A method for detecting Breg cells and Tfh cells in thymoma tissue microenvironment

Abstract

The present invention relates to a method for detecting Breg cells and Tfh cells in a thymoma tissue microenvironment, wherein quantum dots are coupled with specific antibodies, and thymoma tissue slice samples are stained. After the specific antibodies are specifically connected to antigens on Breg cells and / or Tfh cells in the slice sample cells, quantum dots are used to quantify or locate Breg cells and / or Tfh cells. The method of the present invention improves the existing multicolor immunofluorescence technology, and uses a method of carrying antibodies with quantum dots, which eliminates the secondary antibody staining link and improves efficiency; by replacing fluorescent dyes with quantum dots, direct labeling of tissue slices can be achieved, and local positioning of Breg cells and Tfh cells in the thymoma tumor microenvironment is achieved, which is convenient for clarifying their biological behaviors; and the immune status can be evaluated by in vitro detecting the ratio of Breg / Tfh cells in the thymoma tumor microenvironment of the patient.

Description

Technical Field

[0001] The invention belongs to the field of immune cell detection, and in particular relates to a method for detecting Breg cells and Tfh cells in a thymoma tissue microenvironment. Background Art

[0002] The thymus is composed of thymocytes and thymic stromal cells. It is an important lymphoid organ of the body, and its function is closely related to immunity. Thymocytes are T cells at different stages of differentiation, and also include some B cells. Thymic stromal cells include thymic epithelial cells, macrophages, dendritic cells and fibroblasts. Therefore, when thymoma occurs, a very complex thymic microenvironment is formed, which is mainly composed of tumor cells and their surrounding immune and inflammatory cells, tumor-associated fibroblasts and nearby interstitial tissues, microvessels, and various cytokines and chemokines. Factors such as the interaction between multiple immune cells and tumor cells in this environment and the release of various cytokines not only affect tumor cells, but are also closely related to central immune-mediated autoimmune diseases. Clarifying the internal structure of the tumor microenvironment and the biological relationship between various immune cells is very important for the precise treatment of thymoma-related autoimmune diseases.

[0003] Autoimmune diseases are immunological disorders caused by multiple factors, often accompanied by abnormalities in the distribution and function of B cell subsets. At present, research on B cells in bone marrow, peripheral blood and peripheral lymph nodes has made some progress, but the expression and mechanism of action of B cell subsets, especially Breg cells, in the thymic microenvironment are still unclear, which has caused great obstacles to the research on autoimmune diseases with central immunity as the core.

[0004] Existing technologies usually use flow cytometry to detect peripheral blood T cells, B cells and their subsets, but CD20+B cells in peripheral blood will only change under extreme immune conditions. Although this can indicate the severity of the disease, it is not applicable to the early stage of the disease. Although confocal microscopy or multi-label immunofluorescence staining can accurately identify a certain type of multi-marker cell subset, traditional staining technology has defects and poor stability, making it difficult to reveal the important role of Breg cells in central immunity during the occurrence of thymus-related autoimmune diseases. Summary of the invention

[0005] In order to solve the above technical problems, the present invention provides a method for detecting Breg cells and Tfh cells in the microenvironment of thymoma tissue.

[0006] The technical solution adopted by the present invention is: a kit for detecting Breg cells and Tfh cells in the microenvironment of thymoma tissue, including quantum dot-labeled CD5 antibody, quantum dot-labeled CD19 antibody, quantum dot-labeled CD1d antibody, quantum dot-labeled CD4 antibody and quantum dot-labeled CXCR5 antibody.

[0007] Preferably, it comprises quantum dot-coupled antibodies QDs-525-CD5, QDs-585-CD19, QDs-625-CD1d, QDs-525-CD4, QDs-625-CXCR5, antigen repair solution and nucleic acid staining solution.

[0008] Preferably, the step of coupling quantum dots to antibody proteins comprises:

[0009] QDs, EDC, and NHS were mixed at a molar ratio of 1:3000:5500 to activate the quantum dots;

[0010] Adding antibodies to the activated quantum dot solution, with the molar ratio of quantum dots to antibody protein being 1:20-80, and obtaining QDs-Ab conjugates after reaction;

[0011] The supernatant was purified using a PD10 column.

[0012] The kit is used to evaluate the status of immune cells in the thymoma microenvironment for non-disease diagnosis and treatment purposes.

[0013] Preferably, the content and / or location of Breg cells and Tfh cells in thymic tissue sections are labeled by quantum dots.

[0014] Preferably, the immune status in the thymoma microenvironment is evaluated by the ratio of Breg / Tfh cells in thymic tissue.

[0015] Preferably, the specific steps include:

[0016] Thymoma tissue specimens were taken to make tissue sections, which were then baked and dewaxed and dehydrated;

[0017] Perform antigen retrieval on tissue sections;

[0018] Staining the tissue section by using the quantum dot and antibody protein conjugate, when there are multiple quantum dots and antibody protein conjugates, staining is performed in sequence;

[0019] After the quantum dot staining is completed, the cell nucleus is stained, the slides are sealed and photographed;

[0020] The proportion of Breg cells in B cells or the proportion of Tfh cells in T cells were calculated respectively.

[0021] Preferably, the dyeing is performed in the order of QDs-525-CD5, QDs-585-CD19, and QDs-625-CD1d, and each quantum dot dyeing step includes dyeing, incubation, and washing processes;

[0022] Alternatively, the staining is performed in the order of QDs-525-CD4 and QDs-625-CXCR5, and each quantum dot staining step includes staining, incubation and washing processes.

[0023] Preferably, AUC is calculated according to the ROC curve to evaluate the superiority of the Breg / Tfh cell ratio in evaluating the immune status.

[0024] Preferably, when the Breg / Tfh cell ratio of thymoma tissue is lower than 0.825, it is determined to be in an immune active state; when the Breg / Tfh cell ratio of thymoma tissue is lower than 0.626, it is determined that the degree of immune hyperactivity is more serious.

[0025] The advantages and positive effects of the present invention are: the existing multi-color immunofluorescence technology is improved, the method of using quantum dots to carry antibodies is used, the secondary antibody staining step is eliminated, direct labeling is adopted, and the advantages of quantum dots themselves are brought into play, which saves experimental time and improves efficiency;

[0026] By replacing fluorescent dyes with quantum dots, tissue sections can be directly labeled, and Breg cells and Tfh cells can be locally located in the thymoma tumor microenvironment. Both the quantity and position of cells can be detected, making it easier to clarify their biological behavior. By detecting the ratio of Breg / Tfh cells in the patient's thymoma tumor microenvironment in vitro, the patient's immune status can be evaluated from the pathogenesis level, overcoming the disadvantages of previous limited evaluation methods. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 .CD19 + CD5 + CDD1d + Expression of Breg cells in the tumor microenvironment of different groups of thymoma. T group: simple thymoma group; T+MG L Group: thymoma combined with mild myasthenia gravis group; T+MG H Group: thymoma combined with severe myasthenia gravis. Blue pseudocolor: CD19; Green pseudocolor: CD1d; Red pseudocolor: CD5;

[0028] Figure 2 :The spatial relationship of CD19, CD5 and CD1d expression in thymoma microenvironment. T group: simple thymoma group; T+MG LGroup: thymoma combined with mild myasthenia gravis group; T+MG H Group: thymoma combined with severe myasthenia gravis group;

[0029] Figure 3 : Expression of T lymphocytes and Tfh cells in the thymoma microenvironment. T group: simple thymoma group; T+MG L Group: thymoma combined with mild myasthenia gravis group; T+MG H Group: thymoma combined with severe myasthenia gravis. Blue pseudocolor: DAPI; Green pseudocolor: CD4; Red pseudocolor: CXCR5;

[0030] Figure 4 : Statistical results of the expression levels of T lymphocytes and B lymphocytes in the thymoma microenvironment. T group: simple thymoma group; T+MG L Group: thymoma combined with mild myasthenia gravis group; T+MG H Group: thymoma combined with severe myasthenia gravis. The differences of each index among the three groups were detected by one-way ANOVA analysis of variance, and the comparison between the two groups was performed at the same time; the results are shown as Mean±SD, * represents P<0.05, ** represents P<0.01, *** represents P<0.001;

[0031] Figure 5 :The expression levels of Breg cells and Tfh cells in different groups. Lymphocyte populations were selected by FSC and SSC, and then marked with CD19 as B lymphocyte population, followed by marking with CD5 and CD1d to determine Breg cells (Figure A); Lymphocyte populations were selected by FSC and SSC, and then marked with CD4 as T lymphocyte population, followed by marking with CXCR5 and IL-21 to determine Tfh cell subsets (Figure B);

[0032] Figure 6 :Statistical results of Breg / Tfh cell ratio in thymoma combined with MG. Statistical analysis of the expression of Breg / Tfh cell ratio in T group, T+MGL group and T+MGH group; Figure A shows the expression level of Breg / Tfh cell ratio in peripheral blood. Figure B shows the expression level of Breg / Tfh cell ratio in thymus tissue. One-way ANOVA analysis of variance was used to detect the difference between the three groups, and pairwise comparisons were performed at the same time; the results are shown as Mean±SD, * represents P<0.05, ** represents P<0.01, *** represents P<0.001;

[0033] Figure 7 : ROC curve of Breg / Tfh cell ratio in T group and TMG group. DETAILED DESCRIPTION

[0034] The embodiments of the present invention are described below with reference to the accompanying drawings.

[0035] The present invention relates to a method for detecting Breg cells and Tfh cells in a thymoma tissue microenvironment, wherein quantum dots are coupled with specific antibodies, and thymoma tissue slice samples are stained. After the specific antibodies are specifically connected to antigens on Breg cells and / or Tfh cells in the slice sample cells, quantum dots are used to quantify or locate Breg cells and / or Tfh cells. This method replaces the antibody detection method at the gene level or in peripheral blood, and directly detects Breg cells and Tfh cells using thymoma tissue slices as the detection object.

[0036] In certain embodiments of the present invention, the specific antibody protein for Breg cells is one or more of CD5, CD19 and CD1d; the specific antibody for Tfh cells is CD4 and / or CXCR5. Quantum dots of different colors are coupled to antibody proteins respectively, and then used to stain tissue sections, so as to achieve the localization or quantification of Breg cells and Tfh cells. Multicolor immunofluorescence technology uses traditional fluorescent dyes, which have certain defects. The emission spectrum wavelength of traditional dyes is limited; the photostability is poor; the spectrum overlaps easily between different fluorescent dyes; the biocompatibility is poor, and it is not easy to carry out specific labeling; the fluorescence lifetime is short, it is difficult to store for a long time, and the background interference is strong. The use of quantum dots instead of fluorescent dyes can avoid the above problems as much as possible.

[0037] The coupling of quantum dots to antibody proteins includes the following steps:

[0038] Step 1: QDs, EDC, and NHS are mixed at a molar ratio of 1: (2500-3500): (5000-6000), preferably 1: 3000: 5500, diluted with PBS, mixed on a shaker at room temperature, and then the pH is adjusted to 8-9 with PBS to obtain an activated quantum dot solution;

[0039] Step 2: Take an appropriate amount of antibody stock solution, dilute it with PBS buffer, and mix it thoroughly; add antibody to the activated quantum dot solution, the molar ratio of quantum dots to antibody protein is 1:20-80 (1:20, 1:40, 1:80 can be used for labeling respectively), shake it at 4 degrees Celsius in the dark, and obtain QDs-Ab conjugate after reaction;

[0040] Step 3: After the reaction is completed, centrifuge at 8000 rpm to remove possible agglomerated precipitates, retain the supernatant, and purify the supernatant using a PD10 column. The purified product is stored in a refrigerator at 4 degrees Celsius away from light for later use.

[0041] By changing the different feed ratios of quantum dots, required cross-linkers and antibodies, stable loading conditions were developed to achieve stable binding of quantum dots and antibodies. The obtained QDs-Ab can be used immediately, and different colors of quantum dots and different antibodies can be combined according to the steps in the instructions to achieve personalized customization.

[0042] An inappropriate ratio of QDs to Ab will result in poor stability of the obtained complex, easy aggregation, significantly affecting its biocompatibility and making it impossible to use it in biological applications, while an excessive amount of cross-linking agent will cause cross-linking between Abs and aggregation of QDs. It is necessary to precisely control the ratio of QDs, Abs and cross-linking agents to obtain a conjugate with high uniformity (i.e., a conjugate with only one Ab molecule bound to the surface of each nanoparticle) and stability.

[0043] In addition, the purification of the bioconjugate after the coupling of quantum dots and antibodies is currently commonly carried out by gel filtration chromatography and membrane ultrafiltration, and the separation and purification rate is low. The method of the present invention uses PD10 column purification, which effectively improves the purification rate and simplifies the purification steps.

[0044] The specific steps of the detection method of Breg cells and Tfh cells are as follows:

[0045] Step 1: Take thymoma tissue specimens to make tissue sections, bake the tissue sections and dewax and dehydrate them;

[0046] Step 2: Perform antigen repair on tissue sections to facilitate the binding of specific antibodies;

[0047] Step 3: staining the tissue section with the quantum dot and antibody protein conjugate. When there are multiple quantum dots and antibody protein conjugates, the staining is performed in sequence. Each quantum dot staining step includes staining, incubation and washing processes. After fully removing the unbound quantum dots and antibody protein conjugates, the next staining step is performed.

[0048] Step 4: After all quantum dots are stained, the cell nuclei are stained, the slides are sealed and photographed, and the location and number of target cells are analyzed based on the photo information.

[0049] Combining quantum dots with the required antibodies makes up for the shortcomings of traditional fluorescent dyes. At the same time, the special combination method makes the quantum dots and antibodies stably combined to achieve complementary advantages. Quantum dot multi-labeling immunofluorescence technology can effectively observe the expression level and spatial position structure of Breg cells and Tfh cells; the 95% confidence interval of the expression level of each cell subpopulation can be calculated with high accuracy. It can judge the status of autoimmune diseases without combining other markers, especially for early and latent autoimmune diseases. The expression level of Breg cells and Tfh cells in the patient's thymoma tumor microenvironment is detected in vitro to evaluate the immune status.

[0050] The "Breg / B%" or "Tfh / T%" value in the thymoma tumor microenvironment is quantitatively associated with the severity of thymoma combined with autoimmune diseases, especially myasthenia gravis. In addition, in certain embodiments of the present invention, the quantity and location information of Breg cells and Tfh cells can be detected simultaneously, and the quantity and location information of Breg cells and Tfh cells can be analyzed.

[0051] Breg is a subpopulation of B cells that play a negative immunoregulatory role. Inflammation associated with infection or autoimmune response may induce the differentiation and stimulation of Bregs. Although Breg accounts for only about 10% of B cells in healthy individuals, it is essential for maintaining immune tolerance and immune homeostasis. TFH cells are located in the thymic germinal center and regulate B cell proliferation and maturation. These cells contact local B cells through ICOS-ICOSL binding to stabilize their phenotype. In addition, TFH cells secrete IL-4 and IL-21 cytokines, which are essential for the function of the germinal center, promote plasma cell differentiation, and guide the type conversion of IgG1B cells to high-affinity IgE.

[0052] The classic clinical diagnosis and evaluation methods for autoimmune diseases mainly rely on the patient's clinical manifestations, serum antibody level detection, etc. Due to the limitations of sensitivity and specificity, the accuracy of the early assessment of various autoimmune diseases including myasthenia gravis is poor, and there is a lack of effective evaluation methods at the pathogenesis level. By analyzing the ratio of Breg / Tfh cells in thymic tissue, the patient's immune status can be evaluated from the pathogenesis level, overcoming the disadvantages of the limited evaluation methods in the past. Counting Breg cells and Tfh cells by quantum dot multi-labeling immunofluorescence technology is more accurate and more conducive to obtaining an accurate ratio of Breg / Tfh cells in thymic tissue.

[0053] The scheme of the present invention is described below in conjunction with the accompanying drawings, wherein the experimental methods without specific operating steps are all carried out in accordance with the corresponding product instructions, and the instruments, reagents, and consumables used in the examples can all be purchased from commercial companies unless otherwise specified.

[0054] Example 1: Preparation of QDs-Ab conjugates

[0055] The quantum dots used in this embodiment are QDs-525, QDs-585, and QDs-625.

[0056] QDs, EDC, and NHS were mixed at a molar ratio of 1:3000:5500, diluted with PBS, and mixed on a shaker at room temperature for 30 minutes, and then the pH was adjusted to 8-9 with PBS (pH 9.18) to obtain quantum dot activation solution. 100ul of quantum dot activation solution was added to the reaction container. An appropriate amount of antibody stock solution was taken, diluted to 0.01mg / ml with PBS buffer, mixed thoroughly, and added to the above reaction container according to different ratios (reference quantum dots: antibody protein molar ratio of 1:20, 1:40, 1:80 for labeling), and reacted on a shaker at 4 degrees Celsius in the dark for 4 hours.

[0057] After the reaction was completed, centrifuge at 8000 rpm for 3 min to remove possible agglomerated precipitates and retain the supernatant.

[0058] 1% agarose gel electrophoresis was used to detect and analyze, and the best conditions were screened by comparison. The supernatant obtained under the best coupling conditions was purified using a PD10 column, and the purified product was stored in a refrigerator at 4°C away from light for later use.

[0059] Example 2: Detection of Breg cells in the thymoma tumor microenvironment

[0060] According to the method of Example 1, quantum dot-coupled antibodies QDs-525-CD5, QDs-585-CD19, and QDs-625-CD1d were prepared.

[0061] The data of simple thymoma (T), thymoma combined with mild myasthenia gravis (TMG) were collected separately. L ), thymoma combined with severe myasthenia gravis (TMG H ) patients, 30 cases in each group, all with no history of other autoimmune diseases or other tumors. After thymectomy, the thymoma tissue specimens were retained and soaked in 4% formalin for more than 48 hours. The thymoma tissue specimens were made into wax blocks and placed on a microtome to make 4μm tissue sections. The source information of the sections, staining index, date, antibody ratio, etc. were recorded before staining.

[0062] Place the tissue slices in a 60°C oven for 30 minutes for baking. Dewax and dehydrate, transfer the slices to No. 1 100% xylene for 15 minutes, No. 2 100% xylene for 15 minutes, No. 3 100% ethanol for 3 minutes, No. 4 100% ethanol for 3 minutes, No. 5 95% ethanol for 3 minutes, No. 6 75% ethanol for 3 minutes, No. 7 50% ethanol for 3 minutes, and rinse with PBS 3 times for 5 minutes each time.

[0063] Antigen repair was performed on the dewaxed and dehydrated tissue sections. During antigen repair, acidic or alkaline antigen repair solution was selected according to the instructions of the corresponding antibody; the antigen repair solution was added to the container, placed in a microwave oven to heat and boil, and then the tissue sections were placed in the container, and heated on medium-low heat for 10 minutes before being taken out; after the tissue sections naturally cooled to room temperature, they were transferred to the immunohistochemistry washing box, washed with sterile water for 1 minute, and washed with 1×TBST for 2 minutes. The tissue was enclosed with an immunohistochemistry pen so that the entire tissue block on the section was located inside the seal.

[0064] First round of dyeing:

[0065] Blocking: Remove 1× TBST solution, slowly add 10% BSA, and incubate at room temperature for 10 minutes.

[0066] Prepare antibodies: dilute the antibodies with 1×PBS to prepare QDs-525-CD5 quantum dot labeled antibody solution.

[0067] Incubation with the first antibody: remove excess serum, add QDs-525-CD5 quantum dot-labeled antibody, place the tissue sections flat in an immunohistochemistry humidified box, and then place in a 4°C refrigerator overnight.

[0068] Rinse: Rinse three times with a wash bottle containing 1×TBST, 2 minutes each time, to remove unbound QDs-525-CD5 quantum dot-labeled antibodies.

[0069] After the first round of staining, the second round of staining was performed;

[0070] Blocking: Remove 1× TBST solution, slowly add 10% BSA, and incubate at room temperature for 10 minutes.

[0071] Prepare antibodies: dilute the antibodies with 1×PBS to prepare QDs-585-CD19 quantum dot-coupled antibody solution.

[0072] Secondary antibody incubation: remove excess serum, add QDs-585-CD19 quantum dot-coupled antibody, place tissue sections flat in an immunohistochemistry humidified box, and then place in a 4°C refrigerator overnight.

[0073] Rinse: Rinse three times with a wash bottle containing 1×TBST, 2 minutes each time, to remove unbound QDs-585-CD19 quantum dot-coupled antibodies.

[0074] Then carry out the third round of dyeing;

[0075] Blocking: Remove 1× TBST solution, slowly add 10% BSA, and incubate at room temperature for 10 minutes.

[0076] Prepare antibodies: dilute the antibodies with 1×PBS to prepare QDs-625-CD1d quantum dot-coupled antibody solution.

[0077] Incubation with the third antibody: remove excess serum, add QDs-625-CD1d quantum dot-coupled antibody, place the tissue sections flat in an immunohistochemistry humidified box, and then place in a 4°C refrigerator overnight.

[0078] Rinse: Rinse three times with a wash bottle containing 1×TBST, 2 minutes each time, to remove unbound QDs-625-CD1d quantum dot-coupled antibodies.

[0079] After the staining, the tissue sections were stained with nuclei, and 1×DAPI solution was prepared with ddH20 1:100; 1×DAPI solution was added to the tissue sections, incubated at room temperature in the dark for 5 minutes, rinsed with 1×TBST for 2 minutes, and rinsed with ddH2O for 1 minute. After removing the excess liquid on the sections, 30μl of anti-fade mounting medium was added, and then the coverslip was covered. After the tissue sections were stable, the results were observed and photographed using a fluorescence microscope equipped with a 488nm laser, where the green light signal represents CD5, the orange light signal represents CD19, and the red light signal represents CD1d.

[0080] The results are as follows Figure 1 , Figure 2 As shown, compared with T group simple thymoma, T+MG L Group and T+MG H The expression levels of Breg cells in the T+MG group were all decreased. H The expression levels in the group were similar to those in T+MG L The expression trend of CD1d was consistent with the expression level of Breg cells (P=0.0213), while the expression level of CD5 was not significantly different among the three groups. The expression level of CD1d in the thymus tissue of MG was reduced, and the area in the same region as CD19 expression was reduced. L Group and T+MG H In the T+MG group, there were areas of CD19 co-expression with CD5 and CD1d in the thymoma microenvironment. H The co-expression area of ​​CD19 and CD1d decreased in the thymoma group, while there was no significant difference in the co-expression area of ​​CD19 and CD5. There was no significant difference in the expression of CD5 among the groups, but the expression level of CD1d was isotropic with Breg cells in the thymoma microenvironment. Compared with CD5, CD1d may play a more important role in Breg formation.

[0081] Example 3: Detection of Tfh cells in the thymoma tumor microenvironment

[0082] According to the method of Example 1, quantum dot-coupled antibodies QDs-525-CD4 and QDs-625-CXCR5 were prepared.

[0083] Thymoma tumor tissue specimens from patients with simple thymoma, thymoma combined with mild myasthenia gravis, and thymoma combined with severe myasthenia gravis were collected, 30 cases in each group, a total of 90 cases (all specimens were homologous to Example 2), and soaked in 4% formalin for more than 48 hours. Wax blocks were made from thymoma tissue specimens and placed on a microtome to make 4 μm tissue sections. Information such as the source of the sections, staining index, date, and antibody ratio were recorded before staining began.

[0084] Place the tissue slices in a 60°C oven for 30 minutes for baking. Dewax and dehydrate, transfer the slices to No. 1 100% xylene for 15 minutes, No. 2 100% xylene for 15 minutes, No. 3 100% ethanol for 3 minutes, No. 4 100% ethanol for 3 minutes, No. 5 95% ethanol for 3 minutes, No. 6 75% ethanol for 3 minutes, No. 7 50% ethanol for 3 minutes, and rinse with PBS 3 times for 5 minutes each time.

[0085] Antigen repair was performed on the dewaxed and dehydrated tissue sections. During antigen repair, acidic or alkaline antigen repair solution was selected according to the instructions of the corresponding antibody; the antigen repair solution was added to the container, placed in a microwave oven to heat and boil, and then the tissue sections were placed in the container, and heated on medium-low heat for 10 minutes before being taken out; after the tissue sections naturally cooled to room temperature, they were transferred to the immunohistochemistry washing box, washed with sterile water for 1 minute, and washed with 1×TBST for 2 minutes. The tissue was enclosed with an immunohistochemistry pen so that the entire tissue block on the section was located inside the seal.

[0086] First round of dyeing:

[0087] Blocking: Remove 1× TBST solution, slowly add 10% BSA, and incubate at room temperature for 10 minutes.

[0088] Prepare antibodies: dilute the antibodies with 1×PBS to prepare QDs-525-CD4 quantum dot labeled antibody solution.

[0089] Incubation with the first antibody: remove excess serum, add QDs-525-CD4 quantum dot labeled antibody, place the tissue sections flat in an immunohistochemistry humidified box, and then place in a 4°C refrigerator overnight.

[0090] Rinse: Rinse three times with a wash bottle containing 1×TBST, 2 minutes each time, to remove unbound QDs-525-CD4 quantum dot-labeled antibodies.

[0091] Second round of dyeing:

[0092] Blocking: Remove 1× TBST solution, slowly add 10% BSA, and incubate at room temperature for 10 minutes.

[0093] Prepare antibodies: dilute the antibodies with 1×PBS to prepare QDs-625-CXCR5 quantum dot-coupled antibody solution.

[0094] Secondary antibody incubation: remove excess serum, add QDs-625-CXCR5 quantum dot-coupled antibody, place tissue sections flat in an immunohistochemistry humidified box, and then place in a 4°C refrigerator overnight.

[0095] Rinse: Rinse three times with a wash bottle containing 1×TBST, 2 minutes each time, to remove unbound QDs-625-CXCR5 quantum dot-coupled antibodies.

[0096] After the staining, the tissue sections were stained with nuclei, and 1×DAPI solution was prepared with ddH20 1:100; 1×DAPI solution was added to the tissue sections, incubated at room temperature in the dark for 5 minutes, rinsed with 1×TBST for 2 minutes, and rinsed with ddH2O for 1 minute. After removing the excess liquid on the sections, 30μl of anti-fade mounting medium was added, and then the coverslip was covered. After the tissue sections were stable, the results were observed and photographed using a fluorescence microscope equipped with a 488nm laser, where the green light signal represents CD4 and the red light signal represents CXCR5.

[0097] The results are as follows Figure 3 As shown in the figure, CD4 and CXCR5 are marked in the tumor microenvironment. CD4 positive represents T lymphocyte subsets, and double positive of the two indicators represents Tfh cells. Compared with T group simple thymoma, Tfh cells in T+MG L Group and T+MG H The expression level of H The group is also higher than T+MG L group (P=0.0108).

[0098] According to the results obtained in Example 2 and Example 3, the CD19+CD5+CDD1d+ Breg cell subsets and the proportion of the total B cells, the proportion of the CD4+CXCR5+ Tfh cell subsets in the total T cells, the percentage of the CD5+ cell subsets, and the percentage of the CDD1d+ cell subsets in the thymoma tumor microenvironment were counted respectively. All data results were statistically calculated using SPSS 22.0. The quantitative data were displayed by calculating the mean ± standard deviation, and the data were tested for normal distribution. For data that conformed to the normal distribution, two groups of data were tested by two independent sample T tests, and multiple groups of data were tested by one-way analysis of variance; non-parametric test methods were used for data that did not conform to the normal distribution. A P value of less than 0.05 indicates that the differences between the groups are statistically significant. The calculation results are shown in Table 1, and the statistical graphs are shown in Figure 4 shown.

[0099] Table 1 Expression levels of B lymphocytes, T lymphocytes and their related indexes in different groups

[0100]

[0101] Note: T group: simple thymoma group; T+MGL group: thymoma combined with mild myasthenia gravis group; T+MGH group: thymoma combined with severe myasthenia gravis group. Data displayed as Mean±SD One-way ANOVA was used to detect the differences among the three groups, and comparisons were made between the two groups.

[0102] Through this example, the spatial distribution of Breg and Tfh cells in the thymoma tumor microenvironment is more comprehensively demonstrated. Breg cells account for a higher proportion in patients with simple thymoma, and the proportion of Tfh cells gradually increases as the condition of thymoma combined with myasthenia gravis worsens. The dynamic balance between Breg and Tfh cells can indicate the severity of thymoma-related myasthenia gravis.

[0103] The results show that based on quantum dot-mounted antibody technology, the secondary antibody staining step is eliminated, and the local positioning of Breg cells and Tfh cells in the thymoma tumor microenvironment can be more conveniently achieved, which can realize both the detection of quantity and the detection of position, and is convenient for long-term preservation.

[0104] Comparative example: Flow cytometry detection of Breg and Tfh cell subsets in peripheral blood of thymoma patients

[0105] The expression of Breg cells and Tfh cells in peripheral blood was detected by flow cytometry.

[0106] Take out the EDTA peripheral venous anticoagulated blood to be tested (all blood samples are from the same source as in Example 2) from the 4°C refrigerator, mix well, take 500μl with a pipette and add it to a 1.5ml EP tube, then add 500μl sterile 1640 cell culture medium and 2μl PMA lymphocyte stimulator, mix well and place in a 37°C water bath for incubation for 5h. Take out the incubated venous blood suspension and divide it into two parts, one part is used to detect Tfh cells (CD4 + CXCR5 + IL-21 + ) expression level, and the other part was used to detect regulatory B cells (CD19 + CD1d + CD5 + IL10 + ) expression level.

[0107] CD4-FITC and CXCR5-PercP antibodies were added to the EP tube for detecting Tfh cells, and CD5-FITC, CD1d-APC and CD19-PercP antibodies were added to the EP tube for detecting Breg cells, and incubated at room temperature in the dark for 20 minutes.

[0108] Prepare 1× red blood cell lysis solution: add 10× red blood cell lysis solution to a 50ml sterile centrifuge tube, add 9 times the volume of ddH2O, and mix to make 1× red blood cell lysis solution. Use a pipette to add 1ml 1× red blood cell lysis solution to each EP tube, mix well, and incubate at room temperature in the dark for 10 minutes. Place in a centrifuge, strictly balance, centrifuge at 2000r for 5 minutes, discard the supernatant, and keep the cell pellet.

[0109] Add 1× punching agent solution to a 50ml sterile centrifuge tube, and then add 3 times the volume of punching agent solution B to obtain 1× punching agent solution. Use a pipette to add 1ml of 1× cell punching agent solution to each EP tube, mix well, incubate at room temperature in the dark for 30 minutes, then place in a low-speed centrifuge, perform strict balancing, centrifuge at 2000r for 5 minutes, discard the supernatant, and keep the cell pellet.

[0110] Prepare 1× buffer solution: add 10× buffer solution to a 50ml sterile centrifuge tube, then add 9 times the volume of ddH2O, mix well to prepare 1× buffer solution. Add 1ml 1× buffer to an EP tube, mix well, incubate at room temperature in the dark for 20min, then place in a centrifuge, perform strict balancing operation, centrifuge at 2000r for 5min, discard the supernatant, and keep the cell pellet.

[0111] Add intracellular antibody IL-10 to the EP tube for detecting Breg cells, and add intracellular antibody IL-21 to the EP tube for detecting Tfh cells, mix well and incubate at room temperature in the dark for 1 hour; add 1 ml 1×PBS to each EP tube, mix well and place in a centrifuge, perform strict balancing operation, centrifuge at 2000r for 5 minutes, discard the supernatant, and keep the cell pellet. Add 200μl 1×PBS and mix well, then transfer to a flow tube for flow cytometry operation and analysis of the results.

[0112] BD CellQuest TM All data were analyzed using SPSS 22.0. The measurement data were displayed by calculating the mean ± standard deviation, and the data were tested for normal distribution. For data that met the normal distribution, two groups of data were tested by two independent sample T tests, and multiple groups of data were tested by one-way analysis of variance. Non-parametric tests were performed for data that did not meet the normal distribution. A P value less than 0.05 indicated that the difference between the groups was statistically significant. The results are shown in Figure 5 , as shown in Table 2. Compared with patients with simple thymoma, as the condition of thymoma-related myasthenia gravis worsens, the proportion of Breg cells in the patient's peripheral blood gradually decreases, while the proportion of Tfh cells gradually increases.

[0113] Table 2. Expression levels of T and B lymphocyte subsets in peripheral blood

[0114]

[0115] Note: Data are shown as Mean±SD One-way ANOVA was used to detect the differences among the three groups, and comparisons were made between the two groups.

[0116] Example 4: Detection of Breg / Tfh Values ​​in Tissues and Peripheral Blood

[0117] Comparing the data of Example 2 and Example 3 with the comparative example, the obtained Breg / Tfh values ​​in the thymoma tumor microenvironment and peripheral blood were statistically analyzed, and the results were obtained: in the peripheral blood, the Breg / Tfh cell ratios in the three groups were statistically significant (P=0.0420); inter-group detection showed that the Breg / Tfh cell ratio in the T group was higher than that in the T+MG group. L Group and T+MG H group, the difference was statistically significant; T+MG L The Breg / Tfh cell ratio in the group was higher than that in T+MG HIn the thymus tissue, the Breg / Tfh cell ratios in the three groups were statistically different (P=0.006); the Breg / Tfh cell ratios in the T group were higher than those in the T+MG group. L Group and T+MG H Group, T+MG L The Breg / Tfh cell ratio in the T+MG group was higher than that in the T+MG group. H The difference was statistically significant ( Figure 6 The expression levels of Breg / Tfh cell ratio in peripheral blood and thymus tissue of patients are shown in Table 3.

[0118] Table 3. Expression level of Breg / Tfh cell ratio in thymoma combined with MG

[0119]

[0120] Note: Data are shown as Mean±SD One-way ANOVA was used to detect the differences among the three groups, and comparisons were made between the two groups.

[0121] According to the receiver operating characteristic (ROC) curve, the area under the curve (AUC) was calculated to evaluate the superiority of the Breg / Tfh cell ratio in assessing immune status, such as Figure 7 As shown. According to the ROC curve, by calculating Youden's J statistic (J = TPR - FPR), the point with the largest J value among all possible thresholds is selected as the optimal cutoff value. This cutoff value represents the best balance point between sensitivity and specificity of the model and is used for classification decisions.

[0122] The T group and TMG group (including TMG L and TMG H ) to find the best diagnostic threshold for classifying samples into the T group or TMG group, and find the point with the largest J value as the best cutoff value. This cutoff value is used to determine whether the sample belongs to the T group; if the data value is higher than this threshold, it is classified into the T group, otherwise it is classified into the TMG group. Based on this, the best cutoff value of the Breg / Tfh cell ratio of thymoma tissue is 0.825, with a sensitivity (TPR): 83.33%, and a specificity (1 - FPR): 98.33%.

[0123] The same calculation method is used to calculate TMG H and TMG L By comparison, the optimal cutoff value of the Breg / Tfh cell ratio in thymoma tissue was 0.626, with a sensitivity (TPR): 80% and a specificity (1 - FPR): 100%.

[0124] The detection method of the present invention is used to determine the positive reference value of the Breg / Tfh cell ratio: when the ratio is lower than 0.825, the patient can be judged to be in an immune active state. Furthermore, when the ratio is lower than 0.626, the patient's immune hyperactivity can be judged to be more serious.

[0125] Example 2, Example 3 and the comparative example all use blood samples and tissue section samples from the same patient, and the tissue sources are consistent. The results show that the tumor microenvironment of patients with thymoma combined with myasthenia gravis is consistent with the trend of Breg and Tfh cell proportions in peripheral blood. Compared with patients with simple thymoma, as the condition of thymoma-related myasthenia gravis worsens, the Tfh cells in the patient's peripheral blood and tumor microenvironment gradually increase, and the Breg cells gradually decrease. In terms of the overall trend, the expression trend of Breg and Tfh cells in peripheral blood and tumor microenvironment is the same. With regard to the analysis of the Breg / Tfh ratio in the same patient's tissue and peripheral blood, the Breg / Tfh ratio decreases in both the tumor microenvironment and peripheral blood as thymoma-related myasthenia gravis worsens, and the trend is consistent.

[0126] Compared with traditional fluorescent staining methods, stained samples of tissue sections can be preserved for a long time, which is better than the blood samples in the comparison example, and also better than the tissue section samples stained with traditional fluorescent staining; and it can obtain clearer and more accurate results, which can indicate subtle differences, and is especially convenient for evaluating the immune status of patients with mild symptoms.

[0127] The embodiments of the present invention are described in detail above, but the contents described are only preferred embodiments of the present invention and cannot be considered to limit the scope of implementation of the present invention. All equivalent changes and improvements made within the scope of the present invention should still fall within the scope of the patent coverage of the present invention.

Claims

1. A kit for detecting Breg cells and Tfh cells in thymoma tissue microenvironment for the purpose of non-disease diagnosis and treatment to evaluate the state of immune cells in thymoma microenvironment, characterized in that: The kit includes quantum dot-labeled CD5 antibody, quantum dot-labeled CD19 antibody, quantum dot-labeled CD1d antibody, quantum dot-labeled CD4 antibody and quantum dot-labeled CXCR5 antibody; The content and / or location of Breg cells and Tfh cells in thymic tissue sections are labeled by quantum dots; the immune status in the thymoma microenvironment is evaluated by the ratio of Breg / Tfh cells in thymic tissue.

2. The use according to claim 1, characterized in that: The kit includes quantum dot-coupled antibodies QDs-525-CD5, QDs-585-CD19, QDs-625-CD1d, QDs-525-CD4, QDs-625-CXCR5, antigen repair solution and nucleic acid staining solution.

3. The use according to claim 1 or 2, characterized in that: Quantum dot-labeled antibodies were prepared by the following method: QDs, EDC, and NHS were mixed at a molar ratio of 1:3000:5500 to activate the quantum dots; Adding antibodies to the activated quantum dot solution, with the molar ratio of quantum dots to antibody protein being 1:20-80, and obtaining QDs-Ab conjugates after reaction; The supernatant was purified using a PD10 column.

4. The use according to claim 1, characterized in that: The specific steps include: Thymoma tissue specimens were taken to make tissue sections, which were then baked and dewaxed and dehydrated; Perform antigen retrieval on tissue sections; Staining the tissue section by using the quantum dot and antibody protein conjugate, when there are multiple quantum dots and antibody protein conjugates, staining is performed in sequence; After the quantum dot staining is completed, the cell nucleus is stained, the slides are sealed and photographed; The proportion of Breg cells in B cells or the proportion of Tfh cells in T cells were calculated respectively.

5. The use according to claim 4, characterized in that: During staining, the staining was performed in the order of QDs-525-CD5, QDs-585-CD19, and QDs-625-CD1d, and each quantum dot staining step included staining, incubation, and washing processes; Alternatively, the staining is performed in the order of QDs-525-CD4 and QDs-625-CXCR5, and each quantum dot staining step includes staining, incubation and washing processes.

6. The use according to claim 1, characterized in that: The AUC was calculated based on the ROC curve to evaluate the superiority of the Breg / Tfh cell ratio in assessing immune status.

7. The use according to claim 6, characterized in that: When the Breg / Tfh cell ratio of thymoma tissue is lower than 0.825, it is judged to be in an immune active state; when the Breg / Tfh cell ratio of thymoma tissue is lower than 0.626, the degree of immune hyperactivity is judged to be more serious.

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