Use of th40 cell subpopulation in preparing a kit for aiding diagnosis and evaluating systemic lupus erythematosus disease activity

Abstract

The application discloses application of a Th40 cell subpopulation in preparation of a kit for assisting in diagnosis and evaluating SLE disease activity. The application is based on the research result that the inventors first find that the Th40 cell subpopulation is obviously increased in expression in SLE patients with disease activity, and the higher the disease activity is, the higher the proportion of the Th40 cell subpopulation is. Therefore, the Th40 cell subpopulation can be used as one of laboratory immune-related detection indexes for assisting in diagnosis of SLE, and meanwhile, important reference data for evaluating the disease activity of SLE patients are provided.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, and specifically relates to the application of Th40 cell subsets in the preparation of kits for the auxiliary diagnosis and assessment of systemic lupus erythematosus disease activity. Background Technology

[0002] Systemic lupus erythematosus (SLE) is an autoimmune disease caused by pathogenic autoantibodies. Dysfunction of lymphocyte subsets plays an important role in the pathogenesis of SLE, including dysfunction of CD8+ T cells and NK cells, and loss of inhibition of CD4+ cells. + T cell activity continuously stimulates B cell activation, leading to the production of autoantibodies and a persistent autoimmune response. Currently, clinical indicators for assessing SLE disease activity and treatment efficacy are relatively limited, and there are no effective immunomarkers for predicting SLE pathogenesis.

[0003] Currently, the severity of SLE is primarily assessed using the SLEDAI-2000 to evaluate disease activity and severity. However, due to the significant heterogeneity of SLE and the varying organ involvement, SLEDAI-2000 scores differ significantly. Current SLE disease activity assessment tools are limited and lack comprehensive, accurate, and objective laboratory indicators for evaluating disease activity. SLE patients exhibit T-cell immune imbalance. Research has identified several immune indicators related to T-cell activation, and their detection not only explores the mechanisms of abnormal T-cell immunity in SLE patients but also has the potential to serve as clinical indicators for assisting in the diagnosis and assessment of SLE disease severity. Current research on T-cell activation primarily focuses on the mechanisms of abnormal T-cell immunity in SLE patients, lacking specific immune-related indicators characteristic of SLE patients, and even more so, immune indicators for diagnosing and assessing T-cell activation activity. Therefore, a comprehensive understanding of the similarities and differences in T-cell immune changes in SLE patients, especially between severe and mild SLE, holds great promise for future clinical applications in the auxiliary diagnosis of severe SLE. Summary of the Invention

[0004] The primary objective of this invention is to overcome the shortcomings and deficiencies of the prior art and to provide the application of Th40 cell subsets in the preparation of kits for the auxiliary diagnosis and assessment of SLE disease activity.

[0005] Another object of the present invention is to provide a kit for assisting in the diagnosis and assessment of SLE disease activity.

[0006] Another object of the present invention is to provide a method for detecting Th40 cell subsets.

[0007] To achieve the above objectives, the present invention adopts the following technical solution:

[0008] The application of Th40 cell subsets in the preparation of kits for the auxiliary diagnosis and assessment of SLE disease activity is based on the inventors' first discovery that Th40 (CD4+) cells are present in the peripheral blood of SLE patients (SLE patients in this specification include both mild and severe patients). + CD40 + The proportion of T cell subsets is significantly increased in SLE patients, especially in patients with severe SLE.

[0009] The kit includes components, operating procedures, and judgment criteria for detecting Th40 cell subsets;

[0010] The components used to detect Th40 cell subsets are different fluorescently labeled monoclonal antibodies: anti-CD3 antibody, anti-CD4 antibody, and anti-CD40 antibody;

[0011] The operating procedure includes the following steps:

[0012] 1) Process the peripheral blood sample to be tested to form a single-cell suspension;

[0013] 2) Add monoclonal antibodies labeled with different fluorescence to the single-cell suspension obtained in step 1): anti-CD3 antibody, anti-CD4 antibody, and anti-CD40 antibody, mix gently, and incubate in the dark;

[0014] 3) After washing the cells, resuspend them in PBS and analyze them using a flow cytometer to obtain data on the fluorescently labeled Th40 cell subsets;

[0015] The judgment criteria are as follows: when the Th40 cell subset is in CD4 + The median proportion of T cells was 13.30%, which is highly suggestive of SLE disease activity. Among these, the Th40 cell subset was prominent in CD4+. + A median proportion of T cells higher than 13.30% suggests a higher likelihood of severe SLE.

[0016] A kit for assisting in the diagnosis and assessment of SLE disease activity includes components for detecting Th40 cell subsets, such as different fluorescently labeled monoclonal antibodies: anti-CD3 antibody, anti-CD4 antibody, and anti-CD40 antibody.

[0017] The fluorescent labeling of the anti-CD3 antibody is preferably FITC.

[0018] The fluorescent labeling of the anti-CD4 antibody is preferably APC-H7.

[0019] The fluorescent labeling of the anti-CD40 antibody is preferably PE-cy7.

[0020] The kit for assisting in the diagnosis and assessment of SLE disease activity also includes an instruction manual, which describes the operating procedures and judgment criteria for the kit.

[0021] The operating procedure includes the following steps:

[0022] 1) Process the peripheral blood sample to be tested to form a single-cell suspension;

[0023] 2) Add monoclonal antibodies labeled with different fluorescence to the single-cell suspension obtained in step 1): anti-CD3 antibody, anti-CD4 antibody, and anti-CD40 antibody, mix gently, and incubate in the dark;

[0024] 3) After washing the cells, PBS was added to resuspend the cells, and flow cytometry was used to detect the fluorescently labeled Th40 cell subsets.

[0025] The specific steps for processing the peripheral blood sample to be tested described in step 1) are as follows: The peripheral blood sample to be tested is subjected to whole blood red blood cell lysis according to conventional methods, centrifuged to remove the supernatant, washed with PBS, and then cell staining buffer is added to the precipitate obtained by centrifugation to resuspend it to form a single cell suspension.

[0026] The volume of the peripheral blood sample is preferably 200 μL / tube.

[0027] The preferred volume of the cell staining buffer is 50 μL / tube.

[0028] The amount of anti-CD3 antibody added in step 2) is preferably 1 μL of anti-CD3 antibody per 50 μL of single-cell suspension.

[0029] The amount of anti-CD4 antibody added in step 2) is preferably 1 μL of anti-CD4 antibody per 50 μL of single-cell suspension.

[0030] The amount of anti-CD40 antibody added in step 2) is preferably 1 μL of anti-CD40 antibody per 50 μL of single-cell suspension.

[0031] The specific operation of the light-protected incubation in step 2) is to incubate at room temperature in the dark for 15 to 30 minutes; preferably, incubate at room temperature in the dark for 20 minutes.

[0032] The room temperature is 5–35°C; preferably 20–30°C; more preferably 24–26°C.

[0033] The washing described in step 3) is performed using a cell staining buffer.

[0034] The PBS mentioned in step 3) is preferably PBS with a pH of 7.2 to 7.4 and a concentration of 0.01 to 0.1 M; more preferably PBS with a pH of 7.4 and a concentration of 0.01 M.

[0035] The judgment criteria are as follows: when the Th40 cell subset is in CD4 + The median proportion of T cells was 13.30%, indicating a high likelihood of SLE, with the Th40 cell subset accounting for a significant portion of CD4+. + A median proportion of T cells higher than 13.30% suggests a higher likelihood of severe SLE.

[0036] The kit also includes erythrocyte lysis buffer, cell staining buffer, and phosphate buffered solution (PBS) for lysing peripheral blood erythrocytes.

[0037] A method for detecting Th40 cell subsets for non-disease diagnosis or treatment purposes, using the above-mentioned kit, includes the following steps:

[0038] (1) Process the peripheral blood sample to be tested to form a single-cell suspension;

[0039] (2) Add monoclonal antibodies labeled with different fluorescence to the single-cell suspension obtained in step (1): anti-CD3 antibody, anti-CD4 antibody, and anti-CD40 antibody. Mix gently and incubate in the dark.

[0040] (3) After washing the cells, PBS was added to resuspend the cells, and flow cytometry was used to detect the data of the fluorescently labeled Th40 cell subsets.

[0041] The specific steps for processing the peripheral blood sample to be tested in step (1) are as follows: The peripheral blood sample to be tested is subjected to whole blood red blood cell lysis according to conventional methods, centrifuged to remove the supernatant, washed with PBS, and then cell staining buffer is added to the precipitate obtained by centrifugation to resuspend it to form a single cell suspension.

[0042] The preferred volume of peripheral blood sample used is 200 μL / tube.

[0043] The preferred volume of the cell staining buffer is 50 μL / tube.

[0044] The amount of anti-CD3 antibody added in step (2) is preferably 1 μL of anti-CD3 antibody per 50 μL of single cell suspension.

[0045] The fluorescent labeling of the anti-CD3 antibody is preferably FITC.

[0046] The amount of anti-CD4 antibody added in step (2) is preferably 1 μL of anti-CD4 antibody per 50 μL of single cell suspension.

[0047] The fluorescent labeling of the anti-CD4 antibody is preferably APC-H7.

[0048] The amount of anti-CD40 antibody added in step (2) is preferably 1 μL of anti-CD40 antibody per 50 μL of single-cell suspension.

[0049] The fluorescent labeling of the anti-CD40 antibody is preferably PE-cy7.

[0050] The specific operation of the light-protected incubation in step (2) is to incubate at room temperature in the dark for 15 to 30 minutes; preferably, incubate at room temperature in the dark for 20 minutes.

[0051] The room temperature is 5–35°C; preferably 20–30°C; more preferably 24–26°C.

[0052] The washing described in step (3) is performed using a cell staining buffer.

[0053] The PBS mentioned in step (3) is preferably PBS with a pH of 7.2 to 7.4 and a concentration of 0.01 to 0.1 M; more preferably PBS with a pH of 7.4 and a concentration of 0.01 M.

[0054] The aforementioned method for detecting Th40 cell subsets for non-disease diagnosis or treatment purposes is used to study the mechanism of abnormal T-cell immunity in SLE patients by Th40 cell subsets.

[0055] The application specifically includes the following steps: statistical analysis of the expression ratio of the Th40 cell subset. When the median expression ratio of the Th40 cell subset is detected to be higher than 13.30%, it indicates that the patient is more likely to be an SLE patient, and the SLE disease activity can be further analyzed.

[0056] The preferred statistical analysis is the rank-sum test.

[0057] The present invention has the following advantages and effects compared with the prior art:

[0058] (1) The inventors discovered for the first time that the proportion of Th40 cells in the peripheral blood of SLE patients is significantly increased, especially in severe SLE. This can be used as one of the laboratory immune-related test indicators to assist in the diagnosis of SLE, and at the same time, it also provides important reference data for assessing the disease activity of SLE patients.

[0059] (2) This invention provides a method for detecting Th40 cell subsets. This method can quantitatively count the phenotypes of the above-mentioned T cell subsets, and has a very broad application prospect in assisting the diagnosis of SLE and assessing the disease activity of SLE patients. Attached Figure Description

[0060] Figure 1 This is a flow cytometry analysis of the proportions of functional subsets of peripheral blood Th40 cells in SLE patients and healthy controls; where A represents the healthy control group and B represents SLE patients.

[0061] Figure 2 This is a graph showing the proportion of functional subsets of Th40 cells in the peripheral blood of SLE patients and healthy controls.

[0062] Figure 3 This is a graph showing the relationship between the proportion of Th40 cell functional subsets in SLE patients and SLEDAI-2000.

[0063] Figure 4 This is a graph showing the relationship between the proportion of Th40 cell functional subsets and complement C3 in SLE patients.

[0064] Figure 5 This is a graph showing the changes in the proportion of Th40 cell functional subsets before and after treatment in SLE patients. Detailed Implementation

[0065] The present invention will be further described in detail below with reference to the embodiments and accompanying drawings, but the embodiments of the present invention are not limited thereto.

[0066] The anti-CD3 antibody is a fluorescently labeled antibody CD3-FITC, which is FITC-labeled mouse anti-human CD3 (clone number: HIT3a, purchased from Biolegend);

[0067] The anti-CD4 antibody is the fluorescently labeled antibody CD4-APC-H7, which is APC-H7 labeled mouse anti-human CD4 (clone number: RPA-T4, purchased from BD Pharmingen);

[0068] The anti-CD40 antibody is a fluorescently labeled antibody CD40-PE-Cy7, i.e., PE-cy7 labeled mouse anti-human CD40 (clone number: 5C3, purchased from Biolegend);

[0069] Cell staining buffer (purchased from BD Biosciences);

[0070] Red cell lysing buffer (purchased from BD Biosciences).

[0071] Example 1

[0072] The proportion of Th40 cells in T cells was determined using flow cytometry (FCM).

[0073] (1) Blood samples were collected after obtaining informed consent from the patients. All samples were obtained from fasting intravenous EDTA anticoagulated blood in the morning. A total of 24 peripheral blood samples were collected from SLE patients, of which peripheral blood samples were collected from 15 patients one month after treatment. Samples were also collected from 24 healthy individuals. This part of the study protocol has been approved by the ethics committee of our institution. The clinical data of the SLE patients are shown in Table 1.

[0074] (2) Peripheral blood collected from healthy individuals and SLE patients was lysed using erythrocyte lysis buffer. 2 mL of erythrocyte lysis buffer was prepared for every 200 μL of peripheral blood. Lysis was performed at room temperature for 10 min, with gentle pipetting once during lysis. The cells were then centrifuged at 350 g for 5 min, the supernatant was discarded, and 1×PBS was added to 2 mL. The cells were then washed by centrifugation at 350 g, the supernatant was discarded, and 50 μL of cell staining buffer was added to resuspend the cells in a single-cell suspension.

[0075] (3) Flow cytometry was used to detect the proportion of Th40 cell subsets.

[0076] 3.1 Two flow cytometry tubes are required for each sample. One tube is set as the test tube and the other is set as the same type tube. Each tube contains the single-cell suspension prepared in step (2).

[0077] 3.2 Add 1 μL of the corresponding surface analysis fluorescent antibody to the test tube and the isotype tube respectively, including anti-CD3 antibody and anti-CD4 antibody; add 1 μL of anti-CD40 antibody to the test tube, mix gently, and incubate at room temperature in the dark for 20 min.

[0078] 3.3 Add cell staining buffer and wash cells at 350g speed for 5 minutes.

[0079] 3.4 After centrifugation, the supernatant was removed, and the cells were resuspended in 200 μL of PBS. The data were then analyzed using a flow cytometer (FACSVerse). The raw data were analyzed using FlowJo Software. The analyzed data were then summarized and the median of each group was calculated using SPSS 23.0, and the p-value was calculated.

[0080] Flow cytometry results as follows Figure 1 As shown.

[0081] Association Analysis of Th40 Cell Proportion with Disease Activity and Treatment Efficacy: SLEDAI-2000 scores and laboratory indicators were collected from newly diagnosed SLE patients to analyze the correlation between Th40 cell proportion and disease activity and treatment efficacy. In 24 newly diagnosed SLE patients (Table 2), 15 patients were followed up for one month after treatment. Analysis revealed that the median Th40 cell proportion in these 24 newly diagnosed SLE patients was 13.30%. Figure 2As shown, the proportion of Th40 cells in SLE patients was significantly higher than that in the healthy control group, and the difference was statistically significant (P = 0.000); Figure 3 As shown, patients with higher SLEDAI-2000 scores have a higher proportion of Th40 cells, indicating a positive correlation between SLE disease and the proportion of Th40 cells. Complement C3 is an indicator of SLE disease activity; a decrease in C3 is correlated with SLE disease activity. Figure 4 As shown, the proportion of Th40 cells is negatively correlated with C3; Figure 5 As shown, the proportion of Th40 cells decreased significantly after treatment, and the difference in Th40 cells before and after treatment in the same patient was statistically significant (P = 0.005). The method provided by this invention can detect the proportion of Th40 cells, which can serve as one of the laboratory immune-related indicators for the auxiliary diagnosis of SLE. It also provides important reference data for assessing the disease activity of SLE patients.

[0082] Table 1 Clinical data of SLE patients and healthy controls

[0083]

[0084] Table 2. Th40 cell percentage in newly diagnosed SLE patients

[0085] serial number gender age Hb(g / L) <![CDATA[PLT(10 9 / L)]]> <![CDATA[WBC(10 9 / L)]]> <![CDATA[ANA(10 9 / L)]]> Th40 (%) SLE1 F 23 112 80 2.32 1.38 5.66 SLE2 F 25 100.2 237.6 2.89 1.46 1.62 SLE3 F 25 73 60 1.54 1.12 14.2 SLE4 F 38 106 166 2.93 2.07 2.99 SLE5 F 26 59 163 8.22 5.38 76 SLE6 F 56 79 267 3.73 3.09 23 SLE7 M 22 118 85 2.19 1.6 27.3 SLE8 F 26 115 208 3.01 2 25.2 SLE9 F 33 60.7 243.2 4.28 3.76 34.3 SLE10 F 26 65 146 4.45 3.3 3.93 SLE11 F 27 107.6 266.4 2.7 1.48 6.95 SLE12 F 21 76 172 2.41 1.46 38 SLE13 F 24 98 310 2.92 1.8 12.4 SLE14 F 54 77.6 100.2 3.82 2.83 4.41 SLE15 F 22 67 149 3.71 2.63 34.9 SLE16 F 20 83.1 512.2 18.25 16.62 8.23 SLE17 F 23 112.1 73.6 10.34 7.93 33.1 SLE18 M 31 127.2 89.6 3.89 2.72 15.7 SLE19 F 35 131 255 6.51 3.22 39.8 SLE20 F 24 81.1 269 6.49 4.98 9.47 SLE21 F 22 92 122 3.1 1.87 10.5 SLE22 F 28 91 175 3.27 1.68 6.99 SLE23 M 20 105.4 29.3 0.71 16.21 3.08 SLE24 F 53 79 106 5.4 85.4 27.07

[0086] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. The application of Th40 cell subsets in the preparation of a kit for the auxiliary diagnosis and disease activity assessment of systemic lupus erythematosus, characterized by: The kit includes components, operating procedures, and judgment criteria for detecting Th40 cell subsets; The components used to detect Th40 cell subsets are different fluorescently labeled monoclonal antibodies: anti-CD3 antibody, anti-CD4 antibody, and anti-CD40 antibody; The operating procedure includes the following steps: 1) Process the peripheral blood sample to be tested to form a single-cell suspension; 2) Add monoclonal antibodies labeled with different fluorescence to the single-cell suspension obtained in step 1): anti-CD3 antibody, anti-CD4 antibody, and anti-CD40 antibody, mix gently, and incubate in the dark; 3) After washing the cells, resuspend them in PBS and analyze them using a flow cytometer to obtain data on the fluorescently labeled Th40 cell subsets; The judgment criteria are as follows: when the Th40 cell subset is in CD4 + The median proportion of T cells was 13.30%, indicating a high likelihood of SLE, with the Th40 cell subset accounting for a significant portion of CD4+. + The median proportion of T cells is higher than 13.30%, which is more likely to be severe SLE.

2. The application according to claim 1, characterized in that: The kit also includes at least one of the following: erythrocyte lysis buffer, cell staining buffer, and phosphate buffer solution for lysing peripheral blood erythrocytes.

3. The application according to claim 1 or 2, characterized in that: The fluorescent label of the anti-CD3 antibody is FITC; The fluorescently labeled APC-H7 of the anti-CD4 antibody; The anti-CD40 antibody is fluorescently labeled with PE-cy7.

4. The application according to claim 1 or 2, characterized in that: The specific steps for processing the peripheral blood sample to be tested described in step 1) are as follows: The peripheral blood sample to be tested is subjected to whole blood red blood cell lysis according to conventional methods, centrifuged to remove the supernatant, washed with PBS, and then resuspended with cell staining buffer to obtain a single cell suspension.

5. The application according to claim 1 or 2, characterized in that: The volume of the peripheral blood sample is 200 µL / tube; The volume of the cell staining buffer used is 50 µL / tube; The amount of anti-CD3 antibody added in step 2) is 1 µL of anti-CD3 antibody per 50 µL of single-cell suspension; The amount of anti-CD4 antibody added in step 2) is 1 µL of anti-CD4 antibody per 50 µL of single-cell suspension; The amount of anti-CD40 antibody added in step 2) is 1 µL of anti-CD40 antibody per 50 µL of single-cell suspension; The specific operation of the light-protected incubation described in step 2) is to incubate at room temperature in the dark for 15-30 minutes; The washing described in step 3) is performed using a cell staining buffer; The PBS mentioned in step 3) is PBS with a pH of 7.2-7.4 and a concentration of 0.01-0.1M.

6. The application of a method for detecting Th40 cell subsets for purposes other than disease diagnosis or treatment, characterized by: The method for detecting Th40 cell subsets for non-disease diagnosis or treatment purposes is used to study the mechanism of abnormal T-cell immunity in SLE patients by Th40 cell subsets. Includes the following steps: (1) Process the peripheral blood sample to be tested to form a single-cell suspension; (2) Add monoclonal antibodies labeled with different fluorescence to the single-cell suspension obtained in step (1): anti-CD3 antibody, anti-CD4 antibody, and anti-CD40 antibody. Mix gently and incubate in the dark. (3) After washing the cells, PBS was added to resuspend the cells, and flow cytometry was used to detect the data of the fluorescently labeled Th40 cell subsets.

7. The application according to claim 6, characterized in that: The specific steps for processing the peripheral blood sample to be tested in step (1) are as follows: The peripheral blood sample to be tested is subjected to whole blood red blood cell lysis according to conventional methods, centrifuged to remove the supernatant, washed with PBS, and then 50 μL of cell staining buffer is added to the precipitate obtained by centrifugation to resuspend it and form a single cell suspension. The volume of the peripheral blood sample is 200 µL / tube; The volume of the cell staining buffer used is 50 µL / tube; The amount of anti-CD3 antibody added in step (2) is 1 µL of anti-CD3 antibody per 50 µL of single-cell suspension; The amount of anti-CD4 antibody added in step (2) is 1 µL of anti-CD4 antibody per 50 µL of single-cell suspension; The amount of anti-CD40 antibody added in step (2) is 1µL of anti-CD40 antibody per 50µL of single-cell suspension.

8. The application according to claim 6, characterized in that: The specific operation of the light-protected incubation in step (2) is to incubate at room temperature in the dark for 15 to 30 minutes; The washing described in step (3) is performed using a cell staining buffer; The PBS mentioned in step (3) is PBS with a pH of 7.2 to 7.4 and a concentration of 0.01 to 0.1 M.

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