System and method for efficiently inducing regulatory B cells and application of system and method

Abstract

The invention discloses a system and a method for efficiently inducing regulatory B cells and application of the system and the method. The invention provides a system for inducing regulatory B cells, the system comprises a basal culture medium and an inducer, and the system can promote B cells to express IL-10 and reduce the generation of inflammatory factors TNF-alpha at the same time. On the basis, the invention further provides a method for obtaining the regulatory B cells through induction of the system, the obtained regulatory B cells are mainly enriched in CD19 < + > CD1c < + > B cells, and compared with the CD19 < + > CD1c <-> B cells, the regulatory B cells express IL-10 with a higher level. Besides, the CD19 + CD1c + B cell can be used for inhibiting the proliferation of PBMC (peripheral blood mononuclear cells) and reducing the release of effector T cell inflammatory factors, and is used for treating graft-versus-host diseases. The invention contributes to the acquisition of regulatory B cells and the treatment of graft versus host disease.

Description

Technical Field

[0001] The present invention belongs to the technical field of cell induction and culture, and more specifically, relates to a system, method and application of highly efficient induction of regulatory B cells. Background Art

[0002] Graft-versus-host disease (GVHD) is a systemic disease that causes multiple system damage (skin, esophagus, gastrointestinal tract, liver, etc.) after hematopoietic stem cell transplantation and is the main cause of non-recurrence death after surgery. Currently, clinical medication mainly relies on glucocorticoids and immunosuppressants, but their therapeutic effect is poor, and long-term use can cause adverse reactions such as infection.

[0003] Recent studies have found that adoptive infusion of cells with immunomodulatory or regenerative properties can effectively prevent and treat GVHD. Regulatory B cells (Bregs) are a type of cells with immunosuppressive ability. They can inhibit the proliferation of effector T cells and the secretion of inflammatory factors, induce the production of regulatory T cells (Tregs), and play a role in immune tolerance in autoimmune diseases, infections, and transplant-related diseases. Other studies have found that Bregs are closely related to the occurrence and development of GVHD; the number of Bregs in the peripheral blood of GVHD patients is significantly reduced compared with patients without the disease after hematopoietic stem cell transplantation; the lack of Bregs significantly increases the severity of GVHD in mice; umbilical cord blood containing Bregs and hematopoietic stem cells can effectively reduce the incidence of GVHD in patients; the improvement of symptoms in GVHD patients after treatment is accompanied by an increase in the proportion of Bregs. Adoptive infusion of mouse Bregs can effectively improve mouse GVHD. Therefore, cell therapy may be an effective means of preventing and treating GVHD in the future.

[0004] B cells only account for 5-15% of healthy human peripheral blood mononuclear cells, and Bregs account for even less. The proportion of Bregs in human peripheral blood is less than 1%, and it is difficult to obtain Bregs that meet the treatment needs through separation. Although it has been reported that Bregs can be obtained by adding or combining multiple stimulants such as Toll-like receptor agonists, CD40 ligands or feeder cells during in vitro culture to culture and expand B cells. However, the induction effect of the above method is limited, and a sufficient number of Bregs cannot be produced. In addition, due to the lack of suitable purified surface markers, the obtained Bregs cannot meet the needs of further treatment; more importantly, the production of inflammatory factor TNF-α may be increased in this process, resulting in the loss of Bregs' immunoregulatory ability, and even having the effect of promoting inflammation. Therefore, there is an urgent need for a system and method for amplifying and culturing Bregs with high induction efficiency and without increasing the production of inflammatory factors. Summary of the invention

[0005] In view of the defects and shortcomings of the above-mentioned technologies, the present invention provides a system, method and application of highly efficient induction of regulatory B cells.

[0006] The first object of the present invention is to provide a system for inducing regulatory B cells.

[0007] The second object of the present invention is to provide use of the system in inducing regulatory B cells or in preparing a preparation for inducing regulatory B cells.

[0008] The third object of the present invention is to provide a method for inducing and sorting CD19 in vitro + CD1c + B cell approach.

[0009] The above-mentioned purpose of the present invention is achieved through the following technical solutions:

[0010] The present invention provides a system for inducing regulatory B cells in order to solve the problems that the proportion of regulatory B cells in peripheral blood is low, it is difficult to obtain regulatory B cells that meet the treatment needs through separation; B cells are difficult to expand in vitro and are prone to apoptosis; and the current induction scheme cannot produce a sufficient number of regulatory B cells, and while promoting the increase of IL-10, it also induces the production of TNF-α. The system of the present invention can be used to efficiently induce IL-10. + At the same time, the present invention uses high-dimensional multi-color flow cytometry to detect the phenotype of induced regulatory B cells and finds that the induced regulatory B cells highly express CD1c and are related to CD19 + CD1c - Compared with B cells, CD19 + CD1c +B cells express higher levels of IL-10, that is, the system of the present invention can be used to induce regulatory B cells that express higher levels of IL-10. In addition, the CD19 induced by the system of the present invention + CD1c + B cells can inhibit the proliferation of PBMCs and reduce the release of effector T cell inflammatory factors, and are used to treat GVHD. Therefore, the present invention claims protection for the system, method and application of inducing regulatory B cells.

[0011] The present invention provides a system for inducing regulatory B cells, the system comprising a basal culture medium and an inducer, the inducer comprising CpG, and further comprising mesenchymal stem cells (MSC) and / or CHIR-99021; the sequence of the CpG is shown in SEQ ID NO.1.

[0012] Preferably, the inducer is CpG, mesenchymal stem cells and CHIR-99021, as shown in Example 1. When the inducer CpG, mesenchymal stem cells and CHIR-99021 are present in the system at the same time, the induction effect on regulatory B cells is the best.

[0013] Specifically, the final concentration of CpG in the system is 2-10 μg / mL, the final concentration of CHIR-99021 is 4-12 μM, and the quantitative ratio of mesenchymal stem cells to B cells is 1:3-8.

[0014] More specifically, the final concentration of CpG in the system is 3-5 μg / mL, the final concentration of CHIR-99021 is 5-10 μM, and the quantitative ratio of mesenchymal stem cells to B cells is 1:4-6.

[0015] More specifically, the final concentration of CpG in the system was 4 μg / mL, the final concentration of CHIR-99021 was 10 μM, and the quantitative ratio of mesenchymal stem cells to B cells was 1:5.

[0016] Optionally, the basal culture medium is RPMI-1640 complete culture medium.

[0017] Specifically, the regulatory B cells are CD19 + CD1c + of B cells.

[0018] Specifically, the regulatory B cells induced by the present invention are human regulatory B cells. The present invention has found through testing that the regulatory B cells induced by the system of the present invention are mainly enriched in CD19 + CD1c + B cells, whose ability to express IL-10 is similar to that of classical regulatory B cells CD24+ CD27 + B cells are equivalent, but CD19 + CD1c + B cells account for CD19 + The proportion of B cells was significantly higher than that of CD24 + CD27 + B cells, that is, more regulatory B cells can be induced by the system of the present invention, which greatly improves the efficiency of regulatory B cell production and has a higher induction efficiency. The present invention also claims the use of the system in inducing regulatory B cells or in preparing a preparation for inducing regulatory B cells.

[0019] Specifically, the regulatory B cells are CD19 + CD1c + of B cells.

[0020] The present invention also provides a method for inducing and sorting CD19 in vitro + CD1c + The method comprises: inducing the CD19 + B cells were placed in the system for induction culture for 36 to 48 hours, and CD19 cells were sorted using CD1c as a surface marker. + CD1c + B cells.

[0021] Specifically, when the inducer in the system contains mesenchymal stem cells, the mesenchymal stem cells need to be digested one day in advance, and the mesenchymal stem cells are seeded in the well plate at a density of 70% and allowed to adhere to the wall overnight.

[0022] Specifically, the culture conditions are 35-38°C and 5% CO2.

[0023] Specifically, CD19 + CD1c + B cells.

[0024] The present invention has the following beneficial effects:

[0025] The present invention provides a system for inducing regulatory B cells, which can promote B cells to express IL-10 while reducing the production of inflammatory factor TNF-α. On this basis, the present invention also provides a method for inducing regulatory B cells using the system. The present invention uses high-dimensional multi-color flow cytometry to detect the phenotype of induced regulatory B cells and finds that the induced regulatory B cells highly express CD1c and are related to CD19. + CD1c - Compared with B cells, CD19 + CD1c +B cells express higher levels of IL-10. In addition, the present invention converts CD19 + CD1c - B cells were co-cultured with peripheral blood mononuclear cells and effector T cells, respectively, and it was found that they could effectively inhibit the proliferation of peripheral blood mononuclear cells in vitro and reduce the secretion of proinflammatory factors TNF-α and IFN-γ by effector T cells. A humanized mouse GVHD model was further constructed, and it was found that infusion of CD19 + CD1c + B cells can effectively improve the symptoms of GVHD in mice and increase their survival rate. That is, the system and method provided by the present invention improve the induction efficiency of regulatory B cells in vitro, which is helpful for obtaining regulatory B cells and treating GVHD. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 IL-10 is obtained after inducing B cells with different induction systems + The proportion of B cells.

[0027] Figure 2 TNF-α is obtained by inducing B cells with different induction systems + The proportion of B cells.

[0028] Figure 3 CD19 isolated after induction with CpG, MSC and CHIR-99021 + CD1c + B cells and CD24 + CD27 + IL-10 expression capacity of B cells and CD19 + Comparison results of B cell proportions.

[0029] Figure 4 CD19 obtained by sorting after induction with different induction systems + CD1c + B cells and CD19 + CD1c - Results of IL-10 expression ability test of B cells.

[0030] Figure 5 CD19 isolated after induction with CpG, MSC and CHIR-99021 + CD1c + B cells and CD19 + CD1c - Results of the effect of B cells on PBMC proliferation.

[0031] Figure 6 CD19 isolated after induction with CpG, MSC and CHIR-99021+ CD1c + B cells and CD19 + CD1c - The results of the effect of B cells on the release of inflammatory factors by T cells.

[0032] Figure 7 CD19 isolated after induction with CpG, MSC and CHIR-99021 + CD1c + B cells and CD19 + CD1c - B cell therapy outcomes in humanized GVHD mice.

[0033] In the figure, *P<0.05; **P<0.01; ***P<0.001; ns, no significant difference. DETAILED DESCRIPTION

[0034] The present invention is further described below in conjunction with the accompanying drawings and specific examples, but the examples do not limit the present invention in any form. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0035] Unless otherwise specified, the reagents and materials used in the following examples are commercially available.

[0036] The CHIR-99021 used in the examples of the present invention was purchased from selleck with the item number S2924.

[0037] Example 1 Induction and culture of regulatory B cells

[0038] The present invention uses different induction systems to induce and culture the isolated B cells, and the IL-10 + The proportion of B was tested. The specific process is as follows:

[0039] 1. Isolation and culture of peripheral blood mononuclear cells (PBMC)

[0040] PBMCs were isolated by Ficoll density gradient centrifugation. Specifically, peripheral blood was diluted with PBS solution (commercial PBS, biosharp, 21074715) at a volume ratio of 1:4, and an equal amount of Ficoll upper layer was slowly added after sufficient mixing. After weighing and balancing on a balance, the cells were centrifuged at 2800rpm for 25min, with an increase speed of 1 and a decrease speed of 0. After centrifugation, the buffy coat layer was gently aspirated into PBS, centrifuged at 2000rpm for 6min, the supernatant was discarded, and an appropriate amount (5-10mL) of red blood cell lysis buffer was added to each tube for resuspending. The cells were allowed to stand for 3-5min to lyse the red blood cells, and the cells were terminated by filling with PBS solution. The cells were centrifuged at 1500rpm for 5min, the supernatant was discarded, and the precipitate was the isolated PBMCs. The PBMCs were resuspended with RPMI-1640 complete medium (GIBCO, 8123287) for subsequent experiments. Alternatively, the cells were seeded in a T175 culture flask and cultured in a 5% CO2, 37°C incubator for later use.

[0041] 2. B cell sorting

[0042] The isolated PBMCs were filtered through a 200-mesh sieve, washed twice with PBS solution, centrifuged at 1500 rpm for 5 min, and the supernatant was discarded. The cells were resuspended to 1×10 in PBS solution containing 0.1% (m / v mass / volume) BSA. 8 / mL, add anti-human CD19 flow cytometry antibody at a ratio of 1:50 between antibody and cell suspension volume; shake and mix, incubate at room temperature in the dark for 30 minutes; shake and mix again every 10 minutes, add PBS solution to stop; centrifuge at 1500rpm for 5 minutes, discard the supernatant; add an appropriate amount of PBS solution containing 0.1% BSA to resuspend the cell pellet, filter twice with a 200-mesh screen into a flow tube for sorting; collect B cells in a centrifuge tube containing 5mL RPMI-1640 complete medium; centrifuge the collected solution at 1500rpm for 5 minutes, and resuspend the B cells to 1×10 6 / mL, and then carry out subsequent experiments; or plant in T25 culture flasks for later use; the whole sorting process is sterile operation; the obtained B cells are CD19 + B cells.

[0043] 3. Induction of B cells

[0044] The present invention uses different induction systems to induce and culture B cells, corresponding to different experimental groups; the details are as follows:

[0045] Experimental group 1: CpG induction group, the final concentration of CpG in the system was 4 μg / mL; the sequence of the CpG was: 5'-tcgtcgttttgtcgttttgtcgtt-3' (shown in SEQ ID NO. 1), and this example used purchased commercial CpG (ODN 2006, tlrl-2006-5).

[0046] Experimental group 2: CpG and CHIR-99021 induction group, the final concentration of CpG in the system was 4 μg / mL, and the final concentration of CHIR-99021 was 10 μM;

[0047] Experimental group 3: CpG and MSC induction group, the final concentration of CpG in the system was 4 μg / mL, and the ratio of MSC to B cells was 1:5;

[0048] Experimental group 4: CpG, CHIR-99021 and MSC induction group, the final concentration of CpG in the system was 4 μg / mL, the final concentration of CHIR-99021 was 10 μM, and the ratio of MSC to B cells was 1:5;

[0049] The B cell group alone served as the control group and was cultured only with RPMI-1640 complete medium, and the culture conditions were the same as those of the experimental group.

[0050] The specific operations of experimental group 1 and experimental group 2 were as follows: the sorted B cells were resuspended in RPMI-1640 complete medium to 1×10 6 / mL, CpG was added to the experimental group 1 to make the final concentration of 4μg / mL; CpG and CHIR-99021 were added to the experimental group 2 to make the final concentration of CpG 4μg / mL and the final concentration of CHIR-99021 10μM. The specific operations of experimental groups 3 and 4 were as follows: MSCs were digested one day in advance, MSCs were seeded in the well plate at a density of 70%, and adhered to the wall overnight; the sorted B cells were resuspended in RPMI-1640 complete medium to 1×10 6 / mL, and added into the well plate with MSCs pre-plated at a ratio of 5:1. CpG was added to the experimental group 3 to make its final concentration 4μg / mL; CpG and CHIR-99021 were added to the experimental group 4, respectively, to make the final concentration of CpG 4μg / mL, and the final concentration of CHIR-99021 10μM; the experimental groups and the control group were cultured in an incubator at 37℃, 5% CO2 for 48h.

[0051] 4. Detection of IL-10 using flow cytometry intracellular cytokine detection technology + The proportion of B cells

[0052] 5-6 hours before the end of the induction culture, add 2 μL / mL Leukocyte Activation Cocktail for stimulation; transfer the cultured B lymphocytes to the flow tube, fill with flow buffer to wash once, centrifuge at 1500rpm for 5 minutes, and discard the supernatant; add 100 μL flow buffer to each tube to resuspend the cells, add the cell phenotype antibody to be tested according to the recommended concentration in the antibody manual, and shake to mix; incubate at 4℃ in the dark for 30 minutes, and shake to mix once every 10 minutes; after the incubation in the dark, add 200 μL 4% PFA to fix for 20 minutes; fill with flow buffer to wash, centrifuge at 1500rpm for 5 minutes; add 100 μL to each tube 0.2% saponin permeabilization solution, and resuspend the cells, add the intracellular antibody to be tested according to the recommended concentration of the antibody manual, and shake and mix; incubate at 4°C in the dark for 30 minutes, shake and mix once every 10 minutes; after the end of the dark incubation, fill with flow buffer to wash, wash off the excess antibody, centrifuge at 1500rpm for 5 minutes, and discard the supernatant; add 200μL flow buffer to resuspend the cell pellet, and detect on the machine. The names, companies and product numbers of the antibodies used in this example are shown in Table 1.

[0053] Table 1

[0054]

[0055]

[0056] The present invention also uses flow cytometry intracellular cytokine detection technology to detect TNF-α + The proportion of B cells.

[0057] 5. Experimental results

[0058] (1) MSC combined with CHIR-99021 effectively induces regulatory B cells

[0059] The present invention uses the above-mentioned different induction systems to induce and culture the sorted B cells, and at the same time, the IL-10 obtained after induction and culture with different induction systems is induced. + The proportion of B cells was tested and the results were as follows Figure 1 As shown. Figure 1 It can be seen that compared with the CpG induction group, adding MSC or CHIR-99021 to the induction system can significantly increase the expression of IL-10 + The ratio of B cells was significantly increased, and the combined use of MSC and CHIR-99021 could more effectively induce the production of IL-10, indicating that the system of the present invention can efficiently induce regulatory B cells.

[0060] (2) MSC combined with CHIR-99021 promotes B cell secretion of IL-10 while downregulating TNF-α

[0061] The present invention uses the above-mentioned different induction systems to induce and culture the sorted B cells, and at the same time, the TNF-α obtained after induction and culture with different induction systems is induced. + The proportion of B cells was tested and the results were as follows Figure 2 As shown. Figure 2 It can be seen that compared with the CpG induction group, the TNF-α in the CpG, MSC and CHIR-99021 induction groups + The proportion of B cells decreased significantly, indicating that the combination of MSC and CHIR-99021 significantly reduced the secretion of TNF-α by B cells.

[0062] Example 2 Comparison of the ability of different regulatory B cells (Bregs) to secrete IL-10

[0063] 1. CD19 + CD1c + B cells and CD24 + CD27 + B cell comparison

[0064] The present invention is to sort the CD19 obtained after CpG, MSC and CHIR-99021 induction + CD1c + B cells and classical Bregs subsets CD24 + CD27 + The IL-10 expression capacity of B cells was detected, and the proportion of CD19 + The proportion of B cells was analyzed and the results were Figure 3 As shown. Figure 3 It can be seen that CD19 + CD1c + B cells and CD24 + CD27 + The IL-10 expression capacity of B cells is basically the same, but CD24 + CD27 + B cells only have CD19 + 6.32% of B cells, and CD19 + CD1c + B cells occupy CD19 + 19.5% of B cells; indicating that the Bregs induced by the system of the present invention are mainly enriched in CD19 + CD1c + B cells, although their ability to express IL-10 is similar to that of classical Bregs CD24 + CD27 + B cells are equivalent, but CD19 + CD1c+ B cells account for CD19 + The proportion of B cells was significantly higher than that of CD24 + CD27 + B cells, that is, more Bregs can be induced by utilizing the system of the present invention, thereby improving the efficiency of Bregs generation, achieving efficient induction of Bregs, and being more conducive to the subsequent development of their therapeutic potential.

[0065] 2. CD19 + CD1c + B cells and CD19 + CD1c - IL-10 expression in B cells

[0066] The present invention uses traditional flow cytometry to label CD19 cells obtained after induction in different induction systems. + CD1c + B and CD19 + CD1c - The expression of IL-10 in B cells was tested. Figure 4 As shown. Figure 4 It can be seen that the CD19 obtained after induction + CD1c + The level of IL-10 expressed by B cells was significantly higher than that of CD19 + CD1c - B cells; i.e. CD19 induced by CpG, MSC and CHIR-99021 + CD1c + The expression level of IL-10 in B cells was significantly higher than that in other induction systems.

[0067] Example 3CD19 + CD1c + B cells and CD19 + CD1c - Effects of B cells

[0068] 1. CD19 + CD1c + B cells inhibit the proliferation of PBMCs

[0069] (1) Experimental methods

[0070] Take an appropriate amount of PBMC and wash it twice with PBS, centrifuge it at 1500rpm for 5min, discard the supernatant, add 5μM CFSE dye, incubate it at 37℃ in the dark for 10min, and shake and mix it once every 2min; fill it with RPMI-1640 complete medium to terminate the incubation, centrifuge it at 1500rpm for 5min, discard the supernatant, resuspend the precipitate with RPMI-1640 complete medium, and adjust the cell concentration to 1×10 7 100 μL of cell suspension was added to a 96-well plate as a PBMC non-proliferation control. PBMCs were stimulated with 1 μg / mL anti-CD3 monoclonal antibody and 1 μg / mL anti-CD28 monoclonal antibody, and 100 μL was added to a 96-well plate. CD19 cells were collected after co-culture with CpG, MSCs, and CHIR-99021 for 48 hours. + CD1c + B cells and CD19 + CD1c - B cells, count and adjust their concentration to 2 × 10 7 / mL; 100 μL was drawn from each sample and co-cultured with stimulated PBMCs; cells were collected after 96 hours, washed once with flow cytometry buffer, centrifuged at 1500 rpm for 5 min, supernatant discarded, 200 μL of flow cytometry buffer was added to resuspend the cell pellet, and then tested on the machine.

[0071] (2) Experimental results

[0072] The sorted B cells were induced in vitro by CpG, MSC and CHIR-99021 for 48 h, and the induced B cells were obtained. CD19 + CD1c + B cells and CD19 + CD1c - B cells. CFSE-labeled PBMCs were used as the unstimulated group, and PBMCs activated by anti-CD3 and CD28 monoclonal antibodies (CD3 / CD28 mAb) were used as the experimental group. + CD1c + B cells and CD19 + CD1c - B cells were co-cultured with PBMC activated with CD3 / CD28 mAb for 96 h to detect CD19 + CD1c + B cells and CD19 + CD1c - The effect of B cells on PBMC proliferation, the results are as follows Figure 5 As shown. Figure 5 It can be seen that CD3 / CD28 mAb stimulation can significantly promote PBMC proliferation, CD19 +CD1c + B cells, not CD19 + CD1c - B cells can significantly inhibit the proliferation of PBMCs.

[0073] 2. CD19+CD1c + B cells inhibit the release of inflammatory factors by T cells

[0074] The sorted B cells were induced in vitro by CpG, MSC and CHIR-99021 for 48 h, and the induced B cells were obtained. CD19 + CD1c + B cells and CD19 + CD1c - B cells. + After 48 h of co-culture, the release of TNF-α and IFN-γ from T cells was detected. Figure 6 As shown. Figure 6 It can be seen that CD19 + CD1c + B cells and CD19 + CD1c - In the presence of B cells, they can inhibit T cells from secreting proinflammatory factors TNF-α and IFN-γ, but CD19 + CD1c + The inhibitory effect of B cells is significantly stronger than that of CD19 + CD1c + B cells.

[0075] 3. CD19 + CD1c + B cells improve GVHD in mice

[0076] (1) Construction of humanized mouse GVHD model

[0077] The experiment was carried out using immunodeficient mice (B-NDG) raised in an SPF environment for 6 to 8 weeks. The mice were purchased from Zhuhai Baishitong Company, and the experiment was approved by the Experimental Animal Care and Use Committee of Sun Yat-sen University (SYSU-IACUC-2022-000338).

[0078] First, take an appropriate amount of separated PBMCs, filter once with a 200-mesh filter, wash twice with PBS, centrifuge at 1500 rpm for 5 min, discard the supernatant, add an appropriate amount of PBS to resuspend the cell pellet and count the cells. The cell concentration was adjusted to 1×10 8 Each mouse was transplanted with 150 μL of cell suspension (1.5×10 cells / mL) via tail vein injection using an insulin needle. 7The control group was injected with 150 μL PBS solution. The mice were weighed regularly during the period, and a small amount of peripheral blood was drawn on the 18th day after transplantation to detect hCD45 + The number of cells was used to determine whether the model was successful; the mice with successful modeling were randomly divided into 3 groups and injected with 100 μL 1×10 6 CD19 + CD1c + B cells, CD19 + CD1c - The B cell suspension was used as the treatment group, and the treatment control group was injected with 100 μL PBS solution; the survival rate and body weight changes of mice in each group were counted for 60 days.

[0079] (2) Experimental results

[0080] A humanized GVHD mouse model was established by infusing PBMCs into the tail vein of immunodeficient B-NDG mice. After 21 days, CpG, MSCs, and CD19 induced by CHIR-99021 were infused respectively. + CD1c + B cells and CD19 + CD1c - B cells were counted in the healthy group (sham), model group (GVHD), CD19 + CD1c - B cell therapy group (CD1c - ) and CD19 + CD1c + B cell therapy group (CD1c + ) and observed the infiltration of inflammatory cells in target organs. Figure 7 As shown. Figure 7 It is known that injection of CD19 + CD1c + The survival rate of the B cell group was 60%, which was significantly higher than that of the modeling group and CD19 + CD1c - B cell group, and CD19 + CD1c + B cell therapy greatly alleviated the weight loss of GVHD mice, while also reducing the infiltration of inflammatory cells in target organs and alleviating tissue damage.

[0081] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. A system for inducing regulatory B cells, characterized in that: The system comprises a basic culture medium and an inducer, wherein the inducer comprises CpG and also comprises mesenchymal stem cells and / or CHIR-99021; the sequence of the CpG is shown in SEQ ID NO.

1.

2. The system according to claim 1, characterized in that: The inducers are CpG, mesenchymal stem cells and CHIR-99021.

3. The system according to claim 1 or 2, characterized in that: The final concentration of CpG in the system is 2-10 μg / mL, the final concentration of CHIR-99021 is 4-12 μM, and the ratio of the number of mesenchymal stem cells to B cells is 1:3-8.

4. The system according to claim 1 or 2, characterized in that: The basic culture medium is RPMI-1640 complete culture medium.

5. The system according to claim 1 or 2, characterized in that: The regulatory B cells are phenotypically CD19 + CD1c + of B cells.

6. Use of the system according to claim 1 or 2 in inducing regulatory B cells or in preparing a preparation for inducing regulatory B cells.

7. The use according to claim 6, characterized in that: The regulatory B cells are phenotypically CD19 + CD1c + of B cells.

8. In vitro induction and sorting of CD19 + CD1c + B cell method, characterized in that, The CD19 + B cells are placed in the system of claim 1 or 2 and induced and cultured for 36 to 48 hours, and CD19 cells are sorted using CD1c as a surface marker. + CD1c + B cells.

9. The method according to claim 8, characterized in that: The culture conditions were 35-38°C and 5% CO2.

10. The method according to claim 8, characterized in that: CD19 was isolated by flow cytometry + CD1c + B cells.

Images