Detection method for activation of specific T cells after tuberculosis infection and application thereof

Abstract

The invention relates to a method for detecting activation of specific T cells after tuberculosis infection and application of the method. Specifically, the invention provides a reagent combination for detection, and the reagent combination comprises an anti-CD4 antibody (CD4 antibody), an anti-CD154 antibody (CD154 antibody), an IFN-gamma detection antibody and a tuberculosis specific antigen. According to the method, through peripheral whole blood collection, PBMC cell separation, T cell activation (activation), activation termination (blocking), antibody labeling, membrane rupture, fixation, intracellular cytokine dyeing, flow cytometry detection and result analysis and judgment, the ratio of the expression IFN-gamma cells in the CD4 + CD154 + cells can be rapidly and accurately determined, and by combining an ROC curve and a constructed judgment standard, the content of the IFN-gamma cells in the CD4 + CD154 + cells can be determined, and the content of the IFN-gamma cells in the CD4 + CD154 + cells in the CD4 + CD154 + cells in the CD4 + CD154 + cells in the CD4 + CD154 + cells can be determined. And the sample to be detected is judged to be in a positive / negative / gray region, so that whether a patient is infected by the mycobacterium tuberculosis or not can be more accurately diagnosed, especially aiming at a whole blood sample from a special crowd.

Description

Technical Field

[0001] This invention relates to the field of biological diagnostics, and more specifically, to a method for detecting specific T cell activation after tuberculosis infection based on flow cytometry and its application. Background Technology

[0002] Tuberculosis (TB) is a chronic infectious disease caused by Mycobacterium tuberculosis (MTB). It remains one of the top ten causes of death worldwide and is the leading cause of death from a single infectious disease. The World Health Organization (WHO) estimates that approximately one-quarter of the global population (about 2 billion people) carries latent tuberculosis infection (LTBI), and these asymptomatic carriers constitute a large reservoir of active tuberculosis (ATB). TB primarily affects the lungs, causing pulmonary tuberculosis, but it can also lead to bone tuberculosis, kidney tuberculosis, and lymph node tuberculosis.

[0003] Interferon-gamma release assays (IGRAs) represent a significant advancement in modern tuberculosis diagnosis. Their principle is based on detecting the host's T cell immune response to MTB-specific antigens. When the body is infected with MTB, sensitized T lymphocytes produce interferon-gamma upon re-exposure to MTB-specific antigens. IGRAs mimic this process in vitro to detect the infection status. Currently, two main clinically used IGRAs methods are: the QuantiFERON-TB Gold series using enzyme-linked immunosorbent assay (ELISA) and the T-SPOT.TB method using enzyme-linked immunospot assay (ELISPOT).

[0004] The specific antigens used in IGRAs are primarily derived from the early secretory antigen target-6 (ESAT-6) and culture filtrate protein-10 (CFP-10) encoded by the RD1 region gene of MTB. These antigens are absent in BCG and most environmental mycobacteria; therefore, IGRA results are not affected by BCG vaccination history and have significantly higher specificity than the traditional tuberculin skin test (TST). Theoretically, a positive IGRA indicates the presence of MTB-specific memory T cells, suggesting current or past MTB infection.

[0005] In clinical practice, IGRAS is primarily used for screening two groups: for the diagnosis of LTBI and as an adjunct to the diagnosis of active tuberculosis. For active tuberculosis, IGRAS cannot be the sole basis for diagnosis or exclusion, but it can provide important diagnostic reference in the absence of bacteriological evidence (such as smear-negative pulmonary tuberculosis). In LTBI screening, IGRAS is particularly suitable for high-risk groups requiring immunosuppressive therapy, such as patients with rheumatic diseases, organ transplant recipients, and HIV-infected individuals.

[0006] While IGRA is valuable in the diagnosis of tuberculosis infection, severe bacterial infections, active autoimmune diseases, diabetes, and end-stage renal disease can all lead to false negatives. Furthermore, certain medications, such as tricyclic antidepressants, nonsteroidal anti-inflammatory drugs (NSAIDs), and T-cell activation inhibitors, have also been reported to cause false negatives. These factors are prevalent in patients with rheumatic diseases, further complicating the interpretation of IGRA results.

[0007] IGRA also suffers from nonspecific positivity. Although ESAT-6 and CFP-10 are absent in most environmental mycobacteria, five nontuberculous mycobacteria (Mycobacterium Kansas, Mycobacterium marinum, Mycobacterium surgaense, Mycobacterium xanthogenes, and Mycobacterium gastritis) contain the same antigens, which can lead to false positives in IGRA. Furthermore, certain clinical conditions such as pregnancy, chlamydia infection, Crohn's disease, and Alzheimer's disease have been reported to increase interferon-gamma secretion, resulting in false positive results.

[0008] IGRA can produce false positive or false negative results in specific populations, such as patients with psoriasis, an immune-mediated chronic inflammatory disease characterized by overactivation of the Th1 / Th17 immune response, leading to elevated levels of pro-inflammatory cytokines (such as TNF-α, IL-17, and IFN-γ). Since IGRA relies on T cell release of IFN-γ to detect tuberculosis infection, T cells in psoriasis patients may be non-specifically activated to release IFN-γ, potentially leading to false positive results. Many patients with moderate to severe psoriasis are treated with TNF-α inhibitors (such as adalimumab and infliximab) or IL-17 / IL-23 inhibitors (such as secukinumab and ustekinumab). These medications may affect the immune response; for example, TNF-α inhibitors may increase the risk of tuberculosis reactivation, but they may also reduce the sensitivity of IGRA, leading to false negatives. In clinical practice, psoriasis patients should undergo tuberculosis screening (GRA or TST) before receiving biologics, along with chest imaging evaluation. Patients on long-term immunosuppressants should be monitored for tuberculosis symptoms regularly, even if the initial screening is negative. However, these patients may have indeterminate results if their IGRA results frequently show gray areas.

[0009] Furthermore, false positives in IGRA due to fungal infections are not uncommon. The mechanism may be related to immune cross-reactivity and non-specific immune activation: on the one hand, molecular mimicry of fungal antigens (such as Histoplasma capsulatum Hsp60) and Mycobacterium tuberculosis-specific antigens (such as ESAT-6 / CFP-10) can induce cross-reactive T cell activation, promoting the release of IFN-γ; on the other hand, fungal infection triggers systemic Th1 immune polarization by activating pattern recognition receptors (such as Dectin-1 and TLRs), promoting the secretion of cytokines such as IL-12 by monocytes, which in turn non-specifically expands memory T cells, leading to false positives in IGRA. Therefore, IGRA results need to be carefully interpreted in conjunction with clinical context and other testing methods.

[0010] It is worth noting that chronic pulmonary aspergillosis, a fungal infection, often occurs in patients with a history of pulmonary tuberculosis and usually requires simultaneous antifungal and anti-tuberculosis treatment. Therefore, differentiating the efficacy of aspergillosis and tuberculosis treatments and monitoring disease activity are particularly important. Furthermore, chronic aspergillosis infection can further activate the body's Th1 cell immunity, interfering with tuberculosis-specific IGRA test results.

[0011] Therefore, in special populations (such as those with comorbid autoimmune diseases or fungal infections), there is an urgent need to develop a method to identify T cells activated by tuberculosis-specific antigens, in order to assist in clinical diagnosis and medication.

[0012] It should be noted that the information disclosed in the background section above is only used to enhance the understanding of the background of the present invention, and therefore may include information that does not constitute prior art known to those skilled in the art. Summary of the Invention

[0013] The purpose of this invention is to provide a new method for identifying tuberculosis infection based on the ratio of CD154+CD4+ T cells to IFN-γ-expressing cells.

[0014] In a first aspect of the invention, a reagent combination for identifying specific T cell activation after tuberculosis infection is provided, the reagent combination comprising: an anti-CD4 antibody (CD4 antibody), an anti-CD154 antibody (CD154 antibody), an IFN-γ detection antibody, and a tuberculosis-specific antigen.

[0015] In another preferred embodiment, the reagent combination further includes a positive control (positive antigen).

[0016] In another preferred embodiment, the reagent combination also includes a negative control (negative antigen).

[0017] In another preferred embodiment, the negative control is RPMI-1640 culture medium.

[0018] In another preferred embodiment, the positive control is phytohemagglutinin (PHA).

[0019] In another preferred embodiment, the concentration of PHA is 1-10 μg / ml, more preferably 5 μg / ml.

[0020] In another preferred embodiment, the anti-CD4 antibody, the anti-CD154 antibody, and the IFN-γ detection antibody are labeled with different fluorescent markers.

[0021] In another preferred embodiment, the detection antibodies with different fluorescent labels (i.e., anti-CD4 antibody, anti-CD154 antibody, and IFN-γ detection antibody) are used as staining reagents.

[0022] In another preferred embodiment, the fluorescently labeled molecule is selected from the group consisting of: FITC, PE, ECD, PerCP-Cyanine 5.5, PE-Cyanine, 750, APC, Alexa Dyes, Pacific Blue, isotype, or combinations thereof.

[0023] In another preferred embodiment, the staining reagent diluent consists of PBS containing 3% BSA.

[0024] In another preferred embodiment, the tuberculosis-specific antigen is a mixture of ESAT6 and CFP10.

[0025] In another preferred embodiment, the tuberculosis-specific antigen is the antigen detected by Fosun tuberculosis infection T cells.

[0026] In another preferred embodiment, the reagent combination further includes an immune co-stimulatory molecule.

[0027] In another preferred embodiment, the immune co-stimulatory molecule is the CD40 antigen.

[0028] In another preferred embodiment, the reagent combination further includes an inhibitor.

[0029] In another preferred embodiment, the blocking agent is brefeldin A (BFA).

[0030] In another preferred embodiment, the reagent combination further includes a buffer solution.

[0031] In another preferred embodiment, the buffer solution is: complete cell culture medium (RPMI-1640 medium + 10% FBS).

[0032] In another preferred embodiment, the reagent combination further includes: a film-breaking agent (such as BD Cytofix / Cytoperm film-breaking agent) and a film-breaking agent washing solution.

[0033] In a second aspect of the invention, an in vitro detection method for specific T cell activation after tuberculosis infection is provided, the method comprising: mixing detection cells with anti-CD4 antibodies and anti-CD154 antibodies, incubating, detecting by flow cytometry, obtaining detection data, and determining the ratio of IFN-γ-expressing cells in CD4+CD154+ cells based on the detection data.

[0034] In another preferred embodiment, the method includes the following steps:

[0035] (a) Provides detection cells;

[0036] (b) Add anti-CD4 antibody (CD4 antibody) and anti-CD154 antibody (CD154 antibody) to the detection cells in (a) and incubate at room temperature for 15-30 min (preferably 20 min) to obtain incubation product 2;

[0037] (c) Add a membrane-breaking agent to the incubated product 2 in (b) and treat for 20 min to 120 min (preferably 30 min to 110 min, more preferably 40 min to 100 min, even more preferably 50 min to 90 min, and most preferably 60 min), add a membrane-breaking agent washing solution, and obtain the membrane-breaking treated product;

[0038] (d) Add IFN-γ detection antibody to the membrane perforation product in (c) and incubate for 30 min to 120 min (preferably 40 min to 110 min, more preferably 50 min to 100 min, even more preferably 55 min to 90 min, and most preferably 60 min) to obtain incubation product 3;

[0039] (e) The incubation product 3 in (d) was analyzed by flow cytometry to obtain the detection data;

[0040] (f) Determine the ratio of IFN-γ-expressing cells in CD4+CD154+ cells based on the detection data in (e).

[0041] In another preferred embodiment, in step (d), the incubation temperature is 0-10°C, preferably 3°C to 5°C, and most preferably 4°C.

[0042] In another preferred embodiment, the detection cells are PBMC cells that have undergone different pretreatments, including: (i) activation test group: PBMC cells are incubated with CD40 antigen and tuberculosis-specific antigen.

[0043] In another preferred embodiment, the tuberculosis-specific antigen is a mixture of ESAT6 and CFP10 (the antigen detected by Fosun tuberculosis infection T cells).

[0044] In another preferred embodiment, the detection cells further include: (ii) a negative control group: PBMC cells incubated with CD40 antigen and negative antigen.

[0045] In another preferred embodiment, the detection cells further include: (iii) a positive control group: PBMC cells incubated with CD40 antigen and positive antigen.

[0046] In another preferred embodiment, the detection cells include:

[0047] (i) Activation test group: PBMC cells were incubated with CD40 antigen and tuberculosis-specific antigen;

[0048] (ii) Negative control group: PBMC cells were incubated with CD40 antigen and negative antigen; and

[0049] (iii) Positive control group: PBMC cells were incubated with CD40 antigen and positive antigen.

[0050] In another preferred embodiment, the method includes the following steps:

[0051] (1) Provide a sample to be tested, the sample being PBMC cells, and divide the PBMC cells into n parts, where n is a positive integer ≥3 (e.g., 3-10 parts, for example 3, 4, 5, 6 parts);

[0052] (2) Set up a negative control group, a positive control group and an activation test group, and add the PBMC cells to form a test mixture, and stimulate the test mixture to incubate;

[0053] The negative control group, positive control group, and activation test group were tested under the same conditions, with the following differences: In the positive control group, at least one sample of PBMC cells was incubated together with CD40 antigen and positive control (positive antigen); in the activation test group, at least one sample of PBMC cells was incubated together with CD40 antigen and tuberculosis-specific antigen (a mixture of ESAT6 and CFP10); and in the negative control group, at least one sample of PBMC cells was incubated together with CD40 antigen and negative antigen to obtain incubation product 1.

[0054] (3) The test mixtures in the negative control group, positive control group and activation test group were measured to determine the target helper memory T cells in the negative control group, positive control group and activation test group, wherein the helper memory T cells are CD4+CD154+ cells; and the ratio of IFN-γ expressing cells in CD4+CD154+ cells was determined.

[0055] In another preferred embodiment, the positive control (positive antigen) is phytohemagglutinin (PHA).

[0056] In another preferred embodiment, in step (2), the time t1 for incubation together is 5 to 30 hours, more preferably 10 to 20 hours, more preferably 14 to 18 hours, and most preferably 15 to 17 hours (e.g., 16 hours).

[0057] In another preferred embodiment, in step (2), PBMC cells are added to each test tube in the negative control group, positive control group and activation test group, 10% fetal bovine serum + RPMI-1640 medium is added, CD40 antigen is added, and then negative antigen, tuberculosis-specific antigen and positive antigen are added respectively.

[0058] In another preferred embodiment, in step (2), in the negative control group, positive control group and activation test group, PBMC 5×10^6 cells were added to each test tube, 300ul of 10% fetal bovine serum + RPMI-1640 medium was added, 1ul (1μg / ml) of CD40 antigen was added, and then negative antigen, tuberculosis-specific antigen and positive antigen were added respectively.

[0059] In another preferred embodiment, in step (3) of the method, an IFN-γ detection antibody is used to determine the ratio of IFN-γ-expressing cells in CD4+CD154+ cells.

[0060] In another preferred embodiment, in step (3) of the method, CD4+CD154+ cells expressing IFN-γ in the negative control group, positive control group and activation test group are identified by flow cytometry.

[0061] In another preferred embodiment, step (3) of the method further includes: determining the ratio of IFN-γ-expressing cells in CD4+CD154+ cells in the negative control group, positive control group and activation test group.

[0062] In another preferred embodiment, step (3) includes the following steps:

[0063] (3a) Add CD4 antibody and CD154 antibody, and incubate at room temperature for 15-30 min (preferably 20 min) to obtain incubation product 2;

[0064] (3b) Optionally, wash the incubation product 2 obtained in (3a) with PBS;

[0065] (3c) Add a film-breaking agent to the incubation product 2 and treat for 20 min to 120 min (preferably 30 min to 110 min, more preferably 40 min to 100 min, even more preferably 50 min to 90 min, and most preferably 60 min) to obtain the film-breaking product;

[0066] (3d) Optionally, wash the membrane-breaking product obtained in (3c) with a washing solution (such as BD Perm / Wash, catalog number 51-2091KZ, diluted to 1X);

[0067] (3e) Add IFN-γ detection antibody to the membrane perforation product in (3d) and incubate for 30 min to 120 min (preferably 40 min to 110 min, more preferably 50 min to 100 min, even more preferably 55 min to 90 min, and most preferably 60 min) to obtain incubation product 3;

[0068] (3f) Optionally, wash the incubation product 3 obtained in (3e) with a washing solution (such as BD Perm / Wash, catalog number 51-2091KZ, diluted to 1X);

[0069] (3g) The incubation product 3 was analyzed by flow cytometry to determine the ratio of IFN-γ-expressing cells in CD4+CD154+ cells.

[0070] In another preferred embodiment, in step (3e), the incubation temperature is 0-10°C, preferably 3°C to 5°C, and most preferably 4°C.

[0071] In another preferred embodiment, the ratio of IFN-γ-expressing cells to CD4+CD154+ cells is equal to the number of IFN-γ cells / the number of CD4+CD154+ cells * 100%.

[0072] In another preferred embodiment, the method is non-diagnostic and non-therapeutic.

[0073] In a third aspect of the invention, the use of the reagent combination described in the first aspect of the invention for identifying specific T cell activation after tuberculosis infection is provided for preparing a detection kit for detecting the ratio of IFN-γ-expressing cells in CD4+CD154+ cells in PBMCs and / or for tuberculosis infection screening.

[0074] In another preferred embodiment, the tuberculosis infection screening includes tuberculosis screening for the general population and tuberculosis screening for special populations.

[0075] In another preferred embodiment, the special population includes: pregnant women, people infected with chlamydia, people infected with fungi, patients with Crohn's disease, patients with Alzheimer's disease, patients with neurological diseases, patients with psoriasis, patients with vasculitis, and patients with autoimmune diseases.

[0076] In another preferred example, the tuberculosis infection includes: active tuberculosis and latent tuberculosis infection.

[0077] In another preferred example, the kit further includes an instruction manual, and the method described in the second aspect of the present invention is recorded in the instruction manual.

[0078] In the fourth aspect of the present invention, a detection kit is provided, including:

[0079] (1) The reagent combination for identifying the activation of specific T cells after tuberculosis infection described in the first aspect of the present invention; and

[0080] (2) A label or an instruction manual, and the label or the instruction manual indicates that the kit is used for detecting the ratio of IFN-γ-expressing cells in CD4+CD154+ cells in PBMC cells.

[0081] In the fifth aspect of the present invention, a tuberculosis infection screening kit is provided, including:

[0082] (1) Reagents for separating PBMC cells from a whole blood sample;

[0083] (2) The reagent combination for identifying the activation of specific T cells after tuberculosis infection described in the first aspect of the present invention; and

[0084] (3) A label or an instruction manual, and the label or the instruction manual indicates that the kit is used for tuberculosis infection screening; the tuberculosis infection determination criteria in the instruction manual are as follows:

[0085] If the ratio of IFN-γ-positive cells in CD4+CD154+ cells >= 10%, it is determined as positive;

[0086] If the ratio of IFN-γ-positive cells in CD4+CD154+ cells < 2%, it is determined as negative;

[0087] If 2% <= the ratio of IFN-γ-positive cells in CD4+CD154+ cells < 10%, it is determined as the gray zone.

[0088] In another preferred example, the reagents for separating PBMC cells from a whole blood sample include: a culture medium (such as RPMI-1640 culture medium), lymphocyte separation solution, and serum (such as fetal bovine serum, preferably 10% fetal bovine serum).

[0089] In another preferred example, the lymphocyte separation solution is a TBD sample density separation solution.

[0090] In the sixth aspect of the present invention, a method for screening tuberculosis infection is provided, and the method includes:

[0091] (1) Provide a whole blood sample, and the whole blood is peripheral whole blood;

[0092] (2) Isolate PBMC cells from the whole blood sample;

[0093] (3) Use the method described in the second aspect of the present invention to determine the ratio of IFN-γ-expressing cells in PBMC cells among CD4+CD154+ cells;

[0094] (4) Determine the tuberculosis infection result of the whole blood sample:

[0095] If the ratio of IFN-γ-positive cells among CD4+CD154+ cells ≥ 10%, it is determined as positive;

[0096] If the ratio of IFN-γ-positive cells among CD4+CD154+ cells < 2%, it is determined as negative;

[0097] If 2% ≤ the ratio of IFN-γ-positive cells among CD4+CD154+ cells < 10%, it is determined as the gray area.

[0098] In another preferred example, the anticoagulant used for the peripheral whole blood is heparin, lithium heparin, or sodium heparin.

[0099] In another preferred example, the anticoagulant used for the peripheral whole blood does not include EDTA.

[0100] In another preferred example, the peripheral whole blood sample is stored and transported at room temperature (15 - 25°C).

[0101] In another preferred example, the peripheral whole blood sample is sent to the laboratory within 6 hours (preferably 4 hours), and the detection is completed within 72 hours (preferably 36 hours, more preferably 24 hours, still more preferably 12 hours, and most preferably 8 hours).

[0102] In another preferred example, a T-cell extension reagent can be added to the peripheral whole blood sample.

[0103] It should be understood that the above general description and the following detailed description are only exemplary and explanatory, and cannot limit the present invention. Brief Description of the Drawings

[0104] The drawings herein are incorporated into the specification and constitute a part of this specification, showing embodiments consistent with the present invention, and are used together with the specification to explain the principles of the present invention. Obviously, the drawings in the following description are only some embodiments of the present invention, and those of ordinary skill in the art can obtain other drawings based on these drawings without creative efforts.

[0105] Figure 1This is a schematic diagram of flow cytometry results. Tuberculosis-specific T cells are defined as CD4+CD154+IFN-γ+ T cells, and their proportion in the total CD4+ T cells is measured. Left: CD4+CD154+IFN-γ+ T cell proportion <2% indicates cross-reactivity; Right: CD4+CD154+IFN-γ+ T cell proportion >=10% indicates tuberculosis-specific reaction.

[0106] Figure 2 The ROC curve is displayed. Detailed Implementation

[0107] Exemplary embodiments will now be described more fully with reference to the accompanying drawings. However, these exemplary embodiments can be implemented in many forms and should not be construed as limited to the embodiments set forth herein; rather, they are provided so that the invention will be thorough and complete, and the concept of the exemplary embodiments will be fully conveyed to those skilled in the art.

[0108] Unless otherwise defined, all terms and phrases used herein include their meanings as they have in the art, unless explicitly stated otherwise or clearly indicated from the context of their use. While any methods and materials similar to or equivalent to those described herein may be used in carrying out or testing the invention, specific methods and materials are now described.

[0109] Through extensive and in-depth research, the inventors have unexpectedly discovered a method for stimulating PBMCs with tuberculosis-specific antigens and then measuring tuberculosis infection-specific T cells using flow cytometry.

[0110] Specifically, this invention provides a reagent combination for identifying specific T cell activation after tuberculosis infection. This combination includes an anti-CD4 antibody (CD4 antibody), an anti-CD154 antibody (CD154 antibody), an IFN-γ detection antibody, and a tuberculosis-specific antigen. Using this reagent combination, it is possible to rapidly identify whether T cells are tuberculosis-specific T cells, thus providing insights for clinical diagnosis and medication. In this invention, PBMCs isolated from a test sample (e.g., peripheral whole blood) are stimulated with both CD40 antigen and tuberculosis-specific antigen. Flow cytometry is then used to detect the ratio of IFN-γ-expressing cells to CD4+CD154+ cells after tuberculosis-specific activation. Combined with ROC curves and constructed criteria, the test sample is determined to be in the positive / negative / gray zone, thereby providing a more accurate diagnosis of Mycobacterium tuberculosis infection, especially for complex samples from specific populations. This method effectively overcomes the shortcomings of traditional IGRA methods, which are prone to non-specific positive results for complex samples. Based on this, this invention was completed.

[0111] the term

[0112] As used in this invention, the terms “containing,” “comprising,” or “having” include “comprising,” “mainly composed of,” “substantially composed of,” and “composed of”; “mainly composed of,” “substantially composed of,” and “composed of” are subordinate concepts of “containing,” “having,” or “comprising.”

[0113] As used herein, “about,” “approximately,” “roughly,” or “substantially” generally refers to a specific value or range, such as within 20%, preferably within 10%, more preferably within 5%, with a fluctuation of up or down. The values ​​used herein are approximate and mean that, unless expressly stated otherwise, the terms “about,” “approximately,” “roughly,” or “substantially” can be inferred to apply.

[0114] CD40 / CD40L

[0115] CD154, also known as CD40L, is a 33kD type II membrane protein belonging to the TNF family. It is mainly expressed on the surface of activated CD4+ T cells and mast cells. In mice lacking CD40 ligand (CD154), the initiation of antigen-specific T cells is impaired. Co-stimulation with specific antigens and CD40 can induce CD4+ cells to express CD154, which then binds to CD40 to exert its function.

[0116] The ratio of IFN-γ-expressing cells in CD4+CD154+ cells

[0117] CD4+CD154+ cells represent a group of CD4+ helper T cells in the immune system that have been activated by their specific antigens and possess strong auxiliary functions. They are the core coordinators of adaptive immune responses and play a crucial role in anti-infective immunity, vaccine responses, and pathological processes such as autoimmune diseases and allergies. Detection of CD4+ cells... + CD154 + Cells are an important means of assessing the strength and functional status of T-cell immune responses against specific antigens.

[0118] Previous literature has suggested that IFN-γ+CD154+CD4+ cells are antigen-specific T cells after antigen stimulation. Without antigen stimulation, the percentage of IFN-γ+CD154+CD4+ cells is almost zero; after antigen stimulation, the percentage can rise to 5% or even higher. This percentage is correlated with the severity of infection by a specific pathogen and can be further used for diagnosis and differential diagnosis.

[0119] Reagent combination for identifying specific T cell activation after tuberculosis infection

[0120] This invention provides a reagent combination for identifying specific T cell activation after tuberculosis infection.

[0121] Typically, the reagent combination of the present invention includes: an anti-CD4 antibody (CD4 antibody), an anti-CD154 antibody (CD154 antibody), an IFN-γ detection antibody, and a tuberculosis-specific antigen.

[0122] Typically, the tuberculosis-specific antigen is a mixture of ESAT6 and CFP10 (e.g., the antigen for Fosun tuberculosis infection T cell detection).

[0123] Preferably, the reagent combination of the present invention further includes a positive control (positive antigen) and a negative control (negative antigen).

[0124] In this invention, the negative control (negative antigen) is typically a culture medium (such as RPMI-1640 medium), and the positive control (positive antigen) is phytohemagglutinin (PHA).

[0125] Preferably, in the reagent combination of the present invention, the anti-CD4 antibody, the anti-CD154 antibody, and the IFN-γ detection antibody are labeled with different fluorescent markers.

[0126] Preferably, the reagent combination of the present invention further includes an immune co-stimulatory molecule, such as CD40 antigen.

[0127] Preferably, the reagent combination of the present invention further includes an inhibitor (such as BFA).

[0128] Preferably, the reagent combination of the present invention further includes a buffer solution (such as complete cell culture medium).

[0129] Preferably, the reagent combination of the present invention further includes a film-breaking agent (such as BD Cytofix / Cytoperm film-breaking agent).

[0130] In vitro detection method for specific T cell activation after tuberculosis infection

[0131] The present invention also provides an in vitro detection method for specific T cell activation after tuberculosis infection.

[0132] Typically, the method includes: mixing the detection cells with antibodies against CD4 and CD154, incubating them, detecting them by flow cytometry, obtaining detection data, and determining the ratio of IFN-γ-expressing cells in CD4+CD154+ cells based on the detection data.

[0133] The cells to be tested are PBMCs that have undergone different pretreatments, including: (i) activation test group: PBMCs are incubated with CD40 antigen and tuberculosis-specific antigen.

[0134] In a preferred embodiment of the present invention, the detection cells include:

[0135] (i) Activation test group: PBMC cells were incubated with CD40 antigen and tuberculosis-specific antigen;

[0136] (ii) Negative control group: PBMC cells were incubated with CD40 antigen and negative antigen; and

[0137] (iii) Positive control group: PBMC cells were incubated with CD40 antigen and positive antigen.

[0138] Typically, the method of the present invention includes the following steps:

[0139] (a) Provides detection cells;

[0140] (b) Add anti-CD4 antibody (CD4 antibody) and anti-CD154 antibody (CD154 antibody) to the detection cells in (a) and incubate at room temperature for 15-30 min (preferably 20 min) to obtain incubation product 2;

[0141] (c) Add a membrane-breaking agent to the incubated product 2 in (b) and treat for 20 min to 120 min (preferably 30 min to 110 min, more preferably 40 min to 100 min, even more preferably 50 min to 90 min, and most preferably 60 min), add a membrane-breaking agent washing solution, and obtain the membrane-breaking treated product;

[0142] (d) Add IFN-γ detection antibody to the membrane perforation product in (c) and incubate for 30 min to 120 min (preferably 40 min to 110 min, more preferably 50 min to 100 min, even more preferably 55 min to 90 min, and most preferably 60 min) to obtain incubation product 3;

[0143] (e) The incubation product 3 in (d) was analyzed by flow cytometry to obtain the detection data;

[0144] (f) Determine the ratio of IFN-γ-expressing cells in CD4+CD154+ cells based on the detection data in (e).

[0145] In another preferred embodiment of the present invention, the method includes the following steps:

[0146] (1) Provide a sample to be tested, the sample being PBMC cells, and divide the PBMC cells into n parts, where n is a positive integer ≥3 (e.g., 3-10 parts, for example 3, 4, 5, 6 parts);

[0147] (2) Set up a negative control group, a positive control group and an activation test group, and add the PBMC cells to form a test mixture, and stimulate the test mixture to incubate;

[0148] The negative control group, positive control group, and activation test group were tested under the same conditions, with the following differences: In the positive control group, at least one sample of PBMC cells was incubated together with CD40 antigen and positive control (positive antigen); in the activation test group, at least one sample of PBMC cells was incubated together with CD40 antigen and tuberculosis-specific antigen (a mixture of ESAT6 and CFP10); and in the negative control group, at least one sample of PBMC cells was incubated together with CD40 antigen and negative antigen to obtain incubation product 1.

[0149] (3) The test mixtures in the negative control group, positive control group and activation test group were measured to determine the target helper memory T cells in the negative control group, positive control group and activation test group, wherein the helper memory T cells are CD4+CD154+ cells; and the ratio of IFN-γ expressing cells in CD4+CD154+ cells was determined.

[0150] Wherein, the ratio of IFN-γ-expressing cells in CD4+CD154+ cells = number of IFN-γ cells / number of CD4+CD154+ cells * 100%.

[0151] The main advantages of this invention include:

[0152] (1) Simple operation: After separating human PBMCs, co-stimulation is performed, and flow cytometry is used for detection on the second day. There are no complicated operation steps, and the total operation process takes about 36 hours.

[0153] (2) High specificity: It can effectively distinguish between tuberculosis infection and other non-tuberculosis infection-induced non-specific immune enhancement states.

[0154] The present invention will be further illustrated below with reference to specific embodiments.

[0155] It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions as described in *Molecular Cloning: A Laboratory Guide* by Sambrook J. et al. (translated by Huang Peitang et al., Beijing: Science Press, 2002), or according to the manufacturer's recommendations (e.g., product instructions). Unless otherwise stated, percentages and parts are by weight. Unless otherwise specified, the experimental materials and reagents used in the following embodiments are commercially available or can be prepared according to literature methods.

[0156] Example 1

[0157] (I) Sampling and Detection Methods

[0158] 1. Collect peripheral whole blood. Fasting is not required. Anticoagulation with heparin / lithium heparin / sodium heparin is necessary. EDTA anticoagulation is preferred.

[0159] Blood volume requirements:

[0160] 2-3ml for children under 2 years old

[0161] Other patients: 4-6 ml

[0162] 6-8 ml for patients with weakened / suppressed immunity;

[0163] 2. Samples should be stored and transported at room temperature (15-25℃), and must not be frozen or refrigerated;

[0164] 3. Samples should be delivered to the laboratory within 4 hours of collection and tested within 8 hours, or stabilizing solution should be added and testing completed within the corresponding time (T-cell extension reagent);

[0165] 4.1 Patients who have had a blood transfusion or PET-CT scan within the past week may have impaired lymphocyte separation in their blood. It is recommended to wait two weeks before testing.

[0166] 5. Prevent bumps and jostles during transportation to avoid hemolysis of the sample affecting the results;

[0167] 6. Prepare three 15ml centrifuge tubes for each sample. Use a 10ml pipette to draw two 4ml tubes of RPMI-1640 culture medium (Biosharp, catalog number BL303A) and one 4ml tube of lymphocyte separation medium (such as TBD sample density separation medium, catalog number HY2015) (be careful not to get the medium on the tube wall). Insert the used pipettes back into the bag and label them.

[0168] 7. Discard the remaining plasma supernatant after centrifugation using a Pasteur pipette, and resuspend the lymphocytes in one of the tubes with 4 ml of RPMI-1640 medium. Add the medium to 8 ml using a pipette. Slowly add the resuspended lymphocytes to 4 ml of lymphocyte separation medium (such as TBD sample density separation medium, catalog number HY2015) using a Pasteur pipette (tilt the tube of separation medium at 45 degrees, taking care not to disrupt the separation). Centrifuge at 800 g / min, with a speed of 0 for 20 min (to prevent disruption of the separation).

[0169] 8. After centrifugation, the sample separates into four layers (the top layer is plasma and platelets diluted in RPMI-1640 medium, followed by a thin layer of PBMCs, then lymphocyte separation medium (such as TBD sample density separation medium, catalog number HY2015), and finally erythrocytes and granulocytes). Carefully aspirate the PBMCs from the cloud layer using a Pasteur pipette (be careful to aspirate as little lymphocyte separation medium or supernatant as possible to avoid affecting subsequent experiments) and transfer it to another prepared 4ml RPMI-1640 medium. Continue adding RPMI-1640 medium to bring the total volume to 12ml. Centrifuge at 600g / min, vertical speed 9, for 10min.

[0170] 9. After centrifugation, discard the supernatant. The precipitate is PBMC. Resuspend it in 10% fetal bovine serum (Gibco, catalog number A5256701) + RPMI-1640 medium, count the cells and quantify to 1*10^7 / ml.

[0171] 10. Take three PBMC 5×10^6 cells and add 300ul of 10% fetal bovine serum + RPMI-1640 medium, add 1ul (1μg / ml) of CD40 antigen (Biolegend, catalog number 668104), add negative antigen (RPMI-1640 medium), a mixture of tuberculosis-specific antigens ESAT6 and CFP10 (e.g., 150ul of the Fosun Diagnostics I-SPOT.TB, catalog number 1.05.1001 antigen for detecting tuberculosis-infected T cells), and positive antigen (PHA) (e.g., PHA from Fosun Diagnostics I-SPOT.TB, catalog number 1.05.1001); incubate at 37℃ for 16h.

[0172] 11. Add 0.5 μL of blocking agent (1000×) (Biolegend, catalog number 42060), incubate at 37°C for 6 hours, and obtain cells as test cells;

[0173] 12. Flow cytometry: Wash cells twice with PBS, add CD4 antibody (Biolegend, catalog number 300506) and CD154 antibody (Biolegend, catalog number 310824), incubate at room temperature for 20 minutes, wash with PBS, add membrane-permeability agent (BD Cytofix / Cytoperm, catalog number 51-2090KZ) for 1 hour, wash twice with washing buffer (BD Perm / Wash, catalog number 51-2091KZ, diluted to 1X), add 1 μL of IFN-γ detection antibody (Biolegend, catalog number 506507), incubate at 4°C for 1 hour, wash (BD Perm / Wash, catalog number 51-2091KZ), and perform flow cytometry to detect the expression of IFN-γ in CD4+CD154+ cells and determine the ratio of tuberculosis-specific activated cells.

[0174] The flow cytometry detection results are shown in Figure 1 , and the tuberculosis-specific T cells are defined as CD4+CD154+IFN-γ+ T cells, and the proportion of them in total CD4+ T cells is detected. Among them, the proportion of CD4+CD154+IFN-γ+ T cells in the left figure < 2% is defined as cross-reaction, and the proportion of CD4+CD154+IFN-γ+ T cells in the right figure ≥ 10% is defined as tuberculosis-specific reaction.

[0175] (II) ROC curve drawing and determination criterion selection

[0176] In order to clarify the diagnostic value of the ratio of IFN-γ-expressing cells in CD4+CD154+ cells after tuberculosis-specific activation in tuberculosis infection, the inventor included 60 patients who were clinically and laboratory-diagnosed as tuberculosis-negative and 23 patients who were clinically and laboratory-diagnosed as tuberculosis-infected. These 83 people constituted the first cohort (cohort 1). According to the above method, the ratio of IFN-γ-expressing cells in CD4+CD154+ cells in these two groups of people was detected to draw the ROC curve.

[0177] Results ( Figure 2 ) showed that the maximum area under the curve (AUC) was 0.99 and the maximum likelihood ratio was 52.17.

[0178] Determination criterion:

[0179] According to the drawn ROC curve, when the cutoff value corresponding to the maximum likelihood ratio was determined to be 5.94, the diagnostic sensitivity was 86.96% and the specificity was 98.33%; when the cutoff value was 2.5, the diagnostic sensitivity was 100% and the specificity was 90%; when the cutoff value was 10, the diagnostic sensitivity was 69.57% and the specificity was 100%. Therefore, the inventor set the following determination criteria:

[0180] Positive result: The ratio of IFN-γ-positive cells in CD4+CD154+ cells ≥ 10%

[0181] Negative result: The ratio of IFN-γ-positive cells in CD4+CD154+ cells < 2%

[0182] Grey zone result, it is recommended to follow up and observe: 2% ≤ the ratio of IFN-γ-positive cells in CD4+CD154+ cells < 10%.

[0183] Example of detection results in Example 2

[0184] To verify the effectiveness of the method of the present invention and the judgment criteria constructed in Example 1 above in the accurate diagnosis of Mycobacterium tuberculosis infection, the inventors re-enrolled 26 patients with clinical diagnoses of psoriasis / fungal disease / neurological diseases. These 26 patients constituted the second cohort (cohort 2), and the doctors needed to determine whether the patients had tuberculosis infection.

[0185] The results (Table 1) show that, using the current mainstream IGRA detection method, the test results of patients 6-19 all pointed to tuberculosis infection (according to the existing IGRA kit (Fosun Kit I-SPOT.TB, catalog number 1.05.1001), an ISPOT score >9 is considered a possible tuberculosis infection, an ISPOT score <6 is considered negative, and an ISPOT score between 6 and 9 is considered a gray zone). However, there was no other clinical evidence to support tuberculosis infection, which may be due to non-specific activation of T cells under the stimulation of tuberculosis antigen caused by underlying diseases.

[0186] In cases of such complex samples, the method of this invention can effectively distinguish and diagnose more accurately. Examples of detection results are shown in Table 1 below.

[0187] Table 1

[0188]

[0189]

[0190] Therefore, the present invention provides a more accurate method for diagnosing Mycobacterium tuberculosis infection.

[0191] Other embodiments of the invention will readily occur to those skilled in the art upon consideration of the specification and practice of the invention disclosed herein. This application is intended to cover any variations, uses, or adaptations of the invention that follow the general principles of the invention and include common knowledge or customary techniques in the art not disclosed herein. The specification and examples are to be considered exemplary only, and the true scope and spirit of the invention are indicated by the appended claims.

Claims

1. A reagent combination for identifying specific T cell activation after tuberculosis infection, characterized in that, The reagent combination includes: anti-CD4 antibody, anti-CD154 antibody, IFN-γ detection antibody, and tuberculosis-specific antigen.

2. The reagent combination as described in claim 1, characterized in that, The tuberculosis-specific antigen is a mixture of ESAT6 and CFP10.

3. The reagent combination as described in claim 1, characterized in that, The reagent combination also includes: an immune co-stimulatory molecule.

4. The reagent combination as described in claim 1, characterized in that, The reagent combination also includes: an inhibitor.

5. An in vitro detection method for specific T cell activation after tuberculosis infection, characterized in that, The method includes: mixing the detection cells with anti-CD4 antibodies and anti-CD154 antibodies, incubating them, detecting them by flow cytometry, obtaining detection data, and determining the ratio of IFN-γ-expressing cells in CD4+CD154+ cells based on the detection data.

6. The method as described in claim 5, characterized in that, The method includes the following steps: (1) Provide a sample to be tested, the sample being PBMC cells, and divide the PBMC cells into n parts, where n is a positive integer ≥3; (2) Set up a negative control group, a positive control group and an activation test group, and add the PBMC cells to form a test mixture, and stimulate the test mixture to incubate; The negative control group, positive control group, and activation test group were tested under the same conditions, with the following differences: in the positive control group, at least one sample of PBMC cells was incubated with CD40 antigen and positive control; in the activation test group, at least one sample of PBMC cells was incubated with CD40 antigen and tuberculosis-specific antigen; and in the negative control group, at least one sample of PBMC cells was incubated with CD40 antigen and negative antigen to obtain incubation product 1. (3) The test mixtures in the negative control group, positive control group and activation test group are measured to determine the target helper memory T cells in the negative control group, positive control group and activation test group, wherein the antigen-specific helper memory T cells are CD4+CD154+ cells; and the ratio of IFN-γ-expressing cells in CD4+CD154+ cells is determined.

7. The use of the reagent combination for identifying specific T cell activation after tuberculosis infection as described in any one of claims 1-4, characterized in that, This kit is used to prepare a detection kit for detecting the ratio of IFN-γ-expressing cells in CD4+CD154+ cells in PBMCs and / or for tuberculosis infection screening.

8. A test kit, characterized in that, include: (1) The reagent combination for identifying specific T cell activation after tuberculosis infection as described in any one of claims 1-4; and (2) Label or instructions, which indicate that the kit is used to detect the ratio of IFN-γ-expressing cells in PBMC cells to CD4+CD154+ cells.

9. A tuberculosis infection screening kit, characterized in that, include: (1) Reagents for isolating PBMCs from whole blood samples; (2) The reagent combination for identifying specific T cell activation after tuberculosis infection as described in any one of claims 1-4; and (3) Label or instructions for use, which state that the kit is used for tuberculosis infection screening; the tuberculosis infection determination criteria in the instructions for use are as follows: If the ratio of IFN-γ positive cells in CD4+CD154+ cells is >= 10%, it is considered positive; If the percentage of IFN-γ positive cells in CD4+CD154+ cells is <2%, it is considered negative; If 2% <= the ratio of IFN-γ positive cells in CD4+CD154+ cells < 10%, it is determined as the gray zone.

10. A method for screening tuberculosis infection, characterized in that, The method includes: (1) Providing a whole blood sample, and the whole blood is peripheral whole blood; (2) Isolating PBMC cells from the whole blood sample; (3) Determining the ratio of IFN-γ-expressing cells in PBMC cells in CD4+CD154+ cells by the method described in claim 5 or 6; (4) Determining the tuberculosis infection result of the whole blood sample: If the ratio of IFN-γ positive cells in CD4+CD154+ cells >= 10%, it is determined as positive; If the ratio of IFN-γ positive cells in CD4+CD154+ cells < 2%, it is determined as negative; If 2% <= the ratio of IFN-γ positive cells in CD4+CD154+ cells < 10%, it is determined as the gray zone.

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