Methods and reagents for regulating the immunomodulatory function of mesenchymal stem cells

Abstract

The present invention provides a method and reagent for regulating the immunomodulatory function of mesenchymal stem / stromal cells (MSCs). In view of the complexity of immunomodulation embodied by MSCs in the field of scientific research and clinical practice, there is still a bottleneck in its clinical application. The inventors are committed to improving the immunomodulatory effect of MSCs. Based on in-depth research, it is confirmed for the first time that glucocorticoids can regulate MSCs to produce VEGF-C, thereby regulating CD8 + T cell proliferation; TNF-α can cooperate with Dex to regulate VEGF-C. + Changes in the number or activity of T cells are inevitably related to changes in immune function. The present invention provides a new approach for the design of drugs for regulating immunity.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology; more specifically, the present invention relates to a method and reagent for regulating the immunoregulatory function of mesenchymal stem cells. Background Art

[0002] Mesenchymal stem cells (MSCs) are stem cells that exist in most tissue matrices of the body and can be isolated from a variety of tissues, such as bone marrow, fat, and umbilical cord. MSCs have the potential to differentiate into adipocytes, osteoblasts, and chondrocytes. Studies have found that in vitro cultured MSCs have the characteristics of migrating to damaged tissues and participating in the regulation of damage repair. The interaction between inflammation in damaged tissues and MSCs plays an indispensable role in determining the repair characteristics of MSCs. The discovery of cell therapy based on the regulation of inflammatory responses and the participation of MSCs in tissue repair and regeneration has brought the application of stem cells in the treatment of inflammatory diseases into a new era. At present, research on MSCs mainly focuses on the differentiation potential and immunoregulatory function of in vitro expansion of MSCs. Studies have shown that MSCs not only provide the required cell source for tissue reconstruction, but more importantly, they "empower" other cells to promote tissue repair by regulating inflammatory responses. Although MSCs-based cell replacement plays an important role in the treatment of certain specific diseases, recent studies have found that the therapeutic effect of MSCs is mainly attributed to its immunoregulatory function under the regulation of the inflammatory environment. Under the stimulation of inflammatory factors, MSCs produce a large number of immunomodulatory factors, chemokines and growth factors, which regulate the tissue inflammatory microenvironment and tissue stem cells or tissue precursor cells, and promote tissue repair. MSCs have a strong immunosuppressive effect, which can not only inhibit the innate immune response, but also affect the adaptive immune response, but this immunomodulatory function is not innate, but requires the induction of inflammatory factors.

[0003] The immunoregulatory ability of MSCs depends on the types and concentrations of various inflammatory molecules in the microenvironment in which they are located. Different inflammatory states greatly affect the therapeutic effect of MSCs on diseases, suggesting the plasticity of MSCs immunoregulation. Studies have found that under strong inflammatory conditions, MSCs can effectively treat graft versus-host disease (GvHD), but if MSCs are infused on the same day of bone marrow transplantation, that is, before the inflammatory response begins, the therapeutic effect of MSCs is not significant. In addition, MSCs have almost no effect on experimental autoimmune encephalomyelitis in remission. It can be seen that the immunoregulatory ability of MSCs has a strong plasticity and is closely related to the inflammatory state.

[0004] In the pathological process of inflammatory diseases, high levels of inflammatory factors are often closely related to the acute stage of the disease, while in the chronic or remission stage of the disease, inflammatory factors are at relatively low concentrations, which may be the body's self-repair stage. The immunoregulatory function of MSCs at different concentrations of inflammatory factors (IFN-γ and TNF-α) was detected by "chessboard gradient" concentration, and it was found that the dynamic changes in the levels of inflammatory factors can affect the immunoregulatory function of MSCs, causing them to exert immunosuppressive or immunopromoting effects, thus laying the foundation for the study of the plasticity of MSCs immunoregulatory. The main reason is that low levels of inflammatory factors are not enough to induce MSCs to express high levels of inducible nitric oxide synthase (iNOS) or indoleamine-2,3-dioxygenase (IDO) to exert immunosuppressive effects. Instead, they will recruit lymphocytes to their surroundings under the action of a large number of chemokines secreted by MSCs, exacerbating the inflammatory response. Therefore, nitric oxide (NO) and IDO are the "switches" that regulate the immunoregulatory function of MSCs. In the co-culture system of T cells and MSCs activated by low-dose concanavalin A, MSCs also showed similar immune enhancement functions. The above studies suggest that high levels of inflammation stimulate MSCs to exert immunosuppressive functions, while low levels of inflammatory environments stimulate MSCs to exert immune promotion effects. However, the mechanism network regulating MSCs activity in different inflammatory environments has not yet been clarified.

[0005] The immunoregulatory function of MSCs is affected by multiple factors in the inflammatory environment, and immunosuppressants are often used to treat immune system-related diseases, which raises an important scientific question: Do immunosuppressants affect the immunoregulatory function of MSCs? The therapeutic effects of immunosuppressants are similar to those of MSCs, both of which inhibit effector T cells and downregulate inflammatory responses. Based on this, some studies have combined MSCs with commonly used immunosuppressants such as cyclosporine A or dexamethasone (Dex). Cyclosporine A can block T cell activation and is often used to treat organ transplant rejection or autoimmune diseases. MSCs can also induce tolerance in transplanted mice, but the combined use of cyclosporine A reverses the therapeutic effect of MSCs. In addition, TGF-β can also cause MSCs to lose their immunosuppressive function. In general, these findings are of great significance in guiding the clinical application of MSCs.

[0006] The use of allogeneic homohaplotype bone marrow-derived MSCs to treat patients with grade IV acute GvHD (aGvHD) resistant to cyclosporine and steroids marked the beginning of the clinical application of MSCs. Subsequently, other drug-resistant inflammatory diseases such as systemic lupus erythematosus have been successfully cured by MSCs. In such immunosuppressant-resistant patients, persistent inflammation may stimulate the immunosuppressive function of MSCs. In contrast, in patients sensitive to immunosuppressants, combined treatment with MSCs and cyclosporine or steroids has no therapeutic effect. These studies suggest that the immunosuppressive function of MSCs requires strong inflammatory factors to induce, while low concentrations of inflammatory factors can enable them to exert an immunopromoting effect.

[0007] How to effectively improve the immunosuppressive function of MSCs in order to develop more new clinical treatment options based on MSCs is a topic that needs to be studied urgently in this field. Summary of the invention

[0008] The purpose of the present invention is to provide a method and reagent for regulating the immunomodulatory function of mesenchymal stem cells

[0009] In the first aspect of the present invention, there is provided a use of VEGF-C, its downstream signaling pathway or their regulators in the preparation of a pharmaceutical composition for regulating the immunomodulatory effect of MSCs; preferably, the immunomodulatory effect is based on regulating CD8 + The proliferation of T cells plays a role.

[0010] In a preferred embodiment, the pharmaceutical composition further comprises: glucocorticoids; preferably, the glucocorticoids include: dexamethasone (Dex), methylprednisolone, betamethasone, hydrocortisone, prednisone, methylprednisone, beclomethasone propionate, budesonide, and fluticasone propionate.

[0011] In another preferred embodiment, the regulator of VEGF-C or its downstream signaling pathway is a down-regulator, and the immunoregulation is immunosuppression; the immunosuppression includes alleviating or treating CD8 + T cell hyperproliferation-related diseases; preferably including (but not limited to): inhibiting autoimmune diseases, inflammation; more preferably, the CD8 + Diseases associated with excessive T cell proliferation include (but are not limited to): graft-versus-host disease, multiple sclerosis, inflammatory bowel disease, scleroderma, type I diabetes, rheumatoid arthritis, Hashimoto's thyroiditis, psoriasis, systemic lupus erythematosus, and organ damage such as liver damage, lung damage, and digestive tract damage; more preferably, the liver damage includes liver fibrosis or cirrhosis, and the lung damage includes non-fibrosis.

[0012] In another preferred embodiment, the downregulator of VEGF-C or its downstream signaling pathway includes PI3K / AKT pathway inhibitors, TNFR1 downregulators, glucocorticoid receptor downregulators, small molecule compounds that specifically inhibit VEGF-C or its receptors, small molecule compounds that specifically inhibit TNFR1; interfering molecules that specifically interfere with the expression of VEGF-C gene or TNFR1 gene; gene editing reagents that specifically downregulate (including knocking out) VEGF-C or TNFR1; antibodies or ligands that specifically bind to VEGF-C protein or TNFR1 protein, or reagents that specifically block the binding of VEGF-C to its receptor VEGFR3; more preferably, the PI3K / AKT pathway inhibitor includes: pan-PI3K inhibitor LY294002 or AKT inhibitor Afuresertib, the small molecule compound that specifically inhibits VEGF-C receptor is a small molecule compound MAZ51, SAR131675 that inhibits the tyrosine kinase activity of VEGF-C receptor (VEGFR3); antibodies that specifically inhibit VEGFR3.

[0013] In another preferred embodiment, the regulator of VEGF-C or its downstream signaling pathway is VEGF-C or its up-regulator, and the immune regulation is immune enhancement; preferably, the immune enhancement includes promoting CD8 + T cell proliferation; preferably, the immune enhancement includes (but is not limited to): tumor inhibition, inhibition of pathogens (such as viruses or bacteria).

[0014] In another preferred embodiment, the VEGF-C upregulator includes: exogenous VEGF-C or an expression construct or vector containing the VEGF-C coding gene; preferably, the expression construct includes an enhanced promoter, a tissue-specific promoter or an enhancer.

[0015] In another aspect of the present invention, a modified MSCs (or MSCs culture) is provided, into which exogenous VEGF-C or a regulator of its downstream signaling pathway is introduced.

[0016] In a preferred example, the regulator is a downregulator of VEGF-C or its downstream signaling pathway, including PI3K / AKT pathway inhibitors, TNFR1 downregulators, glucocorticoid receptor downregulators, small molecule compounds that specifically inhibit VEGF-C or its receptors, small molecule compounds that specifically inhibit TNFR1; interfering molecules that specifically interfere with the expression of VEGF-C gene or TNFR1 gene; gene editing reagents that specifically downregulate (including knocking out) VEGF-C or TNFR1; antibodies or ligands that specifically bind to VEGF-C protein or TNFR1 protein, or reagents that specifically block the binding of VEGF-C to its receptor VEGFR3; more preferably, the PI3K / AKT pathway inhibitor includes: pan-PI3K inhibitor LY294002 or AKT inhibitor Afuresertib, the small molecule compounds that specifically inhibit VEGF-C receptors are MAZ51, SAR131675; antibodies that specifically inhibit VEGFR3.

[0017] In another preferred embodiment, the regulator is VEGF-C or its upregulator, including exogenous VEGF-C or an expression construct or vector containing the gene encoding the VEGF-C; preferably, the expression construct includes an enhanced promoter, a tissue-specific promoter or an enhancer.

[0018] In another preferred embodiment, the MSCs are MSCs treated with glucocorticoids, and preferably the glucocorticoids include: dexamethasone (Dex), methylprednisolone, betamethasone, hydrocortisone, prednisone, methylprednisone, beclomethasone propionate, budesonide, fluticasone propionate.

[0019] In another aspect of the present invention, the use of the modified MSCs (or MSCs culture) is provided for preparing an immunomodulatory pharmaceutical composition; wherein, MSCs introduced or added with a downregulator of VEGF-C or its downstream signaling pathway are used for immunosuppression; and MSCs introduced or added with an upregulator of VEGF-C or its downstream signaling pathway are used for immunoenhancement.

[0020] In another aspect of the present invention, a pharmaceutical composition or kit for immunosuppression is provided, comprising MSCs and a substance that promotes the immunosuppressive effect of MSCs, wherein the substance is a down-regulator of VEGF-C or its downstream signaling pathway; preferably, the immunosuppression includes alleviating or treating CD8 + T cell hyperproliferation-related diseases; preferably including (but not limited to): inhibiting autoimmune diseases, inflammation; more preferably, the CD8 +Diseases associated with excessive T cell proliferation include (but are not limited to): graft-versus-host disease, multiple sclerosis, inflammatory bowel disease, scleroderma, type I diabetes, rheumatoid arthritis, Hashimoto's thyroiditis, psoriasis, systemic lupus erythematosus, and organ damage such as liver damage, lung damage, and digestive tract damage; more preferably, the liver damage includes liver fibrosis or cirrhosis, and the lung damage includes non-fibrosis.

[0021] In another aspect of the present invention, a pharmaceutical composition or kit for immunosuppression is provided, which comprises the modified MSCs and into which a down-regulator of VEGF-C or its downstream signaling pathway is introduced or added; preferably, the MSCs are MSCs treated with glucocorticoids, and preferably the glucocorticoids include: dexamethasone (Dex), methylprednisolone, betamethasone, hydrocortisone, prednisone, methylprednisone, beclomethasone propionate, budesonide, fluticasone propionate.

[0022] In a preferred embodiment, the pharmaceutical composition or medicine kit further comprises: glucocorticoids; preferably, the glucocorticoids include: dexamethasone (Dex), methylprednisolone, betamethasone, hydrocortisone, prednisone, methylprednisone, beclomethasone propionate, budesonide, and fluticasone propionate.

[0023] In another preferred embodiment, the downregulator of VEGF-C or its downstream signaling pathway includes PI3K / AKT pathway inhibitors, TNFR1 downregulators, glucocorticoid receptor downregulators, small molecule compounds that specifically inhibit VEGF-C or its receptors, small molecule compounds that specifically inhibit TNFR1; interfering molecules that specifically interfere with the expression of VEGF-C gene or TNFR1 gene; gene editing reagents that specifically downregulate (including knocking out) VEGF-C or TNFR1; antibodies or ligands that specifically bind to VEGF-C protein or TNFR1 protein, or reagents that specifically block the binding of VEGF-C to its receptor VEGFR3; more preferably, the PI3K / AKT pathway inhibitor includes: pan-PI3K inhibitor LY294002 or AKT inhibitor Afuresertib, the small molecule compound that specifically inhibits VEGF-C receptor is a small molecule compound MAZ51, SAR131675 that inhibits the tyrosine kinase activity of VEGF-C receptor (VEGFR3); antibodies that specifically inhibit VEGFR3.

[0024] In another aspect of the present invention, a pharmaceutical composition or kit for immune enhancement is provided, comprising MSCs, and a substance that promotes the immune enhancement effect of MSCs; the substance is a VEGF-C upregulator; preferably, the immune enhancement includes: inhibiting tumors, inhibiting pathogens (such as viruses or bacteria); preferably, the VEGF-C upregulator includes: exogenous VEGF-C or an expression construct or vector containing the gene encoding the VEGF-C; preferably, the expression construct includes an enhancing promoter, a tissue-specific promoter or an enhancer.

[0025] In another aspect of the present invention, a pharmaceutical composition or kit for immune enhancement is provided, comprising the modified MSCs and VEGF-C or its upregulator introduced therein; preferably, the MSCs are MSCs treated with glucocorticoids, and preferably the glucocorticoids include: dexamethasone (Dex), methylprednisolone, betamethasone, hydrocortisone, prednisone, methylprednisone, beclomethasone propionate, budesonide, fluticasone propionate.

[0026] In another aspect of the present invention, a method for screening immunomodulatory drugs (including potential drugs) is provided, the method comprising: (1) providing MSCs cells and CD8 + T cells coexist in the system, the MSCs cells express VEGF-C, the CD8 + T cells express VEGFR3; (2) treating the system of claim 1 with the candidate substance, and determining the expression or activity of VEGF-C, the interaction between VEGF-C and VEGFR3, or CD8 + T cell proliferation; (3) Analyze the results of (2), if VEGF-C expression or activity is reduced, the interaction between VEGF-C and VEGFR3 is reduced and / or CD8 + If the proliferation of T cells is inhibited, the candidate substance is an immunosuppressive drug; if the expression or activity of VEGF-C is increased, the interaction between VEGF-C and VEGFR3 is enhanced, and / or CD8 + If T cell proliferation is promoted, the candidate substance is an immunopotentiating drug.

[0027] In another preferred embodiment, the method comprises step (1): in the test group, adding the candidate substance to the system; and / or, step (2) comprises: detecting the expression or activity of VEGF-C, the interaction between VEGF-C and VEGFR3, or CD8 + The T cell proliferation is measured and compared with that of a control group, wherein the control group is a system without adding the candidate substance.

[0028] In another preferred embodiment, the system further comprises the addition of glucocorticoids; preferably, the glucocorticoids include: dexamethasone (Dex), methylprednisolone, betamethasone, hydrocortisone, prednisone, methylprednisone, beclomethasone propionate, budesonide, fluticasone acid.

[0029] In another preferred embodiment, the immunoregulation includes immunosuppression or immunoenhancement.

[0030] In another preferred embodiment, the immunosuppression includes alleviating or treating CD8 + T cell hyperproliferation-related diseases; preferably including (but not limited to): inhibiting autoimmune diseases, inflammation; more preferably, the CD8 + Diseases associated with excessive T cell proliferation include (but are not limited to): graft-versus-host disease, multiple sclerosis, inflammatory bowel disease, scleroderma, type I diabetes, rheumatoid arthritis, Hashimoto's thyroiditis, psoriasis, systemic lupus erythematosus, organ damage such as liver damage, lung damage, and digestive tract damage; more preferably, the liver damage includes liver fibrosis or cirrhosis, and the lung damage includes non-fibrosis;

[0031] In another preferred embodiment, the immune enhancement includes: inhibiting tumors and inhibiting pathogens (such as viruses or bacteria).

[0032] Other aspects of the present invention will be apparent to those skilled in the art in view of the disclosure herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 、Dex through CD8 + T cells reverse the immunosuppressive effect of MSCs. (A) Survival curve of aGvHD mice; C57BL / 6×C3H F1 mice around 8 weeks old were lethally irradiated (13Gry); 24 hours later, whole bone marrow cells (5×10 6 ) and spleen cells (5×10 7 ) were mixed and transplanted into F1 mice via the tail vein; on the 5th and 7th days after bone marrow transplantation, mice received tail vein infusion of BM-MSCs (5×10 5 ), subcutaneous injection of Dex (2 mg / kg, three times a week) or a combination of the two; the mice were observed daily, the survival time of the mice was counted, and the survival curve of the mice was drawn; n = 12 or 16. (B) H&E staining of the liver on day 21; on day 21, the liver was fixed and H&E staining was performed to observe the infiltration of lymphocytes in the liver; the scale length is 50 μm. (C) Splenocyte proliferation; anti-CD3 activated mouse spleen lymphocytes (Spl, 2×10 5 ) alone or in combination with mouse bone marrow-derived MSCs (1×104 ) were co-cultured in a 96-well plate, and different concentrations of Dex were added at the same time. 3 H-labeled thymidine ( 3 H-Tdr) to detect the proliferation of spleen cells. (D) Absolute number of lymphocytes; after the lymphocytes were co-cultured with MSCs for 48 hours, the suspended lymphocytes in the co-culture system were blown out and counted. (E) Flow cytometry analysis of CD4 + and CD8 + Changes in T cell subsets; After lymphocytes and MSCs were co-cultured for 48 hours, the suspended lymphocytes in the co-culture system were stained and CD8 + T and CD4 + The relationship between the proportion of T cells and the concentration of Dex. (FG) CD8 + T cell proliferation and total number; MSCs were compared with naive CD8 + T cells were co-cultured and different concentrations of Dex were added to the system. 3 H-Tdr incorporation method was used to detect T cell proliferation. (HI)CD4 + T cell proliferation and total number. MSCs were compared with naive CD4 + T cells were co-cultured and different concentrations of Dex were added to the system. 3 H-Tdr incorporation was used to detect T cell proliferation. Statistical analysis was performed using Student's t-test (two-tailed) and Log-rank test (Mantel-Cox). Statistical results are expressed as mean ± SEM. *p < 0.05 and **p < 0.01.

[0034] Figure 2 Dex induces MSCs to produce VEGF-C and promotes CD8 + T cell proliferation. (A) CD8 + T cell proliferation. MSCs were incubated with CD8 + T cells were co-cultured and divided into four groups. G1: normal MSCs (G1); G2: Dex-treated group (1 ng / ml); G3: CD8 + T cell co-culture group; G4: Dex treatment and CD8 +T cell co-culture group. Affymetrix 3 expression profile chip was used to analyze the pairwise comparison of the above four groups of cells, and the up-regulated genes of the following groups were obtained: G4 vs G1, G4 vs G2 and G4 vs G3 (the primary selection standard for up-regulated genes here is 2-fold up-regulation). Subsequently, the intersection of the above three groups of up-regulated genes and the corresponding sequences were found using the Venny set analysis method. (B) Venny analysis. The gene ID number to be analyzed was entered into the MAS biological function annotation system for GO analysis to facilitate a detailed understanding of the function, subcellular distribution and signal pathway connection of the target gene. According to the expression change, the genes with higher up-regulated expression among the 22 genes obtained by the Wayne set analysis were listed, and the gene name, subcellular distribution and gene ID number were annotated. (C) Heat map. Some genes with significant up-regulation and down-regulation were selected to make a heat map. Red indicates up-regulation and green indicates down-regulation. (D) List of differentially expressed genes. (E) Vegfc expression level. Freshly isolated spleen cells were stimulated with anti-CD3, and the spleen cell culture supernatant was collected by centrifugation 48 hours later. The spleen cell culture supernatant was added to the MSCs culture system at a ratio of 1:1, and different concentrations of Dex were added at the same time. The culture was continued for 24 hours, and RNA was extracted and RT-PCR was used to detect the changes in mRNA expression in MSCs. (F) Vegfa mRNA expression level. (G) Vegfb mRNA expression level. (H) Vegfd mRNA expression level. (I) Knockdown of MSCs Vegfc expression inhibits Dex-induced CD8 + T cell proliferation. Statistical analysis was performed using Student's t-test (two-tailed). Statistical results are expressed as mean ± SEM. *p < 0.05.

[0035] Figure 3 , VEGF-C exacerbates aGvHD. (A) Schematic diagram of VEGF-C treatment. Neomycin (0.5 mg / ml) was added to the drinking water of mice two days in advance. C57BL / 6×C3H F1 mice aged about 8 weeks were lethally irradiated with 13 Gry (to reduce intestinal toxicity, the irradiation was divided into two times with an interval of 4 hours, the first time was 7 Gry, and the second time was 6 Gry). The next day, whole bone marrow cells (5×10 6 ); or whole bone marrow cells (5×10 6 ) and total spleen cells (5×10 7 ) were mixed and transplanted into F1 mice via the tail vein. (B) Survival curve of aGvHD mice. VEGF-C (10μg / mice / day) was injected subcutaneously 7 to 10 days after bone marrow transplantation for 7 to 10 days. The mice were observed every day and the survival curve was calculated. (C) H&E staining of the lung, liver and colon of aGvHD mice on day 21. (D) Effect of VEGF-C treatment on CD4 + T cells and CD8+ Effect of VEGF-C treatment on the proportion of liver lymphocytes and CD8 T cells in aGvHD mice + Effect of T cell number. 20-21 days after bone marrow transplantation, mice were euthanized, and monocytes in peripheral blood were separated by mouse lymphocyte separation solution, monocytes in liver were separated by Percoll density gradient centrifugation, and lymphocytes in mouse spleen were obtained by grinding and lysing red blood cells. The total number of lymphocytes in the above tissues in each group was counted, and the number of CD8 + Absolute number of T cells. n = 4-6. (GH) Effect of VEGF-C treatment on lymphocytes and CD8 T cells in peripheral blood of aGvHD mice + (IJ) Effect of VEGF-C treatment on the number of splenocytes and CD8 T cells in aGvHD mice + Effect of VEGF-C treatment on the number of CD8 T cells in the liver of aGvHD mice + Effect of EdU incorporation in T cells. Statistical analysis was performed using Student's t-test (two-tailed) and Log-rank test (Mantel-Cox). Statistical results are expressed as mean ± SEM. *p < 0.05, **p < 0.01 and ***p < 0.001.

[0036] Figure 4 VEGF-C promotes CD8 + T cell proliferation. (A) VEGF-C promotes T cell proliferation. MLR reaction: The spleen of (Stimulator) Balb / C mice was removed to obtain a single cell suspension, which was then irradiated with 20Gry to inhibit the proliferation of T cells. The spleen single cell suspension of (Responder) C57BL / 6 mice was purified by magnetic bead sorting to obtain CD8 + T cells. Stimulators (8×10 5 ) and Responders(2×10 5 ) co-culture, different concentrations of VEGF-C were added to the co-culture system, and after 72 hours of culture, 3 H-labeled thymidine was used to detect T cell proliferation. (B) VEGF-C promoted anti-CD3 / 28-activated CD8 + T cell proliferation. Anti-CD3 (2.5 μg / ml) was coated on a 96-well plate overnight, and then purified and separated CD8 + T cells (2×10 5). At the same time, anti-CD28 (1 μg / ml) was added to the suspension to activate T cells. Different concentrations of VEGF-C factor were added to the culture system and cultured for 48 hours. 3 H-Tdr detects T cell proliferation. (C) VEGF-C affects CD8 + Effect of CD69 and CD25 expression on T cells. Cell separation and purification of CD8 + T cells were activated with antiCD3 / 28 and different concentrations of VEGF-C were added. Anti-CD69 and anti-CD25 antibodies and isotype staining were used to detect the expression changes by flow cytometry. (D) Effect of VEGF-C on CD8 + Effects of VEGF-C on CD8 + Effect of IFN-γ expression on T cells. (F) CD8 + The expression of VEGFR2 and VEGFR3 on the surface of T cells. Purified CD8 + T cells were stained with anti-VEGFR2-PE and anti-VEGFR3-APC antibodies and the corresponding isotypes, and the expression of cell surface receptors was detected by flow cytometry. (G) MLR detection of CD8 + T cell proliferation. Among them, Stat3 fl / fl and Stat3 fl / fl CD8 Cre CD8 isolated from mice + T cells are responder cells. (H) Survival curve of aGvHD mice. Stat3 fl / fl and Stat3 fl / fl CD8 Cre Bone marrow cells and spleen cells induce aGvHD in mice. (I) CD8 + T cell proliferation. Mouse CD8 + T cells (2×10 5 ) alone or in combination with mouse bone marrow-derived MSCs (1×10 4 ) were co-cultured in a 96-well plate. Then, Dex (10 ng / ml), VEGF-C (800 ng / ml) and MAZ51 (10 μM) were used for 42 hours. (J) Mouse survival time. The mouse aGvHD model was established. On day 5, mice were injected with MSCs (5×10 6) and Dex (2 mg / kg) or MAZ51 (15 mg / kg / d) treatment (n=7). Statistical analysis was performed using Student's t-test (two-tailed) and Log-rank test (Mantel-Cox). Statistical results are expressed as mean±SEM. *p<0.05, **p<0.01 and ***p<0.001.

[0037] Figure 5 , VEGF-C promotes CD8 + T cell proliferation. (A) GO analysis. CD8 + (B) Heat map of signaling pathways enriched in T cells. CD8 + Expression of cell cycle-related genes in T cells. (C) Effect of VEGF-C on CD8 activated by anti-CD3 / CD28 + Effects of VEGF-C on the expression of Cyclin D1, Cdk2, Cdk4 and Cdk6 in T cells. (D) Effects of VEGF-C on the expression of Cyclin D1, Cdk2, Cdk4 and Cdk6 in T cells activated by PMA+Inomycin + Effects of VEGF-C on the expression of p-p85 / p55, Cdk2, Cdk4 and Cdk6 in T cells. (E) Effects of VEGF-C on the expression of p-p85 / p55, Cdk2, Cdk4 and Cdk6 in T cells. + Effect of VEGFR3 on the expression of p-p85 / p55 and p-Akt in T cells. (F) + (G) Dex does not affect the expression of CD4 + (H) VEGF-C does not affect the expression of Cyclin D1 in T cells activated by anti-CD3 / CD28. + T cells expressed p-p85 / p55, p-Akt and Cyclin D1. (I) Immunoblotting analysis of VEGFR3 fl / fl and VEGFR3 fl / fl CD8 Cre Mouse CD8 + p85 / p55, Akt and their phosphorylation in T cell lysate. (J) Inhibition of PI3K / AKT signaling pathway abolished the effect of VEGF-C on CD8 + Promoting T cell proliferation. Stimulating CD8 with anti-CD3 / CD28 + T cells, CD8 + T cells, and the effects of both on VEGF-C-induced CD8 +Effects on T cell proliferation. Statistical analysis was performed using Student's t-test (two-tailed). Statistical results are expressed as mean ± SEM. *p < 0.05 and **p < 0.01.

[0038] Figure 6 , TNF-α and Dex promote VEGF-C expression. (A) Plasma VEGF-C levels in mice at designated time points during the progression of aGvHD. (B) Plasma VEGF-C levels in patients with aGvHD who are sensitive or insensitive to glucocorticoid treatment. Plasma VEGF-C levels in patients with aGvHD (sensitive) before / after the course of the disease (n=12), patients with aGvHD (insensitive) before / after the course of the disease (n=16), and patients with aGvHD (insensitive) during the course of the disease. (C) VEGF-C concentration in the culture supernatant of MSCs. MSCs were treated with LPS (100 ng / ml) and IFN-γ (10 ng / ml), and the VEGF-C level in the culture supernatant was detected. (D) VEGF-C level in the culture supernatant of MSCs. MSCs were treated with TNFα (10 ng / ml), and the VEGF-C level in the culture supernatant of MSCs was detected by ELISA. (E) Vegfd mRNA expression level. MSCs were treated with Dex (10 ng / ml), TNFα (10 ng / ml) and the combination of Dex (10 ng / ml) and TNFα (10 ng / ml) for 24 hours, and the expression of Vegfd mRNA was detected by RT-PCR. (FG) VEGF-C expression level. MSCs were treated with Dex (10 ng / ml), TNFα (10 ng / ml) and the combination of Dex (10 ng / ml) for 24 hours, and the levels of VEGF-C mRNA and protein were detected by RT-PCR and WB. (H) Vegfc expression level. Tnfr1 expression in MSCs was knocked down by siRNA, and the expression level of Vegfc mRNA in MSCs was detected by TNFα (10 ng / ml). (I) Knockdown of Tnfr1 expression in MSCs inhibited Dex-induced CD8 + T cell proliferation. Anti-CD3 / CD28 activated CD8 + T cells were co-cultured with Ctrl-MSCs or siRNA-Tnfr1 MSCs. +Effect of T cell proliferation. (JK) VEGF-C expression level. MSCs were treated with TNFα (3 ng / ml), Dex (50 ng / ml) and RU486 (1 μM), and the VEGF-C mRNA and protein expression levels in MSCs were detected. (L) VEGF-C expression level. MSCs were treated with different concentrations of Dex (1, 10, 100 ng / ml) and TNFα (5 ng / ml), and the VEGF-C mRNA level in MSCs was detected. Statistical analysis was performed using Student's t-test (two-tailed) and Log-rank test (Mantel-Cox). Statistical results are expressed as mean±SEM. *p<0.05, **p<0.01 and ***p<0.001.

[0039] Figure 7 , dexamethasone and MSCs inhibited melanoma growth. 5 B16 melanoma cells alone or with 2.5×10 5 MSCs were mixed and injected intramuscularly into the outer thigh of C57BL / 6 mice. Five days later, 1 mg / kg of Dex was injected intraperitoneally into the tumor-bearing mice, and Dex was injected every 3 days. After the 12th day of tumor growth, the tumor was dissected, weighed and analyzed. Statistical significance is indicated as: **p<0.01; three independent experiments were repeated, n=7.

[0040] Figure 8 In the Dex and MSCs combination group, there were more CD8 + T cells. (A) Leukocytes in tumor tissue were separated by Percoll density gradient centrifugation, and T cell subsets in tumor tissue were analyzed by flow cytometry. (B) Statistical analysis of CD45 + CD4 in cells + and CD8 + T cell subsets. (C) Total lymphocytes, CD4 + and CD8 + Absolute counts of T cells. In the Dex and MSCs groups, CD8 + The proportion and total number of T cells increased significantly, but CD4 + There was no significant change in T cells. (D) Lymphocytes were isolated from peripheral blood and analyzed for CD4 + and CD8 + T cell subsets. (E) Total lymphocytes and CD8 + T cell counts. Statistical significance is indicated as: *p<0.05; **p<0.01; ***p<0.001.

[0041] Fig. 9The inhibitory effect of Dex and MSCs on tumor growth depends on the immune system and MHC-1-restricted CD8 + T cells. 5×10 5 B16 melanoma cells alone or with 2.5×10 5 MSCs were injected intramuscularly into (A) NOD-SCID mice and (B) β2M - / - The outer thigh of the mouse. Five days later, the tumor-bearing mice were intraperitoneally injected with 1 mg / kg of Dex, and Dex was injected every three days. After the 12th day of tumor growth, the tumor was removed, weighed and analyzed. n = 7 (A), n = 5-7 (B) Three independent experiments were repeated. DETAILED DESCRIPTION

[0042] Given the complexity of MSCs' immunomodulation in both scientific and clinical applications, there is still a bottleneck in their clinical application. The inventors are committed to improving the immunomodulatory effect of MSCs. Based on in-depth research, they have confirmed for the first time that glucocorticoids (such as dexamethasone) can regulate MSCs to produce VEGF-C, thereby regulating CD8 + T cell proliferation; TNF-α can cooperate with Dex to regulate VEGF-C. + Changes in the number or activity of T cells are inevitably related to changes in immune function. The present invention provides a new approach for the design of drugs for regulating immunity.

[0043] the term

[0044] As used herein, the term "autoimmune disease" is any disease in which the immune system mounts an undesirable immune response against itself (e.g., one or more autoantigens). For example, an autoimmune disease involves abnormal destruction of body cells as part of a self-targeted immune response. For another example, the self-destruction manifests as malfunction of an organ (e.g., colon or pancreas). In the present invention, CD8 + "Autoimmune diseases" related to excessive proliferation of T cells.

[0045] As used herein, the down-regulator includes inhibitors, antagonists, blockers, inhibitors of protein / protein, protein / gene (or gene transcript) interaction, and the like.

[0046] As used herein, the up-regulator includes promoters, agonists, activators, enhancers, stabilizers, protein / protein, protein / gene (or gene transcript) interaction promoters, and the like.

[0047] As used herein, the terms "upregulate", "enhance", "enhance", "increase", "promote", "strengthen" and the like are interchangeable and should be used to mean a statistically significant (or significant) upregulation compared to a "control" or normal / conventional state, such as at least 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9% or 10%, preferably at least 15% or 20%, more preferably 25%, 30%, 50%, 80%, 100% or more significant upregulation (such as but not limited to upregulation of gene expression, upregulation of protein levels, upregulation of interactions between proteins, etc.).

[0048] As used herein, the terms "downregulate", "reduce", "lower", "inhibit", "weaken", "block" and the like are interchangeable and should be used to mean a statistically significant (or significant) downregulation compared to a "control" or normal / conventional state, such as at least 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9% or 10%, preferably at least 15% or 20%, more preferably 25%, 30%, 50%, 80%, 100% or more significant downregulation (such as but not limited to downregulation of gene expression, downregulation of protein levels, downregulation of interactions between proteins, etc.).

[0049] As used herein, "exogenous" or "heterologous" refers to the relationship between two or more nucleic acid or protein sequences from different sources, or the relationship between nucleic acids or proteins from different sources and a host. For example, a promoter is exogenous to a gene of interest if the combination of the promoter and the gene of interest sequence does not normally occur in nature. A particular sequence is "exogenous" to a cell or organism into which it is inserted.

[0050] T cell-based immune regulation

[0051] The human immune system has evolved a sophisticated regulatory mechanism that allows it to respond quickly and destructively to microbial invaders while sparing the host's own tissues. Studies have shown that the regulation of this delicate balance depends primarily on the CD4 + T cells and CD8 + T cells. CD4 and CD8 are antigen proteins on T lymphocytes. + T cells are called helper T cells, CD8 + The T cells are called cytotoxic T cells.

[0052] Major histocompatibility complex class I and class II proteins (MHC or HLA in humans) bind peptide antigens and present them on the cell surface for recognition by T lymphocytes expressing special T cell receptors (TCRs) that match specific MHC-peptide combinations. The transmembrane glycoproteins CD8 and CD4 are characteristic of different populations of T lymphocytes whose antigenic responses are restricted by class I and class II MHC molecules, respectively. Both CD8 and CD4 play a major role in the differentiation and selection of T cells during thymic development and in the activation of mature T lymphocytes by antigen-presenting cells. Therefore, CD8 and CD4 are considered to be the main accessory molecules of the T cell receptor. Although CD8 and CD4 are proteins of the immunoglobulin superfamily and determine antigenic restriction by binding to MHC molecules rather than antigenic peptides, the structural basis of their similar functions appears to be very different. Their sequence similarity is low, and CD4 is expressed on the cell surface as a monomer, while CD8 is expressed as an αα homodimer or αβ heterodimer. The immunological role of CD8 in mature cytotoxic lymphocytes is complex. The functions of CTL currently postulated in the art include: first, CD8 plays a role in T cell signaling. Second, an obvious function of CD8 may be to assist cell-cell adhesion, adhering T cells and antigen presenting cells by increasing the affinity of the interaction. Third, CD8 can act as a co-receptor of TCR that participates in the cooperative binding mechanism of the two receptors with MHC / peptide ligands.

[0053] In the present invention, the inventors compared the effects of Dex on MSCs and the upregulation of VEGF-C on CD4 + T cells and CD8 + The results showed that VEGF-C upregulated CD4 + T cells had no significant effect, while CD8 + T cell proliferation increased significantly, leading to an upregulation of total lymphocyte numbers.

[0054] According to the knowledge in this field, CD8 + T cells have the ability to recognize and kill tumor cells. They can also kill cells infected by pathogens (such as viruses or cells) and destroy foreign or abnormal cells. Therefore, when killing tumors and fighting pathogens are the treatment goals, increasing the body's CD8 + T cells are an effective approach.

[0055] On the other hand, CD8 + Excessive proliferation of T cells leads to excessive immunity. Therefore, if immunosuppression (such as alleviating autoimmune diseases) is the treatment goal, reducing the body's CD8 + T cell numbers are an effective approach.

[0056] Immunosuppressive applications

[0057] The present inventors have found that glucocorticoids (such as dexamethasone) promote MSCs to produce VEGF-C, which in turn promotes CD8 + T cell proliferation, thereby inhibiting the immunosuppressive effect of MSCs (for example, inhibiting its therapeutic effect on aGvHD); it was further found that TNF-α cooperated with Dex to promote VEGF-C expression. Therefore, substances that target downregulation of VEGF-C and its related pathways can be used to promote the immunosuppressive effect of MSCs, which is beneficial to the treatment of immune diseases (such as autoimmune diseases, graft-versus-host disease, multiple sclerosis). The immune diseases are generally diseases related to immune hyperimmunity and immune disorders.

[0058] Based on the discovery of the inventors, the present invention provides the use of a down-regulator of VEGF-C or its downstream signaling pathway (including PI3K / AKT pathway and its receptor-related signaling pathway) in the preparation of a pharmaceutical composition for promoting the immunosuppressive effect of MSCs.

[0059] The immunosuppressive effect of the present invention is based on the inhibition of CD8 + T cell proliferation plays a role. Therefore, the immunosuppression includes alleviating or treating CD8 + Diseases related to excessive proliferation of T cells; such diseases include (but are not limited to): inhibition of autoimmune diseases, inflammation, etc.; more specifically include (but are not limited to): inhibition of graft-versus-host disease, etc. CD8 + T cell increase also occurs in multiple sclerosis patients. Therefore, the scheme of the present invention can also be used for the relief or treatment of multiple sclerosis.

[0060] The present invention also provides a pharmaceutical composition for immunosuppression, comprising MSCs and a substance that promotes the immunosuppressive effect of MSCs, wherein the substance is a down-regulator of VEGF-C or its downstream signaling pathway.

[0061] As a preferred embodiment of the present invention, the immunosuppressive pharmaceutical composition further comprises glucocorticoids, including dexamethasone (Dex) and the like.

[0062] In the present invention, the down-regulator of VEGF-C (including its downstream signaling pathway) or TNFR1 gene or protein (including its downstream signaling pathway) refers to any substance that can reduce the activity of VEGF-C or TNFR1 protein, reduce the stability of VEGF-C or TNFR1 gene or protein, down-regulate the expression of VEGF-C or TNFR1 protein, reduce the effective action time of VEGF-C or TNFR1 protein, or inhibit the transcription and translation of VEGF-C or TNFR1 gene, and these substances can be used in the present invention as substances useful for down-regulating VEGF-C or TNFR1, so as to be used for suppressing immunity. For example, the down-regulator is: an interfering RNA molecule or antisense nucleotide that specifically interferes with the expression of VEGF-C or TNFR1 gene; or an antibody or ligand that specifically binds to the protein encoded by VEGF-C or TNFR1 gene, etc.

[0063] It can also downregulate the expression of VEGF-C by affecting the upstream pathway of VEGF-C.

[0064] As an alternative of the present invention, the down-regulator is a small molecule compound targeting VEGF-C or TNFR1. Those skilled in the art can use conventional screening methods in the art to screen such small molecule compounds.

[0065] As an alternative mode of the present invention, the down-regulator is a VEGF-C or TNFR1 specific interfering RNA molecule (shRNA). The use of interfering RNA molecules can significantly down-regulate VEGF-C or TNFR1 in MSCs, which has an effect on immunosuppression. The present invention has no particular restrictions on the preparation method of the interfering RNA molecule, including but not limited to: chemical synthesis, in vitro transcription, etc. The interfering RNA can be delivered into the cell by using an appropriate transfection reagent, or can also be delivered into the cell by using a variety of techniques known in the art.

[0066] As another option of the present invention, the CRISPR / Cas9 system can be used for targeted gene editing to knock out the VEGF-C or TNFR1 gene in MSCs. Common methods for knocking out the VEGF-C or TNFR1 gene include: co-transferring sgRNA or a nucleic acid capable of forming the sgRNA, Cas9 mRNA or a nucleic acid capable of forming the Cas9 mRNA into a targeted region or a targeted cell. After determining the target site, the sgRNA and Cas9 can be introduced into the cell using known methods.

[0067] Application in immune enhancement

[0068] The inventors also found that glucocorticoids (such as dexamethasone) can promote MSCs to produce VEGF-C, which promotes CD8 + T cell proliferation is also beneficial to promote the immune enhancement effect of MSCs, which is beneficial to the treatment of diseases that require immune enhancement, such as tumor treatment. The inventor's research has shown that CD8 + When T cell proliferation is promoted, the total number of lymphocytes in the body can be significantly increased, which is beneficial to the body's tumor-killing effect.

[0069] Based on the present invention, the present invention provides the use of VEGF-C or its upregulator in the preparation of a pharmaceutical composition for promoting the immune enhancement effect of MSCs; preferably, the immune enhancement effect is achieved by promoting CD8 + T cell proliferation plays a role; preferably, the immune enhancement includes but is not limited to: inhibiting tumors, inhibiting pathogens (such as viruses or bacteria), etc.

[0070] As a preferred embodiment of the present invention, the immune enhancing pharmaceutical composition further comprises glucocorticoids, including dexamethasone (Dex) and the like.

[0071] In the present invention, the VEGF-C upregulator includes: exogenous VEGF-C or an expression construct or vector containing a VEGF-C coding gene; preferably, the expression construct includes an enhanced promoter, a tissue-specific promoter or an enhancer.

[0072] In the present invention, the VEGF-C up-regulator includes promoters, agonists, and activators. The "up-regulation" and "promotion" include "up-regulation" and "promotion" of protein activity or "up-regulation" and "promotion" of protein expression, and they are statistically significant "up-regulation" and "promotion". Any substance that can increase the activity of VEGF-C, increase the stability of VEGF-C, up-regulate the expression of the target, increase the effective action time of VEGF-C, regulate the phosphorylation level of VEGF-C and promote protein activation can be used in the present invention as a substance useful for up-regulating VEGF-C. They can be compounds, small chemical molecules, and biological molecules. The biological molecules can be at the nucleic acid level (including DNA, RNA) or at the protein level.

[0073] In one embodiment of the present invention, the expression of VEGF-C protein can be increased by using VEGF-C protein itself or its up-regulator. For example, the up-regulator is an expression vector that over-expresses VEGF-C protein.

[0074] It can also upregulate the expression of VEGF-C by affecting the upstream pathway of VEGF-C.

[0075] Cells, compositions and kits

[0076] MSCs are a group of adult stem cells derived from the mesoderm with high self-renewal and multi-differentiation potentials. They are relatively easy to obtain, and the in vitro expansion and culture technology is mature.

[0077] Based on the new discovery of the inventors, the present invention provides modified MSCs (including MSCs culture). The modification method includes but is not limited to the method used in the present invention. In some embodiments, the cells are modified in vitro or ex vivo.

[0078] By down-regulating VEGF-C or its downstream signaling pathway, the VEGF-C down-regulator is introduced into the modified MSCs.

[0079] By the method of upregulating VEGF-C, the VEGF-C or its upregulator is introduced into the modified MSCs.

[0080] In the present invention, a polynucleotide sequence encoding a regulator of overexpressed VEGF-C or its downstream signaling pathway can be inserted into a recombinant expression vector to transform MSCs. As long as they can replicate and stabilize in MSCs, a variety of plasmids and vectors can be used in the present invention. An important feature of an expression vector is that it usually contains a replication origin, a promoter, a marker gene, and a translation control element. For example, the expression vector can include: a viral vector, a non-viral vector.

[0081] Methods well known to those skilled in the art can be used to construct expression vectors containing polynucleotide sequences of regulators of VEGF-C or its downstream signaling pathway and suitable transcription / translation control signals. These methods include in vitro recombinant DNA technology, DNA synthesis technology, in vivo recombination technology, etc. The DNA sequence can be effectively linked to an appropriate promoter in the expression vector to guide mRNA synthesis. The conversion vector also includes a ribosome binding site for translation initiation and a transcription terminator.

[0082] In some preferred embodiments, the expression vector includes (but is not limited to): adeno-associated virus, lentiviral vector, adenoviral vector, etc. In a more preferred embodiment, the MSCs modified with VEGF-C or its regulator are obtained by transfecting MSCs with a virus that overexpresses VEGF-C or its regulator. For example, an adenoviral vector can be used to overexpress the target gene, and after adenovirus transfection of MSCs, VEGF-C or its regulator is highly expressed in MSCs, which can achieve a higher infection rate. Other viral vectors or non-viral vectors are also applicable.

[0083] As a preferred embodiment of the present invention, the modified MSCs or the culture thereof further comprises treatment with glucocorticoids, and preferably the glucocorticoids include: dexamethasone (Dex) and the like.

[0084] The present invention also provides a composition comprising an effective amount (such as 10 4 -10 9 cells / mL; preferably 10 5 -10 8 cells / mL; better, 10 6 -10 7 cells / ML) of the modified MSCs, and a pharmaceutically acceptable carrier.

[0085] Typically, the cells can be formulated in a non-toxic, inert and pharmaceutically acceptable aqueous carrier medium, wherein the pH is generally about 5-8, preferably, about pH 6-8.

[0086] Pharmaceutically acceptable carriers may include (but are not limited to): saline, buffer, glucose, water, glycerol, ethanol, and combinations thereof. Usually, the pharmaceutical preparation should be matched with the mode of administration. The pharmaceutical composition of the present invention can be prepared in the form of injection, for example, by conventional methods using physiological saline or an aqueous solution containing glucose and other adjuvants. The pharmaceutical composition is preferably manufactured under sterile conditions. The dosage of the active ingredient is a therapeutically effective amount. The pharmaceutical preparation of the present invention can also be prepared into a sustained-release preparation.

[0087] The effective amount of the modified MSCs of the present invention may vary depending on the mode of administration and the severity of the disease to be treated. The selection of the preferred effective amount can be determined by a person of ordinary skill in the art based on various factors (e.g., through clinical trials). The factors include, but are not limited to: pharmacokinetic parameters of the modified MSCs such as bioavailability, metabolism, half-life, etc.; the severity of the disease to be treated, the patient's body weight, the patient's immune status, the route of administration, etc. Generally, several divided doses may be administered daily, or the dose may be reduced proportionally, depending on the requirements of the treatment condition.

[0088] As a preferred embodiment of the present invention, some dosing regimens for animals such as mice are provided in the specific embodiments of the present invention. It is easy for a person skilled in the art to convert the dosage of an animal (such as a mouse) into a dosage suitable for humans. In the art, there are some examples of the ratio of the body surface area of ​​experimental animals and humans commonly used for dosage conversion, such as those listed in the following Table 1.

[0089] Table 1

[0090]

[0091] As an example of the table lookup method of Table 1, for example, the dog dose is 10 mg / kg, and the total dose of a 12 kg dog is 12×10 mg=120 mg; looking up the table above, the intersection of a 70 kg human and a 12 kg dog is 3.1, so the dose of a human (70 kg) = 120 mg×3.1=372 mg. It should be understood that the conversion method between other species and humans can be deduced in the same way.

[0092] In practical application, the pharmaceutical composition can be placed in a medicine box. Therefore, the present invention also provides a medicine box / test kit for regulating immunity, comprising: a container, and recombinant mesenchymal stem cells or cell cultures placed in the container.

[0093] The medicine box / test kit may also contain glucocorticoids and the like.

[0094] In addition, the medicine box / test kit may also contain some auxiliary medication materials, such as injection syringes and the like.

[0095] In addition, the medicine box / test kit may also contain instructions for use, describing the method for regulating immunity based on the combination of compounds of the present invention.

[0096] Screening methods

[0097] Based on the new discovery of the present inventors, a method for screening immunomodulatory drugs (including potential drugs) is also provided, comprising: (1) providing MSCs cells and CD8 + T cells coexist in the system, the MSCs cells express VEGF-C, the CD8 + T cells express VEGFR3; (2) treating the system of claim 1 with the candidate substance, and determining the expression or activity of VEGF-C, the interaction between VEGF-C and VEGFR3, or CD8 + T cell proliferation; (3) Analyze the results of (2), if VEGF-C expression or activity is reduced, the interaction between VEGF-C and VEGFR3 is reduced and / or CD8 + If T cell proliferation is inhibited (significantly reduced / inhibited, such as reduced / inhibited by 10%, 20%, 30%, 50% or more or less), the candidate substance is an immunosuppressive drug; if VEGF-C expression or activity is increased, the interaction between VEGF-C and VEGFR3 is enhanced, and / or CD8 + If T cell proliferation is promoted (significantly improved / enhanced / promoted, such as improved / enhanced / promoted by 10%, 20%, 30%, 50% or more), the candidate substance is an immunoenhancing drug.

[0098] Although VEGF-C is used as the screening target in the above method, it should be understood that screening for VEGF-C upstream / downstream signaling pathways, upstream / downstream genes / proteins is also included in the present invention.

[0099] As a preferred embodiment of the present invention, the system further comprises adding glucocorticoids; preferably, the glucocorticoids include: dexamethasone (Dex) and the like.

[0100] In addition, a control group can be set up to achieve qualitative and quantitative analysis.

[0101] When screening is performed, various techniques well known in the art can be used to determine changes in proteins or their encoding genes and interactions.

[0102] A variety of conventional techniques can be used to identify the transcription or protein expression of genes in the system, including but not limited to oligonucleotide hybridization techniques (such as probes), polymerase chain reaction (PCR), polyacrylamide gel electrophoresis, immunoblotting (such as Western blotting), etc.

[0103] Methods for screening substances acting on a protein or gene or a specific region thereof as a target are well known to those skilled in the art, and all of these methods can be used in the present invention. The candidate substances can be selected from: peptides, polymeric peptides, peptidomimetics, non-peptide compounds, carbohydrates, lipids, antibodies or antibody fragments, ligands, organic small molecules, inorganic small molecules, and nucleic acid sequences, etc. Depending on the type of substance to be screened, those skilled in the art will know how to select an appropriate screening method.

[0104] The substances initially screened out by the above method can constitute a screening library, so that people can eventually screen out substances that are truly useful for affecting the immune regulation of MSCs.

[0105] The present invention is further described below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods in the following examples where specific conditions are not specified are usually carried out according to conventional conditions such as those described in J. Sambrook et al., Molecular Cloning Experiment Guide, 3rd edition, Science Press, 2002, or according to the conditions recommended by the manufacturer.

[0106] Materials and methods

[0107] 1. Cell culture and other solution preparation

[0108] Complete culture medium: DMEM or RPMI1640 culture medium with 10% FBS (volume ratio), 100 U / ml Penicillin, 100 U / ml Streptomycin, 2 mM L-Glutamine, and 50 μM β-mercaptoethanol added.

[0109] Flow cytometry buffer (stored at 4-8°C): 2% FBS + PBS.

[0110] Cell freezing solution: 10% DMSO + 50% FBS + 40% DMEM.

[0111] 2. Cell or animal models

[0112] Mouse melanoma B16 / F0 cells: ATCC.

[0113] iNOS - / - MSCs cells: Mesenchymal stem cell-mediated immunosuppressionoccurs via concerted action of chemokines and nitric oxide (PMID: 18371435).

[0114] β2M - / - Mice: purchased from Jackson Laboratory, USA.

[0115] C57BL / 6×C3H mice: obtained by mating C3H and C57BL / 6.

[0116] C3H mice were obtained from Shanghai Southern Animal Model Center.

[0117] C57BL / 6 (H2b) mice were obtained from the Shanghai Experimental Animal Center, Chinese Academy of Sciences.

[0118] Balb / C (H2d) mice were obtained from the Shanghai Experimental Animal Center, Chinese Academy of Sciences.

[0119] 2. Isolation of Mouse Bone Marrow-derived Mesenchymal Stem Cells

[0120] Isolation, culture and cryopreservation of MSCs from mice: After euthanasia, the femurs of mice were first isolated, and then the bone marrow was completely flushed out with DMEM culture medium until the femurs turned white. The bone marrow cells were centrifuged and then cryopreserved at 2×10 6 / ml concentration and resuspended in DMEM complete culture medium. After 24h of adherent culture, change the medium, and then change the culture medium every 3 days. MSCs will gradually form clones. After the formation of clones, pick a single clone MSCs and plant it in a single well of a 96-well plate to obtain MSCs from a single clone. When the cells proliferate to 80% confluence, subculture them in time. Then add 0.25% trypsin for cell subculture and digest at 37°C, and subculture or freeze at a ratio of 1:3-5.

[0121] 3. Preparation of mouse spleen single cell suspension

[0122] First, euthanize 6-8 week old C57BL / 6 mice and immerse them in 75% ethanol for disinfection. Then use sterile surgical scissors to cut the epidermis and peritoneum of the mouse abdomen in sequence, expose the spleen on the left side of the mouse body, and separate and remove the spleen with forceps. Place the spleen in a 70μm mesh, gently grind it with a sterile 1ml syringe piston, and rinse it with RPMI1640 complete medium to obtain a single cell suspension. Centrifuge at 400g×5min, remove the medium, add 2-5ml of red blood cell lysis buffer and mix it upside down. After the lysis buffer becomes clear after about 1min, add RPMI1640 complete medium to terminate the reaction. Finally, centrifuge at 400g×5min, remove the supernatant, repeat the wash 3 times, resuspend it with RPMI1640, and count it for use.

[0123] 4. 3 H-Tdr lymphocyte proliferation assay

[0124] Thymidine ( 3 H-Tdr) labeled proliferation experiment: 6 h before the end of lymphocyte culture, 3 H-labeled thymidine was added to the cell culture medium at 0.5 μCi per well, and the culture was continued for 6 h before the well plate was frozen to -80°C. After thawing, the cells were collected on glass fiber paper using a vacuum cell harvester, and the CPM value of each well was measured using a β liquid scintillation counter.

[0125] 5. Mouse Graft-versus-Host Disease Model

[0126] Two days before bone marrow transplantation, 0.5 mg / ml neomycin was added to the drinking water of mice. One day before bone marrow transplantation, 8-10 W C57BL / 6×C3H F1 mice were irradiated with 13Gry. 24 h later, 5×10 C57BL / 6 mice were infused into the tail vein. 6 Whole bone marrow cells and 5×10 7 Spleen cells. MSCs were infused into the tail vein 5-7 days after bone marrow transplantation. The mice were observed every day and the survival curve of the mice was calculated. On days 14 and 21 after bone marrow transplantation, the spleen, liver, small intestine, and lung were collected for H&E staining and flow cytometry analysis.

[0127] 6. Mouse melanoma research model

[0128] 5×10 5 Mouse melanoma B16 / F0 cells alone or in combination with 2.5×10 5 MSCs were resuspended in 50 μL HBSS system and injected intramuscularly into the inner thigh of C57BL / 6 mice. The tumor growth was observed every day until the tumor size affected the mouse activity (about 2 to 3 weeks). The mice were euthanized and the melanoma was removed and weighed. After the tumor grew, Dex (1 mg / kg) was injected three times a week.

[0129] 7. Histochemical staining / HE staining

[0130] After the experimental mice were anesthetized, the chest cavity of the mice was opened, and physiological saline was perfused through the heart and then perfused with 4% paraformaldehyde for internal fixation. The lungs, liver, colon, spleen, etc. were then placed in a tube containing 4% paraformaldehyde and fixed again. The fixed tissues were processed in the following steps: (1) Dehydration: 75% alcohol for 1 hour; 95% alcohol for 2 hours, twice; 100% alcohol for 2 hours, repeated once. (2) Transparency: Place the dehydrated tissue in xylene, 60°C, 20 minutes, repeat once. (3) Wax immersion: Place the transparent tissue in melted paraffin, 60°C, 2 hours, repeat once. (4) Embedding. (5) Sectioning: Cut the embedded tissue block into 5μm thick slices. (6) Spreading: Spread the 5μm thick tissue slices on 40°C water to allow the tissue to fully flatten. (7) Slide: After the tissue is flattened, pick up the tissue slices and flatten them on the treated glass slides. (8) Drying: Place the slides on a drying table at 37°C to dry.

[0131] HE staining: according to the conventional method in the art.

[0132] 8. Real-time fluorescence quantitative PCR detection

[0133] RNA was extracted (TIANGEN Cell RNA Extraction Kit); reverse transcription product cDNA was obtained according to TIANGEN TIANScript cDNA First Chain Generation Kit; quantitative PCR was then performed. Mouse Realtime-PCR primers are shown in Table 2.

[0134] Table 2

[0135]

[0136] 9. Protein sample processing and immunoblotting detection

[0137] Protein sample processing and immunoblotting were performed according to conventional methods in the art, wherein the antibody concentrations used were as follows:

[0138] Primary antibodies: anti-β-actin (1:3000); anti-STAT1 (1:3000); anti-p-STAT1 (1:3000);

[0139] Secondary antibody: HRP-anti-rabbit (1:5000); HRP-anti-mouse (1:5000).

[0140] 10. Lentivirus transfection

[0141] (1) 24 hours before transfection, cells were seeded into 12-well plates and 1 mL of complete medium was added to each well. The seeding density should reach 50% the next day.

[0142] (2) Before transfection, thaw the viral particles to room temperature, mix gently, and place on ice. Replace the culture medium with complete culture medium containing 5 μg / mL polybrene, add the viral particles, shake well, and incubate overnight.

[0143] (3) 12 hours after adding the virus, the culture medium was replaced with fresh complete culture medium and incubated overnight.

[0144] (4) When the cells are close to complete fusion, they are digested and passaged at a ratio of 1:3 to 1:5 and cultured for 24-48 hours. The expression of green fluorescent protein is observed under a fluorescence microscope.

[0145] (5) When the cells are close to complete fusion, add Puromycin (3-5 μg / mL) for screening. Then replace the culture medium every 3-4 days. The obtained cells are stable transfected strains.

[0146] 11. CD4 + T and CD8 + T cell isolation

[0147] Mouse spleen single cell suspension was counted and then the mouse CD4 + T and CD8 + The T cell isolation kit was used to obtain purified cells for later use.

[0148] 12. MLR mixed lymphocyte culture

[0149] MLR is an in vitro experimental system for detecting T lymphocyte function. The spleen of (Stimulator) Balb / C mice was removed and irradiated with 20Gry to inhibit the proliferation of T cells therein. At the same time, the spleen single cell suspension of (Responder) C57BL / 6 mice was purified by magnetic bead sorting method to obtain CD8 +T cells. Mix responder and stimulator at a ratio of 1:1, 1:2, 1:4, 1:8 to find the appropriate culture concentration. Add different concentrations of VEGFC at the same time, and detect the proliferation of lymphocytes by 3H-Tdr method after 3-4 days.

[0150] 13. Flow cytometry analysis

[0151] Surface staining: Take 0.5-1×10 cells 6 Resuspend in about 10-20 μl FACS buffer for staining. First, incubate the cells with anti-CD16 / CD32 to block non-specific binding that may be caused by Fc receptors. Then perform surface antibody staining, incubate on ice for 30 minutes, wash twice with PBS, fix or resuspend in PBS again, and perform flow cytometry analysis or intracellular staining.

[0152] Intracellular staining: (1) Stain the cell surface molecules first. (2) After washing once with FACS buffer, add 100μl of Fixation / Permeabilization buffer to each tube and fix overnight at 4℃ or fix at room temperature for 30min. (3) After fixation, the cell specific gravity decreases and the speed needs to be increased. Centrifuge at 600g for 5min, add 200μl of 1×Permeabilization buffer, and wash twice. (4) Centrifuge at 600g for 5min, add intracellular cytokine antibody, and stain on ice for 1 hour away from light. (5) Directly add 200μl of 1×Permeabilization buffer and wash twice. (6) After discarding the supernatant, fix with 4% PFA. (7) After centrifugation, resuspend the cell pellet with 200μl of FACS buffer and analyze it by flow cytometry.

[0153] Example 1: Dexamethasone (Dex) inhibits the therapeutic effect of MSCs on aGvHD

[0154] In order to study the effects of Dex and MSCs on the pathological process of acute graft-versus-host disease (aGvHD), the inventors established a mouse bone marrow transplant aGvHD model that simulates the occurrence of human GvHD. The bone marrow and lymphocytes of C57BL / 6 mice were transplanted into lethally irradiated C57BL / 6×C3H F1 mice to establish a half-matched mixed bone marrow transplant hematopoietic reconstruction aGvHD model. aGvHD mice were treated with tail vein infusion of MSCs, subcutaneous injection of Dex, and co-infusion of MSCs and Dex. The results showed that treatment with MSCs and Dex alone could significantly prolong the survival time of mice, indicating that Dex and MSCs can effectively control the progression of aGvHD ( Figure 1A). However, it is worth noting that co-infusion of Dex and MSCs into mice not only failed to prolong the survival of mice, but significantly aggravated the aGvHD condition of mice ( Figure 1 B). Histological examination results showed that Dex and MSCs alone could significantly inhibit the infiltration of inflammatory cells into the portal vein area. In contrast, co-infusion of Dex and MSCs not only failed to significantly improve inflammatory infiltration, but also promoted the infiltration of inflammatory cells in liver tissue ( Figure 1 B) The above results suggest that Dex blocked the therapeutic effect of MSCs.

[0155] In order to better study the interaction between Dex and MSCs, the inventors introduced an in vitro model of co-culture of MSCs and T cells. MSCs were co-cultured with anti-CD3 activated spleen cells, and different concentrations of Dex were added to the system to detect the effects of MSCs, Dex, and the co-existence of MSCs and Dex on the proliferation of spleen cells in the system. The results showed that Dex had a significant inhibitory effect on the proliferation of spleen cells, and this inhibition was dose-dependent. MSCs also have a strong immunosuppressive effect on T cell proliferation. However, when Dex is present in the co-culture system of T cells and MSCs, the inhibitory effect of MSCs on spleen cells is relieved as the concentration of Dex increases. Under the concentration conditions of 0.1-5ng / ml Dex, the proliferation of spleen cells even exceeds that of the spleen cell control group ( Figure 1 C) In addition to using 3 In addition to detecting spleen cell proliferation by H-Tdr method, the inventors also counted the total number of lymphocytes ( Figure 1 D). The present inventors found that when the concentration of Dex was between 0.5 and 1 ng / ml, the absolute number of lymphocytes in the co-culture system also increased significantly.

[0156] Next, the inventors used flow cytometry to analyze the subcellular populations of lymphocytes in the co-culture system. It can be seen that after adding different gradients of Dex, CD8 + T cell subsets were significantly suppressed, while CD4 + T cells had little effect; similarly, MSCs could also significantly inhibit CD8 + However, when Dex was present in the co-culture system, especially in the concentration window of 0.5-1 ng / ml, CD8 + The proportion of T cells was significantly restored, while CD4 + The proportion of T cells remained unchanged. Flow cytometry data showed that CD8 + The trend of T cell changes is consistent with that of lymphocyte proliferation ( Figure 1 E).

[0157] Next, in order to clarify that Dex mainly promotes CD8 + T cell proliferation reverses MSCs immunosuppression. The inventors used magnetic bead sorting to purify CD8 + T cells and CD4 + The effect of Dex on the proliferation of MSCs and T cells was studied. First, anti-CD3 / 28 was used to activate T cells in the co-culture system. At the same time, different concentrations of Dex were added to the co-culture system to evaluate the effect of Dex on T cell proliferation. The results showed that Dex could only reverse the proliferation of MSCs on CD8 + T cell inhibitory effect ( Figure 1 FG), while CD4 + There was no significant effect on T cell proliferation ( Figure 1 HI).

[0158] Example 2: Dex promotes MSCs to produce VEGF-C

[0159] In order to identify the key molecule that reverses the immunosuppression of MSCs by Dex, the present inventors used an in vitro co-culture system to simulate the interaction between MSCs and CD8 + The interaction between T cells. The previous study of the inventors showed that MSCs can promote immunity in the presence of low concentrations of IFN-γ and TNF-α, TGF-β, CsA or steroids. In this case, iNOS is the switch of MSCs immunosuppression or immunopromotion. By utilizing iNOS - / - MSCs, the inventors found that iNOS-deficient MSCs can still reverse the effect of MSCs on CD8 + T cell inhibitory effect ( Figure 2 A), this data suggests that MSCs may promote CD8 + T cell proliferation.

[0160] Next, RNA-seq was used to analyze the effects of Dex and co-culture treatment on MSCs gene expression. The inventors divided MSCs into four groups: normal MSCs (G1); Dex-treated group (G2); CD8 + T cell co-culture group (G3); Dex and CD8 + T cell co-culture group (G4). The results showed that compared with G1, G2 and G3, 1770, 1765 and 200 genes were upregulated in the G4 group, respectively ( Figure 2B). By using Venny set screening, the inventors identified 22 up-regulated genes (the intersection of three sets). Through research and analysis, the inventors located endothelial growth factor C (VEGF-C). The data showed that the Vegfc gene was significantly up-regulated ( Figure 2 CD).

[0161] The VEGF family has multiple members. The inventors analyzed the expression changes of Vegfa, Vegfb, Vegfc and Vegfd genes in the VEGF family. The inventors used the supernatant of lymphocytes stimulated by anti-CD3 activation to replace the co-cultured T cells. The results showed that Dex and T cell culture supernatant had a synergistic effect on Vegfc gene expression ( Figure 2 E), however, there is no synergistic effect between the two on the expression of other VEGF family genes ( Figure 2 FH).

[0162] Subsequently, the inventors constructed a Vegfc knockdown MSCs cell line (shVegfc-MSCs) and studied the effect of VEGF-C in MSCs on CD8+T cell proliferation. The sequence used for Vegfc knockdown is: (LV3-Vegfc-mus-1400): 5'-GCGAATCGACTGAAGCATTGT-3' (SEQ ID NO: 11).

[0163] The present inventors found that knocking down Vegfc in MSCs significantly inhibited the effect of Dex on CD8 + T cell immune promotion Figure 2 I). This indicates that the Vegfc knockdown sequence LV3-Vegfc-mus-1400 has excellent effects.

[0164] These results demonstrate that Dex promotes CD8 + T cell proliferation.

[0165] Example 3: VEGF-C exacerbates aGvHD progression

[0166] The inventors verified the effect of VEGF-C on the progression of aGvHD in vivo. After myeloablation of C57BL / 6×C3H F1 mice using high-dose radiation, the inventors performed simple bone marrow transplantation (BMT group) and mixed bone marrow and spleen lymphocyte transplantation to construct aGvHD (aGvHD group). The results showed that VEGF-C can significantly aggravate the progression of aGvHD and shorten the survival time of mice ( Figure 3 AB).

[0167] The inventors performed histological staining on mice to observe the lymphocyte infiltration in the liver, lungs and colon. As can be seen from the figure, in the BMT control group, VEGF-C had almost no effect on promoting lymphocyte infiltration. However, in the aGvHD group, the lymphocyte infiltration in the bile ducts and ducts of the liver of the mice in the VEGF-C injection group increased significantly, and the edema and degeneration of hepatocytes increased; the lungs of mice showed a significant increase in inflammatory cell infiltration; in the lower colon, the shortening and reduction of the intestinal crypts, the erosion of mucosal epithelial cells, and the increase of lymphocyte infiltration in the lamina propria indicated that the intestinal damage was aggravated ( Figure 3 C).

[0168] The inventors analyzed the CD8 + T and CD4 + The inventors found that compared with the BMT group, the CD8 + T and CD4 + The proportion of T cells increased significantly, which indicated that the aGvHD course was developing ( Figure 3 D) After VEGF-C infusion, CD8 + The proportion of T cells was significantly upregulated ( Figure 3 D). In addition, the present inventors conducted a study on the total lymphocyte count in peripheral blood, the total lymphocyte count infiltrating the liver, and the CD8 + T cell counts, CD8 infiltrating in the liver + The number of T cells was counted. The results showed that VEGF-C can upregulate lymphocytes and CD8 + Total T cells ( Figure 3 EJ). In addition, the spleen cells in the aGvHD group decreased compared with the BMT group, and VEGF-C could reverse this trend ( Figure 3 IJ). The inventors used EdU incorporation to detect the proliferation of T cells in the liver of aGvHD mice. The results showed that VEGF-C mainly promoted CD8 + T cell proliferation ( Figure 3 K).

[0169] The above data indicate that VEGF-C promotes CD8 + T cell proliferation exacerbates aGvHD progression.

[0170] Example 4: VEGF-C promotes CD8 + T cell proliferation

[0171] The present inventors analyzed the immune-promoting effect of VEGF-C in a one-way mixed lymphocyte reaction (MLR). b ) + T cells are reactive cells, and the Balb / C (H2 d ) Spleen cells are stimulatory cells. Since the major histocompatibility antigens of the responding cells and stimulatory cells are different, the stimulatory cells can provide the first signal and the second signal for T cell activation at the same time. The results show that VEGF-C promotes T cell proliferation in the MLR system in a concentration-dependent manner ( Figure 4 A). Under the conditions of anti-CD3 and anti-CD28 stimulation, VEGF-C can also promote CD8 + T cell proliferation ( Figure 4 B) In addition, VEGF-C treated CD8 + T cells express higher levels of activation markers CD69 and CD25 ( Figure 4 C), but for CD8 + There was no significant effect on T cell apoptosis and IFN-γ expression ( Figure 4 DE).

[0172] Previous studies have shown that VEGFR2 and VEGFR3 are natural receptors for VEGF-C. Therefore, the inventors used flow cytometry to detect CD8 + Expression of VEGFR2 and VEGFR3 on T cells. The results showed that CD8 + T cells highly express VEGFR3 but low expression of VEGFR2 ( Figure 4 F) To further analyze whether VEGF-C promotes CD8 + T cell proliferation, the inventors used the Cre-Loxp system to specifically knock out VEGFR3 in T cells. In the MLR system, VEGFR3-deficient CD8 + T cells do not respond to VEGF-C ( Figure 4 G) VEGFR3 fl / fl CD8 Cre Mouse or VEGFR3 fl / fl When the bone marrow cells and spleen cells of mice were transferred into C57BL / 6×C3H F1 mice to induce aGvHD, mice carrying VEGFR3-deficient T cells had a weaker ability to induce aGvHD and survived longer than the control group ( Figure 4 H). These results suggest that VEGF-C promotes CD8 + T cells proliferate and promote aGvHD.

[0173] Next, the inventors analyzed the effects of VEGFR3 inhibitors on CD8 + T cell proliferation. MAZ51 is a tyrosine kinase inhibitor that can block VEGFR3 kinase activity. + In the co-culture system of T cells and MSCs, the inventors found that blocking the VEGFR3 signaling pathway with MAZ51 could eliminate the CD8 + T cell proliferation ( Figure 4 I). The results showed that MAZ51 treatment could prolong the survival time of aGvHD mice co-administered with MSCs and Dex ( Figure 4 J).

[0174] The above data indicate that Dex promotes CD8 + T cell proliferation.

[0175] Example 5: VEGF-C promotes CD8 + T cell proliferation

[0176] In order to elucidate the role of VEGF-C in promoting CD8 + The molecular mechanism of T cell proliferation, the inventors first studied the CD8 + RNA-seq was performed on CD8 T cells co-cultured with MSCs in the presence of Dex. + T cell "focal adhesion", "cytokine-cytokine receptor interaction" and "actin cytoskeleton" regulation-related pathways were highly enriched ( Figure 5 A). Given that VEGF-C can promote CD8 + The inventors focused on cell proliferation-related genes. The inventors found that after adding Dex to the co-culture system of T cells and MSCs, CD8 + There are 12 cell proliferation-related genes differentially expressed in T cells. Among these genes, Cyclin D1 has the highest expression level ( Figure 5 B). The present inventors further confirmed that under the stimulation of anti-CD3 and anti-CD28, VEGF-C promoted CD8 + T cells express cyclin D1 and cyclin-dependent protein kinases (CDKs), including CDK2, CDK4, and CDK6 ( Figure 5 C) These results indicate that VEGF-C promotes CD8 + T cell proliferation.

[0177] Next, the inventors investigated whether VEGFR3 affects proximal TCR signaling. First, the effect of VEGF-C on CD8 T cells induced by phorbol-12-myristate-13-acetate (PMA) and ionomycin was evaluated. + This activation method can directly act on the PKB and calcium ion signals of the T cell activation pathway and bypass the upstream proximal TCR signal. In the PMA and ionomycin activation system, VEGF-C does not affect the expression of p-p85 / pp-55 and CDKs, which are closely related to T cell activation and proliferation. This suggests that VEGF-C may affect the proximal TCR signal ( Figure 5 D) In ​​the anti-CD3 and anti-CD28 T cell activation systems, PI3K / AKT signaling is crucial for initiating T cell proliferation. The inventors found that VEGF-C treatment can enhance CD8 + PI3K / AKT activity in T cells ( Figure 5 E). At the same time, the inventors analyzed the effect of VEGF-C on CD4 + The inventors found that CD4 + T cells barely express VEGFR3 ( Figure 5 F) and CD8 + Unlike T cells, Dex could not promote CD4 + T cells express Cyclin D1 more Figure 5 G). Effect of exogenous VEGF-C on CD4 + The expression of p-p85 / p-p55, p-Akt and Cyclin D1 in T cells was not significantly affected ( Figure 5 H) In VEGFR3 fl / fl and VEGFR3 fl / fl CD8 Cre In mice, CD8 + Deletion of VEGFR3 in T cells almost completely inhibited VEGF-C-induced p-p85, p-p55, and p-Akt expression ( Figure 5 I). In addition, the present inventors found that pan-PI3K inhibitor LY294002 and AKT inhibitor Afuresertib can treat CD8 + T cells can block VEGF-C-induced CD8 + T cell proliferation ( Figure 5 J). This result indicates that VEGFC overexpressed in MSCs (e.g., induced by Dex) can be downregulated by pan-PI3K inhibitors, thereby further inhibiting CD8 + T cell proliferation.

[0178] Example 6: TNF-α cooperates with Dex to promote VEGF-C expression

[0179] In order to explore the mechanism by which glucocorticoids induce enhanced VEGF-C expression in vivo, the inventors first detected the dynamic changes of VEGF-C in the plasma of aGvHD mice and aGvHD patients that were sensitive or insensitive to glucocorticoid treatment. The results showed that the concentration of VEGF-C in the plasma of aGvHD mice gradually increased ( Figure 6 A). More importantly, the plasma VEGF-C levels of patients with aGvHD who were resistant to steroid therapy (treatment-insensitive) were much higher than those of patients who recovered from aGvHD, regardless of whether these patients were sensitive to glucocorticoid therapy ( Figure 6 B) This significant change suggests that VEGF-C can be induced by inflammation in vivo and is associated with glucocorticoid resistance in patients.

[0180] Subsequently, the inventors studied the effects of several different inflammatory cytokines and TLR agonists on VEGF-C expression. The results showed that LPS significantly upregulated VEGF-C expression in MSCs ( Figure 6 C); TNFα treatment alone can also achieve similar effects ( Figure 6 D); however, IFN-γ failed to induce VEGF-C upregulation ( Figure 6 D). The present inventors found that TNFα and Dex (Combo) can synergistically induce Vegfc expression, but cannot synergistically induce Vegfd expression ( Figure 6 EG, Figure 6 L). Given that TNFα can induce Vegfc expression, the present inventors used small interfering RNA (siRNA) to knock down Tnfr1 expression in MSCs. siRNA sequence (Tnfrsf1a-mus-894 (siRNA2)):

[0181] Sense:5'-CCGCUUGCAAAUGUCACAATT-3'(SEQ ID NO:12);

[0182] Antisense: 5'-UUGUGACAUUUGCAAGCGGTT-3' (SEQ ID NO: 13).

[0183] The results showed that knockdown of Tnfr1 inhibited TNFα-induced Vegfc expression ( Figure 6 H). In addition, Tnfr1 knockdown inhibited Dex-induced CD8 + T cell proliferation ( Figure 6I). In order to verify whether the reaction induced by Dex is through the action of glucocorticoid receptor (GR), the inventors used GR antagonist RU486 to evaluate the role of GR in this process. The inventors found that RU486 completely reversed the expression of VEGF-C induced by Dex ( Figure 6 JK).

[0184] These data suggest that Dex and TNFα can synergistically induce MSCs to express VEGF-C.

[0185] Example 7: Dex and MSCs can inhibit the growth of melanoma

[0186] The study used the B16 melanoma model as the research object. B16 F0 alone or mixed with MSCs was injected into the legs of C57BL / 6 mice by muscle. Dex or PBS control was injected into the mice intraperitoneally starting on the 5th day of tumor growth. The tumors were weighed and analyzed about 12 days later.

[0187] The inventors found that there was no significant change in the growth of tumors in the PBS, Dex and MSCs alone injection groups. However, when Dex was used to treat the MSCs tumor mixed model group, the growth of B16 melanoma was significantly inhibited ( Figure 7 ).

[0188] Example 8: The tumor suppression effect shared by Dex and MSCs is through CD8 + T cell mediated

[0189] In order to further study the effects of Dex and MSCs on anti-tumor immunity, the inventors isolated lymphocytes from mouse peripheral blood and tumor sites and analyzed the cell subtypes in detail. Flow cytometry analysis showed that compared with the tumor group alone, the Dex treatment group alone, and the MSCs group alone, in the Dex and MSCs group, CD8 + The proportion of T cells increased significantly, but CD4 + There was no significant change in T cells ( Figure 8 A, B, D); at the same time, by counting the total lymphocyte count, total CD4 + T and CD8 + The absolute number of T cells, the inventors also found that the increase in the total lymphocyte count was mainly due to CD8 + T cells rather than CD4 + Increased T cells ( Figure 8 C, E).

[0190] In order to verify whether the immune system is involved in the process of "Dex and MSCs inhibiting melanoma", the inventors used severely immunodeficient NOD / SCID mice as recipients. The mice were deficient in T and B cells and had low NK cell function. B16F0 alone or mixed with MSCs was injected intramuscularly into the legs of NOD / SCID mice. Also, Dex or PBS control was injected intraperitoneally into the mice starting from the 5th day of tumor growth. The results showed that although Dex and MSCs can greatly inhibit the tumor growth of wild-type C57 / BL6 mice ( Figure 7 ), but tumor growth was not inhibited in NOD / SCID mice ( Fig. 9 A). Therefore, it is suggested that T, B and NK cells in the immune system are involved in this process.

[0191] In order to verify whether CD8 + T cells are involved in this process, and the study used β2M - / - The mice were used as recipient mice for tumor inoculation and the same experiment was performed. β2 microglobulin (β2M) is one of the components of the first class major histocompatibility antigen complex (MHC-I). If β2M is knocked out, all MHC-I molecules cannot be expressed. During the development of T cells in the thymus, MHC-I molecules restrict CD8 + T cell maturation. - / - In mice, almost all CD8 + The study found that the inhibitory effect of Dex and MSCs on tumors was - / - Blocked in mice ( Fig. 9 B) Therefore, CD8 + T cells mediate the above anti-tumor processes.

[0192] Example 9, Drug Screening

[0193] 1. Screening for potential immunosuppressive substances

[0194] Screening system: Experimental system as described in Example 2: MSCs cells and CD8 + T cells were co-cultured and treated with Dex, at which time VEGF-C expression increased.

[0195] Test group: The screening system is given candidate substances;

[0196] Control group: No candidate substance is administered to the screening system.

[0197] Determination of VEGF-C expression or activity, interaction between VEGF-C and VEGFR3 or CD8 +T cell proliferation; analyze the measurement results, if VEGF-C expression or activity is reduced, VEGF-C and VEGFR3 interaction is reduced and / or CD8 + If the proliferation of T cells is inhibited, the candidate substance is an immunosuppressive drug; if the expression or activity of VEGF-C is increased, the interaction between VEGF-C and VEGFR3 is enhanced, and / or CD8 + If T cell proliferation is promoted, the candidate substance is an immunopotentiating drug.

[0198] All documents mentioned in the present invention are cited as references in this application, just as each document is cited as reference individually. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the claims attached to this application. Sequence Listing <110> Shanghai Institute of Nutrition and Health, Chinese Academy of Sciences <120> Methods and reagents for regulating the immunomodulatory function of mesenchymal stem cells <130> 212086 <160> 13 <170> SIPOSequenceListing 1.0 <210> 1 <211> 20 <212> DNA <213> Primer <400> 1 gctctggctc ctagcaccat 20 <210> 2 <211> twenty one <212> DNA <213> Primer <400> 2 ccaccgatcc acacagagta c 21 <210> 3 <211> twenty three <212> DNA <213> Primer <400> 3 gcacatagag agaatgagct tcc 23 <210> 4 <211> 19 <212> DNA <213> Primer <400> 4 ctccgctctg aaaaggct 19 <210> 5 <211> 20 <212> DNA <213> Primer <400> 5 gccagacagg gttgccatac 20 <210> 6 <211> 22 <212> DNA <213> Primer <400> 6 ggagtgggat ggatgatgtc ag 22 <210> 7 <211> 21 <212> DNA <213> Primer <400> 7 gaggtcaagg cttttgaagg c 21 <210> 8 <211> 21 <212> DNA <213> Primer <400> 8 ctgtcctggt attgagggtg g 21 <210> 9 <211> 19 <212> DNA <213> Primer <400> 9 ttgagcgatc atcccggtc 19 <210> 10 <211> 21 <212> DNA <213> Primer <400> 10 gcgtgagtcc atactggcaa g 21 <210> 11 <211> 21 <212> DNA <213> shVegfc <400> 11 gcgaatcgac tgaagcattg t 21 <210> 12 <211> 21 <212> DNA / RNA <213> siRNA <400> 12 ccgcuugcaa augucacaat t 21 <210> 13 <211> 21 <212> DNA / RNA <213> siRNA <400> 13 uugugacauu ugcaagcggt t 21

Claims

1. A method for screening immunomodulatory drugs, characterized in that: The immunomodulation includes immunosuppression or immunoenhancement, and the method includes: (1) Provide MSCs cells and CD8 + The system of in vitro coexistence of T cells, the MSCs cells express VEGF-C, the CD8 + T cells express VEGFR3; the VEGF-C and VEGFR3 constitute a VEGF-C-VEGFR3 axis; (2) Treating the system described in step (1) with the candidate substance and determining the interaction between VEGF-C and VEGFR3 in the VEGF-C-VEGFR3 axis and the CD8 + T cell proliferation; (3) Analyze the results of (2) and determine if the interaction between VEGF-C and VEGFR3 in the VEGF-C-VEGFR3 axis is reduced and CD8 + If the proliferation of T cells is inhibited, the candidate substance is an immunosuppressive drug; if the interaction between VEGF-C and VEGFR3 in the VEGF-C-VEGFR3 axis is enhanced and CD8 + If T cell proliferation is promoted, the candidate substance is an immune-enhancing drug; The system also includes adding glucocorticoids.

2. The method according to claim 1, characterized in that The glucocorticoid is dexamethasone.

3. The method according to claim 1, characterized in that The immunosuppression is to alleviate or treat CD8 + Diseases associated with T-cell hyperproliferation; graft-versus-host disease.

4. Use of MAZ51, glucocorticoid dexamethasone and MSCs in the preparation of a pharmaceutical composition for inhibiting graft-versus-host disease.

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