T lymphocyte immunophenotype antibody combination, kit and method for flow cytometry detection of MM patient before and after treatment

Abstract

The invention discloses a detection reagent, a kit and a method for detecting T lymphocyte immunophenotype before and after treatment of an MM patient through flow cytometry. Comprising a first antibody combination: an anti-CD3 antibody, an anti-CD45RA antibody, an anti-CD39 antibody, an anti-CD57 antibody, an anti-CCR7 antibody, an anti-CD28 antibody, an anti-CD95 antibody, an anti-TIGIT antibody, an anti-PD-1 antibody and an anti-CD8 antibody; the first group of antibodies comprises an anti-CD3 antibody, an anti-CD45RA antibody, an anti-CD39 antibody, an anti-CD127 antibody, an anti-CD25 antibody, an anti-CD36 antibody, an anti-CCR8 antibody, an anti-NRP1 antibody, an anti-PD-1 antibody and an anti-CD4 antibody, and the third group of antibodies comprises an anti-CD3 antibody, an anti-CD8 antibody, an anti-CD28 antibody, an anti-CD39 antibody, an anti-CD57 antibody, an anti-PD-1 antibody and an anti-TIGIT antibody; the kit is an anti-cTCF-1 antibody, an anti-cTOX antibody and an anti-cKi-67 antibody. The method has the advantages of few detection tubes, high antibody coverage rate, obvious cell grouping and strong systematicness.

Description

Technical Field

[0001] The present invention relates to, in particular to, an antibody combination, a kit and a method for detecting the immunophenotype of T lymphocytes of MM patients before and after treatment by flow cytometry. Background Art

[0002] Multiple myeloma (MM) is a hematological malignancy characterized by abnormal proliferation of malignant plasma cells, which mainly exists in the bone marrow. Its incidence rate is increasing year by year, ranking second among hematological malignancies. Traditional therapies have limited therapeutic effects on MM patients. Most patients will relapse or become refractory. Currently, there is still no cure, which seriously affects the quality of life and life expectancy of patients. T lymphocytes (hereinafter referred to as T cells) are lymphoid stem cells derived from the bone marrow. After differentiation and maturation in the thymus, they become T cells with extremely strong immune activity. T cells can be divided into different subpopulations according to their differentiation stages and functions. They are a group of cells with diverse phenotypes and complex regulatory mechanisms. They are the core effector cells that exert the body's anti-tumor immunity. At the same time, they can also provide a variety of immune target options during anti-tumor immunity, and have shown significant efficacy in recent immunotherapy for MM patients. However, these new therapies also face many challenges, including antigen escape, immunosuppression of the tumor microenvironment, etc., especially tumor-induced T cell dysfunction or exhaustion may be the cause of immune escape and failure of immunotherapy. Therefore, it is particularly important to understand the actual functional status of T cells and immune checkpoints of each subset in the bone marrow of MM patients.

[0003] At present, the immune-related tests for MM patients mainly include the detection of various functional subsets, activated subsets, regulatory subsets, and post-induced secretory functional subsets of T cells in peripheral blood; they have a certain evaluation effect on the immune status of MM patients before and after treatment. However, there are also certain shortcomings. First, MM is a malignant plasma cell abnormal proliferation disease originating from the bone marrow. Therefore, its immune microenvironment exists in the bone marrow. Detecting the functional status of various T cell subsets in the bone marrow can more accurately evaluate the immune microenvironment of the tumor. Second, at present, neither the peripheral blood nor the bone marrow immune function test items are unique combination items serving MM patients, and the specificity is not strong. Furthermore, the immune microenvironment of MM patients is complex, involving many types of lymphocyte subsets and a wide range. It is necessary to include as many antigen marker immunophenotypic analyses as possible to comprehensively monitor and evaluate the immune function status of MM patients before and after treatment.

[0004] Therefore, there is an urgent need to design a multicolor antibody panel that effectively serves the T cell immunophenotyping of MM patients. Summary of the invention

[0005] The purpose of the present invention is to provide a multicolor antibody combination with a small number of detection tubes, high antibody coverage, good detection efficiency, strong precision, obvious cell clustering, and strong systemicity to effectively serve the T cell immune phenotype of MM patients.

[0006] To achieve the above object, the present invention provides an antibody combination for detecting the immunophenotype of T lymphocytes of MM patients before and after treatment by flow cytometry, characterized in that it comprises a first antibody combination, a second antibody combination and a third antibody combination;

[0007] The first antibody combination is anti-CD3, anti-CD45RA, anti-CD39, anti-CD57, anti-CCR7, anti-CD28, anti-CD95, anti-TIGIT, anti-PD-1 and anti-CD8; the corresponding fluorescein labels of these antibodies are in order: Percp-cy5.5, APC, APC-cy7, BV510, BV421, FITC, PE, BV605, PE-cy7, AF-700; preferably, the volume usage ratio of these antibodies is: 3μl: 2μl: 3μl: 2μl: 2μl: 2.5μl: 2μl: 2μl: 2μl: 2μl: 2μl: 3μl;

[0008] The second antibody combination includes the following antibodies: anti-CD3, anti-CD45RA, anti-CD39, anti-CD127, anti-CD25, anti-CD36, anti-CCR8, anti-NRP1, anti-PD-1, and anti-CD4 antibodies. The corresponding fluorescent labels of these antibodies are in the following order: Percp-cy5.5, APC, APC-cy7, BV510, BV421, FITC, PE, BV605, PE-cy7, and AF-700; the volume ratio of these antibodies is: 3μl: 2μl: 3μl: 2μl: 2μl: 2μl: 2μl: 2μl: 2μl: 2μl: 3μl;

[0009] The third group of antibodies includes anti-cell membrane antigen antibodies and anti-intracellular antigen antibodies; the anti-cell membrane antigen antibodies are composed of the following antibodies: anti-CD3, anti-CD8, anti-CD28, anti-CD39, anti-CD57, anti-PD-1, and anti-TIGIT antibodies; the anti-intracellular antigen antibodies are composed of the following antibodies: anti-cTCF-1, anti-cTOX, and anti-cKi-67 antibodies; the corresponding fluorescein labels of these antibodies are, in order: Percp-cy5.5, APC-R700, FITC, APC-cy7, V500, PE-cy7, BV605, PE, APC, and BV421; the volume ratio of these antibodies is: 3μl: 3μl: 2μl: 3μl: 2μl: 2μl: 2μl: 3μl: 3μl: 3μl;

[0010] Furthermore, in the first antibody combination, the volume usage ratio of anti-CD3, anti-CD45RA, anti-CD39, anti-CD57, anti-CCR7, anti-CD28, anti-CD95, anti-TIGIT, anti-PD-1 and anti-CD8 is: 3μl: 2μl: 3μl: 2μl: 2.5μl: 2μl: 2μl: 2μl: 2μl: 2μl: 3μl.

[0011] Furthermore, in the second antibody combination, the volume usage ratio of anti-CD3, anti-CD45RA, anti-CD39, anti-CD127, anti-CD25, anti-CD36, anti-CCR8, anti-NRP1, anti-PD-1, and anti-CD4 antibodies is: 3μl: 2μl: 3μl: 2μl: 2μl: 2μl: 2μl: 2μl: 2μl: 2μl: 3μl.

[0012] Furthermore, in the third antibody combination, the volume ratio of anti-CD3, anti-cTOX, anti-CD39, anti-CD57, anti-cKi-67, anti-CD28, anti-cTCF-1, anti-TIGIT, anti-PD-1, and anti-CD8 antibodies is: 3μl: 3μl: 3μl: 2μl: 3μl: 2μl: 3μl: 2μl: 2μl: 3μl: 3μl: 2μl: 3μl.

[0013] The present invention also protects a T lymphocyte immunophenotype detection kit for detecting MM patients before and after treatment by flow cytometry, characterized in that it contains the antibody combination.

[0014] Furthermore, it also includes hemolysin, membrane permeabilizing agent and phosphate buffered saline (PBS).

[0015] The present invention also provides a method for detecting the immune phenotype of T lymphocytes of MM patients before and after treatment by flow cytometry, characterized in that the antibody combination or the detection kit is used.

[0016] Further, the steps include: the detection steps are as follows:

[0017] S1. Sample preparation: Mix the heparin sodium anticoagulated bone marrow blood of the patient to be tested thoroughly, count the number of cells per microliter of the bone marrow sample using a bulla counting plate, and adjust the cell concentration to 2×10 with 1XPBS. 6 / 100μL;

[0018] S2. Take 3 flow-through sample tubes, add the first antibody combination to tube 1, the second antibody combination to tube 2, and the anti-cell membrane antigen antibody in the third antibody combination to tube 3, then add 100 μL of the diluted bone marrow specimen to each tube, shake well to mix, and incubate at room temperature away from light;

[0019] S3. Hemolysis: Add 2 ml of 1× FACS hemolysin to each of the three tubes after incubation, vortex mix at low speed, stand at room temperature in the dark, centrifuge and wash, and discard the supernatant;

[0020] S4. Washing: Add 2 ml of 1×PBS buffer to tube 1 and tube 2, shake and mix, centrifuge and wash, discard the supernatant; then add 300 μL of 1×PBS to resuspend the cells. If there are clumps, flocs or sediments in the specimen, filter them with a 400-mesh filter before adding, and place them in a refrigerator at 2-8°C and wait for the machine to measure;

[0021] S5. Permeabilization and fixation of intracellular tubes: Add 1 mL of 1x permeabilization agent to tube 3, vortex mix at low speed, and place at 2-8℃ away from light;

[0022] S6. Washing of intracellular tubes: After the resting of tube 3, add 2 mL of 1× PBS, shake and mix, centrifuge and wash, and discard the supernatant;

[0023] S7. Intracellular antibody staining: Add the anti-intracellular antigen antibody in the third antibody combination to tube 3, shake well to mix, and incubate at room temperature in the dark;

[0024] S8. Washing of intracellular tubes: Add 2 mL of 1×PBS to tube 3, shake and mix, centrifuge and wash, and discard the supernatant; add 300 μL of 1×PBS to resuspend the cells. If there are clumps, flocs or sediments in the specimen, filter it with a 400-mesh filter and place it in a refrigerator at 2-8°C to wait for the machine to measure;

[0025] S9. Machine test:

[0026] S10. Data analysis: Flowjo software was used for data analysis.

[0027] Furthermore, the S9. on-machine detection includes instrument setting, calibration and quality control: the BD FACSanto flow cytometer is turned on for preheating and the liquid flow system is turned on, and the baseline and instrument performance tests are run with quality control products. After all of them pass, single-stained samples are prepared according to the fluorescent color matching of this kit, and the appropriate voltage and compensation are determined. A combination template for the antibody expression pattern of healthy people is established.

[0028] According to the set instrument conditions, obtain 100,000 cells per tube. If the test cannot be carried out on the machine in time, add 0.5 ml of 1% paraformaldehyde, mix well and store in a refrigerator at 2-8°C. Complete the test within 24 hours.

[0029] Further, the S10 data analysis is as follows:

[0030] The gating logic of the first antibody combination is to first set the FSC-H / FSC-A live cell gate to remove the adherent doublet cells; display the live cell gate and circle the lymphocytes by FSC-H / SSC-A; establish a CD3 / CD8 scatter plot under the lymphocyte gate to circle CD3+CD8+ T cells and CD3+CD8- CD4+ T cells; establish CD28+ / -, CD39+, CD57+ / -, CD45RA / CCR7 cross gate, CD57 / CD28 cross gate, PD-1 / TIGIT cross gate and

[0031] Scatter plot of helper T lymphocyte subsets of CD45RA+CCR7+CD95+; establish CD28+ / -, CD39+, CD57+ / -, CD45RA / CCR7 cross gate, CD57 / CD28 cross gate, PD-1 / TIGIT cross gate and

[0032] Scatter plot of cytotoxic T cell subsets of CD45RA+CCR7+CD95+;

[0033] The gating logic of the second antibody combination is to first set the FSC-H / FSC-A live cell gate to remove the adherent doublet cells; display the live cell gate and circle the lymphocytes by FSC-H / SSC-A; establish a CD3 / CD4 scatter plot under the lymphocyte gate to circle CD3+CD4+ T cells and CD3+CD4- CD8+ T cells; establish a cross gate of CD36+, CD39+, CD45RA+ / CD45RA-, NRP1 / PD-1, a scatter plot of Treg(CD4+CD25+CD127low / -)+ / -, Treg+CCR8+ and a scatter plot of helper T cell subsets under the CD4+T cell gate; establish a cross gate scatter plot of CD36+, CD39+, CD45RA+ / CD45RA-, NRP1 / PD-1 and a scatter plot of cytotoxic T cell subsets under the CD8+T cell gate;

[0034] The gating logic of the third antibody combination is to first set the FSC-H / FSC-A live cell gate to remove the adherent doublet cells; display the live cell gate and circle the lymphocytes by FSC-H / SSC-A; establish a CD3 / CD8 scatter plot under the lymphocyte gate to circle CD3+CD8+ T cells and CD3+CD8- CD4+ T cells. Under the CD4+ T cell gate, establish CD39+, cKi67+, cTCF-1+, cTOX+ gates and CD28 / CD57 cross gates, PD-1 / TIGIT cross gates and helper T cell subset scatter plots; under the CD8+ T cell gate, establish CD39+, cKi-67+, cTCF-1+, cTOX+, CD28 / CD57 cross gates, PD-1 / TIGIT cross gates and cytotoxic T cell subset scatter plots.

[0035] The first antibody combination includes the following antibodies: anti-CD3, anti-CD8, anti-CD28, anti-CD95, anti-CD45RA, anti-CD39, anti-CCR7, anti-CD57, anti-PD-1, and anti-TIGIT antibodies, and the corresponding fluorescein labels of the combined antibodies are in order: Percp-cy5.5, APC-R700, FITC, PE, APC, APC-cy7, BV421, V500, PE-cy7, and BV605. The first antibody combination is used in one flow tube during detection.

[0036] The second antibody combination includes the following antibodies: anti-CD3, anti-CD4, anti-CD45RA, anti-CD36, anti-CCR8, anti-PD-1, anti-CD39, anti-CD25, anti-CD127, anti-NRP1 antibodies, and the corresponding fluorescein labels of the combination antibodies are in order: Percp-cy5.5, APC-R700, APC, FITC, PE, PE-cy7, APC-cy7, BV421, V500, BV605. The second antibody combination is used in one flow tube during detection.

[0037] The third group of antibodies includes anti-cell membrane antigen antibodies and anti-intracellular antigen antibodies; the anti-cell membrane antigen antibodies are composed of the following antibodies: anti-CD3, anti-CD8, anti-CD28, anti-CD39, anti-CD57, anti-PD-1, and anti-TIGIT antibodies; the anti-intracellular antigen antibodies are composed of the following antibodies: anti-cTCF-1, anti-cTOX, and anti-cKi-67 antibodies; the corresponding fluorescent markers of the combined antibodies are in order: Percp-cy5.5, APC-R700, FITC, APC-cy7, V500, PE-cy7, BV605, PE, APC, and BV421. The third antibody combination is used in one flow tube during detection.

[0038] The fluorescent markers corresponding to the above three combinations include but are not limited to the above markers, and can be selected according to actual conditions and needs in the art.

[0039] The present invention provides a method for detecting the immunophenotyping of each T cell subpopulation in the bone marrow of MM patients before and after treatment by flow cytometry, wherein CD3+CD4+ is a helper T cell subpopulation (Th); CD3+CD8+ is a cytotoxic T cell subpopulation (Tc).

[0040] The cell types of the first antibody combination are:

[0041] CD3+CD4 / 8+CD28+ represents T cell functional subsets; CD3+CD4 / CD8+CD28- represents T cell regulatory subsets; CD3+CD4 / 8+CD57+ represents the marker of T cell aging; CD3+CD4 / 8+CD45RA+CCR7+ represents naive T cells (TN cells); CD3+CD4 / 8+CD45RA-CCR7+ represents central memory T cells (TCM cells); CD3+CD4+ / 8+CD45RA-CCR7- represents effector Memory T cells (TEM cells); CD3+CD4+ / 8+CD45RA+CCR7- represent terminally differentiated effector memory T cells (TEMRA cells); CD3+CD4 / 8+CD45RA+CCR7+CD95+ represent T memory stem cells (TSCM cells); The combination of CD3+CD4 / 8+ with CD39, PD-1+ / -, and TIGIT+ / -, respectively, represents the immune suppression of the immune target CD39 and the immune checkpoints PD-1 and TIGIT in T cells.

[0042] The cell types of the second antibody combination are:

[0043] CD3+CD4 / 8+CD45RA+ / -CD36+ represents the immunosuppression of the immune target CD36 in the initial or memory T cells; CD3+CD4 / 8+CD45RA+ / -CD39+ represents the immunosuppression of the immune target CD39 in the initial or memory T cells;

[0044] The combination of CD3+CD4 / 8+CD45RA+ / - with NRP1+ / - and PD-1+ / - respectively represents the immune suppression of immune checkpoints NRP1 and PD-1 in the initial or memory T cells; CD3+CD4+CD25+CD127low / - represents regulatory T cells (Treg cells) , CCR8 is an immunosuppressive regulatory protein on the surface of Treg cells, and Treg+CCR8+ represents the immune response regulation of Treg cells.

[0045] The cell types of the third antibody combination are:

[0046] CD3+CD4 / 8+cKi-67+ represents the markers of T cell activation and proliferation; CD3+CD4 / 8+cTCF-1+ represents the characteristics of T cell stem cells, which has the effect of resisting T cell exhaustion; CD3+CD4 / 8+cTOX+ represents the markers of T cell exhaustion; CD3+CD4 / 8+CD28-CD57+ represents terminally differentiated effector memory T cells (TEMRA cells);

[0047] CD3+CD4 / 8+CD28+CD57+ represents effector memory T cells (TEM cells); CD3+CD4 / 8+CD28+CD57- represents central memory T cells (TCM cells); CD3+CD4 / 8+CD28-CD57- represents T cell regulatory subsets;

[0048] The CD39+, cTOX+, cKi-67+, and cTCF-1+ subgroups under these subgroups represent their exhaustion, proliferation, and anti-exhaustion conditions.

[0049] The present invention provides a method and kit for detecting the immunophenotype of each subpopulation of T cells in the bone marrow of MM patients before and after treatment by flow cytometry. Those skilled in the art can refer to the scheme of the present invention to make appropriate changes and combinations, including changes in the flexible gate setting of T cells under the same antibody combination, selection and change of the corresponding fluorescent markers of the three antibody combinations according to actual conditions and needs, and flexible arrangement of the intracellular tube membrane rupture fixation time according to the experimental time, and stand at 2-8°C in the dark for 30min-24h.

[0050] The present invention provides a multi-antibody combination kit with 10 colors in 3 tubes in total, which is used for the detection of T cell immune functional subgroups, and comprehensively and systematically performs fine typing of the immune phenotypes of each T cell subgroup in the bone marrow of MM patients. Not only does it use multiple antigens to flexibly set gates for T cells and use multiple antigen markers to group T cells, but it also includes grouping the differentiation subgroups, functional subgroups, and expression of multiple immune checkpoint proteins under each subgroup of T cells, comprehensively and specifically evaluating the patient's immune status, and at the same time grouping the expression of immune checkpoints one by one, better guiding the treatment of clinical anti-tumor immunity, and giving patients more choices and greater possibilities. BRIEF DESCRIPTION OF THE DRAWINGS

[0051] Figure 1 This is a diagram showing the T cell classification results of the first antibody combination of Example 2 ( Figure 1-1 to 1-3);

[0052] Figure 2This is the result diagram of the helper T cell subsets and immune checkpoints of the first antibody combination of Example 2 ( Figure 1-4 to 1-10);

[0053] Figure 3 The cytotoxic T cell subsets and immune checkpoint results of the first antibody combination of Example 2 ( Figure 1-11 to 1-17);

[0054] Figure 4 The T cell classification result diagram of the second antibody combination of Example 2 ( Figure 2-1 to 2-3);

[0055] Figure 5 The result diagram of the helper T cell subsets and immune checkpoints of the second antibody combination of Example 2 ( Figure 2-4 to 2-15);

[0056] Figure 6 The cytotoxic T cell subsets and immune checkpoint results of the second antibody combination of Example 2 ( Figure 2-16 to 2-25);

[0057] Figure 7 This is a diagram showing the T cell classification results of the third antibody combination of Example 2 ( Figure 3-1 to 3-3);

[0058] Figure 8 This is the result diagram of the helper T cell subsets and immune checkpoints of the third antibody combination of Example 2 ( Figure 3-4 to 3-11);

[0059] Fig. 9 The cytotoxic T cell subsets and immune checkpoint results of the third antibody combination of Example 2 ( Figure 3-12 to 3-19);

[0060] Fig.10 This is the T cell classification result diagram of the first antibody combination of Example 3MM initial diagnosis ( Figure 1-1 to 1-3);

[0061] Fig.11 This is the result diagram of the helper T cell subsets and immune checkpoints of the first antibody combination in the initial diagnosis of MM in Example 3 ( Figure 1-4 to 1-10);

[0062] Fig.12 The cytotoxic T cell subsets and immune checkpoint results of the first antibody combination in the initial diagnosis of MM in Example 3 ( Figure 1-11 to 1-17);

[0063] Fig.13This is the T cell classification result diagram of the second antibody combination in the initial diagnosis of MM in Example 3 ( Figure 2-1 to 2-3);

[0064] Fig.14 The result diagram of the helper T cell subsets and immune checkpoints of the second antibody combination in the initial diagnosis of MM in Example 3 ( Figure 2-4 to 2-15);

[0065] Fig.15 The cytotoxic T cell subsets and immune checkpoint results of the second antibody combination in the initial diagnosis of MM in Example 3 ( Figure 2-16 to 2-25);

[0066] Fig.16 This is the T cell classification result diagram of the third antibody combination in the initial diagnosis of Example 3MM ( Figure 3-1 to 3-3);

[0067] Fig.17 This is the result diagram of the helper T cell subsets and immune checkpoints of the third antibody combination in the initial diagnosis of Example 3MM ( Figure 3-4 to 3-11);

[0068] Fig.18 The cytotoxic T cell subsets and immune checkpoint results of the third antibody combination in the initial diagnosis of Example 3MM ( Figure 3-12 to 3-19);

[0069] Fig.19 This is the T cell classification result diagram of the first antibody combination after MM remission in Example 3 ( Figure 1-1 to 1-3);

[0070] Fig. 20 This is the result diagram of the helper T cell subsets and immune checkpoints of the first antibody combination after MM remission in Example 3 ( Figure 1-4 to 1-10);

[0071] Fig.21 The results of the first antibody combination of cytotoxic T cell subsets and immune checkpoints after MM remission in Example 3 are shown in Figure ( Figure 1-11 to 1-17);

[0072] Fig. 22 This is the T cell classification result diagram of the second antibody combination after MM remission in Example 3 ( Figure 2-1 to 2-3);

[0073] Fig.23 This is the result diagram of the helper T cell subsets and immune checkpoints of the second antibody combination after MM remission in Example 3 ( Figure 2-4 to 2-15);

[0074] Fig.24 The results of the cytotoxic T cell subsets and immune checkpoints of the second antibody combination after MM remission in Example 3 ( Figure 2-16 to 2-25);

[0075] Fig.25 This is the T cell classification result diagram of the third antibody combination after MM remission in Example 3 ( Figure 3-1 to 3-3);

[0076] Fig.26 This is the result diagram of the helper T cell subsets and immune checkpoints of the third antibody combination after MM remission in Example 3 ( Figure 3-4 to 3-11);

[0077] Fig. 27 The cytotoxic T cell subsets and immune checkpoint results of the third antibody combination after MM remission in Example 3 ( Figure 3-12 to 3-19). DETAILED DESCRIPTION

[0078] Embodiments of the present invention are described in detail below, and the examples of the embodiments are shown in the accompanying drawings, wherein the same or similar reference numerals represent the same or similar elements or elements with the same or similar functions from beginning to end. The embodiments described below by reference to the accompanying drawings are exemplary, are intended to be used to explain the present invention, and are not to be construed as limitations of the present invention. Those who do not indicate specific techniques or conditions in the embodiments are carried out according to the techniques or conditions described in the documents in this area or according to the product specification. Those who do not indicate the manufacturer of reagents or instruments used are all conventional products that can be obtained commercially.

[0079] The detection steps are as follows:

[0080] 1. Specimen preparation: Mix the patient's bone marrow blood anticoagulated with sodium heparin thoroughly, count the number of cells per microliter of the bone marrow specimen using a bovine abalone counting plate, and adjust the cell concentration to 2×10 with 1XPBS. 6 / 100μL to prepare a single cell suspension.

[0081] 2. Cell membrane surface antigen staining: take 3 flow-through sample tubes, number the tubes 1-2-3 and the corresponding names; use the first antibody combination and the second antibody combination for cell membrane surface antigen staining, add the corresponding fluorescent-labeled antibody premix for cell membrane labeling in the combination according to Table 1 to each tube (3 tubes, tube 1 is added with the antibody combination of group 1 in Table 1, tube 2 is added with the antibody combination of group 2 in Table 1, and tube 3 is added with part of the antibody combination of group 3 in Table 1: anti-CD3, anti-CD8, anti-CD28, anti-CD39, anti-CD57, anti-PD-1, anti-TIGIT antibodies)) and 100 μL of diluted bone marrow specimen, shake well to mix, and incubate at room temperature in the dark for 15 minutes.

[0082] Table 1 Antibodies in each group and their dosage (μl)

[0083]

[0084]

[0085] Note: The above antibody amount is the optimal amount determined after concentration gradient verification of commercial antibodies. Take the above amount of monoclonal antibodies and put them into the flow cytometry tubes numbered 1, 2, and 3 respectively.

[0086] The above antibodies are all commercial antibodies, and the mixed solutions of various antibodies refer to the mixing ratios of the various antibodies when their potencies are substantially equivalent.

[0087] 3. Hemolysis: After taking out, add 2 ml of 1×FACS hemolysin, mix well by low-speed vortexing, stand at room temperature in the dark for 8 minutes, then centrifuge and wash at 1500 rpm for 5 minutes, and discard the supernatant.

[0088] 4. Washing: Add 2 ml of 1×PBS buffer to the cell membrane surface antigen tubes (i.e. tube 1 and tube 2), shake and mix, centrifuge at 1500 rpm for 5 minutes, and discard the supernatant. Add 300 μL of 1×PBS to resuspend the cells (if there are clumps, flocs or sediments in the specimen, filter with a 400-mesh filter), and place in a refrigerator at 2-8°C to wait for the machine to measure.

[0089] 5. Intracellular tube membrane fixation: After the intracellular antigen staining of the third antibody combination is connected to the hemolysis in step 3 (i.e. tube 3 after step 3), add 1 mL of 1x membrane permeabilization agent, mix by low-speed vortexing, and stand at 2-8°C in the dark for 30 min to 24 h.

[0090] 6. Washing of intracellular tubes: After standing, take out and add 2 mL of 1×PBS, shake and mix, centrifuge at 2000 rpm for 5 minutes, and discard the supernatant.

[0091] 7. Intracellular antibody staining: Add the intracellular marker antibodies in Table 1 (i.e., add part of the antibody combination of Group 3 in Table 1 to Tube 3: anti-cTCF-1, anti-cTOX, and anti-cKi-67 antibodies), vortex and mix thoroughly, and incubate at room temperature in the dark for 20 minutes;

[0092] 8. Washing of intracellular tubes: Add 2 mL of 1×PBS, shake and mix, centrifuge at 1500 rpm for 5 minutes, and discard the supernatant. Add 300 μL of 1×PBS to suspend cells (if there are clumps, flocs or sediments in the specimen, filter with a 400-mesh filter), and place in a refrigerator at 2-8°C and wait for measurement on the machine.

[0093] That is, tube No. 1 and tube No. 2 are both put on the machine for inspection and data analysis after the above steps 1-4; tube No. 3 is put on the machine for inspection and data analysis after the above steps 1-3, 5-8.

[0094] Data acquisition steps:

[0095] Instrument setup, calibration and quality control: BD FACSanto flow cytometer was turned on to preheat and the fluid flow system was turned on; quality control product CST (BD FACSDiva TM CS@T Research Beads) run baseline and instrument performance test; after all pass, prepare single staining samples through the fluorescent color matching of this kit to determine the most suitable voltage and compensation. Establish a combination template for the antibody expression pattern of healthy people: Use the flow cytometry detection data of healthy people, set gates to group cells, use FSC-H / SSC-A to set gates to circle the target lymphocyte population, and then analyze the expression of each fluorescent antibody in this cell population. The first antibody combination selects the following antibody pairs: CD8 / CD3, FSC-H / CD28, FSC-H / CD39, FSC-H / CD57, CD45RA / CCR7, CD57 / CD28, PD-1 / T IGIT, FSC-H / CD95; the second antibody combination selected the following antibody pairs: CD3 / CD8, FSC-H / CD36, FSC-H / CD39, FSC-H / CD45RA, NRP1 / PD-1, CD25 / CD127, FSC-H / CCR8; the third antibody combination selected the following antibody pairs: CD3 / CD4, CD39 / Ki-67, cTCF-1 / cTOX, CD57 / CD28, PD-1 / TIGIT.

[0096] According to the set instrument conditions, obtain 100,000 cells per tube. If the test cannot be carried out on the machine in time, add 0.5 ml of 1% paraformaldehyde, mix well and store in a refrigerator at 2-8°C. Complete the test within 24 hours.

[0097] Data analysis system: Flowjo software is used for data analysis

[0098] The gating logic of the first antibody combination is to first set the FSC-H / FSC-A live cell gate to remove the adherent doublet cells; display the live cell gate and circle the lymphocytes by FSC-H / SSC-A; establish a CD3 / CD8 scatter plot under the lymphocyte gate to circle CD3+CD8+ T cells and CD3+CD8- CD4+ T cells; establish CD28+ / -, CD39+, CD57+ / -, CD45RA / CCR7 cross gate, CD57 / CD28 cross gate, PD-1 / TIGIT cross gate and

[0099] Scatter plot of helper T lymphocyte subsets of CD45RA+CCR7+CD95+; establish CD28+ / -, CD39+, CD57+ / -, CD45RA / CCR7 cross gate, CD57 / CD28 cross gate, PD-1 / TIGIT cross gate and

[0100] Scatter plot of cytotoxic T cell subsets of CD45RA+CCR7+CD95+. The logic gate method of immunophenotyping of the first antibody combination is shown in Figures 1 to 3 Shown is the flow cytometry of the primary antibody combination. Figure 1 This is a graph of T cell classification results for the first antibody combination; Figure 2 This is the result diagram of the helper T cell subsets and their immune checkpoints of the first antibody combination; Figure 3 This is the result diagram of cytotoxic T cell subsets and their immune checkpoints in combination with the first antibody.

[0101] The gating logic of the second antibody combination is to first set the FSC-H / FSC-A live cell gate to remove adherent doublet cells; display the live cell gate and circle the lymphocytes by FSC-H / SSC-A; establish a CD3 / CD4 scatter plot under the lymphocyte gate to circle CD3+CD4+ T cells and CD3+CD4- CD8+ T cells; establish a CD36+, CD39+, CD45RA+ / CD45RA-, NRP1 / PD-1 cross gate, Treg(CD4+CD25+CD127low / -)+ / -, Treg+CCR8+ scatter plot and a helper T cell subset scatter plot under the CD4+ T cell gate; establish a CD36+, CD39+, CD45RA+ / CD45RA-, NRP1 / PD-1 cross gate scatter plot and a cytotoxic T cell subset scatter plot under the CD8+ T cell gate. The logic gating method for immunophenotyping of the second antibody combination is shown in Figures 4 to 6 Shown is a flow cytometric plot of the secondary antibody combination. Figure 4 This is a graph of T cell classification results for the second antibody combination; Figure 5 This is a graph showing the helper T cell subsets and their immune checkpoint results for the second antibody combination; Figure 6 This is a clustering diagram of cytotoxic T cell subsets and their immune checkpoints in combination with the second antibody.

[0102] The gating logic of the third antibody combination is to first set the FSC-H / FSC-A live cell gate to remove the adherent doublet cells; display the live cell gate and circle the lymphocytes by FSC-H / SSC-A; establish a CD3 / CD8 scatter plot under the lymphocyte gate to circle CD3+CD8+ T cells and CD3+CD8- CD4+ T cells. Under the CD4+ T cell gate, establish CD39+, cKi67+, cTCF-1+, cTOX+ gates and CD28 / CD57 cross gates, PD-1 / TIGIT cross gates, and helper T cell subset scatter plots; under the CD8+ T cell gate, establish CD39+, cKi-67+, cTCF-1+, cTOX+, CD28 / CD57 cross gates, PD-1 / TIGIT cross gates, and cytotoxic T cell subset scatter plots. The immunophenotyping logic gating method of the third antibody combination is shown in Figure 7-9 Shown is the flow cytometry of the third antibody combination. Figure 7 This is a graph of T cell classification results for the third antibody combination;

[0103] Figure 8 This is a graph showing the helper T cell subsets and their immune checkpoint results for the third antibody combination; Fig. 9 This is a clustering diagram of cytotoxic T cell subsets and their immune checkpoints for the third antibody combination.

[0104] The present invention provides a method and kit for detecting the immunophenotypes of each T cell subpopulation in the bone marrow of MM patients before and after treatment by flow cytometry. Those skilled in the art can refer to the scheme of the present invention to make appropriate changes and combinations.

[0105] The present invention provides a method and a kit for detecting the immunophenotypes of each subgroup of T lymphocytes in the bone marrow of MM patients before and after treatment by flow cytometry. The reagents used in the kit without indicating the manufacturer can be purchased from the regular channel market.

[0106] The fluorescent monoclonal antibodies used in the present invention are CD3-Percp-cy5.5 No. 300428, CD4-Alexa Flour700 No. 300526, CD8-Alexa Flour No. 301028, CD28-FITC No. 302906, CD36-FITC No. 336204, CD95-PE No. 305608, CCR8-PE No. 360604, cTCF-1-PE No. 655208, CD45RA-APC No. 304112, CD39-APC-cy7 No. 328226, CCR7-BV421 No. 353208, CD25-BV421 No. 302630, CD57-BV510 No. 393314, CD127-BV510 No. 3513 32. TIGIT-BV605, Catalog No. 372712, NRP1-BV605, Catalog No. 354532 were purchased from Biolegend; cTOX-APC, Catalog No. 568356, PD-1-PE-cy7, Catalog No. 561272, cKi-67-BV421, Catalog No. 561281 were all purchased from BD; 10×PBS buffer, Catalog No. LR-0106A, and flow cytometry-specific red blood cell lysis buffer (FACS hemolysin), Catalog No. 349202 were purchased from BD; cell membrane disrupter, Catalog No. 00-5123-43, was purchased from eBioscience; BD FACSDiva TM CS@T Research Beads (CST) catalog number 655050 were purchased from BD.

[0107] Example 1 Preparation of kit or detection reagent

[0108] Kit includes:

[0109] The first antibody combination includes the following antibodies: anti-CD3; anti-CD45RA; anti-CD39; anti-CD57; anti-CCR7; anti-CD28; anti-CD95; anti-TIGIT; anti-PD-1; anti-CD8. The corresponding fluorescein labels of the combination antibodies are in order: Percp-cy5.5, APC, APC-cy7, BV510, BV421, FITC, PE, BV605, PE-cy7, AF-700. These antibody combinations are used in one flow tube during detection. The volume usage ratio of these antibodies is: 3μl: 2μl: 3μl: 2μl: 2.5μl: 2μl: 2μl: 2μl: 2μl: 2μl: 3μl; specifically, for example, it can be CD3, 3μl; CD45RA, 2μl; CD39, 3μl; CD57, 2μl; CCR7, 2.5μl; CD28, 2μl; CD95, 2μl; TIGIT, 2μl; PD-1, 2μl; CD8, 3μl.

[0110] The second antibody combination includes the following antibodies: anti-CD3; anti-CD45RA; anti-CD39; anti-CD127; anti-CD25; anti-CD36; anti-CCR8; anti-NRP1; anti-PD-1; anti-CD4 antibodies, and the corresponding fluorescent markers of the combination antibodies are in the following order: Percp-cy5.5, APC, APC-cy7, BV510, BV421, FITC, PE, BV605, PE-cy7, AF-700. These antibody combinations are used in one flow tube during detection. The volume usage ratio of these antibodies is: 3μl: 2μl: 3μl: 2μl: 2μl: 2μl: 2μl: 2μl: 2μl: 3μl; specifically, for example, it can be CD3, 3μl; CD45RA, 2μl; CD39, 3μl; CD127, 2μl; CD25, 2μl; CD36, 2μl; CCR8, 2μl; NRP1, 2μl; PD-1, 2μl; CD4, 3μl.

[0111] The third group of antibodies includes anti-cell membrane antigen antibodies and anti-intracellular antigen antibodies; the anti-cell membrane antigen antibodies are composed of the following antibodies: anti-CD3, anti-CD8, anti-CD28, anti-CD39, anti-CD57, anti-PD-1, and anti-TIGIT antibodies; the anti-intracellular antigen antibodies are composed of the following antibodies: anti-cTCF-1, anti-cTOX, and anti-cKi-67 antibodies; the corresponding fluorescent markers of the combined antibodies are in order: Percp-cy5.5, APC-R700, FITC, APC-cy7, V500, PE-cy7, BV605, PE, APC, and BV421. The third antibody combination is used in one flow tube during detection. Among them, the volume usage ratio of anti-CD3; anti-cTOX; anti-CD39; anti-CD57; anti-cKi-67; anti-CD28; anti-cTCF-1; anti-TIGIT; anti-PD-1; and anti-CD8 antibodies is: 3μl: 3μl: 3μl: 2μl: 3μl: 2μl: 3μl: 2μl: 2μl: 3μl; specifically, for example, it can be: CD3, 3μl; cTOX, 3μl; CD39, 3μl; CD57, 2μl; cKi-67, 3μl; CD28, 2μl; cTCF-1, 3μl; TIGIT, 2μl; PD-1, 2μl; CD8, 3μl.

[0112] It may also include hemolysin, membrane permeabilizing agent and phosphate buffered saline (PBS), which are all commercially available.

[0113] Example 2

[0114] In this example, a normal bone marrow specimen was first selected to perform the flow cytometry T cell immune function assay of the present invention.

[0115] 1. Specimen preparation: Mix the heparin sodium anticoagulated bone marrow specimen thoroughly, count the number of cells per microliter of the bone marrow specimen using a bulla counting plate, and adjust the cell concentration to 2×10 with 1XPBS. 6 / 100μL.

[0116] 2. Cell membrane surface antigen staining: take 3 flow-through sample tubes and number the tubes 1-2-3; use the first antibody combination and the second antibody combination for cell membrane surface antigen staining, add the corresponding fluorescent-labeled antibody premix for cell membrane labeling in the combination according to Table 1 to each tube (3 tubes, tube 1 is added with the antibody combination of group 1 in Table 1, tube 2 is added with the antibody combination of group 2 in Table 1, and tube 3 is added with part of the antibody combination of group 3 in Table 1: anti-CD3, anti-CD8, anti-CD28, anti-CD39, anti-CD57, anti-PD-1, anti-TIGIT antibodies) and 100 μL of diluted bone marrow specimen, shake well to mix, and incubate at room temperature in the dark for 15 minutes.

[0117] 3. Hemolysis: After taking out, add 2 ml of 1×FACS hemolysin, mix well by low-speed vortexing, stand at room temperature in the dark for 8 minutes, then centrifuge and wash at 1500 rpm for 5 minutes, and discard the supernatant.

[0118] 4. Washing: Add 2 ml of 1×PBS buffer to the cell membrane surface antigen tubes (i.e. tube 1 and tube 2), shake and mix, centrifuge at 1500 rpm for 5 minutes, and discard the supernatant. Add 300 μl of 1×PBS to suspend the cells (if there are clumps, flocs or sediments in the specimen, filter them with a 400-mesh filter), and place them in a refrigerator at 2-8°C and wait for the machine to measure.

[0119] 5. Intracellular tube membrane fixation: After the intracellular antigen staining of the third antibody combination is connected to the hemolysis in step 3 (i.e. tube 3 after step 3), add 1 mL of 1x membrane permeabilization agent, mix by low-speed vortexing, and stand at 2-8°C in the dark for 30 min to 24 h.

[0120] 6. Washing of intracellular tubes: After standing, take out and add 2 mL of 1×PBS, shake and mix, centrifuge at 2000 rpm for 5 minutes, and discard the supernatant;

[0121] 7. Intracellular antibody staining: Add the intracellular marker antibodies in Table 1 (i.e., add part of the antibody combination of Group 3 in Table 1 to Tube 3: anti-cTCF-1, anti-cTOX, and anti-cKi-67 antibodies), vortex and mix thoroughly, and incubate at room temperature in the dark for 20 minutes;

[0122] 8. Washing of intracellular tubes: Add 2 mL of 1×PBS, shake and mix, centrifuge at 1500 rpm for 5 minutes, and discard the supernatant. Add 300 μl of 1×PBS to suspend cells (if there are clumps, flocs or sediments in the specimen, filter with a 400-mesh filter), and place in a refrigerator at 2-8°C and wait for measurement on the machine.

[0123] That is: tube 1 and tube 2 are both put on the machine for testing and data analysis after the above steps 1-4; tube 3 is put on the machine for testing and data analysis after the above steps 1-3, 5-8;

[0124] 9. On-machine testing: Turn on the BD FACSanto flow cytometer to preheat and start the liquid flow system. Use the quality control product CST (BDFACSDiva TM CS@T Research Beads) run baseline and instrument performance test, after all passed, establish a combination template of healthy people's antibody expression pattern (use the flow cytometry test data of healthy people, set gates to group cells, use FSC-H / SSC-A to set gates to circle the target lymphocyte population, and then analyze the expression of each fluorescent antibody for this cell population. The first antibody combination selects the following antibody pairs: CD8 / CD3, FSC-H / CD28, FSC-H / CD39, FSC-H / CD57, CD45RA / CCR7, CD57 / CD28, PD-1 / TIGIT, FSC-H / CD95; the second antibody combination selects the following antibody pairs: CD3 / CD8, FSC-H / CD36, FSC-H / CD39, FSC-H / CD45RA, NRP1 / PD-1, CD25 / CD127, FSC-H / CCR8; the third antibody combination selects the following antibody pairs: CD3 / CD4, CD39 / Ki-67, cTCF-1 / cTOX, CD57 / CD28, PD-1 / TIGIT). Each time the sample is loaded, the data is collected. Determine the appropriate voltage and compensation, and prepare the single-stained sample according to the fluorescent color matching of this kit for instrument setting. Use the combination template with established healthy human antibody expression pattern as the loading template. According to the set instrument conditions, obtain 100,000 cells per tube. If the machine cannot be tested in time, add 0.5ml 1% paraformaldehyde, mix well and store in a refrigerator at 2-8℃, and complete the test within 24h.

[0125] 10. Data analysis: Copy the detected data from the instrument and use Flowjo software for data analysis.

[0126] The gating logic of the first antibody combination is to first set the FSC-H / FSC-A live cell gate to remove the adherent doublet cells; display the live cell gate and circle the lymphocytes by FSC-H / SSC-A; establish a CD3 / CD8 scatter plot under the lymphocyte gate to circle CD3+CD8+ T cells and CD3+CD8- CD4+ T cells; establish CD28+ / -, CD39+, CD57+ / -, CD45RA / CCR7 cross gate, CD57 / CD28 cross gate, PD-1 / TIGIT cross gate and

[0127] Scatter plot of helper T lymphocyte subsets of CD45RA+CCR7+CD95+; establish CD28+ / -, CD39+, CD57+ / -, CD45RA / CCR7 cross gate, CD57 / CD28 cross gate, PD-1 / TIGIT cross gate and

[0128] Scatter plot of CD45RA+CCR7+CD95+ cytotoxic T cell subsets.

[0129] The immunophenotyping logic gating method of the first antibody combination is shown in Figure 1-Figure 3 , the first antibody combination cell types are:

[0130] CD3+CD4 / 8+CD28+ represents T cell functional subsets; CD3+CD4 / CD8+CD28- represents T cell regulatory subsets; CD3+CD4 / 8+CD57+ represents the marker of T cell aging; CD3+CD4 / 8+CD45RA+CCR7+ represents naive T cells (TN cells); CD3+CD4 / 8+CD45RA-CCR7+ represents central memory T cells (TCM cells); CD3+CD4+ / 8+CD45RA-CCR7- represents effector Memory T cells (TEM cells); CD3+CD4+ / 8+CD45RA+CCR7- represents terminally differentiated effector memory T cells (TEMRA cells); CD3+CD4 / 8+CD45RA+CCR7+CD95+ represents T memory stem cells (TSCM cells); CD3+CD4 / 8+ combined with CD39, PD-1+ / -, and TIGIT+ / - represent the immune suppression of immune target CD39 and immune checkpoints PD-1 and TIGIT in T cells. See Table 2 for specific experimental results.

[0131] Table 2 Immunophenotyping results of the first antibody combination

[0132]

[0133]

[0134] The gating logic of the second antibody combination is to first set the FSC-H / FSC-A live cell gate to remove adherent doublet cells; display the live cell gate and circle the lymphocytes by FSC-H / SSC-A; establish a CD3 / CD4 scatter plot under the lymphocyte gate to circle CD3+CD4+ T cells and CD3+CD4- CD8+ T cells; establish a CD36+, CD39+, CD45RA+ / CD45RA-, NRP1 / PD-1 cross gate, Treg(CD4+CD25+CD127low / -)+ / -, Treg+CCR8+ scatter plot and a helper T cell subset scatter plot under the CD4+ T cell gate; establish a CD36+, CD39+, CD45RA+ / CD45RA-, NRP1 / PD-1 cross gate scatter plot and a cytotoxic T cell subset scatter plot under the CD8+ T cell gate. The logic gating method for immunophenotyping of the second antibody combination is shown in Figure 4-Figure 6 , the second antibody combination cell types are:

[0135] CD3+CD4 / 8+CD45RA+ / -CD36+ represents the immunosuppression of the immune target CD36 in the initial or memory T cells; CD3+CD4 / 8+CD45RA+ / -CD39+ represents the immunosuppression of the immune target CD39 in the initial or memory T cells;

[0136] The combination of CD3+CD4 / 8+CD45RA+ / - with NRP1+ / - and PD-1+ / - respectively represents the immune suppression of the immune checkpoints NRP1 and PD-1 in the initial or memory T cells; CD3+CD4+CD25+CD127low / - represents regulatory T cells (Treg cells); CCR8 is an immunosuppressive regulatory protein on the surface of Treg cells, and Treg+CCR8+ represents the immune response regulation of Treg cells. The specific experimental results are shown in Table 3.

[0137] Table 3 Immunophenotyping results of the second antibody combination

[0138]

[0139]

[0140] The gating logic of the third antibody combination is to first set the FSC-H / FSC-A live cell gate to remove the adherent doublet cells; display the live cell gate and circle the lymphocytes by FSC-H / SSC-A; establish a CD3 / CD8 scatter plot under the lymphocyte gate to circle CD3+CD8+ T cells and CD3+CD8- CD4+ T cells. Under the CD4+ T cell gate, establish CD39+, cKi67+, cTCF-1+, cTOX+ gates and CD28 / CD57 cross gates, PD-1 / TIGIT cross gates, and helper T cell subset scatter plots; under the CD8+ T cell gate, establish CD39+, cKi-67+, cTCF-1+, cTOX+, CD28 / CD57 cross gates, PD-1 / TIGIT cross gates, and cytotoxic T cell subset scatter plots. The immunophenotyping logic gating method of the third antibody combination is shown in Figure 7-Figure 9 , the third antibody combination cell types are:

[0141] CD3+CD4 / 8+cKi-67+ represents the activation and proliferation of T cells; CD3+CD4 / 8+cTCF-1+ represents the stem cell characteristics of T cells, which has the effect of resisting T cell exhaustion; CD3+CD4 / 8+cTOX+ represents the exhaustion of T cells; CD3+CD4 / 8+CD28-CD57+ represents terminally differentiated effector memory T cells (TEMRA cells);

[0142] CD3+CD4 / 8+CD28+CD57+ represents effector memory T cells (TEM cells); CD3+CD4 / 8+CD28+CD57- represents central memory T cells (TCM cells); CD3+CD4 / 8+CD28-CD57- represents T cell regulatory subpopulations; the CD39+, cTOX+, cKi-67+, and cTCF-1+ subpopulations under these subpopulations represent their exhaustion, proliferation, and anti-exhaustion conditions.

[0143] The specific experimental results are shown in Table 4.

[0144] Table 4 Immunophenotyping results of the third antibody combination

[0145]

[0146]

[0147] From Table 2-4 and Figure 1-3 It can be known that the proportion of each T cell subset and the differentiation pattern of each antigen in normal bone marrow specimens. Although the proportion of each T cell subset is not fixed, the antigen expression of each differentiated subset and the combination of two of them form a regular differentiation pattern, and MM patients can conduct corresponding experiments based on this.

[0148] Example 3

[0149] The flow cytometry T cell immune function assay of the present invention was performed on bone marrow specimens of a patient with MM at the initial diagnosis and after remission.

[0150] Specimens: bone marrow of patients with newly diagnosed MM and patients after remission. The steps and reagents are the same as those in Example 2.

[0151] See Tables 5-7 for specific experimental results and see Tables 5-7 for specific gating logic of flow cytometry for initial diagnosis of bone marrow samples. Figure 10-18 , bone marrow samples after remission Figure 19-27 .

[0152] Table 5 Immunophenotyping results of the first antibody combination

[0153]

[0154]

[0155] The specific experimental results of immunophenotyping of the first antibody combination are shown in Table 5. As can be seen from Table 5, the proportion of lymphocytes in remission patients increased; the proportion of CD3+CD4 / CD8+CD28-T cell regulatory subsets increased; the proportion of CD3+CD8+CD57+, a marker representing Tc cell senescence, increased; the proportion of CD3+CD4 / 8+CD45RA+CCR7+TN cells increased; the proportion of CD3+CD4+ / 8+CD45RA+CCR7- and CD3+CD4 / 8+CD28-CD57+ TEMRA cells increased; and the proportion of CD3+CD4 / 8+CD45RA-CCR7+ and CD3+CD4 / 8+CD28-CD57+ TEMRA cells increased. The proportion of 4 / 8+CD28+CD57+ TCM cells decreased; the proportion of CD3+CD4+ / 8+CD45RA-CCR7-TEM cells decreased; the proportion of CD3+CD4 / 8+CD45RA+CCR7+CD95+TSCM cells decreased; and the combination of CD3+CD4 / 8+ with CD39, PD-1+ / -, and TIGIT+ / -, respectively, represented that the changes in the proportion of T cell immunosuppression of the immune checkpoints CD39, PD-1 and TIGIT were different, especially the proportion of the immune checkpoint PD-1 was significantly decreased.

[0156] Table 6 Immunophenotyping results of the second antibody combination

[0157]

[0158]

[0159] The specific experimental results of the immunophenotyping of the second antibody combination are shown in Table 6. As can be seen from Table 6, the proportion of lymphocytes in remission patients increased; the proportion of immune target CD36 in each T cell subset increased; the proportion of immune target CD39 in Th cells increased; the proportion in Tc cells decreased, and the changes in each Tc cell subset were different; the proportion of Treg cell subsets increased, while Treg+CCR8+ represented a decrease in the proportion of immune response regulation subsets of Treg cells.

[0160] The combination of CD3+CD4 / 8+CD45RA+ / - with NRP1+ / - and PD-1+ / -, respectively, represents that the changes in the proportions of immune checkpoints NRP1 and PD-1 in the initial or memory T cell immunosuppression are different. In particular, it was found that the proportions of NRP1 and PD-1 on various T cell subsets were significantly decreased.

[0161] Table 7 Immunophenotyping results of the third antibody combination

[0162]

[0163]

[0164] The specific experimental results of the immunophenotyping of the third antibody combination are shown in Table 7. As can be seen from Table 7, the proportion of lymphocytes in remission patients increased; the proportion of TEMRA cells with CD3+CD4 / 8+CD28-CD57+ increased; the proportion of TCM cells with CD3+CD4 / 8+CD28+CD57+ decreased; the proportion of immune target CD39 in Th cells increased; the proportion in Tc cells decreased; the proportion of cell proliferation antigen cKi-67 in T cells increased; the proportion of transcription factor cTCF-1 in T cells did not change much; the proportion of transcription checkpoint cTOX in T cells decreased;

[0165] The proportions of CD39+, cTOX+, cKi-67+, and cTCF-1+ in these subgroups varied. In particular, the proportions of cKi-67 and cTCF-1 in each subgroup increased, while the expression of cTOX in CD4+ TEMRA cells increased significantly, while that in CD8+ TEMRA cells decreased.

[0166] From the above results, we can see that the proportion of T cells in this patient increased after treatment remission, while T cells tended to be aged and exhausted; the immune checkpoint clustering results under each T cell subpopulation had the same or different trends, which could enable fine-grained classification of each T cell subpopulation.

[0167] In summary, the analysis method provided by the present invention can perform comprehensive and systematic fine typing of the T cell immune phenotype in the bone marrow of MM patients before and after treatment. It is easy to operate and takes a short time, and can achieve continuous dynamic monitoring of the immune function of MM patients. The method has a flexible gating logic. While grouping the lymphocyte subpopulations, it also has grouping of multiple immune examination targets, which provides good guidance for clinical anti-tumor immune treatment and also provides a clear basis for related clinical drug treatment.

[0168] Although the embodiments of the present invention have been shown and described above, it is to be understood that the above embodiments are exemplary and are not to be construed as limitations on the present invention. A person skilled in the art may change, modify, replace and modify the above embodiments within the scope of the present invention without departing from the principles and intent of the present invention.

Claims

1. A combination of antibodies for detecting T lymphocyte immunophenotypes of MM patients before and after treatment by flow cytometry, characterized in that: including a first antibody combination, a second antibody combination and a third antibody combination; The first antibody combination is anti-CD3, anti-CD45RA, anti-CD39, anti-CD57, anti-CCR7, anti-CD28, anti-CD95, anti-TIGIT, anti-PD-1 and anti-CD8; the corresponding fluorescein labels of these antibodies are in order: Percp-cy5.5, APC, APC-cy7, BV510, BV421, FITC, PE, BV605, PE-cy7, AF-700; preferably, the volume usage ratio of these antibodies is: 3μl: 2μl: 3μl: 2μl: 2μl: 2.5μl: 2μl: 2μl: 2μl: 2μl: 2μl: 3μl; The second antibody combination includes the following antibodies: anti-CD3, anti-CD45RA, anti-CD39, anti-CD127, anti-CD25, anti-CD36, anti-CCR8, anti-NRP1, anti-PD-1, and anti-CD4 antibodies. The corresponding fluorescent labels of these antibodies are in the following order: Percp-cy5.5, APC, APC-cy7, BV510, BV421, FITC, PE, BV605, PE-cy7, and AF-700; the volume ratio of these antibodies is: 3μl: 2μl: 3μl: 2μl: 2μl: 2μl: 2μl: 2μl: 2μl: 2μl: 3μl; The third group of antibodies includes anti-cell membrane antigen antibodies and anti-intracellular antigen antibodies; the anti-cell membrane antigen antibodies are composed of the following antibodies: anti-CD3, anti-CD8, anti-CD28, anti-CD39, anti-CD57, anti-PD-1, and anti-TIGIT antibodies; the anti-intracellular antigen antibodies are composed of the following antibodies: anti-cTCF-1, anti-cTOX, and anti-cKi-67 antibodies; the corresponding fluorescent labels of these antibodies are in order: Percp-cy5.5, APC-R700, FITC, APC-cy7, V500, PE-cy7, BV605, PE, APC, and BV421.

2. The detection reagent according to claim 1, characterized in that In the first antibody combination, the volume ratio of anti-CD3, anti-CD45RA, anti-CD39, anti-CD57, anti-CCR7, anti-CD28, anti-CD95, anti-TIGIT, anti-PD-1 and anti-CD8 is: 3μl: 2μl: 3μl: 2μl: 2.5μl: 2μl: 2μl: 2μl: 2μl: 2μl: 3μl.

3. The detection reagent according to claim 1, characterized in that In the second antibody combination, the volume ratio of anti-CD3, anti-CD45RA, anti-CD39, anti-CD127, anti-CD25, anti-CD36, anti-CCR8, anti-NRP1, anti-PD-1, and anti-CD4 antibodies is: 3μl: 2μl: 3μl: 2μl: 2μl: 2μl: 2μl: 2μl: 2μl: 2μl: 3μl.

4. The detection reagent according to claim 1, characterized in that In the third antibody combination, the volume usage ratio of anti-CD3, anti-cTOX, anti-CD39, anti-CD57, anti-cKi-67, anti-CD28, anti-cTCF-1, anti-TIGIT, anti-PD-1, and anti-CD8 antibodies is: 3μl: 3μl: 2μl: 3μl: 2μl: 2μl: 2μl: 3μl: 3μl: 3μl: 3μl.

5. A kit for detecting the immunophenotype of T lymphocytes in MM patients before and after treatment by flow cytometry, characterized in that: A combination of antibodies comprising any one of claims 1 to 4.

6. The detection kit according to claim 5, characterized in that It also includes hemolysin, membrane permeabilization agent and phosphate buffered saline (PBS).

7. A method for detecting the immunophenotype of T lymphocytes of MM patients before and after treatment by flow cytometry, characterized in that: The detection antibody combination described in any one of claims 1 to 4 is used, or the detection kit described in claim 5 or 6 is used.

8. The method according to claim 7, characterized in that the step include: The detection steps are as follows: S1. Sample preparation: Mix the heparin sodium anticoagulated bone marrow blood of the patient to be tested thoroughly, count the number of cells per microliter of the bone marrow sample using a bulla counting plate, and adjust the cell concentration to 2×10 with 1XPBS. 6 / 100μL; S2. Take 3 flow-through sample tubes, add the first antibody combination to tube 1, the second antibody combination to tube 2, and the anti-cell membrane antigen antibody in the third antibody combination to tube 3, then add 100 μL of the diluted bone marrow specimen to each tube, shake well to mix, and incubate at room temperature away from light; S3. Hemolysis: Add 2 ml of 1× FACS hemolysin to each of the three tubes after incubation, vortex mix at low speed, stand at room temperature in the dark, centrifuge and wash, and discard the supernatant; S4. Washing: Add 2 ml of 1×PBS buffer to tube 1 and tube 2, shake and mix, centrifuge and wash, discard the supernatant; then add 300 μL of 1×PBS to resuspend the cells. If there are clumps, flocs or sediments in the specimen, filter them with a 400-mesh filter before adding, and place them in a refrigerator at 2-8°C and wait for the machine to measure; S5. Permeabilization and fixation of intracellular tubes: Add 1 mL of 1x permeabilization agent to tube 3, vortex mix at low speed, and place at 2-8℃ away from light; S6. Washing of intracellular tubes: After the resting of tube 3, add 2 mL of 1× PBS, shake and mix, centrifuge and wash, and discard the supernatant; S7. Intracellular antibody staining: Add the anti-intracellular antigen antibody in the third antibody combination to tube 3, shake well to mix, and incubate at room temperature in the dark; S8. Washing of intracellular tubes: Add 2 mL of 1×PBS to tube 3, shake and mix, centrifuge and wash, and discard the supernatant; add 300 μL of 1×PBS to resuspend the cells. If there are clumps, flocs or sediments in the specimen, filter it with a 400-mesh filter and place it in a refrigerator at 2-8°C to wait for the machine to measure; S9. On-machine testing: S10. Data analysis: Flowjo software was used for data analysis.

9. The method according to claim 8, characterized in that The S9. on-machine test includes instrument setting, calibration and quality control: the BD FACSanto flow cytometer is turned on for preheating and the liquid flow system is turned on. The baseline and instrument performance tests are run with quality control products. After all of them pass, single-stained samples are prepared according to the fluorescent color matching of this kit, and the appropriate voltage and compensation are determined. Establish a combined template for the antibody expression pattern of healthy people; According to the set instrument conditions, obtain 100,000 cells in each tube. If the test cannot be carried out on the machine in time, add 0.5 ml of 1% paraformaldehyde, mix well and store in a refrigerator at 2-8°C. Complete the test within 24 hours.

10. The method according to claim 8, characterized in that The S10 data analysis is as follows: The gating logic of the first antibody combination is to first set the FSC-H / FSC-A live cell gate to remove the adherent doublet cells; display the live cell gate and circle the lymphocytes by FSC-H / SSC-A; establish a CD3 / CD8 scatter plot under the lymphocyte gate to circle CD3+CD8+ T cells and CD3+CD8- CD4+ T cells; establish CD28+ / -, CD39+, CD57+ / -, CD45RA / CCR7 cross gates under the CD4+ T cell gate , CD57 / CD28 cross gate, PD-1 / TIGIT cross gate and CD45RA+CCR7+CD95+ helper T lymphocyte subset scatter plot; under the CD8+T cell gate, establish CD28+ / -, CD39+, CD57+ / -, CD45RA / CCR7 cross gate, CD57 / CD28 cross gate, PD-1 / TIGIT cross gate and CD45RA+CCR7+CD95+ cytotoxic T cell subset scatter plot; The gating logic of the second antibody combination is to first set the FSC-H / FSC-A live cell gate to remove the adherent doublet cells; display the live cell gate and circle the lymphocytes by FSC-H / SSC-A; establish a CD3 / CD4 scatter plot under the lymphocyte gate to circle CD3+CD4+ T cells and CD3+CD4- CD8+ T cells; establish a cross gate of CD36+, CD39+, CD45RA+ / CD45RA-, NRP1 / PD-1, a scatter plot of Treg(CD4+CD25+CD127low / -)+ / -, Treg+CCR8+ and a scatter plot of helper T cell subsets under the CD4+T cell gate; establish a cross gate scatter plot of CD36+, CD39+, CD45RA+ / CD45RA-, NRP1 / PD-1 and a scatter plot of cytotoxic T cell subsets under the CD8+T cell gate; The gating logic of the third antibody combination is to first set the FSC-H / FSC-A live cell gate to remove the adherent doublet cells; display the live cell gate and circle the lymphocytes by FSC-H / SSC-A; establish a CD3 / CD8 scatter plot under the lymphocyte gate to circle CD3+CD8+ T cells and CD3+CD8- CD4+ T cells. Under the CD4+ T cell gate, establish CD39+, cKi67+, cTCF-1+, cTOX+ gates and CD28 / CD57 cross gates, PD-1 / TIGIT cross gates and helper T cell subset scatter plots; under the CD8+ T cell gate, establish CD39+, cKi-67+, cTCF-1+, cTOX+, CD28 / CD57 cross gates, PD-1 / TIGIT cross gates and cytotoxic T cell subset scatter plots.

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