In-vitro amplification kit for human peripheral blood regulatory T cells as well as use method and application of in-vitro amplification kit

Abstract

The invention discloses an in-vitro amplification kit for human peripheral blood regulatory T cells as well as a use method and application of the in-vitro amplification kit, and belongs to the technical field of cell separation and amplification. In order to solve the technical problems of low efficiency and low purity of an in-vitro amplification method of regulatory T cells in the prior art, the invention provides an in-vitro amplification kit of human peripheral blood regulatory T cells, the kit comprises a culture medium, an activation antibody, an induction factor and an amplification factor; the activated antibody is a CD3 monoclonal antibody and a CD28 monoclonal antibody, the induction factor is a recombinant human TGF-beta monoclonal antibody, an all-transretinoic acid monoclonal antibody and an anti-human IL-6 monoclonal antibody, and the amplification factor is a recombinant human IL-2. The kit provided by the invention comprises the anti-human IL-6 monoclonal antibody, so that CD4 + cells are prevented from differentiating towards Th17, and the differentiation efficiency and purity of regulatory T cells are improved; the kit has the advantages of simple components, low cost, high amplification purity and quantity, simplicity and convenience in operation and high universality, and can meet the dosage of clinical treatment experiments.

Description

Technical Field

[0001] The present invention belongs to the technical field of cell separation and amplification, and in particular relates to an in vitro amplification kit for human peripheral blood regulatory T cells and a use method and application thereof. Background Art

[0002] Regulatory T cells (Tregs) are a type of T cell subset discovered in recent years that can control the body's autoimmune reactivity. They are a cell group that can suppress immune responses and play an important role in immunopathology, graft tolerance, preventing autoimmune responses and maintaining the body's immune balance. They were also called suppressor T cells in the early days. Tregs are CD4 T cells that highly express CD25 and rely on IL-2 for survival. + T cell subsets can be divided into two types according to the different sources of Tregs cell development: CD4 + CD25 + Tregs, natural Tregs (nTregs) and inducible Tregs (iTregs), among which nTregs develop in the thymus and iTregs develop in the periphery.

[0003] Because the number of regulatory T cells in the human body is very small, accounting for only a small proportion of normal CD4 + 5%-10% of T cells; Currently, the regulatory T cells obtained by the reported Tregs in vitro expansion method have technical problems of small number and low purity. Therefore, exploring the in vitro induction and expansion of high-purity regulatory T cells, establishing a more effective cell proliferation culture method, and obtaining functional regulatory T cells in a short period of time are important prerequisites for the smooth implementation of in vitro functional experiments of immune checkpoints and co-stimulatory signal molecule antibody drugs, and are expected to be used for immunoregulation of clinical diseases, such as the treatment of autoimmune diseases and the control and prevention of transplant rejection reactions. Those skilled in the art are eager to develop a simple, high-purity, and high-efficiency Treg in vitro expansion method. Summary of the invention

[0004] The present invention aims to solve the technical problems of low efficiency and low purity in the in vitro amplification method of regulatory T cells in the prior art, and provides an in vitro amplification kit for regulatory T cells of human peripheral blood, and a use method and application thereof.

[0005] One of the purposes of the present invention is to provide an in vitro expansion kit for human peripheral blood regulatory T cells, wherein the in vitro expansion kit comprises: a culture medium, an activation antibody, an induction factor and an expansion factor.

[0006] In a preferred embodiment of the present invention, among the activation antibodies, activation antibody 1 is CD3 monoclonal antibody and activation antibody 2 is CD28 monoclonal antibody.

[0007] In a preferred embodiment of the present invention, among the induction factors, induction factor 1 is recombinant human TGF-β, induction factor 2 is all-trans retinoic acid, and induction factor 3 is anti-human IL-6 monoclonal antibody.

[0008] In a preferred embodiment of the present invention, the amplification factor is recombinant human IL-2.

[0009] In a preferred embodiment of the present invention, the culture medium is GT-T551 serum-free culture medium.

[0010] The second object of the present invention is to provide an in vitro induction and expansion method for human peripheral blood regulatory T cells, the method is carried out using the above-mentioned in vitro expansion kit, and the method specifically comprises the following steps:

[0011] S1: CD4 + T cells, and prepare autologous serum;

[0012] S2: Preparation of initial culture medium: 100 μg CD3 monoclonal antibody, 100 μg CD28 monoclonal antibody, 1 μg recombinant human TGF-β, 5 mL autologous serum prepared above, and 50 mL GT-T551 serum-free culture medium;

[0013] S3: Preparation of expansion medium: 5 mg of all-trans retinoic acid, 5 μg of recombinant human TGF-β, 1 million U of recombinant human IL-2, 50 mL of the above-prepared autologous serum, and 950 mL of GT-T551 serum-free medium;

[0014] S4: 2.5-6×10 7 CD4 obtained from S1 + The T cells were resuspended in 50 mL of the initial culture medium prepared in S2 and cultured in a T75 cell culture flask to obtain human peripheral blood regulatory T cells.

[0015] In a preferred embodiment of the present invention, the specific steps of cell culturing in S4 are:

[0016] S11: On day 1 of culture, recombinant human IL-2 was added to the culture medium to a final concentration of 1000 U / mL;

[0017] S22: On the second day of culture, all-trans retinoic acid was added to the culture medium to a final concentration of approximately 150 μg / mL;

[0018] S33: On the 5th day of culture, when the cell density is ≥ 2×10 6When the number of cells / mL reached 1, the cells were transferred from the T75 cell culture flask to a culture bag for further culture, and 100 mL of the expansion medium prepared in S3 and 25 μg of 167 ng / mL anti-human IL-6 monoclonal antibody were added;

[0019] S44: On the 9th day of culture, when the cell density is ≥ 2 × 10 6 When the cells are 1×10 / mL, dilute the cells to a concentration of 0.5-1×10 6 Pieces / mL;

[0020] S55: On the 11th day of culture, induction factors were added according to the cell proliferation and flow cytometry data;

[0021] S66: On the 14th day of culture, cell samples were taken for flow cytometry analysis to harvest human peripheral blood regulatory T cells.

[0022] 8. In a preferred embodiment of the present invention, the standard for adding the induction factor described in S55 is:

[0023] When the number of cells is less than 2×10 9 When the medium was supplemented with recombinant human TGF-β to a final concentration of 5 ng / mL, all-trans retinoic acid to a final concentration of 5 μg / mL, and recombinant human IL-2 to a final concentration of 1000 U / mL;

[0024] When the number of cells is ≥2×10 9 And the flow cytometry data is CD4 + CD25 + FOXP3 + When the proportion of T cells was ≤60%, recombinant human TGF-β was added to the culture medium to a final concentration of 5 ng / mL and all-trans retinoic acid was added to a final concentration of 5 μg / mL;

[0025] When the number of cells is ≥2×10 9 And the flow cytometry data is CD4 + CD25 + FOXP3 + When the T cell proportion is greater than 60%, no induction factor is needed.

[0026] The third object of the present invention is to provide the use of the above-mentioned in vitro expansion kit in in vitro expansion of human peripheral blood regulatory T cells.

[0027] A fourth object of the present invention is to provide a human peripheral blood regulatory T cell, wherein the human peripheral blood regulatory T cell is obtained by the above-mentioned in vitro induction and expansion method.

[0028] Beneficial effects of the present invention: The present invention provides an in vitro expansion kit for human peripheral blood regulatory T cells, the in vitro expansion kit comprising: a culture medium, an activation antibody, an induction factor and an expansion factor; the activation antibodies are CD3 monoclonal antibody and CD28 monoclonal antibody, the induction factors are recombinant human TGF-β, all-trans retinoic acid, anti-human IL-6 monoclonal antibody, the expansion factor is recombinant human IL-2, and the culture medium is GT-T551 serum-free culture medium.

[0029] The CD3 monoclonal antibody recognizes and interacts with the ε chain of the CD3 molecule on the TCR-CD3 complex, thereby enhancing the CD4 + The CD28 monoclonal antibody interacts with the CD28 molecule on the B7-CD28 complex, thereby providing an effective co-stimulatory signal to stimulate CD4 + The proliferation of T cells; the recombinant human IL-2 is produced by T cells (especially CD4 + T cells) are synthesized after being stimulated by antigens or mitogens, and have the function of promoting lymphocyte growth, proliferation and differentiation; the recombinant human TGF-β is a core regulatory factor for the proliferation and function of regulatory T cells (Tregs for short), and has the function of inducing CD4 + T cells differentiate into Treg cells and increase the concentration of FOXP3. FOXP3 is specifically expressed in CD4 + CD25 + Treg cells and mediate CD4 + CD25 + The development of Treg cells and their function maintenance inhibits differentiation into Th17 cells; the binding of all-trans retinoic acid to its nuclear receptor can promote the TGF-β-dependent FOXP3 + expression, thereby further increasing CD4 + Treg cell response and suppression of CD4 + Differentiation of T cells into Th17.

[0030] Since IL-6 is produced during the activation and proliferation of Treg, the combined action of TGF-β and IL-6 can activate STAT3 and inhibit the expression of FOXP3, thereby blocking Treg differentiation and turning on CD4 +T cells differentiate into Th17 cells, and IL-6 can promote the expansion and activation of T cell populations, including Treg cells. Based on the above two points, it can be seen that IL-6 is required for Treg activation and proliferation. At the same time, there is a Treg / Th17 immune balance in the human body. Excessive IL-6 will increase the inflammatory response, reduce Treg and increase Th17 cells, and the same is true in in vitro culture. Specifically, the present invention adds anti-human IL-6 monoclonal antibody (final concentration = 167ng / mL) on the 5th day of Treg cell culture, thereby satisfying the CD4 + The initial activation of T cells blocked their differentiation into Th17 cells, so that they could only differentiate into Treg cells and proliferate. On the 11th day of Treg cell culture, when the number of cells was ≥2×10 9 And the flow cytometry data is CD4 + CD25 + FOXP3 + When the proportion of T cells is ≤60%, a reagent mainly composed of anti-human IL-6 monoclonal antibody is added to improve the purity of Treg cells.

[0031] In the present invention, TGF-β and all-trans retinoic acid are used to induce and maintain CD4 + T cells differentiate into Treg cells, thereby effectively improving the purity of Treg cells; since all-trans retinoic acid is insoluble in water, the present invention uses a trace amount (10 μL) of dimethyl sulfoxide (DMSO) to dissolve it, and the dissolution effect is better, but too much DMSO will have a negative impact on the cell growth state, so the determination of the amount of DMSO used is more important.

[0032] The present invention provides a kit for in vitro expansion of human peripheral blood regulatory T cells, comprising anti-human IL-6 monoclonal antibodies, to avoid CD4 + The cells differentiated into Th17 direction, improving the differentiation efficiency and purity of regulatory T cells; the other components of this kit are simple, the reagent sources are wide, and the cost is low. The obtained regulatory T cells are of high purity and large quantity. After 14 days of culture, ≥2.5×10 9 The invention provides a method for producing finished cells with a qualified rate of ≥70%, which can meet the dosage of clinical treatment experiments; the method for using the kit has the advantages of simple operation, easy control of conditions, low requirements for equipment, strong versatility, and easy formulation of standard operating procedures; the in vitro expansion method provided by the invention uses the patient's own peripheral blood as the cell source, avoids the risks of immune rejection and infectious diseases from umbilical cord blood or allogeneic blood, and is conducive to clinical application. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 This is a cell flow cytometry image in Example 2;

[0034] Figure 2It is the cell growth curve diagram in Example 2;

[0035] Figure 3 This is the cell flow cytometry detection diagram in Comparative Example 1. DETAILED DESCRIPTION

[0036] Those skilled in the art can refer to the content of this article and appropriately improve the process parameters. It should be particularly noted that all similar replacements and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention. The method and application of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the methods and applications described herein without departing from the content and scope of the present invention to implement and apply the technology of the present invention.

[0037] In order to make the purpose, technical scheme and advantages of the present invention more clear, the present invention is further described in detail below in conjunction with specific embodiments and the accompanying drawings of the specification. The test methods used in the following examples are conventional methods unless otherwise specified, and the materials, reagents, methods and instruments used are conventional materials, reagents, methods and instruments in the art unless otherwise specified, and can be obtained by those skilled in the art through commercial channels.

[0038] Example 1: CD4 + Preparation of T cells

[0039] S1: Collect 100-150 mL of human peripheral blood and obtain 1×10 peripheral blood mononuclear cells (PBMC) using lymphocyte separation medium. 8 indivual;

[0040] S2: CD4 + T cells, main reagents and equipment: EasySep NegativeHuman CD4 Kit, EasySep Violet Magnet, EasySep TM Buffer, 5mL polystyrene test tube; 1×10 8 PBMCs were treated with 1×EasySep TM The buffer was prepared at a concentration of 5 × 10 7 2 mL of cell suspension with a concentration of 100 cells / mL was added to a 5 mL polystyrene test tube;

[0041] S3: Add 50 μL / mL EasysepTM Human CD4+Tcell concentrated mixture to the test tube containing the cell suspension in S2. The ratio of the cell suspension to the concentrated mixture is: 2 mL: 100 μL. Mix well and incubate at 15-25°C for 10 minutes. Vortex the EasysepTM magnetic beads for 30 seconds to ensure that the beads form a uniform suspension without obvious aggregates to obtain a mixed solution.

[0042] S4: Add 100 μL / mL of EasySepTM magnetic beads to the vortexed mixture in S3. The mixing ratio of the cell suspension to the magnetic beads is 2 mL: 200 μL. Mix well and incubate at 15-25°C for 5 minutes. Then add 1× EasySepTM magnetic beads. TM Buffer until the total volume of cell suspension reaches 2.5 mL, gently pipette 2-3 times to mix the cells in the tube, and place the test tube (without cover) in the magnet for 5 minutes;

[0043] S5: Do not separate the magnet and test tube in S4 to maintain the magnetic field to adsorb unwanted cells, then pour the desired cells into a new 5 mL polystyrene test tube for later use. The obtained CD4 + The purity of T cells reached 96%.

[0044] Example 2: Use of an in vitro expansion kit for in vitro expansion of human peripheral blood regulatory T cells

[0045] S1: Preparation of autologous serum: In Example 1, the lymphocyte separation fluid separation method is used to achieve the effect of stratifying human peripheral blood, wherein the upper layer is plasma, the middle layer is PBMC, and the bottom layer is granulocytes and red blood cells; the upper plasma is drawn and placed in a 50mL centrifuge tube, heated at 56°C for 30 minutes to denature and precipitate fibrinogen, cooled to room temperature, and centrifuged at 3800rpm for 10 minutes. The bottom of the test tube is fibrinogen, and the upper yellow clear liquid is autologous serum. The upper clear liquid is drawn and set aside; 60-90mL of autologous serum can be prepared from 100-150mL of human peripheral blood by the above method;

[0046] S2: Preparation of initial culture medium: 100 μg CD3 monoclonal antibody, 100 μg CD28 monoclonal antibody, 1 μg recombinant human TGF-β, 5 mL autologous serum prepared in S1, and 50 mL GT-T551 serum-free culture medium;

[0047] S3: Preparation of expansion medium: 5 mg of all-trans retinoic acid, 5 μg of recombinant human TGF-β, 1 million U of recombinant human IL-2, 50 mL of autologous serum prepared in S1, and 1000 mL of GT-T551 serum-free medium;

[0048] The all-trans retinoic acid is easily degraded by light, air, and room temperature, so it should be kept away from light and quickly handled and prepared as 1 mg / μL DMSO, 10 mg / tube in a -80°C refrigerator for later use; recombinant human TGF-β is stored in a -80°C refrigerator at 1 μg / mL GT-T551 serum-free medium, 1 μg / vial, and should be avoided as much as possible from multiple freeze-thaw cycles;

[0049] S4: 2.5-6×10 7 The CD4+T cells obtained in Example 1 were resuspended in the initial culture medium prepared in 50 mL S2, and placed in a T75 cell culture flask for culture to obtain human peripheral blood regulatory T cells;

[0050] The culturing steps are specifically as follows:

[0051] S11: On day 1 of culture, recombinant human IL-2 was added to the culture medium to a final concentration of 1000 U / mL;

[0052] S22: On the second day of culture, all-trans retinoic acid was added to the culture medium to a final concentration of approximately 150 μg / mL;

[0053] S33: On the 5th day of culture, when the cell density is ≥ 2×10 6 When the number of cells / mL reached 1, the cells were transferred from the T75 cell culture flask to a culture bag for further culture, and 100 mL of the expansion medium prepared in S3 and 25 μg of 167 ng / mL anti-human IL-6 monoclonal antibody were added;

[0054] S44: On the 9th day of culture, when the cell density is ≥ 2 × 10 6 When the cells are 1×10 / mL, dilute the cells to a concentration of 0.5-1×10 6 Pieces / mL;

[0055] S55: On the 11th day of culture, induction factors were added according to the cell proliferation and flow cytometry data;

[0056] The standard for adding induction factors is:

[0057] When the number of cells is less than 2×10 9 When incubating, cell proliferation should be ensured first, and recombinant human TGF-β (final concentration = 5 ng / mL), all-trans retinoic acid (final concentration = 5 μg / mL), and recombinant human IL-2 (final concentration = 1000 U / mL) should be added to the culture medium;

[0058] When the number of cells is ≥2×10 9 And the flow cytometry data is CD4 + CD25 + FOXP3 +When the proportion of T cells is ≤60%, it indicates that the cells proliferate vigorously, but the purity is slightly low. It is necessary to add recombinant human TGF-β (final concentration = 5 ng / mL), all-trans retinoic acid (final concentration = 5 μg / mL), and anti-human IL-6 monoclonal antibody 50 μg to the culture medium to improve the purity;

[0059] When the number of cells is ≥2×10 9 And the flow cytometry data is CD4 + CD25 + FOXP3 + When the proportion of T cells is greater than 60%, it indicates that the cell expansion and purity are good and no induction factor needs to be added;

[0060] S66: On the 14th day of culture, cell samples were taken for flow cytometry analysis to harvest human peripheral blood regulatory T cells.

[0061] The cell growth curve in this embodiment is as follows: Figure 2 As shown, the total number of finished cells obtained by in vitro expansion using the kit provided by the present invention is 3.35×10 9 The results of cell flow cytometry in this embodiment are as follows: Figure 1 As shown, human peripheral blood regulatory T cells (CD4 + CD25 + FOXP3 + The proportion of T cells in the total number of finished cells is ≥70%, indicating that the in vitro expansion kit provided by the present invention has the function of amplifying human peripheral blood regulatory T cells in vitro with high purity and high efficiency.

[0062] Comparative Example 1: Application of an in vitro amplification kit without adding anti-human IL-6 monoclonal antibody in in vitro amplification of human peripheral blood regulatory T cells

[0063] S1: Preparation of autologous serum: In Example 1, the lymphocyte separation fluid separation method is used to achieve the effect of stratifying human peripheral blood, wherein the upper layer is plasma, the middle layer is PBMC, and the bottom layer is granulocytes and red blood cells; the upper plasma is drawn and placed in a 50mL centrifuge tube, heated at 56°C for 30 minutes to denature and precipitate fibrinogen, cooled to room temperature, and centrifuged at 3800rpm for 10 minutes. The bottom of the test tube is fibrinogen, and the upper yellow clear liquid is autologous serum. The upper clear liquid is drawn and set aside; 60-90mL of autologous serum can be prepared from 100-150mL of human peripheral blood by the above method;

[0064] S2: Preparation of initial culture medium: 100 μg CD3 monoclonal antibody, 100 μg CD28 monoclonal antibody, 1 μg recombinant human TGF-β, 5 mL autologous serum prepared in S1, and 50 mL GT-T551 serum-free culture medium;

[0065] S3: Preparation of expansion medium: 50 mg of all-trans retinoic acid, 5 μg of recombinant human TGF-β, 1 million U of recombinant human IL-2, 50 mL of autologous serum prepared in S1, and 1000 mL of GT-T551 serum-free medium;

[0066] The all-trans retinoic acid is easily degraded by light, air, and room temperature, so it should be kept away from light and quickly handled and stored in a -80°C refrigerator at 1 mg / μL DMSO, 10 mg / tube for later use; recombinant human TGF-β is stored in a -80°C refrigerator at 1 μg / mL GT-T551 serum-free medium, 1 μg / vial, and should be avoided as much as possible from multiple freeze-thaw cycles;

[0067] S4: 2.5-6×10 7 CD4 obtained in Example 1 + T cells were resuspended in 50 mL of the initial culture medium prepared in S2 and cultured in a T75 cell culture flask to obtain human peripheral blood regulatory T cells;

[0068] The culturing steps are specifically as follows:

[0069] S11: On day 1 of culture, recombinant human IL-2 was added to the culture medium to a final concentration of 1000 U / mL;

[0070] S22: On the second day of culture, all-trans retinoic acid was added to the culture medium to a final concentration of approximately 150 μg / mL;

[0071] S33: On the 5th day of culture, when the cell density is ≥ 2×10 6 When the number of cells / mL reaches 40, transfer the cells from the T75 cell culture flask into a culture bag for further culture, and add 100 mL of the expansion medium prepared in S3;

[0072] S44: On the 9th day of culture, when the cell density is ≥ 2 × 10 6 When the cells are 1×10 / mL, add the expansion medium prepared in S3 to dilute the cells to a concentration of 0.5-1×10 6 Pieces / mL;

[0073] S55: On the 11th day of culture, induction factors were added according to the cell proliferation and flow cytometry data;

[0074] The standard for adding induction factors is:

[0075] When the number of cells is less than 2×10 9 When incubating, cell proliferation should be ensured first, and recombinant human TGF-β (final concentration = 5 ng / mL), all-trans retinoic acid (final concentration = 5 μg / mL), and recombinant human IL-2 (final concentration = 1000 U / mL) should be added to the culture medium;

[0076] When the number of cells is ≥2×10 9 And the flow cytometry data is CD4 + CD25 + FOXP3 + When the proportion of T cells is ≤60%, it indicates that the cells are proliferating vigorously, but the purity is slightly low, and recombinant human TGF-β (final concentration = 5 ng / mL) and all-trans retinoic acid (final concentration = 5 μg / mL) need to be added to the culture medium;

[0077] When the number of cells is ≥2×10 9 And the flow cytometry data is CD4 + CD25 + FOXP3 + When the proportion of T cells is greater than 60%, it indicates that the cell expansion and purity are good and no induction factor needs to be added;

[0078] S66: On the 14th day of culture, cell samples were taken for flow cytometry to obtain human peripheral blood regulatory T cells.

[0079] The cell growth curve in this comparative example is as follows: Figure 2 As shown, compared with Example 2, in Comparative Example 1, no anti-human IL-6 monoclonal antibody was added, and the total number of finished cells obtained by in vitro expansion was 2.95×10 9 Although there was no significant difference in the total number of cells obtained by in vitro amplification of the kit in Comparative Example 1 and Example 2, the cell numbers both met the standard; however, during the in vitro amplification process of the kit in Comparative Example 1 without adding anti-human IL-6 monoclonal antibody, the value-added slowed down in the later stage, resulting in a lower number of cells after 14 days of in vitro amplification than the number of cells obtained by in vitro amplification of the kit in Example 2.

[0080] The cell flow cytometry results in this comparative example are as follows: Figure 3 As shown in the figure, in the cells amplified in vitro by the kit without adding anti-human IL-6 monoclonal antibody in Comparative Example 1, human peripheral blood regulatory T cells (CD4 + CD25 + FOXP3 + The proportion of T cells in the total number of finished product cells was 14.37%, which failed to meet the standard and was much lower than the 76.01% of finished product cells in Example 2 ( Figure 1 shown).

[0081] It can be seen that the in vitro expansion kit for human peripheral blood regulatory T cells provided by the present invention significantly increases the number of finished cells obtained by in vitro expansion compared to the in vitro expansion method for human peripheral blood regulatory T cells without the addition of anti-human IL-6 monoclonal antibodies, thereby significantly increasing the number and purity of human peripheral blood regulatory T cells in the finished cells.

[0082] The contents not described in detail in the specification of the present invention are well-known technologies for those skilled in the art. Although the present invention has been disclosed as above with preferred embodiments, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the protection scope of the present invention should be based on the definition of the claims.

Claims

1. A kit for in vitro expansion of human peripheral blood regulatory T cells, characterized in that: The in vitro amplification kit comprises: culture medium, activation antibody, induction factor and amplification factor.

2. The in vitro amplification kit according to claim 1, characterized in that Among the activation antibodies, activation antibody 1 is CD3 monoclonal antibody, and activation antibody 2 is CD28 monoclonal antibody.

3. The in vitro amplification kit according to claim 1, characterized in that Among the induction factors, induction factor 1 is recombinant human TGF-β, induction factor 2 is all-trans retinoic acid, and induction factor 3 is anti-human IL-6 monoclonal antibody.

4. The in vitro amplification kit according to claim 1, characterized in that The amplification factor is recombinant human IL-2.

5. The kit according to claim 1, characterized in that The culture medium is GT-T551 serum-free culture medium.

6. A method for inducing and amplifying human peripheral blood regulatory T cells in vitro, characterized in that: The method is carried out using the in vitro amplification kit according to any one of claims 1 to 5, and the method specifically comprises the following steps: S1: CD4 + T cells, and prepare autologous serum; S2: Preparation of initial culture medium: 100 μg CD3 monoclonal antibody, 100 μg CD28 monoclonal antibody, 1 μg recombinant human TGF-β, 5 mL autologous serum prepared in S1, and 50 mL GT-T551 serum-free culture medium; S3: Preparation of expansion medium: 5 mg of all-trans retinoic acid, 5 μg of recombinant human TGF-β, 1 million U of recombinant human IL-2, 50 mL of autologous serum prepared in S1, and 1000 mL of GT-T551 serum-free medium; S4: 2.5-6×10 7 CD4 obtained from S1 + The T cells were resuspended in 50 mL of the initial culture medium prepared in S2 and cultured in a T75 cell culture flask to obtain human peripheral blood regulatory T cells.

7. The in vitro induction amplification method according to claim 6, characterized in that: The culture steps described in S4 are: S11: On day 1 of culture, recombinant human IL-2 was added to the culture medium to a final concentration of 1000 U / mL; S22: On the second day of culture, all-trans retinoic acid was added to the culture medium to a final concentration of approximately 150 μg / mL; S33: On the 5th day of culture, when the cell density is ≥ 2×10 6 When the number of cells / mL reached 1, the cells were transferred from the T75 cell culture flask to a culture bag for further culture, and 100 mL of the expansion medium prepared in S3 and 25 μg of 167 ng / mL anti-human IL-6 monoclonal antibody were added; S44: On the 9th day of culture, when the cell density is ≥ 2 × 10 6 When the cells are 1×10 / mL, dilute the cells to a concentration of 0.5-1×10 6 Pieces / mL; S55: On the 11th day of culture, induction factors were added according to the cell proliferation and flow cytometry data; S66: On the 14th day of culture, cell samples were taken for flow cytometry analysis to harvest human peripheral blood regulatory T cells.

8. The in vitro induction amplification method according to claim 7, characterized in that: The criteria for supplementation of induction factors described in S55 are: When the number of cells is less than 2×10 9 When the medium was supplemented with recombinant human TGF-β to a final concentration of 5 ng / mL, all-trans retinoic acid to a final concentration of 5 μg / mL, and recombinant human IL-2 to a final concentration of 1000 U / mL; When the number of cells is ≥2×10 9 And the flow cytometry data is CD4 + CD25 + FOXP3 + When the proportion of T cells was ≤60%, recombinant human TGF-β was added to the culture medium to a final concentration of 5 ng / mL and all-trans retinoic acid was added to a final concentration of 5 μg / mL; When the number of cells is ≥2×10 9 And the flow cytometry data is CD4 + CD25 + FOXP3 + When the T cell proportion is greater than 60%, no induction factor is needed.

9. Use of the in vitro expansion kit according to any one of claims 1 to 5 for in vitro expansion of human peripheral blood regulatory T cells.

10. A human peripheral blood regulatory T cell, characterized in that: The human peripheral blood regulatory T cells are obtained by using the in vitro induction and expansion method according to any one of claims 6 to 8.

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