A method for enrichment and detection of antigen-specific t cells

Abstract

The present application relates to the field of immunodetection technology, and particularly relates to a method for enriching and detecting antigen-specific T cells. The method for enriching antigen-specific T cells comprises the following steps: stimulating a whole blood sample with a specific antigen, and then sorting T cells in the whole blood sample stimulated by the antigen by using an immunomagnetic bead sorting technology to obtain a T cell sample. The method is based on the strategy of stimulating the antigen first and then sorting the cells, and can quickly and accurately separate T cells from a complex cell system of whole blood, effectively avoiding the complicated operation of traditional sorting methods, realizing automatic sorting, simplifying the operation steps, shortening the sorting time, improving the sorting throughput, and better ensuring the accuracy of subsequent cell detection. The T cells obtained by the above enrichment method can be detected by using an ELISPOT method, and the antigen-specific T cells can be accurately detected.

Description

Technical Field

[0001] This invention relates to the field of immunoassay technology, and more particularly to a method for enriching and detecting antigen-specific T cells. Background Technology

[0002] Antigen-specific T cell detection is an important means of assessing the body's cellular immune status. Among them, the detection of antigen-specific T cells in whole blood has important value in the diagnosis of infectious diseases, vaccine development, cell therapy, gene therapy, and research on autoimmune diseases.

[0003] However, the current sample pretreatment process for detecting antigen-specific T cells in whole blood is relatively cumbersome and lacks automation. Whole blood samples require complex pretreatment to isolate peripheral blood mononuclear cells (PBMCs), and PBMC isolation usually uses density gradient centrifugation, which involves multiple manual steps such as centrifugation, washing, and cell collection. This process is not only cumbersome, but the experience and skill level of the operators can also affect the repeatability and accuracy of the results.

[0004] Common methods for detecting antigen-specific T cells include enzyme-linked immunospot assay (ELISPOT), flow cytometry, and single-cell sequencing. Among these, ELISPOT offers at least the following advantages: it can detect cytokine-secreting cells at extremely low frequencies, with sensitivity 2-3 orders of magnitude higher than traditional ELISA; it allows direct analysis of cytokine secretion at the single-cell level, with each spot corresponding to a single cytokine-secreting cell; it accurately distinguishes different types of cytokine-secreting cells by capturing cytokines with antibodies, demonstrating high specificity; and it can simultaneously analyze multiple samples, making it suitable for large-scale sample analysis. Therefore, ELISPOT, as a core method for detecting antigen-specific T cells in whole blood, is widely used in vaccine research, infectious disease analysis, autoimmune disease research, and cell therapy efficacy evaluation, providing strong support for immunological research and clinical applications. Existing antigen-specific T cell detection methods involve ELISPOT analysis after isolating PBMCs from whole blood, with specific antigen stimulation occurring during the ELISPOT detection process.

[0005] In summary, there is an urgent need to develop a method for the enrichment and detection of whole blood antigen-specific T cells that can be automated and has high accuracy. Summary of the Invention

[0006] This invention provides a method for enriching and detecting antigen-specific T cells.

[0007] To address the problems of existing whole blood antigen-specific T cell enrichment and detection methods, which require prior PBMC separation, are cumbersome, and have low automation, this invention develops a novel antigen-specific T cell enrichment method. This method directly stimulates whole blood samples with specific antigens and automatically sorts activated T cells from the whole blood using specific immunomagnetic beads (i.e., antigen stimulation followed by sorting). Compared to traditional methods (i.e., PBMC separation followed by antigen stimulation), this enrichment method effectively reduces cumbersome whole blood sample pretreatment steps, lowers operational difficulty, improves detection efficiency, reduces the impact of sorting on cell activation, simplifies the selection of cell types, and does not affect the accuracy of subsequent antigen-specific T cell detection. This provides an efficient and convenient method for antigen-specific T cell detection.

[0008] Specifically, the present invention provides the following technical solutions.

[0009] In a first aspect, the present invention provides a method for enriching antigen-specific T cells, the method comprising: firstly stimulating a whole blood sample with a specific antigen, and then using immunomagnetic bead sorting technology to sort the T cells in the whole blood sample after the antigen stimulation treatment to obtain a T cell sample.

[0010] Preferably, the specific antigen is an antigen that can be specifically recognized by T cells and induce T cell activation.

[0011] Preferably, the specific antigen includes one or more selected from microbial specific antigens and tumor specific antigens.

[0012] The microbial-specific antigens include antigens derived from viruses, bacteria, or fungi, and these antigens can be proteins or peptides, etc.

[0013] Preferably, the specific antigen is a bacterial or viral specific antigen.

[0014] In some embodiments of the present invention, as examples, the specific antigen is Mycobacterium tuberculosis specific antigen, influenza A virus specific antigen, cytomegalovirus specific antigen, or Epstein-Barr virus specific antigen. Using bacterial or viral specific antigens to stimulate whole blood samples, followed by immunomagnetic bead sorting of T cells from the stimulated samples, not only eliminates the need for PBMC isolation, improving operational simplicity and automation, but also ensures the accuracy of subsequent antigen-specific T cell detection, with results highly consistent with traditional methods (PBMC isolation).

[0015] The present invention does not have any particular limitation on the type of specific antigens of bacteria or viruses; any known or novel antigen of the bacteria or virus that can stimulate the production of specific T cells can be used. For example, the Mycobacterium tuberculosis specific antigen includes ESAT6 protein or peptide, CFP10 protein or peptide, or a mixture thereof.

[0016] In the above method, the stimulation treatment is: mixing the specific antigen with the whole blood sample and then incubating it.

[0017] Preferably, the incubation time is at least 2 hours, and more preferably 2-18 hours.

[0018] Preferably, the incubation temperature is 35-38°C.

[0019] In the above method, the sorting involves first using T-cell-specific immunomagnetic beads to bind T cells, and then using magnetic sorting technology to separate the T cells specifically bound to the immunomagnetic beads from the whole blood sample.

[0020] The T-cell specific immunomagnetic beads contain T-cell specific antibodies.

[0021] Preferably, the sorting process includes: mixing the stimulated whole blood sample with a sorting buffer and T-cell-specific immunomagnetic beads, followed by a reaction; the reaction temperature is 15-35℃ (preferably 20-30℃). The reaction time is 5-30 minutes.

[0022] Preferably, the volume ratio of the sorting buffer to the whole blood sample is 1:7-2:1.

[0023] Preferably, the immunomagnetic beads have a particle size of 0.4-5 μm. More preferably, they have a particle size of 0.4-1 μm.

[0024] Cells obtained by the enrichment method described above can be used for subsequent cell analysis.

[0025] In a second aspect, the present invention provides a method for detecting antigen-specific T cells, the method comprising: obtaining a T cell sample using the antigen-specific T cell enrichment method described in the first aspect above; and detecting the T cells or the cytokines they secrete.

[0026] Preferably, the T cells or their secreted cytokines are detected using an enzyme-linked immunospot assay.

[0027] In this method, the enzyme-linked immunospot assay no longer uses antigens to stimulate T cells.

[0028] In antigen-specific T-cell detection, existing technologies mostly employ direct ELISA (enzyme-linked immunospot) assays, where the antigen is directly applied to the antibody-coated reaction wells to stimulate the cell sample before proceeding with the standard ELISA detection steps. The enrichment method provided by this invention directly stimulates the whole blood sample with the antigen, followed by T-cell sorting, eliminating the need for additional antigen stimulation during the ELISA detection process.

[0029] Preferably, the detection of the T cells or their secreted cytokines using an enzyme-linked immunospot assay includes: adding the T cell sample to a reaction plate coated with a specific antibody against the antigen-specific T cells or their secreted cytokines, incubating, washing away the cells, adding labeled detection antibody to the reaction wells, and then adding a chromogenic substance to perform a chromogenic reaction.

[0030] Preferably, the incubation time is 3-18 hours.

[0031] In some embodiments of the present invention, the cytokine is IFN-γ; the enzyme-linked immunospot assay is used to detect IFN-γ secreted by antigen-specific T cells.

[0032] The detection methods described above can be used for purposes other than disease diagnosis and treatment, such as detecting the immunization effect of vaccines during the vaccine development process.

[0033] The beneficial effects of this invention include at least the following: This invention provides a method for enriching antigen-specific T cells. This method is based on a strategy of first stimulating the cells with antigen and then sorting them. It rapidly and accurately separates T cells from the complex cellular system of whole blood samples, effectively avoiding the cumbersome operations of traditional sorting methods. Compared to the traditional density gradient centrifugation method (which takes approximately 70 minutes) for separating PBMCs, the cell sorting step of this invention only takes about 20 minutes. The entire sorting process is highly automated, significantly reducing the difficulty of manual operation, simplifying the operation steps, shortening the sorting time, increasing the sorting throughput, and ensuring the accuracy of subsequent cell detection. The T cells sorted using the above enrichment method can be detected using methods such as ELISPOT, enabling single-cell detection of antigen-specific T cells. It can accurately detect the cytokines secreted by antigen-specific T cells, and through cytokine analysis, antigen-specific T cells can be detected more accurately. Attached Figure Description

[0034] To more clearly illustrate the technical solutions in this invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of this invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0035] Figure 1 The results of ELISPOT detection of Mycobacterium tuberculosis antigen-specific T cells in Example 1 of the present invention are shown, wherein a) is an exemplary result of Method 1 and b) is an exemplary result of Method 2. Detailed Implementation

[0036] In a specific embodiment of the present invention, a method for enriching antigen-specific T cells is provided. The method first stimulates a whole blood sample with a specific antigen, then labels T cells with immunomagnetic beads, and then sorts the immunomagnetic bead-labeled T cells from the whole blood sample using magnetic sorting technology.

[0037] Specifically, the enrichment method includes the following steps:

[0038] (1) After adding specific antigens of bacteria or viruses to whole blood samples, incubate for a period of 2-18 hours.

[0039] (2) After incubation, take out the whole blood sample and add immunomagnetic bead sorting buffer and T cell-specific immunomagnetic beads; wherein, the volume ratio of sorting buffer to whole blood is preferably 1:7-2:1; the particle size range of immunomagnetic beads is preferably 0.4-5μm;

[0040] (3) React the mixed solution from step (2) at room temperature for a preferred time of 5-30 minutes. After the reaction is complete, place the solution in a magnetic field for a preferred time of 1-15 minutes. Then discard the remaining portion and resuspend the magnetic bead-cell complex adsorbed in the magnetic field in cell culture medium or other required buffer solution to obtain a T cell suspension.

[0041] The detection of antigen-specific T cells preferably employs the ELISPOT method, which includes the following steps: The T cell suspension obtained by the enrichment method described above is added to an ELISPOT reaction plate pre-embedded with anti-IFN-γ antibody, and incubated under suitable conditions (during incubation, the pre-embedded anti-IFN-γ antibody in the reaction plate specifically captures IFN-γ secreted by antigen-specific T cells). The incubation time can be 3-18 hours. After incubation, washing, enzyme labeling, and color development are performed sequentially, forming visible spots at the bottom of the reaction plate. The number of these spots corresponds to the number of antigen-specific T cells. Since the number of antigen-specific T cells is closely related to the subject's immune status and bacterial or viral infection, the above detection method can be used for disease diagnosis or treatment purposes, such as assessing the subject's immune status or bacterial or viral infection, and can also be used for non-disease diagnosis and treatment purposes, such as vaccine development.

[0042] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of this invention. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention.

[0043] All reagents used in the following examples are commercially available.

[0044] The commercially available kit used in the following examples is the X.DOT kit, Guangdong Xige Biotechnology, 32 doses / kit; the T-pore stimulant of this kit is a mixture of ESAT6 / CFP10 protein and peptides.

[0045] Example 1: Comparison of Antigen-Specific T Cell Detection Methods

[0046] Five mL of heparin sodium anticoagulated blood positive for Mycobacterium tuberculosis was selected, divided into two equal portions, and enriched and detected using methods one and two, respectively:

[0047] Method 1 includes the following steps:

[0048] Add the T-well stimulant from a commercial kit to heparin sodium anticoagulated blood that is positive for Mycobacterium tuberculosis, mix well, and incubate for 6 hours in a cell culture incubator (5% CO2, 37°C). Add an equal volume of sorting buffer (PBS + 0.5% BSA + 1mM EDTA) to the stimulated whole blood sample, and then add T-cell specific immunomagnetic beads (0.4μm particle size) for sorting.

[0049] The specific sorting method is as follows: 2.5 mL of whole blood sample stimulated with antigen, 2.5 mL of sorting buffer, and 50 μL of immunomagnetic beads are added to a reaction tube and continuously stirred to mix. After reacting at room temperature for 10 minutes, the magnetic rod and magnetic rod sleeve are inserted into the solution for adsorption for 2.5 minutes. Then, the sample is transferred to the sorting buffer, the magnetic rod and magnetic rod sleeve are separated, and the target cells adsorbed on the magnetic rod sleeve are released. After 1 minute, the magnetic rod and magnetic rod sleeve are reconnected to adsorb the target cells in the sorting buffer. The magnetic rod and magnetic rod sleeve are transferred to serum-free culture medium, the magnetic rod and magnetic rod sleeve are separated, and the target cells on the magnetic rod sleeve are released into the serum-free culture medium.

[0050] The T cells obtained from the above sorting were counted, and 100,000 cells / well were added to a 96-well plate of the kit and incubated overnight. Then, after washing, enzyme labeling, and color development, the spots at the bottom of the well plate were observed.

[0051] Method 2 includes the following steps:

[0052] PBMCs were sorted from Mycobacterium tuberculosis-positive heparin sodium anticoagulated blood using density gradient centrifugation (for detailed steps, refer to the instructions of the commercial kit). 100,000 sorted cells per well were added to a 96-well plate of the commercial kit, and T-well stimulant was added for co-incubation for 18-22 hours. After washing, enzyme labeling, and color development, the spots at the bottom of the wells were observed.

[0053] Results Analysis: Enrichment and detection were performed on 14 whole blood samples using both Method 1 and Method 2. The results showed that, assuming P ≥ 20 and T ≥ 5 for a positive result, the positive rates of Method 1 and Method 2 were 100% consistent. Here, P refers to the number of spots in the P wells in the EISPOT analysis, and the P well stimulant is a mixture of PMA (phorbol ethyl ester) and PHA (phytohemagglutinin); T refers to the number of spots in the T wells in the EISPOT analysis, and the T well stimulant is a mixture of ESAT6 / CFP10 protein and peptides.

[0054] Figure 1 These are representative results of T-well (i.e., reaction wells where T-well stimulants are added) spots for Method 1 and Method 2.

[0055] Example 2: Effect of different stimulation times on antigen-specific T cell detection

[0056] Five mL of heparin sodium anticoagulated blood positive for Mycobacterium tuberculosis was divided into four aliquots. One aliquot was added with the N-well stimulant from a commercial kit and incubated in a cell culture incubator (5% CO2, 37°C) for 6 h. The other three aliquots were added with the T-well stimulant and incubated in a cell culture incubator (5% CO2, 37°C) for 2, 6, and 18 h, respectively. An equal volume of sorting buffer (PBS + 0.5% BSA + 1 mM EDTA) was added to the stimulated whole blood, followed by the addition of T-cell specific immunomagnetic beads (0.4 μm in diameter) for sorting (method as in Example 1). The sorted T cells were added to a 96-well plate from the kit and incubated overnight. After washing, enzyme labeling, and color development, the spots at the bottom of the wells were observed.

[0057] Table 1. Results of antigen-specific T cell detection in whole blood at 2, 6, and 18 h after stimulation.

[0058]

[0059] Results Analysis: 2h-T, 6h-T, and 18h-T represent stimulation of whole blood with T-well stimulant for 2, 6, and 18 hours, respectively; 6h-N represents stimulation of whole blood with N-well stimulant for 6 hours. N-well stimulant does not contain components that activate T cells, therefore it generally does not produce spots. T-well stimulant is a Mycobacterium tuberculosis-specific antigen, capable of activating Mycobacterium tuberculosis antigen-specific T cells. As shown in Table 1, no spots were observed after 6 hours of N-well stimulant stimulation, indicating that the stimulation-before-sorting method does not produce non-specific spots; more spots were observed after 6 and 18 hours of T-well stimulant stimulation, indicating that the stimulation-before-sorting method can detect antigen-specific T cells. This also indicates that the antigen stimulation time can be 6-18 hours.

[0060] Example 3: Effect of different stimulation times on antigen-specific T cell detection

[0061] Five mL of Mycobacterium tuberculosis-positive heparin sodium anticoagulated blood (samples from a different source than those in Example 2) was selected and divided into five aliquots. T-well stimulants from a commercially available kit were added, and the aliquots were incubated in a cell culture incubator (5% CO2, 37°C) for 2, 3, 4, 6, and 18 h, respectively. An equal volume of sorting buffer (PBS + 0.5% BSA + 1 mM EDTA) was added to the stimulated whole blood, followed by the addition of T-cell specific immunomagnetic beads (0.4 μm particle size) for sorting (method as in Example 1). The sorted T cells were added to a 96-well plate from a commercially available kit and incubated overnight. After washing, enzyme labeling, and color development, the spots at the bottom of the wells were observed.

[0062] Table 2. Results of antigen-specific T cell detection after stimulating whole blood at 2, 3, 4, 6, and 18 hours.

[0063]

[0064] Results Analysis: 2h-T, 3h-T, 4h-T, 6h-T, and 18h-T represent whole blood stimulation with T-pore stimulant at 2, 3, 4, 6, and 18 hours, respectively. The T-pore stimulant is a Mycobacterium tuberculosis-specific antigen that can activate Mycobacterium tuberculosis antigen-specific T cells. As shown in Table 2, T-pore stimulant stimulation of whole blood at 2, 3, 4, 6, and 18 hours resulted in spots, indicating that the method of stimulation followed by sorting can detect antigen-specific T cells, and the antigen stimulation time can be 2-18 hours.

[0065] Example 4: Effect of different IFN-γ capture times on antigen-specific T cell detection

[0066] Five mL of heparin sodium anticoagulated blood positive for Mycobacterium tuberculosis was selected, and T-well stimulant was added. The mixture was incubated in a cell culture incubator (5% CO2, 37℃) for 6 h. An equal volume of sorting buffer (PBS + 0.5% BSA + 1 mM EDTA) was added to the stimulated whole blood sample, followed by the addition of T-cell specific immunomagnetic beads for sorting. The sorted T cells were divided into five aliquots and added to 96-well plates from a commercially available kit. The aliquots were incubated for 3, 6, 9, 12, and 15 h, respectively. After washing, enzyme labeling, and color development, the spots at the bottom of the wells were observed.

[0067] Table 3. Results of antigen-specific T cell detection at IFN-γ capture times of 3, 6, 9, 12, and 15 h.

[0068]

[0069] Results analysis: The T-pore stimulant is a Mycobacterium tuberculosis-specific antigen that can activate Mycobacterium tuberculosis antigen-specific T cells. As shown in Table 3, spots were observed at IFN-γ capture times of 3, 6, 9, 12, and 15 h, indicating that the pre-stimulation followed by sorting method can detect antigen-specific T cells, and the cytokine capture time can be 3-15 h during detection.

[0070] Example 5: Detection of T cells specific to other pathogen antigens

[0071] The ICE peptide library (CT387) consists of 32 peptides derived from influenza A virus, cytomegalovirus, and Epstein-Barr virus. This peptide library was used as a specific antigen, and antigen-specific T cell enrichment and detection were performed according to Method 1 of Example 1. The specific method is as follows:

[0072] 2.5 mL of heparin sodium anticoagulated blood was selected, ICE was added, and incubated in a cell culture incubator (5% CO2, 37℃) for 6 h. An equal volume of sorting buffer (PBS + 0.5% BSA + 1 mM EDTA) was added to the stimulated whole blood sample, and then T cell-specific immunomagnetic beads (0.4 μm particle size) were added for sorting (method as in Example 1).

[0073] The sorted T cells were resuspended in 200 μL of culture medium and added to a 96-well plate of the X.DOT kit at a rate of 100 μL per well. The cells were incubated overnight, and then the spots at the bottom of the wells were observed after washing, enzyme labeling, and color development.

[0074] Table 4 Results of ICE antigen-specific T cell detection

[0075]

[0076] Results Analysis: The ICE peptide library contains specific antigens for influenza A virus, cytomegalovirus, and Epstein-Barr virus, and can activate T cells specific to these viruses. The number of spots in the 8 samples is shown in Table 4, indicating that Method 1 in Example 1 can detect T cells specific to other pathogen antigens.

[0077] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for enriching antigen-specific T cells, characterized in that, The method includes: first, stimulating whole blood samples with a specific antigen, and then using immunomagnetic bead sorting technology to sort T cells in the whole blood samples after antigen stimulation to obtain T cell samples; The method does not include the step of PBMC separation in whole blood samples; The specific antigen is an antigen that can be specifically recognized by T cells and induce T cell activation, and is selected from one or more of microbial specific antigens and tumor specific antigens; The stimulation treatment involves mixing the specific antigen with the whole blood sample and then incubating it.

2. The method for enriching antigen-specific T cells according to claim 1, characterized in that, The specific antigen is a bacterial or viral specific antigen.

3. The method for enriching antigen-specific T cells according to claim 1, characterized in that, The incubation time is at least 2 hours.

4. The method for enriching antigen-specific T cells according to claim 3, characterized in that, The incubation period is 2-18 hours.

5. The method for enriching antigen-specific T cells according to any one of claims 1, characterized in that, The incubation temperature is 35-38℃.

6. The method for enriching antigen-specific T cells according to any one of claims 1 to 5, characterized in that, The sorting process involves first using T-cell-specific immunomagnetic beads to bind T cells, and then using magnetic sorting technology to separate the T cells specifically bound to the immunomagnetic beads from the whole blood sample.

7. The method for enriching antigen-specific T cells according to claim 6, characterized in that, The T-cell-specific immunomagnetic beads contain T-cell-specific antibodies.

8. The method for enriching antigen-specific T cells according to claim 6, characterized in that, The sorting process includes: mixing stimulated whole blood samples with sorting buffer and T cell-specific immunomagnetic beads and then reacting them. The reaction temperature is 15-35°C, and / or the reaction time is 5-30 minutes.

9. The method for enriching antigen-specific T cells according to claim 8, characterized in that, The volume ratio of the sorting buffer to the whole blood sample is 1:7-2:1; And / or, the particle size of the immunomagnetic beads is 0.4-5 μm.

10. A method for detecting antigen-specific T cells, characterized in that, The method includes: T cell samples were obtained using the enrichment method described in any one of claims 1 to 9; The T cells or their secreted cytokines were detected.

11. The method for detecting antigen-specific T cells according to claim 10, characterized in that, The T cells or their secreted cytokines were detected using an enzyme-linked immunospot assay.

12. The method for detecting antigen-specific T cells according to claim 11, characterized in that, The method for detecting the T cells or their secreted cytokines using enzyme-linked immunospot assay includes: adding the T cell sample to a reaction plate coated with a specific antibody against the antigen-specific T cells or their secreted cytokines, incubating, washing away the cells, adding labeled detection antibody to the reaction wells, and then adding a chromogenic substance to perform a chromogenic reaction.

13. The method for detecting antigen-specific T cells according to claim 12, characterized in that, After adding the T cell sample, the incubation time is 3-18 hours.

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