Method for detecting cynomolgus monkey peripheral blood T lymphocyte subtype pSTAT4 and Ki67 signals by flow cytometry

Abstract

The invention provides a method for detecting subtype pSTAT4 and Ki67 signals of peripheral blood T lymphocytes of machin through flow cytometry, and belongs to the technical field of biological detection. According to the method, the integrated analysis of the multi-dimensional functional state of the cynomolgus monkey T lymphocyte is realized, key signal channel activation and proliferation information of different subgroup cells can be synchronously obtained through a single experiment, and the detection comprehensiveness and efficiency are greatly improved. According to the method, an accurate tool capable of directly evaluating the response intensity and proliferation potential of T cells is provided for immune mechanism research based on a machin model, particularly immune therapy or disease research related to regulation and control of pathways such as IL-12 / STAT4 and the like, and the data dimension and reliability of preclinical immunological evaluation are remarkably enhanced.

Description

Technical Field

[0001] This invention relates to the field of biological detection technology, and in particular to a method for detecting pSTAT4 and Ki67 signals in peripheral blood T lymphocytes of cynomolgus monkeys using flow cytometry. Background Technology

[0002] Flow cytometry is a core technique for multi-parameter single-cell analysis in contemporary immunological research. Using fluorescently labeled specific antibodies, it can simultaneously detect multiple molecules on the cell surface and inside, enabling precise segmentation and functional analysis of complex cell populations. In immunology, assessing the functional status of T lymphocytes is crucial, typically involving the detection of their activation, proliferation, and activity of specific signaling pathways.

[0003] pSTAT4 (phosphorylated signal transduction and transcription activator 4) is a key intracellular signal transduction molecule. Its phosphorylation level directly reflects the activation status of cytokine pathways such as IL-12 and is closely related to Th1 cell differentiation and immune response regulation. Ki67 is a nuclear antigen directly related to cell proliferation activity, and its expression level is widely used as an important marker for assessing the cell cycle. In T cell research, understanding both the activation status of its signaling pathways (e.g., through pSTAT4) and its proliferative potential (e.g., through Ki67) is crucial for a comprehensive understanding of its function.

[0004] As a non-human primate with a highly homologous immune system to the human, the cynomolgus monkey is an irreplaceable model for basic immunological research, preclinical evaluation of vaccines and immunotherapeutic drugs. T-cell immune analysis using this model typically requires the identification of major subsets such as CD4⁺ and CD8⁺, and sometimes further analysis of specific subsets such as regulatory T cells. While existing techniques exist for separately detecting phosphorylated proteins (such as pSTAT) or proliferation markers (such as Ki67), and for multi-color subset typing of lymphocytes, a stable and reliable standardized flow cytometry protocol remains a technological gap in the specific species of the cynomolgus monkey. This protocol organically integrates the complex subpopulation of T cells with the simultaneous detection of the two core functional signals, pSTAT4 and Ki67, within the cynomolgus monkey. The lack of such an integrated approach limits researchers' ability to systematically obtain comprehensive information on the multifunctional state of cynomolgus monkey T cells from a single experiment. Summary of the Invention

[0005] The purpose of this invention is to provide a flow cytometry method for detecting pSTAT4 and Ki67 signals in peripheral blood T lymphocyte subtypes of cynomolgus monkeys. This method solves the technical problem of lacking a method in the prior art that can simultaneously and quantitatively detect intracellular pSTAT4 activation signals and Ki67 proliferation signals in peripheral blood of cynomolgus monkeys within the same detection system, targeting key T lymphocyte subpopulations such as CD4⁺, CD8⁺, and regulatory T cells, as well as their differentially expressed PD-1 subpopulations.

[0006] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides a method for detecting T lymphocyte subtypes and their functional status in the peripheral blood of cynomolgus monkeys, comprising the following steps: Peripheral blood samples were collected from cynomolgus monkeys and treated with anticoagulants. The sample was stained using a composition containing CD45, CD3, CD4, CD8, CD25, FoxP3, PD-1, pSTAT4 and Ki-67 specific binding molecules; Flow cytometry was used to analyze the stained samples. Flow cytometry data were analyzed to identify T lymphocytes based on CD45 and CD3 expression, and further analysis was conducted on CD3... + Cellular subpopulations can be distinguished based on CD4 and CD8 expression, and the expression of at least one of the markers PD-1, Ki-67, and pSTAT4 can be analyzed in specific subpopulations.

[0007] Preferably, the specific binding molecule is an antibody, which is a BV510-labeled anti-cynomolgus CD45 antibody, a BB700-labeled anti-human CD3 antibody, a BV786-labeled anti-human CD4 antibody, an Alexa Flour 700-labeled anti-human CD8 antibody, a BV650-labeled anti-human CD25 antibody, an AF488-labeled anti-human PD-1 antibody, a PE-labeled anti-human FoxP3 antibody, an AF647-labeled anti-human pSTAT4 antibody, and a BV421-labeled anti-human Ki-67 antibody.

[0008] Preferably, the specific subgroup analyzed includes CD45. + CD3 + CD4 + T cell subsets.

[0009] Preferably, in CD45 + CD3 + CD4 + Further analysis of CD25 and FoxP3 expression within T cell subsets was conducted to identify regulatory T cell subsets.

[0010] Preferably, when analyzing the streaming data, CD45 is obtained.+ CD3 + CD4 + PD-1 + Ki-67 + Cells, CD45 + CD3 + CD4 + PD-1-Ki-67 + Cells, CD45 + CD3 + CD4 + PD-1 + pSTAT4 + Cells and CD45 + CD3 + CD4 + PD-1-pSTAT4 + Cell percentage and / or average fluorescence intensity.

[0011] Preferably, the specific subgroup analyzed includes CD45. + CD3 + CD8 + T cell subsets.

[0012] Preferably, when analyzing the streaming data, CD45 is obtained. + CD3 + CD8 + PD-1 + Ki-67 + Cells, CD45 + CD3 + CD8 + PD-1-Ki-67 + Cells, CD45 + CD3 + CD8 + PD-1 + pSTAT4 + Cells and CD45 + CD3 + CD8 + PD-1-pSTAT4 + Cell percentage and / or average fluorescence intensity.

[0013] Preferably, prior to the staining step, a step is included in vitro stimulation of a portion of the sample with CD3 and CD28 antibodies as a positive control; the staining step includes sequential surface staining, erythrocyte lysis and fixation, cell membrane rupture, and intracellular staining. In the surface staining step, the sample is mixed with antibodies against CD45, CD3, CD4, CD8, CD25 and PD-1, and incubated at 2℃~8℃ in the dark for 15 minutes to 25 minutes. In the intracellular staining step, the cells after membrane perforation are mixed with antibodies against FoxP3, Ki-67 and pSTAT4, and incubated at 2℃~8℃ in the dark for 80 minutes to 100 minutes.

[0014] Preferably, the amounts of the antibody used in a single test are as follows: 0.8 μL to 1.2 μL of BV510-labeled anti-cynomolgus macaque CD45 antibody; 0.8 μL to 1.2 μL of BB700-labeled anti-human CD3 antibody; 1.6 μL to 2.4 μL of BV786-labeled anti-human CD4 antibody; 0.8 μL to 1.2 μL of Alexa Flour 700-labeled anti-human CD8 antibody; 2.0 μL to 3.0 μL of BV650-labeled anti-human CD25 antibody; 1.6 μL to 2.4 μL of AF488-labeled anti-human PD-1 antibody; 4.0 μL to 6.0 μL of PE-labeled anti-human FoxP3 antibody; 8.0 μL to 12.0 μL of AF647-labeled anti-human pSTAT4 antibody; 0.8 μL to 1.2 μL of BV421-labeled anti-human Ki-67 antibody.

[0015] Preferably, when analyzing the flow cytometry data, lymphocyte populations are circled in the SSC-A / FSC-A scatter plot, and single cell populations are circled in the FSC-H / FSC-A scatter plot, and subsequent analysis is based on the single cell populations; When analyzing the flow cytometry data, the analysis further includes outlining CD45 cells from single-cell populations in the CD45-BV510 / SSC-A scatter plot. + Cell populations, in the CD3-BB700 / SSC-A scatter plot from CD45 + Circle CD45 in the cell population + CD3 + T cell population, in the CD8-Alexa Flour 700 / CD4-BV786 scatter plot from CD45 + CD3 + Circle CD4 in the cell population + Cell population and CD8 + Cell population.

[0016] The beneficial effects of this invention are: This invention enables integrated analysis of the multidimensional functional status of cynomolgus monkey T lymphocytes. A single experiment can simultaneously acquire information on the activation and proliferation of key signaling pathways in different cell subsets, significantly improving the comprehensiveness and efficiency of the detection. This method provides a precise tool for directly assessing the strength and proliferative potential of T cell responses in cynomolgus monkey-based immune mechanism studies, especially in immunotherapy or disease research involving pathways such as IL-12 / STAT4 regulation, significantly enhancing the data dimensionality and reliability of preclinical immunological evaluation. Attached Figure Description

[0017] Figure 1 This is a diagram showing the fluorescence compensation adjustment. Figure 2 Circle the lymphocyte population in the image; Figure 3 Select images for the single-cell phylum; Figure 4 This is a diagram showing the continuous gating analysis of positive cells. 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 Approximately 1.0 mL of blank whole blood was collected from the jugular vein of each monkey. Heparin sodium was used for anticoagulation, and the whole blood was inverted and mixed before use.

[0020] Upper sample tube design (1) Blank control tube: No fluorescent antibody is added; it is used to adjust the voltage.

[0021] (2) Compensation control tubes: BV510 Mouse Anti-NHP CD45, BB700 Mouse Anti-Human CD3, BV786 Mouse Anti-Human CD4, Alexa 700 Mouse Anti-Human CD8, BV650 Mouse Anti-Human CD25, AF488 Mouse Anti-Human PD1, PE Mouse Anti-Human FoxP3, BV421 Mouse Anti-Ki-67, and AF647 Mouse Anti-Human pSTAT4 fluorescent antibodies were added to adjust the compensation between channels.

[0022] (3) Counterstaining tubes: Add BV510 Mouse Anti-NHP CD45, BB700 Mouse Anti-Human CD3, BV786 Mouse Anti-Human CD4, Alexa 700 Mouse Anti-Human CD8, BV650 Mouse Anti-Human CD25, AF488 Mouse Anti-Human PD1, PE Mouse Anti-Human FoxP3, BV421 Mouse Anti-Ki-67, and AF647 Mouse Anti-Human pSTAT4 fluorescent antibodies to determine the feasibility of the protocol.

[0023] (4) Positive control tubes: The positive control, Purified NA / LE Mouse Anti-Human CD3 and Purified NA / LE Mouse Anti-Human CD28 were added to stimulate the samples, and then BV510 Mouse Anti-NHP CD45, BB700 Mouse Anti-Human CD3, BV786 Mouse Anti-Human CD4, Alexa 700 Mouse Anti-Human CD8, BV650 Mouse Anti-Human CD25, AF488 Mouse Anti-Human PD1, PE Mouse Anti-Human FoxP3, BV421 Mouse Anti-Ki-67 and AF647 Mouse Anti-Human pSTAT4 fluorescent antibodies were used to determine the feasibility of the protocol.

[0024] Grouping area settings Lymphocyte populations were selected, and CD45 cells were selected separately. + CD3 + CD45 + CD3 + CD4 + CD45 + CD3 + CD8 + CD45 + CD3 + CD4 + CD25 + FoxP3 + CD45 + CD3 + Ki67 + CD45 + CD3+ CD4 + PD1 + Ki67 + 、CD45 + CD3 + CD4 + PD1-Ki67 + 、CD45 + CD3 + CD4 + PD1 + pSTAT4 + 、CD45 + CD3 + CD4 + PD1-pSTAT4 + 、CD45 + CD3 + CD8 + PD1 + Ki67 + 、CD45 + CD3 + CD8 + PD1-Ki67 + 、CD45 + CD3 + CD8 + PD1 + pSTAT4 + 、CD45 + CD3 + CD8 + PD1-pSTAT4 + 、CD45 + CD3 + CD4 + CD25 + FoxP3 + PD1 + Ki67 + 、CD45 + CD3 + CD4 + CD25 + FoxP3 + PD1-Ki67 + 、CD45 + CD3 + CD4 + CD25 + FoxP3 + PD1 + pSTAT4 + 、CD45 + CD3 + CD4 + CD25+ FoxP3 + PD1-pSTAT4 + Results for positive areas.

[0025] Operating steps (1) Sample addition: 50 μL of anticoagulated whole blood was drawn into flow cytometry tubes; a sample was randomly selected and 50 μL of anticoagulated whole blood was drawn into a flow cytometry tube as a positive control tube.

[0026] (2) Positive control stimulation: 1.0 μL of Purified NA / LE Mouse Anti-Human CD3, Purified NA / LE Mouse Anti-Human CD28 and positive control were added to the positive control tube and stimulated at 37℃±1℃ in the dark for 30 min.

[0027] (3) Surface staining: Add 1.0 μL of BV510 Mouse Anti-NHP CD45, BB700 Mouse Anti-Human CD3, Alexa 700 Mouse Anti-Human CD8 fluorescent antibodies, 2.0 μL of BV786 Mouse Anti-Human CD4, AF488 Mouse Anti-Human PD1 fluorescent antibodies (excluding FMO tubes), and 2.5 μL of BV650 Mouse Anti-Human CD25 fluorescent antibody to each flow cytometry sample tube and positive control tube, mix well, and incubate at 2~8℃ in the dark for 20 min.

[0028] (4) Washing: Add 1000 μL of 1×PBS to wash, centrifuge at 2~8℃, 400×g for 5 min.

[0029] (5) Lysis and fixation of red blood cells: Take out the centrifuged sample, discard the supernatant, dilute RBC Lysis / Fixation Solution (10×) with pure water to RBC Lysis / Fixation Solution (1×), add 1000 μL of RBC Lysis / Fixation Solution (1×) to each tube, mix well, and lyse and fix at 2~8℃ in the dark for about 15 min.

[0030] (6) Centrifuge after lysis: 2~8℃, 400×g, centrifuge for 5 min.

[0031] (7) Washing: Take out the centrifuged sample, discard the supernatant, add 1000 μL of 1×PBS to wash, centrifuge at 2~8℃, 400×g for 5 min.

[0032] (8) Membrane breaking: Take out the centrifuged sample, discard the supernatant, add 1000 μL of Perm Buffer III to each tube, mix well, incubate at 2~8℃ in the dark for 30 min, dilute Perm / Wash Buffer (5×) with pure water to Perm / Wash Buffer (1×), add 2000 μL of Perm / Wash Buffer (1×) to wash, centrifuge at 2~8℃, 400×g for 5 min.

[0033] (9) Washing: Take out the centrifuged sample, discard the supernatant, add 2000 μL of Perm / Wash Buffer (1×) to wash, centrifuge at 2~8℃, 400×g for 5 min.

[0034] (10) Intracellular staining: Take out the centrifuged sample, discard the supernatant, add 5.0 μL PE Mouse Anti-HumanFoxP3, 1.0 μL BV421 Mouse Anti-Ki-67, and 10.0 μL LAF647 Mouse Anti-Human pSTAT4 fluorescent antibody, and incubate at 2~8℃ in the dark for 90 min.

[0035] (11) Washing: Add 2000 μL of Perm / Wash Buffer (1×) to wash, 2~8℃, 400×g, centrifuge for 5min.

[0036] (12) Sample resuspending: Take out the centrifuged sample, discard the supernatant, and add 500 μL of 1×PBS to resuspend.

[0037] (13) Detection: The above-processed samples are detected by flow cytometer.

[0038] Instrument parameter settings: Instrument voltage and threshold The voltage and threshold settings for each detection channel of the flow cytometer are as follows: Forward scatter channel voltage set to 200, threshold set to 120000; Side scatter channel voltage set to 130, threshold not set; B525-FITC fluorescence detection channel voltage set to 150, threshold not set; B690-PC5.5 channel voltage set to 450, threshold not set; Y585-PE channel voltage set to 161, threshold not set; R660-APC channel voltage set to 416, threshold not set; R712-APCA700 channel voltage set to 229, threshold not set; V450-PB channel voltage set to 32, threshold not set; V525-KrO channel voltage set to 18, threshold not set; V660 channel voltage set to 130, threshold not set; V763 channel voltage set to 409, threshold not set. These parameters can be adjusted according to actual detection conditions.

[0039] Instrument fluorescence compensation To eliminate interference caused by spectral overlap in multicolor fluorescence detection, the following fluorescence compensation values ​​were set: The B525-FITC detection channel needs to subtract 0.69% of the signal from the B690-PC5.5 channel, 4.84% from the Y585-PE channel, 0.53% from the R660-APC channel, 2.18% from the R712-APCA700 channel, 0.13% from the V450-PB channel, and 0.10% from the V525-KrO channel. The compensation value for the V660 and V763 channels is 0.00%.

[0040] The B690-PC5.5 detection channel needs to have 2.33% of the signal from the B525-FITC channel, 94.75% from the Y585-PE channel, 28.53% from the R660-APC channel, 1.52% from the R712-APCA700 channel, and the compensation value for the V450-PB channel is 0.00%. It also needs to have 54.70% of the signal from the V525-KrO channel, 42.11% from the V660 channel, and the compensation value for the V763 channel is 0.00%.

[0041] The Y585-PE detection channel needs to have 0.59% of the signal from the B525-FITC channel subtracted, the compensation value for the B690-PC5.5 channel is 0.00%, the signal from the R660-APC channel is 5.04%, the compensation value for the R712-APCA700 channel is 0.00%, the compensation values ​​for the V450-PB and V525-KrO channels are both 0.00%, the compensation value for the V660 channel is 0.00%, and the signal from the V763 channel is subtracted by 0.55%.

[0042] The R660-APC detection channel needs to have 1.26% of the signal from the B525-FITC channel, 2.35% from the B690-PC5.5 channel, 7.30% from the Y585-PE channel, 1.77% from the R712-APCA700 channel, 0.94% from the V450-PB channel, a compensation value of 0.00% for the V525-KrO channel, 90.34% from the V660 channel, and 1.38% from the V763 channel subtracted.

[0043] The compensation value of the R712-APCA700 detection channel for the B525-FITC channel is 0.00%, minus 10.74% of the signal from the B690-PC5.5 channel, minus 6.02% of the signal from the Y585-PE channel, minus 48.82% of the signal from the R660-APC channel, minus 0.63% of the signal from the V450-PB channel, the compensation value for the V525-KrO channel is 0.00%, minus 22.42% of the signal from the V660 channel, and the compensation value for the V763 channel is 0.00%.

[0044] The V450-PB detection channel needs to have 2.63% of the signal from the B525-FITC channel subtracted, the compensation value for the B690-PC5.5 channel is 0.00%, the signal from the Y585-PE channel is subtracted, the compensation value for the R660-APC and R712-APCA700 channels is 0.00%, the signal from the V525-KrO channel is subtracted, the signal from the V660 channel is subtracted, and the signal from the V763 channel is subtracted, totaling 12.29%.

[0045] The V525-KrO detection channel needs to have 7.82% of the signal from the B525-FITC channel, 0.03% from the B690-PC5.5 channel, 1.63% from the Y585-PE channel, and the compensation values ​​for the R660-APC and R712-APCA700 channels are both 0.00%. Additionally, 0.37% of the signal from the V450-PB channel, 6.06% from the V660 channel, and 20.32% from the V763 channel need to be subtracted.

[0046] The V660 detection channel needs to have 2.00% of the signal from the B525-FITC channel, 0.88% from the B690-PC5.5 channel, 3.13% from the Y585-PE channel, and 0.00% from both the R660-APC and R712-APCA700 channels. The compensation value for the V450-PB channel is also 0.00%. Additionally, 2.50% of the signal from the V525-KrO channel and 8.24% from the V763 channel need to be subtracted.

[0047] The V763 detection channel has a compensation value of 0.00% for the B525-FITC channel, minus 10.38% of the signal from the B690-PC5.5 channel, and a compensation value of 0.00% for the Y585-PE, R660-APC, and R712-APCA700 channels, minus 5.94% of the signal from the V450-PB channel, minus 1.38% of the signal from the V525-KrO channel, and minus 24.13% of the signal from the V660 channel.

[0048] Instrument testing template settings (1) Test blank control tube: Adjust the voltage of each channel and set the threshold of the FSC channel.

[0049] (2) Detection of compensation control tubes: Adjust the compensation between channels BV510, BB700, BV786, Alexa 700, BV650, AF488, PE, AF647, and BV421. The difference in average fluorescence intensity (Meadian) values ​​between each channel should be less than 20. Taking the compensation adjustment between channels BV786 and Alexa 700 as an example, the difference in Median values ​​between the Q7-LL quadrant and the Q7-LR quadrant of Alexa 700 should be less than 20. Figure 1 As shown.

[0050] (3) Detection of counterstaining tubes and positive control tubes: Circle the target cell population on the SSC-A / FCS-A dual-parameter scatter plot, such as... Figure 2 Select single-cell populations on the FSC-H / FSC-A two-parameter scatter plot, such as... Figure 3 .

[0051] (4) Circle the positive cell population: On CD45-BV510 / SSC-A, when a single cell population is observed, circle the positive cell population and define it as CD45. + On CD3-B690-PC5.5 / SSC-A, it displays as CD45. + Circle the positive cell population and define it as CD45. + CD3 + CD45 + CD3–. On the CD8-Alexa 700 / CD4-BV786 chart, it is shown up to CD45. + CD3 + Group, circle CD45 + CD3 + CD8 + CD45 + CD3 + CD4 + On the FoxP3-PE / CD25-V660 graph, it shows up to CD45. + CD3 + CD4 + Circle CD45 + CD3 + CD4 + CD25 + FoxP3 + On the Ki67-V450 / PD1-AF488 diagram, it shows CD45. + CD3 + CD4 + Circle CD45+ CD3 + CD4 + PD1 + Ki67 + CD45 + CD3 + CD4 + PD1-Ki67 + On the pSTAT5-APC / PD1-AF488 diagram, it is shown to be CD45. + CD3 + CD4 + Circle CD45 + CD3 + CD4 + PD1 + pSTAT4 + CD45 + CD3 + CD4 + PD1-pSTAT4 + On the Ki67-V450 / PD1-AF488 diagram, it shows CD45. + CD3 + CD8 + Circle CD45 + CD3 + CD8 + PD1 + Ki67 + CD45 + CD3 + CD8 + PD1-Ki67 + On the pSTAT5-APC / PD1-AF488 diagram, it is shown to be CD45. + CD3 + CD8 + Circle CD45 + CD3 + CD8 + PD1 + pSTAT4 + CD45 + CD3 + CD8 + PD1-pSTAT4 + On the Ki67-V450 / PD1-AF488 diagram, it shows CD45. + CD3 + CD4 + CD25 + FoxP3 + Circle CD45 + CD3 +CD4 + CD25 + FoxP3 + PD1 + Ki67 + CD45 + CD3 + CD4 + CD25 + FoxP3 + PD1-Ki67 + On the pSTAT5-APC / PD1-AF488 diagram, it is shown to be CD45. + CD3 + CD4 + CD25 + FoxP3 + Circle CD45 + CD3 + CD4 + CD25 + FoxP3 + PD1 + pSTAT4 + CD45 + CD3 + CD4 + CD25 + FoxP3 + PD1-pSTAT4 + .like Figure 4 .

[0052] Data processing and statistical analysis Samples were tested using a CytoFLEX LX flow cytometer, and the test data were analyzed using the instrument's built-in CytExpert software to obtain the %Parent value as the sample result. The results were then calculated and analyzed using Microsoft Office Excel 2010.

[0053] All calculation results above should be rounded to two decimal places.

[0054] The results are shown in Table 1 below: Table 1. Flow cytometry results of peripheral blood samples from cynomolgus monkeys Time point sample A B C D E F pre-dose Sample 1 0.0296 0.0436 0.0307 0.0285 0.2047 0.0906 Day 3 Sample 1 0.2183 0.1431 0.2772 0.1043 0.5022 0.3324 Day 7 Sample 1 0.5075 0.3868 0.5187 0.2904 0.8843 0.7354 Day 14 Sample 1 0.2915 0.3695 0.3852 0.3028 0.6996 0.8052 Day 21 Sample 1 0.3710 0.2826 0.4719 0.3141 0.8081 0.7116 pre-dose Sample 2 0.0032 0.0768 0.0035 0.0482 0.0124 0.2632 Day 3 Sample 2 0.3093 0.3970 0.2367 0.2268 0.4968 0.8273 Day 7 Sample 2 0.5377 0.8094 0.5819 0.7825 0.8468 0.9456 Day 14 Sample 2 0.2814 0.0475 0.3633 0.0637 0.6891 0.1909 Day 21 Sample 2 0.2535 0.0882 0.2666 0.0770 0.6053 0.3211 pre-dose Sample 3 0.0106 0.0110 0.0123 0.0051 0.0189 0.0227 Day 3 Sample 3 0.7151 0.2897 0.4791 0.3319 0.7663 0.7090 Day 7 Sample 3 0.4223 0.4078 0.5069 0.3305 0.7855 0.6932 Day 14 Sample 3 0.2186 0.1669 0.2325 0.1558 0.6056 0.6084 Day 21 Sample 3 0.6324 0.0624 0.6553 0.0769 0.9125 0.1579 pre-dose Sample 4 0.0467 0.0757 0.0506 0.0565 0.2923 0.1683 Day 3 Sample 4 0.5130 0.5875 0.4981 0.6234 0.8013 0.8959 Day 7 Sample 4 0.5907 0.2168 0.6829 0.2155 0.9145 0.6007 Day 14 Sample 4 0.2558 0.2812 0.2602 0.2350 0.6080 0.7041 Day 21 Sample 4 0.5228 0.2917 0.5758 0.2732 0.8555 0.7213 Note: The column headings in the table are pseudonyms, and their corresponding meanings are as follows: A: CD45 + CD3 + CD4 + Ki67 + % Parent B: CD45 + CD3+ CD4 + pSTAT4 + % Parent C: CD45 + CD3 + CD8 + Ki67 + % Parent D: CD45 + CD3 + CD8 + pSTAT4 + % Parent E: CD45 + CD3 + CD4 + CD25 + FoxP3 + Ki67 + % Parent F: CD45 + CD3 + CD4 + CD25 + FoxP3 + pSTAT4 + % Parent As shown in Table 1, the detection method of this invention can effectively and synchronously quantitatively detect the functional signals of pSTAT4 and Ki67 in different T lymphocyte subsets (CD4⁺ T cells, CD8⁺ T cells, and regulatory T cells) in the peripheral blood of cynomolgus monkeys, and can sensitively capture their dynamic changes at different time points after intervention. The above data demonstrate that the present invention achieves integrated analysis of the multidimensional functional status of cynomolgus monkey T cells: it can clearly distinguish between major subsets and Tregs, and quantitatively assess their proliferative potential (Ki67) and key signaling pathway activation status (pSTAT4) within each subset. The observed general increase in various indicators after drug administration and the differences in response among different samples further prove that this method is suitable for dynamically monitoring immune responses, assessing individual differences and drug efficacy, and provides a stable and reliable multi-parameter functional analysis tool for immunological research and drug evaluation based on the cynomolgus monkey model.

[0055] As demonstrated by the above embodiments, this invention provides a complete and operable flow cytometry detection protocol. This protocol successfully and accurately distinguishes between CD4⁺ T cells, CD8⁺ T cells, and CD4⁺CD25⁺FoxP3⁺ regulatory T cells in the peripheral blood of cynomolgus monkeys, and can further subdivide each subpopulation based on PD-1 expression. Crucially, this protocol stably achieves simultaneous dual-parameter detection of intracellular pSTAT4 and Ki67 signals in all the aforementioned target cell subpopulations. The entire method encompasses standardized steps from sample pretreatment, multicolor antibody combination application, fixation and perforation to instrument acquisition and analysis, confirming its feasibility for simultaneously analyzing T cell subpopulation-specific pSTAT4 and Ki67 signals in a cynomolgus monkey model.

[0056] 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 T lymphocyte subtypes and their functional status in the peripheral blood of cynomolgus monkeys, characterized in that, Includes the following steps: Peripheral blood samples were collected from cynomolgus monkeys and treated with anticoagulants. The sample was stained using a composition containing CD45, CD3, CD4, CD8, CD25, FoxP3, PD-1, pSTAT4 and Ki-67 specific binding molecules; Flow cytometry was used to analyze the stained samples. Flow cytometry data were analyzed to identify T lymphocytes based on CD45 and CD3 expression, and further analysis was conducted on CD3... + Cellular subpopulations can be distinguished based on CD4 and CD8 expression, and the expression of at least one of the markers PD-1, Ki-67, and pSTAT4 can be analyzed in specific subpopulations.

2. The method according to claim 1, characterized in that, The specific binding molecule is an antibody, which is a BV510-labeled anti-cynomolgus monkey CD45 antibody, a BB700-labeled anti-human CD3 antibody, a BV786-labeled anti-human CD4 antibody, an Alexa Flour 700-labeled anti-human CD8 antibody, a BV650-labeled anti-human CD25 antibody, an AF488-labeled anti-human PD-1 antibody, a PE-labeled anti-human FoxP3 antibody, an AF647-labeled anti-human pSTAT4 antibody, and a BV421-labeled anti-human Ki-67 antibody.

3. The method according to claim 1, characterized in that, The specific subgroup analyzed included CD45. + CD3 + CD4 + T cell subsets.

4. The method according to claim 3, characterized in that, In CD45 + CD3 + CD4 + Further analysis of CD25 and FoxP3 expression within T cell subsets was conducted to identify regulatory T cell subsets.

5. The method according to claim 1, characterized in that, When analyzing the streaming data, obtain CD45. + CD3 + CD4 + PD-1 + Ki-67 + Cells, CD45 + CD3 + CD4 + PD-1-Ki-67 + Cells, CD45 + CD3 + CD4 + PD-1 + pSTAT4 + Cells and CD45 + CD3 + CD4 + PD-1-pSTAT4 + Cell percentage and / or average fluorescence intensity.

6. The method according to claim 1, characterized in that, The specific subgroup analyzed included CD45. + CD3 + CD8 + T cell subsets.

7. The method according to claim 6, characterized in that, When analyzing the streaming data, obtain CD45. + CD3 + CD8 + PD-1 + Ki-67 + Cells, CD45 + CD3 + CD8 + PD-1-Ki-67 + Cells, CD45 + CD3 + CD8 + PD-1 + pSTAT4 + Cells and CD45 + CD3 + CD8 + PD-1-pSTAT4 + Cell percentage and / or average fluorescence intensity.

8. The method according to claim 1, characterized in that, Prior to the staining step, a step is included in vitro stimulation of a portion of the samples with CD3 and CD28 antibodies as a positive control; the staining step includes sequential surface staining, erythrocyte lysis and fixation, cell membrane rupture, and intracellular staining. In the surface staining step, the sample is mixed with antibodies against CD45, CD3, CD4, CD8, CD25 and PD-1, and incubated at 2℃~8℃ in the dark for 15 minutes to 25 minutes. In the intracellular staining step, the cells after membrane perforation are mixed with antibodies against FoxP3, Ki-67 and pSTAT4, and incubated at 2℃~8℃ in the dark for 80 minutes to 100 minutes.

9. The method according to claim 2, characterized in that, The amounts of the antibody used in a single test were as follows: 0.8 μL to 1.2 μL of BV510-labeled anti-cynomolgus macaque CD45 antibody; 0.8 μL to 1.2 μL of BB700-labeled anti-human CD3 antibody; 1.6 μL to 2.4 μL of BV786-labeled anti-human CD4 antibody; 0.8 μL to 1.2 μL of Alexa Flour 700-labeled anti-human CD8 antibody; 2.0 μL to 3.0 μL of BV650-labeled anti-human CD25 antibody; 1.6 μL to 2.4 μL of AF488-labeled anti-human PD-1 antibody; 4.0 μL to 6.0 μL of PE-labeled anti-human FoxP3 antibody; 8.0 μL to 12.0 μL of AF647-labeled anti-human pSTAT4 antibody; 0.8 μL to 1.2 μL of BV421-labeled anti-human Ki-67 antibody.

10. The method according to claim 1, characterized in that, When analyzing the flow cytometry data, lymphocyte populations are circled in the SSC-A / FSC-A scatter plot, and single cell populations are circled in the FSC-H / FSC-A scatter plot, and subsequent analysis is based on the single cell populations. When analyzing the flow cytometry data, the analysis further includes outlining CD45 cells from single-cell populations in the CD45-BV510 / SSC-A scatter plot. + Cell populations, in the CD3-BB700 / SSC-A scatter plot from CD45 + Circle CD45 in the cell population + CD3 + T cell population, in the CD8-Alexa Flour 700 / CD4-BV786 scatter plot from CD45 + CD3 + Circle CD4 in the cell population + Cell population and CD8 + Cell population.

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