In-vitro preparation method and application of induced follicular regulatory T cells

Abstract

The invention belongs to the technical field of immunology, and particularly discloses an in-vitro preparation method and application of an induced follicular regulatory T cell iTfr.The in-vitro preparation method comprises the steps that mouse spleen single-cell suspension or healthy human peripheral blood is obtained, peripheral blood mononuclear cells are obtained, then initial CD4 + T cells are obtained through immunomagnetic bead sorting, and iTfr cells are obtained; then carrying out induced differentiation by using an antibody and a specific cytokine, and carrying out continuous stimulation and culture by adopting two different cytokine polarization conditions, so as to obtain the iTfr cell. The invention provides a method for preparing the iTfr cells in vitro, which is expected to obtain an enough number of induced follicular regulatory T cells, and provides an important tool and a treatment means for in vitro research of immune tolerance related diseases such as autoimmune diseases and organ transplantation.

Description

Technical Field

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

[0002] Follicular regulatory T cells (Tfr) are a special subset of regulatory T cells located in the follicular germinal center. Tfr cells play an immunomodulatory role 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 follicular helper T cells and regulatory T cells (Treg) surface molecules, such as PD-1, CXCR5, ICOS, CD25 and CD4. At the same time, Tfr expresses BCL6, the main transcription factor of follicular helper T cells, and FOXP3, the main transcription factor of regulatory T cells.

[0003] In autoimmune diseases, 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. Currently, there is a lack of internationally recognized methods for inducing follicular regulatory T cells, and in vitro research on this T cell subset mainly relies on expensive instruments such as flow cytometry for sorting.

[0004] The origin of follicular regulatory T cells can be either regulatory T cell precursor cells or conventional T cells. Follicular regulatory T cells differentiated from conventional T cells are called induced follicular regulatory T cells (iTfr). iTfr expresses CD38 and can be distinguished by CD38. iTfr cells still retain the ability to assist B cells. At present, there is a lack of further research on the specific role of iTfr cells in maintaining normal human immune function or in diseases. The preparation of iTfr cells in vitro can help us better understand the function of iTfr and its role in the occurrence and development of autoimmune diseases. Summary of the invention

[0005] 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 induced follicular regulatory T cells (iTfr), laying a foundation for in vitro research on induced follicular regulatory T cells and follicular regulatory T cell therapy.

[0006] In order to solve the above technical problems, the present invention discloses an in vitro preparation method and application of iTfr cells. Specifically, after obtaining human peripheral blood or mouse spleen tissue, magnetic beads are used to sort and obtain initial CD4 +T lymphocytes and stimulated the initial T cells to differentiate into CD25 + Tfh then obtains induced follicular regulatory T cells iTfr, the specific technical scheme is as follows: The present invention provides an in vitro preparation method of induced follicular regulatory T cells (iTfr), comprising the following steps: Step 1: Initial CD4 + T lymphocytes, co-stimulatory molecule antibodies, a first cytokine and a culture medium are mixed to form a first culture system for culturing; Step 2: The cells obtained in the first step, the second cytokine, the co-stimulatory molecule antibody and the culture medium are mixed to form a second culture system to culture induced follicular regulatory T cells (iTfr).

[0007] Wherein, in the first step or the second step, the co-stimulatory molecule antibody is a combination of an anti-CD3 antibody and an anti-CD28 antibody; In the first step, the first cytokine comprises one of the following three combinations: (1) Combination of IL-2, IL-6 and IL-21; (2) combination of TGF-β, IL-6, and IL-21; (3) a combination of IL-2, TGF-β, IL-6, and IL-21; In the second step, the second cytokine is a combination of IL-2 and TGF-β.

[0008] Among them, in the first step, the initial CD4 + T lymphocytes were derived from human peripheral blood or mouse spleen tissue, and initial CD4 + T lymphocytes. Preferably, they are derived from human peripheral blood mononuclear cells or mouse spleen lymphocytes; further preferably, the human peripheral blood mononuclear cells are derived from the peripheral blood of healthy volunteers, and the peripheral blood mononuclear cells are obtained by Ficoll density gradient centrifugation; the mouse spleen lymphocytes are derived from wild-type mice such as C57BL / 6J, and are 4-8 weeks old. More preferably, the initial CD4 + T lymphocytes are CD4 + CD45RA + CCR7 + The initial CD4 + T lymphocytes are CD4 + CD44 - CD62L + .

[0009] Among them, in the first step, the initial CD4 +The concentration of T lymphocytes in the first culture system was 1.25~2.5×10 6 Cell / mL; preferably 2.5×10 6 Cell / mL; the concentration of the anti-CD3 antibody in the first culture system is 5 μg / mL, the concentration of the anti-CD28 antibody in the first culture system is 2 μg / mL; the concentration of IL-2 in the first culture system is 0-10 ng / mL; the concentration of TGF-β in the first culture system is 0-5 ng / mL; the concentration of IL-6 in the first culture system is 0-20 ng / mL; the concentration of IL-21 in the first culture system is 20 ng / mL. Preferably, when the initial CD4 + When the T lymphocytes are derived from human peripheral blood, the first cytokine comprises a combination of IL-2 (10 ng / mL), IL-6 (20 ng / mL) and IL-21 (20 ng / mL), or a combination of TGF-β (5 ng / mL), IL-6 (20 ng / mL) and IL-21 (20 ng / mL); when the initial CD4 + When the T lymphocytes are derived from spleen tissue of mice, the first cytokine includes a combination of IL-2 (2.5 ng / mL), TGF-β (5 ng / mL), IL-6 (20 ng / mL) and IL-21 (20 ng / mL).

[0010] Among them, when the initial CD4 + When the T lymphocytes are derived from human peripheral blood, the first cytokine also includes IL-12, and the concentration of IL-12 in the first culture system is 5 ng / mL; when the initial CD4 + When the T lymphocytes are derived from the spleen tissue of mice, cytokine antibodies are also added in the first step for mixing. The cytokine antibodies are a combination of anti-IL-4 antibodies and anti-γ-interferon (IFN-γ) antibodies. The concentration of the anti-IL-4 antibody in the first culture system is 10 μg / mL, and the concentration of the anti-γ-interferon antibody in the first culture system is 10 μg / mL.

[0011] Wherein, 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.

[0012] Wherein, in the second step, the concentration of the anti-CD3 antibody in the second culture system is 5 μg / mL, the concentration of the anti-CD28 antibody in the second culture system is 2 μg / mL, the concentration of IL-2 in the second culture system is 10 ng / mL, and the concentration of TGF-β in the second culture system is 5 ng / mL.

[0013] Wherein, in the first step or the second step, the culture medium is RPMI 1640 culture medium containing 10% v / v fetal bovine serum.

[0014] The volume of the first culture system is equal to the volume of the second culture system.

[0015] Preferably, a method for preparing human iTfr cells in vitro comprises the following steps: Step 1: PBMCs were obtained from human peripheral blood by density gradient centrifugation, followed by magnetic bead separation of naive CD4 + T lymphocytes; the initial CD4 + T lymphocytes were resuspended in RPMI 1640 medium supplemented with 10% v / v fetal bovine serum (FBS); the cells were added to a 96-well plate pre-incubated with anti-CD3 antibody and anti-CD28 antibody, with the number of cells per well being 2.5 × 10 6 Cell / mL, add a combination of cytokines IL-2 (or TGF-β), IL-6, IL12 and IL-21 to the above culture medium, and then culture in a cell culture incubator at 37°C, 5% CO2 for 3-4 days.

[0016] Step 2: Collect the cells obtained in the first step into a centrifuge tube, discard the culture medium after centrifugation, resuspend the cells in RPMI1640 culture medium containing 10% v / v FBS, supplement the culture medium with cytokines IL-2 and TGF-β, add them to a new 96-well plate pre-incubated with anti-CD3 antibodies and anti-CD28 antibodies, and continue to culture in a cell culture incubator at 37°C and 5% CO2 for 3-4 days to obtain iTfr cells.

[0017] Preferably, a method for preparing mouse iTfr in vitro comprises the following steps: Step 1: Wild-type mice were anesthetized with isoflurane and killed by cervical dislocation. The spleen was removed to prepare a cell suspension, and then the initial CD4 + T lymphocytes; the initial CD4 + T lymphocytes were resuspended in RPMI 1640 medium supplemented with 10% v / v FBS; cytokines IL-2, TGF-β, IL-6, and IL-21, anti-IL-4 antibody, and anti-γ-interferon antibody were added to the above medium, and the cells were added to a 96-well plate pre-incubated with anti-CD3 antibody and anti-CD28 antibody, with the number of cells per well being 2.5×10 6 Cell / mL, and then culture in a cell culture incubator at 37°C, 5% CO2 for 3-4 days.

[0018] Step 2: Collect the cells obtained in the first step into a centrifuge tube, discard the culture medium after centrifugation, resuspend the cells in RPMI1640 culture medium containing 10% v / v FBS, supplement the culture medium with cytokines IL-2 and TGF-β, add them to a new 96-well plate pre-incubated with anti-CD3 antibodies and anti-CD28 antibodies, and continue to culture in a cell culture incubator at 37°C and 5% CO2 for 3-4 days to obtain iTfr cells.

[0019] In a second aspect, the present invention provides induced follicular regulatory T cells prepared by the preparation method described in the first aspect.

[0020] In a third aspect, the present invention provides the use of the induced follicular regulatory T cells described in the second aspect in the preparation of drugs for treating autoimmune diseases and drugs for treating organ transplantation.

[0021] Beneficial effects: Tfh in the follicular germinal center can express FOXP3 in the late stage of immune response and acquire the phenotype of follicular regulatory T cells. Inspired by the in vivo research on follicular regulatory T cells, we established a method for obtaining follicular regulatory T cells in vitro through the follicular helper T cell differentiation pathway in this invention. This method is expected to solve the problem of follicular regulatory T cell sorting, which is currently important in the research of follicular regulatory T cells.

[0022] The present invention effectively induces initial CD4 in vitro by selecting common differentiation-inducing costimulatory molecule antibodies, cytokine antibodies and cytokines + T cells to CD4 + FOXP3 + CXCR5 + ICOS + CD38 + The iTfr prepared by this method can be used for mechanism research, and Tfr cell adoptive transfer can also be used to treat autoimmune diseases such as lupus erythematosus, rheumatoid arthritis, etc., as well as immunosuppression in organ transplantation. The specific advantages are as follows: 1) Most of the follicular regulatory T cells obtained by this method express CD38 (i.e., iTfr), a surface molecule that identifies the differentiation of conventional T cells into Tfr. The present invention found that the initial CD4 T cells can be induced to differentiate in vitro by an appropriate ratio of cytokines and cytokine antibodies. + T cells differentiated and a large number of iTfr cells were obtained.

[0023] 2) In this experiment, peripheral blood mononuclear cells were sorted from human peripheral blood or spleen cells from wild-type mice, and then naive T cells were sorted using immunomagnetic beads. Humans only need to add anti-CD3 antibodies, anti-CD28 antibodies, IL-2 (or TGF-β), IL-6, IL-12 and IL-21 to the naive T cells to obtain a large number of FOXP3 Tfr cells. Mice only need to add anti-CD3 antibodies, anti-CD28 antibodies, IL-2, TGF-β, IL-6 and IL-21 to the naive T cells to obtain a large number of FOXP3 Tfr cells. This method is simple, feasible and relatively economical.

[0024] 3) The present invention uses naturally existing cytokines in the body and antibodies to the corresponding targets, which can better simulate the induction differentiation pathway of Tfr in the body and can be used to study the relevant functional mechanisms of iTfr in vitro.

[0025] 4) The present invention provides an in vitro preparation method of iTfr, which has broad application prospects and can be used for immunosuppression in autoimmune diseases and organ transplantation therapy. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] 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.

[0027] Figure 1 This is the result of in vitro induction flow cytometry of human induced follicular regulatory T cells. Figure 1 A in the figure is the initial CD4 + The proportion of T cells differentiated into Tfr cells after 3.5 days of induction, Figure 1 B in the figure is the initial CD4 + The proportion of T cells differentiated into Tfr after 7 days of induction. Figure 1 C in the figure is the initial CD4 + The proportion of Tfr expressing CD38 obtained 7 days after T cell induction is the proportion of iTfr cells.

[0028] Figure 2 The effect of adding IL-2 and / or TGF-β in the first step of induction of human induced follicular regulatory T cells in vitro. Figure 2 A in the figure shows the effect of not adding IL-2 and TGF-β on Tfr induction in the first step. Figure 2 B in the figure is the result of Tfr induced when IL-2 was added in the first step. Figure 2 C in the figure shows the effect of adding TGF-β in the first step on Tfr induction. Figure 2 D in the figure shows the effect of adding IL-2 and TGF-β in the first step on Tfr induction.

[0029] Figure 3 This is the in vitro induction flow cytometry result of mouse induced follicular regulatory T cells, Figure 3 A in the code is CD4 + Expression of FOXP3 in T cells, Figure 3 B in the figure is the proportion of Tfr cells. Figure 3 C in the figure is the proportion of Tfr cells expressing CD38.

[0030] Figure 4 The effect of different concentrations of IL-2 on Tfr induction in the first step of in vitro induction of mouse induced follicular regulatory T cells. Figure 4 A in the figure shows the effect of adding IL-2 (2.5 ng / mL) in the first step on Tfr-induced differentiation. Figure 4 B in the figure shows the effect of adding IL-2 (10 ng / mL) in the first step on Tfr-induced differentiation. Figure 4 C in the figure is the first step to add IL-2 (2.5 ng / mL) to PD-1 + The ratio of Tfr-induced differentiation, Figure 4 D in the figure is the effect of IL-2 (10 ng / mL) on PD-1 in the first step. + The ratio of Tfr-induced differentiation. DETAILED DESCRIPTION

[0031] 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.

[0032] In the following examples, the PBS is a 1× PBS buffer solution with a pH of 7.4.

[0033] Example 1 In vitro induction of human iTfr cells 1. Experimental Materials Peripheral blood of healthy volunteers, immunomagnetic beads (Biolegend, 480042), anti-CD3 antibody (BD, 555329), anti-CD28 antibody (BD, 555725), IL-2 (Peprotech, 200-02-50), IL-6 (Peprotech, 200-06-50), IL-12 (Peprotech, 200-12-10), IL-21 (Peprotech, 200-21), TGF-β (Peprotech, 100-21-10).

[0034] 2. Experimental Methods 1. Acquisition of human follicular regulatory T cells Peripheral blood mononuclear cells (PBMCs) were obtained from the peripheral blood of healthy volunteers and initial CD4 + For T lymphocytes, the sorted cells were resuspended in RPMI 1640 complete medium containing 10% v / v fetal bovine serum and cultured in two steps: Step 1: Add 2.5×10 6 Cell / mL, IL-6 (20 ng / mL), IL-12 (5 ng / mL), IL-21 (20 ng / mL), IL-2 (10 ng / mL), and TGF-β (5 ng / mL) were added to the above culture system to obtain a first culture system with a total volume of 200 μL (the concentrations of the above substances represent the added concentrations in the first culture system), and the cells were cultured in a cell culture incubator at 37°C and 5% CO2 for 3.5 days, and half of the cells were taken for flow cytometry detection.

[0035] Step 2: Collect the remaining cells cultured in the first step, add PBS, centrifuge and discard the culture medium, resuspend the cells in RPMI 1640 complete culture medium containing 10% v / v FBS, add cytokines IL-2 (10 ng / mL) and TGF-β (5 ng / mL), add the cells to a new 96-well plate pre-incubated with anti-CD3 antibody (5 μg / mL) and CD28 antibody (2 μg / mL), and the total volume of the second culture system is 200 μL (the concentrations of each substance represent the added concentrations in the second culture system). Continue to culture in a cell culture incubator at 37°C and 5% CO2 for 3.5 days to obtain induced follicular regulatory T cells (iTfr). Collect the above cells and detect CD4 by flow cytometry. + FOXP3 + ICOS + CXCR5 + The ratio of Tfr.

[0036] 3. Experimental Results like Figure 1 As shown, the initial CD4 + The ratio of Tfr cells after 3.5 days of T lymphocyte induction Figure 1 As shown in A, the initial CD4 + The ratio of Tfr cells after 7 days of T lymphocyte induction Figure 1 As shown in B. Initial CD4 +After 7 days of T cell induction, the proportion of Tfr cells differentiated was significantly greater than that of 3.5 days, and more than 70% of the Tfr cells expressed CD38, i.e., iTfr. Figure 1 As shown in C.

[0037] Example 2 The first cytokine combination for inducing differentiation of human iTfr cells 1. Experimental Materials The experimental materials used in this example are the same as those in Example 1.

[0038] 2. Experimental Methods 1. Acquisition of human follicular regulatory T cells PBMCs were obtained from the peripheral blood of healthy volunteers and initial CD4 + For T lymphocytes, the sorted cells were resuspended in RPMI 1640 complete medium containing 10% v / v fetal bovine serum and cultured in two steps: Step 1: Add 2.5×10 6 Cell / mL, add the following cytokine combination to the above culture system: IL-6 (20 ng / mL), IL-12 (5 ng / mL), IL-21 (20 ng / mL), IL-2 (10 ng / mL), or IL-6 (20 ng / mL), IL-12 (5 ng / mL), IL-21 (20 ng / mL), TGF-β (5 ng / mL), or IL-6 (20 ng / mL), IL-12 (5 ng / mL), IL-21 (20 ng / mL), The total volume of the first culture system obtained was 200 μL (the concentrations of the substances mentioned above all represent the concentrations added in the first culture system), and the cells were cultured in a cell culture incubator at 37° C. and 5% CO 2 for 3.5 days.

[0039] Step 2: The cells cultured in the first step were collected, PBS was added, and the culture medium was discarded after centrifugation. The cells were resuspended in RPMI 1640 complete culture medium containing 10% v / v FBS, and cytokines IL-2 (10 ng / mL) and TGF-β (5 ng / mL) were added. The cells were added to a new 96-well plate pre-incubated with anti-CD3 antibody (5 μg / mL) and CD28 antibody (2 μg / mL). The total volume of the second culture system was 200 μL (the concentrations of the substances mentioned above represent the added concentrations in the second culture system). The cells were cultured in a cell culture incubator at 37°C and 5% CO2 for 3.5 days, and CD4 was detected by flow cytometry. + FOXP3 + ICOS + CXCR5 + The ratio of Tfr reaches the amount.

[0040] 3. Experimental Results like Figure 2 As shown, in the induction of induced follicular regulatory T cells, compared with the induction process without adding IL-2 or TGF-β in the first step (as shown in Figure 2 As shown in A): Adding IL-2 during the first step of induction significantly increased the proportion of Tfr (as shown in Figure 2 As shown in B, the addition of TGF-β during the first step of induction significantly increased the proportion of Tfr (as shown in Figure 2 Therefore, the first step in the induction of induced follicular regulatory T cells requires the presence of cytokines IL-2 and TGF-β. However, the proportion of Tfr in the first step of induction when IL-2 and TGF-β are added simultaneously (the experimental process is the same as in Example 1) is not as high as that when IL-2 or TGF-β is added alone (as shown in Figure 1). Figure 2 as shown in D in the figure).

[0041] Example 3 Induction of mouse iTfr cells in vitro 1. Experimental Materials SPF 4-8 week old female C57BL / 6J mice, immunomagnetic beads (Biolegend, 480040), anti-CD3 antibody (Invitrogen, 16-0031-86), anti-CD28 antibody (Invitrogen, 16-0281-85), IL-2 (Peprotech, 212-12-20), IL-6 (Peprotech, 200-06-50), IL-21 (R&D, 594-ML-010), TGF-β (UA Bioscience, UA040172), anti-IL-4 antibody (Invitrogen, 16-7041-85), anti-γ-interferon antibody (Invitrogen, 14-7311-85).

[0042] 2. Experimental Methods 1. Obtaining mouse follicular regulatory T cells Single cells were obtained from spleen tissue of C57BL / 6J mice and naive CD4 + T cells, and then resuspend the sorted cells in RPMI 1640 complete medium containing 10% v / v FBS and culture them in two steps: Step 1: Add 2.5×10 6 Cell / mL, IL-2 (10 ng / mL), TGF-β (5 ng / mL), IL-6 (20 ng / mL), IL-21 (20 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 first culture system with a total volume of 200 μL (the concentrations of the above substances represent the added concentrations in the first culture system), and the cells were cultured in a cell culture incubator at 37°C and 5% CO2 for 3.5 days.

[0043] Step 2: Collect the cells cultured in the first step, add PBS, centrifuge and discard the culture medium, resuspend the cells in RPMI 1640 complete culture medium containing 10% v / v FBS, add cytokines IL-2 (10 ng / mL) and TGF-β (5 ng / mL), add the cells to a new 96-well plate pre-incubated with anti-CD3 antibody (5 μg / mL) and anti-CD28 antibody (2 μg / mL), and the total volume of the second culture system is 200 μL (the concentrations of each substance represent the added concentrations in the second culture system). Culture in a cell culture incubator at 37°C and 5% CO2 for 3.5 days to obtain induced follicular regulatory T cells.

[0044] The above cells were collected and CD4 + FOXP3 + PD-1 + CXCR5 + Tfr cells and the expression of surface molecule CD38 were detected.

[0045] 3. Experimental Results like Figure 3 As shown, in the induction of follicular regulatory T cells, more than 50% of CD4 + T cells express FOXP3 (such as Figure 3 ), differentiated into CD4 + FOXP3 + PD-1 + CXCR5 + The proportion of Tfr cells is close to 60% (e.g. Figure 3 As shown in B), more than 70% of Tfr in follicular regulatory T cells express CD38, namely iTfr cells. Figure 3 As shown in C.

[0046] Example 4 Effects of different concentrations of IL-2 on obtaining mouse iTfr in the first step 1. Experimental Materials The experimental materials used in this example are the same as those in Example 3.

[0047] 2. Experimental Methods 1. Obtaining mouse follicular regulatory T cells Single cells were obtained from spleen tissue of C57BL / 6J mice and naive CD4 + T cells, and then resuspend the sorted cells in RPMI 1640 complete medium containing 10% v / v FBS and culture them in two steps: Step 1: Add 2.5×10 6Cell / mL, IL-2 (2.5 ng / mL), TGF-β (5 ng / mL), IL-6 (20 ng / mL), IL-21 (20 ng / mL), anti-IL-4 antibody (10 μg / mL), anti-γ-interferon antibody (10 μg / mL) were added to the above culture system, or IL-2 (10 ng / mL), TGF-β (5 ng / mL), IL-6 (20 ng / mL), IL-21 (20 ng / mL), anti-IL-4 antibody (10 μg / mL), anti-γ-interferon antibody (10 μg / mL) were added to obtain a first culture system with a total volume of 200 μL (the concentrations of the above substances all represent the added concentrations in the first culture system), and the cells were cultured in a cell culture incubator at 37°C and 5% CO2 for 3.5 days.

[0048] Step 2: Collect the cells cultured in the first step, add PBS, centrifuge and discard the culture medium, resuspend the cells in RPMI 1640 complete culture medium containing 10% v / v FBS, add cytokines IL-2 (10 ng / mL) and TGF-β (5 ng / mL), add the cells to a new 96-well plate pre-incubated with anti-CD3 antibody (5 μg / mL) and anti-CD28 antibody (2 μg / mL), and the total volume of the second culture system is 200 μL (the concentrations of each substance represent the added concentrations in the second culture system). Culture in a cell culture incubator at 37°C and 5% CO2 for 3.5 days to obtain induced follicular regulatory T cells.

[0049] The above cells were collected and CD4 + FOXP3 + ICOS + CXCR5 + The proportion of Tfr cells and the expression of surface molecules PD-1 and CD38 were detected.

[0050] 3. Experimental Results like Figure 4 As shown, in the first step of inducing follicular regulatory T cells, a relatively low concentration of IL-2 (2.5 ng / mL, as shown in Figure 4 Compared with high concentration of IL-2 (10 ng / mL, as shown in A) Figure 4 In order to obtain a higher proportion of Tfr cells, a relatively low concentration of IL-2 (2.5 ng / mL, as shown in B) was added in the first step. Figure 4 Compared with high concentration of IL-2 (10 ng / mL, as shown in C) Figure 4 PD-1 +The results showed that higher or lower concentrations of IL-2 had no significant effect on the proportion of iTfr. Therefore, based on the above results, a lower concentration of IL-2 (2.5 ng / mL) is required in the first step of inducing iTfr cells.

[0051] The present invention provides an in vitro preparation method of inducible follicular regulatory T cells and the idea and method of their application. There are many methods and ways 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 principle of the present invention. These improvements and modifications should also be regarded as the protection scope of the present invention. All components not specified in this embodiment can be implemented by existing technologies.

Claims

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

2. The in vitro preparation method according to claim 1, characterized in that: In the first step, the initial CD4 + T lymphocytes were derived from human peripheral blood or mouse spleen tissue.

3. The in vitro preparation method according to claim 1, characterized in that: In the first step, the initial CD4 + The concentration of T lymphocytes in the first culture system was 1.25~2.5×10 6 Cells / mL; The concentration of the anti-CD3 antibody in the first culture system is 5 μg / mL, and the concentration of the anti-CD28 antibody in the first culture system is 2 μg / mL; The concentration of IL-2 in the first culture system is 0-10 ng / mL; The concentration of TGF-β in the first culture system is 0-5 ng / mL; The concentration of IL-6 in the first culture system is 0-20 ng / mL; The concentration of IL-21 in the first culture system is 20 ng / mL.

4. The in vitro preparation method according to claim 2, characterized in that: When the initial CD4 + When the T lymphocytes are derived from human peripheral blood, the first cytokine also includes IL-12, and the concentration of IL-12 in the first culture system is 5 ng / mL; When the initial CD4 + When the T lymphocytes are derived from the spleen tissue of mice, cytokine antibodies are also added in the first step for mixing. The cytokine antibodies are a combination of anti-IL-4 antibodies and anti-IFN-γ antibodies. The concentration of the anti-IL-4 antibody in the first culture system is 10 μg / mL, and the concentration of the anti-IFN-γ antibody in the first culture system is 10 μg / mL.

5. 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.

6. The in vitro preparation method according to claim 1, characterized in that: In the second step, the concentration of the anti-CD3 antibody in the second culture system is 5 μg / mL, the concentration of the anti-CD28 antibody in the second culture system is 2 μg / mL; the concentration of IL-2 in the second culture system is 10 ng / mL, and the concentration of TGF-β in the second culture system is 5 ng / mL.

7. The in vitro preparation method according to claim 1, characterized in that: In the first step or the second step, the culture medium is RPMI 1640 culture medium containing 10% v / v fetal bovine serum.

8. The in vitro preparation method according to claim 1, characterized in that: The volume of the first culture system is equal to the volume of the second culture system.

9. Inducible follicular regulatory T cells prepared by the preparation method according to any one of claims 1 to 8.

10. Use of the induced follicular regulatory T cells according to claim 9 in the preparation of drugs for treating autoimmune diseases and drugs for treating organ transplantation.

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