A method for quantitatively analyzing immune cells and functional proteins in systemic lupus erythematosus and its application

Abstract

The present invention discloses a method for quantitatively analyzing the immune cells and functional protein abundances in systemic lupus erythematosus, including fluorescence flow cytometry experiments, sample collection and pretreatment for CyTOF detection, antibody labeling and cell staining for CyTOF analysis, sample use for CyTOF detection, data processing, and statistical analysis. The present invention for the first time describes the phenotypes and functions of immune cell subsets in PBMCs of aSLE and rSLE with different disease activities, and discovers a comprehensive biomarker of multiple parameters that can potentially be related to cell prognosis, namely Tem-like CD8+CD27+CXCR3− T cells, CD27 protein, and is related to the disease prognosis function.

Description

Technical Field

[0001] The present invention belongs to the technical field of disease diagnosis and prognosis rating, and particularly relates to the application of mass cytometry in establishing early diagnosis and prognosis markers for systemic lupus erythematosus. Background Art

[0002] SLE is a classic chronic autoimmune disease, and its main pathological features are the tolerance to nuclear antigens, the deposition of immune complexes in tissues, and multi-organ involvement and damage. Although guidelines for the diagnosis and treatment of SLE are updated based on clinical experience every year, the mortality rate of SLE is still five times higher than that of normal people. The inflammatory response in the disease active state will cause cumulative damage to body tissues. Therefore, timely and effective diagnosis and clinical treatment intervention are of self-evident importance for delaying and controlling the disease progression of SLE and improving the quality of life of patients.

[0003] However, due to the large individual differences in the symptoms of SLE, there are still huge challenges in the early diagnosis of this disease in clinical practice. First, the most frequently occurring symptom is arthritis, that is, joint-related inflammation, with an incidence rate of 85%. However, arthritis is not specific for the diagnosis of SLE. Studies have shown that there are approximately 200 diseases related to arthritis at present, such as gout, fibromyalgia, rheumatoid arthritis, etc. Second, early-stage patients usually show very few clinical symptoms and have a long latency period. In addition, there are some SLE patients with relatively rare symptoms, such as patients with negative antinuclear antibody (ANA), a common biochemical index. These factors together lead to difficulties in the clinical diagnosis of SLE. It is worth noting that patients who are difficult to diagnose clinically are more likely to be severely ill SLE patients in urgent need of treatment.

[0004] By combining the sample injection method of fluorescence flow cytometry and the detection method of ICP-MS, CyTOF can change the signal readout based on fluorescent tags into the signal readout based on the atomic mass of metal elements, and can theoretically detect the expression of more than 40 protein molecules simultaneously. Compared with fluorescent dyes, the single-peak atomic mass signal does not have the interference of spectral signal overlap, and since there is almost no lanthanide metal element in the organism, the background signal is almost 0, and the detection range is wide (75 - 210 Da), and it can theoretically achieve the simultaneous detection of more than 100 proteins, having unique advantages in the immune system analysis of complex diseases.

[0005] Therefore, those skilled in the art are committed to developing a method for studying the cell phenotype, function, and autophagy activity of rSLE blood samples from untreated aSLE and aSLE in remission after treatment based on CyTOF, systematically describing the immune system state under different disease states of SLE and the autophagy activity of specific cell subsets, and using quantitative analysis of the differences between cell subsets to find immune multi-parameter markers related to disease diagnosis and prognosis in SLE. Summary of the Invention

[0006] In view of the above-mentioned defects of the prior art, the technical problem to be solved by the present invention is to use a high-throughput analysis method to find immune multi-parameter markers related to disease diagnosis and prognosis in SLE.

[0007] To achieve the above object, the present invention provides a method for quantitatively analyzing immune cells and functional proteins in systemic lupus erythematosus and its application.

[0008] The first aspect of the present invention is to provide a method for quantitatively analyzing the abundance of immune cells and functional proteins in systemic lupus erythematosus, including the following steps:

[0009] Step 1, fluorescence flow cytometry experiment:

[0010] Based on the SLEDAI standard score, select peripheral blood samples of HCs and SLE after drug treatment, extract PBMCs, perform a blocking experiment before antibody staining, then perform antibody staining, and perform data processing after loading onto the machine.

[0011] Step 2, sample collection and pretreatment for CyTOF detection;

[0012] Step 3, antibody labeling and cell staining for CyTOF analysis;

[0013] Step 4, the sample is used for CyTOF detection and data processing;

[0014] Step 5, statistical analysis.

[0015] Furthermore, the fluorescence flow cytometry experiment in the said Step 2 includes the following steps:

[0016] Step 2.1, design, collect and preprocess samples:

[0017] The preprocessed samples include sample pretreatment for phenotype analysis and sample pretreatment for function analysis;

[0018] Step 2.2, antibody labeling and cell staining for CyTOF analysis;

[0019] Step 2.3, the sample is used for CyTOF detection and data processing.

[0020] Preferably, the sample in step 2.1 is a whole blood sample of HCs, aSLE, and rSLE.

[0021] Furthermore, preferably, the screening is based on the SLEDAI standard score. The definition of aSLE is SLEDAI > 4, and the definition of rSLE is SLEDAI ≤ 4.

[0022] Preferably, the sample pretreatment for phenotypic analysis in step 2.2 is directly completed within 4 hours after leaving the body.

[0023] Preferably, it is characterized in that the sample pretreatment for functional analysis in step 2.2 is directly completed within 10 hours after leaving the body.

[0024] Furthermore, step 3 specifically includes the following steps:

[0025] Step 3.1, Antibody design for CyTOF;

[0026] Step 3.2, CyTOF antibody labeling;

[0027] Step 3.3, Sample cell counting and Pd coding strategy for the sample;

[0028] Step 3.4, Surface antibody staining;

[0029] Step 3.5, Intracellular protein staining step;

[0030] Step 3.6, Ir fixation step.

[0031] The second aspect of the present invention is a biomarker for early diagnosis and prognosis evaluation of systemic lupus erythematosus established by the above method, which is characterized by including CD8+CD27+CXCR3-T cell surface and / or CD27 protein labeling antibodies.

[0032] The third aspect of the present invention is to provide an application of the described method in the early diagnosis or prognosis evaluation of systemic lupus erythematosus.

[0033] The fourth aspect of the present invention is to provide an application of the described biomarker in the early diagnosis and prognosis evaluation of systemic lupus erythematosus.

[0034] The beneficial effects of the present invention are:

[0035] 1) For the first time, the phenotypes and functions of immune cell subsets in PBMCs of aSLE and rSLE with different disease activities were systematically described, so as to more intuitively understand the changes in the immune system during the occurrence and development of the disease from a global perspective. Considering that SLE is a disease with immune heterogeneity, when recruiting samples, we selected untreated newly diagnosed aSLE cases, and rSLE samples were basically follow-up samples of aSLE after a period of drug treatment when the disease condition was relieved, in order to minimize the interference of sample heterogeneity on the experimental results;

[0036] 2) A multi-parameter comprehensive biomarker Tem-like CD8 + CD27 + CXCR3 - T cells were discovered, and their cell proportions, cell activities and prognostic functions are related.

[0037] The concept, specific structure and technical effects of the present invention will be further described below in conjunction with the accompanying drawings to fully understand the purpose, features and effects of the present invention. Brief Description of the Drawings

[0038] Figure 1 is the gating strategy of peripheral blood mononuclear cells of systemic lupus erythematosus detected by fluorescence flow cytometry in a preferred embodiment of the present invention. Detailed Embodiments

[0039] The following introduces multiple preferred embodiments of the present invention with reference to the accompanying drawings of the specification, making its technical content clearer and easier to understand. The present invention can be embodied in many different forms of embodiments, and the protection scope of the present invention is not limited to the embodiments mentioned in the text.

[0040] Example 1: Fluorescence Flow Experiment

[0041] 1. Sample collection: In order to find biomarkers related to disease treatment, the present invention first designed to collect 8 HCs (healthy controls, HCs) and 10 peripheral blood samples of SLE patients after drug treatment from Renji Hospital. All samples were scored based on the SLEDAI criteria given by the American College of Rheumatology.

[0042] 2. The processing process of blood samples mainly includes the following three steps.

[0043] 1) Extraction of PBMCs.

[0044] First, take out the lymphocyte separation solution from the 4°C refrigerator. Add 3 ml to each 15-ml centrifuge tube and equilibrate it to room temperature. The freshly collected blood is diluted with PBS at a concentration of 1:1. Slowly drip 4-8 ml of the diluted blood sample into the centrifuge tube pre-filled with the lymphocyte separation solution. During the addition of blood, tilt the centrifuge tube to the maximum angle so that the blood spreads flat on the surface of the lymphocyte separation solution without mixing with it.

[0045] Then, after sealing the centrifuge tube, place it in a centrifuge to enrich PBMCs (25°C, 400 g, 20 min, with the braking speed set to 5).

[0046] Carefully aspirate the PBMCs that appear as white flocs and are located in the middle of the lymphocyte separation solution and plasma, and transfer them to a new 15-ml centrifuge tube. Add 2-3 times the normal-temperature DMEM medium, mix well, and then place it in a centrifuge (25°C, 400 g, 20 min) for centrifugation to remove the remaining lymphocyte solution.

[0047] 2) Blocking experiment before antibody staining.

[0048] For a cell sample of 1.5×10 6 , first, add 25 μl of Blocking Cocktail (50 μl of Fc R blocking solution diluted in 500 μl of CSB) to each sample, and react at room temperature for 10 min. Do not let the reaction time be too long to avoid affecting the subsequent staining effect.

[0049] 3) Antibody staining process.

[0050] Mix the antibodies according to the optimized ratio for the experiment (1:60 / 1:200 / 1:600), and finally dilute them with CSB to a total volume of 550 μl. Add 25 μl to each sample, and react in the dark at room temperature for 30 min. Then wash the samples 2 times with PBS (25°C, 400 g, 5 min) for fluorescence flow cytometry analysis.

[0051] 3. The data obtained after running on the machine is mainly processed using FlowJo (Treestar Inc.) and Cytobank.

[0052] Example 2. Sample collection and pretreatment for CyTOF detection

[0053] 1. Sample Design and Collection. All samples were obtained from Shanghai Jiao Tong University Renji Hospital, and all SLE samples were scored according to the American College of Rheumatology SLEDAI criteria, with aSLE (active SLE, aSLE) defined as SLEDAI > 4 and rSLE (remission SLE, rSLE) as SLEDAI ≤ 4. This study complied with the ethical requirements of Shanghai Jiao Tong University Renji Hospital, with ethics number

[2017] 201. For the CyTOF experiments, whole blood samples were collected from 8 HCs, 10 aSLE, and 8 rSLE patients.

[0054] In the process of collecting samples, we selected newly diagnosed active SLE patients who had not received drug treatment. In the selection of remitted SLE, we tried to use follow-up samples of aSLE patients whose disease was in remission after treatment. Among them, 7 rSLE patients were follow-up samples of aSLE.

[0055] At the same time, we divided the clinical samples for CyTOF analysis into two categories:

[0056] (1) Directly used for immunophenotyping analysis. The blood was pretreated within 4 hours of ex vivo extraction and placed in a -80°C freezer for subsequent CyTOF staining and on-machine experiments.

[0057] (2) Samples for further functional analysis. For further functional characterization and analysis, some samples were stimulated with lipopolysaccharide and Golgi transport inhibitor (LPS). The experiment was completed within 10 hours of in vitro elution and stored in a -80°C freezer for subsequent use in the machine.

[0058] The strategy for clustering peripheral blood mononuclear cells in systemic lupus erythematosus by fluorescence flow cytometry is as follows: Figure 1 shown.

[0059] 2. Sample preprocessing for phenotypic analysis.

[0060] After being removed from the body, the sample was stored in a blood collection tube (BD) containing heparin and sent to the laboratory for pretreatment 2 to 4 hours after removal from the body. The human peripheral blood lymphocyte separation fluid was equilibrated to room temperature in advance, and the water bath temperature was adjusted to 37°C.

[0061] 1) PBMCs extraction: The operation steps are consistent with the pre-treatment of the fluorescence flow cytometry experiment in Example 1.

[0062] 2) Aspirate the lymphocytes and transfer them to a new 15 ml centrifuge tube. Add 2-3 times the volume of room temperature DMEM medium. Mix well and place in a centrifuge (25°C, 400 g, 20 min) to wash away the remaining lymphocyte fluid.

[0063] 3) Dilute Pt(DMSO solution) with a concentration of 5 mM to 5 μM using DMEM. After centrifugation, discard the supernatant of the cells, resuspend and disperse the cells using a vortex mixer, and then add 1 ml of the diluted Pt (when the total number of cells is less than 10 7 , 1 ml can be used in all cases) to each tube of cells, and react in a water bath for 5 min. Considering that Pt is more inclined to bind to the proteins on dead cells, CyTOF can be used to judge the cell viability based on the signal intensity of Pt in the cells.

[0064] 4) Add cell staining buffer (CSB, 0.5% BSA and 0.20% NaN3 dissolved in 500 ml of PBS) stored on ice to terminate the reaction, and centrifuge to wash away the unreacted Pt (25 °C, 400 g, 5 min).

[0065] 5) Resuspend the cells with 1 ml of CSB, slowly add 1 ml of 3.2% paraformaldehyde (PFA, 3.2 g dissolved in 100 ml of PBS solution) drop by drop, react at room temperature for 10 min, add CSB to terminate the reaction, and centrifuge (25 °C, 400 g, 5 min).

[0066] 6) Resuspend the cells with cell cryopreservation solution (10% DMSO added to CSB), and dilute to 3×10 6 cells / ml, transfer to cryotubes, and store in a -80 °C refrigerator.

[0067] 3. Pretreatment of functional analysis samples.

[0068] The cell pretreatment process before Pt staining is the same as the steps of the phenotypic analysis samples.

[0069] The extracted PBMCs are first resuspended with 300 μl of cell culture medium containing 10% FBS and placed in a 37 °C water bath. Then, take out 10 μl of the cells and mix them with 10 μl of AO / PI dye for counting. According to the counting results of the cell counter, take out 1 - 2×10 6 cells and add them to a 24-well plate, dilute to 1 ml with complete medium, add 2 μl of Leukocyte Activation Cocktail, with BD GolgiPlug to each well plate, gently pipette to mix evenly, and place the mixed 24-well culture plate in a cell culture incubator and culture for 6 h.

[0070] Transfer the cells back into a 15 ml centrifuge tube with 3 ml of DMEM at 37 °C and centrifuge (25 °C, 600 g, 5 min). After centrifugation, the cells are stained with Pt, fixed with PFA, resuspended in freezing medium, and stored in an -80 °C freezer.

[0071] Example 3. Antibody Labeling and Cell Staining for CyTOF Analysis

[0072] 1. Antibody Design for CyTOF

[0073] Although the atomic mass signals of single-peak metal elements do not overlap like fluorescence signals, due to element purity and the presence of metal oxides, there is <5% crosstalk between some metal channels. Generally, metal channels can be divided into three types with preferred protein antibody types:

[0074] (1) Metal channels that are easily interfered with by other metal channels are suitable for labeling antibodies with high expression levels, such as CD45;

[0075] (2) Channels that are likely to interfere with other metal channels are suitable for labeling antibodies with low expression levels, such as CD25;

[0076] (3) Metal channels that are neither interfered with nor interfere with other channels are suitable for labeling antibodies that are relatively difficult to detect, such as nuclear transcription factors.

[0077] Under the condition of meeting the above three principles, according to the existing commercial combinations of labeled metals and antibodies from Fluidigm, the number of antibodies that need to be labeled by oneself can be minimized as much as possible to reduce the workload.

[0078] Based on the above matching principle, finally ensure that the proportion of interference signals in each channel is less than 0.5%.

[0079] 2. CyTOF Antibody Labeling

[0080] During the experimental design process, due to the multiple selectivities of the combination of metal channels and antibodies, in addition to some antibodies pre-labeled by Fluidigm, some antibodies need to be labeled strictly according to the protocol provided by Fluidigm.

[0081] It is mainly divided into three steps, which are briefly introduced as follows.

[0082] 1) Binding and Purification of Metal and Polymer

[0083] Prepare a metal bath at 37 °C in advance.

[0084] First, take out the polymer from -20°C and let it equilibrate to room temperature to avoid its inactivation due to moisture absorption. Centrifuge the taken-out polymer to the bottom to avoid loss of the polymer during the process of opening the lid.

[0085] Subsequently, add 95 ul of L-buffer, then add 5 ul of the corresponding metal element, mix well, and react in a metal bath at 37°C for 30 min. Transfer the mixture of the metal and the polymer to a 3 kDa ultrafiltration tube, add 200 ul of L-buffer, and centrifuge (12000 g, room temperature, 25 min).

[0086] After that, discard the waste liquid, add 400 ul of C-buffer, and centrifuge (12000 g, room temperature, 30 min).

[0087] 2) Reduction and purification of the antibody.

[0088] After heating the metal and the polymer in a metal bath, start the purification of the antigen.

[0089] First, add 100 - 200 ul (100 ug) of the antibody to a 50 kDa ultrafiltration tube, then add R-buffer to make the system up to 500 ul, and centrifuge (12000 g, room temperature, 10 min). At the same time, dilute the TCEP concentration to 4 mM with R-buffer. After centrifugation, discard the supernatant, add 100 ul of TCEP to the antibody, mix well, and react in a metal bath for 30 min. If the activation time is too long, it will cause over-reduction of the antibody, so it must not exceed 30 min.

[0090] After the reaction, add 300 ul of C-buffer, and centrifuge (12000 g, room temperature, 10 min). Repeat the centrifugation once.

[0091] 3) Binding of the purified metal-polymer and the antibody.

[0092] Add 60 ul of C-buffer to the centrifuged metal-polymer, mix well, transfer all to a 50 kDa ultrafiltration tube, and react at 37°C for 90 min after thorough mixing to connect the antibody and the metal-polymer.

[0093] Then add 200 ul of W-buffer for washing, and centrifuge (12000 g, room temperature, 10 min). Repeat the centrifugation 3 times. Add 80 ul of W-buffer to the centrifuged product to recover the product, and use NanoDrop to calculate the absorbance at 280 nm to determine the yield of the recovered product.

[0094] 3. Sample cell counting and Pd coding strategy for the sample.

[0095] Take out the sample to be labeled from the -80°C refrigerator. After thawing, transfer it to a flow tube respectively, and add 2 ml of CSB to each sample. Centrifuge to remove DMSO (600 g, 25°C, 5 min). Resuspend the sample based on the remaining liquid, and take out 10 μl from each tube for cell counting.

[0096] Based on the counting result, take 1.5×10 6 cells from each sample and transfer them to a new flow tube.

[0097] 1) Code the samples according to the Fluidigm operation manual.

[0098] Before the operation, first take out the Pd coding reagent from -20°C and equilibrate it to room temperature. Add 800 μl of Barcode Perm Buffer (1 ml of 10X Barcode Perm Buffe plus 9 ml of PBS) to each tube of cells. At the same time, centrifuge the Pd coding substance that has been equilibrated to room temperature to avoid loss during the process of opening the lid. Add 100 μl of Barcode Perm Buffer to each Pd coding particle tube, and mix it well with the corresponding sample one by one. React at room temperature for 30 min, add CSB to terminate the reaction, centrifuge twice (800 g, 25°C, 5 min). Then add 500 μl of CSB to each tube, transfer all the cells to the same centrifuge tube as fully as possible, and centrifuge (800 g, 25°C, 5 min) for subsequent antibody staining.

[0099] 2) Surface antibody staining.

[0100] According to the pre-experiment results of Fluidigm, 1 μg of commercial antibody is required for every 3×10 6 cells. For 20 samples of 1.5×10 6 cells, it is equivalent to 10 standard tests.

[0101] First, add 500 μl of Blocking Cocktail (46 μl of Fc R blocking solution diluted in 460 μl of CSB), and mix it well with the cells. React at room temperature for 10 min, do not exceed the time to avoid affecting the subsequent antibody staining process.

[0102] During the reaction, for the cocktail solution of surface antibodies, add 10 μl of each of the 20 surface-staining proteins to 310 μl of CSB diluted, mix well, add to the cell sample, and react at room temperature for 30 min. Add 10 ml of CSB stored on ice, and centrifuge twice (800 g, 25 °C, 5 min).

[0103] Table 1 Human-specific antibodies and metal or fluorescent tags required during the experiment

[0104]

[0105]

[0106] 3) Intracellular protein staining steps.

[0107] The entire procedure is carried out according to the experimental protocol provided by eBioscienc. First, fresh permeabilization / fixation working solution and permeabilization buffer need to be prepared.

[0108] Add 10 ml of the permeabilization / fixation working solution to the cells resuspended after centrifugation and transfer to a 50-ml centrifuge tube. Mix well and react for 60 min.

[0109] Then add 30 ml of the permeabilization buffer, centrifuge (800 g, 25 °C, 5 min), 2 times. For the resuspended cells, add 500 μl of Blcoking cocktail again and react for 10 min. At the same time, prepare the cocktail of intracellular antibodies with a total volume of still 500 μl and react for 30 min.

[0110] To ensure the staining effect, the buffer of the cocktail during intracellular staining is replaced with the permeabilization buffer. Then add 30 ml of CSB stored on ice, centrifuge (800 g, 25 °C, 5 min), 2 times.

[0111] 4. Ir fixation step. CyTOF cannot judge the integrity of cells based on scattered light signals. Considering that the metal intensity of Ir is proportional to the amount of DNA, CyTOF chooses to judge the integrity of DNA structure based on the signal intensity of Ir. Dilute Ir in the Fix and Perm solution during cell centrifugation to a final concentration of 125 nM, and then slowly add the mixed Ir solution to the resuspended cell system to 10 ml. Mix well and store in a 4 °C refrigerator for reaction for 12 - 48 h.

[0112] Example 4. Samples for CyTOF detection and data processing

[0113] 1. Sample preparation before loading.

[0114] Add 30 ml of CSB to terminate the Ir staining reaction and centrifuge (800 g, 25°C, 5 min) twice. Then use ultrapure water to wash away excess salts in the sample, repeat the wash and centrifuge 4 times (800 g, 25°C, 5 min). Resuspend the cells in 5 ml of ultrapure water and dilute them to a concentration of 1.5 × 10 6 cells / ml.

[0115] 2. CyTOF data collection.

[0116] When starting the instrument, first connect the pipes and ensure that the nebulizer can see a uniform and continuous spray. Then insert the nebulizer into the connection port and start the startup process.

[0117] After turning on the instrument, start the water washing process to clear the previous residual signal. Then wait for the temperature to stabilize for 15 minutes, replace the water washing with the tuning solution, and perform the instrument signal calibration.

[0118] After tuning is completed, you can add the sensitivity of the EQbeads detection signal. When the detected signal value is within the standard range, wash the injection end with water and set the template required for experimental data collection to prepare to start collecting data.

[0119] Before formally collecting data, the cells in the 50ml large centrifuge tube need to be passed through the top of the flow tube with a filter to filter out large cell aggregates to prevent clogging the injection end, and EQbeads are added at a ratio of 1:5 as an internal reference signal for subsequent data calibration.

[0120] During the data collection process for the sample injection test, ensure that the number of events passing through per second is less than 500. High cell concentrations can easily cause blockage in the injection line. For long-term sample collection, it is recommended to perform signal tuning every 4 hours to avoid signal drift caused by long-term instrument operation.

[0121] 3. Preliminary preprocessing of CyTOF data.

[0122] First, the collected data needs to be standardized based on the internal reference signal of EQbeads. This process can be directly performed based on the preset algorithm in the software (6.7, Fluidigm).

[0123] Next, the decoding process of the sample Pd coding is carried out, which is also directly operated based on the preset algorithm in the software. After data normalization and Pd decoding, each sample will generate a corresponding.fcs file. Before processing the data with the algorithm, we first upload all the.fcs files to cytobank (https: / / community.cytobank.org / , Cytobank, Inc.), remove the signals of double-positive (151Eu and 153Eu) EQ-beads, then screen out the Pt 195 and Pt 196 double-negative live cells, and then manually circle the cells at the place with the highest density of Ir 191 and Ir193 as monocytes by default, and then circle the CD45-positive PBMCs with high expression of 171Yb, and generate a new.fcs file based on the PBMCs for the subsequent algorithm processing process.

[0124] 4. Systematic clustering based on ACCENSE.

[0125] Import all the samples into the directory called by ACCENSE and run based on Matlab version 9.1.0.0418663. According to different k values, the optimized clustering and grouping results are obtained. Finally, k = 30 is selected, and the PBMCs are divided into 43 cell subsets. Then, quantitative and statistical analyses are performed based on the protein expression levels and cell ratios of each cell subset after grouping.

[0126] Example 5. Statistical analysis

[0127] Before performing a significant difference analysis on the protein and cell abundances of all the samples, first analyze whether the data conforms to a normal distribution based on the Kolmogorov-Smirnov test.

[0128] If the data follows a normal distribution, then analyze the differences between two unpaired sample groups based on the two-tailed Welch's t-test, analyze the differences between two paired sample groups based on the two-tailed paired t-test, and analyze the differences among three or more sample groups based on the Tukey multiple comparison test.

[0129] If the data does not follow a normal distribution, then analyze the differences between two unpaired sample groups based on the two-tailed Mann-Whitney test, analyze the differences between two paired sample groups based on the two-tailed Wilcoxon signed-rank test, and analyze the differences among three or more sample groups based on the Kruskal-Wallis test. For the analysis of the correlation between clinical characteristics and CyTOF data, based on the premise that most of the data does not conform to a normal distribution, the Spearman correlation coefficient is used to calculate the correlation matrix.

[0130] Through the implementation of the above embodiments, the following main conclusions are obtained:

[0131] 1. Detection of 27 phenotypic and functional biomarkers in single cells of 8 healthy controls (HCs), 10 active SLE (aSLE), and 8 remission SLE (rSLE) patients was achieved by CyTOF. Using an unsupervised clustering analysis method, the Automatic Classification of Cellular Expression by Nonlinear Stochastic Embedding (ACCENSE), PBMCs were divided into 43 cell subsets;

[0132] 2. Visual description of the immune cell phenotypes across the entire SLE disease spectrum was performed. It was found that the immune profiles of rSLE and HCs were relatively similar, but the immune profile of aSLE was very different from that of HCs and rSLE, and there was also great heterogeneity among aSLE patient samples;

[0133] 3. After quantitative analysis of the 43 cell subsets respectively, in addition to the reported immune differences of Treg cells, it was also found that the proportion of CD8+CD27+CXCR3-T cells in rSLE samples was nearly doubled compared to aSLE;

[0134] 4. Statistical analysis of the expression of function-related proteins (CD27, CD69, Ki67, Atg5, Atg7, and LC3) showed that the cells had upregulated CD69 and decreased CD27 expression in aSLE compared to rSLE, indicating that this cell subset had higher immune activity in aSLE;

[0135] 5. After in vitro co-stimulation of samples with different disease activities with lipopolysaccharide and Golgi transport inhibitors, it was found that aSLE had higher IFNγ expression compared to rSLE, demonstrating that this cell subset had higher immune activity in aSLE. Based on the expression of surface protein markers and the function of secreted cytokines, CD8+CD27+CXCR3-T is a type of effector memory T (Tem) cell;

[0136] 6. Statistical correlation analysis was performed on clinical biochemical test indicators and cell-related parameters (cell ratio and expression of functional markers) of Tem-like CD8+CD27+CXCR3-T cells. It was found that the cell ratio of CD8+CD27+CXCR3-T cells and the expression of CD27 protein were negatively correlated with SLE activity. In addition, the expression of CD69, Atg7, and Atg5 in this group of cells was positively correlated with SLE activity, indicating that this group of cells may be potential markers related to prognosis.

[0137] The preferred specific embodiments of the present invention have been described in detail above. It should be understood that those of ordinary skill in the art can make many modifications and variations based on the concept of the present invention without creative labor. Therefore, all technical solutions that can be obtained by those skilled in the art in the technical field of the present invention through logical analysis, reasoning, or limited experiments based on the concept of the present invention on the basis of the prior art should fall within the protection scope determined by the claims.

Claims

1. A marker for early diagnosis and prognosis assessment of systemic lupus erythematosus, characterized in that, The marker is Tem-like CD8+CD27+CXCR3- T cells; the CD8+CD27+CXCR3- T cells are a multi-parameter and comprehensive marker, and the multi-parameters refer to being labeled by antibodies against CD8 and CD27 proteins simultaneously.

2. Use of a marker as described in claim 1, characterized in that, Determine the cell proportion and cell activity of the marker.

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