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

Abstract

The present invention belongs to the technical field of immunology, and specifically discloses an in vitro preparation method and application of inducible follicular regulatory T cells (iTfr). First, a single-cell suspension of mouse spleen or peripheral blood mononuclear cells from healthy human peripheral blood is obtained, and then naïve CD4 + T cells are obtained by immunomagnetic bead sorting. Then, antibody and specific cytokines are used for induced differentiation, and continuous stimulation and culture are carried out under two different cytokine polarization conditions to obtain iTfr cells. The present invention provides a method for preparing iTfr cells in vitro, which is expected to obtain a sufficient number of inducible follicular regulatory T cells, providing an important tool and treatment means for in vitro research on 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 inducible 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 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 expresses the main transcription factor BCL6 of follicular helper T cells and the main transcription factor FOXP3 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 studies on this T cell subset mainly rely on expensive instruments such as flow sorters for sorting.

[0004] The origin of follicular regulatory T cells can be either regulatory T cell precursors or conventional T cells. Follicular regulatory T cells derived from the differentiation of conventional T cells are called inducible follicular regulatory T cells (iTfr). iTfr expresses CD38 and can be distinguished by CD38. iTfr cells still retain the ability to assist B cells. Currently, the specific role of iTfr cells in maintaining normal human immune function or in diseases lacks further research. Preparing iTfr cells in vitro helps us better understand the function of iTfr and its role in the occurrence and development of autoimmune diseases. Summary of the Invention

[0005] Object of the Invention: The technical problem to be solved by the present invention is to provide an in vitro preparation method and application of inducible follicular regulatory T cells (iTfr) in view of the deficiencies of the prior art, laying a foundation for in vitro research on inducible follicular regulatory T cells and cell therapy of follicular regulatory T cells.

[0006] 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, naive CD4 is obtained by magnetic bead sorting. +T lymphocytes, and by a two-step method to stimulate naive T cells to first differentiate into CD25 + Tfh and then obtain inducible follicular regulatory T cells iTfr. The specific technical solutions are as follows:

[0007] The present invention provides a method for in vitro preparation of inducible follicular regulatory T cells (iTfr), comprising the following steps:

[0008] First step: Mix naive CD4 + T lymphocytes, co-stimulatory molecule antibodies, first cytokines and a culture medium to form a first culture system, and carry out culture;

[0009] Second step: Mix the cells obtained from the first-step culture, second cytokines, co-stimulatory molecule antibodies and a culture medium to form a second culture system, and culture to obtain inducible follicular regulatory T cells (iTfr).

[0010] Among them, in the first step or the second step, the co-stimulatory molecule antibodies are a combination of anti-CD3 antibody and anti-CD28 antibody;

[0011] In the first step, the first cytokines include one of the following three combinations:

[0012] (1) A combination of IL-2, IL-6 and IL-21;

[0013] (2) A combination of TGF-β, IL-6 and IL-21;

[0014] (3) A combination of IL-2, TGF-β, IL-6 and IL-21;

[0015] In the second step, the second cytokines are a combination of IL-2 and TGF-β.

[0016] Among them, in the first step, the naive CD4 + T lymphocytes are derived from human peripheral blood or mouse spleen tissue, and naive CD4 + T lymphocytes are obtained by immunomagnetic bead sorting. 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 obtained by Ficoll density gradient centrifugation; the mouse spleen lymphocytes are derived from wild-type mice such as C57BL / 6J, with an age of 4-8 weeks. More preferably, the naive CD4 + T lymphocytes derived from humans are CD4 + CD45RA + CCR7 + , and the naive CD4 + T lymphocytes derived from mice are CD4+ CD44 - CD62L + 。

[0017] Among them, in the first step, the concentration of the initial CD4 + T lymphocytes in the first culture system is 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 + T lymphocytes are derived from human peripheral blood, the first cytokines include a combination of IL-2 (10 ng / mL), IL-6 (20 ng / mL) and IL-21 (20 ng / mL), or include a combination of TGF-β (5 ng / mL), IL-6 (20 ng / mL) and IL-21 (20 ng / mL); when the initial CD4 + T lymphocytes are derived from mouse spleen tissue, the first cytokines include 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).

[0018] Among them, when the initial CD4 + T lymphocytes are derived from human peripheral blood, the first cytokines further include IL-12, and the concentration of IL-12 in the first culture system is 5 ng / mL; when the initial CD4 + T lymphocytes are derived from mouse spleen tissue, a cytokine antibody is further added for mixing in the first step, and the cytokine antibody is a combination of an anti-IL-4 antibody and an anti-interferon-γ (IFN-γ) antibody. The concentration of the anti-IL-4 antibody in the first culture system is 10 μg / mL, and the concentration of the anti-interferon-γ antibody in the first culture system is 10 μg / mL.

[0019] Among them, in the first step or the second step, for the culture, the culture temperature is 37 °C, the volume concentration of carbon dioxide is 5%, and the culture time is 3 - 4 days.

[0020] Among them, 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.

[0021] Among them, in the first step or the second step, the culture medium is RPMI 1640 medium containing 10% v / v fetal bovine serum.

[0022] Among them, the volume of the first culture system is equal to the volume of the second culture system.

[0023] Preferably, a method for in vitro preparation of human iTfr cells includes the following steps:

[0024] The first step: Obtain PBMC from human peripheral blood by density gradient centrifugation, and then magnetically bead-sort naive CD4 + T lymphocytes; Resuspend the obtained naive CD4 + T lymphocytes with RPMI 1640 medium supplemented with 10% v / v fetal bovine serum (FBS); Add the cells into 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-mentioned medium, and then culture in a cell incubator at 37 °C and 5% CO2 for 3 - 4 days.

[0025] The second step: Collect the cells obtained in the first step into a centrifuge tube, discard the medium after centrifugation, resuspend the cells with RPMI1640 medium containing 10% v / v FBS, supplement the medium with cytokines IL-2 and TGF-β, add them into a new 96-well plate pre-incubated with anti-CD3 antibody and anti-CD28 antibody, and continue to culture in a cell incubator at 37 °C and 5% CO2 for 3 - 4 days to obtain iTfr cells.

[0026] Preferably, a method for in vitro preparation of murine iTfr includes the following steps:

[0027] The first step: Anesthetize and decapitate a wild-type mouse with isoflurane, take its spleen to make a cell suspension, and then magnetically bead-sort naive CD4 + T lymphocytes; Prepare the obtained naive 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-interferon-γ 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 cells / mL, and then cultured in a cell incubator at 37 °C and 5% CO2 for 3 - 4 days.

[0028] Second step: The cells obtained in the first step were collected into a centrifuge tube, the medium was discarded after centrifugation, the cells were resuspended in RPMI1640 medium containing 10% v / v FBS, cytokines IL-2 and TGF-β were supplemented to the medium, and the cells were added to a new 96-well plate pre-incubated with anti-CD3 antibody and anti-CD28 antibody, and continued to be cultured in a cell incubator at 37 °C and 5% CO2 for 3 - 4 days to obtain iTfr cells.

[0029] In a second aspect, the present invention provides induced follicular regulatory T cells prepared by the preparation method described in the first aspect.

[0030] 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 therapeutic drugs for autoimmune diseases and therapeutic drugs for organ transplantation.

[0031] Beneficial effects:

[0032] Tfh in the follicular germinal center can express FOXP3 in the late stage of the immune response and acquire the phenotype of follicular regulatory T cells. The inspiration from the in vivo research on follicular regulatory T cells enabled us to establish a method for obtaining follicular regulatory T cells in vitro through the differentiation pathway of follicular helper T cells in this invention. This method is expected to solve the problem that the current research on follicular regulatory T cells mainly relies on flow sorters for sorting.

[0033] The present invention effectively induces naive CD4 + T cells to differentiate into CD4 + FOXP3 + CXCR5 + ICOS + CD38 + Tfr by selecting common co-stimulatory molecule antibodies, cytokine antibodies and cytokines for induction in vitro. The iTfr prepared by this method can be used for mechanism research, and can also be used for adoptive transfer of Tfr cells to treat autoimmune diseases such as lupus erythematosus and rheumatoid arthritis, as well as immunosuppression in organ transplantation. The specific advantages are as follows:

[0034] 1) Most of the follicular regulatory T cells obtained by this method express CD38 (i.e., iTfr), and this surface molecule identifies the differentiation of conventional T cells into Tfr. The present invention discovers that through an appropriate ratio of cytokines and cytokine antibodies, naive CD4 + T cells can be induced to differentiate in vitro, and a large number of iTfr cells can be obtained.

[0035] 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. For humans, only anti-CD3 antibody, anti-CD28 antibody, IL-2 (or TGF-β), IL-6, IL-12, and IL-21 need to be added to the naive T cells to obtain a large number of Tfr cells expressing FOXP3. For mice, only anti-CD3 antibody, anti-CD28 antibody, IL-2, TGF-β, IL-6, and IL-21 need to be added to the naive T cells to obtain a large number of Tfr cells expressing FOXP3. This method is simple, feasible, and relatively economical.

[0036] 3) The cytokines and antibodies against corresponding targets used in the present invention are all naturally present in the body, which can better mimic the in vivo induction and differentiation pathway of Tfr and can be used for in vitro research on the related functional mechanisms of iTfr.

[0037] 4) The present invention provides a method for in vitro preparation of iTfr, which has broad application prospects and can be used for autoimmune diseases and immunosuppression in organ transplantation treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] The following further describes the present invention in detail with reference to the drawings and specific embodiments, and the above and / or other advantages of the present invention will become clearer.

[0039] Figure 1 is the flow cytometry result of in vitro induction of human induced follicular regulatory T cells. Figure 1 In + , A is the proportion of naive CD4 Figure 1 T cells differentiated into Tfr cells after 3.5 days of induction. + , B is the proportion of naive CD4 Figure 1 T cells differentiated into Tfr after 7 days of induction. + , C is the proportion of CD38-expressing cells in Tfr obtained after 7 days of induction of naive CD4

[0040] Figure 2 shows the effect of adding IL-2 and / or TGF-β in the first step during the in vitro induction of human induced follicular regulatory T cells. Figure 2In this, A is the influence on Tfr induction without adding IL-2 and TGF-β in the first step. Figure 2 In this, B is the Tfr result induced when adding IL-2 in the first step. Figure 2 In this, C is the influence on Tfr induction when adding TGF-β in the first step. Figure 2 In this, D is the influence on Tfr induction when adding IL-2 and TGF-β in the first step.

[0041] Figure 3 It is the flow cytometry result of in vitro induction of murine inducible follicular regulatory T cells, where Figure 3 In this, A is the expression of FOXP3 in CD4 + T cells. Figure 3 In this, B is the proportion of Tfr cells. Figure 3 In this, C is the proportion of CD38-expressing cells in Tfr cells.

[0042] Figure 4 It is the influence of different concentrations of IL-2 on Tfr induction in the first step during the in vitro induction of murine inducible follicular regulatory T cells. Figure 4 In this, A is the influence on Tfr induction and differentiation when adding IL-2 (2.5 ng / mL) in the first step. Figure 4 In this, B is the influence on Tfr induction and differentiation when adding IL-2 (10 ng / mL) in the first step. Figure 4 In this, C is the proportion of PD-1 + Tfr induction and differentiation when adding IL-2 (2.5 ng / mL) in the first step. Figure 4 In this, D is the proportion of PD-1 + Tfr induction and differentiation when adding IL-2 (10 ng / mL) in the first step. Specific implementation mode

[0043] The present invention will be further described below in conjunction with embodiments, but it should not be understood that the above main scope of the present invention is limited to the following embodiments. Without departing from the above technical idea of the present invention, various substitutions and changes made according to the general technical knowledge and conventional methods in the art should be included in the protection scope of the present invention.

[0044] In the following examples, the PBS mentioned is a 1×, pH = 7.4 PBS buffer solution.

[0045] Example 1 In vitro induction of human iTfr cells

[0046] I. Experimental materials

[0047] 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).

[0048] II. Experimental methods

[0049] 1. Obtaining human follicular regulatory T cells

[0050] Peripheral blood mononuclear cells (PBMC) were obtained from the peripheral blood of healthy volunteers, and naive CD4 + T lymphocytes were obtained by immunomagnetic bead sorting. The sorted cells were resuspended in RPMI 1640 complete medium containing 10% v / v fetal bovine serum and cultured by a two-step method:

[0051] First step: Add to a 96-well plate pre-incubated with anti-CD3 antibody (5 μg / mL) and anti-CD28 antibody (2 μg / mL), add 2.5×10 6 Cell / mL, add IL-6 (20 ng / mL), IL-12 (5 ng / mL), IL-21 (20 ng / mL), IL-2 (10 ng / mL), TGF-β (5 ng / mL) to the above culture system. The total volume of the first culture system obtained is 200 μL (the concentrations of the above substances all represent the added concentrations in the first culture system). Culture in a cell incubator at 37°C and 5% CO2 for 3.5 days, and take half of the cells for flow cytometry detection.

[0052] Second step: Collect the remaining cells cultured in the first step, then add PBS and centrifuge to discard the medium. Resuspend the cells again in RPMI 1640 complete medium containing 10% v / v FBS, supplement with cytokines IL-2 (10 ng / mL) and TGF-β (5 ng / mL), and add the cells 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 obtained is 200 μL (the concentrations of the above substances all represent the added concentrations in the second culture system). Continue to culture in a cell 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 proportion of Tfr

[0053] III. Experimental Results

[0054] As Figure 1 shown, the proportion of Tfr cells after 3.5 days of induction of naïve CD4 + T lymphocytes is as shown in A of Figure 1 . The proportion of Tfr cells after 7 days of induction of naïve CD4 + T lymphocytes is as shown in B of Figure 1 . After 7 days of induction of naïve CD4 + T cells, the proportion of differentiated Tfr cells is significantly greater than that after 3.5 days, and more than 70% of the Tfr cells express CD38, that is, iTfr, as shown in C of Figure 1 .

[0055] Example 2 The first cytokine combination for inducing differentiation of human iTfr cells

[0056] I. Experimental Materials

[0057] The experimental materials used in this example are the same as those in Example 1.

[0058] II. Experimental Methods

[0059] 1. Obtaining human follicular regulatory T cells

[0060] PBMC was obtained from the peripheral blood of healthy volunteers, and naïve CD4 + T lymphocytes were obtained by immunomagnetic bead sorting. The sorted cells were resuspended in RPMI 1640 complete medium containing 10% v / v fetal bovine serum and cultured by a two-step method:

[0061] The first step: Add to a 96-well plate pre-incubated with anti-CD3 antibody (5 μg / mL) and anti-CD28 antibody (2 μg / mL), and add 2.5×10 6 Cell / mL to each well. Add the following cytokine combinations to the above culture system:

[0062] IL-6 (20 ng / mL), IL-12 (5 ng / mL), IL-21 (20 ng / mL), IL-2 (10 ng / mL), or

[0063] IL-6 (20 ng / mL), IL-12 (5 ng / mL), IL-21 (20 ng / mL), TGF-β (5 ng / mL), or

[0064] IL-6 (20 ng / mL), IL-12 (5 ng / mL), IL-21 (20 ng / mL),

[0065] The total volume of the first culture system obtained is 200 μL (the concentrations of the various substances described represent the added concentrations in the first culture system), and it is cultured in a cell incubator at 37 °C and 5% CO2 for 3.5 days.

[0066] Step 2: Collect the cells cultured in the first step correspondingly, then add PBS and centrifuge to discard the culture medium. Resuspend the cells again with RPMI 1640 complete medium containing 10% v / v FBS, and supplement and 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). The total volume of the second culture system obtained is 200 μL (the concentrations of the various substances described represent the added concentrations in the second culture system), and it is cultured in a cell incubator at 37 °C and 5% CO2 for 3.5 days. Detect CD4 + FOXP3 + ICOS + CXCR5 + The proportion of Tfr reaches the amount.

[0067] III. Experimental Results

[0068] As Figure 2 shown, in the induction of inducible follicular regulatory T cells, compared with the induction process in the first step without adding either IL-2 or TGF-β (as shown in A in Figure 2 ): Adding IL-2 in the first step of the induction process significantly increases the proportion of Tfr (as shown in B in Figure 2 ), adding TGF-β in the first step of the induction process significantly increases the proportion of Tfr (as shown in C in Figure 2 ). Therefore, the cytokines IL-2 and TGF-β are required to be present in the first step of the induction process of inducible follicular regulatory T cells. However, the proportion of Tfr when adding both IL-2 and TGF-β simultaneously in the first step of the induction process (the experimental process is the same as in Example 1) is not higher than that when adding IL-2 or TGF-β alone (as shown in D in Figure 2 ).

[0069] Example 3 In vitro induction of mouse iTfr cells

[0070] I. Experimental Materials

[0071] SPF-grade female C57BL / 6J mice aged 4 - 8 weeks, 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-interferon-γ antibody (Invitrogen, 14-7311-85).

[0072] II. Experimental methods

[0073] 1. Obtaining follicular regulatory T cells from mice

[0074] Single cells were obtained from the spleen tissue of C57BL / 6J mice, and naive CD4 + T cells were obtained by immunomagnetic bead sorting. Then, the sorted cells were resuspended in RPMI 1640 complete medium containing 10% v / v FBS, and cultured by a two-step method:

[0075] First step: Add to a 96-well plate pre-incubated with anti-CD3 antibody (5 μg / mL) and anti-CD28 antibody (2 μg / mL), add 2.5×10 6 Cell / mL. Add 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-interferon-γ antibody (10 μg / mL) to the above culture system. The total volume of the first culture system is 200 μL (the concentrations of the above substances all represent the added concentrations in the first culture system). Culture in a cell incubator at 37°C and 5% CO2 for 3.5 days.

[0076] Step 2: Collect the cells cultured in the first step correspondingly, then add PBS and centrifuge to discard the culture medium. Resuspend the cells with RPMI 1640 complete medium containing 10% v / v FBS, and supplement with 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). The total volume of the second culture system obtained is 200 μL (the concentrations of the above substances all 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.

[0077] Collect the above cells and detect CD4 by flow cytometry + FOXP3 + PD-1 + CXCR5 + Tfr cells and detect the expression level of surface molecule CD38.

[0078] III. Experimental Results

[0079] As Figure 3 shown, in the induction of induced follicular regulatory T cells, more than 50% of CD4 + T cells express FOXP3 (as shown in A of Figure 3 ), and differentiate into CD4 + FOXP3 + PD-1 + CXCR5 + The proportion of Tfr cells is close to 60% (as shown in B of Figure 3 ), and more than 70% of Tfr in follicular regulatory T cells express CD38, that is, iTfr cells, as shown in C of Figure 3 .

[0080] Example 4 Effect of Different Concentrations of IL-2 in the First Step on Obtaining Mouse iTfr

[0081] I. Experimental Materials

[0082] The experimental materials used in this example are the same as those in Example 3.

[0083] II. Experimental Methods

[0084] 1. Obtaining Mouse Follicular Regulatory T Cells

[0085] Obtain single cells from the spleen tissue of C57BL / 6J mice and obtain naive CD4 +T cells, and then the sorted cells were resuspended in RPMI 1640 complete medium containing 10% v / v FBS, and then cultured by a two-step method:

[0086] First step: Add to a 96-well plate pre-incubated with anti-CD3 antibody (5 μg / mL) and anti-CD28 antibody (2 μg / mL), add 2.5×10 6 Cell / mL to each well, and add 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-interferon-γ antibody (10 μg / mL) to the above culture system, or add 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-interferon-γ antibody (10 μg / mL). The total volume of the first culture system obtained is 200 μL (the concentrations of the above substances all represent the added concentrations in the first culture system), and culture in a cell culture incubator at 37°C and 5% CO2 for 3.5 days.

[0087] Second step: Collect the cells cultured in the first step correspondingly, then centrifuge with PBS and discard the culture medium. Resuspend the cells again in RPMI 1640 complete medium containing 10% v / v FBS, and supplement with 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). The total volume of the second culture system obtained is 200 μL (the concentrations of the above substances all represent the added concentrations in the second culture system), and culture in a cell culture incubator at 37°C and 5% CO2 for 3.5 days to obtain induced follicular regulatory T cells.

[0088] Collect the above cells and detect CD4 + FOXP3 + ICOS + CXCR5 + The proportion of Tfr cells and detect the expression levels of surface molecules PD-1 and CD38.

[0089] III. Experimental results

[0090] As Figure 4 shown, in induced follicular regulatory T cells, a relatively low concentration of IL-2 (2.5 ng / mL) was added in the first step, as Figure 4as shown in A in [reference] obtains a higher proportion of Tfr cells compared to high-concentration IL-2 (10 ng / mL, as shown in Figure 4 B in [reference]), adding relatively low-concentration IL-2 (2.5 ng / mL, as shown in Figure 4 C in [reference]) results in more PD-1 Figure 4 Tfr cells compared to high-concentration IL-2 (10 ng / mL, as shown in + D in [reference], and the results show that neither high nor low concentrations of IL-2 have a significant effect on the proportion of iTfr. Therefore, based on the above results, a lower concentration (2.5 ng / mL) of IL-2 is required in the first step for inducing iTfr cells.

[0091] The present invention provides an in vitro preparation method for inducible follicular regulatory T cells and the idea and method for its application. There are many specific methods and ways to implement this technical solution. The above is only the preferred embodiment of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention. Each component not clearly defined in this embodiment can be realized by existing technologies.

Claims

1. A method for in vitro preparation of induced follicular regulatory T cells, characterized in that, It includes the following steps: Step 1: Mix the initial CD4 + T lymphocytes, costimulatory molecule antibodies, the first cytokine, and a culture medium to form a first culture system, and then conduct the culture; Step 2: Mix the cells obtained in Step 1, the second cytokine, the costimulatory molecule antibody, and the culture medium to form a second culture system, and culture to obtain induced follicular regulatory T cells; Among them, in Step 1 or Step 2, the costimulatory molecule antibody is a combination of anti-CD3 antibody and anti-CD28 antibody; In Step 1, the first cytokine includes one of the following three combinations: (1) A combination of IL-2, IL-6, and IL-21; (2) A combination of TGF-β, IL-6, and IL-21; (3) A combination of IL-2, TGF-β, IL-6, and IL-21; In Step 2, the second cytokine is a combination of IL-2 and TGF-β; Among them, in the first step, the initial CD4 + T lymphocytes are derived from human peripheral blood or mouse spleen tissue; when the initial CD4 + T lymphocytes are derived from human peripheral blood, the first cytokine further includes IL-12; when the initial CD4 + T lymphocytes are derived from mouse spleen tissue, a cytokine antibody is further added for mixing in the first step, and the cytokine antibody is a combination of anti-IL-4 antibody and anti-IFN-γ antibody; In Step 1, 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; At least one of IL-2 or TGF-β is included in the first cytokine.

2. 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 is 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-6 in the first culture system is 20 ng / mL; The concentration of IL-21 in the first culture system is 20 ng / mL.

3. The in vitro preparation method according to claim 1, wherein When the initial CD4 + When the T lymphocytes are derived from human peripheral blood, the first cytokine further includes IL-12, and the concentration of the 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, a cytokine antibody is further added and mixed in the first step. The cytokine antibody is a combination of an anti-IL-4 antibody and an anti-IFN-γ antibody. 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.

4. The in vitro preparation method according to claim 1, wherein, In Step 1 or Step 2, for the culture, 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, wherein In Step 2, the concentration of the anti-CD3 antibody in the second culture system is 5 μg / mL, and 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.

6. The in vitro preparation method according to claim 1, wherein In Step 1 or Step 2, the culture medium is RPMI 1640 medium containing 10% v / v fetal bovine serum.

7. 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.

Images