Method for enhancing Treg cell function and application

Abstract

The invention provides a method for enhancing Treg cell functions and application, and belongs to the technical field of cell engineering. According to the method, hUC-MSCs are irradiated by gamma rays to form feeder layer cells, and then the feeder layer cells and Treg cells are co-cultured to obtain the Treg cells with enhanced cell functions. Through the co-culture of the feeder layer cells and the Treg cells, the inhibition ability of the Treg cells on activation and proliferation of immune cells can be enhanced, and the feeder layer cells and the Treg cells can be used as universal cells in clinical researches of autoimmune diseases, graft versus host diseases and the like.

Description

Technical Field

[0001] The invention belongs to the technical field of cell engineering and relates to a method for enhancing the function of Treg cells and its application. Background Art

[0002] Regulatory T cells (Tregs) are a type of T cell subset that controls the body's own immune reactivity and has an inhibitory effect on immune responses. They play an important role in tumor immunity, transplant tolerance, autoimmune diseases, and the body's autoimmune balance. Treg cells are a double-edged sword for the human immune system and endocrine system. They can suppress excessive immune responses through a series of mechanisms, allowing the body to effectively eliminate antigens or pathogens with minimal self-damage, and play a very important role in maintaining the body's immune tolerance and immune response homeostasis. Therefore, controlling the number and expression of Treg cells can help us control the course of the disease.

[0003] Treg cells exert their immunosuppressive function mainly in two ways. The first way is that Treg cells exert immunomodulatory effects by secreting heterogeneous cytokines, and their target cells are mainly effector T cells (Effector T cells, Teff), dendritic cells (DC), macrophages, B cells and NK cells. The second way is that Treg cells mainly exert their effects through direct cell-to-cell contact. The balance of the human immune system is maintained by Treg cell nuclei and Teff cells, and once the balance between the two is broken, it will lead to the occurrence of various diseases. Therefore, an excessive number of Treg cells will lead to tumors, infectious diseases, etc., while an insufficient number of Treg cells will lead to autoimmune diseases.

[0004] Studies have shown that a decrease in the number of Treg cells or abnormal functions have occurred in a variety of autoimmune diseases. Intervention in the number and function of Treg cells is likely to be a targeted target for the treatment of a variety of autoimmune and inflammatory diseases. As a type of regulatory T cell, CD4+CD25+Treg cells play an important role in the proliferation of autoreactive T cells and the maintenance of immune tolerance. How to enhance the cell function of Treg so that the cells can better treat autoimmune diseases requires a method for preparing feeder layer cells that enhance the function of Treg cells. Summary of the invention

[0005] Based on the above technical problems to be solved, the present invention provides a method and application for enhancing the function of Treg cells.

[0006] To achieve the above object, the present invention adopts the following technical solutions: The present application provides a method for enhancing Treg cell function, the method comprising: S01: CD4+CD25+ double positive cells were positively selected from mononuclear cells using immunomagnetic beads, and Treg cells were induced and cultured using magnetic beads or antibodies coated with anti-human CD3e and anti-human CD28, IL-2, and TGF-β1 to obtain Treg cells.

[0007] Mononuclear cells were extracted from umbilical cord blood or peripheral blood, and the extracted mononuclear cells were sorted using immunomagnetic beads using the EasySep TM human CD4+CD25+T cell sorting kit to facilitate the positive selection of CD4+CD25+ double positive cells. The selected CD4+CD25+ double positive cells were induced and cultured for 14 days at 37°C and 5% CO2 in full saturated humidity to obtain Treg cells. The initial culture density of mononuclear cells was 1×10 8 -5×10 8 Preferably, the concentration of IL-2 is 200 U / mL, and the concentration of TGF-β1 is 2.5 ng / mL.

[0008] IL-2 is interleukin-2, a cytokine produced by activated T cells, which can promote the activation, proliferation and differentiation of lymphocytes, thereby producing Treg cells. TGF-β1 is transforming growth factor-β1, which can promote the differentiation of regulatory T cells and inhibit the over-activation of effector T cells. In some cases, TGF-β1 inhibits IL-2-induced T cell proliferation.

[0009] In this application, the specific process of extracting mononuclear cells from cord blood or peripheral blood includes: Take peripheral blood or cord blood, add 15mL of whole blood to a 50mL centrifuge tube, centrifuge at 3000rpm for 10min, and set aside 1mL of plasma for each tube. Add PBS to the centrifuge tube from which the plasma was taken to a total volume of 15mL and mix well. Add 15mL of lymphocyte separation solution to another 50mL centrifuge tube, and gently add the mixed blood cells and PBS to the lymphocyte separation solution, centrifuge at 1500rpm for 20min, and gradually reduce the speed to prevent vibration from forming a white film. Transfer the intermediate white film to a new 50mL centrifuge tube, add PBS to 40mL and set aside to obtain mononuclear cells.

[0010] S02: IFN-γ and vitamin B6 are added to the culture medium for culturing hUC-MSCs. After the confluence of hUC-MSCs reaches 90%, γ-ray irradiation is performed to form feeder layer cells.

[0011] The arteries, veins and amniotic membranes in the umbilical cord were removed, and hUC-MSCs (English name: Umbilical cord Mesenchymal Stem Cells; Chinese name: Primary umbilical cord mesenchymal stem cells) were cultured at 37°C and 5% CO2 in a fully saturated humidity. After 24 hours of culture, the medium was changed to remove the cells that did not adhere to the wall. When cultured to the P3 generation, IFN-γ at a concentration of 100-400U / mL and vitamin B6 at a concentration of 1.5-4ng / mL were added to the culture medium for culturing hUC-MSCs and cultured until the confluence of hUC-MSCs reached 90%. At this time, the hUC-MSCs were irradiated with γ-rays at a dose of 30-90Gy to form feeder layer cells.

[0012] In the present application, both IFN-γ and vitamin B6 have the ability to enhance immunosuppression. γ-ray irradiation of hUC-MSCs can ensure that the feeder layer cells have no proliferation ability, reduce the risk of Treg cell contamination, and at the same time maintain its nourishing and promoting effect on target Treg cells.

[0013] Preferably, the concentration of IFN-γ is 200 U / mL, the concentration of vitamin B6 is 3 ng / mL, and the irradiation dose of gamma rays is 50 Gy.

[0014] S03: After 14 days of culture, Treg cells were co-cultured with feeder layer cells at a ratio of (4-10):1 for 5 days.

[0015] In this application, co-culture of Treg cells and feeder cells can enhance the cellular function of Treg cells. The principle is as follows: (1) Cell-to-cell interactions and signal transduction Cytokine support: During the co-culture process, feeder layer cells can secrete a variety of cytokines, such as IL-2, TGF-β, etc. These cytokines can promote the proliferation and functional maintenance of Treg cells during the co-culture process. TGF-β can also promote the differentiation and function of Treg cells.

[0016] Cell contact-dependent signaling: Direct contact between feeder cells and Treg cells produces specific ligand interactions, such as CD28-CD80 / CD86, which enhance the suppressive function of Treg cells.

[0017] (2) Metabolic support Nutrient supply: Feeder cells can provide Treg cells with abundant metabolic substrates such as glucose and fatty acids to meet the metabolic needs of Treg cells, so that Treg cells can better maintain their functions in a specific metabolic environment, such as utilizing the metabolism of lactic acid or fatty acids.

[0018] Metabolic reprogramming: Feeder cells can also promote metabolic reprogramming of Treg cells by regulating the local metabolic environment, making them more inclined to utilize oxidative phosphorylation (OXPHOS) rather than glycolysis, thereby enhancing their function.

[0019] (3) Stability and tolerance of the microenvironment Shaping of the immune microenvironment: γ-ray irradiated feeder layer cells can form a stable immune microenvironment, similar to the tolerant microenvironment in the intestinal lamina propria (LP), which supports the function of Treg cells by reducing inflammatory signals and providing necessary nutrients, thereby enhancing the stability and function of Treg cells.

[0020] Interaction of tolerant cells: Feeder layer cells also contain some tolerant cells, such as CD206+ macrophages, etc. The interaction between these cells and Treg cells can further enhance the inhibitory function of Treg cells.

[0021] (4) Phenotypic transformation and functional maturation Phenotypic conversion: With the support of feeder cells, Treg cells may convert from conventional regulatory T cells (cTreg) to effector regulatory T cells (eTreg), and this conversion is accompanied by upregulation of the expression of function-related genes such as IL-10 and GZMB.

[0022] Functional maturation: The signals and microenvironmental conditions provided by the feeder cells can promote the maturation of Treg cells, making them more efficient in their immunosuppressive function.

[0023] (5) Gene expression and stability Stabilization of Foxp3 expression: Feeder cells help maintain the stable expression of the Foxp3 gene in Treg cells by providing a stable microenvironment and signal support, which is the key to Treg cell function.

[0024] Regulation of signaling pathways: Feeder cells enhance the function of Treg cells by affecting signaling pathways such as mTORC1 and IRE1α-XBP1.

[0025] S04: After the co-culture is completed, the cells are washed and collected by centrifugation to obtain Treg cells with enhanced cell function.

[0026] After the co-culture is completed, the cells floating on the surface of the culture medium are collected, and after centrifugation, the collected cells are washed twice with physiological saline to obtain Treg cells with enhanced cell function.

[0027] In addition, the Treg cells prepared by the above method of the present application can be used to inhibit lymphocyte proliferation and activate immune cells.

[0028] The present invention has the following beneficial effects: (1) Co-culture of Treg cells with feeder cells can enhance the differentiation, proliferation and functional maintenance of Treg cells by secreting cellular molecules, providing metabolic support, providing an immune microenvironment, and regulating cell phenotype conversion.

[0029] (2) This method can enhance the inhibitory ability of Treg cells on the activation and proliferation of immune cells.

[0030] (3) The Treg cells prepared by this method can be used as universal cells for clinical research, such as autoimmune diseases, graft-versus-host disease, etc., wherein the immune diseases are autoimmune diseases and graft-versus-host disease, and the autoimmune diseases are multiple sclerosis, systemic lupus erythematosus, type I diabetes, rheumatoid arthritis, inflammatory bowel disease, and one of psoriasis. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 These are the in vitro functional test results of Treg cells in Examples 1-3 and the comparative example, wherein from left to right they are the first group, the second group, the third group, and the fourth group. DETAILED DESCRIPTION

[0032] The technical solution of the present invention is further explained and illustrated by means of specific embodiments below.

[0033] Example 1 The present invention provides a method for enhancing Treg cell function, the method comprising: S101: Mononuclear cells were extracted from umbilical cord blood or peripheral blood, and the extracted mononuclear cells were sorted using immunomagnetic beads using the EasySep TM human CD4+CD25+T cell sorting kit to obtain positively selected CD4+CD25+ double positive cells. The selected CD4+CD25+ double positive cells were induced and cultured for 14 days at 37°C and 5% CO2 with full saturated humidity using 0.5μg / mL magnetic beads coated with anti-human CD3e and anti-human CD28, 200U / mL IL-2, and 2.5ng / mL TGF-β1 to obtain Treg cells. During the culture process, the medium was replenished at half volume every three days, and the medium contained 100U / mL rIL-2.

[0034] S102: hUC-MSCs were cultured, and when cultured to P3, IFN-γ at a concentration of 200 U / mL and vitamin B6 at a concentration of 3 ng / mL were added to the culture medium of hUC-MSCs and cultured until the confluence of hUC-MSCs reached 90%. The hUC-MSCs were irradiated with γ-rays at a dose of 50 Gy to form feeder layer cells.

[0035] S103: Treg cells cultured for 14 days were inoculated into feeder layer cells for co-culture for 5 days.

[0036] S104: After the co-culture is completed, the cells are collected by centrifugation, and the collected cells are washed twice with physiological saline to obtain Treg cells with enhanced cell function.

[0037] Example 2 The present invention provides a method for enhancing Treg cell function, the method comprising: S201: Mononuclear cells were extracted from umbilical cord blood or peripheral blood, and the extracted mononuclear cells were sorted using immunomagnetic beads using the EasySep TM human CD4+CD25+T cell sorting kit to obtain positively selected CD4+CD25+ double positive cells. The selected CD4+CD25+ double positive cells were induced and cultured for 14 days at 37°C and 5% CO2 with full saturated humidity using 0.5μg / mL of antibodies coated with anti-human CD3e and anti-human CD28, 150U / mL of IL-2, and 1.5ng / mL of TGF-β1 to obtain Treg cells. During the culture process, the medium was replenished at half volume every three days, and the medium contained 100U / mL rIL-2.

[0038] S202: hUC-MSCs were cultured, and when cultured to P3, IFN-γ at a concentration of 150 U / mL and vitamin B6 at a concentration of 2 ng / mL were added to the culture medium of hUC-MSCs and cultured until the confluence of hUC-MSCs reached 90%. The hUC-MSCs were irradiated with γ-rays at a dose of 30 Gy to form feeder layer cells.

[0039] S203: The Treg cells cultured for 14 days were inoculated into the feeder layer cells for co-culture for 5 days.

[0040] S204: After the co-culture is completed, the cells are collected by centrifugation, and the collected cells are washed twice with physiological saline to obtain Treg cells with enhanced cell function.

[0041] Example 3 The present invention provides a method for enhancing Treg cell function, the method comprising: S301: Mononuclear cells were extracted from umbilical cord blood or peripheral blood, and the extracted mononuclear cells were sorted using EasySep TM human CD4+CD25+T cell sorting kit with immunomagnetic beads to obtain positively selected CD4+CD25+ double positive cells. The selected CD4+CD25+ double positive cells were induced and cultured for 14 days at 37°C and 5% CO2 with full saturated humidity using 0.5μg / mL magnetic beads coated with anti-human CD3e and anti-human CD28, 350U / mL IL-2, and 3ng / mL TGF-β1 to obtain Treg cells. During the culture process, the medium was replenished at half volume every three days, and the medium contained 100U / mL rIL-2.

[0042] S302: hUC-MSCs were cultured, and when cultured to P3, IFN-γ at a concentration of 350 U / mL and vitamin B6 at a concentration of 4 ng / mL were added to the culture medium of hUC-MSCs and cultured until the confluence of hUC-MSCs reached 90%. The hUC-MSCs were irradiated with γ-rays at a dose of 90 Gy to form feeder layer cells.

[0043] S303: The Treg cells cultured for 14 days were inoculated into the feeder layer cells for co-culture for 5 days.

[0044] S304: After the co-culture is completed, the cells are collected by centrifugation, and the collected cells are washed twice with physiological saline to obtain Treg cells with enhanced cell function.

[0045] Comparative Example The comparative example of the present application provides a method for culturing Treg cells, the method comprising: S01: Mononuclear cells were extracted from umbilical cord blood or peripheral blood, and the extracted mononuclear cells were sorted using EasySep TM human CD4+CD25+T cell sorting kit with immunomagnetic beads to obtain positively selected CD4+CD25+ double positive cells. The selected CD4+CD25+ double positive cells were induced and cultured for 14 days at 37°C and 5% CO2 with full saturation humidity using 0.5μg / mL magnetic beads coated with anti-human CD3e and anti-human CD28, 200U / mL IL-2, and 2.5ng / mL TGF-β1 to obtain Treg cells. During the culture process, the medium was replenished at half volume every three days, and the medium contained 100U / mL rIL-2.

[0046] S02: After 14 days, the Treg cells that covered the bottom of the bottle were inoculated into a culture dish of equal volume. After 5 days, the cells were collected by centrifugation and washed twice with physiological saline to obtain Treg cells.

[0047] The Treg cells collected from Examples 1-3 and the comparative example were counted and detected by flow cytometry to obtain Table 1.

[0048] Table 1: Treg cell counting and flow cytometry results Note: The expression level of CD4+CD25+Foxp3+ is (the number of CD4+CD25+Foxp3+ cells) / (the number of CD4+ cells), and the expression level of CD4+CD25+Foxp3+Helios+ is (the number of CD4+CD25+Foxp3+ Helios+ cells) / (the number of CD4+CD25+Foxp3+ cells).

[0049] As can be seen from Table 1, the changes in the number of cells in Examples 1-3 and the comparative examples are not obvious, and the survival rates are all above 95%, which meets the requirements. The CD4+CD25+Foxp3+ expression levels of the Treg cells in Examples 1-3 are 69%, 60% and 64%, respectively, which are significantly higher than the CD4+CD25+Foxp3+ expression levels of the Treg cells in the comparative examples. In terms of CD4+CD25+Foxp3+Helios+ expression levels, the expression levels of the Treg cells in Example 1 are higher than those in Examples 2 and 3, but the difference is not large; similarly, they are also significantly higher than the CD4+CD25+Foxp3+Helios+ expression levels of the Treg cells in the comparative example. This shows that the use of feeder layer cells to co-culture Treg cells can greatly increase the CD4+CD25+Foxp3+ expression levels and CD4+CD25+Foxp3+Helios+ expression levels.

[0050] In addition, the present invention also performs an in vitro functional test on the Treg cells collected in Examples 1-3 and the comparative example, i.e., the test of the inhibitory ability on lymphocyte proliferation. The specific method of the test is: freshly isolated peripheral blood mononuclear cells are used as effector cells (Effector T cells, Teff), and 2×10 5 Inoculate into 96-well plates. Then, add different amount ratios of Treg cells in Example 1, Example 2, Example 3 and the comparative example for co-culture, wherein the amount of Treg added in the first group is 1:1, and the co-culture time is 24h; the amount of Treg added in the second group is 5:1, and the co-culture time is 24h; the amount of Treg added in the third group is 1:1, and the co-culture time is 48h; the amount of Treg added in the fourth group is 5:1, and the co-culture time is 48h. After the co-culture, cck8 detection was performed respectively, and the detection results are as follows Figure 1 .

[0051] By the attached Figure 1 It can be seen that when Treg:Teff=1:1 and 5:1, the Treg cells in Examples 1-3 all showed significant inhibitory effects on the proliferation of Teff cells, and the inhibitory ability was significantly higher than that of the Treg cells in the control example, which was statistically significant (P<0.05).

[0052] The above description is only the preferred embodiment of the present application and is not intended to limit the present application. For those skilled in the art, the present application may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. A method for enhancing Treg cell function, characterized in that: include: CD4+CD25+ double positive cells were selected from mononuclear cells using immunomagnetic beads, and Treg cells were induced and cultured using magnetic beads or antibodies coated with anti-humanCD3e and anti-human CD28, IL-2, and TGF-β1 to obtain Treg cells; IFN-γ and vitamin B6 were added to the culture medium for culturing hUC-MSCs, and the hUC-MSCs were cultured until their confluence reached 90%, and then irradiated with γ rays to form feeder layer cells; Co-culturing the Treg cells after culturing for 14 days with the feeder layer cells; After the co-culture is completed, the cells are washed and collected by centrifugation to obtain Treg cells with enhanced cell function.

2. The method for enhancing Treg cell function according to claim 1, characterized in that: The concentration of the IL-2 is 100-400 U / mL, and the concentration of the TGF-β1 is 1.5-3 ng / mL.

3. The method for enhancing Treg cell function according to claim 2, characterized in that: The concentration of the IL-2 is 200 U / mL, and the concentration of the TGF-β1 is 2.5 ng / mL.

4. The method for enhancing Treg cell function according to claim 1, characterized in that: The concentration of the IFN-γ is 100-400 U / mL, and the concentration of the vitamin B6 is 1.5-4 ng / mL.

5. The method for enhancing Treg cell function according to claim 4, characterized in that: The concentration of the IFN-γ is 200 U / mL, and the concentration of the vitamin B6 is 3 ng / mL.

6. The method for enhancing Treg cell function according to claim 1, characterized in that: The irradiation dose of the gamma ray is 30-90 Gy.

7. The method for enhancing Treg cell function according to claim 1, characterized in that: The mononuclear cells are selected from cord blood or peripheral blood, and the initial culture density of the mononuclear cells is 1×10 8 -5×10 8 cells / mL.

8. The method for enhancing Treg cell function according to claim 1, characterized in that: The number ratio of the Treg cells and the feeder layer cells during co-culture is (4-10):

1.

9. The Treg cells prepared by the method for enhancing Treg cell function according to any one of claims 1 to 8 are used for inhibiting lymphocyte proliferation and activating immune cells.

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