Applicable population screening method and application of glioma dry-like cell sensitized DC vaccine

Abstract

The invention provides an applicable population screening method and application of a glioma dry-like cell sensitized DC vaccine, peripheral blood of a patient before and after inoculation of the glioma dry-like cell sensitized DC vaccine is obtained, mononuclear cells are extracted, CD8 + T cells are respectively separated from the mononuclear cells through a magnetic bead sorting method, and the CD8 + T cells are separated from the mononuclear cells. The method comprises the following steps: uniformly inoculating CD8 < + > T cells into an ELISPOT cell culture plate coated with an IFN-gamma antibody for culture, respectively setting an experimental group and a positive control group for culture, adding A2B5 < + > glioma cell lysis buffer into the experimental group and adding phytohemagglutinin into the positive control group, and carrying out ELISPOT staining on the cultured cells to obtain the secretion amount of FN-gamma before and after treatment of a patient. According to the secretion amount of the FN-gamma, patients with specific immune response to the glioma dry-like cell sensitization DC vaccine are screened, so that the glioma dry-like cell sensitization DC vaccine is conveniently popularized and applied, and the immune mechanism of the glioma dry-like cell sensitization DC vaccine is researched.

Description

Technical Field

[0001] The present invention relates to the field of glioma treatment, and in particular to a method for screening a population suitable for a glioma stem cell-sensitized DC vaccine and its application. Background Art

[0002] Gliomas are the most common primary cancers of the central nervous system, with approximately 50% of patients developing glioblastoma, the most aggressive form of the disease. Conventional therapies, including surgery, radiation, and drug therapy (usually chemotherapy with temozolomide), have not significantly improved survival outcomes for patients with glioblastoma. Reasons for the lack of progress include the aggressive growth of tumors in vital organs, which limits the effectiveness of local treatments; the protection of tumor cells by the blood-brain barrier; the intrinsic resistance of tumor cells to the induction of cell death; and the fact that tumors do not rely on a single, targetable oncogenic pathway, which poses a challenge to systemic therapy.

[0003] In the field of tumor treatment in recent years, immunotherapy has shown remarkable results. Among them, DC vaccines present tumor-specific antigens to naive T cells by contacting circulating T cells in lymph nodes. After being activated and proliferated, these T cells will be recruited to the tumor area and have a specific killing effect on glioma cells. At present, clinical trials of DC vaccines for gliomas have achieved great success and have shown more reliable characteristics in terms of treatment effectiveness and safety.

[0004] However, each person's immune system status, tumor characteristics, and genetic background are different, which leads to different glioma patients having different responses to glioma DC vaccines. Unlike radiation and chemotherapy, DC vaccines usually require a longer time window to take effect, and local induced immune responses are difficult to distinguish from tumor progression in imaging, which usually leads to early termination of treatment. Therefore, it will be of great significance if the body's response to DC vaccines can be monitored in real time in the clinic, and the treatment plan can be adjusted more reasonably according to the response of the systemic immune response. This will not only help doctors better understand and utilize immunotherapy, but also provide more personalized and effective treatment plans for glioma patients.

[0005] In general, in order to more accurately evaluate the effect of DC vaccines in the treatment of glioma patients and develop individualized and precise treatment strategies for each patient, it is particularly important to detect the tumor-specific systemic immune response induced by DC vaccines. This can not only provide direct evidence for the successful activation of systemic immunity by DC vaccines, but also quantitatively analyze the degree of specific immune response of the body system to tumors. This method can help medical staff to more deeply understand the mechanism of action of DC vaccines, optimize the preparation process of DC vaccines, and thus produce more effective glioma treatment vaccines, which will help to achieve greater breakthroughs in the field of glioma treatment. Summary of the invention

[0006] The purpose of the present invention is to provide a method for screening the applicable population of glioma stem cell-sensitized DC vaccine and its application, by detecting tumor-specific T cells in peripheral blood by enzyme-linked immunosorbent assay to screen the applicable population for which the glioma stem cell-sensitized DC vaccine can produce an immune effect.

[0007] To achieve the above objectives, the present technical solution provides a method for screening the applicable population for glioma stem cell-sensitized DC vaccine, comprising the following steps:

[0008] S1: Obtain peripheral blood from the patient before vaccination with glioma stem cell-sensitized DC vaccine and extract monocytes as comparison cells; obtain peripheral blood from the same patient after vaccination with glioma stem cell-sensitized DC vaccine and extract monocytes as detection cells;

[0009] S2: Separate CD8 from comparison cells and detection cells by magnetic bead sorting + T cells;

[0010] S3: Compare the CD8 + T cells were evenly inoculated into ELISPOT cell culture plates coated with IFN-γ antibodies to obtain control samples. CD8 + T cells were uniformly inoculated into ELISPOT cell culture plates coated with IFN-γ antibodies to obtain test samples;

[0011] S4: Set up an experimental group and a positive control group for the reference sample and the test sample respectively, and add A2B5 to the experimental group + Glioma cell lysate and the addition of phytohemagglutinin to the positive control group, the experimental group of control samples and test samples and the positive control group were cultured under the same conditions;

[0012] S5: Perform ELISPOT staining on the control sample, the experimental group of the test sample and the positive control group respectively. If the secretion of IFN-γ in the experimental group of the test sample is increased compared with the secretion of IFN-γ in the experimental group of the control sample, the current patient belongs to the applicable population.

[0013] The research and development ideas of this application team are as follows:

[0014] After the body is vaccinated with DC vaccines, DC cells carrying tumor-specific antigen epitopes will present antigens to T cells in peripheral lymphoid organs such as lymph nodes, thereby stimulating the activation and clonal proliferation of tumor-specific T cells and allowing them to enter the circulatory system. Therefore, if the expansion of tumor-specific T cells can be detected in the body's peripheral blood, it can be considered as direct evidence that the DC vaccine successfully drives the body's immune system to fight tumors. In addition, some T cells activated and proliferated by DC cells will be transformed into memory T cells. These memory T cells in the circulatory system and tissues play an important role in immune surveillance. When they encounter the same antigen again, they will be immediately activated and clonally proliferate without the need for antigen-presenting cells to present the antigen again. So how to detect the expansion of tumor-specific T cells in the body's peripheral blood? The application team considered using enzyme-linked immunosorbent assay technology for in vitro detection. ELISPOT detection technology is a method that combines cell culture technology and ELISA technology. It can detect cells that secrete antibodies or cytokines in situ. This is an important cellular immunology detection technology. In recent years, ELISPOT technology has been widely used in the analysis and evaluation of peptide-specific T lymphocytes detected from peripheral blood mononuclear cells (PBMC) of patients with infectious diseases, as well as vaccine trials that induce tumor-specific T cells in cancer patients. In terms of the immunogenicity detection of neoantigens in cancer patients, the field of T cell immune function detection reported in the literature usually uses the ELISPOT detection method. This method usually uses in vitro synthesized tumor-specific full-sequence short peptides as peptide libraries and loads them into antigen-presenting cells, namely dendritic cells (DC). Then, the peptide-stimulated dendritic cells are mixed and incubated with T lymphocytes to detect whether the antigenic peptide can specifically activate the immune response. This method mainly detects the specific immune activation effect of the peptide. The application team further studied how to use ELISPOT detection technology to achieve A2B5 + The specific immune activation effect of dendritic cell vaccine sensitized by glioma stem cell lysate is studied. Based on this research idea, this technical solution is proposed.

[0015] This technical solution is used to evaluate the CD8 +The amount of IFN-γ secreted by T cells after being stimulated with A2B5+ tumor cell lysate was detected to screen the population suitable for vaccination with glioma stem cell-sensitized DC vaccine based on the changes in the amount of IFN-γ secretion.

[0016] Specifically, step S1 of this solution includes the following steps:

[0017] Blood samples were obtained from patients before and after vaccination with glioma stem cell-sensitized DC vaccine. PBMC cells were extracted from the peripheral blood of patients before and after vaccination with glioma stem cell-sensitized DC vaccine, respectively. PBMC cells were inoculated in cell culture medium, cultured with complete culture medium, and A2B5 + Glioma cell lysate was stimulated in the presence of rhIL-7, and rhIL-7 was added on the 3rd day and the medium was replaced semi-quantitatively as needed. Cytokines and antigens were supplemented and cultured for 10-21 days to obtain the comparison cells in the peripheral blood before the inoculation of glioma stem-like cell-sensitized DC vaccine and the detection cells in the peripheral blood after the inoculation of glioma stem-like cell-sensitized DC vaccine.

[0018] The steps for extracting PBMC cells from peripheral blood are the same, including the following steps:

[0019] Add the blood sample to the heparin anticoagulation blood collection tube, mix well and transfer to a new centrifuge tube, add an equal volume of normal saline to the new centrifuge tube for dilution and transfer to a new centrifuge tube, add Ficoll-Histopaque separation solution to the new centrifuge tube and centrifuge, take the middle white layer to a new centrifuge tube, add normal saline to the new centrifuge tube for dilution and centrifuge, discard the supernatant after centrifugation and add normal saline to dilute, centrifuge the diluted cells at low speed, discard the supernatant after centrifugation and take the lower layer as PBMC cells.

[0020] The reason why this protocol chooses to extract peripheral blood mononuclear cells (PBMCs) is that PBMCs include a variety of immune cells, such as lymphocytes (T cells, B cells, etc.) and monocytes, so as to facilitate subsequent research on whether the DC vaccine activates the patient's immune system.

[0021] Regarding step S2 of this scheme:

[0022] The cell suspension of the control cells and the cell suspension of the test cells were centrifuged and the supernatant was discarded. Buffer was added to resuspend the cells. CD8 magnetic beads were added to the resuspended control cells and test cells for incubation. After a period of incubation, the cells were centrifuged and the supernatant was discarded. Buffer was added to resuspend the cells. The resuspended control cells and test cells were added to the column for washing and CD8 magnetic beads labeled CD8 were collected. + T cells.

[0023] During the immune response, CD8 + T cells can recognize specific antigen peptide-MHC class I molecule complexes on the surface of cells infected by pathogens or undergoing malignant transformation, and induce tumor cell apoptosis by releasing cytotoxic substances such as perforin and granzyme, thereby exerting a specific killing effect on tumor cells. + The activity and functional status of T cells can directly reflect the effect of specific anti-tumor immune response induced by DC vaccines. + T cells can differentiate into memory CD8 + These memory T cells can exist in the body for a long time. When encountering the same antigen again, they can quickly activate, proliferate and exert immune effects, providing the body with rapid and effective immune protection. + Changes in T cell responses, including memory CD8 + The formation and expansion of T cells can evaluate the immune memory ability induced by the vaccine, which is of great significance for the long-term prevention of tumor recurrence.

[0024] Regarding step S3 of this scheme:

[0025] CD8 + T cells were evenly inoculated into ELISPOT cell culture plates coated with IFN-γ antibodies to obtain control samples. CD8 + T cells were uniformly inoculated into ELISPOT cell culture plates coated with IFN-γ antibodies to obtain test samples. The culture conditions of the reference samples and the test samples were the same, both of which were cultured at 37° C. and 5% CO 2 for 24 hours.

[0026] In some embodiments, ethanol is added to each well of the ELISPOT cell culture plate for pre-wetting, and then washed with deionized water, and then diluted IFN-γ coated antibody is added to each well for coating, and then blocking solution is added to each well for blocking to obtain an ELISPOT cell culture plate coated with IFN-γ antibody, and the CD8 +T cells were evenly inoculated into ELISPOT cell culture plates coated with IFN-γ antibodies to obtain control samples. CD8 + T cells were uniformly inoculated into ELISPOT cell culture plates coated with IFN-γ antibodies to obtain test samples.

[0027] IFN-γ (interferon-γ) is an important cytokine produced by activated T cells and NK cells during the immune response. By coating IFN-γ antibodies, IFN-γ secreted by CD8+T cells during cell culture can be specifically captured, which enables the subsequent accurate detection and quantification of the level of IFN-γ secreted by CD8+T cells after antigen stimulation (such as dendritic cell vaccine stimulation sensitized by A2B5+ glioma cell lysate), thereby reflecting the functional state and immune activation degree of CD8+T cells. IFN-γ plays an important role in immune regulation, antiviral, anti-tumor and other immune processes. In tumor immunotherapy, changes in its secretion level can be used as one of the important indicators for evaluating the intensity and effect of the body's anti-tumor immune response. The detection of a higher level of IFN-γ secretion usually indicates that CD8+T cells are effectively activated and are playing the role of immune surveillance and killing tumor cells, thereby indirectly reflecting the effectiveness of the specific immune response induced by DC vaccines.

[0028] Regarding step S4 of this protocol:

[0029] The experimental groups set in this protocol are used to homogenize CD8 + T cell activity, experimental group used to add A2B5 + In some embodiments, lectin is added to the positive control group at a concentration of 4 ug / mL.

[0030] In some embodiments, the control sample, the experimental group of the test sample, and the positive control group are placed in a carbon dioxide incubator at 37° C. and 5% CO 2 for 24 hours.

[0031] Regarding step S5 of this scheme:

[0032] The control sample and the experimental group and the positive control group of the test sample were ELISPOT stained, and the ELISPOT plate spots were counted to obtain the number of IFN-γ spots. The secretion amount of IFN-γ in the experimental group of the test sample and the secretion amount of IFN-γ in the experimental group of the control sample were obtained according to the number of IFN-γ spots.

[0033] In some embodiments, the cells of the experimental group and the positive control group of the control sample and the test sample are subjected to the following operations to achieve ELISPOT staining: the cells and the culture medium in each well of the ELISPOT culture medium are poured out, the cells are lysed by the hypotonic method, ice-cold deionized water is added, and the ELISPOT plate is placed on ice in an ice bath, each well is washed multiple times with PBST, and the ELISPPOT plate is turned upside down on absorbent paper to dry, biotin-labeled detection antibodies are added to each well for incubation, each well is washed with PBST, the plastic protective layer on the back of the PVDF membrane plate is removed, and the front and back sides of the membrane are washed and covered with a protective layer. , turn the plate upside down on absorbent paper and pat dry, add enzyme-labeled streptavidin to each well for incubation, wash each well with PBST, remove the plastic protective layer on the back of the PVDF membrane plate, wash both sides of the membrane and cover with the protective layer, turn the plate upside down on absorbent paper and pat dry, add AEC color development solution to each well and let it stand, wait for the spots to grow to a suitable size, wash with PBST to terminate the color development process, turn the ELISPPOT plate upside down on absorbent paper, pat dry the tiny water droplets, then remove the protective layer, place it in a ventilated place, let the membrane stand at room temperature to dry naturally, and place the ELISPPOT plate in the ELISPOT automatic reader for counting.

[0034] In some embodiments, if the increase in the secretion of IFN-γ in the experimental group of the test sample is greater than or equal to 1.5 times compared to the secretion of IFN-γ in the experimental group of the control sample, the current patient belongs to the applicable population.

[0035] In order to ensure the accuracy of the experiment, this protocol tested the test samples and control samples in three test groups, each of which included an experimental group and a positive control group, and at least three replicate wells were set in each test group to take the average value of the number of IFN-γ spots in different replicate wells in each test group.

[0036] In some embodiments, the FN-γ spots of the positive control group in each test group are calculated to see whether there is a statistical difference. If there is no statistical difference, the IFN-γ secretion amount in the experimental group of the test sample is continued to be analyzed compared with the IFN-γ secretion amount in the experimental group of the control sample. If there is a statistical difference, the analysis is terminated.

[0037] In some embodiments, A2B5 in step S5 + Glioma cell lysate is A2B5 isolated from tumor tissue collected during surgery. + Glioma cells were obtained after irradiation and repeated freezing and thawing.

[0038] Secondly, this scheme provides an application method for the method for screening the applicable population of the above-mentioned glioma stem cell-sensitized DC vaccine, which can screen patients who produce specific immune responses to the glioma stem cell-sensitized DC vaccine for immunotherapy with the glioma stem cell-sensitized DC vaccine.

[0039] Compared with the prior art, this technical solution has the following characteristics and beneficial effects:

[0040] 1. Accurately screen the applicable population: By testing the amount of IFN-γ secreted by CD8+T cells in the peripheral blood of patients before and after vaccination with glioma stem cell-sensitized DC vaccine after specific antigen stimulation, the immune response of individual patients to the vaccine can be accurately judged, thereby screening out the population suitable for this immunotherapy. This helps to personalize glioma immunotherapy, avoid ineffective treatment for unsuitable patients, and provide more accurate and effective treatment plans for suitable patients, improve the success rate and effectiveness of treatment. In clinical practice, medical resources can be more reasonably allocated to patients who may benefit from glioma stem cell-sensitized DC vaccine treatment, reduce unnecessary medical waste, and improve the utilization efficiency of medical resources.

[0041] 2. Dynamic monitoring of immune response: This program can not only screen the applicable population, but also make longitudinal comparisons of the same patient before and after vaccination, as well as horizontal comparisons between different patients, which helps to gain a deeper understanding of the dynamic process of DC vaccines inducing specific immune responses in vivo, including CD8 + Changes in various stages such as T cell activation, proliferation, and memory cell formation, as well as the relationship between these changes and individual patient characteristics (such as immune system status, tumor characteristics, genetic background, etc.), provide powerful tools and data support for further research on the mechanism of glioma immunotherapy. Through the detection and analysis of a large number of patients, factors that affect the immune effect of DC vaccines can be discovered, thereby providing a basis for the improvement and optimization of vaccines. For example, if it is found that certain patient groups do not respond well to the vaccine, the reasons can be further studied, and the vaccine preparation method, antigen selection or immune adjuvant can be adjusted in a targeted manner to improve the overall efficacy of the vaccine.

[0042] 3. Effectively evaluate the therapeutic effect: IFN-γ secretion is an important detection index, and its changes are closely related to the intensity of the specific immune response induced by DC vaccines, which directly affects the therapeutic effect of glioma. Therefore, this program establishes an effective connection from peripheral blood testing to immune response evaluation and then to prediction of therapeutic effect, providing a simple and reliable method for clinicians to timely evaluate the efficacy of vaccines during treatment. In the early stage of treatment, by detecting the changing trend of IFN-γ secretion, the patient's possible response to vaccine treatment can be predicted, which helps doctors adjust treatment strategies in advance, such as increasing the vaccine dose, combining other treatment methods or strengthening the patient's immune support, thereby improving the overall effect of treatment and the patient's prognosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] Figure 1 This is a T1-weighted MRI follow-up image change diagram of the patient during treatment in Patient Application Example 1.

[0044] Figure 2 This is the ELISPOT test result of IFN-γ in peripheral blood PBMC of the patient during the treatment of the patient application example 1.

[0045] Figure 3 This is the quantitative analysis result diagram of IFN-γ ELISPOT test in patient application example 1, where Figure 3 A is the curve of the change of IFN-γ spot number in the peripheral blood PBMC of the patient during the treatment period. Figure 3 B is the immune response degree change curve. Figure 3 C is a curve chart showing the correlation between the degree of immune response and tumor.

[0046] Figure 4 This is a T1-weighted MRI follow-up image change diagram of the patient during treatment in patient application example 2.

[0047] Figure 5 This is the ELISPOT test result of IFN-γ in peripheral blood PBMC of the patient during the treatment of the patient application example 2.

[0048] Figure 6 This is the quantitative analysis result diagram of IFN-γ ELISPOT test in patient application example 2, where Figure 6 A is the curve of the change of IFN-γ spot number in the peripheral blood PBMC of the patient during the treatment period. Figure 6 B is the immune response degree change curve. Figure 6 C is a curve chart showing the correlation between the degree of immune response and tumor. DETAILED DESCRIPTION

[0049] The following will be combined with the accompanying drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field belong to the scope of protection of the present invention.

[0050] Embodiment 1

[0051] The specific operation examples of the method for screening the applicable population of the glioma stem cell-sensitized DC vaccine provided in this scheme are as follows:

[0052] Step 1: Before and after each treatment with glioma stem cell-sensitized DC vaccine, collect the patient's peripheral blood and extract mononuclear cells (PBMC):

[0053] 1.1. Collect blood samples from patients treated with glioma stem cell-sensitized DC vaccine before and after each treatment into heparin anticoagulation blood collection tubes and mix well;

[0054] 1.2. Transfer the blood sample to a new centrifuge tube and dilute it with an equal volume of saline;

[0055] 1.3. Prepare a new centrifuge tube, add Ficoll-Histopaque separation solution, and tilt the centrifuge tube to 30°;

[0056] 1.4. Density gradient centrifuge the sample prepared in step 1.3 at 2500r / min for 30min, separate it into three layers after centrifugation, aspirate the middle white layer into a new centrifuge tube; dilute with physiological saline, centrifuge at 1500r / min for 10min, discard the supernatant, dilute with physiological saline, and count the cells;

[0057] 1.5. Centrifuge the cells in step 1.4 at low speed at 1200 r / min for 10 min, discard the supernatant, and the lower layer is the PBMC cells;

[0058] 1.6. PBMCs were plated at 5 × 10 6 The cells were evenly plated in a 24-well cell culture plate at a density of 100 cells / well and cultured with complete medium. A2B5+glioma cell lysate (antigen) was used in the presence of rhIL-7 (20

[0059] ng / ml), and on the 3rd day, rhIL-2 (20ng / ml) was added, and semi-quantitative medium was changed as needed, and cytokines and antigens were supplemented at the same time, and cultured for 10-21 days, wherein the monocytes of the peripheral blood of the patients before the vaccination with glioma stem-like cell-sensitized DC vaccine were defined as the comparison cells, and the monocytes extracted from the peripheral blood of the patients after the vaccination with glioma stem-like cell-sensitized DC vaccine were defined as the detection cells.

[0060] Step 2: Isolate CD8 from collected peripheral blood mononuclear cells (control cells and test cells) by magnetic bead sorting + T cells.

[0061] 2.1. Centrifuge the stimulated cell suspension and discard the supernatant. Resuspend the cell pellet and 7 Add 80 μL of buffer to each cell;

[0062] 2.2. Every 10 7 Add 20 μL CD8 magnetic beads to each cell. Mix thoroughly and incubate at 4-8°C for 15 minutes. Wash the cells with 1-2 mL buffer, centrifuge for 10 minutes, and completely remove the supernatant. Resuspend the cells with 500 μL buffer;

[0063] 2.3. Add all the cell suspension to the column and wash the column 3 times with buffer; remove the column from the sorter, add buffer to wash, and immediately use the plunger to push out and collect the cells labeled with magnetic beads.

[0064] And perform cell counting.

[0065] Step 3: In the ELISPOT experiment, the CD8 + T cells were evenly seeded into the experimental culture plate, and then all samples were cultured at 37°C and 5% CO2 for 24 hours.

[0066] 3.1. Pre-wet each well of the ELISPOT cell culture plate with 15 μL of 70% ethanol for 30 seconds, and then wash three times with 100 μL of deionized water to minimize the residual ethanol.

[0067] 3.2. Add 50 μL of diluted coating antibody (IFN-γ) to each well and coat overnight at 4°C. Pour out the coating solution the next day and wash 5 times with PBS. For the last wash, dry on sterile absorbent paper.

[0068] 3.3. Add 200 μL of blocking solution to each well and block at 37°C for 1 hour;

[0069] 3.4. Pour off the blocking solution and, without washing, transfer the CD8 + T cells were evenly plated in a cell culture plate and then cultured at 37°C and 5% CO2 for 24 hours.

[0070] Step 4: Set up three test groups for each sample, each test group includes an experimental group and a positive control group. The positive control group is used to normalize the CD8 + T cell activity, A2B5 was added to the experimental group +Glioma cell lysate (5ug / ml) and positive control group were added at a concentration of 4ug / mL Phytohemagglutinin (Phytohemagglutinin, PHA), cultured in a carbon dioxide incubator at 37°C and 5% CO2 for 24 h;

[0071] Step: 5: Stain the ELISPOT in step 8 and subsequently count the spots on the ELISPOT plate.

[0072] 5.1. Pour the cells and culture medium in the wells, lyse the cells by hypotonic method, add 200 μL of ice-cold deionized water (PBST) to each well, and place the plate on ice for 10 minutes;

[0073] 5.2. Wash each well 10 times with 200 μl PBST. After the last wash, turn the plate upside down on absorbent paper and pat dry.

[0074] 5.3. Add 100 μL of biotin-labeled detection antibody to each well and incubate at 37°C for 1 hour;

[0075] 5.4. Wash each well 5 times with 200 μL PBST. During the last wash, remove the plastic protective layer on the back of the PVDF membrane plate, wash both sides of the membrane at the same time, cover with the protective layer, and turn the plate upside down on absorbent paper and pat dry;

[0076] 5.5. Add 100 μL of enzyme-labeled streptavidin to each well and incubate at 37°C for 1 hour;

[0077] 5.6. Wash each well 5 times with 200 μl PBST. During the last wash, remove the plastic protective layer on the back of the PVDF membrane plate, wash both sides of the membrane at the same time, cover with the protective layer, and turn the plate upside down on absorbent paper and pat dry;

[0078] 5.7. Add 100 μL of AEC colorimetric solution to each well and let stand at room temperature for 20-30 minutes, keeping away from light;

[0079] 5.8. After the spots grow to a suitable size, wash twice with PBST to terminate the color development process. Turn the plate upside down on absorbent paper, pat dry the tiny water droplets, then remove the protective layer, place it in a ventilated place, and let it stand at room temperature for 10-30 minutes to let the membrane dry naturally;

[0080] 5.9. Place the ELISPOT plate in the ELISPOT automatic reader, adjust the appropriate parameters, record the various parameters of the spots, and perform statistical analysis.

[0081] Patient application example 1:

[0082] This protocol uses the actual glioma patients in Huashan Hospital for glioma stem cell-sensitized DC vaccine immunotherapy. The patients underwent T1-weighted enhanced MRI scans before each glioma stem cell-sensitized DC vaccine immunotherapy to dynamically monitor the tumor changes of the glioma. The tumor changes of the patients were obtained as follows: Figure 1 At the same time, before and after receiving the treatment of glioma stem cell-sensitized DC vaccine, the patient's peripheral blood was collected and tested according to the method of Example 1, and the IFN-γ ELISPOT test results of the patient's peripheral blood PBMC during the treatment were obtained as shown in Figure 2 As shown in FIG. 1 , the number of IFN-γ spots in the IFN-γ ELISPOT test results of peripheral blood PBMC after each treatment is shown in FIG. Figure 3 As shown in A, the degree of immune response of patients after each treatment is as follows Figure 3 As shown in B, the correlation between the degree of immune response and the tumor volume of glioma tumors is as follows Figure 3 As shown in C, the quantitative analysis results showed that with multiple injections of DC vaccine, the number of CD8 + The number of T cells gradually increased, the patient's immune response gradually increased, and the tumor volume gradually shrank, eventually disappearing completely and remaining stable for a long time without progression. Further correlation analysis showed that the degree of immunotherapy response in peripheral blood was significantly negatively correlated with tumor volume.

[0083] In this protocol, a value greater than 1.5 compared with the baseline value of ELISPOT test results is defined as a specific immune response. Figure 3 As can be seen in B, in the third course of treatment, a specific anti-tumor immune response was detected in the patient's body. Correspondingly, MRI follow-up showed that the tumor volume began to shrink, further supporting the anti-tumor immune response induced by the DC vaccine. In summary, quantitative analysis of specific anti-tumor T cells in peripheral blood by IFN-γ ELISPOT can be used to dynamically monitor the patient's immune response to DC vaccine treatment, providing an important reference for the evaluation of the efficacy of immunotherapy.

[0084] Patient application example 2:

[0085] This scheme still uses the actual glioma patients in Huashan Hospital for glioma stem cell-sensitized DC vaccine immunotherapy. In this example, the patients were subjected to T1-weighted enhanced MRI scans before each DC vaccine treatment to dynamically monitor tumor changes. The tumor change diagram is shown in FIG. Figure 4 As shown, the patient's tumor volume is increasing.

[0086] At the same time, before and after each course of DC vaccine treatment, peripheral blood of the patient was collected for testing according to the method of Example 1 to obtain the IFN-γ ELISPOT test results of peripheral blood PBMC of the patient during the treatment period. Figure 5 As shown, the Figure 5 Used to evaluate patients' immune response to DC vaccines.

[0087] The number of IFN-γ spots in the IFN-γ ELISPOT test results of peripheral blood PBMC after each treatment is as follows: Figure 6 As shown in A, from Figure 6 The quantitative analysis results of A show that with multiple injections of DC vaccine, the number of CD8+T cells secreting IFN-γ in the patient's peripheral blood did not continue to increase. The application team defined the increase of more than 1.5 times in the ELISPOT test results compared with the baseline value as a specific immune response. It is worth noting that Figure 6 As shown in Figure B, a specific anti-tumor immune response was detected in the patient in the third course of treatment. However, the response failed to maintain in the subsequent courses. In the fourth and fifth courses, the immune response level even dropped below the baseline, indicating that the patient's specific immune response to the DC vaccine was poor. Correspondingly, Figure 4 As shown, follow-up MRI showed that the tumor volume continued to increase, as shown in Figure 6 Further correlation analysis shown in C showed that the response degree of immunotherapy in peripheral blood was significantly negatively correlated with tumor volume.

[0088] In summary, this Example 2 reversely illustrates that the method for screening the applicable population of the glioma stem cell-sensitized DC vaccine proposed in this scheme can screen out patients who are specifically immune to the DC vaccine. This scheme can predict the efficacy of the DC vaccine. The quantitative analysis of specific anti-tumor T cells in the peripheral blood by IFN-γ ELISPOT can be used to dynamically monitor the patient's immune response to DC vaccine treatment. The level of antigen-specific T cells in the peripheral blood is continuously greater than 1.5 compared with the baseline, which is an important prerequisite for the DC vaccine to work, which provides a key reference for the efficacy evaluation of immunotherapy.

[0089] The above embodiments only express several implementation methods of the present application, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the present application. It should be pointed out that, for a person of ordinary skill in the art, several variations and improvements can be made without departing from the concept of the present application, and these all belong to the protection scope of the present application. Therefore, the protection scope of the present application shall be subject to the attached claims.

Claims

1. A method for screening the population suitable for glioma stem cell-sensitized DC vaccine, characterized in that: The following steps are involved: S1: Obtain peripheral blood from the patient before vaccination with glioma stem cell-sensitized DC vaccine and extract monocytes as comparison cells; obtain peripheral blood from the same patient after vaccination with glioma stem cell-sensitized DC vaccine and extract monocytes as detection cells; S2: Separate CD8 from control cells and test cells by magnetic bead sorting + T cells; S3: Compare the CD8 + T cells were evenly inoculated into ELISPOT cell culture plates coated with IFN-γ antibodies to obtain control samples. CD8 + T cells were uniformly inoculated into ELISPOT cell culture plates coated with IFN-γ antibodies to obtain test samples; S4: Set up an experimental group and a positive control group for the reference sample and the test sample respectively, and add A2B5 to the experimental group + Glioma cell lysate and the addition of phytohemagglutinin to the positive control group, the experimental group of control samples and test samples and the positive control group were cultured under the same conditions; S5: Perform ELISPOT staining on the experimental group and positive control group of the control sample and the test sample respectively. If the secretion of IFN-γ in the experimental group of the test sample is increased compared with the secretion of IFN-γ in the experimental group of the control sample, the current patient belongs to the applicable population.

2. The method for screening the applicable population for the glioma stem cell-sensitized DC vaccine according to claim 1, characterized in that: In step S5, ELISPOT plate spot counting is performed to obtain the number of IFN-γ spots, and the secretion amount of IFN-γ in the experimental group of the test sample and the secretion amount of IFN-γ in the experimental group of the control sample are obtained according to the number of IFN-γ spots.

3. The method for screening the applicable population for the glioma stem cell-sensitized DC vaccine according to claim 1, characterized in that: If the increase in the secretion amount of IFN-γ in the experimental group of the test sample is greater than or equal to 1.5 times the secretion amount of IFN-γ in the experimental group of the control sample, the current patient belongs to the applicable population.

4. The method for screening the applicable population for the glioma stem cell-sensitized DC vaccine according to claim 1, characterized in that: A2B5 in step S5 + Glioma cell lysate is A2B5 isolated from tumor tissue collected during surgery. + Glioma cells were obtained after irradiation and repeated freezing and thawing.

5. The method for screening the applicable population for the glioma stem cell-sensitized DC vaccine according to claim 1, characterized in that: Calculate whether there is a statistical difference in the FN-γ spots of the positive control group in each test group. If there is no statistical difference, continue to analyze the IFN-γ secretion in the experimental group of the test sample compared with the IFN-γ secretion in the experimental group of the control sample. If there is a statistical difference, terminate the analysis.

6. The method for screening the applicable population for the glioma stem cell-sensitized DC vaccine according to claim 1, characterized in that: In step S1, blood samples of the patient before and after the glioma stem cell-sensitized DC vaccine are obtained, and PBMC cells are extracted from the peripheral blood of the patient before and after the glioma stem cell-sensitized DC vaccine is inoculated, and the PBMC cells are inoculated in a cell culture medium, cultured in a complete culture medium, and A2B5 is added. + Glioma cell lysate was stimulated in the presence of rhIL-7, and rhIL-7 was added on the 3rd day and the medium was replaced semi-quantitatively as needed. Cytokines and antigens were supplemented and cultured for 10-21 days to obtain the comparison cells in the peripheral blood before the inoculation of glioma stem-like cell-sensitized DC vaccine and the detection cells in the peripheral blood after the inoculation of glioma stem-like cell-sensitized DC vaccine.

7. The method for screening the applicable population for the glioma stem cell-sensitized DC vaccine according to claim 1, characterized in that: In step S2, the cell suspension of the comparison cells and the cell suspension of the test cells are centrifuged and the supernatant is discarded, a buffer is added to resuspend the cells, CD8 magnetic beads are added to the resuspended comparison cells and test cells for incubation, centrifuged after a period of incubation and the supernatant is discarded, a buffer is added to resuspend the cells, the resuspended comparison cells and test cells are added to the column for washing and CD8 magnetic beads labeled CD8 are collected. + T cells.

8. The method for screening the applicable population for the glioma stem cell-sensitized DC vaccine according to claim 1, characterized in that: In step S3, the culture conditions of the reference sample and the test sample are the same, that is, they are cultured at 37° C. and 5% CO 2 for 24 hours.

9. The method for screening the applicable population for the glioma stem cell-sensitized DC vaccine according to claim 1, characterized in that: In step S4, phytohemagglutinin was added to the positive control group at a concentration of 4 ug / mL, and the control sample, the experimental group of the test sample and the positive control group were placed in a carbon dioxide incubator at 37° C. and 5% CO 2 for 24 h.

10. An application method of a method for screening a population suitable for a glioma stem cell-sensitized DC vaccine, characterized in that: It is used to screen patients who have a specific immune response to glioma stem cell-sensitized DC vaccine in order to carry out immunotherapy with glioma stem cell-sensitized DC vaccine.

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