldrich, stimulation was performed in the presence or absence of mesenchymal stem cell culture medium and rhHGF. In addition, anti-CD3/CD28 bead-stimulated CD4 ⁺ T cells were co-cultured with rhHGF-treated HLA-mismatched CD4 ⁺ cells in different ratios. After 72 hours, CFSE-labeled cells (5 x 10 ⁵ cells/well) were collected and washed twice with 0.1% BSA. As described above, the analysis of the number of cell divisions was performed by FACS.

Cytokine array

Analysis was performed on conditioned medium collected from mesenchymal stem cell culture plates (including co-culture with CD14 ⁺ cells or not), and cytokine profiles were determined by human quantitative antibody arrays (purchased from RayBiotech) and following the manufacturer's recommended protocol. Briefly, the test membrane and the blocking buffer were placed at room temperature for 30 minutes, and then the sample was added and left at room temperature for 90 minutes. Then, the test membrane was washed three times with the washing buffer I at room temperature. The test membrane was washed twice with Wash Buffer II for 5 minutes each time; the biotin-conjugated antibody was placed at room temperature for 90 minutes. Finally, the test membrane was washed and co-localized with horseradish catalase for 2 hours at room temperature, and then co-located with the detection buffer for 2 minutes. Detection was performed with a fluorescence detector (GenePix 4000B Microarray Scanner, available from Axon Instruments, Inc.), and the data was digitized and subjected to image analysis (GenePix Pro software, available from Axon Instruments, Inc.). The relative protein concentration was obtained by subtracting the background staining and normalizing the positive control panel of the same test membrane.

RNA silencing test

The mesenchymal stem cells secreted HGF RNA-based tests using two different muting HGF- interfering RNA (referred to as ^{HGF siRNA) (Stealth Select RNAi TM} siRNA, available from Gibco-Invitrogen), and measured according to the manufacturer recommended procedure and using the manufacturing Negative control group recommended by the firm (StealthTM negative control). Using Lipofectamine ^TM RNAiMAX (available from Gibco-Invitrogen), and according to manufacturer's recommended procedures, the siRNA transfection mesenchymal stem cells. Briefly, the cells were cultured in serum-free medium for 24 hours, the transfection mixture was added for 4-6 hours, and transferred to complete medium. After 3 days of culture, the culture medium was detected by ELISA to confirm in the mesenchymal stem cells. HGF inhibition effect. HGF and mononuclear cells produced by mesenchymal stem cells in conditioned medium were assayed by enzyme-linked immunosorbent assay (ELISA) using HGF and IL-10 as described in the previous literature and according to the manufacturer's instructions (purchased from R&D systems). IL-10 produced by the cell/cell strain.

Immunophenotypic test

The cells (peripheral blood leukocytes, CD14 ⁺ cells, and blood cancer cell lines) were stained with different surface markers as described in the literature, and analyzed by BDFACSCalibur (purchased from BD Biosciences). The antibodies used in the flow cytometry analysis were purchased from BD Biosciences except that the anti-c-Met antibody was purchased from eBiosciences. An appropriate FITC-conjugated and PE-conjugated isotype immunoglobulin control group was used for each analysis.

Intracellular interleukin staining

IFN-γ, IL-4 and IL13 produced by anti-CD3/CD28-stimulated CD4 ⁺ T cells (including co-culture with HGF-treated CD14 ⁺ cells) were measured 72 hours after culture. The cells were purchased at 50 μg/ml PMA (phorbol 12-myristate 13-acetate, purchased from Sigma-Aldrich), 0.745 μg/ml ionomycin (purchased from Sigma-Aldrich), and 4 μM of montanin (purchased from PMA). Re-dissolved in the presence of BioLegend) for 4-6 hours. Next, cells were co-localized with anti-human IFN-γ FITC (purchased from BioLegend), anti-human IL-4 PE (purchased from BioLegend), or anti-human IL13 PE (purchased from BD Biosciences) at 4 °C. minute. FITC-conjugated and PE-conjugated immunoglobulins were used in parallel as a control group. The cells were washed twice and suspended in PBS-BSA for FACS analysis.

In vivo testing of recombinant mouse HGF

Mice (C57BL/6) were injected intravenously with 200 ng of recombinant mouse HGF (rmHGF, purchased from Peprotech) diluted in 150 μl of PBS. After 8 days, bone marrow cells were removed from the mouse femur by PBS lavage; and the spleen was ground at 4 ° C with a 3-ml syringe head and filtered through a nylon mesh. The resulting cell suspension was centrifuged, the red blood cells were lysed, and the resulting single cell suspension was washed. Using magnetic bead separation (auto-MACS), CD45R/B220 PE (purchased from Biolegend, clone: RA3-6B2), CD90.2 PE (purchased from eBioscience, clone: 53-2.1) and anti-PE beads were used, respectively. To remove B-lymphocytes and T-lymphocytes from splenocytes. The cell suspension was co-located with anti-mouse CD11b PE (purchased from BioLegend) and anti-mouse IL10 PerCPCy5.5 (purchased from BD Biosciences) and detected by FACS.

RNA isolation and reverse transcription-polymerase chain reaction (RT-PCR)

Total RNA was extracted with Trizol reagent (purchased from Gibco-Invitrogen), and RT-PCR was performed as described in the literature, using the following PCR primer: the forward derivative of IL-10 was 5 ' -CTGTGAAAACAAGAGCAAGGC-3 ' , and The primer was 5 ' -GAAGCTTCTGTTGGCTCCC-3 ' ; and the forward primer of β-actin (positive control group) was 5 ' -TGGCACCACCTTCTACAATGAGC-3 ' , and the reverse primer was 5 ' -GCACAGCTTCTCCTTAATGTCACGC-3 ' . Each experiment is triple-covered. Relative quantification of mRNA was obtained by optical density analysis using ImageJ software (NIH) and normalization with β-actin.

Western blot analysis

The protein is extracted from the cells as described in the literature of the predecessor. The major anti-systems that antagonize ERK1/2, STAT3, and α-tubulin were purchased from Santa Cruz Biotechnology, and antagonized the major resistances of phospho-ERK1/2, phospho-STAT3, p38 MAPK, phospho-p38 MAPK, Akt, and phospho-Akt. The system was purchased from Cell Signaling Technologies.

Statistical Analysis

Statistical analysis was performed with SPSS software (purchased from SPSS Inc., version 19.0), Student's t-test was used for alignment between the two groups, and ANOVA was used for alignment between the plural groups. All data were expressed as mean ± SEM and statistical significance was defined as p < 0.05. All experiments were at least triple-over.

Example 2 (1) HGF can inhibit the proliferation of peripheral blood leukocytes induced by anti-CD3/28-activation

As shown in Example 1, mesenchymal stem cells exhibited strong immunoregulatory effects on T lymphocytes, and the effects thereof were regulated by secretory factors. As shown in Figure 7A, the mesenchymal stem cell conditioned medium (MSC-CM) inhibits peripheral blood leukocyte proliferation, which is activated by PHA (which preferentially stimulates monocytes), or is anti-CD3/28 (specifically Stimulate T cells) activation. Compared with PHA-stimulated peripheral blood leukocytes, MSC-CM showed a slightly stronger inhibitory effect on anti-CD3/28-stimulated peripheral blood leukocytes. In addition, when anti-CD3/28-stimulated peripheral blood leukocytes are co-cultured with mesenchymal stem cells, the cytokine profile will change, and the Th1 inflammatory cytokine (pro-inflammatory Th1 milieu)-IFN-γ and TNF-α is a representative, and more immunomodulatory milieu - represented by IL-10 (Fig. 7B).

In different cytokines, chemokines, and matrix-related molecular assays (Fig. 7C), HGF is the most secreted amount in MSC-CM. Next, rhHGF is added to the activated peripheral blood leukocytes. Surprisingly, rhHGF inhibits the proliferation of peripheral blood leukocytes induced by anti-CD3/28-activation, but not for PHA-stimulated peripheral blood leukocytes (Fig. 7D), indicating that the immunosuppressive effect of HGF is directed against T cells. Moreover, the inhibitory effect of HGF on peripheral blood leukocytes was not dose-dependent. Further, the HGF inhibition test of mesenchymal stem cells by siRNA showed the results of ELISA detection as shown in Fig. 7E. When anti-CD3/28-activated peripheral blood leukocytes were co-cultured with siHGF-mesenchyelia stem cells, the immunosuppressive effect was reduced to a significant extent (Fig. 7F). In addition, the medium of HGF-siRNA mesenchymal stem cells partially restored the proliferation inhibition effect of peripheral blood leukocytes (data not shown). These results show that HGF secreted by mesenchymal stem cells can inhibit the proliferation of peripheral blood leukocytes induced by anti-CD3/28-activation.

Example 2-2(2) Activated CD4 ⁺ Cellular HGF-regulated immune regulation, CD14 ⁺ Monocytes are necessary

HGF is a ligand for c-Met and c-Met is a tyrosine kinase. According to the aforementioned HGF, the anti-CD3/28-activated peripheral blood leukocytes are inhibited, but the PHA-activated peripheral blood leukocytes are not, indicating that the immunomodulatory effect of HGF is directed against T cells. However, c-Met was not expressed on primary T cells (data not shown in the specification), and Jurkat cells, that is, T cell blood cancer cell lines were also absent (Fig. 8A). It has been reported that T cells do not exhibit c-Met, but monocyte lines exhibit a small amount of c-Met; monocytes are CD14 ⁺ cells, including about 10% of human peripheral blood leukocytes, and human monocyte blood cells are known. The major CD14 ⁺ monocytes from strain U937 and the peripheral blood leukocytes of the provider showed c-Met (Figs. 8A and 8B).

The above results show that the effect of HGF on T cells may require monocytes as an intermediate. To test this hypothesis, CD14 ⁺ cells were removed from peripheral blood leukocytes, followed by rhHGF to anti-CD3/28-activated peripheral blood leukocytes. As shown in Fig. 8C, in the case where CD14+ cells were removed, the inhibitory effect of HGF on the proliferation of peripheral blood leukocytes stimulated by anti-CD3/28 was completely eliminated.

Monoclonal cells screened for CD14 ⁺ -beads were treated with rhHGF and co-cultured with anti-CD3/28-stimulated CD4+ cells. As shown in Fig. 8D, the proliferative effect of T cells can be inhibited by HGF-treated CD14 ⁺ cells, and the inhibition increases with the ratio of the target cells: target cells (CD14 ⁺ to CD4 ⁺ cells). However, the dose of HGF had no effect on the inhibition of monocyte regulation, as in Figure 7D. The anti-CD3/28-activated peripheral blood leukocytes and the HGF-treated CD14 ⁺ monocyte co-culture system altered the cytokine profile from a higher Th1 towards a Th2 environment (Fig. 8E). CD4 ⁺ Th1 (IFN-γ) and intracellular FACS analysis of T cells Th2 (IL-4 and IL- 13) The intracellular product display, CD4 ⁺ T cells were treated with HGF- of CD14 ⁺ cells by co-culture increases IL -4 and IL-13 production; conversely, IFN-γ is reduced. These data show that HGF directly interacts with CD14 ⁺ monocytes, which in turn alters the immune function of T cells.

Example 2 (3) HGF can significantly induce IL-10 production

Monocytes are the main component of cellular influx in an inflammatory environment and can differentiate into dendritic cells or macrophages depending on environmental stimuli to provide proinflammatory or anti-inflammatory properties. To investigate whether HGF can regulate the differentiation of CD14 ⁺ monocytes, it was analyzed to differentiate into dendritic cell lines or macrophage cell lines with changes in surface marker expression. After HGF treatment, the expression levels of the markers CD11b, CD11c and HLA-DR associated with dendritic cells, or the marker CD206 associated with macrophages were unchanged (Fig. 9A). In addition, the performance of DC-SIGN, CD68 and CD73 was not measured. However, the fraction of CD16-cells increased slightly (Fig. 9B), and the subpopulation of CD14 ⁺ monocytes has been reported to be immunomodulatory.

In Figure 10A, quantitative cytokine analysis of CD14 ⁺ cells co-cultured with mesenchymal stem cells showed a significant decrease in the production of inflammatory precursor interleukins (including IFN-γ and TNF-α), while anti-inflammatory mediators (IL-10) is significantly increased. This data shows that the immunosuppressive effect of mesenchymal stem cells can be derived from the reduction of IFN-γ and the increase in IL-10 production. To further confirm the interaction between HGF and increased IL-10 production by monocytes, primary CD14 ⁺ cells were co-cultured with rhHGF and the concentration of IL-10 contained in the medium was measured. As shown in Figure 10B, the selection medium of HGF-treated CD14 ⁺ cells and MSC-CM-treated CD14 ⁺ cells all had high IL-10 production above the baseline.

mHGF was injected into wild type B6 mice for in vivo testing of the above effects. As shown in Fig. 10C, HGF induced a slight increase in IL-10-producing monocyte populations in splenocytes, but not in bone marrow cells or peripheral blood (data not shown). Accordingly, HGF is associated with the production of IL-10 by CD14 ⁺ monocytes.

Example 2 (4) HGF induces IL-10 secretion via phosphorylation of ERK1/2

Further study the mechanism by which HGF induces monocyte to produce IL-10. Mononuclear cell line U937 was treated with HGF and tested for changes in IL-10 gene expression. As shown in Figure 11A, the increase in IL-10 transcript peaked at 6 hours of rhHGF treatment, indicating a direct relationship between HGF and IL-10 production. HGF initiates many intracellular signaling pathways and their downstream pathways. PI3K/AKT, Ras/ERK1/2, p38/MAPKs, and STAT3 are also involved in the regulation of IL-10 gene expression. As shown in Figure 11B, phosphorylation of ERK1/2, STAT3, and p38/MAPK was increased after treatment with HGF in U937 cell line, but AKT was not. To further investigate these three pathways, U937 cells were treated with HGF and specific inhibitors simultaneously. Inhibitors include: U0126 (p-ERK1/2 inhibitor), SB203580 (p38/MAPK) Formulation), and cpd188 (pSTAT3 inhibitor). Results As shown in Figures 11C and 11D, IL-10 gene expression decreased in a dose-dependent manner in the U0126 and SB203580 groups, but only ERK1/2 inhibitors caused a significant decrease in IL-10 protein production after HGF treatment. . This result shows that IL-10 production of HGF-regulated monocytes is regulated by ERK1/2.

As shown in Figure 12, HGF (especially mesenchymal stem cell secretors) acts on CD14 ⁺ monocytes to regulate the function of allogeneic T cells and increase IL-10 via ERK1/2. The HGF secreted by mesenchymal stem cells directly acts on CD14 ⁺ monocytes, which represent the receptor for HGF, c-Met, to inhibit the proliferation of activated CD4 ⁺ cells. Such CD14 ⁺ monocytes are required for the inhibitory effect of HGF secreted by mesenchymal stem cells on activated CD4 ⁺ cells, and the inhibitory effect includes increasing immunoregulatory CD14 ⁺ CD16 ^- monocytes, inducing IL-10 production, The cytokine profile of T cells is regulated by Th1 to Th2. In terms of mechanism, HGF induces IL-10 production by CD14 ⁺ monocytes via the ERK1/2 pathway. The above examples show the mechanism of strong immunomodulatory properties of mesenchymal stem cells which are not known in the prior art, and emphasize the important role that immunoregulatory monocytes play in altering T cell immune function.

It is worth noting that the inhibitory effect of HGF on T cell proliferation is not dose-dependent, probably because c-Met (HGF receptor) on monocytes is low in expression, so the threshold of receptor saturation is relatively low.

HGF induces phosphorylation of ERK1/2 and p38MAPK in monocytes, and HGF-induced IL-10 production is only blocked by ERK1/2 inhibitors, indicating Ras/Raf signaling pathway and HGF-induced monocyte secretion. Related to IL-10. ERK1/2 is responsible for the phosphorylation and activation of several transcription factors that control IL-10 expression in immune cells, including MAF, JUN, GATA3, and SMAD4, not the receptor for p38. Therefore, these transcription factors may play a key role in the production of IL-10 by HGF-induced monocytes.

The above example demonstrates that HGF can induce an immunomodulatory phenotype directly and very rapidly in CD14 ⁺ monocytes (within 3 days) and that the cells remain unattached, indicating that differentiation has not yet begun. Circulating peripheral blood mononuclear cells of a single population (as CD14 ⁺⁺ CD16 ^-) can produce high amounts of anti-inflammatory cytokines, including IL-10. Circulating CD14 ⁺ monocytes are the majority of components of peripheral blood leukocytes, and if immunoregulatory monocytes can be rapidly induced, these leukocytes can make a more significant contribution to the overall amount of circulating immune regulatory cells.

The above description of the specific embodiments is intended to be illustrative of the invention, and is not intended to limit the invention. It will be understood by those skilled in the art that various changes or modifications may be made to the present invention without departing from the scope of the appended claims.

Figure 1 shows the characteristics of mesenchymal stem cells (BM-MSCs) and placenta-derived mesenchymal stem cells (P-MSCs), Figure 1A: surface markers; Figure 1B: Three-lineage differentiation of BM-MSCs and P-MSCs Type, Osteo: Osteogenic, Chondro: Chondrogenic, Adipo: Adipogenic; Scale: 200 mm.

Figure 2 shows that in peripheral blood leukocytes (PBLs), mesenchymal stem cells amplify the number of functional CD14 ^- CD11b ⁺ CD33 ⁺ bone marrow-derived suppressor cells (MDSCs). Figure 2A: CD14 ^- CD11b ⁺ CD33 ⁺ cells are increased from peripheral blood leukocytes by mesenchymal stem cells, and allogeneic peripheral blood leukocytes (30 suppliers) are cultured alone or co-cultured with mesenchymal stem cells (BM-mesenchyelia) There are three providers of stem cells and P-mesenchyelia stem cells). Figure 2B: Quantitative percentage of CD11b ⁺ or CD14 ^- CD11b ⁺ CD33 ⁺ cells co-cultured with peripheral blood leukocytes, compared with p<0.05 for peripheral blood leukocytes culture alone. Figure 2C: Inhibitory function of mesenchymal stem cells induced by mesenchymal stem cells. Allogeneic peripheral blood leukocytes (T, labeled cells) were stained with CFSE (carboxyfluorescein suceinimidyl ester) to assess cell division, with or without mesenchymal stem cells. The expanded bone marrow-derived suppressor cells (E, active cells) were stimulated with anti-CD3/28 (a-CD3/28), wherein the addition was in a different E:T ratio. PBL cell division was assessed by flow cytometry, and the right panel is quantified, p < 0.05. Figure 2D: Expression of inducible nitric oxide synthase (iNOS), Figure 2E: MSC-expanded bone marrow-derived suppressor cell arginase-1 (ARG1), negative selection of CD14 ^- cells by PBL, Cultured alone or co-cultured with mesenchymal stem cells, further stained with CD11b, CD33, and iNOS (Fig. 2D) or ARG1 (Fig. 2E). Figure 2F: Increased CD4 ⁺ CD25 ^High regulatory T cells (T _regs ) by MSC-expanded bone marrow-derived suppressor cells, and MSC-expressing bone marrow-derived suppressor cells expressing CD14 ^- CD11b ⁺ CD33 ⁺ (FACS) ), and co-cultured with anti-CD3/28-stimulated allogeneic PBL in different ratios, and evaluated the induction of T cells with high expression of CD4 ⁺ CD25, the right picture is the quantitative result, compared with PBL/a-CD3/28 p<0.05.

Figure 3 shows that leaf stem cell expansion between bone marrow-derived suppressor cells is regulated by secreted hepatocyte growth factor (HGF). Figure 3A: Bone marrow-derived suppressor cells are stimulated by mesenchymal stem cells and are regulated by secreted factors. Co-culture of allogeneic PBL with mesenchymal stem cells, whether directly contacted (MSC) or separated by culture plates (TW), produced amplification of CD14 ^- CD11b ⁺ CD33 ⁺ bone marrow-derived suppressor cells; ^* , p <0.05 and PBL Comparison by individual culturers; ns: not significant. Figure 3B: Analysis of high-level secretory factors of mesenchymal stem cells by interleukin quantitative array; IL: interleukin, LAP: latent peptide, SDF: matrix-derived factor. Figure 3C: Addition of exogenous recombinant HGF to PBL increased the number of bone marrow-derived suppressor cells ( ^* , p < 0.05 linear trend). Figure 3D: Test of iNOS (upstream) and ARG1 (downstream) expression in HGF-amplified bone marrow-derived suppressor cells. Figure 3E: Inhibition of anti-CD3/28-stimulated PBL proliferation by HGF-amplified bone marrow-derived suppressor cells, cell division was stained with CFSE, and analyzed by flow cytometry. Figure 3F: Inhibition of HGF secretion by mesenchymal stem cells by HGF-specific small interfering RNA (siRNA) will abolish the expansion of bone marrow-derived suppressor cells. Allogeneic peripheral blood leukocytes were co-cultured with mesenchymal stem cells (from 3 providers) and transfected with unlabeled small interfering RNA (siRNA Ctrl) or HGF specific siRNA (siHGF) and subjected to CD14 ^- CD11b ⁺ CD33 ⁺ cells. Evaluation; ^* , p < 0.05 compared with mesenchymal stem cells (left panel) or PBL culture alone (right panel).

Figure 4 shows that HGF secreted by cancer cells and administration of HGF in mice increase the number of bone marrow-derived suppressor cells. Figure 4A: The amount of HGF secreted by mesenchymal stem cells, MG63 (osteosarcoma cell line), embryonic stem cell-derived leaf precursor cells (EMP), and JEG-3 (chorionic cancer cell line); Figure 4B: Four cell types and ploidy proliferation of bone marrow-derived suppressor cells after PBL co-culture; ^* , p <0.05 linear trend. Figure 4C: Recombinant HGF (100 ng) was injected into C57BL/6 mice by tail vein, and CD11b ⁺ Gr1 ⁺ cells in peripheral blood, spleen and bone marrow were evaluated one day and three days after injection; ^* , p < 0.05 compared with PBS.

Figure 5 shows that bone marrow-derived suppressor cell lines expanded by HGF are regulated by their receptor c-met and increased STAT3 phosphorylation. Figure 5A: Flow cytometry analysis of c-met manifestations on bone marrow-derived suppressor cells (12 providers) and provides a graph of quantified mean fluorescence intensity (MFI); ^* , p < 0.05 versus isotype immunoglobulin The protein control group was compared (Ctrl). Figure 5B: Correlation of c-met with HGF-regulated bone marrow-derived suppressor cell expansion. Peripheral blood leukocytes were blocked with homotypic immunoglobulin (IsoAb) or c-met blocking antibody (anti-c-Met), treated with recombinant HGF (20 ng/ml), and the increase in CD14 ^- CD11b ⁺ CD33 ⁺ cells was assessed. Figure 5C: STAT3 is associated with HGF-regulated bone marrow-derived suppressor cell expansion. Recombinant HGF (20 ng/ml) was added to CD14 ^- white blood cells, and CD11b, CD33, and phosphorylated STAT3 (pSTAT3) were stained and evaluated by flow cytometry (value in the upper right corner is MFI). Quantitative MFI chart; ^* , p <0.05 linear trend. Figure 5D: Recombinant HGF (20 ng/ml) and different doses of cpd188 (STAT3 inhibitor) were added to PBL, and CD11b, CD33, and phosphorylated STAT3 (pSTAT3) were stained by flow cytometry. The evaluation provided a quantitative proportion of CD14 ^- CD11b ⁺ CD33 ⁺ pSTAT3 ⁺ cells; a, p < 0.05 compared with PBL culture alone; b, p < 0.05 linear trend.

Figure 6 shows the mechanism by which HGF regulates the expansion of bone marrow-derived suppressor cells.

Figure 7 shows the inhibitory effect of HGF secreted by leaf stem cells between placenta-derived cells on the proliferation of anti-CD3/28-activated allogeneic peripheral blood leukocytes. Figure 7A: Peripheral blood leukocytes, cultured alone (unstimulated; black column), stimulated with PHA (white column), or treated with anti-CD3/28 activated beads (grey column), and treated with MSC-CM or No; or Figure 7D: recombinant human HGF (rhHGF); n = 15. The proliferative effect of stimulated peripheral blood leukocytes was set to 100%, which was defined by flow cytometry analysis of >90% of peripheral blood leukocytes stained by CFSE to produce cell division; in Figure 7A: a, p < 0.01 and b, p<0.05 compared with the control group; c, p<0.05 and PHA-activated peripheral blood leukocytes and treated with MSC-CM. In Figure 7D: a, p <0.05; b, p < 0.05 with PHA-activated peripheral blood leukocytes and treated with HGF. Figure 7B: Cytokine quantitative array detection of cytokines, produced from mesenchymal stem cells (black bars), peripheral blood leukocytes (white bars), peripheral blood leukocytes and mesenchymal stem cells co-culture (grey bars), anti-CD3/ 28 activated peripheral blood leukocytes (slanted column), and peripheral blood leukocytes activated by anti-CD3/28 were co-cultured with mesenchymal stem cells (spotted column), cultured for 3 days, n=1. Figure 7C: Quantitative array detection of cytokines secreted by cytokines and matrix-related factors in mesenchymal stem cells. Figure 7E: ELISA assay results for HGF production in conditioned medium of mesenchymal stem cells, leaf stem cells treated with HGF-siRNA, and leaf stem cells treated with unlabeled siRNA, n=10. Figure 7F: Proliferation of peripheral blood leukocytes activated by anti-CD3/28, cultured alone or co-cultured with siRNA transfected leaf stem cells for 72 hours, analyzed by flow cytometry CFSE, n=9; a, p <0.05 was compared with peripheral blood leukocytes and mesenchymal stem cells co-cultured with anti-CD3/28 activated peripheral blood. All experimental data were derived from the average of 3 independent experiments and are expressed as mean ± SEM unless otherwise stated; , p <0.01; ^* , p < 0.001, unless otherwise stated, compared with the control group.

Figure 8 shows that CD14 ⁺ monocytes are required for the immunomodulatory effects of HGF on T lymphocyte function. Figures 8A & 8B: c-Met performance, in T lymphocyte strain (Jurkat), monocyte strain (U937), and primary CD14 ⁺ cells purified from peripheral blood leukocytes (B; n = 7); MFI, firefly Average light intensity. Figure 8C: Peripheral blood leukocytes (white bars) or peripheral blood leukocytes (black bars) from which CD14 ⁺ cells were removed were cultured alone or stimulated with anti-CD3/28 beads, treated with rhHGF or not; n=15. The degree of proliferation of peripheral blood leukocytes treated with rhHGF was expressed as the relative percentage of peripheral blood leukocytes that were anti-CD3/28-activated by peripheral blood leukocytes or CD14 ⁺ cells removed; a, p<0.01; b, p<0.05 Compared to anti-CD3/28-activated peripheral blood leukocytes treated with HGF. Figure 8D: Proliferation of CD4 ⁺ T cells, cultured alone or stimulated with anti-CD3/28 beads, treated with rhHGF-treated CD14 ⁺ cells or not, cultured for 72 hours (proportion of CD14 ⁺ to CD4 ⁺ T cells: 1 /10, 1/5, 1/2, 1/1). Proliferation of stimulated CD4 ⁺ T cells was set to 100%, which was defined as flow cytometry analysis of CF90-stained CD4 ⁺ T cells with >90% cell division; a, p < 0.05 linear trend. Figure 8E: Intracellular production of IFN- γ , IL-4 and IL-13 by anti-CD3/28-activated CD4 ⁺ T cells co-cultured with rhHGF-treated CD14 ⁺ cells. The total number of T cells was set to 100%. All experimental data were from the average of 3 independent experiments and were expressed as mean ± SEM unless otherwise stated; #, p <0.05; ^* , p < 0.001, unless otherwise stated, compared to the control group.

Figure 9A shows dendritic cell (DC)- and macrophage-related surface molecules expressed on CD14 ⁺ cells, treated with rhHGF (grey and diagonal columns), not treated with rhHGF (black bars), with flow cytometry The instrument was analyzed for 72 hours of culture. The MFI expression of the isotope control group (white column) was set to 1. All experimental data were from the average of 3 independent experiments and are expressed as mean ± SEM. Figure 9B shows the performance of CD14 and CD16 on peripheral blood leukocytes, treated with rhHGF or not, analyzed by flow cytometry, n=8.

Figure 10 shows that HGF increases the amount of IL-10 production in monocytes. Figure 10A: Cytokine profile of Th1/2, CD14 ⁺ cells were cultured for 3 days alone (white column), co-cultured with mesenchymal stem cells for 3 days (black bars), and detected by cytokine quantitative array (n = 1). Figure 10B: IL-10 production by CD14 ⁺ cells, treated with rhHGF, MSC-CM, siHGF-MSC-CM or not, measured by ELISA. Figure 10C: IL-10-producing monocyte population (IL10 ⁺ CD11b ⁺ cells) of bone marrow and spleen cells of mice receiving rmHGF injection. The total number of cells was set to 100%. All experimental data were from the average of 3 independent experiments and were expressed as mean ± SEM; #, p <0.05; unless otherwise stated, all were compared to the control group.

Figure 11 shows that HGF-induced IL-10 production by CD14 ⁺ monocytes is regulated by the ERK1/2 pathway. Figure 11A: IL-10 expression of rhHGF-treated U937 cells was detected by RT-PCR at predetermined time points, and β-actin was used as an internal control group. Figure 11B: Western blotting detection of phosphorylated (p-)ERK1/2, p-STAT3, p-p38, and p-AKT in rhythoma-treated U937 cells, with ERK1/2, STAT3 , p38, AKT, and α-tubulin were used as internal control groups. Figure 11C: IL-10 gene expression of U937 cells treated with rhHGF and pretreated with ERK1/2 inhibitor U0126, p38 MAPK inhibitor SB203580, or STAT3 inhibitor cpd188, detected by RT-PCR. Figure 11D: IL-10 protein expression of U937 cells pretreated with inhibitor U0126 or SB203580 and treated with rhHGF (black bars) or not treated with rhHGF (white bars); n = 3; data by mean ± SEM which performed; , p <0.01; ^* , p <0.001, compared with the control group.

Figure 12 shows the mechanism by which HGF induces IL-10 production in CD14 ⁺ monocytes.

Claims (12)

A method for producing immunoregulatory cells in vitro comprises: treating peripheral monocytes with hepatocyte growth factor to induce differentiation of the peripheral monocytes into immunoregulatory leukocytes; wherein the concentration of the hepatocyte growth factor is 3 40 ng/ml; and wherein the immunoregulatory white blood cell line comprises a bone marrow-derived suppressor cell, a monocyte, or a combination thereof, and the cell labeled by the bone marrow-derived suppressor cell is labeled CD14 ^- CD11b ⁺ CD33 ⁺ , the single core The cells expressed by the cells are labeled as CD14 ⁺ CD16 ^- IL10 ⁺ . The method of claim 1, wherein the peripheral monocyte cell line is derived from a mammal. The method of claim 1, wherein the hepatocyte growth factor is a recombinant protein or a natural protein. The method of claim 3, wherein the hepatocyte growth factor is derived from a leaf stem cell. The method of claim 1, wherein the immunoregulatory leukocyte line inhibits proliferation of activated allogeneic lymphocytes. The method of claim 1, wherein the bone marrow-derived suppressor cell line produces arginase and nitric oxide synthase. The method of claim 1, wherein the bone marrow-derived suppressor cell line induces regulatory T cells. The method of claim 1, wherein the monocyte cell line produces IL-10. The method of claim 1, wherein the monocyte cell line produces an anti-inflammatory cytokine. The method of claim 1, wherein the monocyte cell line modulates an immune response such that it predominantly targets a Th2-dominant response. An immunoregulatory cell prepared according to the method of claim 1 of the patent application, comprising CD14 ^- CD11b ⁺ CD33 ⁺ bone marrow-derived suppressor cells and CD14 ⁺ CD16 ^- IL10 ⁺ monocytes. The method of claim 1, wherein the step of treating peripheral monocytes with hepatocyte growth factor is via contact.