Method for evaluating immune cell activity

Abstract

The application provides an immune cell activity evaluation method, and belongs to the technical field of immunology. The method carries out pre-stimulation on immune cells by using a cytokine mixed solution containing IL-6 and TNF-α, and then carries out co-culture of the pretreated peripheral blood and a logarithmic growth phase target cell suspension, carries out staining and dark incubation, and then carries out detection by using a flow cytometer to calculate the target cell apoptosis ratio. Meanwhile, the mononuclear cells are separated and induced into immune effector cells and are expanded and cultured, the fluorescently labeled target cells are co-incubated with the immune effector cells, and then the killing rate of the immune effector cells on the target cells is calculated. On this basis, the target cell suspension and the immune cell suspension are mixed and incubated according to the effector-target ratio, PI staining solution is added for incubation, and finally the flow cytometer is used to detect the target cell apoptosis rate. Compared with the prior art, the sensitivity of the application is significantly improved, the results have strong comparability, the detection and evaluation process is simple and efficient, and there is no safety risk.

Description

Technical Field

[0001] This invention relates to the field of immunology, specifically to a method for evaluating the activity of immune cells. Background Technology

[0002] Immune cell activity is a key indicator of the body's immune function, directly reflecting the activity level of the immune system. By detecting immune activity, it's possible to determine whether immune function is normal, whether there is hypoactivity or overactivation, thus assessing the immune status. Based on the results of immune cell activity, doctors can develop personalized treatment plans. For example, immune-enhancing therapy may be considered for those with weakened immune function, while immunosuppression strategies may be adopted for those with overactive immune systems. Furthermore, abnormal immune cell activity is associated with various diseases, including recurrent infections, tumors, and autoimmune diseases (such as systemic lupus erythematosus and rheumatoid arthritis). Regular assessments help detect potential problems early and intervene promptly. During tumor treatment, chronic infections, or immunotherapy, dynamic monitoring of changes in immune cell activity can assess the effectiveness of treatment and allow for timely adjustments to the treatment plan.

[0003] In existing technologies, immune function assays primarily examine the functional status of the immune system, including indicators such as the number and activity of immune cells, immunoglobulin levels, and complement system function. Common tests include lymphocyte subset analysis, immunoglobulin quantification, complement C3 / C4 measurement, and autoantibody screening. Tests targeting immune cell activity mainly include natural killer cell activity assays, lymphocyte transfer assays, and immunoglobulin assays. Natural killer cells, in particular, proliferate in the blood surrounding the site of injury when the body is damaged. They then present immune cells and antigens from the injured site to B cells, leading to B cell activation, division, and ultimately the formation of killer cells. This is a crucial aspect of immunocytology examination. The lymphocyte transfer assay involves culturing specific cells from lymphocyte tissue and allowing them to react specifically to different antigens at different developmental stages. The cells present the antigens to T cells, which then react with macrophages or lymphocytes to clear the antigens—a process known as lymphocyte transfer. This test can determine the development, maturation, and function of lymphocytes, thereby assessing the normality of the body's immune function. Immunoglobulins are a class of substances that possess antibody activity or chemical structure and are similar to antibody molecules. Immunoglobulin assays can detect the levels of immunoglobulins in the body, thereby identifying the presence of diseases such as immunodeficiency or hyperimmune dysfunction.

[0004] In addition, commonly used methods for evaluating immune cell activity include lymphocyte proliferation assays, cytokine level measurement, flow cytometry analysis, cytotoxicity assays, T-cell receptor library sequencing, immunospot assays, and delayed-type hypersensitivity skin tests. Among these, the lymphocyte proliferation assay induces lymphocyte proliferation through stimuli such as PHA, ConA, or specific antigens, and employs... 3 H-TdR incorporation assay and CFSE staining combined with flow cytometry are used to quantify proliferation levels, reflecting the functional status of T / B cells. Cytokine levels are measured using techniques such as ELISA and Luminex multifactor assay to measure the concentrations of cytokines such as IFN-γ, IL-2, and TNF-α, indirectly reflecting the activation status of immune cells. Flow cytometry can simultaneously detect CD4+. + CD8 + The assay measures the proportion of isotropic lymphocyte subsets, as well as viability indicators such as apoptosis, mitochondrial membrane potential, and cell cycle. Cytotoxicity assays are used to evaluate the cytotoxic activity of immune cells and CTLs.

[0005] In summary, current conventional evaluation methods lack sufficient sensitivity. ELISA and flow cytometry have limited ability to detect low-abundance proteins or subclinical immune dysregulation states, making it difficult to capture early or weak immune responses. Furthermore, the comparability of results is generally poor, with significant variations between different laboratories, platforms, or reagent batches. Some new technologies lack unified standards, making cross-study comparisons difficult. Additionally, current evaluation methods are highly dependent on cell viability. For example, with ELISApot, if cell viability is below 90%, the experimental results may be completely invalid; apoptotic or dead cells significantly interfere with spot formation and background signals. Moreover, some methods pose risks of radioactivity and toxicity. 51 Taking the Cr release method as an example, although it has high sensitivity, it involves radioactive materials, is dangerous to operate, and has complex waste disposal, posing significant safety hazards. Summary of the Invention

[0006] This invention aims to address the technical deficiencies of existing technologies by providing a method for evaluating immune cell activity, thereby solving technical problems such as insufficient sensitivity and poor comparability of results in conventional evaluation methods.

[0007] To achieve the above technical objectives, the present invention adopts the following technical solution: A method for evaluating immune cell activity includes the following steps: 1) Peripheral blood was collected and mixed with a cytokine solution to pre-stimulate immune cells; target cells in the logarithmic growth phase were cultured and a suspension was prepared; in a multi-well cell culture plate, the same volume of target cell suspension and pretreated peripheral blood was added to each well of the experimental group, and the same volume of target cell suspension and complete culture medium was added to each well of the control group; the culture plate was placed in an incubator for culture; all liquid in the wells was transferred to EP tubes, the wells were washed with physiological saline, and the samples were combined; PBS was added, the supernatant was discarded by centrifugation, and the cells were resuspended; the same volume of staining solution was added to the experimental group and the control group, and the cells were mixed by pipetting and incubated in the dark; flow cytometry was used for detection; 2) Mononuclear cells were isolated from peripheral blood, induced into immune effector cells, and expanded and cultured. Fluorescently labeled tumor cells were used as target cells, and immune effector cells were co-incubated with target cells. After co-incubation, cell products were collected, and the number of fluorescently labeled positive cells in the co-incubation products was detected using a fluorescent cell counter. The killing rate of target cells was calculated. 3) Tumor cell suspension and immune cell suspension were cultured separately; the tumor cell suspension and immune cell suspension were mixed according to the effector-target ratio to form the experimental group, and the tumor cell suspension was used as the control group. The experimental group and the control group were incubated in an incubator. The cells were transferred to centrifuge tubes and washed with PBS. PI staining solution was added for incubation. The apoptosis rate of tumor cells in the experimental group and the control group was detected by flow cytometry.

[0008] Preferably, the cytokine mixture in step 1) is a mixed stimulating solution of IL-6 and TNF-α; the concentration of both IL-6 and TNF-α stock solutions is 0.1 mg / mL, and the diluent is DMEM complete medium containing 0.1% BSA; the preparation method includes: taking 0.1 μL of IL-6 stock solution and 0.2 μL of TNF-α stock solution, adding them to 999.7 μL of DMEM complete medium containing 0.1% BSA, and mixing well.

[0009] Preferably, the target cells in step 1) are HCT116 cells, and the target cell culture medium is DMEM or RPMI-1640, containing 10% fetal bovine serum, 1% penicillin antibiotics, and non-essential amino acids and growth factors.

[0010] As a preferred method, the resuscitation of target cells includes: removing the cryovial from liquid nitrogen and quickly placing it in a 37°C water bath until thawed; transferring the cell suspension to a centrifuge tube containing pre-warmed culture medium and centrifuging at 1000 rpm for 5 minutes to remove DMSO; resuspending the cells and seeding them in a culture flask, and changing the culture medium after 24 hours to remove dead cells.

[0011] As a preferred method, the passage of target cells includes: when the cell density reaches 80%-90%, passage is performed: discard the old culture medium, wash with PBS; cover the cell layer with a thin layer of 0.25% trypsin-EDTA digestion, incubate at 37°C for 1-3 minutes; add serum-containing culture medium to stop digestion, pipette into a single-cell suspension and then divide into flasks according to the ratio.

[0012] Preferably, in step 2), mononuclear cells are isolated by the following method: peripheral blood anticoagulated with heparin, EDTA, or citrate is collected and diluted with an equal volume of PBS or sterile balanced salt solution; the diluted blood is slowly stacked along the tube wall onto Ficoll layering solution preheated to 18-20°C, maintaining a clear interface, centrifuged, and the white membrane layer is aspirated with a sterile pipette and transferred to a new centrifuge tube; 5-10 times the volume of PBS is added, the cells are resuspended, the supernatant is discarded, and the cells are resuspended in RPMI 1640 containing 10% FBS or PBS.

[0013] Preferably, in step 2), the fluorescently labeled tumor cells are constructed by the following method: using lentivirus as a vector, the luciferase gene is cloned into a eukaryotic expression system, the constructed expression system is introduced into tumor cells in the logarithmic growth phase by liposome transfection or lentivirus infection, puromycin is added for screening, and the process continues for 7 to 14 days until all untransfected cells die.

[0014] Preferably, in step 2), the co-incubation culture system is RPMI 1640 or IMDM medium containing 10% FBS, the ratio of immune effector cells to target cells in co-incubation is 2:1, and the incubation time is 4-6 hours.

[0015] Preferably, in step 3), the tumor cell suspension is prepared by the following method: the tumor cells to be resuscitated are transferred into a constant temperature water bath, the cryovial is shaken, the cell suspension is transferred to RPMI 1640, mixed, centrifuged, the supernatant is discarded, the cells are resuspended in complete culture medium, and added to T-25 cell culture flask at a ratio of 1:5. After mixing, the cells are incubated until the cell density reaches 85%~90%.

[0016] Preferably, the PI staining solution in step 3) is prepared by the following method: take 500 μL of 500 μg / mL RNase A, add 250 μL of 1 mg / mL PI, and add deionized water to 10 mL to make the final concentration: PI 25 μg / mL, RNase A 25 μg / mL.

[0017] This invention provides a method for evaluating immune cell activity. The method first pre-stimulates immune cells with a cytokine mixture containing IL-6 and TNF-α. Then, pre-treated peripheral blood is co-cultured with a suspension of target cells in the logarithmic growth phase. After staining and incubation in the dark, the apoptosis rate of target cells is calculated using flow cytometry. Simultaneously, isolated mononuclear cells are induced into immune effector cells and expanded. Fluorescently labeled target cells are then co-incubated with immune effector cells to calculate the killing rate of target cells by immune effector cells. Based on this, the target cell suspension and immune cell suspension are mixed and incubated according to the effector-target ratio, followed by incubation with PI staining solution. Finally, the apoptosis rate of target cells is detected by flow cytometry. Compared with existing technologies, this invention significantly improves sensitivity, provides highly comparable results, and the detection and evaluation process is simple, efficient, and poses no safety risks. Attached Figure Description

[0018] Figure 1 This is a graph showing the evaluation results of the immune cell killing activity of Example 1 and Comparative Example 1. Detailed Implementation

[0019] The specific embodiments of the present invention will be described in detail below. To avoid excessive and unnecessary detail, well-known structures or functions will not be described in detail in the following embodiments. The approximate language used in the following embodiments is for quantitative purposes, indicating that a certain degree of variation in quantity is permissible without changing the basic function. Unless otherwise defined, the technical and scientific terms used in the following embodiments have the same meaning as commonly understood by those skilled in the art to which this invention pertains.

[0020] Example 1 Peripheral blood was collected and mixed with a cytokine solution to pre-stimulate immune cells. HCT116 cell suspension in logarithmic growth phase was cultured and prepared. In multi-well cell culture plates, the experimental group received the same volume of HCT116 cell suspension and pre-treated peripheral blood per well, while the control group received the same volume of HCT116 cell suspension and complete culture medium per well. The culture plates were placed in an incubator. All liquid in the wells was transferred to EP tubes, and the wells were washed with physiological saline before merging the samples. PBS was added, the supernatant was discarded by centrifugation, and the cells were resuspended. The experimental and control groups received the same volume of staining solution, were mixed by pipetting, and incubated in the dark. Flow cytometry was used for detection. Peripheral blood was collected to isolate mononuclear cells, which were induced into immune effector cells and expanded. Fluorescently labeled tumor cells were used as HCT116 cells, and immune effector cells were co-incubated with HCT116 cells. After co-incubation, cell products were collected, and the number of fluorescently labeled positive cells in the co-incubation products was detected using a fluorescence cell counter. The killing rate of HCT116 cells was calculated. Tumor cell suspensions and immune cell suspensions were cultured separately. The tumor cell suspensions and immune cell suspensions were mixed according to the effector-target ratio to form the experimental group, and the tumor cell suspension was used as the control group. The experimental group and the control group were incubated in an incubator. The cells were transferred to centrifuge tubes, washed with PBS, and then incubated with PI staining solution. The apoptosis rate of tumor cells in the experimental group and the control group was detected by flow cytometry. The calculated killing rate of immune cells on HCT116 cells was 69.52%, and the apoptosis rate of HCT116 cells was 55.41%.

[0021] The cytokine mixture was a mixed stimulating solution of IL-6 and TNF-α; the concentration of both IL-6 and TNF-α stock solutions was 0.1 mg / mL, and the diluent was DMEM complete medium containing 0.1% BSA; the preparation method included: taking 0.1 μL of IL-6 stock solution and 0.2 μL of TNF-α stock solution, adding them to 999.7 μL of DMEM complete medium containing 0.1% BSA, and mixing well.

[0022] HCT116 cells are cultured in DMEM or RPMI-1640, containing 10% fetal bovine serum, 1% penicillin-dextrose antibodies, non-essential amino acids, and growth factors. The resuscitation method for HCT116 cells includes: removing the cryovials from liquid nitrogen and quickly placing them in a 37°C water bath until thawed; transferring the cell suspension to centrifuge tubes containing pre-warmed medium and centrifuging at 1000 rpm for 5 minutes to remove DMSO; resuspending the cells and seeding them in culture flasks, changing the medium after 24 hours to remove dead cells. The passage method for HCT116 cells includes: passage when the cell density reaches 80%-90%; discarding the old medium and washing with PBS; covering the cells with a thin layer of 0.25% trypsin-EDTA digestion and incubating at 37°C for 1-3 minutes; adding serum-containing medium to stop digestion, resuspending the cells into single-cell suspensions, and then aliquoting into flasks according to the specified ratio. Mononuclear cells were isolated using the following method: peripheral blood anticoagulated with heparin, EDTA, or citrate was collected and diluted with an equal volume of PBS or sterile balanced salt solution; the diluted blood was slowly stacked along the tube wall onto Ficoll layering medium preheated to 18-20°C, maintaining a clear interface; centrifuged; the white membrane layer was aspirated with a sterile pipette and transferred to a new centrifuge tube; 5-10 times the volume of PBS was added, the cells were resuspended, the supernatant was discarded, and the cells were resuspended in RPMI 1640 containing 10% FBS or PBS.

[0023] Fluorescently labeled tumor cells were constructed using the following method: The luciferase gene was cloned into a eukaryotic expression system using lentivirus as a vector. The constructed expression system was then transfected into tumor cells in logarithmic growth phase via liposome transfection or lentivirus infection. Puromycin was added for selection, and the process continued for 7–14 days until all untransfected cells died. The co-incubation culture system was RPMI 1640 or IMDM medium containing 10% FBS. The ratio of immune effector cells to HCT116 cells was 2:1, and the incubation time was 4–6 hours.

[0024] The tumor cell suspension was prepared as follows: Tumor cells to be resuscitated were transferred to a constant temperature water bath, the cryovials were shaken, the cell suspension was transferred to RPMI 1640, mixed, centrifuged, and the supernatant was discarded. The cells were resuspended in complete culture medium and added to a T-25 cell culture flask at a ratio of 1:5. After mixing, the cells were incubated statically until the cell density reached 85%–90%. The PI staining solution was prepared as follows: 500 μL of 500 μg / mL RNase A was added, along with 250 μL of 1 mg / mL PI. Deionized water was added to bring the final concentration to 10 mL, resulting in a final concentration of PI 25 μg / mL and RNase A 25 μg / mL.

[0025] Comparative Example 1 Resuscitation and culture of target cells HCT116: Remove the cells to be resuscitated and transfer them to a constant temperature water bath. Shake the cryovial to completely thaw the contents. Open the cryovial cap and transfer the cell suspension to a centrifuge tube containing RPMI 1640 culture medium. Mix well and centrifuge. After centrifugation, discard the supernatant and resuspend the cells in complete culture medium. Count the number of viable cells and transfer them to a T-25 cell culture flask at a ratio of 1:5. Mix well and transfer to a 5% CO2 incubator for static culture. After 48 hours, when the cell density reaches 80%-90%, the cells are ready to be used as target cells.

[0026] Preparation of target cells HCT116: Transfer cells from culture flasks to centrifuge tubes, centrifuge, discard the supernatant after centrifugation, add RPMI 1640 medium to resuspend and adjust the concentration to 1×105 cells / ml.

[0027] Preparation of effector cells: Take out the cultured CIK cells, centrifuge them, discard the supernatant after centrifugation, add CIK cell culture medium, and dilute the concentration to 1×106 cells / ml, 2×106 cells / ml, and 4×106 cells / ml as effector cells of different concentrations.

[0028] Plate formation: Seed cells into well plates, add equal amounts of effector cells and target cells to the experimental wells according to different effector cell and target cell ratios, and set up effector cell control wells and target cell control wells. Place the well plates with added liquid in a 37°C, 5% CO2 incubator for 24 h.

[0029] Killing assay: Remove the plate, add 20 μl of CCK-8 to each well, mix well, and then incubate at 37°C in a 5% CO2 incubator for 4 hours. Afterward, measure the OD value at 450 nm using a microplate reader. Calculate the killing rate of effector cells using the following formula: Killing rate (%) = [1 - (OD value of experimental wells - OD value of effector cell control wells) / OD value of target cell control wells] × 100%. The calculated killing rate of immune cells against HCT116 cells was 68.87%, and the apoptosis rate of HCT116 cells was 55.36%. Therefore, the evaluation results of the killing activity of immune cells in Example 1 and Comparative Example 1 are basically consistent, and the method in Example 1 is accurate and effective.

[0030] The embodiments of the present invention have been described in detail above, but the content described is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the scope of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for evaluating the activity of immune cells, characterized in that, Includes the following steps: 1) Peripheral blood was collected and mixed with a cytokine solution to pre-stimulate immune cells; target cells in the logarithmic growth phase were cultured and a suspension was prepared; in a multi-well cell culture plate, the same volume of target cell suspension and pretreated peripheral blood was added to each well of the experimental group, and the same volume of target cell suspension and complete culture medium was added to each well of the control group; the culture plate was placed in an incubator for culture; all liquid in the wells was transferred to EP tubes, the wells were washed with physiological saline, and the samples were combined; PBS was added, the supernatant was discarded by centrifugation, and the cells were resuspended; the same volume of staining solution was added to the experimental group and the control group, and the cells were mixed by pipetting and incubated in the dark; flow cytometry was used for detection; 2) Mononuclear cells were isolated from peripheral blood, induced into immune effector cells, and expanded and cultured. Fluorescently labeled tumor cells were used as target cells, and immune effector cells were co-incubated with target cells. After co-incubation, cell products were collected, and the number of fluorescently labeled positive cells in the co-incubation products was detected using a fluorescent cell counter. The killing rate of target cells was calculated. 3) Tumor cell suspension and immune cell suspension were cultured separately; the tumor cell suspension and immune cell suspension were mixed according to the effector-target ratio to form the experimental group, and the tumor cell suspension was used as the control group. The experimental group and the control group were incubated in an incubator. The cells were transferred to centrifuge tubes and washed with PBS. PI staining solution was added for incubation. The apoptosis rate of tumor cells in the experimental group and the control group was detected by flow cytometry.

2. The method for evaluating immune cell activity according to claim 1, characterized in that, The cytokine mixture solution mentioned in step 1) is a mixed stimulation solution of IL-6 and TNF-α; the concentration of both IL-6 and TNF-α stock solutions is 0.1 mg / mL, and the diluent is DMEM complete medium containing 0.1% BSA; the preparation method includes: taking 0.1 μL of IL-6 stock solution and 0.2 μL of TNF-α stock solution, adding them to 999.7 μL of DMEM complete medium containing 0.1% BSA, and mixing well.

3. The method for evaluating immune cell activity according to claim 1, characterized in that, The target cells mentioned in step 1) are HCT116 cells, and the target cell culture medium is DMEM or RPMI-1640, containing 10% fetal bovine serum, 1% penicillin antibiotics, and non-essential amino acids and growth factors.

4. The method for evaluating immune cell activity according to claim 3, characterized in that, The resuscitation method for target cells includes: removing the cryovial from liquid nitrogen and quickly placing it in a 37°C water bath until thawed; transferring the cell suspension to a centrifuge tube containing pre-warmed culture medium and centrifuging at 1000 rpm for 5 minutes to remove DMSO; resuspending the cells and seeding them into culture flasks, and changing the culture medium after 24 hours to remove dead cells.

5. The method for evaluating immune cell activity according to claim 4, characterized in that, The method for passage of target cells includes: when the cell density reaches 80%-90%, passage is performed: discard the old culture medium, add PBS to wash; cover the cell layer with a thin layer of 0.25% trypsin-EDTA digestion, incubate at 37°C for 1-3 minutes; add serum-containing culture medium to stop digestion, pipette into a single-cell suspension and then divide into flasks according to the ratio.

6. The method for evaluating immune cell activity according to claim 1, characterized in that, In step 2), mononuclear cells are isolated using the following method: peripheral blood anticoagulated with heparin, EDTA, or citrate is collected and diluted with an equal volume of PBS or sterile balanced salt solution; the diluted blood is slowly stacked along the tube wall onto Ficoll layering medium preheated to 18-20°C, maintaining a clear interface, centrifuged, and the white membrane layer is aspirated with a sterile pipette and transferred to a new centrifuge tube; 5-10 times the volume of PBS is added, the cells are resuspended, the supernatant is discarded, and the cells are resuspended in RPMI 1640 containing 10% FBS or PBS.

7. The method for evaluating immune cell activity according to claim 1, characterized in that, In step 2), the fluorescently labeled tumor cells are constructed by the following method: using lentivirus as a vector, the luciferase gene is cloned into a eukaryotic expression system, the constructed expression system is introduced into tumor cells in the logarithmic growth phase by liposome transfection or lentivirus infection, puromycin is added for screening, and the process continues for 7 to 14 days until all untransfected cells die.

8. The method for evaluating immune cell activity according to claim 1, characterized in that, In step 2), the co-incubation culture system is RPMI 1640 or IMDM medium containing 10% FBS, the ratio of immune effector cells to target cells in co-incubation is 2:1, and the incubation time is 4-6 hours.

9. The method for evaluating immune cell activity according to claim 1, characterized in that, In step 3), the tumor cell suspension is prepared by the following method: the tumor cells to be resuscitated are transferred into a constant temperature water bath, the cryovial is shaken, the cell suspension is transferred to RPMI 1640, mixed, centrifuged, the supernatant is discarded, the cells are resuspended in complete culture medium, and added to T-25 cell culture flask at a ratio of 1:

5. After mixing, the cells are incubated until the cell density reaches 85%~90%.

10. The method for evaluating immune cell activity according to claim 1, characterized in that, In step 3), the PI staining solution is prepared as follows: Take 500 μL of 500 μg / mL RNase A, add 250 μL of 1 mg / mL PI, and add deionized water to 10 mL to make the final concentration: PI 25 μg / mL, RNase A 25 μg / mL.

Images