Use of monoclonal antibody 2f5 in autoimmune diseases and regulation of b cells

Abstract

The application discloses application of monoclonal antibody 2F5 in autoimmune diseases and regulation of B cells, wherein the monoclonal antibody 2F5 is obtained from peripheral blood of SLE, and it is first proved that the monoclonal antibody 2F5 can promote B cell proliferation, activation and differentiation through primary cell and cell line experiment models. The antibody provided by the application is completely humanized, has clear targeting, can well simulate a blood microenvironment of high-titer autoantibodies in peripheral blood of an autoimmune disease patient, is an ideal tool for understanding biological functions of the autoantibodies and studying regulation of B cell activation and differentiation by the autoantibodies, and is helpful to deepen understanding of mechanisms of immune disorders, to make it possible to treat immunopathological diseases caused by abnormal differentiation of B cells, and to lay an important foundation for developing a new target for treating autoimmune diseases and tumors by targeting pathogenic B cells.

Description

Technical Field

[0001] The present invention relates to the technical field of monoclonal antibody preparation and immunological detection, and in particular to the application of monoclonal antibody 2F5 in autoimmune diseases and regulating B cells. Background Art

[0002] Autoimmune diseases (AIDs) are a class of diseases caused by the immune system's erroneous response to self-antigens. This pathological condition results in the immune system attacking the body's own tissues, organs, or cellular components, ultimately causing organ damage or dysfunction. In recent years, the incidence of various autoimmune diseases has been on the rise, particularly among young and middle-aged women, and has become a leading cause of death. Currently, there are nearly 100 known autoimmune diseases, among the more common of which include systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), primary Sjögren's syndrome (pSS), and Graves' disease (GD).

[0003] B cells are the primary cells of humoral immunity, achieving immune responses by producing memory B cells that rapidly respond to subsequent antigen exposure and plasma cells that continuously secrete antibodies. These B cell subsets maintain long-term humoral immunity and host protection, preventing recurrent infection. Abnormal B cell activation and differentiation play a key role in the pathogenesis of autoimmune diseases and are a significant factor in the increased overall cancer risk in patients with autoimmune diseases. B cells participate directly or indirectly in the autoimmune process by presenting antigens to T cells and producing proinflammatory cytokines or autoantibodies. Abnormal B cell activation may be associated with factors such as the breakdown of immune tolerance and an imbalance in the Th1 / Th2 cell balance. It is also influenced by imbalances in the regulation of signaling molecules within B cells, such as Toll-like receptors (TLRs). CD11c is present in healthy individuals, patients with autoimmune diseases, and (chronic) infections. + Tbet + B cells have upregulated genes related to antibody secreting cell (ASC) differentiation. Mouse experiments showed that CD11c + Tbet + B cells are involved in preventing chronic viral infection. In healthy individuals, CD11c is highly expressed in + Tbet +B cells are primed for ASC differentiation and, when cultured in the presence of B cell antigen receptor ligation, TLR9 ligands, and interleukin-21, secrete increased levels of IgM and IgG. These cells can function as a separate germinal center (GC)-independent memory-like population and participate in protective immunity against infection or immunization. Autoantibodies are a double-edged sword, playing a role in both protective immunity against infection and in the development of autoimmune diseases. However, although high titers of autoantibodies in the blood are a hallmark of many autoimmune diseases, their specific impact on B cell differentiation remains unclear.

[0004] The current method of using cytokines (such as interleukin-21) to regulate B cells has a small effective concentration window of cytokines and is prone to toxic side effects; while the method of using genome modification technology (such as knocking out the transcription factor Pax5) to regulate B cells has high technical requirements and has the risk of "off-target"; therefore, a simple and quick method is urgently needed to regulate them. Summary of the Invention

[0005] The first objective of the present invention is to address the deficiencies of the prior art and provide the use of monoclonal antibody 2F5 in the preparation of a drug for treating autoimmune diseases. The monoclonal antibody 2F5 can specifically bind to the DEAD box helicase 5 (DDX5) protein and is an autoantibody, that is, an antibody against the body's own tissues, organs, cells and their components.

[0006] In order to achieve the above object, the present invention adopts the following technical solutions:

[0007] Use of monoclonal antibody 2F5 in the preparation of a medicament for treating autoimmune diseases caused by abnormal B cell differentiation. In monoclonal antibody 2F5, the complementarity determining regions (CDR1, CDR2, and CDR3) of the light chain 2F5 are represented by LCDR1, LCDR2, and LCDR3, respectively; the LCDR1 sequence is QSVSSN, the LCDR2 sequence is GAS, and the LCDR3 sequence is QQYNNWPRT. The complementarity determining regions (CDR1, CDR2, and CDR3) of the heavy chain 2F5 are represented by HCDR1, HCDR2, and HCDR3, respectively; the HCDR1 sequence is GFTFSDYS, the HCDR2 sequence is ITSSSGYT, and the HCDR3 sequence is ARVRSSWGPIDS.

[0008] Preferably, the monoclonal antibody 2F5 promotes B cell proliferation, activation and differentiation.

[0009] The second object of the present invention is to provide the use of the monoclonal antibody 2F5 in the preparation of a preparation for promoting B cell proliferation, activation or differentiation.

[0010] Preferably, the B cells include RAMOS, OCI-LY-19 and primary B cells.

[0011] A third object of the present invention is to provide a method for specifically inducing B cell proliferation, activation or differentiation for non-diagnostic and non-therapeutic purposes, the method comprising the following steps:

[0012] The purified monoclonal antibody 2F5 was diluted to prepare a 2F5 antibody diluent, and then the diluted solution was used to treat B cells.

[0013] Preferably, the B cells include RAMOS, OCI-LY-19 and primary B cells.

[0014] A fourth object of the present invention is to provide a composition for treating autoimmune diseases, characterized in that the composition comprises one or more of the following ingredients:

[0015] (a) Monoclonal antibody 2F5;

[0016] (b) nucleic acid encoding monoclonal antibody 2F5;

[0017] (c) Expression vector encoding the monoclonal antibody 2F5 nucleic acid.

[0018] Compared with the prior art, the present invention has the following beneficial effects:

[0019] This study utilizes the monoclonal autoantibody 2F5, obtained from SLE peripheral blood, to demonstrate for the first time its ability to promote B cell proliferation, activation, and differentiation in primary and cell line experimental models, expanding its application in immune regulation. This fully humanized, well-targeted antibody can closely mimic the blood microenvironment of high-titer autoantibodies in the peripheral blood of patients with autoimmune diseases, making it an ideal tool for clarifying the biological functions of autoantibodies and studying their regulation of B cell activation and differentiation.

[0020] The method of the monoclonal antibody 2F5 targeted stimulation of B cells in the present invention induces the differentiation of specific B cell subsets, reduces the impact on non-target cells, has strong specificity, high controllability, better safety and economy, and antibody-mediated B cell activation can occur rapidly, providing the possibility of rapid induction of immune responses. The present invention achieves precise regulation of B cell differentiation, making therapeutic intervention in immunopathological diseases caused by abnormal B cell differentiation possible, and lays an important foundation for the development of new targets targeting pathogenic B cells to treat autoimmune diseases and tumors. At the same time, a small amount of the correct antibody can also significantly enhance the responsiveness of low-affinity B cells, thereby generating high-affinity antibodies. Therefore, the precise regulation of B cell responses of the present invention is of great significance for vaccine design, improving vaccine effectiveness, and blocking the spread of infectious diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 Figure 2 shows the effects of 2F5 antibody on the proliferation of PBMCs and B cell lines. A: 2F5 antibody promoted PBMC proliferation, with the optimal proliferation time being 48 hours; B: Both 2F5 supernatant and 2F5 antibody promoted OCI-LY-19 cell proliferation, with the optimal proliferation time being 48 hours; C: Both 2F5 supernatant and 2F5 antibody promoted RAMOS cell proliferation, with the optimal proliferation time being 24 hours. ****P < 0.0001, ***P < 0.001, **P < 0.01, *P < 0.05, ns P > 0.05.

[0022] Figure 2 Figure 2 shows the effect of 2F5 antibody on B cell activation. (A) Surface fluorescence intensity of OCI-LY-19 cells treated with 2F5 antibody; (B) Surface fluorescence intensity of RAMOS cells treated with 2F5 antibody. Scale bar: 50 μm.

[0023] Figure 3 Figure 2: 2F5 antibody supernatant stimulates the activation of two B cell lines. (A) Surface fluorescence intensity of OCI-LY-19 cells treated with 2F5 supernatant, as assessed by indirect immunofluorescence assay; (B) Surface fluorescence intensity of RAMOS cells treated with 2F5 supernatant, as assessed by indirect immunofluorescence assay. Scale bar: 50 μm.

[0024] Figure 4 The results show that 2F5 antibody promotes the activation of primary B cells. The scale bar indicates 50 μm.

[0025] Figure 5 Figure 2 shows the effects of 2F5 antibody on the differentiation of PBMCs and RAMOS cells. A: The proportion of CD19+CD27+CD38+ B cells in PBMCs increased after stimulation with purified 2F5 antibody (*P < 0.05, ns P > 0.05). B: Both purified 2F5 antibody and IgG antibody promoted cell surface CD138 expression, i.e., B cell differentiation. However, compared with IgG, 2F5 had a more significant effect in promoting B cell differentiation.

[0026] Figure 6 Figure 2 shows the morphological effects of the 2F5 antibody on two B cell lines. A: Wright-Giemsa staining of OCI-LY-19 cells treated with the 2F5 antibody; B: Wright-Giemsa staining of RAMOS cells treated with the 2F5 antibody. Scale bar: 50 μm.

[0027] Figure 7To detect the effect of 2F5 antibody on the differentiation of primary B cells by ELISA method, **P < 0.01, *P < 0.05, nsP > 0.05. DETAILED DESCRIPTION

[0028] The present application is further described below in conjunction with the examples and the accompanying drawings.

[0029] The experimental methods described in the following examples are all routine methods unless otherwise specified; the reagents and materials described are all commercially available unless otherwise specified.

[0030] 1. Experimental materials

[0031] 1.1 Experimental cells

[0032] (1) RAMOS (human B lymphoma cell): suspension growth, high expression of CD38 can secrete IgM, expression of membrane type and secreted immunoglobulin. Purchased from Guangzhou Geniue Cell Center.

[0033] (2) OCI-LY-19 (human diffuse large B cell lymphoma cell): suspension growth, high expression of CD38. As a kind of diffuse large B cell lymphoma cell line, it is currently often used as an experimental cell to explore the mechanism of diffuse large B cell lymphoma in scientific research. Purchased from Guangzhou Geniue Cell Center.

[0034] (3) PBMCs (Peripheral Blood Mononuclear Cells) were derived from healthy volunteers, 4 males and 6 females, aged 18-26 years old. The volunteers were all in the fasting state in the morning, and 30 mL of peripheral elbow venous blood was drawn, using ethylenediaminetetraacetic acid (EDTA) anticoagulation. The research subjects all comply with the principle of informed consent, and signed the informed consent form.

[0035] 1.2 Experimental reagents and equipment

[0036] RPMI-1640 (Thermofisher, 11875093), human peripheral blood mononuclear cell extraction kit (Solarbio, P8680), human primary B cell extraction kit (Thermofisher, 11351D), human IgM ELISA kit (ABclonal, RK00097), CCK-8 kit (Dojindo, CK04), CD80 rabbit monoclonal antibody (ABclonal, A25138), FITC-labeled goat anti-rabbit IgG secondary antibody (ABclonal, AS011), PE-CD19 antibody (Lianke Biotechnology, F11019A02), APC-CD27 antibody (Abcam, ab239293), FITC-CD38 antibody (Lianke Biotechnology, F1103801)

[0037] 2. Experimental Methods

[0038] 2.1 Preparation of 2F5 Antibody

[0039] A fully human monoclonal antibody library was established using human B cell cloning technology. ELISA reaction plates were coated with purified DDX5 protein as the antigen at 4°C overnight. The reaction plates were then blocked with 4% BSA / PBST for 1 hour at room temperature. After blocking, hybridoma cell culture supernatant from the antibody library was added to the reaction plates and reacted at room temperature for 1 hour. The plates were washed three times with PBST for 5 minutes each. Goat anti-human horseradish peroxidase-labeled secondary antibody was added and reacted at room temperature for 1 hour. The plates were washed three times with PBST-PBS for 5 minutes each. Hybridoma cell lines with positive reactions were screened by color development, namely 2F5. The plates were then expanded and the serum-free cell culture supernatant was collected. Purified monoclonal antibody 2F5 was obtained after purification with a protein A affinity column, desalting, and concentration.

[0040] The amino acid sequence of the monoclonal antibody 2F5 is shown in Tables 1 and 2 below:

[0041] Table 1 CDR (complementarity determining region) amino acid sequence of monoclonal antibody 2F5

[0042]

[0043] Table 2 Amino acid sequence of the variable region of monoclonal antibody 2F5

[0044] 2.2 2F5 and IgG Antibody Configuration

[0045] (1) Take a 15 mL centrifuge tube and add the prepared 1640 medium. Take a tube of purified 2F5 antibody (1 mg / mL, 480 μL) and add 120 / 240 μL of 2F5 antibody to the centrifuge tube to make an 80 / 160 μM antibody diluent. Mix evenly. Filter through a 0.22 nm bacterial filter and store in a 4°C refrigerator.

[0046] (2) Take a 15 mL centrifuge tube, add the prepared 1640 medium, take a tube of IgG antibody (10 mg / mL, 45 μL), add 15 / 30 μL of IgG antibody to the centrifuge tube to make an 80 / 160 μM antibody diluent, and mix evenly. Filter through a 0.22 nm bacterial filter membrane and store in a 4°C refrigerator.

[0047] 2.3 Configuration Grouping

[0048] (1) 2F5 group (80 μM): 120 μL of 2F5 stock solution was added to 9.88 mL of 1640 culture medium to dilute to 80 μM 2F5 antibody diluent.

[0049] (2) 2F5 group (160 μM): 240 μL of 2F5 stock solution was added to 9.76 mL of 1640 culture medium to dilute to 160 μM 2F5 antibody diluent.

[0050] (3) IgG group (80 μM): 15 μL of IgG stock solution was added to 12.485 mL of 1640 culture medium to dilute to 80 μM IgG antibody diluent.

[0051] (4) IgG group (80 μM): 30 μL of IgG stock solution was added to 12.47 mL of 1640 culture medium to dilute to 160 μM IgG antibody diluent.

[0052] (5) NC group (normal culture in 1640 medium).

[0053] (6) Control group: only secondary antibody was added without primary antibody.

[0054] 2.4 Indirect immunofluorescence

[0055] (1) Coating: Coat a 48-well plate with poly-D-lysine (200 μg / mL) and incubate at 37°C for more than 1 hour.

[0056] (2) Inoculate cells and place in a 37°C cell culture incubator for 24 hours. After the cells settle to the bottom, carefully aspirate the supernatant. Add different treatment solutions to each well, gently shake the cells to mix, and then place the cells in the incubator for another 24 hours (primary B cells are cultured for 48 hours).

[0057] (3) Carefully aspirate the supernatant and heat in a metal bath at 100°C for 5-10 minutes (bake dry). Wash once with PBS.

[0058] (4) Fixation: Add 200 μL of 4% paraformaldehyde to each well for fixation and let it stand at room temperature for 10 min.

[0059] (5) Washing: Add 600 μL PBST to each well and wash twice for 5 min each time, then add 600 μL PBS and wash once for 5 min at room temperature.

[0060] (6) Blocking: Add 200 μL of blocking solution (10% FBS, 2% BSA in PBS) to each well for blocking and place on a shaker for 1 hour to shake evenly.

[0061] (7) Add primary antibody: Add primary antibody (Rb CD80 mAb) diluted 1:100 with PBS, place in a sealed bag, and refrigerate at 4°C overnight.

[0062] (8) Washing: Add 600 μL PBST to each well and wash twice for 5 min each, and then wash once with PBS for 5 min at room temperature.

[0063] (9) Add secondary antibody: Add fluorescent secondary antibody (goat anti-rabbit IgG-FITC) diluted with PBS 1:100 (be careful to avoid light) and place on a shaker for 2-3 hours to allow the antibody to fully contact the cells.

[0064] (10) Washing: Add 600 μL PBST to each well and wash twice for 5 min each, then wash once with PBS for 5 min at room temperature.

[0065] (11) Nuclear staining: Add 100 μL Hoechest staining solution (1:10000) to each well, protect from light, and incubate at room temperature for 10 min.

[0066] (12) Washing: Add 600 μL PBST to each well and wash twice for 5 min each, then wash once with PBS for 5 min at room temperature.

[0067] (13) Sealing: Aspirate the liquid in the wells and add 50 μL of sealing medium (50% glycerol in PBS) to each well.

[0068] (14) Observation under fluorescence microscope

[0069] 2.5 Cell Counting Kit-8 (CCK-8)

[0070] (1) Add 200 μL PBS to the outermost circle of the 96-well plate to prevent the target well liquid from evaporating.

[0071] (2) Cell counting: Concentrate the cell density to 1 x 10 3 cells / μL.

[0072] (3) Add 100 μL of different group treatment liquid to each well, and suck 10 μL (treated to 10 μL according to the required concentration) cell stock solution into the well.

[0073] (4) Place the 96-well plate in a 37°C incubator for 12 / 24 / 48 h.

[0074] (5) Add 10 μL CCK-8 reagent to each well. (Note: avoid light)

[0075] (6) After incubation in a 37°C incubator for 4 h, detect at a wavelength of 450 nm using a microplate reader. (Detect within 5 min)

[0076] 2.6 Wright-Giemsa Staining

[0077] (1) Cell smears

[0078] ① Cells were cultured in a 6-well plate for 5 d, then 1 mL syringe was used to suck the cells into a 1.5 mL EP tube and centrifuged. The supernatant was discarded, 1 mL PBS was added for washing, and centrifuged.

[0079] ② The supernatant was sucked out, and about 20 μL liquid was left in each tube. 200 μL syringe was used to suck out and evenly smear on the center of the adsorptive glass slide in a circle, then placed in the oven to dry.

[0080] ③ Add 4% paraformaldehyde on the glass slide for fixation (cover the cells), and discard after 15 min. Wash with PBS for 2 min (cover the cells), and discard. Dry in a 37°C oven.

[0081] (2) Staining

[0082] ① Add Wright-Giemsa A liquid (about 0.5 mL-0.8 mL) to the smear, and let the dye cover the entire specimen, stain for 1 min.

[0083] ② Add Wright-Giemsa B liquid on top of A liquid (add 2-3 times the amount of A liquid), blow a gentle breeze with an ear bulb to create ripples on the liquid surface, and mix the two liquids thoroughly, stain for 3-10 min.

[0084] ③ Wash with water (do not pour out the dye solution first when rinsing, it should be rinsed away with running water to prevent sediment from settling on the specimen).

[0085] ④ Drying and microscopic examination.

[0086] 2.7 Enzyme Linked Immunosorbent Assay (ELISA)

[0087] (1) Sample preparation: Cells were cultured in a 6-well plate for 24 h. The cell culture dish was then removed and placed under a microscope for observation. The cells were centrifuged at 1000 rpm for 5 min. The supernatant was discarded and each well was treated with a different group of solutions. The cells were then cultured again at 37°C in a cell culture incubator for 5-7 days. The cells from different groups were collected in a centrifuge tube and centrifuged at 1000 rpm for 5 min. The supernatant was collected.

[0088] (2) Dilute the sample and standard in a certain ratio using the sample / standard diluent (R1). Then remove the microwell strip from the plate frame.

[0089] (3) Add 350 μL of 1× washing buffer to each well, let it stand for 40 s, and then discard the liquid in the well. This step is repeated three times.

[0090] (4) Add 100 μL of sample / standard diluent (R1) to the blank well.

[0091] (5) Add 100 μL of standards / samples of different concentrations to the other wells, seal the wells with the provided sealing film, and incubate at 37°C for 2 h.

[0092] (6) Prepare biotinylated antibody (1×) 15 min before use

[0093] (7) Discard the liquid in the wells and repeat the washing steps in step 3.

[0094] (8) Add biotinylated antibody working solution (100 μL / well) to each well, cover with a new sealing film, and incubate at 37°C for 1 h.

[0095] (9) Prepare streptavidin-HRP (1×) 15 min before use.

[0096] (10) Add streptavidin-HRP working solution (100 μL / well) to each well, cover with a new sealing film, and incubate at 37°C for 30 minutes.

[0097] (11) Preheat the microplate reader.

[0098] (12) Discard the liquid in the wells and repeat the washing steps in step 3.

[0099] (13) Add TMB substrate (100 μL / well) to the wells and incubate at 37°C in the dark for 15-20 min.

[0100] (14) Add stop solution (50 μL / well) and immediately place in a microplate reader for detection.

[0101] 2.8 Collection of Human Peripheral Blood and Isolation of PBMCs

[0102] (1) Take 5 mL of venous blood from a healthy individual and place it in a purple-capped EDTA anticoagulant tube. Dilute the whole blood with an equal volume of whole blood and tissue diluent or PBS.

[0103] (2) Take a 15 mL sterile centrifuge tube, first add 3.6 mL of reagent A, then add 2.4 mL of reagent D, so that the two form a gradient interface (the volume ratio of reagent A: reagent D is 3:2, and the total amount of reagent is equal to the volume of the diluted blood sample). Note that the stratification of the two reagents must be clear.

[0104] (3) Use a Pasteur pipette to spread the diluted blood evenly over the surface of the separation solution, making sure the interface between the two liquids is clear. (Because of the density difference between the two, a distinct stratified interface will form.)

[0105] (4) Centrifuge at room temperature in a swing-out rotor at 500-800 × g for 20-30 min.

[0106] (5) After centrifugation, obvious stratification will appear: the first layer is the plasma layer; the second layer is the white mononuclear cell layer; the third layer is the transparent reagent D liquid layer; the fourth layer is the translucent reagent A liquid layer; and the fifth layer is the red blood cell layer.

[0107] (6) Carefully transfer the second layer of white flocculent mononuclear cells to another sterile 15 mL centrifuge tube, add 10 mL of cell washing solution or PBS, invert to mix, and centrifuge at 250 × g for 10 min.

[0108] (7) Discard the supernatant, resuspend the cells in 5 mL of cell washing buffer or PBS, and centrifuge at 250 × g for 10 min.

[0109] (8) Repeat step 7.

[0110] (9) Discard the supernatant and resuspend the cells for later use.

[0111] 2.9 Extraction of primary B lymphocytes

[0112] (1) Use an appropriate volume of ImunoSep TM Resuspend PBMCs in Buffer and adjust the cell concentration to 1×10 8 cells / mL and prepared into a single-cell suspension.

[0113] (2) Place the required number of cells in a 15 mL centrifuge tube, but do not exceed 2 × 10 8 indivual.

[0114] (3) Add 20 μL of sorting reagent A to every 100 μL of cell suspension. Mix well by vortexing or pipetting five times with a 1 mL pipette tip, and incubate at room temperature for 10 min.

[0115] (4) Adding ImunoSep cells TM Buffer was added to 4 mL, cells were washed, and then centrifuged at 300 × g for 5 min at room temperature.

[0116] (5) Discard the supernatant and use ImunoSep TM Resuspend cells in Buffer to their original volume.

[0117] (6) Add 20 μL of sorting reagent B to every 100 μL of cell suspension, mix well using a vortexer or pipette five times with a 1 mL pipette tip, and incubate at room temperature for 5 min.

[0118] (7) Add ImunoSep TM Add Buffer to 2.5 mL. Mix thoroughly by pipetting up and down three times with a 1 mL pipette tip. Be careful not to use a vortexer to mix.

[0119] (8) Insert the centrifuge tube containing the cell suspension into the magnet, allowing the bottom of the tube to pass through the hole at the bottom of the magnet until it touches the work surface. Let it stand at room temperature for 5 minutes.

[0120] (9) Keep the centrifuge tube in the magnet, lift the magnet and the centrifuge tube together, and quickly pour the supernatant into the 50 mL centrifuge tube. (The time should not exceed 2 seconds)

[0121] 2.10 Flow cytometry

[0122] (1) The cells were cultured in a 6-well plate for 24 h, then centrifuged and the medium was changed and different treatment solutions were added. The cells were then placed back into the cell culture incubator and cultured for 5-7 days (the 6-well plate was filled with approximately 2×10 cells). 6 Cells from different groups were collected in centrifuge tubes, centrifuged at 1000 rpm for 5 min, and the supernatant was discarded.

[0123] (2) Wash the cells with 1 mL Wash Buffer (PBS containing 0.1% BSA) and mix thoroughly using a 1 mL pipette tip. Centrifuge at 1000 rpm for 5 min and discard the supernatant.

[0124] (3) Add Fc receptor blocker (5 μL / 106 Use a 1 mL pipette tip to mix the blocking agent and sample cells evenly and let it stand at room temperature for 10 minutes.

[0125] (4) After blocking, no washing is required. Add fluorescently labeled primary antibodies (PE-CD19, FITC-CD38, APC-CD27) to each centrifuge tube according to the antibody instructions and mix well using a vortex.

[0126] (5) Place the sample in a 4°C refrigerator and incubate for 35 min.

[0127] (6) After incubation, add Wash Buffer (2 mL per 15 mL centrifuge tube), centrifuge at 1000 rpm for 5 min, and discard the supernatant. Repeat the washing process 3 times.

[0128] (7) Resuspend the cells in 500 μL of washing buffer. Detect on a flow cytometer and analyze the results.

[0129] 3. Results

[0130] 3.1 2F5 promotes the proliferation of PBMC and B cell lines

[0131] The CCK8 assay was used to examine the effects of 2F5 antibody on the proliferation of PBMC cells and different B lymphocyte cell lines. PBMC cells, OCI-LY-19 cells, and RAMOS cells were treated with 2F5 supernatant or purified 2F5 antibody, respectively. The results showed that compared with the NC control group, both 2F5 antibody and IgG antibody promoted PBMC cell proliferation, but the 2F5 antibody group was more potent than the IgG control group, and the optimal time point for 2F5 antibody proliferation was 48 hours ( Figure 1 A). Further investigation of the effect of 2F5 antibody on the proliferation of different B lymphocyte cell lines revealed that 2F5 supernatant promoted the proliferation of OCI-LY-19 cells, and this promoting effect was independent of the components of HT culture medium. 2F5 antibody also promoted the proliferation of OCI-LY-19 cells, and the optimal time point for the proliferation of 2F5 antibody was 48h ( Figure 1 B). In addition, both 2F5 supernatant and 2F5 antibody can promote the proliferation of RAMOS cells, and the optimal time point for the proliferation of 2F5 antibody is 24h ( Figure 1 C).

[0132] 3.2 2F5 promotes B cell lineage activation

[0133] The indirect immunofluorescence method was used to evaluate the effect of 2F5 purified antibody on the activation state of different B lymphocyte lines. OCI-LY-19 cells and RAMOS cells were treated with 2F5 purified antibody. The results showed that both cells treated with 2F5 purified antibody and IgG antibody exhibited significant green fluorescence compared with the blank control group. In particular, the cells treated with 2F5 purified antibody showed more specific green fluorescence signals, which were mainly distributed on the cell membrane, while the green fluorescence signals of the IgG control group were uniformly distributed in the cells, and the overall fluorescence intensity of the IgG control group was lower than that of the 2F5 antibody group. Figure 2 It is suggested that both 2F5 purified antibody (80 μM) and IgG antibody (80 μM) can promote the expression of CD80 on the cell surface and activate B cell lines, but compared with the IgG control group, 2F5 purified antibody shows more significant effect in promoting B cell activation. In addition, 2F5 supernatant can also promote the expression of CD80 on the membrane surface of the two B cell lines, that is, promote the activation of B cell lines, and this promoting effect is not related to the components of HT medium. Figure 3

[0134] 3.3 2F5 promotes the activation of primary B cells

[0135] The indirect immunofluorescence method was used to detect the effect of 2F5 purified antibody on the activation of primary B cells, and the primary B cells were treated with 2F5 purified antibody (160 μM). The results showed that compared with the blank control group, the primary B cells cultured with 2F5 antibody, IgG antibody and 1640 medium all had green fluorescence signals on the membrane surface, but the fluorescence intensity had obvious differences. Compared with the IgG control group and the NC control group, the B cells treated with 2F5 antibody showed more significant and specific fluorescence signals, which were distributed on the cell membrane surface in the form of star points and high brightness, which was consistent with the typical biological characteristics of the activation marker protein CD80 on the cell membrane surface. Figure 4 It is suggested that the expression of activation marker CD80 on the membrane surface of primary B cells treated with 2F5 antibody increases significantly, and the cell activation is more obvious.

[0136] 3.4 2F5 promotes the differentiation of PBMC, B cell lines and primary B cells

[0137] Flow cytometry was used to detect the effect of 2F5 on the differentiation of PBMC cells and RAMOS cells. PBMC cells and RAMOS cells were treated with 2F5 purified antibody. The results showed that in PBMC cells, compared with the NC control group, the proportion of CD19 + CD27 + CD38 + B cells in the 2F5 antibody group increased significantly, and the proportion of CD19 + ​CD27 + CD38 + There was no significant difference in the proportion of B cells ( Figure 5 A). This indicates that after treatment with 2F5 purified antibody, B cell differentiation is more obvious, and the proportion of plasma cells is larger. In RAMOS cells, compared with the NC control group, both the 2F5 antibody group and the IgG control group showed a significant increase in the expression of CD138, a B cell surface differentiation marker, but the expression level of the IgG control group was slightly lower than that of the 2F5 antibody group ( Figure 5 B). This suggests that both 2F5 purified antibody and IgG antibody have the ability to promote the expression of CD138 on the surface of B cells and promote B cell differentiation, but compared with IgG, 2F5 is more effective in promoting B cell differentiation; In addition, the results of Giemsa staining showed that ( Figure 6 ), compared with the NC control group and the IgG control group, OCI-LY-19 cells and RAMOS cells treated with 2F5 purified antibody showed significant morphological changes: the cell volume became larger, the nucleus was darkly stained, occupying most or the entire cell and biased to one side. These characteristics are typical manifestations of the B cell differentiation process. Therefore, the significant changes in the morphology of the two B cell lines also suggest that the 2F5 antibody (80μM) may have induced the differentiation of the two B cell lines. In order to further explore the effect of 2F5 antibody on the differentiation of primary B cells, we quantitatively detected the secretion level of IgM in the culture supernatant by ELISA, and the results showed that ( Figure 7 Compared with the NC control group, the IgM content in the cell culture supernatant of both the 2F5 antibody group and the IgG control group increased, but the IgM content in the cell culture supernatant of the 2F5 antibody group was significantly higher than that of the IgG group. These results confirm the important role of 2F5 in the differentiation of PBMCs, B cell lines, and primary B cells.

Claims

1. A method for specifically inducing proliferation or differentiation of peripheral blood mononuclear cells for non-diagnostic and non-therapeutic purposes, characterized in that: The method comprises the following steps: The purified monoclonal antibody 2F5 was diluted to prepare a 2F5 antibody diluent, and then used to treat peripheral blood mononuclear cells; wherein the monoclonal antibody 2F5: The amino acid sequence of the complementarity determining region CDR1 of the light chain 2F5 is: QSVSSN; The amino acid sequence of the complementarity determining region CDR2 of the light chain 2F5 is: GAS; The amino acid sequence of the complementarity determining region CDR3 of the light chain 2F5 is: QQYNNWPRT; The amino acid sequence of the complementarity determining region CDR1 of the heavy chain 2F5 is: GFTFSDYS; The amino acid sequence of the complementarity determining region CDR2 of the heavy chain 2F5 is: ITSSSGYT; The amino acid sequence of the complementarity determining region CDR3 of the heavy chain 2F5 is: ARVRSSWGPIDS.

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