CN1314414A - New activated T cell surface antigen of molecular weight 140 KDa - Google Patents

Abstract

A new activated T cell surface antigen with a molecular weight of 140kDa, involving biotechnology. Immune cell membrane molecules have been widely used in the diagnosis, prevention and treatment of some clinical diseases. The invention uses activated T cells obtained from mixed lymphocyte culture to immunize BALB/c mice, prepares splenocyte suspension, fuses with myeloma cell NS-1, and selectively cultures with HAT. The A2 monoclonal antibody was obtained after screening the hybridoma supernatant. Analysis of the activated T cell surface antigen recognized by the monoclonal antibody shows that it has a unique distribution, expression and function, and its molecular weight is 140kDa.

Description

A new activated T cell surface antigen with a molecular weight of 140kDa

The present invention relates to biotechnology.

The body's immune system is composed of central lymphoid organs, peripheral lymphoid organs, immune cells and immune molecules. The immune response process depends on the interaction between cells in the immune system, including direct contact between cells and the interaction through the release of cytokines or other mediators. The material basis for mutual recognition between immune cells or between cells and media is immune cell membrane molecules. The study of immune cell membrane molecules is of great significance for in-depth understanding of the immune response mechanism, as well as for the diagnosis, prevention and treatment of certain clinical diseases. As of August 2000, the US FDA has approved five monoclonal antibodies that recognize immune cell membrane molecules for the treatment of clinical diseases, and dozens of other monoclonal antibodies are undergoing clinical trials.

A new surface antigen of activated T cells with a molecular weight of 140kDa of the present invention is uniquely distributed in CD4 ⁺ , CD8 ⁺ , TCRγδ ⁺ T cells activated by mixed lymphocyte reaction and activated NK cells, while in other activation pathways (such as solid phase CD3 stimulation, PHA, PMA and PMA+A23187 stimulation), activated T cells did not express this molecule. It can cooperate with CD3 molecule to stimulate T cell activation. Its unique distribution, expression pattern and function indicate that it is a new molecule.

Technical route of the present invention:

Activated T cells obtained from mixed lymphocyte culture were used to immunize BALB/c mice. Three days after booster immunization, the spleen was removed to prepare spleen cell suspension, which was fused with myeloma cell NS-1 and selectively cultured with HAT. Through indirect immunofluorescence labeling and flow cytometry, hybridoma supernatants capable of binding to activated T cells obtained from mixed lymphocyte culture were screened, fusion cells secreting positive antibodies were cloned, and A2 monoclonal antibody was obtained. The surface antigens of activated T cells recognized by the A2 monoclonal antibody were analyzed by immunofluorescence staining, flow cytometry and redirected cytotoxicity experiments, showing that they had unique distribution, expression rules and functions. Its molecular weight was identified as 140kDa by biotin labeling, immunoprecipitation and chemiluminescence.

The activated T cell surface antigen with a molecular weight of 140kDa is expressed in the activated T cell subset TCRγδ ^{+ T cells, therefore, it can be used as an activation marker of TCRγδ + T} cells, and has a role in the research of transplant immunity and clinical treatment of transplant rejection. Significance.

The specific implementation manner of technical route of the present invention:

1. Mixed lymphocyte culture (MLC) Aseptically extract 10 ml of normal human peripheral blood, anticoagulate with heparin, and separate peripheral blood mononuclear cells (PBMC) by density gradient centrifugation. 2 ml of complete medium contains 1.5×10 ⁶ PBMC and 0.5 ×10 ⁶ 3000Rad irradiated Daudi cells were cultured in a 24-well plate in a 37°C, 5% CO ₂ incubator for 7 days to obtain activated T cells.

2. Intraperitoneal injection of 1×10 ⁷ activated T cells obtained from cell immunization mixed lymphocyte culture to immunize BALB/c mice, immunized twice in the same way within 4 weeks, and 1 week later, 1×10 ⁷ activated T cells were injected into the tail vein for booster immunization 3 days later, the spleen was taken for fusion.

3. Preparation of Monoclonal Antibody Three days after the booster immunization, the spleen was taken, the spleen cell suspension was prepared, fused with myeloma cell NS-1, and selectively cultured with HAT. Through indirect immunofluorescence labeling and flow cytometry, hybridoma supernatants capable of binding to activated T cells obtained from mixed lymphocyte culture were screened, fusion cells secreting positive antibodies were cloned, and A2 monoclonal antibody was obtained.

4. Study on the Distribution of Membrane Molecule p140 Recognized by A2 Monoclonal Antibody

4.1 Activation of human PBMC: Obtain human PBMC by the same method as 1., culture 2×106 cells in 2ml of complete medium in a 24-well plate, add PHA (4 μg/ml), PMA (40ng/ml), PMA (40ng/ml) respectively /ml)+A23187 (0.9 μg/ml), and another well was coated with CD3 mAb (coated with a concentration of 8 μg/ml). The PMA and PMA+A23187 groups were cultured for 48 hours, the PHA and CD3 mAb groups were cultured for 72 hours, and the PBMCs stimulated by PHA were maintained for 12 days by adding rHuIL-2 (100u/ml). At the same time, a group of mixed lymphocyte cultured cells were maintained for 12 days by adding rHuIL-2 (100u/ml). rHuIL-2 (100u/ml) was added to the mixed lymphocyte culture cells used for the following immunological double-color analysis on the first day of stimulation.

4.2 Immunofluorescence staining and flow cytometry analysis: adjust the concentration of various cells to 1×10 ⁷ /ml with complete medium containing 10% calf serum, take 50 μl of cell suspension and add 50 μl of pre-diluted 1:20 (Diluted with DPBS) In the tube of inactivated normal rabbit serum, add different mAbs, shake fully, act at 4°C for 30 min, and wash twice with washing solution. Discard the supernatant, add the working concentration of goat anti-mouse FITC-labeled secondary antibody (diluted 1:20), shake fully, act at 4°C for 30 min, wash twice with washing solution, add appropriate amount of fixative, and detect with FCM. In the direct immunofluorescence two-color experiment, adjust the cell concentration to 1×10 ⁷ /ml, take 50 μl of cell suspension and add 50 μl of 1:20 diluted inactivated normal rabbit serum in advance, incubate at 4°C for 10 minutes, and then add PE to each tube and FITC-labeled specific antibody, and then continue to incubate at 4°C for 30 minutes, and finally wash 3 times, add an appropriate amount of fixative, and detect with FCM.

4.3 Results obtained: The immune cell membrane molecule p140 recognized by the A2 monoclonal antibody is not expressed in resting peripheral blood mononuclear cells (PBMC), but is selectively expressed on the surface of T cells activated by mixed lymphocyte culture. Other activation methods such as solid-phase CD3 stimulation, PHA, PMA and PMA+A23187 stimulation could not induce its expression. PHA-stimulated PBMCs did not express p140 until day 12, while T cells activated by mixed lymphocyte culture expressed higher expression from day 3. Flow cytometry analysis found that p140 molecules were expressed on the surface of activated T and NK cells, especially in TCRγδ ⁺ T cells.

5. Investigate the functions involved in the p140 molecule

5.1 Redirection cytotoxicity test: P815 cells in logarithmic growth phase were collected, labeled with ⁵¹ Cr (100 μCi/1×10 ⁶ cells), incubated at 37° C. for 2 h, and shaken every 15 min. After labeling, wash 3 times, adjust the concentration of target cells to 1×10 ⁶ /ml, add to 96-well U-shaped plate, 100 μl/well. The activated lymphocytes 7 days after MLC were collected as effector cells, and the cell concentration was adjusted to 1×10 ⁶ /ml. Take 600 μl of cell suspension from each group and add corresponding antibodies to incubate at 37°C for 30 minutes, centrifuge, resuspend with 600 μl of complete medium, perform multiple dilutions, and add them to 96-well U-shaped plates containing labeled P815 cells. 100 μl/well, cultured in a 37°C, 5% CO ₂ incubator for 4 hours, collected the supernatant, and detected the cpm value with a gamma counter.

5.2 The results obtained: p140 molecule has a synergistic effect with CD3 molecule, and when the effect-to-target ratio is 10:1 and 5:1, the cytotoxic activity stimulated by CD3 is increased by 12.1% and 9.2%, respectively.

6. Identifying the molecular weight of the p140 molecule

6.1 Biotin labeling, immunoprecipitation and chemiluminescence: Collect 1×10 ⁷ JM cells in the logarithmic growth phase, operate according to the steps described in the biotin labeling and immunoprecipitation kit, and transfer the obtained samples to On the PVDF membrane, use the POD-labeled Avidin chemiluminescence kit for exposure and color development.

6.2 Obtained results: The molecular weight of the p140 molecule was finally identified as 120-140 kDa.

Claims (2)

1. A new activated T cell surface antigen with a molecular weight of 140kDa is characterized in that: the activated T cells obtained by mixed lymphocyte culture are used to immunize BALB/c mice, and 3 days after the booster immunization, the spleen is taken to prepare a spleen cell suspension, and Myeloma cells NS-1 were fused and selectively cultured with HAT; through indirect immunofluorescence labeling and flow cytometry, hybridoma supernatants that could combine with activated T cells obtained from mixed lymphocyte culture were screened to secrete positive antibody fusion cells It was cloned and A2 monoclonal antibody was obtained: the surface antigen of activated T cells recognized by A2 monoclonal antibody was analyzed by immunofluorescence staining, flow cytometry and redirection cytotoxicity experiments, which showed that it had a unique distribution, expression pattern and Function, identified by biotin labeling, immunoprecipitation and chemiluminescence, its molecular weight is 140kDa. 2. According to claim 1, a new activated T cell surface antigen with a molecular weight of 140kDa is characterized in that: the cell surface antigen is uniquely distributed in CD4 ⁺ , CD8 ⁺ , TCRγδ ⁺ T cells activated by mixed lymphocyte reaction and activated T cells. NK cells, while in other activation pathways (such as solid-phase CD3 stimulation, PHA, PMA and PMA+A23187 stimulation), the surface of activated T cells does not express this molecule, which can co-stimulate T cell activation with CD3 molecules, and its molecular weight is 140kDa.