CN116430055A - T cell immunological state marker combination for evaluating chronic pathological state of T lymphocyte of disease and application thereof - Google Patents

T cell immunological state marker combination for evaluating chronic pathological state of T lymphocyte of disease and application thereof Download PDF

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CN116430055A
CN116430055A CN202310435512.XA CN202310435512A CN116430055A CN 116430055 A CN116430055 A CN 116430055A CN 202310435512 A CN202310435512 A CN 202310435512A CN 116430055 A CN116430055 A CN 116430055A
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cell
disease
marker combination
tim3
lag3
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陈国纯
陈慧慧
谭重庆
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Second Xiangya Hospital of Central South University
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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

T cell immunological state marker combination for evaluating chronic pathological state of T lymphocyte of disease and application thereof
Technical Field
The invention relates to the field of disease evaluation, in particular to a T cell immunological state marker combination for evaluating chronic pathological states of T lymphocytes of diseases.
Background
Chronic diseases (infections, tumors, metabolic diseases, cardiovascular diseases, nervous system diseases, etc.) are the most major factors threatening the global health of humans, and chronic systemic inflammatory response is one of the key pathophysiological mechanisms of all chronic diseases. T cells are the most important effector cells that regulate immune homeostasis in humans, can be classified into various subtypes according to their surface markers and functions, and are in the process of dynamic switching. The continuous activation state of T cells can cause systemic inflammatory injury by secreting a large amount of cytokines, and mediate the occurrence and development processes of various chronic diseases; on the other hand, T cells in constant activation continuously secrete large amounts of cytokines, ultimately leading to depletion of cell function. Once the T cell function is depleted, the immune system is permanently and severely damaged, the body is hypo-resistant, and life-threatening severe infections are liable to occur. Timely and accurate assessment of T cell immunophenotype and functional homeostasis and adjustment of therapeutic regimens are significant clinical problems to be solved urgently.
The method for evaluating the immune function and inflammatory response index of the patient, which is commonly used by clinicians, mainly depends on the traditional blood biochemical detection project (such as blood convention, immunoglobulin, C-reactive protein and the like), and has the defects of low sensitivity and specificity, easiness in being influenced by different disease factors and the like. Flow cytometry is a novel inspection technology which is developed and popularized in the medical field in recent years, and commonly evaluates immune function indexes including basic parting detection of CD3+ T cells, and the flow cytometry detection can screen, separate and detect the quantity and proportion of the T cells of a patient, but clinical practice finds that a plurality of shortages of improvement exist in the current flow cytometry detection scheme. Firstly, the existing common flow cytometry parting detection indexes are limited to the surface markers (CD 3, CD4 and CD 8) of the T cells, and can only prompt the total number or proportion of the whole T cells to change, so that the biological function change and the chronic activation state of various T cell subsets are difficult to be reflected finely, and a clinician cannot be guided to judge the T cell functions and the immune homeostasis of a patient in time.
Infections have been the most serious threat to human health. Normal T cell immunity is one of the most important lines of defense against invasion by various foreign pathogens. In various disease states, patients may develop chronic activation of long-term T cells and ultimately lead to the occurrence of T cell function depletion; at the same time, various clinical immune intervention treatment schemes also have significant effects on T cell function. T cell dysfunction can lead to the occurrence of fatal infections. Therefore, timely and accurate assessment of a patient's T cell chronic activation state is critical to guiding a clinician in formulating or adjusting a treatment regimen, especially for patients requiring long-term treatment with immunosuppressant regimens.
Clinicians have long been accustomed to using blood routine, immunoglobulin, etc. test methods to determine a patient's level of immunity. Recently, along with the popularization of clinical application of flow cytometry, the detection of two different T cells of CD4+ and CD8+ can be realized. Although some patients with obvious hypoimmunity (such as obvious leucopenia, hypoIgG blood disease, CD4/CD8 low value, etc.) can be screened in the existing biochemical detection projects to a certain extent, most patients cannot have leucopenia or IgG level reduction in early stage or even long time of immune function impairment. This is because immune cells including T cells have been significantly abnormal in their biological activity before they undergo 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, the protection function of clearing the external pathogenic bacteria is obviously impaired, and the above detection technology cannot reflect the actual situation of low organism immunity in time, and cannot meet the requirements of modern clinical medicine.
The multiparameter flow cytometry can combine the fluorescent labeled antibody with target protein expressed by T cell, detect the fluorescent intensity produced by the cell to obtain the expression level of the target protein, and subdivide one great class of T cell into several T cell subsets with different functions. The multi-parameter flow cytometry can detect a plurality of surface markers of cells at one time, so that the number, the proportion and the biological functions of T cells are systematically evaluated, and systematic judgment is made on the disease state, the infection risk, the drug curative effect and the like of a patient. However, since the number of T cell surface proteins (markers) is large, and the difference in cell biological functions reflected by different empty combinations of the various protein expressions is significant, the variation is particularly large during disease progression and drug treatment, and the possibility of combination variation in geometric progression is formed. How to find expression combinations with clinical diagnostic significance and to rapidly and accurately analyze the functional activity of T cells according to the expression level thereof is still a clinical problem which is not solved at present.
Disclosure of Invention
In order to solve the problems, the invention provides a T cell immunological state marker combination for evaluating the chronic pathological state of disease T lymphocytes, and the invention covers the function evaluation, the disease activity evaluation, the treatment effect, the clinical prognosis and the prognosis prediction of a wide range of T cell subsets by detecting the combination.
In order to achieve the above object, the present invention provides the following technical solutions:
the present invention provides a T cell immunological status marker combination for assessing a chronic pathological status of a disease T lymphocyte, comprising any of CD3, CD4, CD8, CD16, CD25, CD45RA, CD45RO, CD56, CD62L, CD, CD69, CD127, CD154, CCR7, CTLA4, igM, FOXP3, TIM3, LAG3, PD1, PDL1 and PS6 (Phospho-S6 Ribosomal Protein).
Preferably, the disease comprises one or more of a primary glomerular disease, an autoimmune disease, an infection, a metabolic disease, a disease of the blood system, and a pharmaceutical kidney injury.
Preferably, the primary glomerular disease comprises one or more of a micro-lesion, focal segmental sclerosis, membranous nephropathy, and IgA nephropathy;
the autoimmune disease includes one or more of systemic lupus erythematosus, sjogren's syndrome, rheumatoid arthritis, and mixed connective tissue disease;
The infection includes one or more of tuberculosis and a novel coronavirus infection;
the metabolic disease includes one or more of diabetes, obesity, and calcification defenses;
the blood system diseases include one or more of amyloidosis, monoclonal immunoglobulin blood, multiple myeloma, lymphoma and thrombotic microangiopathy.
Preferably, when evaluating the immune function of a glucocorticoid-treated patient, the T cell immunological marker combination comprises CD3, CD4, CD8, CD19, CD25, CD127, FOXP3, TIM3, LAG3, CTLA4, and PS6.
Preferably, when detecting a microscopic lesion, the T cell immunological marker combination comprises CD3, CD4, CD8, CD25, CD127, FOXP3 and PS6.
Preferably, when detecting systemic inflammatory lesions caused by tuberculosis infection, the T cell immunological marker combination comprises CD45, CD3, CD4, CD8, CD25, TIM3, LAG3, PD1, PDL1 and CTLA4.
Preferably, the T cell immunological marker combination comprises CD3, CD4, CD8, CD25, CD127, FOXP3, TIM3 and LAG3 when detecting systemic inflammatory lesions, especially acute kidney lesions, caused by novel coronavirus infection.
Preferably, when evaluating the chronic pathological status of T cells of different glomerular diseases, the T cell immunological status marker combination comprises CD3, CD4, CD8, CD25, CD127, TIM3, LAG3, CTLA4, FOXP3 and PS6.
The diseases include lupus nephritis, micro lesions, focal segmental glomerulosclerosis and membranous nephropathy.
The invention also provides application of the T lymphocyte immunological state marker combination in preparation of a reagent composition for evaluating chronic pathological state of T lymphocytes.
The product is a reagent composition for evaluating a T cell immunological state of a chronic pathological state of a disease T lymphocyte, comprising a fluorochrome-conjugated antibody directed against a marker combination comprising: any of CD3, CD4, CD8, CD16, CD25, CD45RA, CD45RO, CD56, CD62L, CD, CD69, CD127, CD154, CCR7, CTLA4, igM, FOXP3, TIM3, LAG3, PD1, PDL1 and PS 6;
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 the emission wavelength of the fluorescent dye are in the range of 300nm-810nm, including but not limited to disodium 4-acetamido-4 '-isothiocyanatestilbene-2, 2' -disulfonic acid; acridine and derivatives thereof such as acridine, acridine orange, acridine yellow, acridine red, and isothiocyanate acridine; 5- (2-aminoethylamino) -1-naphthalenesulfonic acid sodium salt (EDANS); 4-amino-N- [3- (vinylsulfonyl) phenyl ] naphthalimide-3, 5-disulfonate (fluorescein VS); n- (4-aniline-1-naphthyl) maleimide; 2-aminobenzamide; brilliant yellow; coumarin and derivatives such as coumarins, 7-acetoxy-4-methylcoumarin (AMC, coumarin 120), 7-amino-4-trifluoromethylcoumarin (coumarin 151); cyan pigments and derivatives such as tetrabromo tetrachlorofluorescein, cy3, cy5, cy5.5, and Cy7;4', 6-diamidino-2-phenylindole (DAPI); bromophthalic-trimesic-red; 7-diethylamino-3- (4' -isothiocyanatophenyl) -4-methylcoumarin; diethylaminocoumarin; diethylenetriamine pentaacetic acid ester; 4,4 '-diisothiocyanodihydrostilbene-2, 2' -disulfonic acid; 4,4 '-diisothiocyano-2, 2' -stilbenedisulfonic acid; dansyl chloride (DNS, dansyl chloride); 4- (4' -dimethylaminophenylazo) benzoic acid (DABCYL); 4-dimethylaminoazobenzene-4' -thioisocyanate (DABITC); eosin and derivative such as eosin and eosin isothiocyanate; erythrosine and derivative organisms such as erythrosine and erythrosine isothiocyanate; ethidium; luciferins 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), chlorotriazinyl fluorescein, naphthyl fluorescein and qflitc (XRITC); fluorescent amine; IR144; IR1446; green Fluorescent Protein (GFP); reef Coral Fluorescent Protein (RCFP); lizheimine TM; lisamine rhodamine, fluorescein; malachite green isothiocyanate; 4-methylumbelliferone; o-cresol peptide; nitrotyrosine; pararosaniline; nile red; green state oregon; phenol red; b-phycoerythrin; phthalic dicarboxaldehyde; the middlenaphthalene and derivative organisms such as middlenaphthalene, pyrene butyric acid and succinimidyl 1-pyrene butyric acid; reactive Red 4 (Cibacron bright Red 3B-A); rhodamine and derivatives such as 6-carboxy-X-Rhodamine (ROX), 6-carboxyrhodamine (R6G), 4, 7-dichloro rhodamine Lissamine, rhodamine B sulfonyl chloride, rhodamine (Rhod), rhodamine B, rhodamine 123, rhodamine X isothiocyanate, sulfonylrhodamine B, sulfonylrhodamine 101, sulfonylchloromercuride of sulfonylrhodamine 101 (red, texas), 6-carboxytetramethyl rhodamine (TAMRA), tetramethyl rhodamine, and Tetramethyl Rhodamine Isothiocyanate (TRITC); riboflavin; rosolic acid and terbium chelate complexes; xanthenes; or a combination thereof. Other fluorophores known to those skilled in the art, or combinations thereof, may also be used.
Preferably, the fluorescent dye comprises: FITC, PE, perCP, PE-Cy7, FITC, PE-Cy5, perCP, PE-Cy7, alexa Fluor 488, AF647, APC-Cy7, BV421 and BV510.
The invention also provides a kit for use in the invention for functional assessment of T cell subsets, activity assessment of disease, therapeutic effect, clinical outcome and prognosis prediction comprising a reagent composition or at least one set of reagent compositions according to any of the preceding claims, optionally together with instructions for use, buffers and/or control samples.
The kit is used for evaluating the activity level of the disease, treating effect, clinical prognosis and prognosis prediction. The diseases include one or more of primary glomerular diseases, autoimmune diseases, infections, metabolic diseases, blood system diseases and pharmaceutical kidney injuries.
Preferably, the primary glomerular disease comprises one or more of a micro-lesion, focal segmental sclerosis, membranous nephropathy, and IgA nephropathy;
the autoimmune disease includes one or more of systemic lupus erythematosus, sjogren's syndrome, rheumatoid arthritis, and mixed connective tissue disease;
The infection includes one or more of tuberculosis and a novel coronavirus infection;
the metabolic disease includes one or more of diabetes, obesity, and calcification defenses;
the blood system diseases include one or more of amyloidosis, monoclonal immunoglobulin blood, multiple myeloma, lymphoma and thrombotic microangiopathy.
The invention also provides a multicolor flow cytometry method for function evaluation of T cell subsets, activity evaluation of diseases, treatment effect, clinical prognosis and prognosis prediction, which comprises the following steps:
(a) Preparing or providing a biological sample comprising T lymphocytes;
(b) Contacting the sample with a reagent composition for evaluating the T cell immunological status of a chronic pathological state of disease T lymphocytes as described above;
(c) Analyzing T lymphocytes in the sample in a flow cytometer; and
(d) The obtained data is stored and analyzed.
The polychromatic flow cytometry method may further comprise any of the following steps:
(e) Lysing the red blood cells;
(f) Isolating the mononuclear cells and preparing a suspension;
(g) Blocking cell surface Fc receptors (e.g., membrane disruption treatment prior to cytokine or nuclear transcription factor detection);
(h) Incubating with fluorescent antibody;
(i) Washing the free fluorescent antibody and detecting the free fluorescent antibody on a machine;
(j) Fluorescence compensation adjustment;
(k) One or more of blank, isotype, FMO, negative and positive control groups were set up.
Wherein the sample comprises peripheral blood, bone marrow, tissue samples, or other types.
Preferably, the tissue sample is selected from lymph node, gland, spleen or liver.
Preferably, the body fluid is selected from the group consisting of cerebrospinal fluid, vitreous humor, synovial fluid, fine needle aspirate, pleural effusion and ascites.
Preferably, the sample is peripheral blood.
The reagent composition can measure markers in serum samples, wherein the markers comprise: any of CD3, CD4, CD8, CD16, CD25, CD45RA, CD45RO, CD56, CD62L, CD44, CD69, CD127, CD154, CCR7, CTLA4, igM, FOXP3, TIM3, LAG3, PD1, PDL1, 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 comprises data acquisition, analysis and recording means, for example using a computer, wherein a plurality of data channels record from each detector data of the light scattering and the fluorescence emitted by each particle as it passes through the sensing region. The analysis system classifies and counts particles, each of which is presented as a series of digitized parameter values.
In the flow cytometric analysis of particles using the methods of the present invention, the flow cytometric analyzer may be configured to trigger at selected parameters to distinguish the target particles from background and noise. "trigger" refers to a preset threshold for detecting a parameter. It is generally used as a means of detecting the passage of particles through a laser beam. Detection of an event exceeding a predetermined threshold for a selected parameter triggers the collection of light scattering and fluorescence data for the particle. No data is collected for particles or other components in the assay medium whose response is below a threshold value. The triggering parameter may be the detection of forward scattered light caused by the particle passing through the beam. The flow cytometer then detects and collects light scattering and fluorescence data of the particles.
The beneficial effects of the invention are as follows:
(1) Functional assessment covering a broad T cell subpopulation by detection of the combination;
(2) The accurate typing and immunocompetence detection of the T cells are rapidly realized, and the chronic activation state of the T cells is evaluated with high sensitivity and high specificity;
(3) The system evaluates the severity of the disease, the infection risk and the influence of the medicine on the cellular immunity function;
(4) The operation is simple, the result is easy to understand, and the popularization and the application in basic medical institutions are easy.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are required to be used in the embodiments will be briefly described below.
FIG. 1, example of the clinical application of T lymphocyte subpopulation in the case of glucocorticoid treatment of MCD patients and whether or not to co-infect. 370 cases of glomerular disease patients were analyzed by flow cytometry under different groupings (hormone-free, hormone-infected), wherein a was a T lymphocyte subpopulation (CD 45, CD3, CD4, CD8, TIM3, LAG3, CTLA 4) selection strategy profile, as shown in the figures, all markers were tested using domestic standard clinical laboratory selection and gating parameters; b is the proportion of cd3+ T cells in different groups of patients; c is the proportion of cd3+cd4+ T cells in different groupings; d is the proportion of cd3+cdd8+t cells in different groupings; e is the proportion of CD3+TIM3+T cells in the different groupings; f is the proportion of cd3+lag3+ T cells in different groupings; g is the proportion of cd3+ ctla4+ T cells in different groups (< 0.05P < 0.01P < 0.001P <0.0001 ns, no statistical difference).
FIG. 2, fine detection of regulatory T lymphocytes early on in the judgment of recurrence and growth of glomerular disease (micro lesions, MCD)Application instance in phase mitigation. Wherein a is a selection strategy profile of T lymphocyte subpopulations (CD 45, CD3, CD4, CD8, CD25, FOXP 3), as shown all markers were detected using domestic standard clinical laboratory selection and gating parameters; b is FOXP3 expression level of CD4+ T cells (FOXP 3) in different groups including Healthy Control (HC), non-kidney-comprehensive micro-lesions (NNS-MCD), sustained-remission micro-lesions (SR-MCD) and early recurrent micro-lesions (ER-MCD) + ,FOXP3 medium ,FOXP3 high ) Layering analysis results; c is FOXP3 expression level (FOXP 3) at various time points and under various disease states + ,FOXP3 medium ,FOXP3 high ) Correlation contrast analysis of (×p)<0.05,**P<0.01,***P<0.001,****P<0.0001, ns, no statistical difference).
FIG. 3 shows an example of the application of the regulatory T lymphocytes combined with PS6 protein detection in early diagnosis of recurrence of glomerular disease (micro lesions, MCD) and long-term remission. Wherein a is a flow cytometry detection of cd4+ T cell PS6 protein average fluorescence intensity (MFI) representative results in different cohorts, including healthy control (Ctrl), non-nephrotic syndrome (NNS), nephrotic Syndrome (NS), complete Remission (CR), and Early Relapse (ER); b is CD4+PS6 before treatment for different group people high T cell relative ratio; CD4+PS6 after 2 months of MCD complete remission with hormonal treatment high The ratio of T cell changes compared to pre-treatment; d is CD4+PS6 of MCD patient with Early Recurrence (ER) high The ratio of T cells to change upon complete remission for 2 months of treatment; e is cd4+ps6 at 12 months in MCD patients with sustained remission high The ratio of T cells to total remission at 2 months of treatment was varied. F is a receiver operating characteristic curve (ROC curve) for judging early recurrence of MCD by using CD4+FOXP3+T cell proportion; g is CD4+FOXP3 medium Judging the ROC curve of early recurrence of MCD by the T cell proportion; h is CD4+PS6 high Judging the ROC curve of early recurrence of MCD by the T cell proportion; i is the CD4+FOXP3 medium And CD4+PS6 high T cell ratio in combination with determination of the ROC curve for early recurrence of MCD.
FIG. 4, example of the clinical application of the fine detection of T lymphocyte subpopulations in the novel infection-associated Acute Kidney Injury (AKI) by coronaviruses. The method for the combined detection of T lymphocyte subpopulations (CD 3, CD4, CD8, CD25, CD127, LAG3 and TIM 3) is as previously described, wherein a is the flow cytometry analysis of patients with cd3+tim3+t cell subpopulations at a proportion of minimal lesions (MCD), minimal lesions combined with acute kidney injury (MCD-AKI), drug-related AKI and novel coronavirus infection-related AKI; b is CD3+LAG3+T cell subgroup proportion result analysis; c is CD4+CD25+CD127 low T cell proportion results analysis (×p)<0.05,**P<0.01,***P<0.001,****P<0.0001)。
FIG. 5, CD45, CD3, CD4, CD8, CD25, TIM3, LAG3, PD1, PDL1 and CTLA4 in combination analysis were used in systemic inflammatory lesions of tuberculosis infection. Wherein a is a selection strategy profile and outcome for each T cell subpopulation prior to treatment; b is a strategy profile and results for the sorting of each T cell subpopulation after treatment. C-G was a dynamic detection of the proportion of cd3+tim3+, cd3+ctla4+, cd3+lag3+, cd3+pd1+ and cd4+cd25+ T cell subsets at different time points following 12 months.
FIG. 6, examples of clinical applications of CD3+ TIM3+ and CD3+ LAG3+ T cell subsets assays in the assessment of severe Systemic Lupus Erythematosus (SLE) disease. The methods of detection of T lymphocyte subpopulations (CD 3, LAG3 and TIM 3) were as described previously, incorporating >1000 flow cytometry assay results analyses, including systemic lupus erythematosus (SLE, n=409), micro lesions (MCD, n=97), focal segmental glomerulosclerosis (FSGS, n=67), membranous nephropathy (MN, n=252), igA nephropathy (IgAN, n=227). Wherein A-E is the proportion of representative case CD3+TIM3+T cell subsets; a is the change process that the proportion of CD3+TIM3T cells is obviously increased to a normal range after 1 patient suffering from severe SLE complicated with lupus encephalopathy and lupus nephritis is effectively treated; b is 1 patient with severe disease recurrence SLE; c is 1 patient with SLE in rest period; d is 1 drug AKI patient; e is 1 CKD stage 5 patient; f is the comparative analysis of the ratio of CD3+TIM3+T cells of SLE patients to other primary glomerular patients; g is the comparative analysis of CD3+LAG3+T cell ratio of SLE patients and other primary glomerular patients; correlation analysis of H for cd3+tim3+ and cd3+lag3+ T cell subsets; i and J are CD3+TIM3+ (I) and CD3+LAG3+T cell (J) subsets, respectively, and are related to assays for evaluating traditional clinical indicators of SLE disease activity, including complement (C1 q, C3, C4), autoantibodies (ds-DNA), urine detection (urine albumin, urine total protein, urine red blood cells), serum creatinine (Scr), and C-reactive protein (CRP).
Detailed Description
The present invention provides a T cell immunological status marker combination for assessing a chronic pathological status of a disease T lymphocyte, comprising any of CD3, CD4, CD8, CD16, CD25, CD45RA, CD45RO, CD56, CD62L, CD, CD69, CD127, CD154, CCR7, CTLA4, igM, FOXP3, TIM3, LAG3, PD1, PDL1 and PS 6. In the present invention, the disease preferably includes one or more of a primary glomerular disease, an autoimmune disease, an infection, a metabolic disease, a blood system disease and a pharmaceutical kidney injury. In the present invention, the primary glomerular disease preferably includes one or more of a minute lesion, focal segmental sclerosis, membranous nephropathy, and IgA nephropathy; the autoimmune disease preferably comprises one or more of systemic lupus erythematosus, sjogren's syndrome, rheumatoid arthritis, and mixed connective tissue disease; the infection preferably comprises one or more of tuberculosis and a novel coronavirus infection; the metabolic disease preferably comprises one or more of diabetes, obesity and calcification defenses;
the hematological disorders preferably include one or more of amyloidosis, monoclonal immunoglobulin blood, multiple myeloma, lymphoma, and thrombotic microangiopathy.
In the present invention, when evaluating the immune function of a glucocorticoid-treated patient, the T cell immunological marker combination preferably includes CD3, CD4, CD8, TIM3, LAG3, CTLA4, PD1, and PDL1.
In the present invention, when detecting a microscopic lesion, the T cell immunological marker combination preferably includes CD3, CD4, CD8, PS6, CD25, CD127 and FOXP3.
In the present invention, when detecting systemic inflammatory lesions 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 T cell chronic pathological status of different glomerular diseases, the T cell immunological status marker combination preferably includes CD3, CD4, CD8, CD25, CD127, TIM3, LAG3, CTLA4 and PS6.
In the present invention, when evaluating the effect of disease treatment, the T cell immunological marker combination preferably includes CD3, CD4, CD8, CD25, CD62L, CD, CD69, CD127, FOXP3, TIM3, LAG3, CTLA4 and PS6.
In the present invention, the disease preferably includes one or more of lupus nephritis, a minute lesion, focal segmental glomerulosclerosis, and membranous nephropathy.
The invention also provides application of the T cell immunological state marker combination in preparation of a reagent for evaluating chronic pathological state of T lymphocytes.
According to the invention, through flow cytometry detection of 3386 clinical patients, different combinations of 106T cell markers are screened and verified, wherein cell surface marker proteins, cytokines, nuclear transcription factors and key signal channel molecules are covered, and the detection indexes specifically comprise: bcl-6, CCR10, CCR3, CCR4, CCR5, CCR6, CCR7, CD127, CD137, CD152, CD154, CD16, CD161, CD21, CD23, CD25, CD27, CD28, CD3, CD38, CD4, CD40L, CD44, 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, ki67, KLRB1, LAG3, LEF1, lymphotoxin, NCAM1, NKG 1.1, 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 V.alpha.24, TCR.beta.11, TCR γ/δ, TGF-beta, TIM1, TIM3, TNF-beta.
Multichannel flow cytometry detection the labeled antibodies are selected according to different monoclonal antibody sources, excitation light wavelengths and fluorescein spectral characteristics, including FITC, PE, perCP, PE-Cy7, FITC, PE-Cy5, perCP, PE-Cy7, alexa Fluor 488, AF647, APC-Cy7, BV421 and BV510, and conventional multichannel flow cytometry can be implemented in the present scheme, and the used flow cytometry detection related instruments, antibodies or reagents can be purchased or self-made directly from the commercial companies BD Biosciences, bioLegend, beckman and Cell Signaling Technology. The detection operation steps comprise: extracting 1-2ml of peripheral blood, lysing erythrocytes, separating mononuclear cells, preparing suspension, sealing cell surface Fc receptor (such as membrane rupture treatment for cytokine or nuclear transcription factor detection), incubating with fluorescent coupling antibody for 30 min, washing free fluorescent antibody, performing on-machine detection, completing fluorescent compensation adjustment, and analyzing detection result. All detection indexes are strictly set up in blank, isotype, FMO, negative and positive control groups.
Phenotypic and functional analyses of peripheral blood immune cells from 3386 patients were performed by multi-channel flow cytometry, and 23T cell immunological markers and combinations were finally established from the above 106 flow cell indices reflecting T lymphocyte chronic pathological states of different diseases, including CD3, CD4, CD8, CD16, CD25, CD45RA, CD45RO, CD56, CD62L, CD44, CD69, CD127, CD154, CCR7, CTLA4, igM, FOXP3, TIM3, LAG3, PD1, PDL1, PS6. The invention further verifies through the long-term follow-up (12 months) of more than 1500 clinical patients, and proves that the marker combination scheme can effectively evaluate the chronic pathological state of T cells, the active degree of diseases, the treatment effect and the clinical prognosis, and has important clinical guidance value. The invention is clinically verified in a plurality of diseases, comprising: primary glomerular disease (micro lesions, focal segmental sclerosis, membranous nephropathy, igA nephropathy), autoimmune disease (systemic lupus erythematosus, sjogren's syndrome, rheumatoid arthritis, mixed connective tissue disease), infection (tuberculosis, novel coronavirus infection), metabolic disease (diabetes, obesity, calcification defenses), blood system disease (amyloidosis, monoclonal immunoglobulin blood, multiple myeloma, lymphoma, thrombotic microangiopathy) and pharmaceutical kidney injury.
The invention also provides application of the T cell immunological state marker combination in preparing a disease auxiliary diagnosis or prognosis prediction product.
The product may be a kit comprising antibodies to 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 to CD3, CD4, CD8, TIM3, LAG3 and CTLA4;
preferably, antibodies to CD3, CD4, CD8, CD25, CD127 and FOXP 3;
preferably, antibodies to CD3, CD4, CD8, CD25, CD127, FOXP3 and PS 6;
preferably, antibodies to CD3, CD4, CD8, CD25, CD127, LAG3 and TIM 3;
preferably, antibodies to CD45, CD3, CD4, CD8, CD25, TIM3, LAG3, PD1, PDL1 and CTLA 4.
The kit further comprises a marker capable of measuring in a serum sample, the marker comprising: 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 comprises a fluorescent dye, wherein the absorption wavelength and the emission wavelength of the fluorescent dye are in the range of 300nm-810nm, and the fluorescent dye comprises disodium 4-acetamido-4 '-isothiocyanatestilbene-2, 2' -disulfonic acid; acridine and derivatives thereof such as acridine, acridine orange, acridine yellow, acridine red, and isothiocyanate acridine; 5- (2-aminoethylamino) -1-naphthalenesulfonic acid sodium salt (EDANS); 4-amino-N- [3- (vinylsulfonyl) phenyl ] naphthalimide-3, 5-disulfonate (fluorescein VS); n- (4-aniline-1-naphthyl) maleimide; 2-aminobenzamide; brilliant yellow; coumarin and derivatives such as coumarins, 7-acetoxy-4-methylcoumarin (AMC, coumarin 120), 7-amino-4-trifluoromethylcoumarin (coumarin 151); cyan pigments and derivatives such as tetrabromo tetrachlorofluorescein, cy3, cy5, cy5.5, and Cy7;4', 6-diamidino-2-phenylindole (DAPI); bromophthalic-trimesic-red; 7-diethylamino-3- (4' -isothiocyanatophenyl) -4-methylcoumarin; diethylaminocoumarin; diethylenetriamine pentaacetic acid ester; 4,4 '-diisothiocyanodihydrostilbene-2, 2' -disulfonic acid; 4,4 '-diisothiocyano-2, 2' -stilbenedisulfonic acid; dansyl chloride (DNS, dansyl chloride); 4- (4' -dimethylaminophenylazo) benzoic acid (DABCYL); 4-dimethylaminoazobenzene-4' -thioisocyanate (DABITC); eosin and derivative such as eosin and eosin isothiocyanate; erythrosine and derivative organisms such as erythrosine and erythrosine isothiocyanate; ethidium; luciferins 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), chlorotriazinyl fluorescein, naphthyl fluorescein and qflitc (XRITC); fluorescent amine; IR144; IR1446; green Fluorescent Protein (GFP); reef Coral Fluorescent Protein (RCFP); lizheimine TM; lisamine rhodamine, fluorescein; malachite green isothiocyanate; 4-methylumbelliferone; o-cresol peptide; nitrotyrosine; pararosaniline; nile red; green state oregon; phenol red; b-phycoerythrin; phthalic dicarboxaldehyde; the middlenaphthalene and derivative organisms such as middlenaphthalene, pyrene butyric acid and succinimidyl 1-pyrene butyric acid; reactive Red 4 (Cibacron bright Red 3B-A); rhodamine and derivatives such as 6-carboxy-X-Rhodamine (ROX), 6-carboxyrhodamine (R6G), 4, 7-dichloro rhodamine Lissamine, rhodamine B sulfonyl chloride, rhodamine (Rhod), rhodamine B, rhodamine 123, rhodamine X isothiocyanate, sulfonylrhodamine B, sulfonylrhodamine 101, sulfonylchloromercuride of sulfonylrhodamine 101 (red, texas), 6-carboxytetramethyl rhodamine (TAMRA), tetramethyl rhodamine, and Tetramethyl Rhodamine Isothiocyanate (TRITC); riboflavin; rosolic acid and terbium chelate complexes; xanthenes; or a combination thereof. Other fluorophores known to those skilled in the art, or combinations thereof, may also be used.
Preferably, the fluorescent dye comprises: FITC, PE, perCP, PE-Cy7, FITC, PE-Cy5, perCP, PE-Cy7, alexa Fluor 488, AF647, 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 specific marker combinations, kits, and flow cytometry methods of the present invention, the present invention is described in detail below with reference to the examples, but they should not be construed as limiting the scope of the present invention.
Example 1
The use of CD3, CD4, CD8, TIM3, LAG3 and CTLA4 in combination assays for the evaluation of immune function in patients with various diseases and above long-term glucocorticoid therapy.
The method comprises the following steps: peripheral blood of the patient was collected by 5ml, centrifuged at 2000 r/min for 30 minutes by Ficoll density gradient centrifugation, and Peripheral Blood Mononuclear Cells (PBMC) were isolated. 70-90% of PBMC are lymphocytes (70-90%), with CD3+ T cells representing the vast majority of lymphocytes (45-70%). By extracting PBMC, further analysis can be effectively performed on CD3+CD4+T cells, and the influence of other blood components on the test result is avoided. The gating, blank control and analysis methods of flow cytometry detection are shown in FIG. 1A. According to the conventional clinical flow cytometry detection method, the method uses 2-5 multiplied by 10 6 The cells were resuspended at a concentration of cells/mL and primary antibodies (0.1-10. Mu.g/mL) were added according to the recommended reference range of the instructions and incubated at room temperature or 4℃for 30-60 minutes in the absence of light. In the embodiment, the detection is performed by adopting a flow cytometry with more than 4 channels, a commercial company can provide a plurality of primary antibodies or secondary antibodies with fluorescent labels, the antibody selection principle is that 1 kind of fluorescein is adopted in each channel, the fluorescein collocation between the channels is reasonably distributed according to the antigen expression intensity, and the fluorescein with small spectrum overlapping is selected, so that an optimized fluorescein antibody collocation scheme can be adopted.
The results of the study on 370 patients with glomerular disease (figure 1) demonstrate that long-term hormone treatment significantly reduces the proportion of cd3+cd4+ T cells, and relatively increases the proportion of cd3+cd8+ T cells, resulting in a significant reduction in the ratio of CD4/CD8, especially in combination with infection, which can be used to clinically evaluate the risk of reduced cellular immune function caused by hormone treatment. At the same time, further combinatorial analysis demonstrated that both cd3+tim3+, cd3+lag3+ and cd3+ctla4+ T cells were significantly elevated in hormone-treated patients, especially in patients with hormone-treated infection. The combined analysis that the above index is increased by 50% compared with the normal value range or 25% compared with the individual baseline value can early indicate that the hormone leads to the reduction of the cellular immunity function and the risk of infection is increased. The combination has proved to have high specificity and significance through multiple diseases, large samples and long-term clinical follow-up study. At present, no clinical alternative similar to the combination exists at home and abroad.
Example 2
Use of a CD3, CD4, CD8, CD25, CD127 and FOXP3 combination assay for early diagnosis of complete remission, early recurrence and long-term remission of glomerular disease (micro lesions, MCD).
Flow cytometry detection routine manipulation methods are described in example 1, wherein the present protocol involves cd4+foxp3+ highly homologous (> 95%) to cd4+cd25+cd127-T cells. FOXP3 is an expressed factor in the nucleus and requires cell fixation and membrane rupture prior to antibody incubation. The above index was used as a regulatory T cell marker.
As shown in fig. 2, for a long-term follow-up of 23 patients with nephrotic syndrome for 12 months, the proportion of peripheral blood T cell subsets was examined at 0,2,4,8, 12 months and the time points of early recurrence, respectively, and T cells were analyzed in layers according to FOXP3 expression levels. As shown by the research results of different populations of Healthy Control (HC), non-kidney syndrome micro-lesions (NNS-MCD), sustained release (SR-MCD) and early recurrence (ER-MCD), the proportion change of regulatory T cell subset is closely related to the treatment outcome of nephrotic syndrome, wherein FOXP3 + And FOXP3 medium Two groups of expression are increased earlier>25%) suggests effective treatment and complete remission of the disease; compared with the prior art, the two are reduced >25%) prompting diseaseEarly recurrence of the disease; FOXP3 in patients with long-term stable sustained remission + And FOXP3 medium The proportion of the subgroups did not change significantly before and after. Thus, the combination of assays is highly specific and sensitive to the prediction of early remission, early recurrence and sustained remission of MCD disease. At present, no clinical alternative similar to the combination exists at home and abroad.
Example 3
Use of a CD3, CD4, CD8, CD25, CD127, FOXP3 and PS6 combination assay for complete remission, early relapse and long term remission after judgment of glomerular disease (micro lesions, MCD) treatment. Flow cytometry detection routine procedure was as described in example 1 above.
As shown in FIG. 3, long-term follow-up results for 12 months for 23 patients with nephrotic syndrome confirm that CD4+PS6 high The T cell fraction was significantly higher in nephrotic syndrome MCD patients (SR-MCD and ER-MCD) than in control group (HC) and non-nephrotic syndrome MCD patients (NNS-MCD), especially in early relapsing patients (ER-MCD); further continuous dynamic test results confirm that CD4+PS6 is fully relieved after treatment high T cell proportion is significantly reduced; cd4+ps6 at the time of recurrence high T cell proportion is re-increased; while sustained remission (12 months) of CD4+PS6 high There was no significant change in T cell ratio from complete remission (2 months). CD4+PS6 high T cell ratio variation>25%) is an effective indicator suggesting disease outcome. Analysis by receiver operating characteristic curve (ROC curve) using CD4+FOXP3 + 、CD4+FOXP3 medium And CD4+PS6 high Area under the curve (AUC) of T cell subpopulation versus predicted early recurrence of MCD was 0.79,0.79 and 0.89, respectively, whereas CD4+ FOXP3 was combined medium And CD4+PS6 high The AUC of the T cells reaches 0.92, the indexes have the characteristics of high sensitivity and high specificity, and the advantages of the combined analysis are more remarkable.
The above results confirm that CD4+FOXP3 + 、CD4+FOXP3 medium 、CD4+PS6 high The T cell proportion can be used as an effective prediction index for improvement, long-term stability and early recurrence of MCD patients, which is an important clinical problem to be solved urgently. The combination is subjected to long-term clinical treatmentFollow-up studies confirm high specificity and significance. At present, no clinical alternative similar to the combination exists at home and abroad.
Example 4
Use of a CD3, CD4, CD8, CD25, CD127, LAG3 and TIM3 combination assay in novel acute kidney injury associated with coronavirus infection. Flow cytometry detection routine procedure As described in example 1, 17 cases of clinical diagnosis were analyzed for results of flow cytometry detection in patients with novel coronavirus infection-associated Acute Kidney Injury (AKI), and CD3+TIM3+ and CD4+CD25+CD127 of novel coronavirus infection-associated AKI were compared with minimal lesions (MCD), minimal lesion combined acute kidney injury (MCD-AKI) and drug-associated AKI low T cell proportion increases significantly, while cd3+lag3+t cell proportion decreases. The above results suggest that the combined analysis of CD3+TIM3+ and CD4+CD25+CD127 low T cell proportion increase>Normal 25%) and decrease in the proportion of CD3+LAG3+T cells>The normal value of 10%) has important clinical guidance value for judging the risk of combining AKI multi-organ injury of patients with new crown infection (especially old high risk group). At present, no clinical alternative similar to the combination exists at home and abroad.
Example 5
Use of CD45, CD3, CD4, CD8, CD25, TIM3, LAG3, PD1, PDL1 and CTLA4 in combination assays for systemic inflammatory lesions of tuberculosis infection. Conventional flow cytometry detection procedures as described in example 1 above showed that chronic infections resulted in significant increases in the proportion of inhibitory cd3+tim3+t and cd3+ctla4+ T cells, with no significant changes in other T cell markers, as evidenced by the 12 months follow-up of cases of severe nuclear infection combined with renal, hepatic, peritoneal multi-visceral inflammatory responses; after anti-tuberculosis treatment, the changes of CD3+TIM3+T and CD3+CTLA4+T cell subsets are highly consistent with pathological changes of multi-organ diffuse inflammatory cell infiltration, and key T cell subtype changes can be sensitively detected through the combination, the disease is reflected in a specific way, and the disease is closely related to the treatment effect. The combination is proved to have high specificity and significance by long-term clinical follow-up study. At present, no clinical alternative similar to the combination exists at home and abroad.
Example 6
The use of cd3+ tim3+ and cd3+ LAG3+ T cell subpopulation assays for evaluating severity, therapeutic efficacy, and remission status of systemic lupus erythematosus disease. Routine procedure for flow cytometry detection as described in example 1 above, long-term follow-up of the case cohort and systematic analysis of flow cytometry detection results for >1000 patients demonstrated that systemic lupus erythematosus nephritis (n=409) was compared to primary glomerular disease, including: the proportion of micro lesions (n=97), focal segmental glomerulosclerosis (n=67), membranous nephropathy (n=252) and IgA nephropathy (n=227) of which the proportion of cd3+tim3+ and cd3+lag3+ T cell subsets is significantly increased, the changes of the two are highly consistent and are closely related to the severity of the disease, the treatment response and the long-term stable state of the disease, and the increase of the proportion of the two (> 25% of normal value or > 50% of baseline value) can be used as an effective index for evaluating the severity of the systemic lupus erythematosus nephritis disease through the correlation analysis with a plurality of traditional clinical indexes. The combination is verified by a large number of clinical samples, has high specificity for evaluating the disease severity degree, the treatment effect and the disease alleviation of SLE, and has important guiding value in the clinical diagnosis and treatment process of severe SLE (lupus encephalopathy and lupus nephritis). At present, no clinical alternative similar to the combination exists at home and abroad.
Although the foregoing embodiments have been described in some, but not all, embodiments of the invention, it should be understood that other embodiments may be devised in accordance with the present embodiments without departing from the spirit and scope of the invention.

Claims (10)

1. A T cell immunological status marker combination for assessing a chronic pathological status of a disease T lymphocyte, comprising any of CD3, CD4, CD8, CD25, CD45RA, CD45RO, CD62L, CD, CD127, CD154, CCR7, CTLA4, igM, FOXP3, TIM3, LAG3, PD1, PDL1 and PS 6.
2. The T cell immunological status marker combination according to claim 1, wherein the disease comprises one or more of a primary glomerular disease, an autoimmune disease, an infection, a metabolic disease, a disease of the blood system and a pharmaceutical kidney injury.
3. The T cell immunological status marker combination according to claim 2, wherein the primary glomerular disease comprises one or more of a micro-lesion, focal segmental sclerosis, membranous nephropathy, and IgA nephropathy;
the autoimmune disease includes one or more of systemic lupus erythematosus, sjogren's syndrome, rheumatoid arthritis, and mixed connective tissue disease;
The infection includes one or more of tuberculosis and a novel coronavirus infection;
the metabolic disease includes one or more of diabetes, obesity, and calcification defenses;
the blood system diseases include one or more of amyloidosis, monoclonal immunoglobulin blood, multiple myeloma, lymphoma and thrombotic microangiopathy.
4. The T cell immune state marker combination of claim 1, wherein when evaluating glucocorticoid treatment patient immune function, the T cell immune state marker combination comprises CD3, CD4, CD8, TIM3, LAG3, CTLA4, PD1, and PDL1.
5. The T cell immune state marker combination according to claim 1 or 3, wherein when detecting a micro-lesion, the T cell immune state marker combination comprises CD3, CD4, CD8, CD25, CD127, FOXP3 and PS6 (Phospho-S6 Ribosomal Protein).
6. The T cell immune state marker combination of claim 1 or 3, wherein when detecting systemic lupus erythematosus, the T cell immune state marker combination comprises CD3, CD4, CD8, CD25, CD127, FOXP3, TIM3, LAG3, and CTLA4;
Preferably, when detecting systemic inflammatory lesions caused by infection, the T cell immunological marker combination comprises CD45, CD3, CD4, CD8, CD25, TIM3, LAG3, PD1, PDL1 and CTLA4;
preferably, when evaluating the T cell chronic pathological status of different glomerular diseases, the T cell immunological status marker combination comprises CD3, CD4, CD8, CD25, TIM3, LAG3 and CTLA4;
preferably, when evaluating the effect of treatment of a disease comprising one or more of lupus nephritis, micro-lesions, focal segmental glomerulosclerosis and membranous nephropathy, the T-cell immunological marker combination comprises CD3, CD4, CD8, CD25, CD62L, CD44, CD69, CD127 and FOXP 3.
7. Use of a T cell immunological status marker combination according to any one of claims 1 to 6 for the preparation of a reagent for assessing a chronic pathological state of a disease T lymphocyte.
8. A reagent composition for assessing a T cell immunological state of a chronic pathological state of a disease T lymphocyte, comprising a fluorochrome-conjugated antibody directed against a marker combination comprising: any of CD3, CD4, CD8, CD16, CD25, CD45RA, CD45RO, CD56, CD62L, CD, CD69, CD127, CD154, CCR7, CTLA4, igM, FOXP3, TIM3, LAG3, PD1, PDL1 and PS6,
Preferably, when detecting systemic lupus erythematosus, the T cell immunological marker combination comprises CD3, CD4, CD8, CD25, CD127, FOXP3, TIM3, LAG3, and CTLA4;
preferably, when detecting systemic inflammatory lesions caused by infection, the T cell immunological marker combination comprises CD45, CD3, CD4, CD8, CD25, TIM3, LAG3, PD1, PDL1 and CTLA4;
preferably, when evaluating the T cell chronic pathological status of different glomerular diseases, the T cell immunological status marker combination comprises CD3, CD4, CD8, CD25, TIM3, LAG3 and CTLA4;
preferably, when evaluating the effect of treatment of a disease comprising one or more of lupus nephritis, micro-lesions, focal segmental glomerulosclerosis and membranous nephropathy, the T-cell immunological marker combination comprises CD3, CD4, CD8, CD25, CD62L, CD44, CD69, CD127 and FOXP 3;
the absorption wavelength and/or emission wavelength of the fluorescent dye is in the range of 300nm to 810nm, preferably the fluorescent dye comprises: FITC, PE, perCP, PE-Cy7, FITC, PE-Cy5, perCP, PE-Cy7, alexa Fluor 488, AF647, APC-Cy7, BV421 and BV510.
9. A kit for functional assessment of T cell subsets, activity assessment of disease, therapeutic effect, clinical outcome and prognosis prediction, comprising a buffer, a fluorescent dye and an antibody against a marker combination comprising: any of CD3, CD4, CD8, CD16, CD25, CD45RA, CD45RO, CD56, CD62L, CD, CD69, CD127, CD154, CCR7, CTLA4, igM, FOXP3, TIM3, LAG3, PD1, PDL1 and PS 6;
Preferably, the kit comprises fluorescent dye-conjugated antibodies to a marker combination comprising: any of CD3, CD4, CD8, CD16, CD25, CD45RA, CD45RO, CD56, CD62L, CD, CD69, CD127, CD154, CCR7, CTLA4, igM, FOXP3, TIM3, LAG3, PD1, PDL1 and PS 6;
preferably, the absorption wavelength and/or emission wavelength of the fluorescent dye is in the range of 300nm to 810nm, and further preferably, the fluorescent dye includes: FITC, PE, perCP, PE-Cy7, FITC, PE-Cy5, perCP, PE-Cy7, alexa Fluor 488, AF647, APC-Cy7, BV421 and BV510.
10. A multi-color flow cytometry method for functional assessment of T cell subsets, activity assessment of disease, therapeutic effect, clinical outcome and prognosis, comprising the steps of:
(a) Preparing or providing a biological sample comprising T lymphocytes;
(b) Contacting the sample with a reagent composition for evaluating a T cell immunological state of a chronic pathological state of a disease T lymphocyte as described in the preceding claims;
(c) Analyzing T lymphocytes in the sample in a flow cytometer; and
(d) Storing and analyzing the obtained data;
preferably, the polychromatic flow cytometry method further comprises any of the following steps:
(e) Lysing the cells;
(f) Isolating the mononuclear cells and preparing a suspension;
(g) Blocking cell surface Fc receptors;
(h) Incubating with fluorescent antibody;
(i) Washing the free fluorescent antibody and detecting the free fluorescent antibody on a machine;
(j) Fluorescence compensation adjustment;
(k) One or more of blank, isotype, FMO, negative and positive control groups were established.
CN202310435512.XA 2023-04-21 2023-04-21 T cell immunological state marker combination for evaluating chronic pathological state of T lymphocyte of disease and application thereof Pending CN116430055A (en)

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109254147A (en) * 2018-10-12 2019-01-22 东莞暨南大学研究院 Human peripheral blood immune cell function fully assesses kit and appraisal procedure
US20210139601A1 (en) * 2017-05-25 2021-05-13 The Broad Institute, Inc. Lymphocyte antigen cd5-like (cd5l) monomer, homodimer, and interleukin 12b (p40) heterodimer agonists and methods of use thereof
CN113125718A (en) * 2021-04-16 2021-07-16 浙江普罗亭健康科技有限公司 45 antibody kit for monitoring human immune state and application

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20210139601A1 (en) * 2017-05-25 2021-05-13 The Broad Institute, Inc. Lymphocyte antigen cd5-like (cd5l) monomer, homodimer, and interleukin 12b (p40) heterodimer agonists and methods of use thereof
CN109254147A (en) * 2018-10-12 2019-01-22 东莞暨南大学研究院 Human peripheral blood immune cell function fully assesses kit and appraisal procedure
CN113125718A (en) * 2021-04-16 2021-07-16 浙江普罗亭健康科技有限公司 45 antibody kit for monitoring human immune state and application

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