CN116430055B - A combination of T cell immunological markers for evaluating the chronic pathological state of disease T lymphocytes and its application - Google Patents

Abstract

The invention provides a T cell immunological state marker combination for evaluating chronic pathological states of T lymphocytes of diseases and application thereof, and relates to the field of disease evaluation. Including any of CD3, CD4, CD8, FOXP3, CD25, CD127, TIM3, PS6, etc. The invention covers the functional evaluation of a wide range of T cell subsets by detecting combinations; the invention rapidly realizes accurate parting and immunocompetence detection of the T cells, and evaluates the chronic activation state of the T cells with high sensitivity and high specificity; the system evaluates the influence of the severity of the disease, the treatment effect, early recurrence, infection risk and medicines on the cellular immunity function; the invention has simple operation and easily understood result, and is easy to popularize and apply in basic medical institutions.

Description

A combination of T cell immunological markers for evaluating the chronic pathological state of T lymphocytes in diseases and its application

Technical Field

The present invention relates to the field of disease evaluation, and in particular to a combination of T cell immunological state markers for evaluating the chronic pathological state of disease T lymphocytes.

Background technique

Chronic diseases (infection, tumors, metabolic diseases, cardiovascular diseases, nervous system diseases, etc.) are the most important factors threatening global human health, and chronic systemic inflammatory response is one of the key pathophysiological mechanisms of all chronic diseases. T cells are the most important effector cells for regulating human immune homeostasis. They can be divided into many different subtypes according to their surface markers and functions, and are in a dynamic conversion process. The continuous activation of T cells can cause systemic inflammatory damage by secreting a large number of cytokines, mediating the occurrence and development of a variety of chronic diseases; on the other hand, T cells in continuous activation continue to secrete a large number of cytokines, eventually leading to cell function exhaustion. Once T cell function is exhausted, the immune system will suffer persistent and severe damage, the body's resistance will be low, and life-threatening severe infections will occur easily. Timely and accurate assessment of T cell immune phenotype and functional homeostasis and adjustment of treatment plans are major clinical issues that need to be solved urgently.

The methods commonly used by clinicians to evaluate patients' immune function and inflammatory response indicators mainly rely on traditional blood biochemical test items (such as blood routine, immunoglobulin, C-reactive protein, etc.), which generally have shortcomings such as low sensitivity and specificity, and are easily affected by different disease factors. Flow cytometry is a new inspection technology that has been promoted in the medical field in recent years. Common indicators for evaluating immune function include basic typing detection of CD3+T cells. Flow cytometry can screen, separate, and detect the number and proportion of T cells in patients. However, clinical practice has found that the current flow cytometry detection scheme has many shortcomings that need to be improved. First, the existing commonly used flow cytometry typing detection indicators are limited to T cell surface markers (CD3, CD4, CD8), which can only indicate changes in the total number or proportion of overall T cells. It is difficult to accurately reflect the changes in the biological functions of various T cell subsets and their chronic activation state, and cannot guide clinicians to make timely judgments on patients' T cell function and immune homeostasis.

Infection has always been the most serious threat to human health. Normal T cell immunity is one of the most important lines of defense against the invasion of various foreign pathogens. In a variety of disease states, patients may experience long-term chronic activation of T cells, which may eventually lead to T cell exhaustion; at the same time, various clinical immune intervention treatment programs also have a significant effect on T cell function. Abnormal T cell function may lead to fatal infections. Therefore, timely and accurate assessment of the patient's chronic T cell activation status is crucial to guide clinicians in formulating or adjusting treatment plans, especially for patients who need long-term use of immunosuppressant regimens. It is a major life-threatening issue.

For a long time, clinicians have been accustomed to using blood routine, immunoglobulin and other test methods to judge the patient's immunity level. Recently, with the promotion of clinical application of flow cytometry, it is possible to detect two different T cells, CD4+ and CD8+. Although these existing biochemical test items can screen out some patients with obvious low immunity to a certain extent (such as obvious leukopenia, low IgG blood, low CD4/CD8 values, etc.), the vast majority of patients will not have a decrease in leukocyte or IgG levels in the early stage of immune dysfunction or even for a long period of time. This is because immune cells, including T cells, have obvious abnormal cell biological activity before pathological damage such as apoptosis (a programmed death process). At this time, although the number of immune cells (T cells, etc.) is still within the normal range, its protective function of clearing foreign pathogens has been significantly impaired. The above detection technologies cannot reflect the actual situation of low immunity in a timely manner, and can no longer meet the requirements of modern clinical medicine.

Multi-parameter flow cytometry can detect the fluorescence intensity produced by cells based on the binding of fluorescently labeled antibodies to target proteins expressed by T cells to obtain the expression level of target proteins, and then subdivide a large class of T cells into multiple T cell subsets with different functions based on the different expression levels of target proteins in different cells. Multi-parameter flow cytometry can detect multiple surface markers of cells at one time, thereby systematically evaluating the number, proportion and biological function of T cells, and making systematic judgments on patients' disease status, infection risk and drug efficacy. However, due to the large number of T cell surface proteins (markers) and the significant differences in cell biological functions reflected by different spatiotemporal combinations of various protein expressions, especially during disease progression and drug treatment, the changes are greater, forming a geometric series of possible combinations. How to discover expression combinations with clinical diagnostic significance and quickly and accurately analyze T cell functional activity based on their expression levels is still an unresolved clinical problem.

Summary of the invention

In order to solve the above problems, the present invention provides a combination of T cell immunological state markers for evaluating the chronic pathological state of disease T lymphocytes. By detecting the combination, the present invention covers the functional evaluation of a wide range of T cell subsets, evaluation of disease activity, treatment effects, clinical outcomes and prognosis prediction.

In order to achieve the above object, the present invention provides the following technical solutions:

The present invention provides a combination of T cell immunological markers for evaluating the chronic pathological state of disease T lymphocytes, including any several of CD3, CD4, CD8, CD16, CD25, CD45, CD45RA, CD45RO, CD56, CD62L, CD44, CD69, CD127, CD154, CCR7, CTLA4, IgM, FOXP3, TIM3, LAG3, PD1, PDL1 and PS6 (Phospho-S6 Ribosomal Protein).

Preferably, the disease includes one or more of primary glomerular disease, autoimmune disease, infection, metabolic disease, blood system disease and drug-induced renal injury.

Preferably, the primary glomerular disease includes one or more of minimal change disease, focal segmental sclerosis, membranous nephropathy and IgA nephropathy;

The autoimmune disease includes one or more of systemic lupus erythematosus, Sjögren's syndrome, rheumatoid arthritis and mixed connective tissue disease;

The infection includes one or more of tuberculosis and novel coronavirus infection;

The metabolic disease includes one or more of diabetes, obesity and calciphylaxis;

The blood system disease includes one or more of amyloidosis, monoclonal immunoglobulinemia, multiple myeloma, lymphoma and thrombotic microangiopathy.

Preferably, when evaluating the immune function of patients treated with glucocorticoids, the T cell immunological state marker combination includes CD3, CD4, CD8, CD19, CD25, CD127, FOXP3, TIM3, LAG3, CTLA4 and PS6.

Preferably, when detecting minimal lesions, the T cell immunological status marker combination includes CD3, CD4, CD8, CD25, CD127, FOXP3 and PS6.

Preferably, when detecting systemic inflammatory damage caused by tuberculosis infection, the T cell immunological marker combination includes CD45, CD3, CD4, CD8, CD25, TIM3, LAG3, PD1, PDL1 and CTLA4.

Preferably, when detecting systemic inflammatory damage caused by novel coronavirus infection, especially acute kidney injury, the T cell immunological marker combination includes CD3, CD4, CD8, CD25, CD127, FOXP3, TIM3 and LAG3.

Preferably, when evaluating the chronic pathological state of T cells in different glomerular diseases, the T cell immunological state marker combination includes CD3, CD4, CD8, CD25, CD127, TIM3, LAG3, CTLA4, FOXP3 and PS6.

The diseases include one or more of lupus nephritis, minimal change disease, focal segmental glomerulosclerosis and membranous nephropathy.

The present invention also provides the use of the combination of T lymphocyte immunological state markers described in the above technical solution in the preparation of a reagent composition for evaluating the chronic pathological state of disease T lymphocytes.

The product is a reagent composition for evaluating the T cell immunological state of chronic pathological state of disease T lymphocytes, which comprises fluorescent dye-coupled antibodies against the following marker combination, wherein the marker combination comprises any of CD3, CD4, CD8, CD16, CD25, CD45, CD45RA, CD45RO, CD56, CD62L, CD44, CD69, CD127, CD154, CCR7, CTLA4, IgM, FOXP3, TIM3, LAG3, PD1, PDL1 and PS6;

Preferably, the markers are CD3, CD4, CD8, TIM3, LAG3 and CTLA4;

Preferably, the markers are CD3, CD4, CD8, CD25, CD127 and FOXP3;

Preferably, the markers are CD3, CD4, CD8, CD25, CD127, FOXP3 and PS6;

Preferably, the markers are CD3, CD4, CD8, CD25, CD127, LAG3 and TIM3;

Preferably, the markers are CD45, CD3, CD4, CD8, CD25, TIM3, LAG3, PD1, PDL1 and CTLA4;

The absorption wavelength and emission wavelength range of the fluorescent dye are 300nm-810nm, including but not limited to disodium 4-acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic acid; acridine and its derivatives such as acridine, acridine orange, acridine yellow, acridine red and acridine isothiocyanate; 5-(2-aminoethylamino)-1-naphthalenesulfonic acid sodium salt (EDANS); 4-amino-N-[3-(vinylsulfonyl)phenyl]naphthalimide-3,5-disulfonate (fluorescent yellow VS); N-(4-anilino-1-naphthyl)maleimide; 2-aminobenzamide; brilliant yellow; coumarin and its derivatives such as coumarin, 7-acetoxy-4-methylcoumarin (AMC, coumarin 120), 7-amino-4-trifluoromethylcoumarin (coumarin 120), 4-amino-1-naphthalenesulfonic acid sodium salt (EDANS); 4-amino-N-[3-(vinylsulfonyl)phenyl]naphthalimide-3,5-disulfonate (fluorescent yellow VS); N-(4-anilino-1-naphthyl)maleimide; 2-aminobenzamide; brilliant yellow; coumarin and its derivatives such as coumarin, 7-acetoxy-4-methylcoumarin (AMC, coumarin 120), 4-amino-1-naphthalenesulfonic acid sodium salt (EDANS); 4-amino-N-[3-(vinylsulfonyl)phenyl]naphthalimide-3,5-disulfonate (fluorescent yellow VS); N-(4-anilino-1-naphthyl)maleimide; 2-aminobenzamide; brilliant yellow; coumarin and its derivatives such as coumarin, 4-methylcoumarin (AMC, coumarin 120), 4-amino- 151); cyanine and derivatives such as tetrabromotetrachlorofluorescein, Cy3, Cy5, Cy5.5, and Cy7; 4',6-diamidino-2-phenylindole (DAPI); bromopyrogallol red; 7-diethylamino-3-(4'-isothiocyanatophenyl)-4-methylcoumarin; diethylaminocoumarin; diethylenetriamine pentaacetate; 4,4'-diisothiocyanatodihydrostilbene-2,2'-disulfonic acid; 4,4'-diisothiocyanato-2,2'-stilbene disulfonic acid; dansyl chloride (DNS, dansyl chloride); 4-(4'-dimethylaminophenylazo)benzoic acid (DABCYL); 4-dimethylaminoazobenzene-4'-thioisocyanate (DABITC); eosin and derivatives such as eosin and eosin isothiocyanate; erythrosine and derivatives such as erythrosine and erythrosine isothiocyanate; ethidium; fluorescein and derivatives such as 5-carboxyfluorescein (FAM), 5-(4,6-dichlorotriazine)aminofluorescein (DTAF), 2'7'-dimethoxy-4'5'-dichloro-6-carboxyfluorescein (JOE), fluorescein isothiocyanate (FITC), chlorotriazineylfluorescein, naphthylfluorescein and QFITC (XRITC); fluorescamine; IR144; IR1446; green fluorescent protein (GFP); reef coral fluorescent protein (RCFP); lissamine TM; lissamine rhodamine, fluorescein yellow; malachite green isothiocyanate; 4-methylumbelliferone; o-cresyl peptide; nitrotyrosine; pararosaniline; Nile red; Oregon green; phenol red; B-phycoerythrin; o-Phthaldehyde; pyrene and derivatives such as pyrene, pyrenebutyric acid and succinimidyl 1-pyrenebutyric acid; Reactive Red 4 (CibacronTM Brilliant Red 3B-A); Rhodamine and derivatives such as 6-carboxy-X-rhodamine (ROX), 6-carboxyrhodamine (R6G), 4,7-dichlororhodamine lissamine, rhodamine B sulfonyl chloride, rhodamine (Rhod), rhodamine B, rhodamine 123, rhodamine X isothiocyanate, sulforhodamine B, sulforhodamine 101, sulfonyl chloride derivative of sulforhodamine 101 (Texas Red), 6-carboxytetramethylrhodamine (TAMRA), tetramethylrhodamine and tetramethylrhodamine isothiocyanate (TRITC); riboflavin; rhodamine and terbium chelate derivatives; xanthene; or a combination thereof. Other fluorophores or combinations thereof known to those skilled in the art may also be used.

Preferably, the fluorescent dyes include: FITC, PE, PerCP, PE-Cy7, FITC, PE-Cy5, PerCP, PE-Cy7, Alexa Fluor 488, AF647, APC, APC-Cy7, BV421 and BV510.

The present invention also provides a kit for evaluating the function of T cell subsets, evaluating the activity of a disease, predicting treatment effects, clinical outcomes and prognosis, comprising a reagent composition or at least one group of reagent compositions as described in any one of the preceding claims, optionally further comprising instructions for use, a buffer and/or a control sample.

The kit is used for evaluating the activity level of a disease, the therapeutic effect, the clinical outcome and the prognosis prediction. The disease includes one or more of primary glomerular disease, autoimmune disease, infection, metabolic disease, blood system disease and drug-induced renal injury.

Preferably, the primary glomerular disease includes one or more of minimal change disease, focal segmental sclerosis, membranous nephropathy and IgA nephropathy;

The autoimmune disease includes one or more of systemic lupus erythematosus, Sjögren's syndrome, rheumatoid arthritis and mixed connective tissue disease;

The infection includes one or more of tuberculosis and novel coronavirus infection;

The metabolic disease includes one or more of diabetes, obesity and calciphylaxis;

The blood system disease includes one or more of amyloidosis, monoclonal immunoglobulinemia, multiple myeloma, lymphoma and thrombotic microangiopathy.

The present invention also provides a multicolor flow cytometry method for functional evaluation of T cell subsets, evaluation of disease activity, treatment effect, clinical outcome and prognosis prediction, which comprises the following steps:

(a) preparing or providing a biological sample containing T lymphocytes;

(b) contacting the sample with the above-mentioned reagent composition for evaluating the T cell immunological state of chronic pathological state of disease T lymphocytes;

(c) analyzing T lymphocytes in the sample in a flow cytometer; and

(d) Storing and analyzing the acquired data.

The multicolor flow cytometry method may further comprise any of the following steps:

(e) lysing red blood cells;

(f) isolating mononuclear cells and preparing a suspension;

(g) Blocking cell surface Fc receptors (for example, permeabilization is performed first for cytokine or nuclear transcription factor detection);

(h) fluorescent antibody incubation;

(i) washing free fluorescent antibodies and detecting them on an instrument;

(j) Fluorescence compensation adjustment;

(k) One or more of the following operations: establishing blank, isotype, FMO, negative and positive control groups.

The sample includes peripheral blood, bone marrow, tissue sample, or other types.

Preferably, the tissue sample is selected from lymph nodes, adenoids, spleen or liver.

Preferably, the body fluid is selected from the group consisting of cerebrospinal fluid, vitreous fluid, synovial fluid, fine needle aspirate, pleural effusion and ascites.

Preferably, the sample is peripheral blood.

The reagent composition can measure markers in serum samples, and the markers include any of CD3, CD4, CD8, CD16, CD25, CD45, CD45RA, CD45RO, CD56, CD62L, CD44, CD69, CD127, CD154, CCR7, CTLA4, IgM, FOXP3, TIM3, LAG3, PD1, PDL1, and PS6;

Preferably, the markers are CD3, CD4, CD8, TIM3, LAG3 and CTLA4;

Preferably, the markers are CD3, CD4, CD8, CD25, CD127 and FOXP3;

Preferably, the markers are CD3, CD4, CD8, CD25, CD127, FOXP3 and PS6;

Preferably, the markers are CD3, CD4, CD8, CD25, CD127, LAG3 and TIM3;

Preferably, the markers are CD45, CD3, CD4, CD8, CD25, TIM3, LAG3, PD1, PDL1 and CTLA4.

The flow cytometric analysis method further includes a data acquisition, analysis and recording device, such as a computer, wherein a plurality of data channels record the light scattering and fluorescence data emitted by each particle when it passes through the sensing area from each detector. The analysis system classifies and counts the particles, wherein each particle is presented as a series of digitized parameter values.

In the process of flow cytometric analysis of particles using the method of the present invention, the flow cytometer can be set to trigger at a selected parameter to distinguish the target particles from the background and noise. "Trigger" refers to a preset threshold for a detection parameter. It is generally used as a means to detect the passage of a particle through a laser beam. The detection of an event exceeding the preset threshold of the selected parameter triggers the collection of light scattering and fluorescence data for the particle. For particles or other components in the assay medium whose response is below the threshold, their data is not collected. The trigger parameter can be the detection of forward scattered light caused by the particle passing through the light beam. The flow cytometer then detects and collects the light scattering and fluorescence data of the particle.

The beneficial effects of the present invention are:

(1) Covering the functional evaluation of a wide range of T cell subsets through a combination of tests;

(2) Rapidly achieve accurate typing and immune activity detection of T cells, and evaluate the chronic activation state of T cells with high sensitivity and specificity;

(3) systematically evaluate disease severity, infection risk, and the effects of drugs on cellular immune function;

(4) The operation is simple, the results are easy to understand, and it is easy to promote and apply in primary medical institutions.

BRIEF DESCRIPTION OF THE DRAWINGS

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required to be used in the embodiments are briefly introduced below.

Figure 1. An example of clinical application of detailed detection of T lymphocyte subsets in patients with MCD treated with glucocorticoids and with or without concurrent infection. Results of flow cytometry analysis of 370 patients with glomerular diseases in different groups (no hormone and no infection, hormone and no infection, no hormone infection, hormone infection). A is the sorting strategy diagram of T lymphocyte subsets (CD45, CD3, CD4, CD8, TIM3, LAG3, CTLA4). As shown in the figure, all markers were detected using domestic standard clinical laboratory sorting and gating parameters; B is the proportion of CD3+T cells in patients in different groups; C is the proportion of CD3+CD4+T cells in different groups; D is the proportion of CD3+CDD8+T cells in different groups; E is the proportion of CD3+TIM3+T cells in different groups; F is the proportion of CD3+LAG3+T cells in different groups; G is the proportion of CD3+CTLA4+T cells in different groups (*P<0.05, **P<0.01, ***P<0.001, ****P<0.0001, NS, no statistical difference).

Figure 2. An example of the application of detailed detection of regulatory T lymphocytes in early judgment of recurrence and long-term remission of glomerular disease (minimal change disease, MCD). A is the sorting strategy diagram of T lymphocyte subsets (CD45, CD3, CD4, CD8, CD25, FOXP3). As shown in the figure, all markers were detected using domestic standard clinical laboratory sorting and gating parameters; B is the stratified analysis results of FOXP3 expression levels (FOXP3 + , FOXP3 medium , FOXP3 high ) of CD4+T cells in different groups, including healthy controls (HC), non-nephrotic syndrome minimal change disease (NNS-MCD), sustained remission minimal change disease (SR-MCD ⁾ and early relapse minimal change disease (ER-MCD) ^{; C is the correlation comparative analysis of FOXP3 expression levels (FOXP3 + , FOXP3 medium , FOXP3 high )} at different time points and disease states (*P<0.05, **P<0.01, ***P<0.001, ****P<0.0001, NS, no statistical difference).

Figure 3. Application example of regulatory T lymphocyte combined with PS6 protein detection in early judgment of relapse and long-term remission of glomerular disease (minimal change disease, MCD). A is the representative result of flow cytometry detection of mean fluorescence intensity (MFI) of PS6 protein of CD4+T cells in different groups of people, including healthy control (Ctrl), non-nephrotic syndrome (NNS), nephrotic syndrome (NS), complete remission (CR) and early relapse (ER); B is the relative proportion of CD4+PS6 ^high T cells in different groups of people before treatment; C is the change ratio of CD4+PS6 ^high T cells after complete remission of MCD for 2 months of hormone treatment compared with that before treatment; D is the change ratio of CD4+PS6 ^high T cells in MCD patients with early relapse (ER) compared with complete remission after 2 months of treatment; E is the change ratio of CD4+PS6 ^high T cells in MCD patients with sustained remission at 12 months compared with complete remission after 2 months of treatment. F is the receiver operating characteristic curve (ROC curve) for judging the early recurrence of MCD by using the CD4+FOXP3+T cell ratio; G is the ROC curve for judging the early recurrence of MCD by using the CD4+FOXP3 ^medium T cell ratio; H is the ROC curve for judging the early recurrence of MCD by using the CD4+PS6 ^high T cell ratio; I is the ROC curve for judging the early recurrence of MCD by using the combined CD4+FOXP3 ^medium and CD4+PS6 ^high T cell ratios.

Figure 4. Clinical application example of fine detection of T lymphocyte subsets in acute kidney injury (AKI) associated with novel coronavirus infection. The combined detection method of T lymphocyte subsets (CD3, CD4, CD8, CD25, CD127, LAG3 and TIM3) is as described above, where A is the flow cytometric analysis of the proportion of CD3+TIM3+T cell subsets in patients with minimal change disease (MCD), minimal change disease combined with acute kidney injury (MCD-AKI), drug-related AKI and novel coronavirus infection-related AKI; B is the analysis of the proportion of CD3+LAG3+T cell subsets; C is the analysis of the proportion of CD4+CD25+CD127 ^low T cells (*P<0.05, **P<0.01, ***P<0.001, ****P<0.0001).

Figure 5. Application of combined analysis of CD45, CD3, CD4, CD8, CD25, TIM3, LAG3, PD1, PDL1 and CTLA4 in systemic inflammatory damage caused by tuberculosis infection. A is the sorting strategy diagram and results of each T cell subset before treatment; B is the sorting strategy diagram and results of each T cell subset after treatment. C-G are the proportions of CD3+TIM3+, CD3+CTLA4+, CD3+LAG3+, CD3+PD1+ and CD4+CD25+T cell subsets detected dynamically at different time points during the 12-month follow-up.

Figure 6. Clinical application example of CD3+TIM3+ and CD3+LAG3+ T cell subset detection in the evaluation of severe systemic lupus erythematosus (SLE). The detection methods of T lymphocyte subsets (CD3, LAG3, and TIM3) were described previously, and >1000 cases of flow cytometric analysis were included, including systemic lupus erythematosus (SLE, n = 409), minimal change disease (MCD, n = 97), focal segmental glomerulosclerosis (FSGS, n = 67), membranous nephropathy (MN, n = 252), and IgA nephropathy (IgAN, n = 227). Among them, A–E are the proportions of CD3+TIM3+T cell subsets in representative cases; A is a patient with severe SLE combined with lupus encephalopathy and lupus nephritis, in which the proportion of CD3+TIM3 T cells increased significantly and returned to the normal range after effective treatment; B is a patient with severe SLE with relapse; C is a patient with quiescent SLE; D is a patient with drug-induced AKI; E is a patient with CKD Stage 5 patients; F is a comparative analysis of the proportion of CD3+TIM3+T cells between SLE patients and other primary glomerular patients; G is a comparative analysis of the proportion of CD3+LAG3+T cells between SLE patients and other primary glomerular patients; H is a correlation analysis between CD3+TIM3+ and CD3+LAG3+T cell subsets; I and J are correlation analyses of CD3+TIM3+ (I) and CD3+LAG3+T cell (J) subsets with traditional clinical indicators for evaluating SLE disease activity, including complement (C1q, C3, C4), autoantibodies (ds-DNA), urine tests (urine albumin, urine total protein, urine red blood cells), serum creatinine (Scr) and C-reactive protein (CRP).

Detailed ways

The present invention provides a combination of T cell immunological markers for evaluating the chronic pathological state of disease T lymphocytes, including any of CD3, CD4, CD8, CD16, CD25, CD45, CD45RA, CD45RO, CD56, CD62L, CD44, CD69, CD127, CD154, CCR7, CTLA4, IgM, FOXP3, TIM3, LAG3, PD1, PDL1 and PS6. In the present invention, the disease preferably includes one or more of primary glomerular disease, autoimmune disease, infection, metabolic disease, blood system disease and drug-induced renal injury. In the present invention, the primary glomerular disease preferably includes one or more of minimal change disease, focal segmental sclerosis, membranous nephropathy and IgA nephropathy; the autoimmune disease preferably includes one or more of systemic lupus erythematosus, Sjögren's syndrome, rheumatoid arthritis and mixed connective tissue disease; the infection preferably includes one or more of tuberculosis and novel coronavirus infection; the metabolic disease preferably includes one or more of diabetes, obesity and calciphylaxis;

The blood system disease preferably includes one or more of amyloidosis, monoclonal immunoglobulinemia, multiple myeloma, lymphoma and thrombotic microangiopathy.

In the present invention, when evaluating the immune function of patients treated with glucocorticoids, the T cell immunological state marker combination preferably includes CD3, CD4, CD8, TIM3, LAG3, CTLA4, PD1 and PDL1.

In the present invention, when detecting minimal lesions, the T cell immunological state marker combination preferably includes CD3, CD4, CD8, PS6, CD25, CD127 and FOXP3.

In the present invention, when detecting systemic inflammatory damage caused by tuberculosis infection, the T cell immunological marker combination preferably includes CD45, CD3, CD4, CD8, CD25, TIM3, LAG3, PD1, PDL1 and CTLA4.

In the present invention, when evaluating the chronic pathological state of T cells in different glomerular diseases, the T cell immunological state marker combination preferably includes CD3, CD4, CD8, CD25, CD127, TIM3, LAG3, CTLA4 and PS6.

In the present invention, when evaluating the therapeutic effect of a disease, the T cell immunological state marker combination preferably includes CD3, CD4, CD8, CD25, CD62L, CD44, CD69, CD127, FOXP3, TIM3, LAG3, CTLA4 and PS6.

In the present invention, the disease preferably includes one or more of lupus nephritis, minimal change disease, focal segmental glomerulosclerosis and membranous nephropathy.

The present invention also provides the use of the combination of T cell immunological state markers described in the above technical solution in the preparation of a reagent for evaluating the chronic pathological state of disease T lymphocytes.

The present invention screened and verified different combinations of 106 T cell markers through flow cytometric detection of 3386 clinical patients, which covered cell surface marker proteins, cytokines, nuclear transcription factors and key signal pathway molecules. The test indicators specifically included: Bcl-6, CCR10, CCR3, CCR4, CCR5, CCR6, CCR7, CD127, CD137, CD152, CD154, CD16, CD161, CD21, CD23, CD25, CD27, CD28, CD3, CD38, CD4, CD40L, CD44, CD45, CD45RA, CD45RO, CD56, CD58, CD62L, CD69, CD8, CD80, CD86, CD95, CTLA-4, CXCR3, CXCR5, EOMES, FAS, FOXO1, FOXO3, FOXO4, FOXP1, FOXP3, GATA3,Granzyme,GZMA,HLA-DR,ICOS,IFN-gamma,IgD,IgM,IL-10,IL-12,IL-13,IL-17,IL-18,IL-2,IL-21,IL-22,IL-25,IL-26,IL-4,IL-5,IL-9,IL23R,IL2RA,IL2RB,IRF4,ITGAE,ITGAL,Ki 67,KLRB1,LAG3,LEF1,Lymphotoxin,NCAM1,NK1.1,NKG2D,OX40,p-mTOR,PD1,PDL1,PECAM1,Perforin,PRDM1,PS6,PTGDR2,PU.1/Spi1,RORC,RORγt,SELL,STAT1,STAT4,STAT5,T-bet,TBX21,TCF7,TCR Vα24, TCR Vβ11, TCRγ/δ, TGF-beta, TIM1, TIM3, TNF-alpha, TNF-beta.

Multi-channel flow cytometry detection selects labeled antibodies according to different monoclonal antibody sources, excitation light wavelengths and fluorescent spectral characteristics, including FITC, PE, PerCP, PE-Cy7, FITC, PE-Cy5, PerCP, PE-Cy7, Alexa Fluor 488, AF647, APC, APC-Cy7, BV421 and BV510. Conventional multi-channel flow cytometers can complete this protocol. The flow cytometry detection related instruments, equipment, antibodies or reagents used can be purchased directly from commercial companies BD Biosciences, BioLegend, Beckman and Cell Signaling Technology or made by yourself. The detection operation steps include: extracting 1-2ml peripheral blood, lysing red blood cells, separating mononuclear cells and preparing suspension, blocking cell surface Fc receptors (if cytokine or nuclear transcription factor detection is performed first, membrane permeabilization treatment is performed), incubating with fluorescent conjugated antibodies for 30 minutes, washing free fluorescent antibodies and detecting on the machine, completing fluorescence compensation adjustment, and analyzing the detection results. All test indicators have strict blank, Isotype, FMO, negative and positive control groups.

The phenotype and function of peripheral blood immune cells of 3386 patients were analyzed by multi-channel flow cytometry, and finally 23 T cell immunology markers and combinations were established from the above 106 flow cytometric indicators to reflect the chronic pathological state of T lymphocytes in different diseases, including CD3, CD4, CD8, CD16, CD25, CD45, CD45RA, CD45RO, CD56, CD62L, CD44, CD69, CD127, CD154, CCR7, CTLA4, IgM, FOXP3, TIM3, LAG3, PD1, PDL1, PS6. The present invention is further verified by long-term follow-up (>12 months) of more than 1500 clinical patients, confirming that the above marker combination scheme can effectively evaluate the chronic pathological state of T cells and the degree of disease activity, therapeutic effect and clinical outcome, and has important clinical guidance value. The present invention has been clinically verified in a variety of diseases, including: primary glomerular diseases (minimal change disease, focal segmental sclerosis, membranous nephropathy, IgA nephropathy), autoimmune diseases (systemic lupus erythematosus, Sjögren's syndrome, rheumatoid arthritis, mixed connective tissue disease), infections (tuberculosis, new coronavirus infection), metabolic diseases (diabetes, obesity, calciphylaxis), blood system diseases (amyloidosis, monoclonal immunoglobulinemia, multiple myeloma, lymphoma, thrombotic microangiopathy) and drug-induced renal injury, etc.

The present invention also provides the use of the T cell immunological state marker combination described in the above technical solution in the preparation of disease auxiliary diagnosis or prognosis prediction products.

The product may be a kit comprising antibodies against CD3, CD4, CD8, CD16, CD25, CD45, CD45RA, CD45RO, CD56, CD62L, CD44, CD69, CD127, CD154, CCR7, CTLA4, IgM, FOXP3, TIM3, LAG3, PD1, PDL1, PS6,

Preferably, antibodies against CD3, CD4, CD8, TIM3, LAG3 and CTLA4;

Preferably, antibodies against CD3, CD4, CD8, CD25, CD127 and FOXP3;

Preferably, antibodies against CD3, CD4, CD8, CD25, CD127, FOXP3 and PS6;

Preferably, it is an antibody against CD3, CD4, CD8, CD25, CD127, LAG3 and TIM3;

Preferably, it is an antibody against CD45, CD3, CD4, CD8, CD25, TIM3, LAG3, PD1, PDL1 and CTLA4.

The kit also includes markers capable of measuring serum samples, wherein the markers include: CD3, CD4, CD8, CD16, CD25, CD45, CD45RA, CD45RO, CD56, CD62L, CD44, CD69, CD127, CD154, CCR7, CTLA4, IgM, FOXP3, TIM3, LAG3, PD1, PDL1, PS6,

Preferably, CD3, CD4, CD8, TIM3, LAG3 and CTLA4;

Preferably, CD3, CD4, CD8, CD25, CD127 and FOXP3;

Preferably, CD3, CD4, CD8, CD25, CD127, FOXP3 and PS6;

Preferably, CD3, CD4, CD8, CD25, CD127, LAG3 and TIM3;

Preferably, CD45, CD3, CD4, CD8, CD25, TIM3, LAG3, PD1, PDL1 and CTLA4.

The kit also includes a fluorescent dye, the absorption wavelength and emission wavelength of which are in the range of 300nm-810nm, including but not limited to disodium 4-acetylamino-4'-isothiocyanatostilbene-2,2'-disulfonic acid; acridine and its derivatives such as acridine, acridine orange, acridine yellow, acridine red and acridine isothiocyanate; 5-(2-aminoethylamino)-1-naphthalenesulfonic acid sodium salt (EDANS); 4-amino-N-[3-(vinylsulfonyl)phenyl]naphthalimide-3,5-disulfonate (fluorescent yellow VS); N-(4-anilino-1-naphthyl)maleimide; 2-aminobenzamide; brilliant yellow; coumarin and its derivatives such as coumarin, 7-acetoxy-4-methylcoumarin (AMC, coumarin 120), 7-amino-4-tris(vinyl)-1-naphthalenesulfonic acid sodium salt (EDANS); Fluoromethylcoumarin (Coumaran 151); cyanines and derivatives such as tetrabromotetrachlorofluorescein, Cy3, Cy5, Cy5.5, and Cy7; 4',6-diamidino-2-phenylindole (DAPI); bromopyrogallol red; 7-diethylamino-3-(4'-isothiocyanatophenyl)-4-methylcoumarin; diethylaminocoumarin; diethylenetriamine pentaacetate; 4,4'-diisothiocyanatodihydrostilbene-2,2'-disulfonic acid; 4,4'-diisothiocyanato-2,2'-stilbene disulfonic acid; dansyl chloride (DNS, dansyl chloride); 4-(4'-dimethylaminophenylazo)benzoic acid (DABCYL); 4-dimethylaminoazobenzene-4'-thioisocyanate (DABITC); eosin and derivatives such as eosin and eosin isothiocyanate esters; erythrosine and derivatives such as erythrosine and erythrosine isothiocyanate; ethidium; fluorescein and derivatives such as 5-carboxyfluorescein (FAM), 5-(4,6-dichlorotriazine)aminofluorescein (DTAF), 2'7'-dimethoxy-4'5'-dichloro-6-carboxyfluorescein (JOE), fluorescein isothiocyanate (FITC), chlorotriazineylfluorescein, naphthylfluorescein and QFITC (XRITC); fluorescamine; IR144; IR1446; green fluorescent protein (GFP); reef coral fluorescent protein (RCFP); lissamine TM; lissamine rhodamine, fluorescein yellow; malachite green isothiocyanate; 4-methylumbelliferone; o-cresyl peptide; nitrotyrosine; pararosaniline; Nile red; Oregon green; phenol red; B-phycoerythrosine Protein; o-phthalaldehyde; pyrene and derivatives such as pyrene, pyrenebutyric acid and succinimidyl 1-pyrenebutyric acid; Reactive Red 4 (CibacronTM Brilliant Red 3B-A); Rhodamine and derivatives such as 6-carboxy-X-rhodamine (ROX), 6-carboxyrhodamine (R6G), 4,7-dichlororhodamine lissamine, rhodamine B sulfonyl chloride, rhodamine (Rhod), rhodamine B, rhodamine 123, rhodamine X isothiocyanate, sulforhodamine B, sulforhodamine 101, sulfonyl chloride derivative of sulforhodamine 101 (Texas Red), 6-carboxytetramethylrhodamine (TAMRA), tetramethylrhodamine and tetramethylrhodamine isothiocyanate (TRITC); riboflavin; rhodamine and terbium chelate derivatives; xanthene; or a combination thereof. Other fluorophores or combinations thereof known to those skilled in the art may also be used.

Preferably, the fluorescent dyes include: FITC, PE, PerCP, PE-Cy7, FITC, PE-Cy5, PerCP, PE-Cy7, Alexa Fluor 488, AF647, APC, APC-Cy7, BV421 and BV510.

The kit also includes reagents for performing flow cytometric analysis. Examples of reagents include buffers for at least one of reconstitution and dilution of the detectable molecule, buffers for contacting a control composition or cell sample with one or more binding components, wash buffers, control beads, fluorescent beads for flow cytometer calibration, and combinations thereof.

In order to further illustrate the application of the marker combination, kit, and flow cytometry method described in the present invention, the present invention is described in detail below in conjunction with examples, but they should not be construed as limiting the scope of protection of the present invention.

Example 1

Application of combined analysis of CD3, CD4, CD8, TIM3, LAG3 and CTLA4 in the evaluation of immune function in patients with the above diseases treated with long-term glucocorticoids.

The method is as follows: 5 ml of peripheral blood is extracted from the patient, and peripheral blood mononuclear cells (PBMC) are separated and extracted by Ficoll density gradient centrifugation at a speed of 2000r/min for 30 minutes. 70-90% of PBMCs are lymphocytes (70-90%), of which CD3+T cells account for the vast majority of lymphocytes (45-70%). By extracting PBMC, CD3+CD4+T cells can be effectively further analyzed to avoid the influence of other blood components on the test results. The gate setting, blank control and analysis method of flow cytometric detection are shown in Figure 1 A. According to the conventional clinical flow cytometer detection method, the cells are resuspended at a concentration of 2-5×10 ⁶ cells/mL, and the primary antibody (0.1-10μg/mL) is added according to the reference value range recommended in the instructions and incubated at room temperature or 4°C in the dark for 30-60 minutes. This embodiment uses a flow cytometer with more than 4 channels for detection. Commercial companies can provide a variety of primary antibodies or fluorescein secondary antibodies with self-contained fluorescent labels. The principle of antibody selection is to use one fluorescein for each channel. The fluorescein combination between channels is reasonably allocated according to the strength of antigen expression and the fluorescein with small spectral overlap is selected. An optimized fluorescein-antibody combination scheme can be used.

The results of a study of 370 patients with glomerular diseases (Figure 1) confirmed that long-term hormone therapy significantly reduced the proportion of CD3+CD4+T cells and relatively increased the proportion of CD3+CD8+T cells, resulting in a significant decrease in the CD4/CD8 ratio, especially when combined with infection. The above indicators can be used to clinically evaluate the risk of low cellular immunity caused by hormone therapy. At the same time, further joint analysis confirmed that CD3+TIM3+, CD3+LAG3+ and CD3+CTLA4+T cells were significantly increased in patients with hormone therapy, especially in patients with infection during hormone therapy. The combined analysis of the above indicators, which increased by 50% over the normal range or 25% over the individual baseline value, can indicate that hormones cause a decrease in cellular immunity and an increased risk of infection. This combination has been proven to be highly specific and significant by multiple disease types, large samples, and long-term clinical follow-up studies. At present, there are no clinical alternatives similar to this combination at home and abroad.

Example 2

Application of combined analysis of CD3, CD4, CD8, CD25, CD127 and FOXP3 in early judgment of complete remission, early relapse and long-term remission of glomerular disease (minimal change disease, MCD).

The conventional manipulation method of flow cytometry detection is described in Example 1, wherein the CD4+FOXP3+ and CD4+CD25+CD127-T cells involved in this scheme are highly homologous (>95%). FOXP3 is a factor expressed in the nucleus, and cell fixation and membrane permeabilization are required before antibody incubation. The above indicators are used as regulatory T cell markers.

As shown in Figure 2, 23 patients with nephrotic syndrome were followed up for 12 months. The proportion of peripheral blood T cell subsets was detected at 0, 2, 4, 8, 12 months and the time point of early relapse, and T cells were stratified and analyzed according to the expression level of FOXP3. The results of the study on healthy controls (HC), non-nephrotic minimal change disease (NNS-MCD), sustained remission (SR-MCD) and early relapse (ER-MCD) confirmed that the change in the proportion of regulatory T cell subsets was closely related to the treatment outcome of nephrotic syndrome. Among them, the increase in the expression of FOXP3 ⁺ and FOXP3 ^medium groups (>25%) indicated that the treatment was effective and the disease was completely relieved; while the decrease in both groups (>25%) indicated early relapse of the disease; in patients with long-term stable and sustained remission, the proportion of FOXP3 ⁺ and FOXP3 ^medium subgroups did not change significantly before and after. Therefore, this detection combination has high specificity and sensitivity for predicting early remission, early relapse and sustained remission of MCD disease. At present, there is no clinical alternative similar to this combination at home and abroad.

Example 3

Application of combined analysis of CD3, CD4, CD8, CD25, CD127, FOXP3 and PS6 in judging complete remission, early relapse and long-term remission after treatment of glomerular disease (minimal change disease, MCD). The conventional operation method of flow cytometry detection is as described in Example 1 above.

As shown in Figure 3, the long-term follow-up results of 23 patients with nephrotic syndrome for 12 months confirmed that the proportion of CD4+PS6 ^high T cells in patients with nephrotic syndrome MCD (SR-MCD and ER-MCD) was significantly higher than that in the control group (HC) and non-nephrotic syndrome MCD patients (NNS-MCD), especially in patients with early relapse (ER-MCD). The increase was more obvious; further continuous dynamic detection results confirmed that the proportion of CD4+PS6 ^high T cells decreased significantly when complete remission occurred after treatment; the proportion of CD4+PS6 ^high T cells increased again when relapse occurred; and the proportion of CD4+PS6 ^high T cells in sustained remission (12 months) did not change significantly from that in complete remission (2 months). Changes in the proportion of CD4+PS6 ^high T cells (>25%) are effective indicators of disease outcome. Through receiver operating characteristic curve (ROC curve) analysis, the areas under the curve (AUC) of CD4+FOXP3 ⁺ , CD4+FOXP3 ^medium and CD4+PS6 ^high T cell subsets for predicting early recurrence of MCD were 0.79, 0.79 and 0.89, respectively, while the AUC of combined analysis of CD4+FOXP3 ^medium and CD4+PS6 ^high T cells reached 0.92. All of the above indicators have the characteristics of high sensitivity and high specificity, and the advantage of combined analysis is more significant.

The above results confirm that the proportion of CD4+FOXP3 ⁺ , CD4+FOXP3 ^medium , and CD4+PS6 ^high T cells can be used as an effective predictor of improvement, long-term stability, and early relapse in MCD patients, which is an important clinical problem that needs to be solved urgently. This combination has been proven to be highly specific and significant through long-term clinical follow-up studies. At present, there are no clinical alternatives similar to this combination at home and abroad.

Example 4

Application of combined analysis of CD3, CD4, CD8, CD25, CD127, LAG3 and TIM3 in acute kidney injury associated with novel coronavirus infection. The conventional operation method of flow cytometry detection is described in the previous Example 1. The flow cytometry detection results of 17 patients with clinical diagnosis of acute kidney injury (AKI) associated with novel coronavirus infection were analyzed. Compared with minimal change disease (MCD), minimal change disease combined with acute kidney injury (MCD-AKI) and drug-related AKI, the proportion of CD3+TIM3+ and CD4+CD25+CD127 ^low T cells in new crown-related AKI was significantly increased, while the proportion of CD3+LAG3+T cells decreased. The above results suggest that the combined analysis of the increase in the proportion of CD3+TIM3+ and CD4+CD25+CD127 ^low T cells (>25% of normal value) and the decrease in the proportion of CD3+LAG3+T cells (>10% of normal value) has important clinical guidance value for judging the risk of multiple organ damage in patients with new crown infection (especially the elderly high-risk population) combined with AKI. Currently, there are no clinical alternatives similar to this combination either at home or abroad.

Example 5

The application of CD45, CD3, CD4, CD8, CD25, TIM3, LAG3, PD1, PDL1 and CTLA4 combined analysis in systemic inflammatory damage caused by tuberculosis infection. Flow cytometry detection routine operation method As described in the previous embodiment 1, the results of 12 months of follow-up of cases of severe tuberculosis infection combined with kidney, liver, and peritoneal multi-organ inflammatory response confirmed that chronic infection caused a significant increase in the proportion of suppressive CD3+TIM3+T and CD3+CTLA4+T cells, and no significant changes were observed in other T cell markers; after anti-tuberculosis treatment, the changes in CD3+TIM3+T and CD3+CTLA4+T cell subsets were highly consistent with the pathological changes of diffuse inflammatory cell infiltration in multiple organs, and the combination could sensitively detect changes in key T cell subtypes, specifically reflecting the disease outcome, and was closely related to the treatment effect. The combination has been confirmed to have high specificity and significance through long-term clinical follow-up studies. At present, there are no clinical alternatives similar to this combination at home and abroad.

Example 6

Application of CD3+TIM3+ and CD3+LAG3+T cell subset detection in evaluating the severity, treatment effect and remission status of systemic lupus erythematosus. The conventional operation method of flow cytometry detection is as described in the previous Example 1. Long-term follow-up of the case cohort and systematic analysis of the flow cytometric detection results of >1000 patients have confirmed that the proportions of CD3+TIM3+ and CD3+LAG3+T cell subsets in systemic lupus erythematosus nephritis (n=409) are significantly increased compared with primary glomerular diseases, including: minimal change disease (n=97), focal segmental glomerulosclerosis (n=67), membranous nephropathy (n=252), and IgA nephropathy (n=227). The changes in the two are highly consistent and are closely related to the severity of the disease, treatment response and long-term stable state of the disease. The correlation analysis with multiple traditional clinical indicators confirmed that the increase in the proportion of the two (>25% of the normal value or >50% of the baseline value) can be used as an effective indicator for evaluating the severity of systemic lupus erythematosus nephritis. This combination has been tested on a large number of clinical samples and is highly specific for evaluating the severity, treatment effect and disease remission of SLE. It has important guiding value in the clinical diagnosis and treatment of severe SLE (lupus encephalopathy and lupus nephritis). Currently, there is no clinical alternative similar to this combination at home and abroad.

Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims (4)

1. Application of CD3, CD4, CD8, CD25, CD127, FOXP3 and PS6 in the preparation of a combined analysis kit for early judgment of complete remission, early relapse and long-term remission of nephrotic syndrome MCD. 2. The use according to claim 1, characterized in that: The kit also includes a buffer and a fluorescent dye. 3. The use according to claim 2, characterized in that the absorption wavelength and/or emission wavelength range of the fluorescent dye is 300nm-810nm. 4. The use according to claim 2 or 3, characterized in that the fluorescent dyes include: FITC, PE, PerCP, PE-Cy7, FITC, PE-Cy5, PerCP, PE-Cy7, Alexa Fluor488, AF647, APC, APC-Cy7, BV421 and BV510.