Method for detecting cynomolgus monkey activated memory T cells and tissue resident memory T cells by flow cytometry

Abstract

The invention provides a method for detecting cynomolgus monkey activated memory T cells and tissue resident memory T cells through flow cytometry, and belongs to the technical field of biological detection. According to the invention, a stable and reliable flow cytometry detection method is established by optimizing a specific fluorescent antibody combination and a detection process. The method has the direct effects that a plurality of key T cell subgroups including activated memory T cells and tissue resident memory T cells can be clearly identified at one time from peripheral blood and nasal mucosa samples of cynomolgus monkeys, the detection result is accurate, and the repeatability is good. The establishment of the method provides key technical support for comprehensive and deep evaluation of immune response, especially local immune memory of mucous membrane parts in preclinical drug safety and immune efficacy evaluation, and has important application value for research and development of biological medicine products such as vaccines and immunomodulators.

Description

Technical Field

[0001] This invention relates to the field of biodetection technology, and in particular to a flow cytometry method for detecting activated memory T cells and tissue-resident memory T cells in cynomolgus monkeys. Background Technology

[0002] Flow cytometry is a technique for rapid, multi-parameter quantitative analysis and sorting of cells or other biological particles suspended in a fluid. This technique utilizes fluorescently labeled antibodies that bind to specific antigens on or inside cells. As cells pass through a detector one by one, the instrument identifies and counts the cells' physical and chemical properties and phenotype by detecting their scattered light and fluorescence signals. In the fields of immunology research and drug development, flow cytometry has become an indispensable tool for analyzing complex immune cell populations.

[0003] T lymphocytes are the core of adaptive immunity and can be divided into different subsets based on their surface markers and functions. Among them, memory T cells are formed after the body's first exposure to an antigen and initiate a rapid and strong immune response upon re-encountering the same antigen, forming the basis of long-term immune protection. The memory T cell population is highly heterogeneous, including memory cells circulating in the blood and peripheral lymphoid organs, as well as tissue-resident memory T cells that reside in specific tissues and do not participate in recirculation. Activated memory T cells typically highly express early activation markers such as CD69, while tissue-resident memory T cells characteristically express molecules such as CD103, anchoring them to epithelial tissues.

[0004] The cynomolgus monkey, as an important non-human primate laboratory animal, is widely used in biomedical research, particularly in immunology, infectious disease models, and preclinical efficacy and safety evaluations. In-depth assessment of its immune system, especially its mucosal immune response, is crucial for the development of respiratory vaccines, inhaled formulations, and other products. However, due to the complex and overlapping phenotypes of different T cell subsets, simultaneously analyzing multiple key subsets, especially in mucosal samples with limited cell counts and complex backgrounds, such as nasal lavage fluid, presents technical challenges. Existing detection methods often struggle to detect CD4+ within a single sample tube. + and CD8 + The systematic and precise differentiation and quantification of activated memory subsets and tissue-resident memory subsets in T cells has limited the ability to comprehensively assess the immune response. Summary of the Invention

[0005] The purpose of this invention is to provide a flow cytometry method for detecting activated memory T cells and tissue-resident memory T cells in cynomolgus monkeys, solving the problem that existing technologies cannot simultaneously and accurately analyze CD4 in multiple samples such as peripheral blood and nasal lavage fluid from cynomolgus monkeys. + With CD8 +Technical issues related to activated memory T cells and tissue-resident memory T cells within T cell subsets.

[0006] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides a method for detecting activated memory T cells and tissue-resident memory T cells in cynomolgus monkeys using flow cytometry, the method comprising the following steps: Samples of cynomolgus monkeys were collected and treated with anticoagulants. The sample was mixed with a fluorescent antibody and then stained. Flow cytometry was used to analyze the stained samples. Flow cytometry data were analyzed to distinguish between CD4-positive and CD8-positive T cell subsets, and further detection was performed on CD4-positive T cell subsets containing CD44-positive CD69-positive activated memory T cells and CD44-positive CD103-positive CD69-positive tissue-resident memory T cells, as well as CD8-positive T cell subsets containing CD44-positive CD69-positive activated memory T cells and CD44-positive CD103-positive CD69-positive tissue-resident memory T cells. The fluorescent antibody combination includes BV510 Mouse Anti-NHP CD45 antibody, BB700 Mouse Anti-Human CD3 antibody, BUV395 Mouse Anti-Human CD4 antibody, Alexa Fluor 700 anti-human CD8a antibody, PE Anti-Human CD69 antibody, Human CD44 / Pgp-1 APC antibody, and CD103 Monoclonl Antibody FITC antibody.

[0007] Preferably, the cynomolgus monkey samples include peripheral blood samples and nasal irrigation fluid samples.

[0008] Preferably, the peripheral blood sample is treated with heparin sodium anticoagulation.

[0009] Preferably, the nasal irrigation fluid sample is processed by centrifugation to obtain cells.

[0010] Preferably, the amount of the BV510 Mouse Anti-NHP CD45 antibody used is 0.5-2 μL per test; The dosage of the BB700 Mouse Anti-Human CD3 antibody is 0.5-2 μL per test.

[0011] Preferably, the amount of the BUV395 Mouse Anti-Human CD4 antibody used is 0.5-2 μL per test; The dosage of the Alexa Fluor 700 anti-human CD8a antibody is 0.5-2 μL per test.

[0012] Preferably, the amount of the PE Anti-Human CD69 antibody used is 1-4 μL per test; The dosage of the Human CD44 / Pgp-1 APC antibody is 2-5 μL per test; The amount of CD103 Monoclonl Antibody FITC antibody used is 1-4 μL per test.

[0013] Preferably, a flow cytometer is used for detection, wherein the FSC channel voltage of the flow cytometer is set to 200-300 and the threshold is set to 50000-100000.

[0014] Preferably, the analysis steps include gate logic analysis, namely, circling the leukocyte population on the FSC-A / SSC-A scatter plot, circling the single cell population on the FSC-A / FSC-H scatter plot, circling the T cell population on the CD45 and CD3 positive scatter plot, circling the CD4 positive T cell and CD8 positive T cell subsets on the CD4 and CD8 positive scatter plot, and circling activated memory T cells and tissue-resident memory T cells on the CD44, CD69 and CD103 positive scatter plot.

[0015] Preferably, the method includes using a blank control tube, a compensation control tube, and a fluorescence minus one control tube.

[0016] The beneficial effects of this invention are: This invention establishes a stable and reliable flow cytometry detection method by optimizing specific fluorescent antibody combinations and detection procedures. Its direct effect is the ability to clearly identify multiple key T cell subsets, including activated memory T cells and tissue-resident memory T cells, in a single, simultaneous analysis of peripheral blood and nasal mucosa samples from cynomolgus monkeys. The detection results are accurate and reproducible. This method provides crucial technical support for comprehensively and thoroughly evaluating immune responses, particularly local immune memory at the mucosal site, in preclinical drug safety and immunogenicity assessments. It also has significant application value for the research and development of biopharmaceutical products such as vaccines and immunomodulators. Attached Figure Description

[0017] Figure 1 A schematic diagram showing the monochrome compensation adjustment of the Y585-PE-A and B525-FITC-A channels; Figure 2 A graph showing the subpopulation parameters of T cells in counterstained samples; Figure 3Gating the white blood cell population (WBC) in the FSC-A / SSC-A scatter plot; Figure 4 scatter plot of LD R763-APCA750-A / SSC-A to show viable cell populations (LD, suitable for nasal irrigation fluid samples); Figure 5 Gate the scatter plot for FSC-A / FSC-H to show single cell populations (suitable for nasal irrigation fluid samples); Figure 6 Gate the scatter plot of FSC-A / FSC-H to show single cell populations (applicable to peripheral blood samples); Figure 7 A comprehensive overview of multi-step gating analysis of T cells and their activated memory and tissue-resident memory subsets; Figure 8 This is a logic diagram for the subpopulation classification of T cells in counterstained samples. Detailed Implementation

[0018] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0019] Example Preparation of blank whole blood and nasal lavage fluid samples from cynomolgus monkeys: About 2.0 mL of blank whole blood was collected from the veins of the limbs, anticoagulated with heparin sodium, and temporarily stored at room temperature; about 2.0 mL of nasal lavage fluid was collected through nasal irrigation, temporarily stored at room temperature, and transported to the laboratory within 2 hours.

[0020] Operating steps Nasal irrigation solution (1) Sample processing: The nasal irrigation fluid sample was centrifuged under the following conditions: temperature 20℃, speed 400×g, time 5min. The centrifuged sample was removed, the supernatant was discarded, and the remaining cells at the bottom of the tube were vortexed and mixed. The sample was then stored at 2-8℃ for later use.

[0021] (2) Reagent preparation: Prepare the Fixable Viability Stain 780 (LIVE / DEAD) dye by mixing 1×PBS buffer with Fixable Viability Stain 780 (LIVE / DEAD) dye (diluted 400 times).

[0022] (3) Adding samples: Take 200µL of diluted Fixable Viability Stain 780 dye into each flow cytometry tube, add all the remaining nasal irrigation fluid cells from the bottom of the tube into the corresponding flow cytometry detection tube, vortex to mix, and incubate at 15~25℃ in the dark for 10min.

[0023] (4) Centrifugation after washing: Take out the incubated samples, add 1000µL of 1×PBS buffer to each tube, and vortex to mix. Centrifugation conditions: temperature 20℃, speed 400×g, time 5 min.

[0024] (5) Add antibody: Take 1µL of CD45, 1µL of CD3, 1µL of CD4, 1µL of CD8, 2µL of CD69, 5µL of CD44, and 2µL of CD103 fluorescent antibody into the corresponding flow cytometry single staining tubes and FMO(CD69) / FMO(CD44) / FMO(CD103), vortex to mix, and incubate at 15~25℃ in the dark for 20 min.

[0025] (6) Centrifugation after washing: Take out the incubated sample and add 1000µL of 1×PBS buffer, vortex to mix. Centrifugation conditions: temperature 20℃, speed 400×g, time 5min.

[0026] (7) Sample testing: Take out the centrifuged sample, discard the supernatant, add 500µL of 1×PBS buffer to resuspend, vortex to mix, and store temporarily in the dark for testing.

[0027] Peripherally anticoagulated whole blood (1) Add antibody: Take 1µL of CD45, 1µL of CD3, 1µL of CD4, 1µL of CD8, 2µL of CD69, 5µL of CD44, and 2µL of CD103 fluorescent antibodies into the corresponding flow cytometry single staining tubes and FMO(CD69) / FMO(CD44) / FMO(CD103), and then prepare mixed antibodies according to the above ratio. Take 13µL into the corresponding flow cytometry counterstaining tube, vortex mix, and store at 2-8℃ in the dark for later use.

[0028] (2) Add samples: After each sample is inverted and mixed, 50µL of anticoagulated whole blood is drawn into the corresponding negative control tube, single staining tube, FMO tube and counterstaining tube, vortexed and mixed, and incubated at 15~25℃ in the dark for 20 min.

[0029] (3) Add lysis buffer: Add 1000µL of 1×NH4Cl red blood cell lysis buffer at a ratio of 1:20 to the sample volume, vortex to mix, and incubate at 15~25℃ in the dark for 15min until the blood is clear.

[0030] (4) Centrifugation after pyrolysis: Centrifugation conditions: temperature 20℃, speed 400×g, time 5min.

[0031] (5) Centrifugation after washing: Remove the centrifuged sample, discard the supernatant, add 1000µL of 1×PBS buffer, and vortex to mix. Temperature 20℃, speed 400×g, time 5min.

[0032] (6) Sample testing: Take out the centrifuged sample, discard the supernatant, add 500µL of 1×PBS buffer to resuspend, vortex to mix, and store temporarily in the dark for testing.

[0033] Antibody titers were optimized using CD44 antibody as an example, and the dosage of other antibodies was determined based on similar principles.

[0034] Operating steps: (1) Add antibody: Take 1 μL, 2 μL, 3 μL, 4 μL and 5 μL of Human CD44 / Pgp-1 APC fluorescent antibody respectively and add them to the corresponding numbered flow cytometry tubes. Keep them at 2-8℃ in the dark for later use.

[0035] (2) Add sample: Take 50 μL of anticoagulated whole blood and add it to the flow cytometry tube with the corresponding sample number. Vortex mix and incubate at 15~25℃ in the dark for 20 min.

[0036] (3) Lysis of red blood cells: Add 1000 μL of 1×NH4Cl red blood cell lysis buffer to the flow cytometer tube at a ratio of 1:20 of the sample volume. After vortexing and mixing, lyse the blood at 15~25℃ in the dark for about 15 min until the blood is clear.

[0037] (4) Centrifugation after lysis: Centrifuge at 15~25℃ and 400×g for 5 min.

[0038] (5) Centrifugation after washing: Take out the centrifuged sample, discard the supernatant, add 1000 μL of 1×PBS buffer to wash, and centrifuge at 15~25℃ and 400×g for 5 min.

[0039] (6) Sample testing: Take out the centrifuged sample, discard the supernatant, add 500 μL of 1×PBS buffer to resuspend the sample, and test it on the machine.

[0040] Calculation formula The recommended starting point for the highest final concentration is 5 μL. Set up 5 concentration gradients, with 2 replicates for each concentration. Take the average of the median fluorescence intensity of the negative and positive cell populations, and the average of the relative standard deviation (rSD) of the fluorescence intensity of the negative cell population. Calculate the staining index (SI) at different concentrations, and select the concentration corresponding to the highest SI as the optimal antibody dosage. Data processing and statistical analysis Samples were analyzed using a CytoFLEX LX flow cytometer, and the data were analyzed using the instrument's built-in CytExpert software. The %Parent value was obtained as the sample result, and the analysis results were saved and printed.

[0041] In one detection in this embodiment, the detection parameters of the flow cytometer (instrument number: SEC-0008) were set as follows: Voltage and threshold settings: forward scattering angle channel voltage was set to 266, threshold was set to 80000; side scattering angle channel voltage was set to 236, no threshold was set; B525-FITC channel voltage was set to 188; Y585-PE channel voltage was set to 161; R660-APC channel voltage was set to 416; Y712-APCA700 channel voltage was set to 315; V525-KrO channel voltage was set to 18; V610 channel voltage was set to 98; NUV450 channel voltage was set to 10.

[0042] Compensation settings: The compensation settings for the B525-FITC channel are as follows: 1.68% for the Y585-PE channel, 0.29% for the R660-APC channel, and 0.00% for other channels. The compensation settings for the Y585-PE channel are as follows: -0.24% for the R712-APC700 channel, 31.12% for the V610 channel, and 0.00% for other channels. The compensation settings for the R660-APC channel are as follows: 0.52% for the R712-APC700 channel, -0.73% for the V525-KrO channel, 0.33% for the V610 channel, and 0.00% for other channels. The compensation settings for the R712-APC700 channels are as follows: 21.75% for the R660-APC channel, -0.41% for the V525-KrO channel, 0.04% for the V610 channel, and 0.00% for other channels. The compensation settings for the V525-KrO channels are as follows: 0.04% for the R660-APC channel, 0.35% for the V610 channel, and 0.00% for other channels. The compensation settings for the V610 channels are as follows: 4.76% for the Y585-PE channel, 5.35% for the R712-APC700 channel, 103.28% for the V525-KrO channel, and 0.00% for other channels. The compensation settings for the NUV450 channels are as follows: 0.09% for the R660-APC channel, 3.70% for the V525-KrO channel, 0.60% for the V610 channel, and 0.00% for other channels.

[0043] Grouping settings and gate logic Group settings (1) Test blank control tube: Adjust the voltage of each channel and set the threshold of the FSC channel.

[0044] (2) Detection of compensation control tubes: Adjust the compensation between channels of BV510 Mouse Anti-NHP CD45, BV605 Mouse Anti-Human CD3, BUV395 Mouse Anti-Human CD4, Alexa Fluor 700 anti-human CD8a, PEAnti-Human CD69, Human CD44 / Pgp-1 APC antibody, and CD103 Monoclonl Antibody FITC. The difference in average fluorescence intensity (Mean) between channels should be less than 20. Taking the compensation adjustment between Y585-PE-A and B525-FITC channels as an example, the difference in Mean value between the Q1-LL quadrant and Q1-LR quadrant of B525-FITC should be less than 20. Figure 1 , Figure 2 .

[0045] (3) Detection of counterstaining tubes: Circle the target cell population on the FSC-A / SSC-A dual-parameter scatter plot, defining it as WBC, such as... Figure 3 On the LD R763-APCA750-A / SSC-A two-parameter scatter plot, WBCs are displayed; the negative cell population is circled and defined as LD. Figure 4 (This graph is only available for nasal irrigation fluid samples); On the FSC-A / FSC-H two-parameter scatter plot, single-cell populations are circled and defined as "Single cells" up to the LD level. Figure 5 (This image is only available for nasal irrigation fluid samples); On the FSC-A / FSC-H dual-parameter scatter plot, WBCs are displayed, and single-cell populations are circled and defined as "Single cells," as shown below. Figure 6 (This graph is only available for peripheral blood samples); On the V525-KrO-A / SSC-A two-parameter scatter plot, Single cells are shown, and the positive cell population is circled and defined as LYM; On the V610-A / SSC-A two-parameter scatter plot, the LYM population is shown, and the positive cell population is circled and defined as CD45. + CD3 + On the NUV450-A / R712-APCA700-A two-parameter scatter plot, the value is displayed up to CD45. + CD3 + Circle the positive cell populations and define them as CD45. + CD3 + CD8 + and CD45 + CD3 + CD4 + On the R660-APC-A / SSC-A two-parameter scatter plot, the value is displayed up to CD45. +CD3 + CD4 + Circle the positive cell population and define it as CD45. + CD3 + CD4 + CD44 + On the B525-FITC-A / Y585-PE-A two-parameter scatter plot, it is shown up to CD45. + CD3 + CD4 + CD44 + Circle the positive cell populations and define them as CD45. + CD3 + CD4 + CD44 + CD69 + CD45 + CD3 + CD4 + CD44 + CD103 + and CD45 + CD3 + CD4 + CD44 + CD103 + CD69 + On the R660-APC-A / SSC-A two-parameter scatter plot, the value is displayed up to CD45. + CD3 + CD8 + Circle the positive cell population and define it as CD45. + CD3 + CD8 + CD44 + On the B525-FITC-A / Y585-PE-A two-parameter scatter plot, it is shown up to CD45. + CD3 + CD8 + CD44 + Circle the positive cell populations and define them as CD45. + CD3 + CD8 + CD44 + CD69 + CD45 + CD3 + CD8 + CD44 + CD103 + and CD45 + CD3 + CD8 + CD44+ CD103 + CD69 + ,like Figure 7 .

[0046] The diagram of T cell subset grouping and gating analysis of counterstained samples is shown below. Figure 8 As shown.

[0047] The method of this invention was used to test peripheral blood and nasal lavage fluid samples from cynomolgus monkeys, and the results are shown in Table 4.

[0048] Table 4. Flow cytometry results of key T cell subsets in peripheral blood and nasal lavage fluid of cynomolgus monkeys. (Data is expressed as a percentage, showing the detection effect of the method of the present invention on different samples) Animal Number Sample type Detection time point Total T cells <![CDATA[CD4 + Tm]]> <![CDATA[CD8 + Tm]]> <![CDATA[CD4 + Trm]]> <![CDATA[CD8 + Trm]]> 1F001 peripheral blood Pre-dose 25.30 83.06 13.36 17.14 1.62 1M001 peripheral blood Pre-dose 20.88 71.09 25.78 8.27 0.82 1M002 peripheral blood Pre-dose 24.58 68.46 25.38 6.08 18.29 2F001 peripheral blood Pre-dose 26.96 74.58 20.34 1.75 18.89 2M001 peripheral blood Pre-dose 28.01 81.01 16.46 4.30 3.32 3M001 peripheral blood Pre-dose 5.81 75.00 9.62 2.16 4.33 1F001 Nasal irrigation solution Day 2 5.02 95.45 4.55 23.08 0.00 1M001 Nasal irrigation solution Day 2 3.67 100.00 0.00 8.33 0.00 1M002 Nasal irrigation solution Day 2 10.31 74.47 25.53 25.93 0.00 2F001 Nasal irrigation solution Day 2 25.85 86.05 13.95 22.22 0.00 3F001 Nasal irrigation solution Day 2 4.79 86.67 13.33 6.50 4.07 3M001 Nasal irrigation solution Day 2 6.54 85.42 14.58 3.85 0.55 Note: 1. Animals are numbered 1, 2, and 3, representing low, medium, and high doses, respectively; M represents males and F represents females. 2. The cell subsets in the table are defined as follows: "Total T cells" refers to CD45-positive and CD3-positive T lymphocytes; "CD45-positive and CD3-positive T lymphocytes" refers to CD45-positive and CD3-positive T lymphocytes. + Tm and CD8 + "Tm" refers to CD44-positive and CD69-positive activated memory T cells in the CD4-positive and CD8-positive T cell subsets, respectively; "CD4" refers to CD4+ CD69-positive activated memory T cells. + "Trm" and "CD8" + "Trm" refers to CD44-positive, CD103-positive, and CD69-positive tissue-resident memory T cells from the CD4-positive and CD8-positive T cell subsets, respectively. See the Examples section of the instruction manual for the calculation of each percentage (%).

[0049] In sample number 1F001, CD45 + CD3 + T cells account for 25.30% of lymphocytes, of which CD4+ + CD44 in subgroup + CD69 + The proportion of activated memory T cells was 83.06%, CD8+ + CD44 in subgroup + CD103 + CD69 + The proportion of tissue-resident memory T cells was 1.62%. Similarly, in sample 1M001, CD4+... + CD44 in subgroup + CD69 + The proportion of activated memory T cells was 71.09%, CD8 + CD44 in subgroup +CD103 + CD69 + The proportion of tissue-resident memory T cells was 0.82%. These data confirm that the method of the present invention can simultaneously and accurately detect activated memory T cells and tissue-resident memory T cells in peripheral blood and nasal lavage fluid samples.

[0050] As can be seen from the above embodiments, the present invention provides an optimized flow cytometry detection scheme and reagent combination. This scheme can be successfully applied to peripheral blood and nasal lavage fluid samples from cynomolgus monkeys, stably detecting CD4. + With CD8 + T cell subsets, and further effectively distinguished CD44 from them. + CD69 + Activation of memory T cells and CD44 + CD103 + CD69 + Tissue-resident memory T cells. This method demonstrates good feasibility and provides a reliable tool for systematically assessing the cellular immune response status of cynomolgus monkeys.

[0051] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for detecting activated memory T cells and tissue-resident memory T cells in cynomolgus monkeys using flow cytometry, characterized in that, The method includes the following steps: Samples of cynomolgus monkeys were collected and treated with anticoagulants. The sample was mixed with a fluorescent antibody and then stained. Flow cytometry was used to analyze the stained samples. Flow cytometry data were analyzed to distinguish between CD4-positive and CD8-positive T cell subsets, and further detection was performed on CD4-positive T cell subsets containing CD44-positive CD69-positive activated memory T cells and CD44-positive CD103-positive CD69-positive tissue-resident memory T cells, as well as CD8-positive T cell subsets containing CD44-positive CD69-positive activated memory T cells and CD44-positive CD103-positive CD69-positive tissue-resident memory T cells. The fluorescent antibody combination includes BV510 Mouse Anti-NHP CD45 antibody, BB700 Mouse Anti-Human CD3 antibody, BUV395 Mouse Anti-Human CD4 antibody, Alexa Fluor 700 anti-human CD8a antibody, PE Anti-Human CD69 antibody, Human CD44 / Pgp-1 APC antibody and CD103 Monoclonl Antibody FITC antibody.

2. The method as described in claim 1, characterized in that, The cynomolgus monkey samples included peripheral blood samples and nasal irrigation fluid samples.

3. The method as described in claim 2, characterized in that, The peripheral blood samples were treated with heparin sodium anticoagulation.

4. The method as described in claim 2, characterized in that, Cells were obtained from the nasal irrigation fluid sample by centrifugation.

5. The method as described in claim 1, characterized in that, The dosage of the BV510 Mouse Anti-NHP CD45 antibody is 0.5-2 μL per test; The dosage of the BB700 Mouse Anti-Human CD3 antibody is 0.5-2 μL per test.

6. The method as described in claim 1, characterized in that, The dosage of the BUV395 Mouse Anti-Human CD4 antibody is 0.5-2 μL per test; The dosage of the Alexa Fluor 700 anti-human CD8a antibody is 0.5-2 μL per test.

7. The method as described in claim 1, characterized in that, The dosage of the PE Anti-Human CD69 antibody is 1-4 μL per test; The dosage of the Human CD44 / Pgp-1 APC antibody is 2-5 μL per test; The amount of CD103 Monoclonl Antibody FITC antibody used is 1-4 μL per test.

8. The method as described in claim 1, characterized in that, The detection was performed using a flow cytometer, with the FSC channel voltage set to 200-300 and the threshold set to 50000-100000.

9. The method as described in claim 1, characterized in that, The analysis steps include gate logic analysis, namely, circling the leukocyte population on the FSC-A / SSC-A scatter plot, circling the single cell population on the FSC-A / FSC-H scatter plot, circling the T cell population on the CD45 and CD3 positive scatter plot, circling the CD4 positive T cell and CD8 positive T cell subsets on the CD4 and CD8 positive scatter plot, and circling activated memory T cells and tissue-resident memory T cells on the CD44, CD69 and CD103 positive scatter plot.

10. The method as described in claim 1, characterized in that, The method includes using blank control tubes, compensation control tubes, and fluorescence minus one control tubes.

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