A method for inducing differentiation of memory B cells specifically and its application.

Abstract

This invention provides a method for specifically inducing the differentiation of memory B cells using blood. [Solution] The present invention relates to a method for inducing differentiation of memory B cells specifically and its application, and more specifically, to a method for inducing differentiation of memory B cells specifically, comprising the steps of treating a biological sample obtained from an individual with an anti-CD3 antibody or ligand and an anti-CD28 antibody or ligand, and to a method for detecting memory B cells specific to a particular antigen using the same.

Description

Technical Field

[0001] This application claims priority based on Korean Patent Application No. 10-2020-0008914, filed on January 22, 2020, and the entire specification thereof is a reference of this application.

[0002] It relates to a method for inducing differentiation specific to memory B cells and its utilization. More specifically, it relates to a method for inducing differentiation specific to memory B cells, which includes treating a biological sample obtained from an individual with an anti-CD3 antibody or ligand; and an anti-CD28 antibody or ligand, and a method for detecting memory B cells specific to a specific antigen using the same.

Background Art

[0003] When our body is attacked by pathogens such as viruses and bacteria, or experiences an antigen of a component that is not in our own body such as a vaccine, our body comes to have an immune memory process against these. This is called a memory response. In our body, when exposed again to pathogens or vaccine antigens by the formed memory, related immune responses occur more strongly and quickly. The decisive immune cells that lead such immune memory responses are B cells and T cells, which play important roles in acquired immunity. Therefore, by separating and analyzing these immune cells well, it becomes possible to measure the amount and quality of immune memory each human has, which of course serves as a criterion for judging what immune situations our body has had in the past, and also makes it possible to predict what immune defense mechanisms will operate when pathogens invade in the future.

[0004] However, in clinical practice, the complex and difficult limitations of cytodiagnostic techniques make it difficult for the majority of medical professionals to easily maintain high-level immunological information for each patient. Currently, the most widely used indirect method is to measure antibodies, which are thought to be products of immune memory cell activity, in the blood. However, this conventional method has the disadvantage that the degree of antibody response maintenance varies depending on the type of pathogen and the host, making it difficult to use during specific periods of disease when antibodies are not formed.

[0005] Methods for directly measuring immune memory cells also have significant limitations, primarily because immune memory cells exist in very small numbers within the body. Furthermore, even blood, which is useful for diagnosis, is mostly inhabited by immune organs (spleen, lymph nodes, intestines), requiring invasive sample acquisition techniques and complex, indirect imaging medical technologies.

[0006] On the other hand, according to Clinical and Experimental Immunology 177:333-340, while methods for treating cells with various differentiation factor cocktails are known for analyzing antigen-specific memory B cells, these methods have limitations in that they can induce differentiation in naive B cells that have never been exposed to the antigen, leading to nonspecific antigen-antibody reactions.

[0007] Therefore, there is a need for a method that avoids invasive methods requiring close access to immune organs, uses blood, which is the easiest to obtain, and specifically differentiates only memory B cells with great precision, thereby confirming the presence of memory B cells specific to a particular antigen. [Overview of the Initiative] [Problems that the invention aims to solve]

[0008] As a result of diligent research to develop a method for specifically inducing the differentiation of memory B cells using blood obtained by a non-invasive method, the inventors have obtained from an individual We discovered that treating a biological sample with anti-CD3 antibody or ligand and anti-CD28 antibody or ligand specifically induces differentiation of memory B cells, leading to the completion of the present invention.

[0009] Therefore, the object of the present invention is to provide a differentiation induction method specific to memory B cells, comprising the step of treating a biological sample obtained from an individual with an anti-CD3 antibody or ligand and an anti-CD28 antibody or ligand.

[0010] Another object of the present invention is to provide a method for detecting antigen-specific memory B cells, comprising the steps of (a) treating a biological sample obtained from an individual with an anti-CD3 antibody or ligand and an anti-CD28 antibody or ligand; (b) treating the biological sample with an antigen; and (c) detecting an antibody specifically bound to the antigen.

[0011] Another object of the present invention is to provide a differentiation induction composition specific to memory B cells, comprising an anti-CD3 antibody or ligand and an anti-CD28 antibody or ligand.

[0012] Another object of the present invention is to provide a differentiation induction composition specific to memory B cells, comprising an anti-CD3 antibody or ligand and an anti-CD28 antibody or ligand.

[0013] Another object of the present invention is to provide a differentiation-inducing composition specific to memory B cells, comprising a substance from an anti-CD3 antibody or ligand and an anti-CD28 antibody or ligand.

[0014] Another object of the present invention is to provide the use of anti-CD3 antibodies or ligands and anti-CD28 antibodies or ligands for preparing differentiation-inducing agents specific to memory B cells. [Means for solving the problem]

[0015] To achieve the aforementioned objectives of the present invention, the present invention provides a method for inducing differentiation of memory B cells, comprising the step of treating a biological sample obtained from an individual with an anti-CD3 antibody or ligand and an anti-CD28 antibody or ligand.

[0016] To achieve another objective of the present invention, the present invention provides a method for detecting antigen-specific memory B cells, comprising the steps of (a) treating a biological sample obtained from an individual with an anti-CD3 antibody or ligand and an anti-CD28 antibody or ligand; (b) treating the biological sample with an antigen; and (c) detecting an antibody specifically bound to the antigen.

[0017] To achieve another objective of the present invention, the present invention provides a differentiation-inducing composition specific to memory B cells, comprising an anti-CD3 antibody or ligand and an anti-CD28 antibody or ligand.

[0018] Furthermore, the present invention provides a differentiation induction composition specific to memory B cells, comprising an anti-CD3 antibody or ligand and an anti-CD28 antibody or ligand.

[0019] Furthermore, the present invention provides a differentiation induction composition specific to memory B cells, comprising a parenchyma from an anti-CD3 antibody or ligand and an anti-CD28 antibody or ligand.

[0020] To achieve another objective of the present invention, the present invention provides the use of anti-CD3 antibodies or ligands and anti-CD28 antibodies or ligands for preparing formulations for differentiation induction specific to memory B cells.

[0021] The present invention will be described in detail below.

[0022] According to one embodiment of the present invention, it was confirmed that when PBMCs derived from blood obtained from an individual were treated with an anti-CD3 antibody or ligand and an anti-CD28 antibody or ligand, naive B cell differentiation was not induced, but differentiation specific to memory B cells was induced.

[0023] Thus, the present invention provides a method for inducing differentiation specific to memory B cells, which includes a step of treating a biological sample obtained from an individual with an anti-CD3 antibody or ligand and an anti-CD28 antibody or ligand.

[0024] In the present invention, the “individual” is not particularly limited in terms of its type, and may be, for example, a human, ape, dog, cat, rabbit, guinea pig, rat, mouse, cow, sheep, pig or goat, and preferably may be a human.

[0025] In particular, the individual may be a subject for analyzing whether it has a history of past infection with bacteria or virus.

[0026] In the present invention, the “biological sample” is not particularly limited in terms of its type as long as it is a sample obtained from the individual, and may be selected, for example, from the group consisting of whole blood, plasma, serum, peripheral blood mononuclear cells (PBMC), urine, feces, saliva, sputum, sweat, tears, tissues and combinations thereof. Preferably, it may be selected from the group consisting of whole blood, plasma, serum, PBMC and combinations thereof, and most preferably may be PBMC.

[0027] In the present invention, the “anti-CD3 antibody” or “anti-CD28 antibody” means a specific protein molecule directed against the antigenic site of CD3 or CD28 expressed on the surface of CD4+ T cells. For the purpose of the present invention, the antibody means an antibody that specifically binds to CD3 or CD28 and activates CD4+ T cells, and includes all polyclonal antibodies, monoclonal antibodies and recombinant antibodies.

[0028] The antibody of the present invention includes functional fragments of the antibody molecule in addition to the complete form having two full-length light chains and two full-length heavy chains. The functional fragment of the antibody molecule means a fragment that retains at least the antigen-binding function, and includes, but is not limited to, Fab, F(ab’), F(ab’)2 and Fv.

[0029] In the present invention, the term "ligand" means any type of molecule capable of binding to CD3 or CD28. Preferably, the ligand means any type of molecule that binds to CD3 or CD28 and activates CD4+ T cells.

[0030] In the present invention, the term "T cell" refers to a T lymphocyte as defined in the art, and may include thymocytes, immature T lymphocytes, mature T lymphocytes, quiescent T lymphocytes, or activated T lymphocytes.

[0031] In the present invention, "CD4+ T cells" refer to a subset of T cells that express CD4 on their surface and are associated with cell-mediated immune responses. They are characterized by a post-stimulation secretion profile, which may include the secretion of cytokines such as IFN-γ, TNF-α, IL-2, IL-4, and IL-10.

[0032] In the present invention, "specific" means that, among the various immune cells present in a biological sample, particularly naive B cells and memory B cells, it induces the differentiation of only memory B cells.

[0033] In the present invention, the "memory B cell" refers to one of the B cell subtypes that are used to exchange with memory B cells and are formed in germinal centers after primary infection. The memory B cell accelerates the immune response when secondary infection occurs with the same pathogen and generates a potent antibody-mediated immune response.

[0034] In this invention, the term "naive B cell" refers to a B cell that has not been exposed to an antigen. When a naive B cell is exposed to an antigen, it differentiates into memory B cells and plasma cells that secrete antibodies.

[0035] In the present invention, "specific differentiation induction of memory B cells" means converting memory B cells that do not secrete antibodies into plasma cells that secrete antibodies against specific antigens. When memory B cells differentiate into plasma cells, they express IgG and IgM that respond to specific antigens programmed in their respective memories.

[0036] On the other hand, according to one embodiment of the present invention, it was confirmed that the differentiation of memory B cells is significantly improved by further removing CD8+ T cells from a biological sample obtained from an individual before treating the sample with an anti-CD3 antibody or ligand and an anti-CD28 antibody or ligand.

[0037] Therefore, the method provided by the present invention may further be characterized by removing CD8+ T cells from the biological sample before treating the biological sample obtained from an individual with an anti-CD3 antibody or ligand and an anti-CD28 antibody or ligand.

[0038] In this invention, the term "CD8+ T cells" refers to a subpopulation of T cells that express CD8 on their surface, are MHC class I-restrictive, and act as cytotoxic T cells. The "CD8" molecule is a differentiation antigen found on thymocytes and on cytotoxic and suppressive T lymphocytes. The CD8 antigen is a member of the immunoglobulin supergene lineage and is an associative recognition element in the interaction of major histocompatibility complex class I restriction.

[0039] There are no particular limitations on the method for removing the CD8+ T cells in the present invention, but they may be removed by methods such as flow cytometry, FACS (fluorescence activated cell sorting), magnetic beads, or columns. The separated PBMCs may be treated with antibodies or beads that react with CD8 molecules on the cell surface, and the cells may be selectively removed using FACS or columns.

[0040] Furthermore, the present invention provides a method for detecting antigen-specific memory B cells, comprising the steps of (a) treating a biological sample obtained from an individual with an anti-CD3 antibody or ligand and an anti-CD28 antibody or ligand; (b) treating the biological sample with an antigen; and (c) detecting an antibody specifically bound to the antigen.

[0041] The present invention relates to a method for detecting whether there are antibodies that specifically recognize a particular antigen among the antibodies secreted by differentiated memory B cells, using the aforementioned differentiation induction method for memory B cells.

[0042] Since memory B cells present in a biological sample obtained from an individual are formed after the individual has been exposed to a specific antigen in the past, in step (a), by differentiating the memory B cells present in the biological sample into plasma cells that secrete antibodies and reacting them with the specific antigen, it is possible to confirm whether memory B cells for the specific antigen are present in the biological sample. In other words, the individual has been exposed to bacteria containing the antigen in the past... Alternatively, confirming whether a patient has previously been infected with the virus can sometimes help establish a rapid treatment strategy.

[0043] The aforementioned step (a) can be understood similarly by referring to the explanation above.

[0044] Step (b) in the present invention is a step in which an antigen-antibody reaction is induced between the antibody secreted by memory B cells that have differentiated into antibody-secreting plasma cells in step (a) and the target antigen.

[0045] In step (b) of the present invention, the term "antigen" is not particularly limited in type, as long as it is expressed by a pathogenic substance, more specifically, by bacteria or viruses.

[0046] The antigen may include peptides, proteins, nucleic acids, sugars, pathogens, attenuated pathogens, inactivated pathogens, viruses, virus-like particles (VLPs), cells, or cell fragments.

[0047] Non-limiting examples of the aforementioned "antigen" include tuberculosis antigen, anthrax antigen, HAV (Hepatitis A virus) antigen, HBV (Hepatitis B virus) antigen, HCV (Hepatitis C virus) antigen, HIV (human immunodeficiency virus) antigen, influenza virus antigen, HSV (Herpes simplex virus) antigen, Hib (Haemophilus influenzae type b) antigen, Neisseria meningitidis antigen, Corynebacterium diphtheria antigen, Bordetella pertussis antigen, Clostridium tetani antigen, HPV (human papilloma virus) antigen, Varicella virus, Enterococcus antigen, Staphylococcus aureus antigen, and Klebsiella pneumoniae antigen. Antigens of pneumonia, antigens of Acinetobacter baumannii, antigens of Pseudomonas aeruginosa, antigens of Enterobacter, and Helicobacter pylori It may also contain antigens of pylori, malaria, dengue virus, MERS virus, Zika virus, Orientia tsutsugamushi, severe fever with thrombocytopenia syndrome (SFTS) bunyavirus, Japanese encephalitis virus, severe acute respitaroty syndrome-coronavirus (SARS-CoV), severe acute respitaroty syndrome-coronavirus (SARS-CoV-2), Ebola virus, hepatitis C virus, hepatitis B virus, acute respiratory syndrome virus, West Nile virus, stomatitis vesiculosum virus, Newcastle disease virus, or pneumococcus.

[0048] Step (c) is a step to confirm whether an antigen-antibody reaction has occurred between the antigen treated in step (b) and the antibodies secreted by memory B cells differentiated from the biological sample.

[0049] In step (c) above, the method for detecting the presence or absence of an antigen-antibody reaction can be any method known in the industry without limitation, and non-limiting examples may include ELISPOT, ELISA, competitive ELISA, magnetic beads, or immunochromatographic assays, preferably the method for detecting the presence or absence of an antigen-antibody reaction may be ELISPOT.

[0050] If an antibody specifically bound to the antigen is detected in step (c) above, it can be determined that the individual has a history of infection with a pathogenic substance that expresses the antigen, more specifically, with bacteria or a virus.

[0051] Furthermore, the present invention provides a differentiation induction composition specific to memory B cells, comprising an anti-CD3 antibody or ligand and an anti-CD28 antibody or ligand.

[0052] In addition to anti-CD3 antibodies or ligands and anti-CD28 antibodies or ligands, the compositions of the present invention may further include factors necessary for the survival of memory B cells, for example, a suitable medium (i.e., Minimal Essential Medium (Media) or RPMI Medium (Media) 1640, or X-vivo 5, (Lonza)) which may contain serum (i.e., fetal bovine or human serum), cytokines, or any other additives for the growth of cells known to those skilled in the art. Other additives for the growth of cells include, but are not limited to, surfactants, plasmamenates, and reducing agents such as N-acetylcysteine ​​and 2-mercaptoethanol. The culture medium may contain, but is not limited to, RPMI 1640, A1M-V, DMEM, MEM, a-MEM, F-12, X-Vivo 1, and added amino acids, along with Optimizer, X-Vivo 20, sodium pyruvate, and vitamins, serum-free, or supplemented with an appropriate amount of serum (or plasma), or a defined set of hormones and / or sufficient amounts of cytokines for the growth and expansion of T cells.

[0053] The present invention provides the use of anti-CD3 antibodies or ligands and anti-CD28 antibodies or ligands for preparing differentiation-inducing formulations specific to memory B cells.

[0054] In this invention, the term "comprising" is used interchangeably with "containing" or "characterizing" and does not exclude any additional components or process steps not mentioned in the composition or method. The term "consisting of" means excluding any additional elements, steps, or components not otherwise specified. The term "essentially consisting of" means, within the scope of the composition or method, including any components or steps not substantially affecting its basic properties, in addition to the described components or steps. [Effects of the Invention]

[0055] According to the method of the present invention, only memory B cells present in a biological sample can be specifically differentiated, and the presence or absence of a history of infection with a pathogenic substance containing a specific antigen in the individual that provided the biological sample can be analyzed very accurately and rapidly. [Brief explanation of the drawing]

[0056] [Figure 1] Figure 1 shows the results of detecting secreted IgM and IgG using ELISPOT after treating isolated PBMCs with anti-CD3 antibody and anti-CD8 antibody (immunoreceptor activation treatment group: anti-CD3 antibody and anti-CD8 antibody treatment group). [Figure 2] Figure 2 shows the results of detecting secreted IgM and IgG using ELISPOT after removing CD4+ T cells or CD8+ T cells from isolated PBMCs and treating them with anti-CD3 and anti-CD8 antibodies. [Figure 3a] , [Figure 3b] Figures 3a and 3b show the results of detecting secreted IgM and IgG using ELISPOT after removing IgD or IgG from PBMCs from which CD8+ T cells had been removed, followed by treatment with anti-CD3 and anti-CD8 antibodies. [Figure 4]Figure 4 shows the results of isolating naive B cells from PBMCs, treating them with the polyclonal activators TLR7 / 8 agonist (R848) and TLR9 agonist (CPG) at 1 μg / ml, and then measuring the amount of antibodies using the ELISPOT experiment. [Figure 5] Figure 5 shows the results of isolating PBMCs from the blood of monkeys with or without experience of exposure to the MELS antigen, removing CD8+ T cells, and then treating them with (i) anti-CD3 antibody and anti-CD8 antibody (immunoreceptor activation group), (ii) TLR7 / 8 agonist (R848), or (iii) TLR9 agonist (CPG), and confirming the presence or absence of IgM and IgG secretion using ELISPOT (immunoreceptor activation group: anti-CD3 antibody and anti-CD8 antibody treatment group, TLR agonist 1: TLR7 / 8 agonist (R848), TLR agonist 2: TLR9 agonist (CPG)). [Figure 6a] , [Figure 6b] Figures 6a and 6b show the results of isolating PBMCs from the blood of monkeys that had or had not been exposed to dengue virus antigen (Figure 6a) or coronavirus virus antigen (Figure 6b), removing CD8+ T cells, treating them with anti-CD3 antibody and anti-CD8 antibody (immunoreceptor activation group), and confirming the presence or absence of IgM and IgG secretion using ELISPOT (immunoreceptor activation group: anti-CD3 antibody and anti-CD8 antibody treatment group). [Modes for carrying out the invention]

[0057] The present invention will be described in detail below with reference to the following examples. However, the following examples are for illustrative purposes only and do not limit the present invention.

[0058] Example 1: Isolation of PBMCs (peripheral blood mononuclear cells) Blood was isolated from normal human and non-human primates. After mixing 1×PBS with the blood in an EDTA tube, the layers were slowly separated into 4 ml of Ficoll-Hypaque (Lymphoprep; Axis-Shield, 1114545). After slow centrifugation at 4000 rpm for 20 minutes, the white band containing PBMCs was collected using a pipette once the layers separated. 1×PBS was added again and centrifugation was performed at 1500 rpm for 5 minutes, and the supernatant was removed. After this, 1 ml of culture medium was added to loosen the cells. The cell count was calculated using an Olympus R1 automated cell counter.

[0059] Example 2: Differentiation induction of memory B cells by treatment with anti-CD3 antibody or ligand and anti-CD28 antibody or ligand

[0060] (1) Measurement of antibody secretion by treatment of PBMCs with anti-CD3 antibody or ligand and anti-CD28 antibody or ligand. The PBMCs isolated in Example 1 were treated with an anti-CD3 antibody or ligand, and an anti-CD28 antibody or ligand. The PBMCs (1 × 10⁶) separated in Example 1 were placed in a 96-well plate coated with 1 μg / ml of anti-CD3 antibody (Clone OKT3, Biolegend Cat. No. 317302), and the PBMCs were further separated in Example 1. 5 Cells were placed in each well and treated with 1 μg / ml of anti-CD28 antibody (Clone CD28.2, BD Cat. No. 555725). The cells were then incubated at 37°C in a 5% CO2 incubator for 3-4 days.

[0061] Since the antibodies secreted from memory B cells consist of IgG and IgM types, the amounts of IgM and IgG secreted from the PBMC sample were then measured using the ELISPOT method.

[0062] Specifically, 50 µl each of anti-IgG and IgM antibodies at 10 μg / ml were placed in plates and coated at 4°C for 24 hours. PBMCs stimulated with anti-CD3 and anti-CD28 antibodies were placed in the coated plates and reacted at 37°C in a 5% CO2 incubator for 4 hours. Subsequently, anti-IgG-FITC antibody and IgM-Biotin antibody (Southern Biotech) were reacted at room temperature for 2 hours, and FITC-HRP and Strep-AP (CTL) were reacted at room temperature for 1 hour. Finally, a blue reducing agent was added, and after reacting at room temperature for 15 minutes, IgM and IgG levels were measured.

[0063] The results for this are shown in Figure 1. As shown in Figure 1, treatment of PBMCs with anti-CD3 antibody or ligand and anti-CD28 antibody or ligand significantly increased the secretion of IgG and IgM antibodies compared to PBMCs that were not treated with anti-CD3 antibody or ligand and anti-CD28 antibody or ligand. In other words, it can be expected that treatment with anti-CD3 antibody or ligand and anti-CD28 antibody or ligand induced the differentiation of memory B cells, and that the secretion of IgG and IgM in the differentiated B cells increased.

[0064] (2) Differentiation induction of memory B cells in PBMCs from which CD4+ T cells or CD8+ T cells have been removed In experiment (1) described above, to optimize the conditions for inducing differentiation of memory B cells when treated with anti-CD3 antibody or ligand and anti-CD28 antibody or ligand, CD4+ T cells or CD8+ T cells were removed from PBMCs, and then treated with anti-CD3 antibody or ligand and anti-CD28 antibody or ligand in the same manner as in the experiment described above. CD4+ T cells or CD8+ T cells were removed from PBMCs using the following methods, respectively.

[0065] Specifically, PBMC-CD4 T cells (PBMCs from which CD4+ T cells have been removed) and PBMC-CD8 T cells (PBMCs from which CD8+ T cells have been removed) were separated using anti-CD4 microbead kits and anti-CD8 microbead kits (Miltenyi Biotech, Auburn, Calif.). PBMC(1×10) 7 Add 80 μl of buffer (0.5% BSA and 2 mM EDTA in PBS) to the cells, add 20 μl of CD4 microbeads or CD8 microbeads, mix well, and react at 4°C for 15 minutes. Stop the reaction, After centrifugation at 1500 rpm for 5 minutes, PBMC-CD4 T cells or PBMC-CD8 T cells that cannot be attached to a magnet were obtained using 10 ml of buffer and an LS MACS column (Miltenyi Biotech, Auburn, Calif).

[0066] The results for this are shown in Figure 2. As shown in Figure 2, PBMCs from which CD4+ T cells were removed did not secrete any antibodies even when treated with anti-CD3 antibody or ligand and anti-CD28 antibody or ligand. However, PBMCs from which CD8+ T cells were removed showed a significantly increased amount of antibody secretion compared to PBMCs when treated with anti-CD3 antibody or ligand and anti-CD28 antibody or ligand.

[0067] Therefore, the following experiments were performed using PBMCs from which CD8+ T cells had been removed.

[0068] (3) Confirmation of whether the differentiation induction is specific to memory B cells. In conventional techniques, the substances used to induce differentiation of memory B cells are polyclonal activators. Therefore, they differentiate not only memory B cells but also naive B cells, making it impossible to specifically differentiate only B cells that possess actual immune memory against a particular antigen.

[0069] Therefore, the inventors confirmed whether a method of treating PBMCs with an anti-CD3 antibody or ligand and an anti-CD28 antibody or ligand specifically differentiates only memory B cells and not naive B cells.

[0070] Specifically, cells showing IgD and IgG from the cell surface corresponding to PBMCs were removed, and the remaining cells were isolated using BD FACS Aria II. Subsequently, the isolated cells (1X10) were placed in a 96-well plate coated the previous day with 1 μg / ml of anti-CD3 antibody (Clone OKT3, Biolegend Cat. No. 317302). 5 Cells were placed in each well, treated with 1 μg / ml of anti-CD28 antibody (Clone CD 28.2, BD Cat. No. 555725), and then incubated at 37°C for 3-4 days in 5% solution. After reacting in a CO2 incubator, the amount of antibody was measured using an ELISPOT experiment.

[0071] The results for this are shown in Figures 3a and 3b. As confirmed in Figure 3a, in PBMCs from which cells expressing IgD on the surface were removed, treatment with anti-CD3 antibody or ligand and anti-CD28 antibody or ligand still increased the secretion of IgM and IgG antibodies. On the other hand, as confirmed in Figure 3B, in PBMCs from which cells expressing IgG on the surface were removed, treatment with anti-CD3 antibody or ligand and anti-CD28 antibody or ligand did not increase the secretion of IgM and IgG antibodies at all.

[0072] In other words, naive B cells expressing IgD on their surface did not differentiate into antibody-secreting cells even when treated with anti-CD3 antibody or ligand and anti-CD28 antibody or ligand, while only memory B cells expressing IgG on their surface were differentiated by treatment with anti-CD3 antibody or ligand and anti-CD28 antibody or ligand.

[0073] The polyclonal activator, a substance used in previous studies, differentiates naive B cells that have not experienced the antigen and produces surface IgM antibodies that are not associated with memory of the specific antigen.

[0074] Therefore, the inventors confirmed the presence or absence of such nonspecific reactions by treating PBMCs with TLR agonists, which are typically known as polyclonal activators.

[0075] Specifically, IgD+CD27- cells, which exhibit naive B cell characteristics on the surface of cells corresponding to PBMCs, were isolated using BD FACS Aria II. Subsequently, the isolated cells (1 × 10⁶) were placed in a 96-well plate. 4 Cells were placed in each well and treated with 1 μg / ml of TLR7 / 8 agonist (R848) and TLR9 agonist (CPG). After incubation for 3-4 days at 37°C in a 5% CO2 incubator, the amount of antibody was measured using an ELISPOT experiment.

[0076] The results for this are shown in Figure 4. As shown in Figure 4, a significant increase in IgM secretion was observed in naive B cells treated with the TLR7 / 8 agonist (R848) and the TLR9 agonist (CPG). This confirms that polyclonal activators can induce differentiation even in naive B cells that are not associated with antigen-specific memory.

[0077] Example 3: Detection of memory B cells specific to a particular antigen After establishing a method for specifically differentiating only memory B cells in PBMCs using the methods described in Examples 1 and 2, the following evaluation was performed to determine whether the biological samples isolated from actual subjects had been exposed to a specific antigen using the aforementioned method.

[0078] Specifically, PBMCs (5 × 10¹³) of monkeys that had undergone a third inoculation into the thigh muscle with the MERS virus antigen protein and adjuvant were isolated. By the method of the present invention, the monkey PBMCs (5 × 10¹³) isolated in a 96-well plate coated the previous day were treated with 1 μg / ml of anti-CD3 antibody (Clone SP34, BD Cat. No. 557052). 5 Cells were placed in wells and treated with 1 μg / ml of anti-CD28 antibody (Clone CD28.2, BD Cat. No. 555725), then incubated at 37°C for 3-4 days in a 5% CO2 incubator. Alternatively, instead of anti-CD3 and anti-CD28 antibodies, the isolated monkey PBMCs were treated with a polyclonal activator, such as a TLR7 / 8 agonist (R848, TLR agonist 1) or a TLR9 agonist (CP After treatment with G, TLR agonist 2), the mixture was reacted in a 37°C, 5% CO2 incubator for 3-4 days. Subsequently, ELISPOT was performed after coating the cells with a protein specific to MERS.

[0079] The results for this are shown in Figure 5. As can be seen in Figure 5, among the PBMCs of monkeys in which memory B cell differentiation was induced by the method of the present invention (immunoreceptor activation treatment group), IgG (red numbers) and IgM (blue numbers), which specifically recognize the MERS antigen, were accurately detected in the PBMCs of two monkeys that had been exposed to MERS virus, while they were not detected in the PBMCs of two monkeys that had not been exposed to MERS virus. On the other hand, in the PBMCs of monkeys in which memory B cell differentiation was induced by the method of the present invention, the secretion of IgG-type antibodies that specifically recognize the MERS antigen was detected at a significantly higher level than in the groups treated with the polyclonal activator TLR7 / 8 agonist (R848, TLR agonist 1) or TLR9 agonist (CPG, TLR agonist 2).

[0080] Furthermore, experiments were conducted using PBMCs (Patient-Based Microorganisms) of monkeys that had undergone a third inoculation into the thigh muscle using the same method as described above, and monkeys infected with the coronavirus (Severe acute respiratory syndrome-coronavirus; SARS-CoV-2). The results showed that in PBMCs of monkeys that had been exposed to each viral antigen, IgG (red numbers) and IgM (blue numbers) that specifically recognize dengue virus antigen (a) or coronavirus antigen (b) were accurately detected, while antibodies were not detected in PBMCs of monkeys that had not been exposed to each viral antigen. [Industrial applicability]

[0081] According to the method of the present invention, only memory B cells present in a biological sample can be specifically differentiated, and the presence or absence of a history of infection with a pathogenic substance containing a specific antigen in the individual that provided the biological sample can be analyzed with great accuracy and speed, making it highly applicable to industry.

Claims

1. A method for inducing differentiation of memory B cells, comprising the step of treating peripheral blood mononuclear cells (PBMCs) obtained from an individual with anti-CD3 antibody and anti-CD28 antibody.

2. The method according to claim 1, further comprising the step of removing CD8+ T cells from PBMCs obtained from an individual before treating the PBMCs with an anti-CD3 antibody and an anti-CD28 antibody.

3. The method according to claim 1, characterized in that the memory B cells are differentiated and secrete IgG and IgM.

4. (a) A step of treating peripheral blood mononuclear cells (PBMCs) obtained from an individual with anti-CD3 antibody and anti-CD28 antibody; (b) the step of treating the PBMC with an antigen; and (c) A method for detecting antigen-specific memory B cells, comprising the step of detecting an antibody specifically bound to the antigen.

5. The method according to claim 4, further comprising the step of removing CD8+ T cells from the PBMCs obtained from the individual before treating the PBMCs with anti-CD3 antibody and anti-CD28 antibody in step (a).

6. The aforementioned antigens include Mycobacterium tuberculosis antigen, Bacillus anthrax antigen, HAV (Hepatitis A virus) antigen, HBV (Hepatitis B virus) antigen, HCV (Hepatitis C virus) antigen, HIV (human immunodeficiency virus) antigen, influenza virus antigen, HSV (Herpes simplex virus) antigen, Hib (Haemophilus influenzae type b) antigen, Neisseria meningitidis antigen, Corynebacterium diphtheria antigen, Bordetella pertussis antigen, Clostridium tetani antigen, HPV (human papilloma virus) antigen, Varicella virus, Enterococcus antigen, Staphylococcus aureus antigen, and Klebsiella pneumoniae antigen. Antigens of pneumonia, Acinetobacter baumannii, Pseudomonas aeruginosa, Enterobacter, Helicobacter pylori, malaria, dengue virus, MERS virus, Zika virus, Orientia tsutsugamushi, severe fever with thrombocytopenia syndrome (SFTS) bunyavirus, Japanese encephalitis virus, and SAS coronavirus (severe acute respirator). The method according to claim 4, characterized in that it is one or more antigens selected from the group consisting of the antigen of SARS-CoV syndrome virus, SARS-CoV-2 (severe acute respiroty syndrome-corona virus), Ebola virus, hepatitis C virus, hepatitis B virus, acute respiratory syndrome virus, West Nile virus, stomatitis vesiculosum virus, Newcastle disease virus, and pneumococcus.

7. The method according to claim 4, characterized in that the detection of antigen-specifically bound antibodies in step (c) is performed by a plurality of methods selected from the group consisting of ELISPOT, ELISA, competitive ELISA, magnetic beads, and immunochromatographic assay.

8. If an antibody specifically bound to the antigen is detected in step (c), then step (c) The method according to claim 4, further comprising the step of using the results of the first step as a reference for determining whether the individual has a history of infection with bacteria or viruses that express the antigen.

9. A composition for inducing differentiation into memory B cells, containing anti-CD3 antibody and anti-CD28 antibody.

10. Use of anti-CD3 and anti-CD28 antibodies for the production of differentiation-inducing formulations for memory B cells.

Images