Molecular marker for immunotherapy prediction of targeting gonocyte and application of molecular marker

Abstract

The invention relates to a molecular marker for immunotherapy prediction of a targeting shower cell and application of the molecular marker, and belongs to the technical field of molecular biology. The invention provides a molecular marker for immunotherapy prediction of a targeting shower cell. The molecular marker comprises CD8, CD4, PD-1, Foxp3, CD19 and CD3, wherein the CD8, the CD4, the PD-1, the Foxp3, the CD19 and the CD3 are used for immunotherapy prediction; or comprises a CD45, a CD45RA, a CD45RO, a CD25, a CCR7 and a CD127; or comprises CD3, LAG3, Tim3, ICOS, CD69 and CD103; or comprises a CD79A, a CD79B, a CD20, a CD21 and a CD138; or the CD56, the CD161, the CD57, the CD94 and the Granzyme B are included; the invention relates to a CD56, CD161, CD57, CD94 and Granzyme B. The CD56, the CD161, the CD57, the CD94 and the Granzyme B. The molecular marker can accurately distinguish shower cell groups and interaction.

Description

Technical Field

[0001] The present invention relates to molecular markers for predicting immunotherapy targeting lymphoid cells and their applications, and belongs to the technical field of molecular biology. Background Art

[0002] Immunotherapy is a treatment method that, based on immunological principles, artificially enhances or inhibits the immune function of the body when the immune function is low or hyperactive, so as to achieve the purpose of treating diseases. There are many methods of immunotherapy, which are applicable to the treatment of a variety of diseases. Among them, tumor immunotherapy aims to activate the human immune system and rely on the body's own immune function to kill cancer cells and tumor tissues. For example, the immune checkpoint blockade therapy based on programmed death receptor and its ligand can inhibit the binding of the programmed death receptor and its ligand, thereby enhancing the aggressiveness of the host immune system against tumor cells, and then playing a role in treating tumors. Different from the previous surgery, chemotherapy, radiotherapy and targeted therapy, the target of immunotherapy is not tumor cells and tissues, but the body's own immune system, such as lymphoid cells.

[0003] Lymphoid cells are a type of white blood cells and an important part of the immune system. They play important roles in immune monitoring and immune response in the human body. Lymphoid cells can be divided into several categories, including T cells, B cells and natural killer cells (NK cells). These cells work together in the immune system to protect the body from pathogens and abnormal cells. The functions and subsets of lymphoid cells can be identified and classified by specific proteins (such as CD4, CD8, etc.) on the surface. These cells play important roles in processes such as immune monitoring, immune regulation and immune killing. The study of lymphoid cells helps to deeply understand the working principle of the immune system and provides important references for the prevention, diagnosis and treatment of diseases.

[0004] Lymphoid immune checkpoints refer to a series of molecules and their interaction networks that play key roles in the activation, proliferation and function regulation of lymphoid cells, especially T cells and B cells. These checkpoints are part of the fine regulation within the immune system, aiming to balance the effectiveness and tolerance of the immune response, avoid the occurrence of autoimmune diseases, and at the same time ensure the effective clearance of pathogens and abnormal cells. The dysregulation of lymphoid immune checkpoints, whether overactivation or inhibition, is closely related to a variety of disease states, including cancer, autoimmune diseases, chronic infections and allergic reactions, etc. Therefore, through the detection of lymphoid immune checkpoints, it is possible to well obtain information related to the changes in disease states after treatment based on lymphoid cell populations and their interactions, and then effectively predict the treatment effects of drugs or treatment regimens.

[0005] However, the existing detection of lymphoid immune checkpoints is very single, and the information on lymphoid immune checkpoints related to disease state changes that can be obtained is also very single, resulting in limited prediction effects and making it difficult to comprehensively and well predict and evaluate the efficacy of immunotherapeutic drugs targeting lymphoid cell immune checkpoints. For example, immunohistochemical detection of lymphoid immune checkpoints only targeting programmed death receptor 1 (PD-1) and programmed death ligand 1 (PD-L1) cannot comprehensively analyze the tumor microenvironment, and thus cannot effectively predict the tumor treatment effect of immunotherapeutic drugs targeting PD-1 / PD-L1. If sufficient clinical samples can be collected and sufficient immunohistochemical detection of different lymphoid immune checkpoints in the clinical samples can be carried out, it may be possible to achieve comprehensive and good efficacy prediction and evaluation of immunotherapeutic drugs targeting lymphoid cell immune checkpoints. However, for clinical samples, tissue samples are particularly precious, and traditional immunohistochemical techniques are relatively wasteful of samples. At the same time, immunohistochemical analysis belongs to qualitative analysis, and its judgment mostly depends on experience, and its analysis results will be different and inaccurate. Therefore, the scheme of carrying out sufficient immunohistochemical detection of different lymphoid immune checkpoints in clinical samples is not realistic.

[0006] Multiplex immunofluorescence immunohistochemistry (mIHC) technology can more accurately determine tissue cell types, spatial distribution and interactions between cells based on fewer tissue samples, and can well make up for the defects of immunohistochemical techniques. Detecting different lymphoid immune checkpoints in clinical samples based on multiplex immunofluorescence immunohistochemistry technology may be able to achieve comprehensive and good efficacy prediction and evaluation of lymphoid cell immune checkpoint drugs. However, the number of protein marks that can be labeled on a tissue section by multiplex immunofluorescence immunohistochemistry technology is also limited. Therefore, how to select appropriate protein marks to more precisely distinguish lymphoid cell populations and their interactions is the key to achieving comprehensive and good efficacy prediction and evaluation of lymphoid cell immune checkpoint drugs. Summary of the Invention

[0007] To solve the above problems, the present invention provides a molecular marker for immunotherapy prediction targeting lymphoid cells, and the molecular marker includes CD8 protein, CD4 protein, PD-1 protein, Foxp3 protein, CD19 protein and CD3 protein; Alternatively, the molecular marker includes CD45 protein, CD45RA protein, CD45RO protein, CD25 protein, CCR7 protein and CD127 protein; Alternatively, the molecular marker includes CD3 protein, LAG3 protein, Tim3 protein, ICOS protein, CD69 protein and CD103 protein; Alternatively, the molecular marker includes CD79A protein, CD79B protein, CD20 protein, CD21 protein and CD138 protein; Alternatively, the molecular markers include CD20 protein, CXCR5 protein, CD27 protein, CD38 protein, IGD protein, and IGM protein; Alternatively, the molecular markers include CD56 protein, CD161 protein, CD57 protein, CD94 protein, and Granzyme B protein.

[0008] In one embodiment of the present invention, the lymphoid cells include T lymphocytes, B lymphocytes, and / or NK cells.

[0009] In one embodiment of the present invention, the molecular markers consist of CD8 protein, CD4 protein, PD-1 protein, Foxp3 protein, CD19 protein, and CD3 protein; Alternatively, the molecular markers consist of CD45 protein, CD45RA protein, CD45RO protein, CD25 protein, CCR7 protein, and CD127 protein; Alternatively, the molecular markers consist of CD3 protein, LAG3 protein, Tim3 protein, ICOS protein, CD69 protein, and CD103 protein; Alternatively, the molecular markers consist of CD79A protein, CD79B protein, CD20 protein, CD21 protein, and CD138 protein; Alternatively, the molecular markers consist of CD20 protein, CXCR5 protein, CD27 protein, CD38 protein, IGD protein, and IGM protein; Alternatively, the molecular markers consist of CD56 protein, CD161 protein, CD57 protein, CD94 protein, and Granzyme B protein.

[0010] The present invention also provides the use of a reagent for detecting the above-mentioned molecular markers in the preparation of a product for predicting immunotherapy that targets lymphoid cells.

[0011] In one embodiment of the present invention, the reagent includes a first antibody; The first antibody includes an anti-CD8 protein antibody, an anti-CD4 protein antibody, an anti-PD-1 protein antibody, an anti-Foxp3 protein antibody, an anti-CD19 protein antibody, and an anti-CD3 protein antibody; Alternatively, the first antibody includes an anti-CD45 protein antibody, an anti-CD45RA protein antibody, an anti-CD45RO protein antibody, an anti-CD25 protein antibody, an anti-CCR7 protein antibody, and an anti-CD127 protein antibody; Alternatively, the first antibody includes an anti-CD3 protein antibody, an anti-LAG3 protein antibody, an anti-Tim3 protein antibody, an anti-ICOS protein antibody, an anti-CD69 protein antibody, and an anti-CD103 protein antibody; Alternatively, the first antibody includes an anti-CD79A protein antibody, an anti-CD79B protein antibody, an anti-CD20 protein antibody, an anti-CD21 protein antibody, and an anti-CD138 protein antibody; Alternatively, the first antibody includes an anti-CD20 protein antibody, an anti-CXCR5 protein antibody, an anti-CD27 protein antibody, an anti-CD38 protein antibody, an anti-IGD protein antibody, and an anti-IGM protein antibody; Alternatively, the first antibody includes an anti-CD56 protein antibody, an anti-CD161 protein antibody, an anti-CD57 protein antibody, an anti-CD94 protein antibody, and an anti-Granzyme B protein antibody.

[0012] In one embodiment of the present invention, the first antibody is composed of an anti-CD8 protein antibody, an anti-CD4 protein antibody, an anti-PD-1 protein antibody, an anti-Foxp3 protein antibody, an anti-CD19 protein antibody, and an anti-CD3 protein antibody; Alternatively, the first antibody is composed of an anti-CD45 protein antibody, an anti-CD45RA protein antibody, an anti-CD45RO protein antibody, an anti-CD25 protein antibody, an anti-CCR7 protein antibody, and an anti-CD127 protein antibody; Alternatively, the first antibody is composed of an anti-CD3 protein antibody, an anti-LAG3 protein antibody, an anti-Tim3 protein antibody, an anti-ICOS protein antibody, an anti-CD69 protein antibody, and an anti-CD103 protein antibody; Alternatively, the first antibody is composed of an anti-CD79A protein antibody, an anti-CD79B protein antibody, an anti-CD20 protein antibody, an anti-CD21 protein antibody, and an anti-CD138 protein antibody; Alternatively, the first antibody is composed of an anti-CD20 protein antibody, an anti-CXCR5 protein antibody, an anti-CD27 protein antibody, an anti-CD38 protein antibody, an anti-IGD protein antibody, and an anti-IGM protein antibody; Alternatively, the first antibody is composed of an anti-CD56 protein antibody, an anti-CD161 protein antibody, an anti-CD57 protein antibody, an anti-CD94 protein antibody, and an anti-Granzyme B protein antibody.

[0013] In one embodiment of the present invention, the reagent further includes a second antibody that can specifically bind to the first antibody and a fluorophore working solution; each antibody corresponds to a fluorophore working solution of a specific wavelength.

[0014] In one embodiment of the present invention, the fluorophore working solution includes an Opal 520 working solution, an Opal 570 working solution, an Opal 620 working solution, an Opal 690 working solution, an Opal 480 working solution, and / or an Opal 780 working solution.

[0015] In one embodiment of the present invention, the product includes an immunohistochemistry kit.

[0016] In one embodiment of the present invention, the immunohistochemistry kit is based on multiplex immunofluorescence immunohistochemistry (mIHC) technology.

[0017] The present invention also provides a product for predicting immunotherapy targeting lymphoid cells, and the product contains reagents for detecting the above-mentioned molecular markers.

[0018] In one embodiment of the present invention, the reagent includes a first antibody; The first antibody includes an anti-CD8 protein antibody, an anti-CD4 protein antibody, an anti-PD-1 protein antibody, an anti-Foxp3 protein antibody, an anti-CD19 protein antibody, and an anti-CD3 protein antibody; Alternatively, the first antibody includes an anti-CD45 protein antibody, an anti-CD45RA protein antibody, an anti-CD45RO protein antibody, an anti-CD25 protein antibody, an anti-CCR7 protein antibody, and an anti-CD127 protein antibody; Alternatively, the first antibody includes an anti-CD3 protein antibody, an anti-LAG3 protein antibody, an anti-Tim3 protein antibody, an anti-ICOS protein antibody, an anti-CD69 protein antibody, and an anti-CD103 protein antibody; Alternatively, the first antibody includes an anti-CD79A protein antibody, an anti-CD79B protein antibody, an anti-CD20 protein antibody, an anti-CD21 protein antibody, and an anti-CD138 protein antibody; Alternatively, the first antibody includes an anti-CD20 protein antibody, an anti-CXCR5 protein antibody, an anti-CD27 protein antibody, an anti-CD38 protein antibody, an anti-IGD protein antibody, and an anti-IGM protein antibody; Alternatively, the first antibody includes an anti-CD56 protein antibody, an anti-CD161 protein antibody, an anti-CD57 protein antibody, an anti-CD94 protein antibody, and an anti-Granzyme B protein antibody.

[0019] In one embodiment of the present invention, the first antibody consists of an anti-CD8 protein antibody, an anti-CD4 protein antibody, an anti-PD-1 protein antibody, an anti-Foxp3 protein antibody, an anti-CD19 protein antibody, and an anti-CD3 protein antibody; Alternatively, the first antibody consists of an anti-CD45 protein antibody, an anti-CD45RA protein antibody, an anti-CD45RO protein antibody, an anti-CD25 protein antibody, an anti-CCR7 protein antibody, and an anti-CD127 protein antibody; Alternatively, the first antibody consists of an anti-CD3 protein antibody, an anti-LAG3 protein antibody, an anti-Tim3 protein antibody, an anti-ICOS protein antibody, an anti-CD69 protein antibody, and an anti-CD103 protein antibody; Alternatively, the first antibody is composed of an anti-CD79A protein antibody, an anti-CD79B protein antibody, an anti-CD20 protein antibody, an anti-CD21 protein antibody, and an anti-CD138 protein antibody; Alternatively, the first antibody is composed of an anti-CD20 protein antibody, an anti-CXCR5 protein antibody, an anti-CD27 protein antibody, an anti-CD38 protein antibody, an anti-IGD protein antibody, and an anti-IGM protein antibody; Alternatively, the first antibody is composed of an anti-CD56 protein antibody, an anti-CD161 protein antibody, an anti-CD57 protein antibody, an anti-CD94 protein antibody, and an anti-Granzyme B protein antibody.

[0020] In one embodiment of the present invention, the reagent further includes a second antibody that can specifically bind to the first antibody and a fluorescein working solution; each antibody corresponds to a fluorescein working solution of a specific wavelength.

[0021] In one embodiment of the present invention, the product includes an immunohistochemistry kit.

[0022] In one embodiment of the present invention, the immunohistochemistry kit is based on multiplex immunofluorescence immunohistochemistry (mIHC) technology.

[0023] The present invention also provides an immunohistochemistry detection method targeting lymphoid cells. The immunohistochemistry detection method is not for the purpose of diagnosing and treating diseases. The immunohistochemistry detection method uses the above product to perform immunohistochemistry detection on a sample to be tested.

[0024] In one embodiment of the present invention, the immunohistochemistry detection method is based on multiplex immunofluorescence immunohistochemistry (mIHC) technology.

[0025] In one embodiment of the present invention, the immunohistochemistry detection method includes a multiplex immunohistochemistry staining step; the multiplex immunohistochemistry staining step includes: in the order of molecular markers (the order here is randomly sorted from the antigen proteins in the molecular markers and there is no specific order), respectively using the first antibody corresponding to the molecular marker and the fluorescein working solution, repeatedly performing blocking, primary antibody incubation, secondary antibody incubation, fluorescence incubation, and antigen repair after labeling on the tissue section of the sample to be tested; The blocking includes: dropping a blocking reagent on the tissue section of the sample to be tested until the tissue sample is completely covered and then incubating; The primary antibody incubation includes: dropping the first antibody corresponding to the molecular marker on the tissue section until the tissue section is completely covered and then incubating; after the incubation ends, wash the tissue section; The secondary antibody incubation includes: dropping the second antibody corresponding to the first antibody on the tissue section until the tissue section is completely covered and then incubating; after the incubation ends, wash the tissue section; The fluorescence incubation includes: dropping a fluorescein working solution corresponding to a molecular marker onto a tissue section until the tissue section is completely covered, and then incubating; after the incubation ends, washing the tissue section; The antigen retrieval after labeling includes: soaking the tissue section in an antigen retrieval reagent, heating it, and then cooling it; after the cooling ends, washing the tissue section.

[0026] In one embodiment of the present invention, before the multiple immunohistochemical staining step, the immunohistochemical detection method further includes a blocking step and an antigen retrieval step before labeling; The blocking step includes: dropping a blocking reagent onto the tissue section until the tissue sample is completely covered, and then incubating it in the dark; after the incubation ends, washing the tissue section; The antigen retrieval step before labeling includes: soaking the tissue section of the sample to be tested in an antigen retrieval reagent, heating it, and then cooling it; after the cooling ends, washing the tissue section.

[0027] In one embodiment of the present invention, after the multiple immunohistochemical staining step, the immunohistochemical detection method further includes a coverslipping step and a detection step; The coverslipping step includes: dropping a coverslipping agent onto the tissue section; after the dropping ends, covering it with a coverslip to perform coverslipping; The detection step includes: scanning the tissue section to obtain an immunofluorescence image, and obtaining the immunohistochemical result of the tissue section according to the immunofluorescence image.

[0028] In one embodiment of the present invention, the fluorescein working solution includes Opal 520 working solution, Opal 570 working solution, Opal 620 working solution, Opal 690 working solution, Opal 480 working solution, and / or Opal 780 working solution.

[0029] In one embodiment of the present invention, the corresponding relationship between the molecular marker and the fluorescein working solution includes: CD8 protein corresponds to Opal 480 working solution; CD4 protein corresponds to Opal 520 working solution; PD-1 protein corresponds to Opal 570 working solution; Foxp3 protein corresponds to Opal 620 working solution; CD19 protein corresponds to Opal 690 working solution; CD3 protein corresponds to Opal 780 working solution; Alternatively, the corresponding relationship between the molecular marker and the fluorescein working solution includes: CD45 protein corresponds to Opal 480 working solution; CD45RA protein corresponds to Opal 520 working solution; CD45RO protein corresponds to Opal 570 working solution; CD25 protein corresponds to Opal 620 working solution; CCR7 protein corresponds to Opal 690 working solution; CD127 protein corresponds to Opal 780 working solution; Alternatively, the corresponding relationship between the molecular marker and the fluorescein working solution includes: CD3 protein corresponds to Opal 480 working solution; LAG3 protein corresponds to Opal 520 working solution; Tim3 protein corresponds to Opal 570 working solution; ICOS protein corresponds to Opal 620 working solution; CD69 protein corresponds to Opal 690 working solution; CD103 protein corresponds to Opal 780 working solution; Alternatively, the corresponding relationship between the molecular marker and the fluorescein working solution includes: CD79A protein corresponds to Opal 520 working solution; CD79B protein corresponds to Opal 570 working solution; CD20 protein corresponds to Opal 620 working solution; CD21 protein corresponds to Opal 690 working solution; CD138 protein corresponds to Opal 780 working solution; Alternatively, the corresponding relationship between the molecular marker and the fluorescein working solution includes: CD20 protein corresponds to Opal 480 working solution; CXCR5 protein corresponds to Opal 520 working solution; CD27 protein corresponds to Opal 570 working solution; CD38 protein corresponds to Opal 620 working solution; IGD protein corresponds to Opal 690 working solution; IGM protein corresponds to Opal 780 working solution; Alternatively, the corresponding relationship between the molecular marker and the fluorescein working solution includes: CD56 protein corresponds to Opal 520 working solution; CD161 protein corresponds to Opal 570 working solution; CD57 protein corresponds to Opal 620 working solution; CD94 protein corresponds to Opal 690 working solution; Granzyme B protein corresponds to Opal 780 working solution.

[0030] In one embodiment of the present invention, in the multiple immunohistochemical staining step, when reaching the round corresponding to the molecular marker corresponding to the Opal 780 working solution, the first antibody corresponding to the molecular marker and the Opal 780 working solution are used to sequentially perform blocking, primary antibody incubation, secondary antibody incubation, signal amplification staining, post-labeling antigen repair, fluorescence incubation, and nuclear staining on the tissue section of the sample to be tested; The signal amplification staining includes: dropping the signal amplification staining working solution on the tissue section until it completely covers the tissue section and then incubating; after the incubation ends, washing the tissue section (the Opal 780 working solution needs to use the signal amplification staining working solution as a bridge to jointly complete the labeling of the antigen protein with the signal amplification staining working solution); The nuclear staining includes: dropping the nuclear staining reagent on the tissue section until it completely covers the tissue section and then incubating; after the incubation ends, washing the tissue section.

[0031] In one embodiment of the present invention, the signal amplification staining working solution includes an opal tsa-dig working solution.

[0032] In one embodiment of the present invention, the blocking reagent is a hydrogen peroxide solution with a concentration of 2% - 5% (where "%" refers to w / v, g / 100 mL).

[0033] In one embodiment of the present invention, the blocking reagent is a hydrogen peroxide solution with a concentration of 3% (where "%" refers to w / v, g / 100 mL).

[0034] The present invention also provides a drug screening model for screening immunotherapeutic drugs targeting lymphoid cells, which includes an information collection module, a data analysis module, and a prediction module; The information collection module is used to detect and collect immunohistochemical results related to the above-mentioned molecular markers in the experimental subject after administering the immunotherapeutic drug to be screened; The data analysis module is used to analyze the immunohistochemical results obtained in the information collection step to obtain analysis results related to the lymphoid cell immune checkpoint; The prediction module is used to classify the immunotherapeutic drug to be screened as effective or ineffective according to the analysis results obtained by the data analysis module.

[0035] In one embodiment of the present invention, the experimental subject is a cell or an experimental animal.

[0036] The present invention also provides a drug screening method for screening immunotherapeutic drugs targeting lymphoid cells, which includes: administering the immunotherapeutic drug to be screened to an experimental subject; obtaining immunohistochemical results related to the above-mentioned molecular markers in the experimental subject after administering the immunotherapeutic drug to be screened; and judging whether the immunotherapeutic drug to be screened is effective or ineffective according to the immunohistochemical results.

[0037] In one embodiment of the present invention, the method includes: administering the immunotherapeutic drug to be screened to an experimental subject; using the above-mentioned immunohistochemical detection method to obtain immunohistochemical results related to the above-mentioned molecular markers in the experimental subject after administering the immunotherapeutic drug to be screened; and judging whether the immunotherapeutic drug to be screened is effective or ineffective according to the immunohistochemical results.

[0038] In one embodiment of the present invention, the experimental subject is a cell or an experimental animal.

[0039] The present invention also provides the application of the above-mentioned molecular markers or the above-mentioned products in immunohistochemical detection targeting lymphoid cells or screening of immunotherapeutic drugs targeting lymphoid cells, and this application is not for the purpose of diagnosing and treating diseases.

[0040] The technical solution of the present invention has the following advantages: The present invention provides a molecular marker for predicting immunotherapy that targets lymphoid cells. The molecular marker includes CD8 protein, CD4 protein, PD-1 protein, Foxp3 protein, CD19 protein, and CD3 protein; alternatively, the molecular marker includes CD45 protein, CD45RA protein, CD45RO protein, CD25 protein, CCR7 protein, and CD127 protein; alternatively, the molecular marker includes CD3 protein, LAG3 protein, Tim3 protein, ICOS protein, CD69 protein, and CD103 protein; alternatively, the molecular marker includes CD79A protein, CD79B protein, CD20 protein, CD21 protein, and CD138 protein; alternatively, the molecular marker includes CD20 protein, CXCR5 protein, CD27 protein, CD38 protein, IGD protein, and IGM protein; alternatively, the molecular marker includes CD56 protein, CD161 protein, CD57 protein, CD94 protein, and Granzyme B protein. Research shows that the molecular marker can more precisely distinguish lymphoid cell populations and their interactions. Therefore, immunohistochemical detection of the molecular marker can achieve comprehensive and good efficacy prediction and evaluation of immunotherapy drugs targeting lymphoid cell immune checkpoints.

[0041] The molecular marker can generally be applied to the following clinical scenarios: First, immune regulation disorder diseases: In immune regulation disorder diseases such as autoimmune diseases (such as rheumatoid arthritis, systemic lupus erythematosus, etc.), the proportion and activity of different lymphocyte subsets can be studied through these molecular markers, thereby helping to understand the pathogenesis of the diseases.

[0042] Second, malignant tumors: These molecular markers are obtained by screening tumors as targets and have good detection effects for tumors. In the tumor microenvironment, the infiltration and activity of lymphocytes such as T cells, B cells, and regulatory T cells (Treg cells) can be studied through these molecular markers, thereby helping to evaluate the immunotherapy potential of tumors.

[0043] Third, infectious diseases: In infectious diseases such as viral infections or other bacterial infections, the responses of immune cells can be studied through these molecular markers, including the activation of CD8 + T cells, the expression of PD-1, etc., which helps to understand the host immune response.

[0044] Fourth, organ transplantation: In transplantation immunology, the infiltration and activity of lymphocytes such as T cells, B cells, and regulatory T cells (Treg cells) can be studied through these molecular markers, thereby helping to evaluate the risk of transplant rejection.

[0045] Generally speaking, multiplex immunofluorescence staining for these molecular markers has important applications in studying the pathogenesis of immune-related diseases, tumor immunology, infection immunology, etc. In pathological research, they can provide useful information about the distribution, activation status, and interactions of lymphocyte subsets. Description of the Drawings

[0046] Figure 1 : Immunohistochemical staining image obtained using the molecular marker combination of Example 1-1.

[0047] Figure 2 : Figure 1 Cell type annotation image corresponding to the obtained immunohistochemical staining image.

[0048] Figure 3 : Figure 1 Cell type heat map corresponding to the obtained immunohistochemical staining image.

[0049] Figure 4 : Cell proportion map obtained using the molecular marker combination of Example 1-1.

[0050] Figure 5 : Box plot of the significant differences in cell proportions obtained using the molecular marker combination of Example 1-1 among different groups. Figure 5 In, A (Group A) refers to the CD25-positive region, and B (Group B) refers to the CD25-negative region.

[0051] Figure 6 : Cell neighborhood mapping diagram obtained using the molecular marker combination of Example 1-1.

[0052] Figure 7 : Figure 6 Cell component heat map in the obtained cell neighborhood.

[0053] Figure 8 : Box plot of the differences in the proportions of different cell neighborhoods obtained using the molecular marker combination of Example 1-1 among different groups. Figure 8 In, A (Group A) refers to the CD25-positive region, and B (Group B) refers to the CD25-negative region.

[0054] Figure 9 : Cell interaction map obtained using the molecular marker combination of Example 1-1.

[0055] Figure 10 : Figure 9 Cell interaction pair quantity heat map obtained.

[0056] Figure 11: Immunohistochemical staining images obtained using the molecular marker combination of Example 2-1.

[0057] Figure 12 : Figure 11 The obtained immunohistochemical staining images correspond to cell type annotation images.

[0058] Figure 13 : Figure 11 The obtained immunohistochemical staining images correspond to cell type heatmaps.

[0059] Figure 14 : Cell proportion maps obtained using the molecular marker combination of Example 2-1.

[0060] Figure 15 : Box plots of the significant differences in cell proportions obtained using the molecular marker combination of Example 2-1 between different groups. Figure 15 In it, A (Group A) refers to the CD3-positive enriched region, and B (Group B) refers to the CD3-positive sparse region.

[0061] Figure 16 : Cell neighborhood mapping diagrams obtained using the molecular marker combination of Example 2-1.

[0062] Figure 17 : Figure 16 Heatmaps of cell components in the obtained cell neighborhoods.

[0063] Figure 18 : Box plots of the differences in the proportions of different cell neighborhoods obtained using the molecular marker combination of Example 2-1 between different groups. Figure 18 In it, A (Group A) refers to the CD3-positive enriched region, and B (Group B) refers to the CD3-positive sparse region.

[0064] Figure 19 : Cell interaction diagrams obtained using the molecular marker combination of Example 2-1.

[0065] Figure 20 : Figure 19 Heatmaps of the number of obtained cell interaction pairs.

[0066] Figure 21 : Immunohistochemical staining images obtained using the molecular marker combination of Example 2-1.

[0067] Figure 22 : Figure 21 The obtained immunohistochemical staining images correspond to cell type annotation images.

[0068] Figure 23 : Figure 21The heatmap of cell types corresponding to the obtained immunohistochemical staining images.

[0069] Figure 24 : The cell proportion map obtained using the molecular marker combination of Example 3-1.

[0070] Figure 25 : The box plot of the significant differences in the cell proportions obtained using the molecular marker combination of Example 3-1 among different groups. Figure 25 In it, A (Group A) refers to the CD79A-positive enriched region, and B (Group B) refers to the CD79A-positive sparse region.

[0071] Figure 26 : The cell neighborhood mapping map obtained using the molecular marker combination of Example 3-1.

[0072] Figure 27 : Figure 26 The heatmap of cell components in the obtained cell neighborhood.

[0073] Figure 28 : The box plot of the differences in the proportions of different cell neighborhoods obtained using the molecular marker combination of Example 3-1 among different groups. Figure 28 In it, A (Group A) refers to the CD79A-positive enriched region, and B (Group B) refers to the CD79A-positive sparse region.

[0074] Figure 29 : The cell interaction map obtained using the molecular marker combination of Example 3-1.

[0075] Figure 30 : Figure 29 The heatmap of the number of obtained cell interaction pairs.

[0076] Figure 31 : The immunohistochemical staining image obtained using the molecular marker combination of Example 4-1.

[0077] Figure 32 : Figure 31 The cell type annotation image corresponding to the obtained immunohistochemical staining image.

[0078] Figure 33 : Figure 31 The heatmap of cell types corresponding to the obtained immunohistochemical staining images.

[0079] Figure 34 : The cell proportion map obtained using the molecular marker combination of Example 4-1.

[0080] Figure 35:Box plot of the significant differences in the proportion of cells obtained using the molecular marker combination of Example 4-1 among different groups. Figure 35 In it, A (Group A) refers to the B cell enrichment region, and B (Group B) refers to the T cell enrichment region.

[0081] Figure 36 :Cell neighborhood mapping diagram obtained using the molecular marker combination of Example 4-1.

[0082] Figure 37 : Figure 36 Heat map of cell components in the obtained cell neighborhood.

[0083] Figure 38 :Box plot of the differences in the proportion of different cell neighborhoods obtained using the molecular marker combination of Example 4-1 among different groups. Figure 38 In it, A (Group A) refers to the B cell enrichment region, and B (Group B) refers to the T cell enrichment region.

[0084] Figure 39 :Cell interaction diagram obtained using the molecular marker combination of Example 4-1.

[0085] Figure 40 : Figure 39 Heat map of the number of obtained cell interaction pairs.

[0086] Figure 41 :Immunohistochemical staining image obtained using the molecular marker combination of Example 5-1.

[0087] Figure 42 : Figure 41 Cell type annotation image corresponding to the obtained immunohistochemical staining image.

[0088] Figure 43 : Figure 41 Heat map of cell types corresponding to the obtained immunohistochemical staining image.

[0089] Figure 44 :Cell proportion diagram obtained using the molecular marker combination of Example 5-1.

[0090] Figure 45 :Box plot of the significant differences in the proportion of cells obtained using the molecular marker combination of Example 5-1 among different groups. Figure 45 In it, A (Group A) refers to the Granzyme B positive region, and B (Group B) refers to the Granzyme B negative region.

[0091] Figure 46: Cell neighborhood mapping diagram obtained using the molecular marker combination of Example 5-1.

[0092] Figure 47 : Figure 46 Heat map of cell components in the obtained cell neighborhood.

[0093] Figure 48 : Box plot of the differences in the proportions of different cell neighborhoods obtained using the molecular marker combination of Example 5-1 among different groups. Figure 48 In it, A (Group A) refers to the Granzyme B-positive region, and B (Group B) refers to the Granzyme B-negative region.

[0094] Figure 49 : Cell interaction map obtained using the molecular marker combination of Example 5-1.

[0095] Figure 50 : Figure 49 Heat map of the number of obtained cell interaction pairs.

[0096] Figure 51 : Concordance index of the multivariate prognostic COX model for each molecular marker combination.

[0097] Figure 52 : Akaike information criterion of the multivariate prognostic COX model for each molecular marker combination. Detailed implementation manners

[0098] The following examples are provided to better further understand the present invention. They are not limited to the described best implementation manners, and do not limit the content and protection scope of the present invention. Any product that is the same as or similar to the present invention obtained by anyone under the inspiration of the present invention or by combining the features of the present invention with other existing technologies falls within the protection scope of the present invention.

[0099] For those not specifying specific experimental steps or conditions in the following examples, the operations or conditions of the conventional experimental steps described in the literature in this field can be followed. For the reagents or instruments not indicating the manufacturer, they are all conventional reagent products that can be obtained through commercial purchase.

[0100] The corresponding relationship between the protein names and gene names involved in the following examples is shown in Table 1.

[0101] Table 1 Corresponding table of protein names and gene names

[0102] Example 1-1: A molecular marker for predicting immunotherapy targeting lymphoid cells This embodiment provides a molecular marker for predicting immunotherapy that targets lymphoid cells, and the molecular marker is composed of CD8 protein, CD4 protein, PD-1 protein, Foxp3 protein, CD19 protein, and CD3 protein.

[0103] Example 2-1: A multiplex immunohistochemistry kit targeting lymphoid cells This embodiment provides a multiplex immunohistochemistry kit targeting lymphoid cells. The multiplex immunohistochemistry kit includes an antigen retrieval reagent, a blocking reagent, a blocking solution, a primary antibody, a secondary antibody, a fluorophore working solution, a signal amplification staining working solution, a nuclear staining reagent, and a mounting medium; The antigen retrieval reagent is Tris-EDTA antigen retrieval solution (purchased from Solarbio); The blocking reagent is a 3% (where % refers to w / v, g / 100 mL) hydrogen peroxide solution; The blocking solution is a blocking solution (purchased from Akoya); The primary antibody is composed of an anti-CD8 protein antibody (purchased from Biolegend, model C8 / 144B), an anti-CD4 protein antibody (purchased from Sidaite, model SDT-R163), an anti-PD-1 protein antibody (purchased from CST, model D4W2J), an anti-Foxp3 protein antibody (purchased from Sidaite, model SDT-R064), an anti-CD19 protein antibody (purchased from Abcam, model EPR5906), and an anti-CD3 protein antibody (purchased from CST, model D7A6E); The secondary antibody is Anti-Ms+Rb HRP antibody; The fluorophore working solution is provided by the Opal Polaris™ 7-Color manual immunohistochemistry kit (NEL861001KT), and is composed of an Opal 480 fluorophore working solution corresponding to the anti-CD8 protein antibody, an Opal 520 fluorophore working solution corresponding to the anti-CD4 protein antibody, an Opal 570 fluorophore working solution corresponding to the anti-PD-1 protein antibody, an Opal 620 fluorophore working solution corresponding to the anti-Foxp3 protein antibody, an Opal 690 fluorophore working solution corresponding to the anti-CD19 protein antibody, and an Opal 780 fluorophore working solution corresponding to the anti-CD3 protein antibody; The signal amplification staining working solution is provided by the Opal Polaris™ 7-Color manual immunohistochemistry kit (NEL861001KT), and is the opal tsa-dig fluorophore working solution; The nuclear staining reagent is DAPI staining solution (purchased from Akoya); The mounting medium is an anti-fluorescence quenching mounting medium (purchased from Beyotime).

[0104] Example 3-1: An immunohistochemical detection method targeting lymphoid cells This example provides an immunohistochemical detection method targeting lymphoid cells. The immunohistochemical detection method uses the multiplex immunohistochemical kit of Example 2-1 to perform immunohistochemical detection on the sample to be tested. The immunohistochemical detection method includes the following steps: 1. Tissue section dewaxing step Bake the tissue section in an oven at 62°C for 1.5 h. After baking, immerse the tissue section in xylene I for 10 min, then take out the tissue section and immerse it in xylene II for 10 min. After immersion, take out the tissue section and immerse it in absolute ethanol I, absolute ethanol II, 98% (v / v) ethanol, 95% (v / v) ethanol, and 85% (v / v) ethanol for 5 min in sequence. After immersion, take out the tissue section and wash it 3 times with DEPC water for 5 min each time; 2. Tissue fixation step Drop 4% (w / v, g / 100 mL) paraformaldehyde (purchased from Beyotime) on the tissue section until it completely covers the tissue sample, and incubate at room temperature (25°C) for 20 min. After incubation, wash the tissue section 3 times with DEPC water for 2 min each time; 3. Blocking step Drop the blocking reagent on the tissue section until it completely covers the tissue sample, and incubate at room temperature (25°C) in the dark for 25 min. After incubation, wash the tissue section 3 times with PBS buffer for 5 min each time; 4. Antigen retrieval step before labeling Immerse the tissue section in a staining jar containing the antigen retrieval reagent, and place it in a rice cooker. Start timing when the water boils for 10 minutes before boiling, and then turn to keep warm and time for 30 min for heating treatment. After heating, cool naturally at room temperature (25°C). After cooling, wash the tissue section 3 times with 1×TBST solution (purchased from Solarbio) for 2 min each time; 5. Multiplex immunohistochemical staining step According to the order of molecular markers, use the first antibody corresponding to the molecular marker and the fluorescent working solution respectively, and repeat the steps of blocking, primary antibody incubation, secondary antibody incubation, fluorescence incubation, and antigen retrieval after labeling on the tissue section in sequence. The specific process is as follows: Blocking: Use a histochemical pen to outline the tissue specimen part, drop the blocking reagent on the tissue section until it completely covers the tissue sample, and incubate at room temperature (25°C) for 10 min. After incubation, drain the blocking solution; Primary antibody incubation: Drop the primary antibody corresponding to the molecular marker onto the tissue section until the tissue section is completely covered, and then incubate it in the dark at room temperature (25°C) for 1 h; after the incubation, wash the tissue section 3 times with 1×TBST solution, 2 min each time; Secondary antibody incubation: Drop the secondary antibody corresponding to the primary antibody (Anti-Ms+Rb HRP antibody) onto the tissue section until the tissue section is completely covered, and then incubate it in the dark at room temperature (25°C) for 15 min; after the incubation, wash the tissue section 3 times with 1×TBST solution, 2 min each time; Fluorescence incubation: Drop the working solution of the fluorophore corresponding to the molecular marker onto the tissue section until the tissue section is completely covered, and then incubate it in the dark at room temperature (25°C) for 15 min; after the incubation, wash the tissue section 3 times with 1×TBST solution, 2 min each time; Antigen retrieval after labeling: Immerse the tissue section in a staining jar containing the antigen retrieval reagent, place it in a rice cooker before boiling, start timing for 10 minutes when the water boils, and then switch to keep-warm timing for 30 min for heating treatment; after the heating, let it cool naturally at room temperature (25°C); after cooling, rinse the tissue section once with 1×TBST solution for 2 min; Complete the immunohistochemical staining rounds of CD4 protein, PD-1 protein, Foxp3 protein, CD19 protein and CD8 protein in sequence according to the above operations; among them, the anti-CD4 protein antibody corresponding to CD4 protein is diluted 400 times with the blocking solution before use, the anti-PD-1 protein antibody corresponding to PD-1 protein is diluted 50 times with the blocking solution before use, the anti-Foxp3 protein antibody corresponding to Foxp3 protein is diluted 250 times with the blocking solution before use, the anti-CD19 protein antibody corresponding to CD19 protein is diluted 50 times with the blocking solution before use, the anti-CD8 protein antibody corresponding to CD8 protein is diluted 30 times with the blocking solution before use, and the secondary antibody does not need to be diluted; When performing the immunohistochemical staining round of CD3 protein, use the primary antibody corresponding to CD3 protein and the working solution of the fluorophore, and perform blocking, primary antibody incubation, secondary antibody incubation, signal amplification staining, antigen retrieval after labeling, fluorescence incubation and nuclear staining on the tissue section of the sample to be tested in sequence. The specific process is as follows; Blocking: Use a histochemical pen to outline the tissue specimen part, drop the blocking reagent onto the tissue section until the tissue sample is completely covered, and then incubate it at room temperature (25°C) for 10 min; after the incubation, drain the blocking solution; Primary antibody incubation: Drop the primary antibody corresponding to the molecular marker onto the tissue section until the tissue section is completely covered, and then incubate it in the dark at room temperature (25°C) for 1 h; after the incubation, wash the tissue section 3 times with 1×TBST solution, 2 min each time; Secondary antibody incubation: Drop the secondary antibody corresponding to the primary antibody (Anti-Ms+Rb HRP antibody) onto the tissue section until the tissue section is completely covered, and incubate in the dark at room temperature (25°C) for 15 min; after incubation, wash the tissue section 3 times with 1×TBST solution, 2 min each time; Signal amplification staining: Drop the signal amplification staining working solution onto the tissue section until the tissue section is completely covered, and incubate in the dark at room temperature (25°C) for 10 min; after incubation, wash the tissue section 3 times with 1×TBST solution, 2 min each time; Antigen retrieval after labeling: Immerse the tissue section in a staining jar containing antigen retrieval reagent, place it in a rice cooker before boiling, start timing for 10 minutes when the water boils, then switch to keep warm and time for 30 min for heating treatment; after heating, let it cool naturally at room temperature (25°C); after cooling, rinse the tissue section 1 time with 1×TBST solution, 2 min; Fluorescence incubation: Drop the fluorescein working solution corresponding to the molecular marker onto the tissue section until the tissue section is completely covered, and incubate in the dark at room temperature (25°C) for 15 min; after incubation, wash the tissue section 3 times with 1×TBST solution, 2 min each time; Nuclear staining step: Drop the nuclear staining reagent onto the tissue section until the tissue section is completely covered, and incubate at room temperature (25°C) for 5 min; after incubation, rinse the tissue section 1 time with 1×TBST solution, 2 min, and wash the tissue section 1 time with 1×TBST solution, 2 min; Complete the immunohistochemical staining rounds of CD3 protein according to the above operations; among them, the anti-CD3 protein antibody corresponding to CD3 protein is diluted 100 times with the blocking solution before use, and the secondary antibody does not need to be diluted; 6. Mounting step First wipe the edge of the tissue section clean, then drop the mounting agent onto the tissue section, and finally cover it with a coverslip for mounting; 7. Detection step Scan the tissue section to obtain an immunofluorescence image, and obtain the immunohistochemical results of the tissue section according to the immunofluorescence image.

[0105] Example 1-2: A molecular marker for predicting immunotherapy targeting lymphoid cells This example provides a molecular marker for predicting immunotherapy targeting lymphoid cells, and the molecular marker is composed of CD45 protein, CD45RA protein, CD45RO protein, CD25 protein, CCR7 protein and CD127 protein.

[0106] Example 2-2: A multiplex immunohistochemistry kit targeting lymphoid cells This example provides a multiplex immunohistochemistry kit targeting lymphoid cells. Based on Example 2-1, the first antibody in the multiplex immunohistochemistry kit is replaced with: The first antibody consists of an anti-CD45 protein antibody (purchased from CST, model D9M8I), an anti-CD45RA protein antibody (purchased from Biolegend, model HI100), an anti-CD45RO protein antibody (purchased from Biolegend, model UCHL1), an anti-CD25 protein antibody (purchased from Abcam, model EPR6452), an anti-CCR7 protein antibody (purchased from Abcam, model EPR23192-57), and an anti-CD127 protein antibody (purchased from Abcam, model EPR23747-333); The fluorophore working solution is replaced with: The fluorophore working solution is provided by the Opal Polaris™ 7-Color Manual Immunohistochemistry Kit (NEL861001KT) and consists of an Opal 480 fluorophore working solution corresponding to the anti-CD45 protein antibody, an Opal 520 fluorophore working solution corresponding to the anti-CD45RA protein antibody, an Opal 570 fluorophore working solution corresponding to the anti-CD45RO protein antibody, an Opal 620 fluorophore working solution corresponding to the anti-CD25 protein antibody, an Opal 690 fluorophore working solution corresponding to the anti-CCR7 protein antibody, and an Opal 780 fluorophore working solution corresponding to the anti-CD127 protein antibody.

[0107] Example 3-2: An immunohistochemical detection method targeting lymphoid cells This example provides an immunohistochemical detection method targeting lymphoid cells. Based on Experimental Example 3-1, the multiplex immunohistochemistry kit of Example 2-2 is used to perform immunohistochemical detection on the sample to be tested. Among them, the order of molecular markers in the multiplex immunohistochemistry staining step is: CD45 protein, CD45RA protein, CD45RO protein, CD25 protein, CCR7 protein, and CD127 protein. Among them, the anti-CD45 protein antibody is diluted 100 times with the blocking solution before use, the anti-CD45RA protein antibody is diluted 400 times with the blocking solution before use, the anti-CD45RO protein antibody is diluted 200 times with the blocking solution before use, the anti-CD25 protein antibody is diluted 100 times with the blocking solution before use, the anti-CCR7 protein antibody is diluted 100 times with the blocking solution before use, and the anti-CD127 protein antibody is diluted 35 times with the blocking solution before use.

[0108] Example 1-3: A molecular marker for immunotherapy prediction targeting lymphoid cells This embodiment provides a molecular marker for predicting immunotherapy that targets lymphoid cells, and the molecular marker is composed of CD3 protein, LAG3 protein, Tim3 protein, ICOS protein, CD69 protein, and CD103 protein.

[0109] Example 2-3: A multiplex immunohistochemistry kit for targeting lymphoid cells This embodiment provides a multiplex immunohistochemistry kit for targeting lymphoid cells. Based on Example 2-1, the first antibody is replaced with: The first antibody is composed of an anti-CD3 protein antibody (purchased from CST, model D7A6E), an anti-LAG3 protein antibody (purchased from Abcam, model SP346), an anti-Tim3 protein antibody (purchased from Abcam, model EPR22241), an anti-ICOS protein antibody (purchased from Abcam, model SP98), an anti-CD69 protein antibody (purchased from Abcam, model EPR21841), and an anti-CD103 protein antibody (purchased from Abcam, model EPR4166(2)); The fluorophore working solution is replaced with: The fluorophore working solution is provided by the Opal Polaris™ 7-Color Manual Immunohistochemistry Kit (NEL861001KT), and is composed of an Opal 480 fluorophore working solution corresponding to the anti-CD3 protein antibody, an Opal 520 fluorophore working solution corresponding to the anti-LAG3 protein antibody, an Opal 570 fluorophore working solution corresponding to the anti-Tim3 protein antibody, an Opal 620 fluorophore working solution corresponding to the anti-ICOS protein antibody, an Opal 690 fluorophore working solution corresponding to the anti-CD69 protein antibody, an opal tsa-dig fluorophore working solution corresponding to the anti-CD103 protein antibody, and an Opal 780 fluorophore working solution.

[0110] Example 3-3: An immunohistochemistry detection method for targeting lymphoid cells This embodiment provides an immunohistochemical detection method targeting lymphoid cells. The immunohistochemical detection method is based on Experimental Example 3-1 and uses the multiplex immunohistochemical kit of Embodiment 2-3 to perform immunohistochemical detection on the sample to be tested. Among them, the order of molecular markers in the multiplex immunohistochemical staining step is: CD3 protein, LAG3 protein, Tim3 protein, ICOS protein, CD69 protein, and CD103 protein. Among them, the anti-CD3 protein antibody is diluted 100 times with the blocking solution before use, the anti-LAG3 protein antibody is diluted 50 times with the blocking solution before use, the anti-Tim3 protein antibody is diluted 100 times with the blocking solution before use, the anti-ICOS protein antibody is diluted 25 times with the blocking solution before use, the anti-CD69 protein antibody is diluted 50 times with the blocking solution before use, and the anti-CD103 protein antibody is diluted 50 times with the blocking solution before use.

[0111] Example 1-4: A molecular marker for immunotherapy prediction targeting lymphoid cells This embodiment provides a molecular marker for immunotherapy prediction targeting lymphoid cells, and the molecular marker is composed of CD79A protein, CD79B protein, CD20 protein, CD21 protein, and CD138 protein.

[0112] Example 2-4: A multiplex immunohistochemical kit targeting lymphoid cells This embodiment provides a multiplex immunohistochemical kit targeting lymphoid cells. The multiplex immunohistochemical kit is based on Embodiment 2-1, and the primary antibody is replaced with: The primary antibody is composed of an anti-CD79A protein antibody (purchased from NOVUS, model HM57), an anti-CD79B protein antibody (purchased from Abcam, model SP240), an anti-CD20 protein antibody (purchased from Biolegend, model C20Mab-60), an anti-CD21 protein antibody (purchased from Abcam, model SP186), and an anti-CD138 protein antibody (purchased from Abcam, model EPR6454); The fluorescent working solution is replaced with: The fluorescent working solution is provided by the Opal Polaris™ 7-Color Manual Immunohistochemistry Kit (NEL861001KT) and is composed of an Opal 480 fluorescent working solution corresponding to the anti-CD79A protein antibody, an Opal 520 fluorescent working solution corresponding to the anti-CD79B protein antibody, an Opal 570 fluorescent working solution corresponding to the anti-CD20 protein antibody, an Opal 620 fluorescent working solution corresponding to the anti-CD21 protein antibody, an opal tsa-dig fluorescent working solution corresponding to the anti-CD138 protein antibody, and an Opal 780 fluorescent working solution.

[0113] Example 3-4: An immunohistochemical detection method targeting lymphoid cells This example provides an immunohistochemical detection method targeting lymphoid cells. The immunohistochemical detection method is based on Experiment Example 3-1, and the multiplex immunohistochemical kit of Example 2-4 is used to perform immunohistochemical detection on the sample to be tested. Among them, the order of molecular markers in the multiplex immunohistochemical staining step is: CD79B protein, CD79A protein, CD20 protein, CD21 protein, and CD138 protein. Among them, the anti-CD79B protein antibody is diluted 50 times with the blocking solution before use, the anti-CD79A protein antibody is diluted 150 times with the blocking solution before use, the anti-CD20 protein antibody is diluted 100 times with the blocking solution before use, the anti-CD21 protein antibody is diluted 100 times with the blocking solution before use, and the anti-CD138 protein antibody is diluted 50 times with the blocking solution before use.

[0114] Example 1-5: A molecular marker for immunotherapy prediction targeting lymphoid cells This example provides a molecular marker for immunotherapy prediction targeting lymphoid cells. The molecular marker consists of CD20 protein, CXCR5 protein, CD27 protein, CD38 protein, IGD protein, and IGM protein.

[0115] Example 2-5: A multiplex immunohistochemical kit targeting lymphoid cells This example provides a multiplex immunohistochemical kit targeting lymphoid cells. The multiplex immunohistochemical kit is based on Example 2-1, and the primary antibody is replaced with: The primary antibody consists of anti-CD20 protein antibody (purchased from Biolegend, model C20Mab-60), anti-CXCR5 protein antibody (purchased from Abcam, model EPR23463-30), anti-CD27 protein antibody (purchased from Abcam, model EPR8569), anti-CD38 protein antibody (purchased from Ancam, model EPR4106), anti-IGD protein antibody (purchased from Abcam, model EPR6146), and anti-IGM protein antibody (purchased from ABclonal, model polyclonal); The fluorescein working solution is replaced with: The fluorescein working solution is provided by the Opal Polaris™ 7-Color Manual Immunohistochemistry Kit (NEL861001KT), and is composed of the Opal 480 fluorescein working solution corresponding to the anti-CD20 protein antibody, the Opal 520 fluorescein working solution corresponding to the anti-CXCR5 protein antibody, the Opal 570 fluorescein working solution corresponding to the anti-CD27 protein antibody, the Opal 620 fluorescein working solution corresponding to the anti-CD38 protein antibody, the Opal 690 fluorescein working solution corresponding to the anti-IGD protein antibody, the opal tsa-dig fluorescein working solution corresponding to the anti-IGM protein antibody, and the Opal 780 fluorescein working solution.

[0116] Examples 3-5: An immunohistochemical detection method targeting lymphoid cells This example provides an immunohistochemical detection method targeting lymphoid cells. The immunohistochemical detection method is based on Experimental Example 3-1, and the multiplex immunohistochemistry kit of Example 2-5 is used to perform immunohistochemical detection on the sample to be tested. Among them, the order of molecular markers in the multiplex immunohistochemical staining step is: CD20 protein, CXCR5 protein, CD27 protein, CD38 protein, IGD protein, and IGM protein. Among them, the anti-CD20 protein antibody is diluted 100 times with the blocking solution before use, the anti-CXCR5 protein antibody is diluted 100 times with the blocking solution before use, the anti-CD27 protein antibody is diluted 100 times with the blocking solution before use, the anti-CD38 protein antibody is diluted 50 times with the blocking solution before use, the anti-IGD protein antibody is diluted 100 times with the blocking solution before use, and the anti-IGM protein antibody is diluted 100 times with the blocking solution before use.

[0117] Examples 1-6: A molecular marker for immunotherapy prediction targeting lymphoid cells This example provides a molecular marker for immunotherapy prediction targeting lymphoid cells, and the molecular marker is composed of CD56 protein, CD161 protein, CD57 protein, CD94 protein, and Granzyme B protein.

[0118] Examples 2-6: A multiplex immunohistochemistry kit targeting lymphoid cells This example provides a multiplex immunohistochemistry kit targeting lymphoid cells. The multiplex immunohistochemistry kit is based on Example 2-1, and the primary antibody is replaced with: The first antibody is composed of an anti-CD56 protein antibody (purchased from ZhengNeng, model polyclonal), an anti-CD161 protein antibody (purchased from Abcam, model EPR26340-6), an anti-CD57 protein antibody (purchased from BD, model NK-1), an anti-CD94 protein antibody (purchased from Abcam, model EPR21003), and an anti-Granzyme B protein antibody (purchased from Abcam, model EPR20129-217); Replace the fluorescein working solution with: The fluorescein working solution is provided by the Opal Polaris™ 7-Color Manual Immunohistochemistry Kit (NEL861001KT) and consists of an Opal 480 fluorescein working solution corresponding to the anti-CD56 protein antibody, an Opal 520 fluorescein working solution corresponding to the anti-CD161 protein antibody, an Opal 570 fluorescein working solution corresponding to the anti-CD57 protein antibody, an Opal 620 fluorescein working solution corresponding to the anti-CD94 protein antibody, an opaltsa-dig fluorescein working solution corresponding to the anti-Granzyme B protein antibody, and an Opal 780 fluorescein working solution.

[0119] Examples 3-6: An immunohistochemical detection method targeting lymphoid cells This example provides an immunohistochemical detection method targeting lymphoid cells. The immunohistochemical detection method is based on Experimental Example 3-1 and uses the multiplex immunohistochemistry kit of Examples 2-6 to perform immunohistochemical detection on the sample to be tested. Among them, the order of molecular markers in the multiplex immunohistochemical staining step is: CD161 protein, CD56 protein, CD57 protein, CD94 protein, and Granzyme B protein. Among them, the anti-CD161 protein antibody is diluted 50 times with the blocking solution before use, the anti-CD56 protein antibody is diluted 100 times with the blocking solution before use, the anti-CD57 protein antibody is diluted 500 times with the blocking solution before use, the anti-CD94 protein antibody is diluted 50 times with the blocking solution before use, and the anti-Granzyme B protein antibody is diluted 400 times with the blocking solution before use.

[0120] Experimental Example 1: Performance verification of molecular markers and multiplex immunohistochemistry kit This experimental example verified the performance of molecular markers and the multiplex immunohistochemistry kit. The verification process is as follows: Using tissue sections of intestinal tissue as samples, after immunohistochemical detection of the tissue sections using the immunohistochemical detection methods of Examples 3-1 to 3-6 respectively, cell segmentation was performed using Qupath (v0.5.0), and the detection results were analyzed using R (4.2) to obtain the lymphoid cell populations, cell spatial distribution, and interaction relationships between cells in the tissue. The detection and analysis results are shown in Figures 1 to 50 .

[0121] It can be seen from Figures 1 to 50 that: The molecular marker combinations of Examples 1-1 to 1-6 can accurately localize and target lymphoid cells; can carefully complete the subdivision and quantitative cell component analysis of lymphoid cells such as T cells, B cells, and NK cells; can perform spatial neighborhood analysis on specific subsets of lymphoid cells in space, analyzing the distribution of cell types and cell components in the neighborhood; can analyze the cell interaction situation of lymphoid subset cells in space, analyzing the interaction and avoidance between different cell types, so as to explore the mechanism of interaction between cells; can be applied in immunohistochemical detection targeting lymphoid cells or screening of immunotherapeutic drugs targeting lymphoid cells. It can be seen that only the molecular marker combinations in Examples 1-1 to 1-6 can more accurately determine the lymphoid cell populations, cell spatial distribution, and interaction between cells in the tissue with a small sample size, and can achieve a comprehensive and good efficacy prediction and evaluation of immunotherapeutic drugs targeting lymphoid cell immune checkpoints.

[0122] Experimental Example 2: Influence of the selection of molecular markers on the detection effect of lymphoid cells This experimental example explored the influence of the selection of molecular markers on the detection effect of lymphoid cells. The experimental process is as follows: Using two sets of molecular marker combinations (control combination 1 to control combination 2) composed of common lymphoid cell protein markers as controls, the molecular marker combinations of Examples 1-1 to 1-6 were verified. During the verification stage, a large number of sample data containing prognostic survival information were collected from the The Cancer Genome Atlas (TCGA) database. Finally, through screening, a total of 418 available sample data were obtained. The Gene Set Variation Analysis (GSVA) algorithm was used to perform functional enrichment analysis on each gene in the molecular marker combination, systematically calculating the pathway activity scores of each gene set. Using the GSVA scores of the genes in the 8 sets of combinations as features, eight groups of multivariate survival analysis regression models (COX models) were constructed (the corresponding relationships between the proteins and genes in the molecular marker combination are shown in Table 2). To comprehensively evaluate the model performance, a dual verification system was adopted: the matching degree between the model prediction and the observed survival time was measured by the Concordance Index (C-Index), and at the same time, the Akaike Information Criterion (AIC) was combined to evaluate the simplicity and goodness of fit of the model. The evaluation results are shown in Figures 51 to 52 .

[0123] It can be seen from Figures 51 to 52 that: compared with the control combination 1 to control combination 2, the C-Index scores of the molecular marker combinations of Examples 1-1 to 1-6 have a significant increase, and at the same time, the decrease in AIC is significant, proving that the models constructed using the molecular marker combinations of Examples 1-1 to 1-6 have better performance for prognostic survival prediction, thus verifying that the molecular marker combinations of Examples 1-1 to 1-6 have better prediction effects for prognostic survival prediction.

[0124] Table 2 Corresponding relationship table between each protein and gene in the molecular marker combination

[0125] Obviously, the above examples are only for clear illustration and not a limitation of the implementation manner. For those of ordinary skill in the art, other different forms of changes or variations can be made based on the above description. It is not necessary and impossible to list all the implementation manners here. And the obvious changes or variations derived therefrom are still within the protection scope of the present invention.

Claims

1. A molecular marker targeting lymphoid cells for immunotherapy prediction, characterized in that, The molecular markers include CD8 protein, CD4 protein, PD-1 protein, Foxp3 protein, CD19 protein, and CD3 protein; Alternatively, the molecular markers include CD45 protein, CD45RA protein, CD45RO protein, CD25 protein, CCR7 protein, and CD127 protein; Alternatively, the molecular markers include CD3 protein, LAG3 protein, Tim3 protein, ICOS protein, CD69 protein, and CD103 protein; Alternatively, the molecular markers include CD79A protein, CD79B protein, CD20 protein, CD21 protein, and CD138 protein; Alternatively, the molecular markers include CD20 protein, CXCR5 protein, CD27 protein, CD38 protein, IGD protein, and IGM protein; Alternatively, the molecular markers include CD56 protein, CD161 protein, CD57 protein, CD94 protein, and Granzyme B protein.

2. The molecular marker according to claim 1, wherein The molecular markers consist of CD8 protein, CD4 protein, PD-1 protein, Foxp3 protein, CD19 protein, and CD3 protein; Alternatively, the molecular markers consist of CD45 protein, CD45RA protein, CD45RO protein, CD25 protein, CCR7 protein, and CD127 protein; Alternatively, the molecular markers consist of CD3 protein, LAG3 protein, Tim3 protein, ICOS protein, CD69 protein, and CD103 protein; Alternatively, the molecular markers consist of CD79A protein, CD79B protein, CD20 protein, CD21 protein, and CD138 protein; Alternatively, the molecular markers consist of CD20 protein, CXCR5 protein, CD27 protein, CD38 protein, IGD protein, and IGM protein; Alternatively, the molecular markers consist of CD56 protein, CD161 protein, CD57 protein, CD94 protein, and Granzyme B protein.

3. Use of a reagent for detecting the molecular markers according to claim 1 or 2 in the preparation of a product for predicting immunotherapy that targets lymphoid cells.

4. A product for immunotherapy prediction targeting lymphoid cells, characterized in that, The product contains a reagent for detecting the molecular markers according to claim 1 or 2.

5. The product according to claim 4, characterized in that, The reagent includes a first antibody; The first antibody includes an anti-CD8 protein antibody, an anti-CD4 protein antibody, an anti-PD-1 protein antibody, an anti-Foxp3 protein antibody, an anti-CD19 protein antibody, and an anti-CD3 protein antibody; Alternatively, the first antibody includes an anti-CD45 protein antibody, an anti-CD45RA protein antibody, an anti-CD45RO protein antibody, an anti-CD25 protein antibody, an anti-CCR7 protein antibody, and an anti-CD127 protein antibody; Alternatively, the first antibody includes an anti-CD3 protein antibody, an anti-LAG3 protein antibody, an anti-Tim3 protein antibody, an anti-ICOS protein antibody, an anti-CD69 protein antibody, and an anti-CD103 protein antibody; Alternatively, the first antibody includes an anti-CD79A protein antibody, an anti-CD79B protein antibody, an anti-CD20 protein antibody, an anti-CD21 protein antibody, and an anti-CD138 protein antibody; Alternatively, the first antibody includes an anti-CD20 protein antibody, an anti-CXCR5 protein antibody, an anti-CD27 protein antibody, an anti-CD38 protein antibody, an anti-IGD protein antibody, and an anti-IGM protein antibody; Alternatively, the first antibody includes an anti-CD56 protein antibody, an anti-CD161 protein antibody, an anti-CD57 protein antibody, an anti-CD94 protein antibody, and an anti-Granzyme B protein antibody.

6. The product according to claim 5, wherein, The first antibody consists of an anti-CD8 protein antibody, an anti-CD4 protein antibody, an anti-PD-1 protein antibody, an anti-Foxp3 protein antibody, an anti-CD19 protein antibody, and an anti-CD3 protein antibody; Alternatively, the first antibody consists of an anti-CD45 protein antibody, an anti-CD45RA protein antibody, an anti-CD45RO protein antibody, an anti-CD25 protein antibody, an anti-CCR7 protein antibody, and an anti-CD127 protein antibody; Alternatively, the first antibody consists of an anti-CD3 protein antibody, an anti-LAG3 protein antibody, an anti-Tim3 protein antibody, an anti-ICOS protein antibody, an anti-CD69 protein antibody, and an anti-CD103 protein antibody; Alternatively, the first antibody consists of an anti-CD79A protein antibody, an anti-CD79B protein antibody, an anti-CD20 protein antibody, an anti-CD21 protein antibody, and an anti-CD138 protein antibody; Alternatively, the first antibody consists of an anti-CD20 protein antibody, an anti-CXCR5 protein antibody, an anti-CD27 protein antibody, an anti-CD38 protein antibody, an anti-IGD protein antibody, and an anti-IGM protein antibody; Alternatively, the first antibody consists of an anti-CD56 protein antibody, an anti-CD161 protein antibody, an anti-CD57 protein antibody, an anti-CD94 protein antibody, and an anti-Granzyme B protein antibody.

7. An immunohistochemical detection method targeting lymphoid cells, characterized in that, The immunohistochemical detection method is not for the purpose of diagnosing and treating diseases. The immunohistochemical detection method uses the product according to any one of claims 4 to 6 to perform immunohistochemical detection on a sample to be tested.

8. A drug screening model for screening immunotherapeutic drugs targeting lymphoid cells, characterized in that, The drug screening model includes an information collection module, a data analysis module, and a prediction module; The information collection module is used to detect and collect immunohistochemical results related to the molecular markers according to claim 1 or 2 after an experimental subject is administered with a drug to be screened for immunotherapy; The data analysis module is used to analyze the immunohistochemical results obtained in the information collection step to obtain analysis results related to lymphoid cell immune checkpoints; The prediction module is used to classify the drug to be screened for immunotherapy as effective or ineffective according to the analysis results obtained by the data analysis module.

9. A drug screening method for screening immunotherapeutic drugs targeting lymphoid cells, characterized in that, The method includes: administering a drug to be screened for immunotherapy to an experimental subject; obtaining immunohistochemical results related to the molecular markers according to claim 1 or 2 after the experimental subject is administered with the drug to be screened for immunotherapy; and judging whether the drug to be screened for immunotherapy is effective or ineffective according to the immunohistochemical results.

10. Use of the molecular marker according to claim 1 or 2 or the product according to any one of claims 4 to 6 in immunohistochemical detection targeting lymphoid cells or screening of immunotherapeutic drugs targeting lymphoid cells, characterized in that, The application is not for the purpose of diagnosing and treating diseases.

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