A method of in vitro inducing proliferation of breg cells

Abstract

The application discloses a method for inducing Breg cell proliferation in vitro. The method comprises the following steps: (1) culturing B cells derived from mammals in a culture medium containing CD40 antibodies; (2) adding SARS-CoV-2 virus antigens to the culture system, wherein the SARS-CoV-2 virus antigens are selected from one or more of N protein, S protein specific epitope peptide segments or S1 subunits containing RBD regions of S protein; and (3) after 24-96 hours of culture, the induction of Breg cells is achieved. The application uses SARS-CoV-2 specific protein antigens to significantly improve the proportion of B cell conversion to Breg cells, and realizes the specific induction of Breg (CD19 + IL-10 + ) cell proliferation and differentiation under in vitro conditions.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to a method for inducing Breg cell proliferation in vitro. Background Technology

[0002] Regulatory B cells (Breg) are CD19 cells with immunosuppressive effects. + B cell subsets, characterized primarily by IL-10 secretion, are crucial for maintaining immune homeostasis. In autoimmune diseases such as rheumatoid arthritis and systemic lupus erythematosus, Breg cells often exhibit decreased numbers and weakened function, significantly influencing disease progression. Adoptive infusion of Breg cells or induction of Breg cell proliferation in vivo can effectively alleviate disease symptoms and slow disease progression. Therefore, researching methods to induce Breg cell proliferation is an important strategy for the prevention and treatment of these diseases and has significant clinical value.

[0003] Viral proteins are the most important factors influencing the body's immune system. Some can enhance the body's immune response, leading to a cytokine storm, while others can help the virus suppress the immune response, thereby increasing viral survival and infection efficiency. Whether viral proteins can effectively induce an increase in the number and function of Breg cells remains unclear and warrants further investigation. The N protein (Nucleocapsid) of the SARS-CoV-2 virus is located inside the virus, intertwined with the viral genomic RNA, and constitutes the largest proportion of the virus's structural proteins. The S protein (Spike protein) is the most important surface membrane protein of coronaviruses, responsible for viral binding to host cell membrane receptors and membrane fusion, containing two subunits, S1 and S2. S1 contains a receptor-binding domain (RBD) responsible for recognizing cellular receptors. S2 contains elements required for membrane fusion. Viral structural proteins such as the N and S proteins have been shown to play important roles in infection and immunity, but current technologies mostly focus on their roles in viral invasion, replication, and antibody production. Whether these proteins can induce an increase in the number and function of Breg cells remains unclear. This deficiency limits our in-depth understanding of the pathogenic mechanisms of the virus and also restricts the exploration and development of related immune intervention strategies. Summary of the Invention

[0004] To address the shortcomings of existing technologies, this invention proposes a method for inducing Breg cell proliferation in vitro. This invention specifically induces B cells to differentiate into Breg cells in vitro using a specific SARS-CoV-2 antigen, achieving efficient acquisition of Breg cells and endowing them with significant immunosuppressive function, thus providing a new technical solution for cell therapy of immune-related diseases.

[0005] This invention provides a method for inducing Breg cell proliferation in vitro, comprising the following steps: (1) B cells derived from mammals were cultured in a medium containing CD40 antibody; (2) Add SARS-CoV-2 viral antigen to the culture system, wherein the SARS-CoV-2 viral antigen is selected from one or more of the N protein, the S protein specific epitope peptide, or the S1 subunit of the S protein containing the RBD region; (3) After culturing for 24 to 96 hours, Breg cells were proliferated.

[0006] In some embodiments, the amino acid sequence of the N protein is as described in SEQ ID NO.1, the amino acid sequence of the S protein specific epitope peptide is as described in SEQ ID NO.2, and the amino acid sequence of the S1 subunit of the S protein containing the RBD region is as described in SEQ ID NO.3.

[0007] In some embodiments, the concentration of the CD40 antibody is 0.5~1.5 μg / mL.

[0008] In some implementations, the mammal is a human or a mouse.

[0009] In some embodiments, the B cells are derived from peripheral blood mononuclear cells or spleen B cells.

[0010] In some embodiments, the amount of the SARS-CoV-2 viral protein antigen used is 0.2~10 μg / mL.

[0011] In some embodiments, the Breg cells are CD19. + IL - 10 + Phenotype cells.

[0012] In some embodiments, the concentration of the CD40 antibody is 0.5~1.5 μg / mL.

[0013] The present invention also provides the application of the method in the preparation of Breg cell proliferation inducers.

[0014] In summary, compared with the prior art, the present invention achieves the following technical effects: 1. This invention uses a specific SARS-CoV-2 protein antigen to significantly increase the proportion of B cells transforming into Breg cells, achieving specific induction of Breg (CD19) cells under in vitro conditions. + IL-10 + Cell proliferation and differentiation.

[0015] 2. The present invention induces an increase in IL-10 secretion in Breg cells, accompanied by upregulation of anti-inflammatory factors (IL-10, TGF-β) and downregulation of pro-inflammatory factors (IFN-γ, TNF-α, etc.), which significantly enhances the immunomodulatory function of B cells. Attached Figure Description

[0016] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0017] Figure 1 This invention relates to the mechanism and technical route for B cell response to SARS-CoV-2 viral protein antigens. Figure 2 Example 1 of this invention illustrates how SARS-CoV-2 virus N and S proteins promote the proliferation of mouse B cells. A shows the dilution of B cells due to V450 proliferation after mouse spleen cells were stained with V450 and stimulated with different doses of N protein (NP) along with CD40 antibody for 72 hours. B shows the statistical results of the dilution of B cells due to V450 proliferation after mouse B cells were stained with V450 and stimulated with NP, SAG (S protein-specific epitope peptide), SRBD (S1 subunit of S protein containing RBD region) along with CD40 antibody for 48 hours. C represents representative flow cytometry results, under the same treatment conditions as B. Compared with Ctrl, P < 0.001.

[0018] Figure 3 Example 1 of this invention describes how SARS-CoV-2 virus N and S proteins induce an increase in the proportion of mouse B10 cells; A represents the proportion of mouse B cells in B cells after 48 hours of stimulation with NP, SAG (S protein-specific epitope peptide), SRBD (S1 subunit of S protein containing RBD region), and CD40 antibody; B represents the statistical results of Figure A. Compared with Ctrl, P < 0.01.

[0019] Figure 4 Example 1 of the present invention describes the effects of SARS-CoV-2 virus N and S proteins on B10 cells in human peripheral blood mononuclear cells.

[0020] Figure 5Example 2 of this invention illustrates the effect of SARS-CoV-2 viral N protein on the expression of important factors and receptors in B cells. Mouse B cells were stimulated with NP along with CD40 antibody and cultured for 48 hours. Total RNA was collected from the cells for RT-qPCR analysis. The vertical axis represents the difference in CT values ​​between the PCR normalized to the internal control and the control, while the upper part of the horizontal axis indicates upregulation of expression. Detailed Implementation

[0021] To enable those skilled in the art to better understand the present invention, the technical solutions in the embodiments of the present invention are clearly and completely described. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort should fall within the scope of protection of the present invention.

[0022] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, all materials and reagents used are commercially available.

[0023] Example 1: The role of SARS-CoV-2 viral N and S proteins in B cell antibody diversity 1. Effects of SARS-CoV-2 viral proteins on mouse B cell growth The company's synthesized SARS-CoV-2 viral N protein, SAG (S protein-specific epitope peptide), and SRBD (S1 subunit containing the RBD region) were used to treat 2×10⁻⁶ cells at different doses (0.2 µg / mL, 2 µg / mL). 6 / mL mouse spleen CD19 + B cells (containing 1 µg / mL CD40 antibody in the culture medium). After culturing for 43 hours, LPS (10 µg / mL), PMA (50 ng / mL), ionomycin (500 ng / mL), and monensin (2 µM) (L+PIM) were added. After culturing for another 5 hours, the cells were collected, and the growth of B cells was assessed by cell counting and viability testing.

[0024] like Figure 2 As shown, 2 µg / mL of SARS-CoV-2 virus N protein and S protein can promote the proliferation of mouse B cells and significantly accelerate the growth rate of B cells, suggesting that the two antigens have a significant role in regulating B cell activity.

[0025] 2. Effects of SARS-CoV-2 viral proteins on the proportion of B10 cells in mice 2 × 100 mouse spleens 6 / mL CD19 +B cells were cultured with CD40 antibody, and different doses (2µg / mL~4µg / mL) of N protein, S1 subunit of S protein containing RBD region, or S protein-specific epitope peptide were added. After 48 hours of culture, L+PIM was added, and the cells were cultured for another 5 hours. Flow cytometry was then used to detect B10 cells (CD40-positive cells). + IL - 10 + The proportion of B cells.

[0026] like Figure 3 As shown, both N and S proteins significantly increased the proportion of B10 cells in the spleen of mice, with significant differences compared to the control group, and the increase was greater than 50%, suggesting that the SARS-CoV-2 protein antigen can effectively induce the generation of B10 cells.

[0027] 3. Effects of SARS-CoV-2 viral proteins on human B10 and B / T cell ratio Take 2×10 6 / mL of healthy human peripheral blood mononuclear cells (PBMCs) were stimulated with N protein, SAG (S protein-specific epitope peptide), and SRBD (S1 subunit containing the RBD region) along with CD40 antibody and cultured for 48 hours. Subsequently, the CD19 activity of B10 cells was measured. + The proportion of B cells, cell viability, and CD19 + B cells and CD4 + The proportion of T cells in PBMCs.

[0028] like Figure 4 As shown, N and S proteins significantly increased the proportion of B10 cells in human PBMCs, with an increase of more than 60%, while also improving overall cell viability.

[0029] Example 2: Mechanism by which SARS-CoV-2 viral protein antigens affect B10 cell formation 1. Purified mouse spleen B cells (2×10⁻⁶) 6 Cells were cultured for 48 hours with 2 µg / mL SARS-CoV-2 viral N protein (NP) and 1 µg / mL CD40 antibody. Total RNA was extracted from the cells, and the transcriptional levels of key cytokines such as IL-6, IL-10, IL-1β, IFN-γ, TNF-α, and IL-12α were detected by RT-qPCR. The effect of N protein on the expression of B cell cytokines and receptors was analyzed.

[0030] The results are as follows Figure 5As shown, the effects of the SARS-CoV-2 virus N protein on important factors and receptors of B cells were significantly different, significantly increasing the expression of IL-6, IL-10 and IL-1β, but decreasing the expression of IFN-γ, TNF-α and IL-12α.

[0031] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

[0032] sequence list SEQ ID NO.1 MSDNGPQNQRNAPRITFGGPSDSTGSNQNGERSGARSKQRRPQGLPNNTASWFTALTQHGKEDLKFPRGQGVPINTNSSPDDQIGYYRRATRRIRGGDGKDLSPRWYFYYLGTGPEAGLPYGANKDGIIWVATEGALNTPKDHIGTRNPANNAAIVLQLPQGTTLPKGFYAEGSRGGSQASSRSSSRSRNSSRNSTPGSSRGTSPAR MAGNGGDAALALLLLDRLNQLESKMSGKGQQQQGQTVTKKSAAEASKKPRQKRTATKAYNVTQAFGRRGPEQTQGNFGDQELIRQGTDYKHWPQIAQFAPSASAF FGMSRIGMEVTPSGTWLTYTGAIKLDDKDPNFKDQVILLNKHIDAYKTFPPTEPKKDKKKKADETQALPQRQKKQQTVTLLPAAADLDDFSKQLQQSMSSADSTQA SEQ ID NO.2 SEQ ID NO.3 RVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNF

Claims

1. A method for inducing Breg cell proliferation in vitro, characterized in that, Includes the following steps: (1) B cells derived from mammals were cultured in a medium containing CD40 antibody; (2) Add SARS-CoV-2 viral antigen to the culture system, wherein the SARS-CoV-2 viral antigen is selected from one or more of the N protein, the S protein specific epitope peptide, or the S1 subunit of the S protein containing the RBD region; (3) After culturing for 24 to 96 hours, Breg cells were proliferated.

2. The method according to claim 1, characterized in that, The amino acid sequence of the N protein is as described in SEQ ID NO.1, the amino acid sequence of the specific epitope peptide of the S protein is as described in SEQ ID NO.2, and the amino acid sequence of the S1 subunit of the S protein containing the RBD region is as described in SEQ ID NO.

3.

3. The method according to claim 1, characterized in that, The concentration of the CD40 antibody is 0.5~1.5 μg / mL.

4. The method according to claim 1, characterized in that, The mammals referred to are humans or mice.

5. The method according to claim 1, characterized in that, The B cells are derived from peripheral blood mononuclear cells or spleen B cells.

6. The method according to claim 1, characterized in that, The dosage of the SARS-CoV-2 viral protein antigen is 0.2~10 μg / mL.

7. The method according to claim 1, characterized in that, The Breg cells are CD19. + IL - 10 + Phenotype cells.

8. The use of the method according to any one of claims 1 to 7 in the preparation of Breg cell proliferation inducers.

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