In vitro preparation method and application of natural follicular regulatory T cells

Abstract

The present invention relates to the field of immunology technology, and discloses an in vitro preparation method of natural follicular regulatory T cells and its application. The present invention first uses magnetic beads to sort wild-type mouse spleen cells to obtain initial CD4 + T lymphocytes are then induced to differentiate with antibodies and specific cytokines, and two different cytokine polarization conditions are used for continuous stimulation and culture to obtain natural follicular regulatory T cells. The present invention provides a method for preparing natural follicular regulatory T cells in vitro, which is expected to obtain a sufficient number of natural follicular regulatory T cells, providing an important tool and treatment for the study of immune tolerance-related diseases such as autoimmune diseases and organ transplantation.

Description

Technical Field

[0001] The present invention relates to the technical field of immunology, and in particular to an in vitro preparation method and application of natural follicular regulatory T cells. Background Art

[0002] Follicular Regulatory T Cells (Tfr) are a special subset of regulatory T cells located in the germinal center of follicles. Tfr cells exert immunomodulatory effects by inhibiting the help of follicular helper T cells (Tfh) to B cells and directly acting on B cells. Follicular regulatory T cells co-express surface molecules of follicular helper T cells and regulatory T cells (Treg), such as PD-1, CXCR5, ICOS, CD25, and CD4. At the same time, Tfr cells express BCL6, the main transcription factor of follicular helper T cells, and FOXP3, the main transcription factor of regulatory T cells.

[0003] In autoimmune diseases, such as rheumatoid arthritis, systemic lupus erythematosus, and primary Sjögren's syndrome, the number of follicular regulatory T cells and the ratio of regulatory T cells to helper T cells decrease, and this decrease in ratio is positively correlated with disease activity. At present, the research on follicular regulatory T cells mainly focuses on the cell regulatory mechanism. Due to the lack of internationally recognized in vitro induced differentiation methods, in vitro research on this T cell subset mainly relies on expensive instruments such as flow sorters for sorting. In addition, the content of this type of cell in the body is very small, so it is difficult to obtain a large number of follicular regulatory T cells. Developing a method that can obtain follicular regulatory T cells by in vitro induced differentiation, which has cost advantages, is of great significance for the study and understanding of this group of cells.

[0004] One method known at present is to use T cell activation and amplification magnetic beads, cytokines IL-2, TGF-β and the Chinese herbal medicine ingredient baicalin to induce co-induction (refer to Chinese patent CN107177547A); another method is to obtain it through anti-TCR-β antibodies, anti-CD3 antibodies and cytokines (PMID: 32711255). The former method uses activated magnetic beads to enrich T cells and induces differentiation through baicalin. However, baicalin is not a component of the human body, it is expensive and difficult to obtain in large quantities, and baicalin-induced differentiation is difficult to simulate the process of follicular regulatory T differentiation in vivo. The latter method is difficult to obtain high-purity Tfr cells (only about 20%), which limits the wide application of Tfr cell in vitro mechanism research and cell therapy.

[0005] The origin of follicular regulatory T cells can be either regulatory T cell precursors or conventional T cells. Current research has gradually denied the view that follicular regulatory T cells are derived only from regulatory T cell precursors. Follicular regulatory T cells differentiated from regulatory T cells are called natural follicular regulatory T cells (nTfr), while follicular regulatory T cells differentiated from conventional T cells are called induced follicular regulatory T cells (iTfr). Inducible follicular regulatory T cells express CD38, while natural follicular regulatory T cells do not express CD38. Among them, nTfr cells can be used for adoptive transfer to treat autoimmune diseases such as lupus erythematosus, rheumatoid arthritis, etc., as well as immunosuppression in organ transplantation. In the prior art, a large number of studies focus on the induced differentiation of Tfr cells, and there is a lack of in vitro induced differentiation methods for nTfr cells, which limits the wide application of in vitro mechanism research of nTfr cells and cell therapy. Summary of the invention

[0006] Purpose of the invention: The technical problem to be solved by the present invention is to address the deficiencies in the prior art and to provide an in vitro preparation method and application of natural follicular regulatory T (nTfr) cells in mice, laying a foundation for in vitro research on nTfr cells and cell therapy of follicular regulatory T cells.

[0007] In order to achieve the above technical objectives, the present invention discloses an in vitro preparation method and application of nTfr cells. The specific technical scheme is as follows:

[0008] An in vitro preparation method of natural follicular regulatory T (nTfr) cells comprises the following steps:

[0009] Step 1: Initial CD4 + T lymphocytes are mixed with co-stimulatory molecule antibodies and the first cytokine and cultured;

[0010] Step 2: The cells obtained by culture in the first step are mixed with the co-stimulatory molecule antibody, the second cytokine and the cytokine antibody to culture natural follicular regulatory T cells.

[0011] Among them, in the first step, the initial CD4 + T lymphocytes are obtained by magnetic bead sorting of spleen cells of mice. Preferably, the mice are wild C57BL / 6J mice, aged 4-6 weeks. Further preferably, wild-type mice are anesthetized with isoflurane and killed by cervical dislocation. The spleen is taken out to prepare a cell suspension, and the cell suspension is purified by Biolegend Mouse Naïve CD4 + T sorting magnetic beads to sort initial CD4 + More preferably, the initial CD4 + T lymphocytes are CD4 + CD44- CD62L + cell.

[0012] In the first step, the co-stimulatory molecule antibody is one of the following three combinations:

[0013] (1) A combination of anti-CD3 antibody and anti-CD28 antibody;

[0014] (2) Combination of anti-TCR-β antibody and anti-CD28 antibody;

[0015] (3) A combination of anti-CD3 antibody, anti-TCR-β antibody, and anti-CD28 antibody;

[0016] A combination of an anti-CD3 antibody and an anti-CD28 antibody is preferred.

[0017] The first cytokine is a combination of IL-2 and TGF-β.

[0018] Among them, in the first step of the culture system, the initial CD4 + The concentration of T lymphocytes is 1.25-2.5×10 6 Cells / mL; the concentration of the anti-CD3 antibody is 0-5 μg / mL, the concentration of the anti-TCR-β antibody is 0-5 μg / mL, and the concentration of the anti-CD28 antibody is 2 μg / mL; the concentration of the IL-2 is 2-15 ng / mL, and the concentration of the TGF-β is 1-10 ng / mL; preferably, the initial CD4 + The concentration of T lymphocytes was 2.5×10 6 Cells / mL, the concentration of anti-CD3 antibody was 5 μg / mL, the concentration of anti-TCR-β antibody was 0 μg / mL, the concentration of anti-CD28 antibody was 2 μg / mL; the concentration of IL-2 was 5-10 ng / mL, and the concentration of TGF-β was 2.5-5 ng / mL.

[0019] In the first step or the second step, the culture temperature is 37° C., the carbon dioxide volume concentration is 5%, and the culture time is 3-4 days. Preferably, the culture time is 3.5 days.

[0020] Preferably, in the first step or the second step, the mixing is performed in a culture medium, and the culture medium is an RPMI 1640 culture medium containing 10% v / v fetal bovine serum (FBS).

[0021] Preferably, the cells cultured in the first step contain regulatory T cells, i.e., CD4 + CD25 + CD127 low / - cell.

[0022] More preferably, the first step comprises: + T lymphocytes were resuspended in RPMI 1640 medium containing 10% FBS; the resuspended cells were added to a 96-well plate pre-incubated with anti-CD3 antibodies and anti-CD28 antibodies, and cytokines IL-2 and TGF-β were added to the above medium and cultured.

[0023] Wherein, in the second step, the second cytokine is a combination of IL-2, TGF-β, IL-6 and IL-21; the cytokine antibody is a combination of anti-interleukin 4 (IL-4) antibody and anti-γ-interferon antibody. The costimulatory molecule antibody in the second step has the same composition and concentration as the costimulatory molecule antibody in the first step, preferably a combination of anti-CD3 antibody and anti-CD28 antibody, the concentration of anti-CD3 antibody in the culture system in the second step is 5 μg / mL, and the concentration of anti-CD28 antibody in the culture system in the second step is 2 μg / mL.

[0024] In the culture system of the second step, the concentration of IL-2 is 2.5-10 ng / mL, the concentration of TGF-β is 5-10 ng / mL, the concentration of IL-6 is 20 ng / mL, the concentration of IL-21 is 20 ng / mL, the concentration of anti-IL-4 antibody is 10 μg / mL, and the concentration of anti-γ-interferon antibody is 10 μg / mL.

[0025] The total volume of the culture system in the first step is the same as the total volume of the culture system in the second step.

[0026] Preferably, the specific steps of the second step are: collecting the cells obtained in the first step into a centrifuge tube, discarding the culture medium after centrifugation, resuspending the cells with RPMI 1640 culture medium containing 10% FBS, adding the obtained cell resuspension to a new 96-well plate pre-incubated with anti-CD3 antibodies and anti-CD28 antibodies, supplementing the culture medium with cytokines IL-2, TGF-β, IL-6 and IL-21, and culturing to obtain nTfr cells.

[0027] In a second aspect, the present invention provides natural follicular regulatory T cells prepared by the preparation method described in the first aspect. Preferably, the natural follicular regulatory T cells are CD4 + FOXP3 + CXCR5 + PD-1 + CD38 - cell.

[0028] In a third aspect, the present invention provides the use of the natural follicular regulatory T cells described in the second aspect in the preparation of autoimmune disease drugs and organ transplantation therapeutic drugs. Preferably, the autoimmune diseases include but are not limited to lupus erythematosus, rheumatoid arthritis and Sjögren's syndrome.

[0029] Beneficial effects:

[0030] The present invention effectively induces initial CD4 in vitro by selecting differentiation-inducing costimulatory molecule antibodies, cytokine antibodies and cytokines. + T cells to CD4 + FOXP3 + CXCR5 + PD-1 + CD38 - nTfr cell differentiation, this method is expected to solve the problem of relying on flow cytometry for sorting in current follicular regulatory T cell research. The nTfr cells prepared by this method can be used for mechanism research, and can also be used for nTfr cell adoptive transfer to treat autoimmune diseases such as lupus erythematosus, rheumatoid arthritis, etc., as well as immunosuppression in organ transplantation. The specific advantages are as follows:

[0031] 1) Most of the follicular regulatory T cells obtained by this method do not express CD38, that is, nTfr cells. The surface molecule CD38 is used to identify nTfr cells and iTfr cells. The present invention found that the initial CD4 + T cells differentiated and a large number of nTfr cells were obtained (>80% nTfr cells).

[0032] 2) In this study, we isolated naive CD4 + T cells, only in the initial CD4 + By adding anti-CD3 antibodies, anti-CD28 antibodies, IL-2, TGF-β, IL-6, IL-21, anti-IL-4 antibodies, and anti-γ-interferon antibodies to T cells, a large number of Tfr cells can be obtained, and the Tfr cell content can reach about 50%. This method is simple, feasible, and relatively economical.

[0033] 3) The present invention uses naturally existing cytokines in the body and antibodies to corresponding targets, which can better simulate the induced differentiation of nTfr cells in the body and can be used for the study of relevant functional mechanisms of nTfr cells in vitro.

[0034] 4) Tfr cells have the function of inhibiting the auxiliary effect of Tfh cells on B cells, as well as directly inhibiting the function of B cells. However, Tfr cells decrease in autoimmune diseases, and this decrease is positively correlated with the activity of the disease. Treg cells have certain therapeutic effects in maintaining immunosuppression in organ transplantation and treating autoimmune diseases. As a special subset of regulatory T cells, Tfr cells can regulate the immune response of germinal centers. The present invention provides an in vitro preparation method of nTfr cells, which can obtain a large number of nTfr cells (>80%). This method has broad application prospects and can be used to treat autoimmune diseases such as lupus erythematosus, rheumatoid arthritis, Sjögren's syndrome, etc., as well as for immunosuppression in organ transplantation treatment, such as kidney transplantation and liver transplantation. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] The present invention will be further described in detail below in conjunction with the accompanying drawings and specific embodiments, and the above and / or other advantages of the present invention will become more clear.

[0036] Figure 1 Flow cytometry was used to detect the effects of different co-stimulatory molecule antibody activation methods on the proportion of Tfr cells. Figure 1 A in the figure is activated with anti-CD3 antibody, anti-TCR-β antibody and anti-CD28 antibody; Figure 1 B in the above is activated by anti-CD3 antibody and anti-CD28 antibody; Figure 1 C in the figure is activated by anti-TCR-β antibody and anti-CD28 antibody; Figure 1 D in the figure is a bar graph of the proportion of Tfr cells obtained by three activation methods, wherein CD3+TCR-β indicates activation with anti-CD3 antibody, anti-TCR-β antibody and anti-CD28 antibody, CD3 indicates activation with anti-CD3 antibody and anti-CD28 antibody, and TCR-β indicates activation with anti-TCR-β antibody and anti-CD28 antibody.

[0037] Figure 2 This is a flow cytometric test of the effects of IL-6 and IL-21 in the first step of the two-step method on nTfr cells. Figure 2 A in the figure indicates the proportion of Tfr cells induced by only IL-2 and TGF-β in the first step; Figure 2 B in the figure is the proportion of Tfr cells induced by cytokines IL-2, TGF-β, and IL-6; Figure 2 C in the figure is the proportion of Tfr cells induced by cytokines IL2, TGF-β, and IL-21; Figure 2 D in the figure is the ratio of nTfr cells in Tfr cells when the cytokines in the first step are only IL-2 and TGF-β. DETAILED DESCRIPTION

[0038] The present invention is further described below in conjunction with the embodiments, but it should not be understood that the above subject scope of the present invention is limited to the following embodiments. Without departing from the above technical ideas of the present invention, various substitutions and changes are made according to the common technical knowledge and customary methods in the field, which should all be included in the protection scope of the present invention.

[0039] In the following examples, the experimental materials used are as follows: SPF grade 4-6 week old female C57BL / 6J mice, immunomagnetic beads (Biolegend, 480040), anti-CD3 antibody (Invitrogen, 16-0031-86), anti-CD28 antibody (Invitrogen, 16-0281-85), anti-TCR-β antibody (Bioxcell, BE0102), anti-ICOS antibody (Biolegend, 313539) IL-2 (Peprotech, 212-12-20), IL-6 (Peprotech, 216-16-50), IL-21 (R&D, 594-ML-010), TGF-β (UABioscience, UA040172), anti-γ-interferon antibody ( Invitrogen, 14-7311-85), anti-IL4 antibody (Invitrogen, 16-7041-85).

[0040] In the following examples, the Tfr cells are CD4 + FOXP3 + PD-1 + CXCR5 + Tfr cells, the expression of CD38 in Tfr cells is confirmed by flow cytometry, and the nTfr cells are cells that do not express CD38 in the Tfr cells.

[0041] Example 1 Selection of in vitro activation methods for mouse Tfr cells

[0042] Single cells were obtained from spleen tissue of wild-type C57BL / 6J mice and naive CD4 + T lymphocytes (CD4 + CD44 - CD62L + The sorted cells were then resuspended in RPMI 1640 complete medium containing 10% v / v fetal bovine serum (FBS) and added to a 96-well plate pre-incubated with co-stimulatory molecule antibodies. 2.5×10 6 Cells / mL, including the following three pre-incubation methods (activation methods) of co-stimulatory molecule antibodies:

[0043] (1) Incubation with anti-CD3 antibody (5 μg / mL), anti-TCR-β antibody (5 μg / mL), and anti-CD28 antibody (2 μg / mL);

[0044] (2) Incubation with anti-CD3 antibody (5 μg / mL) and anti-CD28 antibody (2 μg / mL);

[0045] (3) Incubation with anti-TCR-β antibody (5 μg / mL) and anti-CD28 antibody (2 μg / mL).

[0046] IL-2 (10 ng / mL), TGF-β (0.5 ng / mL), anti-IL-4 antibody (10 μg / mL), and anti-γ-interferon antibody (10 μg / mL) were added to the above culture system to obtain a total volume of 200 μL (the concentrations of the substances described represent the added concentrations in the culture system), and cultured in a cell culture incubator at 37°C and 5% v / v CO2 for 3.5 days.

[0047] The above-cultured cells were collected accordingly, then added with PBS (1×, pH=7.4), centrifuged and discarded the culture medium, and the cells were resuspended in RPMI 1640 complete culture medium containing 10% v / v FBS, and the cells were added to a new 96-well plate pre-incubated with anti-CD3 antibody (5 μg / mL) and anti-CD28 antibody (2 μg / mL), and cytokines IL-2 2 ng / mL), TGF-β (0.5 ng / mL), IL-6 (25 ng / mL), IL-21 (25 ng / mL), anti-IL-4 antibody (10 μg / mL), anti-ICOS antibody (5 μg / mL), and anti-γ-interferon antibody (10 μg / mL) were added (the total culture system in the second step was 200 μL, and the concentrations of the above substances all represent the added concentrations in the culture system) and cultured in a cell culture incubator at 37°C and 5% v / v CO2 for 3.5 days to obtain Tfr cells.

[0048] The above cells were collected and CD4 + FOXP3 + PD-1 + CXCR5 + The proportion of Tfr cells. Figure 1 As shown, all three activation methods can activate the initial CD4 + T cells, making the initial CD4 + T cells are transformed from initial CD4 + T cells differentiate into Tfr cells.

[0049] Example 2 Effects of different induction conditions on in vitro differentiation of mouse Tfr cells

[0050] Single cells were obtained from spleen tissue of C57BL / 6 and naive CD4 + T lymphocytes (CD4 + CD44 - CD62L + Cells), then the sorted cells were resuspended in RPMI 1640 complete medium containing 10% fetal bovine serum to obtain a cell resuspension, and cultured in two steps:

[0051] Step 1: Add the obtained cell resuspension to a 96-well plate pre-incubated with co-stimulatory molecule antibodies (the concentration added in the culture system is 5 μg / mL anti-CD3 antibody and 2 μg / mL anti-CD28 antibody), and add 1.25×10 6 Cells / mL, cytokines IL-2 and TGF-β were added, and the total volume of the culture system was 200 μL. The cells were cultured in a cell culture incubator at 37°C and 5% v / vCO2 for 3.5 days to obtain regulatory T cells (CD4 + CD25 high CD127 low / - cells) of the cell fluid.

[0052] Step 2: Collect all the cells obtained from the first step of the culture, add PBS (1×, pH=7.4), centrifuge and discard the culture medium, resuspend the cells with RPMI 1640 complete culture medium containing 10% v / v FBS, add the obtained cell resuspension to a new 96-well plate pre-incubated with anti-CD3 antibody (5 μg / mL) and anti-CD28 antibody (2 μg / mL), and add cytokines IL-2, TGF-β, IL-6, IL-21, anti-IL-4 antibody (10 μg / mL), and anti-γ-interferon antibody (10 μg / mL). The total volume of the culture system in the second step is 200 μL (the concentrations of each substance represent the added concentrations in the culture system). Table 1 shows the added concentrations of various cytokines and antibodies. After culturing in a cell culture incubator at 37°C and 5% v / v CO2 for 3.5 days, Tfr cells can be obtained. Collect the above cells and detect CD4 by flow cytometry. + FOXP3 + PD-1 + CXCR5 + Tfr cell ratio.

[0053] In this example, the concentrations of various cytokines added in the two-step method were screened, as shown in Table 1.

[0054] Table 1 Concentrations of various cytokines added in the two-step culture

[0055]

[0056] The experimental results are shown in Table 2. Different proportions of follicular regulatory T cells were obtained by using different concentrations of cytokines IL-2 and TGF-β in the two-step method, among which,

[0057] The first step described in experimental group 1 was IL-2 (10 ng / mL) and TGF-β (5 ng / mL), and the second step was IL-2 (2.5 ng / mL) and TGF-β (5 ng / mL);

[0058] The first step described in experimental group 15 was IL-2 (5 ng / mL) and TGF-β (2.5 ng / mL), and the second step was IL-2 (5 ng / mL) and TGF-β (5 ng / mL);

[0059] The first step described in experimental group 16 was IL-2 (5 ng / mL) and TGF-β (2.5 ng / mL), and the second step was IL-2 (5 ng / mL) and TGF-β (10 ng / mL).

[0060] The combination of the above three cytokines is the most ideal condition compared with other concentrations.

[0061] Table 2 The proportion of Tfr cells in different groups

[0062]

[0063] Example 3 Exploration of cytokine combinations in mouse nTfr cells

[0064] Single cells were obtained from spleen tissue of C57BL / 6J and naive CD4 + T lymphocytes (CD4 + CD44 - CD62L + Cells), then the sorted cells were resuspended in RPMI 1640 complete medium containing 10% v / v FBS to obtain a cell resuspension, and cultured in two steps:

[0065] Step 1: Add the obtained cell resuspension to a 96-well plate pre-incubated with co-stimulatory molecule antibodies (the concentration added in the culture system is 5 μg / mL anti-CD3 antibody and 2 μg / mL anti-CD28 antibody), and add 2.5×10 6 Cells / mL; then add various cytokine combinations at the following concentrations:

[0066] (1) A combination of IL-2 (10 ng / mL), TGF-β (5 ng / mL), and IL-6 (20 ng / mL), or

[0067] (2) a combination of IL-2 (10 ng / mL), TGF-β (5 ng / mL), and IL-21 (20 ng / mL), or

[0068] (3) a combination of IL-2 (10 ng / mL), TGF-β (5 ng / mL), IL-6 (20 ng / mL), and IL-21 (20 ng / mL), or

[0069] (4) Combination of IL-2 (10 ng / mL) and TGF-β (5 ng / mL),

[0070] The total volume of the culture system was 200 μL and cultured in a cell culture incubator at 37°C and 5% v / v CO2 for 3.5 days.

[0071] Step 2: Collect the above cultured cells accordingly, add PBS (1×, pH=7.4), centrifuge and discard the culture medium, resuspend the cells with RPMI 1640 complete culture medium containing 10% v / v FBS, and add the obtained cell resuspension to a new 96-well plate pre-incubated with anti-CD3 antibody (added concentration 5 μg / mL) and anti-CD28 antibody (added concentration 2 μg / mL), and add cytokines IL-2 (added concentration 10 ng / mL), TGF-β (added concentration 5 ng / mL), IL-6 (added concentration 20ng / mL), IL-21 (added concentration 20 ng / mL), anti-IL-4 antibody (added concentration 10 μg / mL), and anti-γ-interferon antibody (added concentration 10 μg / mL). The total volume of the culture system is 200 μL. Culture in a cell culture incubator at 37°C and 5% v / v CO2 for 3.5 days to obtain Tfr cells. Collect the above cells and detect CD4 by flow cytometry. + FOXP3 + PD-1 + CXCR5 + Tfr cells, and the proportion of nTfr cells was obtained by detecting whether CD38 was expressed.

[0072] The results are as follows Figure 2 As shown, in the first step of inducing nTfr, the addition of cytokine IL-6 resulted in a decrease in the proportion of Tfr cells obtained ( Figure 2 A and Figure 2 B in the figure), adding cytokine IL-21 in the first step will also lead to a decrease in the proportion of Tfr cells ( Figure 2 A and Figure 2 C). When only IL-2 and TGF-β were added in the first step, the expression of CD38 in the obtained Tfr cells was detected, as shown in FIG. Figure 2 As shown in D, the proportion of nTfr cells in Tfr cells reached more than 80%.

[0073] In summary, the preferred cytokine combination for inducing nTfr cells is IL-2 and TGF-β in the first step, and IL-2, TGF-β, IL-6, and IL-21 in the second step.

[0074] The present invention provides an in vitro preparation method of natural follicular regulatory T cells and an idea and method for their application. There are many methods and approaches to implement the technical solution. The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention. All components not specified in this embodiment can be implemented using existing technologies.

Claims

1. A method for preparing natural follicular regulatory T cells in vitro, characterized in that: The steps include: Step 1: Initial CD4 + T lymphocytes are mixed with co-stimulatory molecule antibodies and the first cytokine and cultured; Step 2: mixing the cells cultured in the first step with the co-stimulatory molecule antibody, the second cytokine and the cytokine antibody to culture natural follicular regulatory T cells; Wherein, in the first step, the co-stimulatory molecule antibody is one of the following three combinations: (1) A combination of an anti-CD3 antibody and an anti-CD28 antibody; (2) a combination of anti-TCR-β antibody and anti-CD28 antibody; (3) a combination of anti-CD3 antibody, anti-TCR-β antibody, and anti-CD28 antibody; The first cytokine is a combination of IL-2 and TGF-β; In the second step, the second cytokine is a combination of IL-2, TGF-β, IL-6 and IL-21; the cytokine antibody is a combination of anti-interleukin 4 antibody and anti-γ-interferon antibody.

2. The in vitro preparation method according to claim 1, characterized in that: In the first step, the initial CD4 + T lymphocytes were obtained by magnetic bead sorting of mouse spleen cells.

3. The in vitro preparation method according to claim 1, characterized in that: In the first step of the culture system, the initial CD4 + The concentration of T lymphocytes is 1.25-2.5×10 6 Cells / mL; the concentration of the anti-CD3 antibody is 0-5μg / mL, the concentration of the anti-TCR-β antibody is 0-5μg / mL, and the concentration of the anti-CD28 antibody is 2μg / mL; the concentration of the IL-2 is 2-15ng / mL, and the concentration of the TGF-β is 1-10ng / mL.

4. The in vitro preparation method according to claim 1, characterized in that: In the first step or the second step, the culture temperature is 37° C., the volume concentration of carbon dioxide is 5%, and the culture time is 3-4 days.

5. The in vitro preparation method according to claim 1, characterized in that: In the culture system of the second step, the concentration of IL-2 is 2.5-10 ng / mL, the concentration of TGF-β is 5-10 ng / mL, the concentration of IL-6 is 20 ng / mL, the concentration of IL-21 is 20 ng / mL, the concentration of anti-interleukin 4 antibody is 10 μg / mL, and the concentration of anti-γ-interferon antibody is 10 μg / mL.

6. The in vitro preparation method according to claim 1, characterized in that: The total volume of the culture system in the first step is the same as the total volume of the culture system in the second step.

Images