Macaca fascicularis T cell and activation, proliferation and depletion detection method and kit thereof

Abstract

The invention provides cynomolgus monkey T cells as well as a detection method and a kit for activation, proliferation and depletion of the cynomolgus monkey T cells, and belongs to the technical field of biological detection. According to the invention, the cynomolgus monkey T cells and the activation, proliferation and depletion detection method thereof are constructed by using fluorescent antibody combinations of specific types and proportions, and early, middle and late activation and depletion states and proliferation capacity of cynomolgus monkey T lymphocytes can be accurately and rapidly detected at the same time. A verification experiment proves that the detection method provided by the invention has excellent detection accuracy and relatively high intra-batch precision and inter-batch precision, has important significance for evaluating the influence of the medicine on the cynomolgus monkey immune system in a non-clinical research stage of a new medicine, is favorable for deeply understanding the influence of the medicine on the cynomolgus monkey immune response, and has a good application prospect. Important data is provided for safety and effectiveness evaluation of the medicine.

Description

Technical Field

[0001] The present invention relates to the field of biological detection technology, and in particular to a method and a kit for detecting cynomolgus monkey T cells and activation, proliferation and exhaustion thereof. Background Art

[0002] T cell activation is a core process in the adaptive immune system and is essential for the body's immune defense. When resting naive T cells encounter corresponding antigens, they will rapidly undergo clonal expansion and differentiate into different effector cell types. The significance of T cell activation is to initiate and regulate adaptive immune responses and protect the body from pathogens and tumors.

[0003] T cell exhaustion refers to a dysfunctional state of T cells characterized by an impaired ability to respond to infected cells or tumors. In chronic infection states, the continued presence of invading pathogens leads to prolonged antigenic stimulation, resulting in T cell dysfunction. In the tumor environment, T cells are exhausted due to chronic antigen exposure, resulting in reduced effector function. Characteristics of T cell exhaustion include loss of effector function, increased and sustained expression of inhibitory receptors, altered epigenetic and transcriptional profiles, and changes in metabolic patterns. In the tumor microenvironment, T cell exhaustion is considered a key obstacle in tumor immunotherapy.

[0004] T cell proliferation is a key component of the adaptive immune system and is of great significance in the amplification of immune responses, the formation of immune memory, the maintenance of immune diversity, immune regulation, tumor immune surveillance, the effectiveness of vaccination, the control of chronic infections, and the balance of autoimmune diseases. Detection and monitoring of T cell activation, exhaustion, and proliferation provide basic data for a comprehensive assessment of the effects of drugs on the immune system, and facilitate a deeper understanding of the working mechanism of the immune system, including the initiation, regulation, and termination of immune responses.

[0005] The cynomolgus monkey has a high degree of similarity with humans in physiological and metabolic mechanisms, and is often used as a model animal for biological products in the non-clinical research stage of new drugs to help clarify the pharmacological and toxicological characteristics. Therefore, it is of great significance to monitor cynomolgus monkey T cells and their activation, proliferation and exhaustion. Summary of the invention

[0006] The object of the present invention is to provide a method and a kit for detecting cynomolgus monkey T cells and their activation, proliferation and depletion, which have high accuracy.

[0007] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0008] The present invention provides a method for detecting cynomolgus monkey T cells and activation, proliferation and depletion thereof, comprising the following steps:

[0009] Take a combination of antibodies 1 labeled with different fluorescence and mix them with the sample to be tested for incubation;

[0010] Process the samples and obtain the test data through flow cytometry;

[0011] Obtaining cynomolgus monkey T cells and their activation, proliferation and exhaustion conditions according to the detection data;

[0012] The sample processing includes intracellular staining, and the intracellular staining includes taking a combination of antibodies 2 with different fluorescent labels and mixing and incubating them with the sample to be detected;

[0013] The antibody combination 1 comprises:

[0014] CD45 fluorescent antibody to label leukocytes;

[0015] CD3 fluorescent antibody to label total T lymphocytes;

[0016] CD4 fluorescent antibody to label helper T lymphocytes;

[0017] CD8 fluorescent antibody to label cytotoxic T lymphocytes;

[0018] CD25 fluorescent antibody to label regulatory T cells;

[0019] CD69 fluorescent antibody to label early activated T cells;

[0020] HLA-DR fluorescent antibody to label late activated T cells;

[0021] PD-1 fluorescent antibody was used to label the exhausted cells.

[0022] Preferably, the fluorescent label of the CD45 fluorescent antibody is FITC;

[0023] The fluorescent marker of the CD3 fluorescent antibody is PercP;

[0024] The fluorescent label of the CD4 fluorescent antibody is BUV395;

[0025] The fluorescent marker of the CD8 fluorescent antibody is BV605;

[0026] The fluorescent marker of the CD25 fluorescent antibody is APC;

[0027] The fluorescent marker of the CD69 fluorescent antibody is R718;

[0028] The fluorescent label of the HLA-DR fluorescent antibody is PE-Cy7;

[0029] The fluorescent marker of the PD-1 fluorescent antibody is BV785.

[0030] Preferably, obtaining the detection data includes data acquisition and data analysis;

[0031] The data analysis includes selecting CD45 + CD3 + 、CD45 + CD3 + CD4 + 、CD45 + CD3 + CD8 + 、CD45 + CD3 + CD4 + CD25 + Foxp3 + 、CD45 + CD3 + Ki67 + 、CD45 + CD3 + CD4 + Ki67 + 、CD45 + CD3 + CD8 + Ki67 + 、CD45 + CD3 + CD4 + CD25 + FoxP3 + Ki67 + 、CD45 + CD3 + CD69 + 、CD45 + CD3 + CD4 + CD69 + 、CD45 + CD3 + CD8 + CD69 + 、CD45 + CD3 + CD4 + CD25 + FoxP3 + CD69 + 、CD45 + CD3 + HLA-DR + 、CD45 + CD3 + CD4 + HLA-DR + 、CD45+ CD3 + CD8 + HLA-DR + 、CD45 + CD3 + CD4 + CD25 + FoxP3 + HLA-DR + 、CD45 + CD3 + PD-1 + 、CD45 + CD3 + CD4 + PD-1 + 、CD45 + CD3 + CD8 + PD-1 + 、CD45 + CD3 + CD4 + CD25 + FoxP3 + PD-1 + 、CD45 + CD3 + CD25 + 、CD45 + CD3 + CD4 + CD25 + 、CD45 + CD3 + CD8 + CD25 + Subgroup expression.

[0032] Preferably, the antibody combination 2 includes a Foxp3 fluorescent antibody for marking regulatory T cells and a Ki67 fluorescent antibody for marking proliferating cells.

[0033] Preferably, the fluorescent label of the Foxp3 fluorescent antibody is PE;

[0034] The fluorescent label of the Ki67 fluorescent antibody is BV421.

[0035] The present invention also provides a kit for detecting cynomolgus monkey T cells and their activation, proliferation and depletion, wherein the kit comprises the following fluorescently labeled antibodies:

[0036] CD45 fluorescent antibody to label leukocytes;

[0037] CD3 fluorescent antibody to label total T lymphocytes;

[0038] CD4 fluorescent antibody to label helper T lymphocytes;

[0039] CD8 fluorescent antibody to label cytotoxic T lymphocytes;

[0040] CD25 fluorescent antibody to label regulatory T cells;

[0041] CD69 fluorescent antibody to label early activated T cells;

[0042] HLA-DR fluorescent antibody to label late activated T cells;

[0043] PD-1 fluorescent antibody to label exhausted cells;

[0044] Foxp3 fluorescent antibody to label regulatory T cells;

[0045] Ki67 fluorescent antibody to label proliferating cells;

[0046] The fluorescent label of the CD45 fluorescent antibody is FITC;

[0047] The fluorescent marker of the CD3 fluorescent antibody is PercP;

[0048] The fluorescent label of the CD4 fluorescent antibody is BUV395;

[0049] The fluorescent marker of the CD8 fluorescent antibody is BV605;

[0050] The fluorescent marker of the CD25 fluorescent antibody is APC;

[0051] The fluorescent marker of the CD69 fluorescent antibody is R718;

[0052] The fluorescent label of the HLA-DR fluorescent antibody is PE-Cy7;

[0053] The fluorescent label of the CD16 fluorescent antibody is APC-Cy7;

[0054] The fluorescent marker of the PD-1 fluorescent antibody is BV785.

[0055] The fluorescent label of the Foxp3 fluorescent antibody is PE;

[0056] The fluorescent label of the Ki67 fluorescent antibody is BV421.

[0057] Preferably, the volume ratio of the CD45 fluorescent antibody for marking leukocytes: the CD3 fluorescent antibody for marking total T lymphocytes: the CD4 fluorescent antibody for marking helper T lymphocytes: the PD-1 fluorescent antibody for marking exhausted cells: the HLA-DR fluorescent antibody for marking late activated T cells: the CD8 fluorescent antibody for marking cytotoxic T lymphocytes: the CD69 fluorescent antibody for marking early activated T cells: the CD25 fluorescent antibody for marking regulatory T cells: the Foxp3 fluorescent antibody for marking regulatory T cells: the Ki67 fluorescent antibody for marking proliferating cells is 4.5-5.5:4.5-5.5:0.5-1.5:0.5-1.5:0.5-1.5:2-3:2-3:1.5-2.5:4.5-5.5:0.5-1.5.

[0058] Beneficial effects of the present invention:

[0059] The present invention constructs a method for detecting cynomolgus monkey T cells and their activation, proliferation and exhaustion by using a combination of fluorescent antibodies of specific types and ratios, which can simultaneously and accurately and rapidly detect the early, middle and late activation, exhaustion state and proliferation capacity of cynomolgus monkey T lymphocytes. Verification experiments have shown that the detection method provided by the present invention has excellent detection accuracy, high intra-batch precision and inter-batch precision, and can be used to evaluate the effects of drugs on the immune system of cynomolgus monkeys in the non-clinical research stage of new drugs. It is of great significance, helps to deeply understand the effects of drugs on the immune response of cynomolgus monkeys, and provides important data for the safety and effectiveness evaluation of drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0060] Figure 1 Circled diagram of cynomolgus monkey T cells and their activation, proliferation and depletion for Example 1.

[0061] Figure 2 Schematic diagram of the linear results of Example 1①;

[0062] Figure 3 Schematic diagram of the linear results of Example 1②;

[0063] Figure 4 Schematic diagram of the linear results of Example 1③. DETAILED DESCRIPTION

[0064] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0065] The sources of the reagents used in the embodiments of the present invention are shown in Table 1 below:

[0066] Table 1 Example reagent information

[0067]

[0068] Example 1 Detection of T cells in cynomolgus monkeys and their activation and proliferation About 2.0 mL of venous blood was collected from the lower limbs of each cynomolgus monkey and anticoagulated with sodium heparin. The instrument voltage and threshold are shown in Table 2 below:

[0069] Table 2 Instrument voltage and threshold information

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[0072] The instrument compensation is shown in Table 3 below:

[0073] Table 3 Instrument compensation information

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[0078] Group area settings

[0079] Select the leukocyte population and lymphocyte population, select CD45 + CD3 + 、CD45 + CD3 + CD4 + 、CD45 + CD3 + CD8 + 、CD45 + CD3 + CD4 + CD25 + Foxp3 + 、CD45 + CD3 + Ki67 + 、CD45 + CD3 + CD4 + Ki67 + 、CD45 + CD3 + CD8 + Ki67 + 、CD45 + CD3 + CD4 + CD25 + FoxP3 + Ki67+ 、CD45 + CD3 + CD69 + 、CD45 + CD3 + CD4 + CD69 + 、CD45 + CD3 + CD8 + CD69 + 、CD45 + CD3 + CD4 + CD25 + FoxP3 + CD69 + 、CD45 + CD3 + HLA-DR + 、CD45 + CD3 + CD4 + HLA-DR + 、CD45 + CD3 + CD8 + HLA-DR + 、CD45 + CD3 + CD4 + CD25 + FoxP3 + HLA-DR + 、CD45 + CD3 + PD-1 + 、CD45 + CD3 + CD4 + PD-1 + 、CD45 + CD3 + CD8 + PD-1 + 、CD45 + CD3 + CD4 + CD25 + FoxP3 + PD-1 + 、CD45 + CD3 + CD25 + 、CD45 + CD3 + CD4 + CD25 +、CD45 + CD3 + CD8 + CD25 + Results for positive areas.

[0080] Detection steps: (1) Antibody preparation: add 5 μL of FITC Mouse Anti-NHP CD45, PercPMouse Anti-Human CD3, 1 μL of BUV395 Mouse Anti-Human CD4, BV785 anti-human CD279 (PD-1), PE-Cy7 Mouse Anti-Human HLA-DR, 2.5 μL of BV605 Mouse Anti-Human CD8, R718 Mouse Anti-Human CD69, and 2 μL of APC Anti-Hu / NHP CD25 fluorescent antibody to prepare a mixed antibody.

[0081] (2) Sample addition: Add 20 μL of mixed antibody to each flow cytometry tube, then add 50 μL of whole blood sample, mix well, and incubate at 15-25°C in the dark for 20 min.

[0082] (3) Lysis of red blood cells: Add 1000 μL of 1× NH4Cl red blood cell lysis buffer at a ratio of 1:20 to the flow tube, mix well, and lyse in a dark environment at 15-25°C for 15 min.

[0083] (4) Centrifugation: 15-25°C, 400 g, for 5 min.

[0084] (5) Washing: Discard the supernatant and add 1000 μL of 1× PBS for washing.

[0085] (6) Centrifugation: 15-25°C, 400 g, for 5 min.

[0086] (7) Fixation and membrane permeabilization: Discard the supernatant, add 1000 μL Tf 1× Fix / Perm Buffer, and incubate at 2-8°C in the dark for 45 min.

[0087] (8) Washing and centrifugation: Add 1000 μL Tf 1× Perm / Wash Buffer, centrifuge at 2-8°C, 400 g for 5 min.

[0088] (9) Washing and centrifugation: Discard the supernatant, add 2000 μL Tf 1× Perm / Wash Buffer, centrifuge at 2-8°C, 400 g, and centrifuge for 5 min.

[0089] (10) Intracellular staining: Discard the supernatant, pipette 1 μL BV421 Mouse Anti-Ki-67 and 5 μL PeAnti-Human Foxp3 fluorescent antibody into flow cytometry tubes, and incubate at 2-8°C in the dark for 45 min.

[0090] (11) Washing and centrifugation: Add 2000 μL Tf 1× Perm / Wash Buffer, centrifuge at 2-8°C, 400 g for 5 min.

[0091] (12) Resuspend: Discard the supernatant and add 500 μL 1× PBS buffer.

[0092] (13) Detection: Flow cytometer is used to detect the processed samples.

[0093] The detection method in Example 1 was verified to examine the precision and linearity of the analytical method.

[0094] Intra-batch precision: 4 samples were analyzed in the same analysis batch, and each sample was repeated 3 times. The CV of each measured value was calculated, and the CV range was 0.01-23.14%, which was less than 25% and met the acceptance criteria. The specific results are shown in Table 4.

[0095] Inter-batch precision: Three samples were analyzed in two independent analytical batches within one day, and each sample was repeated three times. The CV of the average value of each measured value of each sample in the two batches was calculated, and the CV range was 0.19-16.65%, which was less than 25%, meeting the acceptance criteria. The specific results are shown in Table 5.

[0096] Linearity: A high-concentration sample was stained with CD45 alone and completely stained, and then the CD45 alone and completely stained samples were serially diluted in a proportional gradient (1:9, 2:8, 3:7, 4:6, 5:5, 6:4, 7:3, 8:2, 9:1, 10:0), and each diluted sample was measured twice. The mean value of the high-value sample was used as the benchmark result, and the theoretical results of each series of diluted samples were calculated based on the dilution ratio as the expected value. The average value of the test results was taken as the measured value. The horizontal axis X represents the expected value of each sample, and the vertical axis Y represents the measured value of each sample. Linear regression statistics were performed to obtain Y=bX+a, the slope was obtained, and the determination coefficient R was calculated. 2 The measured value was compared with the expected value to calculate the recovery rate. The slope, determination coefficient recovery rate (%) all met the standards. The results are shown in Table 6.

[0097] Table 4 Intra-batch precision verification results

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[0111] Table 5 Results of inter-batch precision verification

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[0131] Table 6 Linearity verification results

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[0135] The results showed that the intra-batch precision CV ranged from 0.01 to 23.14%, the inter-batch precision CV ranged from 0.19 to 16.65%, the linear slope ranged from 0.9832 to 0.9874, and the R 2 The range of was 0.9995-0.9997, and the recovery range was 97.93-105.89, indicating that the bioanalytical method can be used for the detection of cynomolgus macaque T cells and their activation, proliferation and depletion with high detection accuracy.

[0136] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for detecting cynomolgus monkey T cells and activation, proliferation and depletion thereof, characterized in that: The following steps are involved: Take a combination of antibodies 1 labeled with different fluorescence and mix them with the sample to be tested for incubation; Process the samples and obtain the test data through flow cytometry; Obtaining cynomolgus monkey T cells and their activation, proliferation and exhaustion conditions according to the detection data; The sample processing includes intracellular staining, and the intracellular staining includes taking a combination of antibodies 2 with different fluorescent labels and mixing and incubating them with the sample to be detected; The antibody combination 1 comprises: CD45 fluorescent antibody to label leukocytes; CD3 fluorescent antibody to label total T lymphocytes; CD4 fluorescent antibody to label helper T lymphocytes; CD8 fluorescent antibody to label cytotoxic T lymphocytes; CD25 fluorescent antibody to label regulatory T cells; CD69 fluorescent antibody to label early activated T cells; HLA-DR fluorescent antibody to label late activated T cells; PD-1 fluorescent antibody was used to label the exhausted cells.

2. The detection method according to claim 1, characterized in that: The fluorescent label of the CD45 fluorescent antibody is FITC; The fluorescent marker of the CD3 fluorescent antibody is PercP; The fluorescent label of the CD4 fluorescent antibody is BUV395; The fluorescent marker of the CD8 fluorescent antibody is BV605; The fluorescent marker of the CD25 fluorescent antibody is APC; The fluorescent marker of the CD69 fluorescent antibody is R718; The fluorescent label of the HLA-DR fluorescent antibody is PE-Cy7; The fluorescent marker of the PD-1 fluorescent antibody is BV785.

3. The detection method according to claim 1, characterized in that: The obtaining of detection data includes data acquisition and data analysis; The data analysis includes selecting CD45 + CD3 + 、CD45 + CD3 + CD4 + 、CD45 + CD3 + CD8 + 、CD45 + CD3 + CD4 + CD25 + Foxp3 + 、CD45 + CD3 + Ki67 + 、CD45 + CD3 + CD4 + Ki67 + 、CD45 + CD3 + CD8 + Ki67 + 、CD45 + CD3 + CD4 + CD25 + FoxP3 + Ki67 + 、CD45 + CD3 + CD69 + 、CD45 + CD3 + CD4 + CD69 + 、CD45 + CD3 + CD8 + CD69 + 、CD45 + CD3 + CD4 + CD25 + FoxP3 + CD69 + 、CD45 + CD3 + HLA-DR + 、CD45 + CD3 + CD4 + HLA-DR + 、CD45 + CD3 + CD8 + HLA-DR + 、CD45 + CD3 + CD4 + CD25 + FoxP3 + HLA-DR + and CD45 + CD3 + PD-1 + and CD45 + CD3 + CD4 + PD-1 + and CD45 + CD3 + CD8 + PD-1 + and CD45 + CD3 + CD4 + CD25 + FoxP3 + PD-1 + and CD45 + CD3 + CD25 + and CD45 + CD3 + CD4 + CD25 + and CD45 + CD3 + CD8 + CD25 + Expression status of subsets.

4. The detection method according to any one of claims 1 to 3, characterized in that: The antibody combination 2 includes a Foxp3 fluorescent antibody for marking regulatory T cells and a Ki67 fluorescent antibody for marking proliferating cells.

5. The detection method according to claim 4, characterized in that: The fluorescent label of the Foxp3 fluorescent antibody is PE; The fluorescent label of the Ki67 fluorescent antibody is BV421.

6. A kit for detecting cynomolgus monkey T cells and their activation, proliferation and depletion, characterized in that: The kit includes the following fluorescently labeled antibodies: CD45 fluorescent antibody to label leukocytes; CD3 fluorescent antibody to label total T lymphocytes; CD4 fluorescent antibody to label helper T lymphocytes; CD8 fluorescent antibody to label cytotoxic T lymphocytes; CD25 fluorescent antibody to label regulatory T cells; CD69 fluorescent antibody to label early activated T cells; HLA-DR fluorescent antibody to label late activated T cells; PD-1 fluorescent antibody to label exhausted cells; Foxp3 fluorescent antibody to label regulatory T cells; Ki67 fluorescent antibody to label proliferating cells; The fluorescent label of the CD45 fluorescent antibody is FITC; The fluorescent marker of the CD3 fluorescent antibody is PercP; The fluorescent marker of the CD4 fluorescent antibody is BUV395; The fluorescent marker of the CD8 fluorescent antibody is BV605; The fluorescent marker of the CD25 fluorescent antibody is APC; The fluorescent marker of the CD69 fluorescent antibody is R718; The fluorescent label of the HLA-DR fluorescent antibody is PE-Cy7; The fluorescent label of the CD16 fluorescent antibody is APC-Cy7; The fluorescent marker of the PD-1 fluorescent antibody is BV785. The fluorescent label of the Foxp3 fluorescent antibody is PE; The fluorescent label of the Ki67 fluorescent antibody is BV421.

7. The kit according to claim 6, characterized in that The volume ratio of the CD45 fluorescent antibody for marking leukocytes: the CD3 fluorescent antibody for marking total T lymphocytes: the CD4 fluorescent antibody for marking helper T lymphocytes: the PD-1 fluorescent antibody for marking exhausted cells: the HLA-DR fluorescent antibody for marking late activated T cells: the CD8 fluorescent antibody for marking cytotoxic T lymphocytes: the CD69 fluorescent antibody for marking early activated T cells: the CD25 fluorescent antibody for marking regulatory T cells: the Foxp3 fluorescent antibody for marking regulatory T cells: the Ki67 fluorescent antibody for marking proliferating cells is 4.5-5.5:4.5-5.5:0.5-1.5:0.5-1.5:0.5-1.5:2-3:2-3:1.5-2.5:4.5-5.5:0.5-1.5.

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