CN118956747B - In vitro preparation method and application of natural follicular regulatory T cells - Google Patents
In vitro preparation method and application of natural follicular regulatory T cells Download PDFInfo
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Abstract
The invention relates to the technical field of immunology, and discloses an in-vitro preparation method and application of a natural follicular regulatory T cell. The invention firstly utilizes magnetic beads to sort wild mouse spleen cells to obtain initial CD4 + T lymphocytes, then uses antibodies and specific cytokines to conduct induced differentiation, and adopts two different cytokine polarization conditions to continuously stimulate and culture, thereby obtaining natural follicular regulatory T cells. The invention provides a method for preparing natural follicular regulatory T cells in vitro, which is hopeful to obtain enough natural follicular regulatory T cells and provides an important tool and a treatment means for researching autoimmune diseases, organ transplantation and other immune tolerance related diseases.
Description
Technical Field
The invention relates to the technical field of immunology, in particular to an in-vitro preparation method and application of natural follicular regulatory T cells.
Background
Follicular regulatory T cells (Follicular Regulatory T Cell, tfr) are regulatory T cells of a specific subset of populations that are located in the follicular germinal center. Tfr cells exert an immunomodulatory effect by inhibiting the assistance of follicular helper T cells (Follicular HELPER T CELL, tfh) on B cells, as well as acting directly on B cells. Follicular regulatory T cells co-express surface molecules of follicular helper T cells and regulatory T cells (Regulatory T Cell, treg), such as PD-1, CXCR5, ICOS, CD25, and CD4. Meanwhile, tfr cells express BCL6, the major transcription factor of follicular helper T cells, and FOXP3, the major transcription factor of regulatory T cells.
In autoimmune diseases such as rheumatoid arthritis, systemic lupus erythematosus, primary sjogren'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. Currently, studies of follicular regulatory T cells have focused mainly on the cell regulatory mechanisms. Because of the lack of internationally accepted in vitro induced differentiation methods, in vitro studies of this T cell subset have relied primarily on noble instruments such as flow sorters for sorting. In addition, the content of such cells in the body is very small, and thus it is difficult to obtain a large number of follicular regulatory T cells. The development of a method for obtaining the follicular regulatory T cells by in vitro induced differentiation has the advantages of cost and has important significance for the research and understanding of the cells.
One of the methods known at present is the co-induction of the cytokine IL-2, TGF-beta and baicalin, a Chinese herbal component, by using T cell activation and amplification magnetic beads (refer to China patent CN 107177547A), and the other is the method of obtaining by adding cytokines to anti-TCR-beta antibodies and anti-CD 3 antibodies (PMID: 32711255). The former method enriches T cells with activated magnetic beads and induces differentiation by baicalin, however baicalin is not a self-component of the human body, 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 Tfr cells with high purity (only about 20 percent), and has limited the wide application in Tfr cell in-vitro mechanism research, cell therapy and the like.
The origin of follicular regulatory T cells may be either regulatory T cell precursors or conventional T cells. Current studies gradually deny the notion that follicular regulatory T cells originate only from regulatory T cell precursors. Follicular regulatory T cells differentiated from regulatory T cells are referred to as natural follicular regulatory T cells (nTfr), while follicular regulatory T cells differentiated from conventional T cells are referred to as inducible follicular regulatory T cells (iTfr). Inducible follicular regulatory T cells express CD38, whereas natural follicular regulatory T cells do not express CD 38. Wherein nTfr cells are useful for adoptive transfer for the treatment of autoimmune diseases such as lupus erythematosus, rheumatoid arthritis, and the like, and immunosuppression in organ transplants. In the prior art, a great deal of research is focused on the induction and differentiation of Tfr cells, and the lack of an in-vitro induction and differentiation method of nTfr cells limits the wide application of in-vitro mechanism research of nTfr cells, cell treatment and the like.
Disclosure of Invention
The invention aims to solve the technical problem of providing an in-vitro preparation method and application of a mouse natural follicular regulatory T (nTfr) cell aiming at the defects of the prior art, and lays a foundation for in-vitro research of nTfr cells and cell treatment of the follicular regulatory T cells.
In order to achieve the technical purpose, the invention discloses an in-vitro preparation method and application of nTfr cells. The specific technical scheme is as follows:
An in vitro method for preparing natural follicular regulatory T (nftr) cells, comprising the steps of:
firstly, mixing initial CD4 + T lymphocytes with a co-stimulatory molecule antibody and a first cytokine, and culturing;
And step two, mixing the cells obtained by the culture in the step one with the costimulatory molecule antibodies, the second cytokines and the cytokine antibodies, and culturing to obtain the natural follicular regulatory T cells.
Wherein, in the first step, the initial CD4 + T lymphocyte is obtained by sorting spleen cells of a mouse by magnetic beads, preferably, the mouse is a wild C57BL/6J type mouse, and the week age is 4-6 weeks. Further preferably, wild type mice are sacrificed by cervical removal by isoflurane anesthesia, their spleens are taken to make a cell suspension, and the primary CD4 + T cells are sorted using bioleged murine Na gamma CD4 + T sorting magnetic beads. More preferably, the naive CD4 + T lymphocytes are CD4 +CD44-CD62L+ cells.
In the first step, the co-stimulatory molecule antibodies are one of the following three combinations:
(1) A combination of an anti-CD 3 antibody and an anti-CD 28 antibody;
(2) A combination of an anti-TCR- β antibody and an anti-CD 28 antibody;
(3) A combination of an anti-CD 3 antibody, an anti-TCR- β antibody, and an anti-CD 28 antibody;
Preferably a combination of an anti-CD 3 antibody and an anti-CD 28 antibody.
The first cytokine is a combination of IL-2 and TGF-beta.
Wherein, in the culture system of the first step, the initial CD4 + T lymphocyte concentration is 1.25-2.5X10 6 Cells/mL, the anti-CD 3 antibody concentration is 0-5 mug/mL, the anti-TCR-beta antibody concentration is 0-5 mug/mL, the anti-CD 28 antibody concentration is 2 mug/mL, the IL-2 concentration is 2-15 ng/mL, the TGF-beta concentration is 1-10 ng/mL, preferably, the initial CD4 + T lymphocyte concentration is 2.5X10 6 Cells/mL, the anti-CD 3 antibody concentration is 5 mug/mL, the anti-TCR-beta antibody concentration is 0 mug/mL, the anti-CD 28 antibody concentration is 2 mug/mL, the IL-2 concentration is 5-10 TGF ng/mL, and the TGF-beta concentration is 2.5-5 ng/mL.
In the first step or the second step, the culture temperature is 37 ℃, the carbon dioxide volume concentration is 5%, and the culture time is 3-4 days. Preferably, the incubation time is 3.5 days.
Preferably, in the first or second step, the mixing is performed in a medium which is RPMI 1640 medium containing 10% v/v Fetal Bovine Serum (FBS).
Preferably, the cells obtained by the culture in the first step contain regulatory T cells, i.e., CD4 +CD25+CD127low/- cells.
Further preferably, the specific steps of the first step are that the initial CD4 + T lymphocyte is resuspended in RPMI 1640 medium containing 10% FBS, the resuspended cells are added into a 96-well plate which is pre-incubated with anti-CD 3 antibody and anti-CD 28 antibody, and cytokines IL-2 and TGF-beta are added into the medium and cultured to obtain the antigen.
Wherein in the second step, the second cytokine is the combination of IL-2, TGF-beta, IL-6 and IL-21, and the cytokine antibody is the combination of anti-interleukin 4 (IL-4) antibody and anti-gamma-interferon antibody. The composition and concentration of the co-stimulatory molecule antibodies in the second step are the same as those of the co-stimulatory molecule antibodies in the first step, preferably a combination of anti-CD 3 antibodies and anti-CD 28 antibodies, the concentration of anti-CD 3 antibodies in the second step being 5. Mu.g/mL and the concentration of anti-CD 28 antibodies in the second step being 2. Mu.g/mL.
In the second step of the culture system, the concentration of IL-2 is 2.5-10 ng/mL, the concentration of TGF-beta 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 mug/mL, and the concentration of anti-gamma-interferon antibody is 10 mug/mL.
Wherein 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.
Preferably, the specific steps of the second step are that the cells obtained in the first step are collected into a centrifuge tube, the culture medium is discarded after centrifugation, the cells are resuspended by using RPMI 1640 culture medium containing 10% FBS, the obtained cell resuspension is added into a new 96-well plate which is pre-incubated with anti-CD 3 antibody and anti-CD 28 antibody, and cytokines IL-2, TGF-beta, IL-6 and IL-21 are supplemented into the culture medium, and nTfr cells are obtained after culture.
In a second aspect, the invention provides a natural follicular regulatory T cell prepared by the method of preparation of the first aspect. Preferably, the natural follicular regulatory T cells are CD4 +FOXP3+CXCR5+PD-1+CD38- cells.
In a third aspect, the present invention provides the use of a natural follicular regulatory T cell according to the second aspect for the preparation of a medicament for the treatment of autoimmune diseases and for the treatment of organ transplantation. Preferably, the autoimmune diseases include, but are not limited to, lupus erythematosus, rheumatoid arthritis, and sjogren's syndrome.
The beneficial effects are that:
The method effectively induces the initial CD4 + T cells to differentiate into CD4 +FOXP3+CXCR5+PD-1+CD38- nTfr cells in vitro by selecting the induced differentiation costimulatory molecule antibody, the cytokine antibody and the cytokine, is expected to solve the difficult problem of sorting by a flow sorter in the current research of follicular regulatory T cells, and the nTfr cells prepared by the method can be used for mechanism research and also can be used for nTfr cells adoptive transfer to treat autoimmune diseases, such as lupus erythematosus, rheumatoid arthritis and the like, and immunosuppression in organ transplantation. The method has the specific advantages that:
1) The surface molecule of the follicular regulatory T cells obtained by the method does not express CD38, namely nTfr cells, and CD38 is used for identifying nTfr cells and iTfr cells. The present inventors have found that by appropriate ratios of cytokines, cytokine antibodies and co-stimulatory molecule antibodies, primary CD4 + T cells can be induced to differentiate in vitro and a large number of nTfr cells (> 80% nTfr cells) can be obtained.
2) According to the experiment, the initial CD4 + T cells are sorted from the spleen of the mouse, and a large number of Tfr cells can be obtained by adding the anti-CD 3 antibody, the anti-CD 28 antibody, the IL-2, the TGF-beta, the IL-6, the IL-21, the anti-IL-4 antibody and the anti-gamma-interferon antibody into the initial CD4 + T cells, wherein the Tfr cell content can reach about 50 percent, and the method is simple and feasible and is economical.
3) The invention adopts the cell factor naturally existing in the body and the antibody of the corresponding target spot, can better simulate the induced differentiation of nTfr cells in the body, and can be used for researching the in vitro related functional mechanism of nTfr cells.
4) Tfr cells have the function of inhibiting the helper effect of Tfh cells on B cells and directly inhibiting B cells. However, tfr cells are reduced in autoimmune diseases, which is positively correlated with disease activity. Treg cells have certain curative effects in maintaining immunosuppression, autoimmune disease treatment and the like in organ transplantation. Tfr cells, as a specific subset of regulatory T cells, regulate the immune response of germinal centers. The invention provides an in vitro preparation method of nTfr cells, which can obtain a large number of nTfr cells (> 80%). The method has wide application prospect, and can be used for treating autoimmune diseases such as lupus erythematosus, rheumatoid arthritis, sjogren syndrome and the like, and for immunosuppression in organ transplantation treatment such as kidney transplantation, liver transplantation and the like.
Drawings
The foregoing and/or other advantages of the invention will become more apparent from the following detailed description of the invention when taken in conjunction with the accompanying drawings and detailed description.
FIG. 1 flow-through assay of the effect of different co-stimulatory molecule antibody activation patterns on the proportion of Tfr cells. A in FIG. 1 is a bar graph of Tfr cell ratios obtained by three activation methods, namely, activation with anti-CD 3 antibody, anti-TCR-beta antibody and anti-CD 28 antibody, B in FIG. 1 is activation with anti-CD 3 antibody and anti-CD 28 antibody, C in FIG. 1 is activation with anti-TCR-beta antibody and anti-CD 28 antibody, and D in FIG. 1 is a bar graph of Tfr cell ratios obtained by three activation methods, wherein CD3+TCR-beta represents activation with anti-CD 3 antibody, anti-TCR-beta antibody and anti-CD 28 antibody, CD3 represents activation with anti-CD 3 antibody and anti-CD 28 antibody, and TCR-beta represents activation with anti-TCR-beta antibody and anti-CD 28 antibody.
FIG. 2 shows the effect of IL-6 and IL-21 on nTfr cells in the first step of a two-step flow assay. The A in FIG. 2 is the ratio of the cytokines in the first step to IL-2 and TGF-beta induced Tfr cells only, the B in FIG. 2 is the ratio of the cytokines IL-2, TGF-beta and IL-6 induced Tfr cells, the C in FIG. 2 is the ratio of the cytokines IL2, TGF-beta and IL-21 induced Tfr cells, and the D in FIG. 2 is the ratio of nTfr cells in the Tfr cells when the cytokines in the first step are only IL-2 and TGF-beta.
Detailed Description
The present invention will be further described with reference to the following examples, but it should not be construed that the scope of the present invention is limited to the examples. Various substitutions and alterations are made according to the general technical knowledge and the conventional methods in the field without departing from the technical idea of the present invention, and all such substitutions and alterations are included in the protection scope of the present invention.
In the following examples, SPF grade 4-6 week female C57BL/6J mice, immunomagnetic beads (Biolegend, 480040), anti-CD 3 antibodies (Invitrogen, 16-0031-86), anti-CD 28 antibodies (Invitrogen, 16-0281-85), anti-TCR-. Beta.antibodies (Bioxcell, BE 0102), 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（UA Bioscience,UA040172）、 anti-gamma-interferon antibodies (Invitrogen, 14-7311-85), anti-IL 4 antibodies (Invitrogen, 16-7041-85) were used.
In the following examples, the Tfr cells were CD4 +FOXP3+PD-1+CXCR5+ Tfr cells, the expression of CD38 in Tfr cells was confirmed by flow experiments, and the nTfr cells were cells that did not express CD38 in the Tfr cells.
Example 1 selection of in vitro activation mode of mouse Tfr cells
Single Cells were obtained from spleen tissue of wild C57BL/6J mice, primary CD4 + T lymphocytes (CD 4 +CD44-CD62L+ Cells) were obtained by immunomagnetic bead sorting, and then the sorted Cells were resuspended in RPMI 1640 complete medium containing 10% v/v Fetal Bovine Serum (FBS), added to 96-well plates pre-incubated with co-stimulatory molecule antibodies, 2.5x10 6 Cells/mL were added per well, including the following three pre-incubation modes (activation modes) of co-stimulatory molecule antibodies:
(1) anti-CD 3 antibody (5. Mu.g/mL), anti-TCR-. Beta.antibody (5. Mu.g/mL) and anti-CD 28 antibody (2. Mu.g/mL) were incubated;
(2) Incubation of anti-CD 3 antibody (5. Mu.g/mL) and anti-CD 28 antibody (2. Mu.g/mL);
(3) anti-TCR-beta antibody (5. Mu.g/mL) and anti-CD 28 antibody (2. Mu.g/mL).
IL-2 (10 ng/mL), TGF-. Beta.0.5/ng/mL, anti-IL-4 antibody (10. Mu.g/mL), and anti-gamma-interferon antibody (10. Mu.g/mL) were added to the above culture system, and the total volume of the resulting culture system was 200. Mu.L (the concentrations of the respective substances represent the added concentrations in the culture system), and cultured in a cell culture incubator at 37℃and 5% v/v CO 2 for 3.5 days.
The cultured cells were collected correspondingly, and after centrifugation with PBS (1X, pH=7.4), the medium was discarded, the cells were resuspended in RPMI 1640 complete medium containing 10% v/v FBS, and the cells were added to a new 96-well plate pre-incubated with anti-CD 3 antibody (5. Mu.g/mL) and anti-CD 28 antibody (2. Mu.g/mL), supplemented with cytokine IL-2 ng/mL), TGF- β (0.5 ng/mL), IL-6 (25 ng/mL), IL-21 (25 ng/mL), anti-IL-4 antibody (10. Mu.g/mL), anti-ICOS antibody (5. Mu.g/mL), anti-gamma-interferon antibody (10. Mu.g/mL) (the total culture system in the second step was 200. Mu.L, and the concentrations of each of the substances represent the added concentrations in the culture system) were cultured in a cell incubator at 37℃and 5% v/v CO 2 for 3.5 days, thereby obtaining Tfr cells.
The cells were collected and the proportion of CD4 +FOXP3+PD-1+CXCR5+ Tfr cells was examined by flow cytometry. As a result, as shown in FIG. 1, all three activation modes activated primary CD4 + T cells, allowing the primary CD4 + T cells to differentiate from primary CD4 + T cells into Tfr cells under the action of various cytokines and various antibodies.
Example 2 Effect of different Induction conditions on in vitro induced differentiation of mouse Tfr cells
Single cells were obtained from C57BL/6 spleen tissue, primary CD4 + T lymphocytes (CD 4 +CD44-CD62L+ cells) were obtained by immunomagnetic bead sorting, and then the sorted cells were resuspended in RPMI 1640 complete medium containing 10% fetal bovine serum to obtain a cell resuspension, which was subjected to two-step culture:
The first step was to add the obtained cell suspension to a 96-well plate previously incubated with CO-stimulatory molecule antibodies (anti-CD 3 antibody at an added concentration of 5. Mu.g/mL and anti-CD 28 antibody at 2. Mu.g/mL in a culture system) and 1.25X10 6 Cells/mL each well followed by the addition of cytokines IL-2 and TGF-. Beta.to the wells, and to culture the resulting cell suspension in a cell culture incubator at 37℃and 5% v/v CO 2 for 3.5 days to obtain a cell solution containing regulatory T Cells (CD 4 +CD25highCD127low/- Cells).
And a second step of collecting all cells obtained by the first step, adding PBS (1×, pH=7.4), centrifuging to remove the culture medium, re-suspending the cells with RPMI 1640 complete culture medium containing 10% v/v FBS, adding the obtained cell re-suspension into a 96-well plate newly pre-incubated with anti-CD 3 antibody (5 μg/mL) and anti-CD 28 antibody (2 μg/mL), supplementing cytokines IL-2, TGF- β, IL-6, IL-21, anti-IL-4 antibody (10 μg/mL) and anti-gamma-interferon antibody (10 μg/mL), culturing in a cell culture box for 3.5 days at 37 ℃ and 5% v/v CO 2, wherein the total volume of the culture system of the second step is 200 μl (the concentration of each substance represents the additive concentration in the culture system), and the additive concentrations of the various cytokines and the antibody shown in Table 1 are added, thereby obtaining the r cells. The cells were collected and the proportion of CD4 +FOXP3+PD-1+CXCR5+ Tfr cells was examined by flow cytometry.
The concentrations of the various cytokines added in the two-step method were screened in this example, as shown in Table 1.
TABLE 1 additive concentrations of various cytokines in two-step culture
The experimental results are shown in Table 2, and different proportions of follicular regulatory T cells were obtained by varying concentrations of cytokines IL-2 and TGF-beta in a two-step process,
First step IL-2 (10 ng/mL), TGF- β (5 ng/mL), second step IL-2 (2.5 ng/mL), TGF- β (5 ng/mL) as described in Experimental group 1;
First step IL-2 (5 ng/mL), TGF- β (2.5 ng/mL), second step IL-2 (5 ng/mL), TGF- β (5 ng/mL) as described in experimental group 15;
First step IL-2 (5 ng/mL), TGF- β (2.5 ng/mL), second step IL-2 (5 ng/mL), TGF- β (10 ng/mL) as described in experimental group 16.
The combination of the three cytokines is the most ideal condition compared with other concentrations.
TABLE 2 ratio of Tfr cells in different groups
Example 3 exploration of combinations of mouse nTfr cytokines
Single cells were obtained from C57BL/6J spleen tissue, primary CD4 + T lymphocytes (CD 4 +CD44-CD62L+ cells) were obtained by immunomagnetic bead sorting, and then the sorted cells were resuspended in RPMI 1640 complete medium containing 10% v/v FBS to obtain a cell resuspension, which was subjected to two-step culture:
The first step is to add the resulting cell suspension to a 96-well plate pre-incubated with co-stimulatory molecule antibodies (anti-CD 3 antibody added at a concentration of 5. Mu.g/mL and anti-CD 28 antibody added at a concentration of 2. Mu.g/mL in the culture system) at 2.5X10 6 Cells/mL per well, and to add various combinations of cytokines at the following additive concentrations:
(1) IL-2 (10 ng/mL), TGF-beta (5 ng/mL) and IL-6 (20 ng/mL), or
(2) IL-2 (10 ng/mL), TGF-beta (5 ng/mL) and IL-21 (20 ng/mL), or
(3) IL-2 (10 ng/mL), TGF-beta (5 ng/mL), IL-6 (20 ng/mL), and IL-21 (20 ng/mL), or
(4) A combination of IL-2 (10 ng/mL) and TGF-beta (5 ng/mL),
The total volume of the resulting culture system was 200. Mu.L, and cultured in a cell incubator at 37℃for 3.5 days with 5% v/v CO 2.
And secondly, correspondingly collecting the cultured cells, adding PBS (1X, pH=7.4), centrifuging, discarding the culture medium, re-suspending the cells with RPMI 1640 complete culture medium containing 10% v/v FBS, adding the obtained cell re-suspension into a new 96-well plate which is pre-incubated with anti-CD 3 antibody (added with 5 mug/mL) and anti-CD 28 antibody (added with 2 mug/mL), supplementing the cell factors IL-2 (added with 10 ng/mL), TGF-beta (added with 5 ng/mL), IL-6 (added with 20 ng/mL), IL-21 (added with 20 ng/mL), anti-IL-4 antibody (added with 10 mug/mL) and anti-gamma-interferon antibody (added with 10 mug/mL), and culturing the obtained cell re-suspension in a cell culture box with 37 ℃ and 5% v/v CO 2 for 3.5 days to obtain Tfr cells. The cells were collected, examined by flow cytometry for CD4 +FOXP3+PD-1+CXCR5+ Tfr cells, and the proportion of nTfr cells was obtained by detecting whether CD38 was expressed.
As a result, as shown in FIG. 2, in the induction nTfr, the addition of the cytokine IL-6 in the first step resulted in a decrease in the proportion of Tfr cells obtained (A in FIG. 2 and B in FIG. 2), and the addition of the cytokine IL-21 in the first step resulted in a decrease in the proportion of Tfr cells (A in FIG. 2 and C in FIG. 2). When only IL-2 and TGF-beta are added in the first step, whether CD38 in Tfr cells is expressed or not is detected, and as shown by D in figure 2, the proportion of nTfr cells in Tfr cells reaches more than 80%.
In summary, it is preferred that the cytokine combination inducing nTfr cells is IL-2 and TGF-beta in the first step, IL-2, TGF-beta, IL-6, IL-21 in the second step.
The invention provides an in vitro preparation method and an application thought and method of a natural follicular regulatory T cell, and a method and a way for realizing the technical scheme are numerous, the above is only a preferred embodiment of the invention, and it should be pointed out that a plurality of improvements and modifications can be made to those skilled in the art without departing from the principle of the invention, and the improvements and modifications are also considered as the protection scope of the invention. The components not explicitly described in this embodiment can be implemented by using the prior art.
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