A flow cytometry kit and monitoring method for monitoring tumor-related immune microenvironment

Abstract

The present invention relates to the field of flow cytometry, and more specifically, to a flow cytometry kit and a monitoring method for monitoring tumor-related immune microenvironment. The kit of the present invention includes 30 antibodies for monitoring 30 items, and combined with a full-spectrum flow cytometer, all the above tests can be completed in one panel, which is convenient, fast, time-saving, labor-saving, and easy to operate.

Description

Technical Field

[0001] The present invention relates to the field of flow cytometry, and in particular to a flow cytometry kit and a monitoring method for monitoring tumor-related immune microenvironment. Background Art

[0002] The tumor microenvironment (TME) is composed of tumor cells, stromal cells, immune cells and their secretory factors, vascular endothelial cells and extracellular matrix (ECM) and other components that colonize normal tissues. It plays an important role in tumor progression, immune escape and drug resistance mechanisms. Initially, these immune cells and related matrix components recruited and activated by tumor cells can form an anti-tumor inflammatory microenvironment to hinder the occurrence and development of tumors. After continuous tumor antigen stimulation and immune activation response, the relevant effector cells in the microenvironment are in a state of exhaustion, resulting in the inability to perform normal functions, thereby generating an immunosuppressive microenvironment to promote the occurrence and development of tumors. Studies in recent years have shown that immunotherapy targeting TME can stimulate or restore the inherent tumor suppression ability of the immune system, reshape the immune microenvironment, and produce anti-tumor responses. Therefore, monitoring the individualized TME characteristics of tumor patients can be used to adjust and optimize immunotherapy strategies.

[0003] The immune process includes three stages: immune clearance, immune balance, and immune escape. In the immune clearance stage, innate immunity and adaptive immunity work together to quickly eliminate tumor cells. If tumor cell mutations cannot be effectively identified, they may enter the balance stage and survive with the tumor. If under continuous immune pressure, tumor cell mutations are no longer recognized by adaptive immunity, are insensitive to immune effector mechanisms, or induce the tumor microenvironment to an immunosuppressive state, tumor cells enter the escape stage, and tumor growth is no longer blocked by the immune system, leading to immune tolerance.

[0004] The immune cells involved in the immune process include innate immune phagocytes, dendritic cells, natural killer cells, etc. Adaptive immune cells include T cells and B cells. Each type of cell is divided into different subgroups, which perform different functions. Innate immunity and adaptive immunity, T cells and B cells jointly participate in the immune process of tumor clearance and development through synergy, balanced regulation of activation and inhibition, expression of inhibitory receptors and other mechanisms.

[0005] Flow cytometry (FCM) is a technique that directly detects single cells labeled with fluorescent signals to achieve high-speed quantitative analysis and sorting of cells. The main advantages are fast measurement speed, multi-parameter detection, and integration of multiple disciplines and technologies such as optics, electronics, fluid mechanics, cell chemistry, immunology, lasers and computers. It is both a cell analysis technology and an accurate sorting technology. At present, it has a wide range of applications in the field of clinical medicine and can be used for immunophenotyping and immunological research of blood tumors. From the advent of 1-laser 2-color flow in 1980 to the advent of 3-laser 13-color flow in 2000, the rapid development of flow technology has greatly promoted the application and development of clinical immunology monitoring. The advent of full-spectrum flow in 2017 has increased the detection channels of three-laser flow from 13 to 38 in traditional flow, and can achieve more than 25-color monitoring in one tube, greatly improving the amount of detection information, diagnostic accuracy and depth of immunology detection.

[0006] Because the tumor-related immune microenvironment involves a large number of immune cells and cell subsets, numerous inhibitory receptors, fewer traditional flow cytometry channels, expensive antibodies, and insufficient commercial fluorescent dyes, previous tests have not only been insufficient in detection depth, but also difficult to detect all the target cells. The lack of detection breadth and depth makes it difficult to screen out cells that have a clear impact on the occurrence and development of specific tumors for testing and monitoring, and it is even more difficult to adjust clinical treatment strategies in a timely and individualized manner based on this.

[0007] In view of this, the present invention is proposed. Summary of the invention

[0008] The first invention object of the present invention is to provide a flow cytometry kit for monitoring tumor-related immune microenvironment.

[0009] The second invention objective of the present invention is to provide a method for using the kit.

[0010] The third invention objective of the present invention is to provide a method for monitoring tumor-related immune microenvironment.

[0011] In order to achieve the purpose of the invention, the technical solution adopted is:

[0012] The present invention provides a flow cytometry kit for monitoring tumor-related immune microenvironment, wherein the kit contains antibodies respectively labeled with different fluorescent substances, wherein the antibodies include antibodies for monitoring immunosuppression-related antibodies, antibodies for T subgroup classification, and antibodies for monitoring immune activation;

[0013] The antibodies used to monitor immunosuppression include: anti-2B4 antibody, anti-BTLA antibody, anti-CD160 antibody, anti-LAG-3 antibody, anti-VISTA antibody, anti-CD95 antibody, anti-CTLA4 antibody, anti-PD-1 antibody, anti-TIGIT antibody and anti-TIM3 antibody;

[0014] The antibodies used for T subgroup classification include: anti-CCR6 antibody, anti-CD127 antibody, anti-CD183 antibody, anti-CD25 antibody, anti-CD28 antibody, anti-CD3 antibody, anti-CD4 antibody, anti-CD45RA antibody, anti-CD62L antibody, anti-CD8 antibody, anti-EOMES antibody, anti-T-bet antibody, anti-TCRab antibody and anti-TCRrd antibody;

[0015] The antibodies used to monitor immune activation include: anti-CD38 antibody, anti-CD39 antibody, anti-CD69 antibody, anti-CD80 antibody, anti-CD86 antibody and anti-HLA-DR antibody.

[0016] Optionally, the anti-2B4 antibody is connected to fluorescein RB730, the anti-BTLA antibody is connected to fluorescein RR664, the anti-CD160 antibody is connected to fluorescein RR600, the anti-LAG-3 antibody is connected to fluorescein RV496, the anti-VISTA antibody is connected to fluorescein RB780, the anti-CD95 antibody is connected to fluorescein RV520, the anti-CTLA4 antibody is connected to fluorescein RR778, the anti-PD-1 antibody is connected to fluorescein RV805, the anti-TIGIT antibody is connected to fluorescein RV444, the anti-TIM3 antibody is connected to fluorescein RB515, the anti-CCR6 antibody is connected to fluorescein RR678, the anti-CD127 antibody is connected to fluorescein RB667, the anti-CD183 antibody is connected to fluorescein RR705, the anti-CD25 antibody is connected to fluorescein RV480, and the anti-CD28 antibody is connected to fluorescein RV73 4. The anti-CD3 antibody is connected to fluorescein RV780, the anti-CD4 antibody is connected to fluorescein RV624, the anti-CD45RA antibody is connected to fluorescein RV526, the anti-CD62L antibody is connected to fluorescein RB697, the anti-CD8 antibody is connected to fluorescein RV650, the anti-EOMES antibody is connected to fluorescein RV681, the anti-T-bet antibody is connected to fluorescein RV656, the anti-TCRab antibody is connected to fluorescein RB608, the anti-TCRrd antibody is connected to fluorescein RB574, the anti-CD38 antibody is connected to fluorescein RB670, the anti-CD39 antibody is connected to fluorescein RB710, the anti-CD69 antibody is connected to fluorescein RV600, the anti-CD80 antibody is connected to fluorescein RB713, the anti-CD86 antibody is connected to fluorescein RV540, and the anti-HLA-DR antibody is connected to fluorescein RV645.

[0017] Optionally, each of the antibodies is packaged separately, and the concentration of the antibody is 80-120 μg / ml, preferably 100 μg / ml.

[0018] Optionally, the packaging volume of each of the antibodies is 0.5 mL; the volume of each of the antibodies used in each sample to be tested is 5 μl.

[0019] Optionally, anti-CD95 antibody, anti-CTLA4 antibody, anti-PD-1 antibody, anti-TIGIT antibody, TIM3 antibody, anti-2B4 antibody, anti-BTLA antibody, anti-CD160 antibody, anti-LAG-3 antibody, anti-VISTA antibody, anti-CCR6 antibody, anti-CD127 antibody, anti-CD183 antibody, anti-CD25 antibody, anti-CD28 antibody, anti-CD3 antibody, anti-CD4 antibody, anti-CD45RA antibody, anti-CD62L antibody, anti-CD8 antibody, anti-TCRab antibody, anti-TCRrd antibody, anti-CD38 antibody, anti-CD39 antibody, anti-CD69 antibody, anti-CD80 antibody, anti-CD86 antibody and anti-HLA-DR antibody are membrane antibodies, and anti-EOMES antibody and anti-T-bet antibody are cytoplasmic antibodies.

[0020] The present invention relates to a method for using the kit, which at least comprises the following steps:

[0021] (1) Take two flow cytometry tubes and mark them as sample tube and control tube;

[0022] (2) First add Brilliant Stain Buffer to the sample tube, and then add a cell membrane antibody connected with fluorescein; the cell membrane antibodies include anti-CD95 antibody, anti-CTLA4 antibody, anti-PD-1 antibody, anti-TIGIT antibody, TIM3 antibody, anti-2B4 antibody, anti-BTLA antibody, anti-CD160 antibody, anti-LAG-3 antibody, anti-VISTA antibody, anti-CCR6 antibody, anti-CD127 antibody, anti-CD183 antibody, anti-CD25 antibody, anti-CD28 antibody, anti-CD3 antibody, anti-CD4 antibody, anti-CD45RA antibody, anti-CD62L antibody, anti-CD8 antibody, anti-TCRab antibody, anti-TCRrd antibody, anti-CD38 antibody, anti-CD39 antibody, anti-CD69 antibody, anti-CD80 antibody, anti-CD86 antibody and anti-HLA-DR antibody;

[0023] (3) Add the sample to be tested to the sample tube and the control tube respectively, and incubate to allow the antibody to bind to the cell antigen;

[0024] (4) Add red blood cell lysis buffer to the sample tube and control tube, centrifuge, and wash;

[0025] (5) Add membrane permeabilization agent 1 to the sample tube and control tube respectively, incubate and then wash;

[0026] (6) Add membrane permeabilization agent 2 to the sample tube and control tube respectively, incubate and then wash;

[0027] (7) adding cytoplasmic antibodies linked to fluorescein to the sample tube and incubating; the cytoplasmic antibodies include anti-EOMES antibodies and anti-T-bet antibodies;

[0028] (8) Wash, mix and inspect on a machine.

[0029] Optionally, all operations in the method of use are carried out at room temperature.

[0030] The present invention also relates to a method for monitoring a tumor-associated immune microenvironment, comprising at least the following steps:

[0031] (1) Take two flow cytometry tubes and mark them as sample tube and control tube;

[0032] (2) First add Brilliant Stain Buffer to the sample tube, then add membrane antibody;

[0033] (3) Add the sample to be tested to the sample tube and the control tube respectively and incubate;

[0034] (4) Add red blood cell lysis buffer to the sample tube and control tube, centrifuge, and wash;

[0035] (5) Add membrane permeabilization agent 1 to the sample tube and control tube respectively, incubate and then wash;

[0036] (6) Add membrane permeabilization agent 2 to the sample tube and control tube respectively, incubate and then wash;

[0037] (7) Add cytoplasmic antibodies to the sample tube and incubate;

[0038] (8) Cleaning, mixing, and testing;

[0039] (9) Analyze data.

[0040] The present invention has at least the following beneficial effects:

[0041] The kit of the present invention includes 30 antibodies, and monitors 30 items, including CD4+T / CD8+T / initial T / effector T / central memory T / effector memory T / stem cell memory T / rescible exhausted T / irrescible exhausted T / Treg / TH1 / TH2 / TH17 / abT / rdT / aging T / T activation / Treg activation / T inhibition / inhibitory receptors associated with tumor progression. Combined with a full-spectrum flow cytometer, all the above tests can be completed with one panel, which is convenient, fast, time-saving, labor-saving, and easy to operate. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1 is a two-dimensional dot plot of flow cytometry of Example 2;

[0043] Figure 2 is a two-dimensional dot plot of flow cytometry of Example 2;

[0044] Figure 3is a two-dimensional dot plot of flow cytometry of Example 2;

[0045] Figure 4 is a two-dimensional dot plot of flow cytometry of Example 2;

[0046] Figure 5 is a two-dimensional dot plot of flow cytometry of Example 2;

[0047] Figure 6 is a two-dimensional dot plot of flow cytometry of Example 2;

[0048] Figure 7 This is a two-dimensional dot plot of flow cytometry in Example 2.

[0049] The present invention is further described below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present application and are not used to limit the scope of the present invention. DETAILED DESCRIPTION

[0050] The present invention is further described below by examples and comparative examples, which are only used to illustrate the present invention and are not limited to the following examples. Any modification or equivalent replacement of the technical solution of the present invention without departing from the spirit and scope of the technical solution of the present invention shall be included in the protection scope of the present invention.

[0051] The embodiment of the present invention proposes a flow cytometry kit for monitoring tumor-related immune microenvironment, which uses full-spectrum flow cytometry, designs a 30-color 1-tube panel, and a total of 30 antibodies to deeply detect T cell subpopulations, T cell activation status, and the expression of some inhibitory receptors, monitor the expression of all T cell subpopulations and related functions of tumor patients in different periods and different course changes of immune status, and deeply monitor T cell subpopulations and functions in the immune microenvironment of tumor patients in combination with full-spectrum flow cytometer. It is routinely used for monitoring specific time nodes of clinical tumor patients, providing a reference for timely adjustment of clinical immunotherapy programs, optimization of individualized program design, and immune resistance research.

[0052] Specifically, the flow cytometry kit of the embodiment of the present invention contains antibodies respectively labeled with different fluorescent dyes, including antibodies for monitoring immunosuppression-related antibodies, antibodies for T subgroup classification, and antibodies for monitoring immune activation, so that immunosuppression and activation can be systematically monitored and T subgroups can be classified at the same time.

[0053] Antibodies used to monitor immunosuppression include: anti-2B4 antibody, anti-BTLA antibody, anti-CD160 antibody, anti-LAG-3 antibody, anti-VISTA antibody, anti-CD95 antibody, anti-CTLA4 antibody, anti-PD-1 antibody, anti-TIGIT antibody, and anti-TIM3 antibody. Antibodies related to immunosuppression are used to monitor whether immune function is suppressed, resulting in disease failure or progression.

[0054] Antibodies used to monitor immune activation include: anti-CD38 antibody, anti-CD39 antibody, anti-CD69 antibody, anti-CD80 antibody, anti-CD86 antibody, and anti-HLA-DR antibody. Among them, anti-CD38 antibody is used to monitor T cell activation, anti-CD39 antibody is used to monitor early T activation, anti-HLA-DR antibody is used to monitor mid-stage T activation, anti-CD25 antibody is used to monitor early T activation, and anti-CD69 antibody, anti-CD80 antibody, and anti-CD86 antibody are all used to monitor activation status. This allows for a more comprehensive detection of activation, which is used to observe whether T cell activation is normal when the body encounters antigen stimulation, and whether the anti-infection and anti-tumor functions are normal.

[0055] Antibodies used for T cell subset classification include: anti-CCR6 antibody, anti-CD127 antibody, anti-CD183 antibody, anti-CD25 antibody, anti-CD28 antibody, anti-CD3 antibody, anti-CD4 antibody, anti-CD45RA antibody, anti-CD62L antibody, anti-CD8 antibody, anti-EOMES antibody, anti-T-bet antibody, anti-TCRab antibody, and anti-TCRrd antibody; classification antibodies are used to distinguish T cell subsets. The functions of T cell subsets and each antibody are shown in Table 1:

[0056] Table 1

[0057]

[0058]

[0059]

[0060] The monitoring result of the kit of the present invention can be verified by using freeze-dried human T / B / NK lymphocyte national reference product, and the verification result is: the accuracy rate is 100%.

[0061] Specifically, the corresponding relationship between the fluorescein and the antibody binding in the kit of the embodiment of the present invention is shown in Table 2:

[0062] Table 2

[0063]

[0064]

[0065] In the embodiment of the present invention, the intensity of the fluorescent dye of the corresponding intensity is matched according to the intensity of antibody expression. This panel is optimized in the experiment using full-spectrum multicolor flow cytometry and can be used directly in the clinic without preliminary experiments. It will not lead to the inability to classify T cell subpopulations or inaccurate test results due to inappropriate expression of antibody intensity during detection.

[0066] Specifically, each antibody in the embodiment of the present invention is packaged separately, and the concentration of the antibody is 80-120 μg / ml, preferably 100 μg / ml. The packaging volume of each antibody is 0.5 mL; the volume of each antibody used for each sample to be tested is 5 μl. The antibodies are stored in a PBS buffer solution supplemented with 0.1 wt% sodium azide.

[0067] Other reagents required for the experiment include: Brilliant Stain Buffer (BD, purchase link: https: / / www.bdbiosciences.com / cn / solrSearch?text=566349#); Permeabilization Reagent 1 and Permeabilization Reagent 2 (IntraPrepPermeabilization Reagent, from Beckman Coulter, purchase link: https: / / www.beckmancoulter.cn / bls_search / flowreagents / search / ); PBS; Red blood cell lysis buffer (Lyse TM Lysing Buffer, from BD Pharm, the purchase link is https: / / www.bdbiosciences.com / cn / applications / research / stem-cell-research / stem-cell-buffers-and-ancillary-reagents / lysing-buffer / p / 555899).

[0068] The specific method of using the kit of the embodiment of the present invention is:

[0069] 1. Preparation of reagents:

[0070] 1. Take two flow tubes and mark them as sample tube and control tube;

[0071] 2. Add 10 mL of Brilliant Stain Buffer to the sample tube, then add 5 mL of cell membrane antibody and mix thoroughly;

[0072] 3. Add 100 mL of the sample to be tested to the sample tube and control tube respectively, mix thoroughly, and incubate at room temperature for 15 minutes to allow the antibody to bind to the cell antigen;

[0073] 3. After mixing again, add 4 mL of red blood cell lysis solution to the sample tube and control tube respectively, and lyse at room temperature for 10 minutes;

[0074] 4. Centrifuge at 1500 rpm for 5 min, discard the supernatant, and mix thoroughly;

[0075] 5. Add 4 mL of PBS to the sample tube and control tube respectively, centrifuge at 1500 rpm for 5 min, discard the supernatant, and mix thoroughly;

[0076] 6. Add 50-100 μL of membrane permeabilization agent 1 to the sample tube and control tube respectively, incubate at room temperature in the dark for 10 minutes, and mix thoroughly;

[0077] 7. Add 4 mL of PBS to the sample tube and control tube respectively, centrifuge at 1500 rpm, discard the supernatant, and mix well;

[0078] 8. Add 250 μL of membrane permeabilization agent to the sample tube and control tube respectively, and gently mix the samples with a pipette, and incubate at room temperature away from light for 5 minutes;

[0079] 9. Add cytoplasmic antibodies to the sample tube, mix thoroughly, incubate at room temperature in the dark for 15 minutes to allow the antibody to bind to the cell antigen, and mix well;

[0080] 10. Add 4 mL of PBS to the sample tube and control tube respectively, centrifuge at 1500 rpm, and discard the supernatant;

[0081] 11. Add appropriate amount of PBS to the sample tube and control tube respectively, mix well, filter through a 300-mesh filter, and wait for testing.

[0082] 2. Machine testing:

[0083] The prepared sample tubes and control tubes were directly detected by Cytek full-spectrum flow cytometer, and the data were analyzed using the instrument's own analysis system.

[0084] By monitoring the percentage of target cells in the sample to be tested, and comparing it with the reference value, we can see whether the proportion of the detected cell subpopulation is higher or lower than the reference value, thereby evaluating the immune status of tumor patients.

[0085] 3. Result output.

[0086] The embodiment of the present invention also relates to a method for monitoring a tumor-associated immune microenvironment, which comprises at least the following steps:

[0087] 1. Take two flow tubes and mark them as sample tube and control tube;

[0088] 2. Add 10 mL of Brilliant Stain Buffer to the sample tube, then add 5 mL of membrane antibody and mix thoroughly;

[0089] 3. Add 100 mL of the sample to be tested to the sample tube and control tube respectively, mix thoroughly, and incubate at room temperature for 15 minutes to allow the antibody to bind to the cell antigen;

[0090] 3. After mixing again, add 4 mL of red blood cell lysis solution to the sample tube and control tube respectively, and lyse at room temperature for 10 minutes;

[0091] 4. Centrifuge at 1500 rpm for 5 min, discard the supernatant, and mix thoroughly;

[0092] 5. Add 4 mL of PBS to the sample tube and control tube respectively, centrifuge at 1500 rpm for 5 min, discard the supernatant, and mix thoroughly;

[0093] 6. Add 50-100 μL of membrane permeabilization agent 1 to the sample tube and control tube respectively, incubate at room temperature in the dark for 10 minutes, and mix thoroughly;

[0094] 7. Add 4 mL of PBS to the sample tube and control tube respectively, centrifuge at 1500 rpm, discard the supernatant, and mix well;

[0095] 8. Add 250 μL of membrane permeabilization agent to the sample tube and control tube respectively, and gently mix the samples with a pipette, and incubate at room temperature away from light for 5 minutes;

[0096] 9. Add cytoplasmic antibodies to the sample tube, mix thoroughly, incubate at room temperature in the dark for 15 minutes to allow the antibody to bind to the cell antigen, and mix well;

[0097] 10. Add 4 mL of PBS to the sample tube and control tube respectively, centrifuge at 1500 rpm, and discard the supernatant;

[0098] 11. Add appropriate amount of PBS to the sample tube and control tube respectively, mix well, filter through a 300-mesh filter, and wait for testing.

[0099] 12. Data analysis: Use the instrument's own software to analyze data, and compare the obtained data on the percentage of each target cell occupying the nucleus with the data of normal people or the first diagnosis of the test subject (i.e., the reference value) to see whether the proportion of the detected cell subpopulation is higher or lower than the reference value, so as to evaluate the immune status of the tumor patient;

[0100] Specifically, the determination of reference values ​​refers to setting a number of specifications and standards among the healthy population in a region, sampling and surveying the population, and calculating the mean value (X) and standard deviation (S) of the measurement results through statistical processing. Usually X is positioned as the reference value, and X±2S is set as the reference range.

[0101] Example 1

[0102] A flow cytometry kit for monitoring tumor-associated immune microenvironment, the specific composition of which is shown in Table 3.

[0103] Table 3

[0104]

[0105]

[0106] Example 2

[0107] The peripheral blood sample of the subject to be tested is used as the test sample, and the kit of Example 1 is used for detection to obtain a two-dimensional dot map of flow cytometry as shown in FIG. Figure 1 to Figure 7 (The meaning of the gate is shown in Table 5). The instrument's own software is used for data analysis, and a table as shown in Table 4 is automatically generated. The percentage of each target cell occupying the nucleus is compared with the reference value to see whether the proportion of the detected cell subpopulation is higher or lower than the reference value, thereby evaluating the immune status of the subject.

[0108] The test results of the subject are shown in Table 4.

[0109] Table 4

[0110]

[0111]

[0112]

[0113] Table 5

[0114]

[0115]

[0116]

[0117] Although the present application is disclosed as above with preferred embodiments, it is not intended to limit the claims. Any technical personnel in this field may make several possible changes and modifications without departing from the concept of the present application. Therefore, the scope of protection of the present application shall be based on the scope defined by the claims of the present application.

Claims

1. A flow cytometry kit for monitoring tumor-associated immune microenvironment, characterized in that: The kit contains antibodies respectively labeled with different fluoresceins, and the antibodies include antibodies for monitoring immunosuppression, antibodies for T subgroup classification, and antibodies for monitoring immune activation; The antibodies used to monitor immunosuppression include: anti-2B4 antibody, anti-BTLA antibody, anti-CD160 antibody, anti-LAG-3 antibody, anti-VISTA antibody, anti-CD95 antibody, anti-CTLA4 antibody, anti-PD-1 antibody, anti-TIGIT antibody and anti-TIM3 antibody; The antibodies used for T subgroup classification include: anti-CCR6 antibody, anti-CD127 antibody, anti-CD183 antibody, anti-CD25 antibody, anti-CD28 antibody, anti-CD3 antibody, anti-CD4 antibody, anti-CD45RA antibody, anti-CD62L antibody, anti-CD8 antibody, anti-EOMES antibody, anti-T-bet antibody, anti-TCRab antibody and anti-TCRrd antibody; The antibodies used to monitor immune activation include: anti-CD38 antibody, anti-CD39 antibody, anti-CD69 antibody, anti-CD80 antibody, anti-CD86 antibody and anti-HLA-DR antibody; The anti-2B4 antibody is connected to fluorescein RB730, the anti-BTLA antibody is connected to fluorescein RR664, the anti-CD160 antibody is connected to fluorescein RR600, the anti-LAG-3 antibody is connected to fluorescein RV496, the anti-VISTA antibody is connected to fluorescein RB780, the anti-CD95 antibody is connected to fluorescein RV520, the anti-CTLA4 antibody is connected to fluorescein RR778, the anti-PD-1 antibody is connected to fluorescein RV805, the anti-TIGIT antibody is connected to fluorescein RV444, the anti-TIM3 antibody is connected to fluorescein RB515, the anti-CCR6 antibody is connected to fluorescein RR678, the anti-CD127 antibody is connected to fluorescein RB667, the anti-CD183 antibody is connected to fluorescein RR705, the anti-CD25 antibody is connected to fluorescein RV480, the anti-CD28 antibody is connected to fluorescein RV734, The anti-CD3 antibody is connected to fluorescein RV780, the anti-CD4 antibody is connected to fluorescein RV624, the anti-CD45RA antibody is connected to fluorescein RV526, the anti-CD62L antibody is connected to fluorescein RB697, the anti-CD8 antibody is connected to fluorescein RV650, the anti-EOMES antibody is connected to fluorescein RV681, the anti-T-bet antibody is connected to fluorescein RV656, the anti-TCRab antibody is connected to fluorescein RB608, the anti-TCRrd antibody is connected to fluorescein RB574, the anti-CD38 antibody is connected to fluorescein RB670, the anti-CD39 antibody is connected to fluorescein RB710, the anti-CD69 antibody is connected to fluorescein RV600, the anti-CD80 antibody is connected to fluorescein RB713, the anti-CD86 antibody is connected to fluorescein RV540, and the anti-HLA-DR antibody is connected to fluorescein RV645.

2. The kit according to claim 1, characterized in that Each of the antibodies is packaged separately, and the concentration of the antibodies is 80-120 μg / ml.

3. The kit according to claim 2, characterized in that Each of the antibodies was packaged separately, and the concentration of the antibodies was 100 μg / ml.

4. The kit according to claim 3, characterized in that The packaging volume of each of the antibodies is 0.5 mL; the volume of each of the antibodies used in each sample to be tested is 5 μL.

5. The kit according to claim 1, characterized in that Anti-CD95 antibody, anti-CTLA4 antibody, anti-PD-1 antibody, anti-TIGIT antibody, TIM3 antibody, anti-2B4 antibody, anti-BTLA antibody, anti-CD160 antibody, anti-LAG-3 antibody, anti-VISTA antibody, anti-CCR6 antibody, anti-CD127 antibody, anti-CD183 antibody, anti-CD25 antibody, anti-CD28 antibody, anti-CD3 antibody, anti-CD4 antibody, anti-CD45RA antibody, anti-CD62L antibody, anti-CD8 antibody, anti-TCRab antibody, anti-TCRrd antibody, anti-CD38 antibody, anti-CD39 antibody, anti-CD69 antibody, anti-CD80 antibody, anti-CD86 antibody and anti-HLA-DR antibody are cell membrane antibodies; Anti-EOMES antibodies and anti-T-bet antibodies are cytoplasmic antibodies.

6. Use of an antibody composition in preparing a kit for detecting tumor-associated immune microenvironment, characterized in that: The antibody composition contains antibodies respectively labeled with different fluoresceins, and the antibodies include antibodies for monitoring immunosuppression, antibodies for T subgroup classification, and antibodies for monitoring immune activation; The antibodies used to monitor immunosuppression include: anti-2B4 antibody, anti-BTLA antibody, anti-CD160 antibody, anti-LAG-3 antibody, anti-VISTA antibody, anti-CD95 antibody, anti-CTLA4 antibody, anti-PD-1 antibody, anti-TIGIT antibody and anti-TIM3 antibody; The antibodies used for T subgroup classification include: anti-CCR6 antibody, anti-CD127 antibody, anti-CD183 antibody, anti-CD25 antibody, anti-CD28 antibody, anti-CD3 antibody, anti-CD4 antibody, anti-CD45RA antibody, anti-CD62L antibody, anti-CD8 antibody, anti-EOMES antibody, anti-T-bet antibody, anti-TCRab antibody and anti-TCRrd antibody; The antibodies used to monitor immune activation include: anti-CD38 antibody, anti-CD39 antibody, anti-CD69 antibody, anti-CD80 antibody, anti-CD86 antibody and anti-HLA-DR antibody; The anti-2B4 antibody is connected to fluorescein RB730, the anti-BTLA antibody is connected to fluorescein RR664, the anti-CD160 antibody is connected to fluorescein RR600, the anti-LAG-3 antibody is connected to fluorescein RV496, the anti-VISTA antibody is connected to fluorescein RB780, the anti-CD95 antibody is connected to fluorescein RV520, the anti-CTLA4 antibody is connected to fluorescein RR778, the anti-PD-1 antibody is connected to fluorescein RV805, the anti-TIGIT antibody is connected to fluorescein RV444, the anti-TIM3 antibody is connected to fluorescein RB515, the anti-CCR6 antibody is connected to fluorescein RR678, the anti-CD127 antibody is connected to fluorescein RB667, the anti-CD183 antibody is connected to fluorescein RR705, the anti-CD25 antibody is connected to fluorescein RV480, the anti-CD28 antibody is connected to fluorescein RV734, The anti-CD3 antibody is connected to fluorescein RV780, the anti-CD4 antibody is connected to fluorescein RV624, the anti-CD45RA antibody is connected to fluorescein RV526, the anti-CD62L antibody is connected to fluorescein RB697, the anti-CD8 antibody is connected to fluorescein RV650, the anti-EOMES antibody is connected to fluorescein RV681, the anti-T-bet antibody is connected to fluorescein RV656, the anti-TCRab antibody is connected to fluorescein RB608, the anti-TCRrd antibody is connected to fluorescein RB574, the anti-CD38 antibody is connected to fluorescein RB670, the anti-CD39 antibody is connected to fluorescein RB710, the anti-CD69 antibody is connected to fluorescein RV600, the anti-CD80 antibody is connected to fluorescein RB713, the anti-CD86 antibody is connected to fluorescein RV540, and the anti-HLA-DR antibody is connected to fluorescein RV645.

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