Application of CD4+CD8 + double-positive T cell in improvement of poor immune reconstruction of HIV infected patient

Abstract

The invention discloses an application of CD4 + CD8 + double-positive T cells in improvement of poor immune reconstitution of HIV (human immunodeficiency virus) infected patients, and relates to the technical field of biological pharmacy, in particular to the application of preparation of the CD4 + CD8 + double-positive T cells in the poor immune reconstitution of the HIV infected patients. The number of peripheral blood CD4 + T cells of HIV infected patients is increased, the immune system of the patients is improved, and the probability of possible opportunity infection and other related diseases of the patients is reduced, so that the morbidity and mortality of the patients are reduced.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biopharmaceutical technology, and particularly relates to CD4 + CD8 + Application of double-positive T cells in improving incomplete immune reconstitution of HIV infected patients. BACKGROUND

[0002] HIV is the main pathogen causing acquired immunodeficiency syndrome (AIDS), which endangers the health of infected persons. HIV virus is mainly divided into two types: HIV-1 type and HIV-2 type. Antiretroviral therapy (ART) can effectively reduce the viral load of HIV infected patients, increase the CD4 + T cell level in patients, and improve the survival rate of infected patients. After ART, the recovery degree of immune cells of different patients is significantly different.

[0003] However, about 20%-40% of patients cannot restore CD4 + T cells to normal level (≥ 500 cells / μL) after 2 years of ART treatment, and such patients are called incomplete immune reconstitution. The probability of infection with pathogenic microorganisms such as Pneumocystis carinii is increased in such patients due to low immunity of the body. Over time, patients can develop severe immune dysfunction and AIDS-related and non-related metabolic syndrome, liver disease, kidney disease, cardiovascular disease, etc., increasing the morbidity and mortality of infected patients. Therefore, incomplete immune reconstitution affects the treatment effect of ART and the prognosis of HIV infection.

[0004] CD4 + CD8 + Double-positive (DP) T cells are a specific stage in the differentiation process of T cells, which are derived from CD4 + helper T cells and CD8 + cytotoxic T cells. CD4 + CD8 + DP T cells (CD4 + CD8 + The function of double-positive T cells is mainly related to cytotoxicity and inhibition of immune response. In the process of acute HIV infection, CD4 + CD8 + DPT cells have higher proliferation activity. CD4 + CD8 +The increase in the number of DP T cells is accompanied by an increase in the expression of cytotoxic markers and a lower secretion of TNF-α, which indicates that CD4 + CD8 + The DP T cells are capable of modulating the immune response after HIV infection, reducing the probability of opportunistic infections and other related diseases in the patient, thus reducing the morbidity and mortality of the patient. SUMMARY

[0005] The present application aims to provide a CD4 + CD8 + The application of the double positive T cells in improving the poor immune reconstruction of HIV infected patients, increasing the number of CD4 + T cells in the peripheral blood of HIV infected patients, improving the immune system of the patient and reducing the probability of opportunistic infections and other related diseases that the patient can have, thus reducing the morbidity and mortality of the patient.

[0006] To solve the above technical problems, the present application adopts the following technical solutions: detecting CD4 + CD8 + The application of the double positive T cells in improving the poor immune reconstruction of HIV infected patients in the peripheral blood sample.

[0007] The further detection steps include: extraction of peripheral blood mononuclear cells, cell recovery, preparation of flow cytometry samples.

[0008] Further, the extraction of peripheral blood mononuclear cells is: peripheral blood mononuclear cells are extracted by the reagent of human peripheral blood lymphocyte separation medium, and trypan blue solution is added to the extracted peripheral blood mononuclear cells. After counting, the cells are resuspended with cell freezing solution and stored in liquid nitrogen.

[0009] Further, the cell freezing solution is 90% newborn calf serum + 10% dimethyl sulfoxide.

[0010] Further, the cell recovery is: the frozen cells are thawed and recovered by water bath, and then centrifuged with phosphate buffered solution.

[0011] Further, the preparation of the flow cytometry sample is: Step 1: centrifuge the recovered cells in a centrifuge tube, and remove the supernatant after centrifugation; Step 2: resuspend the precipitate collected after centrifugation with phosphate buffered solution, and centrifuge at room temperature. Remove the supernatant after centrifugation; Step 3: resuspend the precipitate collected after centrifugation with phosphate buffered solution, add the antibody of the cell viability detection kit, and incubate and stain in the dark; Step 4: centrifuge the dyed PBMC to remove the supernatant, resuspend the precipitate with cell staining buffer, add CD3 + , CD4 + , CD8 + flowing antibodies to incubate in the dark; Step 5: add fresh fixed membrane breaking buffer to the PBMC dyed on the cell surface, resuspend the cell precipitate by gun blowing, and incubate in the dark after fixing; Step 6: centrifuge the fixed PBMC to remove the supernatant, add membrane breaking washing buffer to the precipitated cells, and remove the supernatant after centrifugation; Step 7: add membrane breaking washing buffer and intracellular antibodies of granzyme B, T-box transcription factor 21, and nuclear proliferation antigen to the PBMC sample, vortex, and incubate in the dark; Step 8: wash the incubated sample with membrane breaking washing buffer, centrifuge to remove the supernatant; Step 9: resuspend the washed cells with cell staining buffer, filter, and use for machine detection to analyze cell types.

[0012] Further: in step 4, after the PBMC dyed on the cell surface is completed, resuspend with cell staining solution, wash by blowing, centrifuge to remove the supernatant, then resuspend with cell staining solution, filter, and use for machine detection to obtain the proportion and number of CD3 + T cells, CD4 + T cells, CD8 + T cells, CD4 + CD8 + DP T cells, and CD4 + CD8 + DP T cells expressing granzyme B, T-box transcription factor 21, and nuclear proliferation antigen.

[0013] Further: in step 4, gently tap the tube wall regularly during the incubation period.

[0014] Further: in step 5, vortex regularly during the incubation period.

[0015] Compared with the prior art, the present application has at least one of the following beneficial effects: (1) In the present application, by elucidating the application of CD4 + CD8 + double positive T cells in improving the poor immune reconstruction of HIV infected patients, CD4 + CD8 + DP T cells are prepared to be applied to improve the poor immune reconstruction of HIV infected patients, and to improve and restore the CD4 +T cell level, thus restoring the immune function of the patient and improving the quality of life of the HIV infected patient. BRIEF DESCRIPTION OF DRAWINGS

[0016] Figure 1 CD4 + T cells, CD8 + T cells, CD4 + CD8 + DP T cells and CD4 + CD8 + Flow cytometry gating strategy of CD4 Figure 2 CD4 + T cells, CD8 + T cells, CD4 + CD8 + DP T cells + T cells and the number of cells per ml of peripheral blood; Figure 3 CD4 + CD8 + DP T cells and the number of cells per ml of peripheral blood; Figure 4 CD4 + CD8 + DP T cells and the number of CD4 + T cells per ml of peripheral blood. DETAILED DESCRIPTION

[0017] In order to make the objects, technical solutions and advantages of the present application clearer, the present application will be further described in detail below with reference to the accompanying drawings and examples. It should be understood that the specific examples described herein are only used to explain the present application and are not intended to limit the present application.

[0018] Example 1 I. HIV infected patient enrollment and recruitment The study intends to collect the relevant information and medical history of healthy individuals and HIV infected patients, such as name, gender, nationality, birthplace, age, diagnosis time, ART medication regimen, etc. Based on the laboratory test results (blood routine, blood biochemistry, liver and kidney function, CD3 + T, CD4 + T, CD8 + T cell count) of the patients, healthy individuals (HCs), HIV-1 infected patients with good immune reconstruction (IRs) and patients with poor immune reconstruction (INRs) who meet the conditions for entering the study are screened. All participants need to provide a written informed consent before participating in the study, and the approval of the Clinical Research Ethics Committee of the First Affiliated Hospital of Kunming Medical University is obtained. The research plan of this project has been approved by the hospital ethics committee, and the project is carried out in accordance with the relevant guidelines and regulations.

[0019] 1. Inclusion criteria for enrolled patients: (1) Male and female, aged 30-55 years old; (2) Confirmed HIV infection; (3) ART treatment time at least 2 years or more; (4) No viral load detected for more than 2 consecutive years without viral rebound in between; (5) Patient / relative informed consent to participate in this study; (6) ART treatment regimen is tenofovir disoproxil (TDF) + lamivudine (3TC) + efavirenz (EFV) or zidovudine (AZT) + 3TC + nevirapine (NVP).

[0020] 2. Identification criteria for HIV infected patients with good and poor immune reconstruction: (1) Patients with good immune reconstruction (IRs): peripheral blood CD4 + T cells > 500 / μL; (2) Patients with poor immune reconstruction (INRs): peripheral blood CD4 + T cells < 350 / μL, or CD4 + T cell growth fold (after treatment / before treatment) < 1.2.

[0021] 3. Exclusion criteria for HIV infected patients entering the study: (1) Age below 30 years old and above 55 years old; (2) Currently infected with hepatitis virus, syphilis, sexually transmitted diseases, herpes simplex virus and other pathogens without cure; (3) Infected with tuberculosis; (4) Patients with severe liver, kidney, heart and brain dysfunction, or with severe complications such as hypertension, diabetes, coronary heart disease, etc.; patients with severe acute infection and without control, or with purulent and chronic infection, and patients with delayed wound healing; patients with uncontrolled central nervous metastatic tumor, with obvious intracranial hypertension or neuropsychiatric symptoms; (5) Poor adherence to ART.

[0022] II. Sample collection Peripheral blood samples of HIV-infected patients treated with ART from 2016 to 2023 were collected, including 10 cases of good immune reconstitution and 5 cases of poor immune reconstitution. Peripheral blood samples of each patient were collected in 2 EDTA-2K blood collection tubes, and the total volume of the 2 blood collection tubes was 8 mL. The clinical data and immune index characteristics of HIV-infected patients with good immune reconstitution and poor immune reconstitution are shown in Table 1, wherein IRs (Immunological responders) represent HIV-infected patients with good immune reconstitution, and INRs (Immunological non-responders) represent patients with poor immune reconstitution.

[0023] Table 1. Clinical data and immune index table Note: p <0.05 indicates that the data is statistically significant, represented by *, and p <0.01 indicates that the data is extremely statistically significant, represented by ** III. Extraction of patient peripheral blood PBMC The patient peripheral blood PBMC (peripheral blood mononuclear cells) was extracted using the "human peripheral blood lymphocyte separation medium" reagent of Solarbio Company (Solarbio Company, item number: P8610), and the operation was completed according to the operation manual of the reagent.

[0024] Among them, 90 μL of peripheral blood PBMC was extracted from each patient, 10 μL of 0.04% trypan blue solution was added, and after accurate counting using a cell counter, the cells were resuspended with cell freezing solution (90% newborn calf serum + 10% DMSO (dimethyl sulfoxide)) and stored in liquid nitrogen. Before detection, it was recovered again.

[0025] IV. Cell recovery All frozen cell samples were quickly thawed and recovered using a 37 ℃ water bath, and then the PBMC sample of each patient was added to 10 mL of PBS (phosphate buffered solution) and centrifuged twice for flow cytometry sample preparation.

[0026] V. Preparation of flow cytometry samples Step 1: 1 mL of the resuscitated cells were added to a 1.5 mL centrifuge tube, and the cells were centrifuged at 400 g for 5 minutes, and the supernatant was removed after centrifugation.

[0027] Step 2: The precipitate collected after centrifugation in step 1 was resuspended with 1 mL of phosphate buffer solution (PBS), and centrifuged at 400 g for 5 minutes at room temperature, and the supernatant was removed after centrifugation.

[0028] Step 3: The precipitate collected after centrifugation in step 2 was resuspended with 1 mL of PBS, and then 1 μL of antibody of cell viability detection kit Zombie UV™ Fixable Viability Kit (produced by BioLegend, USA) was added, and incubated at 37 ℃ for 7 minutes in the dark. The dye for detecting cell viability was bound to the resuspended PBMCs by incubation and staining.

[0029] Step 4: After centrifugation at 400 g for 8 minutes, the supernatant was removed, and then the precipitate was resuspended with 100 μL of cell staining buffer Cell Stain Buffer, and then 1 μL of CD3 + (CD3-APC-Cy7, #557757, BD, USA), CD4 + (CD4-FITC, #317408, BioLegend, USA) and CD8 + (CD8-PE-Cy7, #301012, BioLegend, USA) flow antibodies were added, and incubated at 4 ℃ for 40 minutes in the dark (the tube wall was tapped every 10 minutes during incubation).

[0030] At the same time, 1 ×10 6 cells of the PBMC sample after step 4 were detected for the proportion and number of CD3 + T cells, CD4 + T cells, CD8 + T cells, CD4 + CD8 + T cells, and the proportion and number of DP T cells were detected, and the specific steps were as follows: After the PBMCs were stained on the surface of the cells after step 4, 1 mL of cell staining solution Cell Stain Buffer was added for resuspension, and after washing by blowing, the supernatant was removed after centrifugation at 400 g for 8 minutes. Then 200 μL of Cell Stain Buffer was added for resuspension, and after filtration with a 200-mesh nylon mesh, the flow cytometer was used for detection to obtain the percentage and number of cells, and the proportion and number of cells were analyzed by Flowjov10.9.0 software.

[0031] To detect CD4 + CD8 + DP T cells express Granzyme B, T-box transcription factor 21 (T-bet), and nuclear proliferation antigen (Ki-67) positive cell population ratio, PBMC samples after step 4 operation and the rest of the unused cell staining buffer CellStain Buffer washing, follow-up step 5 to step 9 operation.

[0032] Step 5: Add 1 mL of freshly prepared 1x Fix / Perm Buffer to the PBMC in step 4 after cell surface staining, resuspend the cells by gun blowing, and incubate the sample at 4°C in the dark for 45 minutes (vortex for 5 seconds every 10 minutes during incubation).

[0033] Step 6: Centrifuge the PBMC fixed in step 5 at 400g for 8 minutes, remove the supernatant, and add 1 mL of 1x Perm / Wash Buffer to the precipitated cells. Then centrifuge at 400g for 8 minutes again, and remove the supernatant.

[0034] Step 7: Add 80 μL of 1x Perm / Wash Buffer and 1 μL of intracellular antibody of Granzyme B, T-box transcription factor 21 (T-bet), and nuclear proliferation antigen (Ki-67) to the PBMC sample after step 6, vortex the tube gently for 10 seconds, and incubate at 4°C in the dark for 50 minutes (vortex for 5 seconds every 10 minutes during incubation).

[0035] Step 8: Add 1 mL of 1x Perm / Wash Buffer to the sample after step 7 to wash the cells, then centrifuge at 400g for 8 minutes and remove the supernatant.

[0036] Step 9: Resuspend the cells after step 8 washing by adding 200 μL of Cell Stain Buffer, filter through a 200-mesh nylon mesh, and use for flow cytometry to detect CD4 + CD8 + The proportion of DP T cells and the number of cells expressing the above three markers per milliliter of peripheral blood were detected by flow cytometry. The proportion and cell number detection results obtained by flow cytometry were analyzed using Flowjo v10.9.0 software. The main subgroups analyzed included CD4 + T cells, CD8 + T cells, CD4 + CD8 +DP T cells, expressing the cytotoxic molecule CD4 + CD8 + DPT cells (CD4) + CD8 + GranzymeB + CD4, which mediates type I cellular immune responses + CD8 + DP T cells (CD4) + CD8 + T-bet + ), proliferating CD4 + CD8 + DP T cells (CD4) + CD8 + Ki-67 + ).

[0037] VI. Statistical Analysis of Data Statistical analysis was performed using GraphPad Prism version 10.0. Comparisons between groups were statistically tested using the Unpaired T-test. p A value <0.05 indicates statistical significance, indicated by an asterisk (*). p <0.01 indicates that the data is highly statistically significant, denoted by **.

[0038] VII. Correlation Analysis CD4 counts were obtained from peripheral blood of HIV-infected patients with different levels of immune reconstitution. + CD8 + DP T cells, CD4 + CD8 + GranzymeB + DP T cells, CD4 + CD8 + T-bet + DP T cells and CD4 + CD8 + Ki-67 + The number of DP T cells is related to the patient's peripheral blood CD4 count. + Correlation analysis was performed on the number of T cells to analyze peripheral blood CD4 counts in HIV-infected patients with different degrees of immune reconstitution. + CD8 + DP T cells and CD4 cells expressing cytotoxicity, type I cellular immune responses, and proliferative function. + CD8 + The relationship between the number of DPT cells and the degree of immune reconstitution recovery in patients.

[0039] VIII. Results (1) Figure 1 CD4 in HIV-infected patients with different immune reconstitution groups + T cells, CD8 + T cells, CD4 + CD8 + DP T cells and CD4 cells expressing protein markers of cytotoxicity (Granzyme B), type I response (T-bet), and cell proliferation (Ki-67). + CD8 + Flow cytometry gating strategy diagram of DP T cell positive cell population.

[0040] Figure 1 The left-hand image is a flow cytometry quadrature plot. This plot is derived from cell samples obtained after steps 3 and 4 of the flow cytometry sample preparation process. Viable cells were first screened using a cell viability assay kit, and then incubated with CD3+ cells in step 4. + CD4 + CD8 + The detection results obtained from flow cytometry antibody testing, from Figure 1 In the left-hand diagram, CD4 can be... + T cells ( Figure 1 (Left image, bottom right image), CD8 + T cells ( Figure 1 (Top left image in the left image), CD4 + CD8 + DP T cells ( Figure 1 (From the left image to the top right image) The percentage of cells is statistically analyzed. Based on the volume of peripheral blood extracted from each patient and the number of PBMCs isolated from the patient, the number of these cell subpopulations per milliliter of peripheral blood can be calculated.

[0041] To further clarify CD4 + CD8 + The immune function of DP T cells was assessed by treating PBMC samples, followed by fixation, permeabilization, and washing in steps 5 and 6. In step 7, the cells were stained with granzyme B, T-box transcription factor 21 (T-bet), and nuclear proliferation antigen (Ki-67). After washing, the stained cells were analyzed. The two columns on the right of the graph represent CD4+ cells obtained after passing through the quartile gate diagram on the left. + CD8 + Representative flow cytometry plots of GranzymeB, T-bet, and Ki-67 protein or transcription factor expression levels in DP T cells in HIV-infected patients with good and poor immune reconstitution. The rectangles represent the positive cell percentage for each protein or transcription factor, thus characterizing CD4+ expression. + CD8 + DPT cytotoxicity, mediating type I cellular immune responses, and immune functions related to cell proliferation.

[0042] (2) The peripheral blood collected from 10 HIV infected patients with good immune reconstitution and 5 patients with poor immune reconstitution, and after extracting PBMC, the sample preparation steps 1 to 4 of flow cytometry were operated, and the detection results were obtained after the flow cytometry antibody CD3 + , CD4 + , CD8 + staining and machine detection. After each sample detection, according to the four quadrant diagram of the left side of the figure, the proportion of CD4 + T cells, CD8 + T cells, and CD4 + CD8 + DP T cells in CD3 + T cells and the number of cells can be calculated. Figure 1

[0043] As can be seen from the figure, compared with HIV infected patients with good immune reconstitution, the proportion of CD4 + T cells in CD3 + T cells, the number of CD4 + T cells per mL of peripheral blood of patients with poor immune reconstitution were significantly reduced (P<0.05, P<0.01), and the proportion of CD8 + T cells in CD3 + T cells was significantly increased (P<0.05), which reflected the typical state of poor immune reconstitution of patients with poor immune reconstitution; the percentage of CD4 + CD8 + DP T cells in CD4 + T cells and the number of CD4 + CD8 + DP T cells per mL of peripheral blood of patients with poor immune reconstitution were significantly reduced (P<0.05) compared with patients with good immune reconstitution. Figure 2 p <0.05, p <0.01), and the proportion of CD8 + T cells in CD3 + T cells was significantly increased (P<0.05), which reflected the typical state of poor immune reconstitution of patients with poor immune reconstitution; the percentage of CD4 + CD8 + DP T cells in CD4 + T cells and the number of CD4 + CD8 + DP T cells per mL of peripheral blood of patients with poor immune reconstitution were significantly reduced (P<0.05) compared with patients with good immune reconstitution. p <0.05). p

[0044] (3) The peripheral blood collected from 10 HIV infected patients with good immune reconstitution and 5 patients with poor immune reconstitution, and after extracting PBMC, the sample preparation steps 1 to 9 of flow cytometry were operated, and the cell surface was stained by flow cytometry antibodies CD3 + , CD4 + , CD8 + , and then after the fixation, membrane breaking and washing of steps 5 to 6, Granzyme B, T-box transcription factor 21 (T-bet), and nuclear proliferation antigen (Ki-67) were used for staining in step 7, and after the remaining washing process, the stained cells were detected by machine. After each sample detection, according toFigure 1 The two columns on the right present the flow cytometry results, from which CD4 can be calculated. + CD8 + The percentage of DP T cells expressing cytotoxic molecules (Granzyme B), mediating type I immune responses (T-bet), and proliferative markers (Ki-67) positive cells (per CD4+). + CD8 + The proportion of DP T cells, and the CD4+ expression of these three immune function markers. + CD8 + The number of DP T.

[0045] like Figure 3 As shown, although HIV infection patients with good and poor immune reconstitution express cytotoxic molecules (Granzyme B), mediate type I immune responses (T-bet), and proliferative markers (Ki-67) CD4+, they also express these markers. + CD8 + DP T cells (CD4) + CD8 + GranzymeB + DP T cells, CD4 + CD8 + T-bet + DP T cells, CD4 + CD8 + Ki-67 + DP T cells account for a significant portion of CD4+. + CD8 + There was no significant difference in the proportion of DP T cells. p >0.05), but patients with poor immune reconstitution expressed cytotoxic molecules, mediated type I immune responses, and proliferating CD4 per mL of peripheral blood. + CD8 + The number of DP T cells was significantly lower in patients with good immune reconstitution. p <0.05 indicates impaired function of DPT cells in patients with poor immune reconstitution, including cytotoxicity, mediating type I cellular immune responses, and proliferation.

[0046] (4) According to Figure 2 CD4 levels were found in peripheral blood PBMC samples from 10 patients with good immune reconstitution and 5 patients with poor immune reconstitution. + T cell count, and Figure 3 The statistical analysis revealed that CD4+ expresses cytotoxic molecules (Granzyme B), mediates type I immune responses (T-bet), and is a biomarker for proliferation (Ki-67). + CD8 +DP T cell number, correlation coefficient of each patient was calculated, and the correlation graph was made.

[0047] As shown in Figure 4 Figure 2, the CD4 + CD8 + DP T cell, CD4 + CD8 + Granzyme B + DP T cell, CD4 + CD8 + T-bet + DP T cell, and CD4 + CD8 + Ki-67 + The number of DP T cells and the number of CD4 + T cells in individual patients showed a significant positive correlation, and the correlation coefficients were 0.52, 0.38, 0.52, 0.63, p respectively, all of which were less than 0.05, indicating that CD4 + CD8 + DP T cells and their CD4 + CD8 + DP T cells have a close positive correlation with the degree of immune recovery of patients undergoing ART treatment.

[0048] In summary, through correlation analysis, it is found that the cytotoxicity, mediation of type I cellular immune response, and cell proliferation function of the cells have a positive promoting effect on the immune recovery status of HIV-infected patients.

[0049] Although the present application has been described with reference to the explanatory embodiments thereof, it is to be understood that many other modifications and embodiments will be apparent to those skilled in the art, which will fall within the principles and spirit of the present application. More specifically, many variations and modifications will be possible in the components constituting the subject combination layout and / or the layout itself, within the scope of the present application disclosure, drawings and claims. In addition to the variations and modifications of the components and / or the layout, other uses will be apparent to those skilled in the art.

Claims

1. Detect CD4 + CD8 + Application of double-positive T cells in peripheral blood samples to improve poor immune reconstitution in HIV-infected patients.

2. The application according to claim 1, characterized in that: The detection steps include: extraction of peripheral blood mononuclear cells, cell resuscitation, and preparation of flow cytometry samples.

3. The application according to claim 2, characterized in that: The extraction of peripheral blood mononuclear cells is as follows: peripheral blood mononuclear cells are extracted using reagents from human peripheral blood lymphocyte separation fluid. Trypan blue solution is added to the extracted peripheral blood mononuclear cells, and after counting, they are resuspended in cell cryopreservation solution and stored in liquid nitrogen.

4. The application according to claim 3, characterized in that: The cell cryopreservation solution is 90% newborn calf serum + 10% dimethyl sulfoxide.

5. The application according to claim 2, characterized in that: The cell revival process involves thawing and reviving frozen cells in a water bath, followed by centrifugation with the addition of phosphate buffer solution.

6. The application according to claim 2, characterized in that: Preparation of the flow cytometry samples: Step 1: Add the revived cells to a centrifuge tube and centrifuge. After centrifugation, remove the supernatant. Step 2: The precipitate collected after centrifugation is resuspended in phosphate buffer solution and centrifuged at room temperature. The supernatant is discarded after centrifugation. Step 3: After centrifugation, the collected precipitate is resuspended in phosphate buffer solution, and the antibody from the cell viability assay kit is added. The mixture is then incubated in the dark for staining. Step 4: After staining, centrifuge the PBMCs to remove the supernatant, resuspend the pellet in cell staining buffer, and add CD3+. + CD4 + CD8 + Flow cytometry antibody incubation in the dark; Step 5: Add freshly prepared fixation and permeabilization buffer to the stained PBMCs on the cell surface, resuspend the cell pellet by pipetting, and then incubate in the dark for fixation. Step 6: After fixation, centrifuge the PBMCs to remove the supernatant, add cell permeabilization washing buffer to the precipitated cells, centrifuge again, and remove the supernatant. Step 7: Add membrane perforation washing buffer and intracellular antibodies against granzyme B, T-box transcription factor 21, and nuclear proliferation antigen to the PBMC sample, vortex, and incubate in the dark. Step 8: Wash the cells with the incubated sample using cell permeabilization washing buffer, centrifuge, and remove the supernatant. Step 9: After washing, the cells are resuspended in cell staining buffer, filtered, and then used for instrumental analysis to determine cell type.

7. The application according to claim 6, characterized in that: In step 4, after PBMCs are stained on the cell surface, the cell staining solution is added for resuspending. After washing by pipetting, the supernatant is removed by centrifugation, followed by resuspending with cell staining solution, filtration, and then used for instrumental detection to obtain CD3. + T cells, CD4 + T cells, CD8 + T cells, CD4 + CD8 + DP T cells, and CD4 + CD8 + The proportion and number of DP T cells expressing granzyme B, T-box transcription factor 21, and nuclear proliferation antigen.

8. The application according to claim 6, characterized in that: In step 4, the tube wall is gently tapped periodically during the incubation period.

9. The application according to claim 6, characterized in that: In step 5, vortex treatment is performed periodically during the incubation period.

Images