Application of GPR56 in preparation of product for screening or auxiliary diagnosis of primary sicca syndrome

Abstract

The invention discloses an application of GPR56 in preparation of a product for screening or auxiliary diagnosis of primary sicca syndrome, and claims to protect any one of the following applications of a substance for detecting GPR56 or targeting GPR56: application in diagnosis of primary sicca syndrome or preparation of a product for diagnosis of primary sicca syndrome, application of GPR56 in screening or auxiliary diagnosis of primary sicca syndrome, and application of GPR56 in screening or auxiliary diagnosis of primary sicca syndrome. The invention also relates to an application in screening the primary sicca syndrome or preparing a product for screening the primary sicca syndrome. Application in preparation of a product for promoting expression of granzyme B; the invention is applied to preparation of a product for enhancing the killing function of T cells. The GPR56 and the GZMB are highly positively correlated, GPR56 + T cells are stimulated by further adopting an antibody of the GPR56, and expression of the GZMB can be activated by activating the GPR56, so that the GPR56 serving as a target spot can be used for improving the toxicity of the T cells.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular, to the application of GPR56 in the preparation of primary Sjögren's syndrome screening or auxiliary diagnosis products. Background Art

[0002] Cytotoxic T cells (CTLs) are one of the important immune effector cells of the adaptive immune response and have been shown to play an important role in the host's immune response to pathogens, tumors, and transplanted tissues. They can destroy virus-infected and malignantly transformed cells before the spread of viral infection or the occurrence of cancer. Cytotoxic T cells include CD8 CTLs as well as CD4 CTLs. The main mechanism of target cell destruction mediated by CD8+CTLs includes the delivery of cytolytic effector molecules, including granzymes and perforins, which are stored in the cytoplasmic granules of CTLs and released during CTL-mediated target cell lysis. The target cells of CD4 CTLs are limited to antigen-presenting cells expressing MHC class II, which are able to secrete granzyme B (GZMB) and perforin and kill target cells in an MHC class II-restricted manner. Recently, CD4 CTLs have also been found in human peripheral blood, and CD4 CTLs have also been detected in anti-tumor responses, showing complementary functions to CD8 CTLs. Accurate and rapid evaluation of CTL cells in clinical diagnosis is of great significance for the accurate diagnosis of various diseases. However, cell surface markers specific to CD4 CTLs have not yet been discovered. Discovering surface molecules that can characterize CTL cells is very important for improving the diagnostic efficiency of CTL cells and for classifying CTL cells for therapeutic research.

[0003] Genetic plasticity (GPL) is used to describe the changes in gene expression levels under different experimental conditions. According to the plasticity of genes, genes can be divided into high plasticity genes and low plasticity genes. The former suggests that their expression levels are easily affected by experimental conditions (such as different individuals, different stimuli and treatments, disease states, tissue sources, etc.) in a broad sense and change significantly; the latter indicates that under various experimental conditions, genes generally tend to be highly expressed, or lowly expressed / no expressed. Genetic plasticity can be quantitatively evaluated by GPL scoring, and all plastic genes constitute the plasticity group (plasticitome). Genetic plasticity analysis based on transcriptomics big data can not only evaluate and identify marker molecules of specific immune cells, but also quickly discover new immune cell subsets based on high plasticity genes. The concept of virtual sorting proposed for high plasticity genes is to use virtual methods to analyze big data samples, such as identifying co-expressed and mutually exclusive expressed genes, to obtain functional clues of immune cell subsets and possible positive or negative regulatory factors.

[0004] Granzyme B (GZMB) in CD4 + T cells are highly plastic molecules. Therefore, based on the large number of CD4 + T cell transcriptomic data identified co-expressed molecules of GZMB through virtual sorting, among which the cell membrane surface protein is expected to serve as a specific marker molecule for CD4 CTL. Summary of the invention

[0005] The present invention aims to provide a GPR56 that can be used as a marker and to provide a substance for detecting GPR56.

[0006] The present invention claims the use of a substance for detecting GPR56 in any of the following:

[0007] A1) Use in the diagnosis of primary Sjögren's syndrome or in the preparation of a product for the diagnosis of primary Sjögren's syndrome;

[0008] A2) Use in screening for primary Sjögren's syndrome or in preparing a product for screening for primary Sjögren's syndrome;

[0009] A3) Use in early diagnosis of lung adenocarcinoma or in the preparation of a product for early diagnosis of lung adenocarcinoma;

[0010] A4) Use in screening for early lung adenocarcinoma or in preparing a product for screening for early lung adenocarcinoma;

[0011] A5) Use in preparing a product for promoting the expression of granzyme B;

[0012] A6) Application in the preparation of products that enhance the killing function of T cells.

[0013] Wherein, the substance for detecting GPR56 is the following B1) or B2):

[0014] B1) a reagent or an instrument for detecting the proportion of cells expressing GPR56 in T cells;

[0015] B2) Reagents or instruments for detecting the GPR56 protein content in T cells.

[0016] Wherein, the T cells are CD4+T cells or CD8+T cells in peripheral blood.

[0017] Wherein, the substance for detecting GPR56 includes an antibody that binds to GPR56.

[0018] The present invention also claims a kit, which comprises the above-mentioned substance for detecting GPR56.

[0019] Wherein, the detection sample of the kit is peripheral blood.

[0020] The above-mentioned kit may be used in any of the following applications and should also be within the scope of protection of the present invention:

[0021] A1) Use in the diagnosis of primary Sjögren's syndrome or in the preparation of a product for the diagnosis of primary Sjögren's syndrome;

[0022] A2) Use in screening for primary Sjögren's syndrome or in preparing a product for screening for primary Sjögren's syndrome;

[0023] A3) Use in early diagnosis of lung adenocarcinoma or in the preparation of a product for early diagnosis of lung adenocarcinoma;

[0024] A4) Use in screening for early lung adenocarcinoma or in preparing a product for screening for early lung adenocarcinoma;

[0025] A5) Use in preparing a product for promoting the expression of granzyme B;

[0026] A6) Application in the preparation of products that enhance the killing function of T cells.

[0027] The application of GPR56 as a target in the preparation of products that promote the expression of granzyme B or in the preparation of products that enhance the killing function of T cells should also be within the scope of protection of the present invention. For example, the application of drugs such as agonistic antibodies targeting GPR56 or small molecules that promote the activity of GPR56 to promote the expression of granzyme B; the application of increasing the expression of GPR56 in T cells by means of genetic engineering, cell engineering, drug intervention, etc.; and also including the application of using antibody blocking and small molecule inhibitors to inhibit the activity of GPR56 to reduce the toxicity of T cells in view of the relationship between GPR56 and granzyme B, such as reducing the damage of cytotoxic T cells to their own tissue cells by targeting GPR56 as a target in autoimmune diseases.

[0028] The present invention also provides a method for screening or diagnosing primary Sjögren's syndrome, which comprises detecting the proportion or content of cells expressing GPR56 protein in CD4+T cells of a patient to be tested, thereby determining whether the patient to be tested is a patient with primary Sjögren's syndrome.

[0029] The present invention also provides a method for screening or diagnosing early lung adenocarcinoma, which comprises detecting the proportion or content of cells expressing GPR56 protein in CD4+T cells of a patient to be tested, thereby determining whether the patient to be tested is a patient with early lung adenocarcinoma.

[0030] The present invention finds that GPR56 is highly positively correlated with GZMB through a virtual sorting method, and in previous studies, it was found that GRP56+T cells and GZMB+T cells in human peripheral blood T cells highly overlap. After further stimulation of GPR56+T cells with GPR56 antibodies, it was found that activation of GPR56 can activate the expression of GZMB, suggesting that GPR56 can be used as a target to increase the toxicity of T cells. Therefore, the present invention aims to protect the use of this target in the preparation of products that promote the expression of granzyme B, as well as the use of this target in the preparation of products that enhance the killing function of T cells. In addition, based on the regulatory relationship between GPR56 and granzyme B, the present invention also protects intervention measures against this target, such as the use of products that reduce the killing function of T cells through measures such as blocking antibodies or small molecule inhibitors. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 To analyze and compare the positive proportions of GPR56 in peripheral blood CD4 and CD8 cells between normal subjects and newly diagnosed pSS patients.

[0032] Figure 2 ROC curve analysis of the diagnostic value of the proportion of GPR56 in CD4+T cell subsets for screening early pSS patients.

[0033] Figure 3 This is a comparison of the GPR56-positive proportions in peripheral blood CD4 and CD8 cells and their four subsets between normal subjects and patients with early lung adenocarcinoma.

[0034] Figure 4 The ROC curve shows the diagnostic value of the proportion of GPR56 in T cell subsets for screening patients with early lung adenocarcinoma (LUAD). Tn, naive T cells; TCM, central memory T cells; TEM, effector memory T cells; TEMRA, effector T cells.

[0035] Figure 5 Stimulation of CD4+GPR56+ T cells (GPR56+ cells in CD4+ T cells) with anti-GPR56 resulted in a significant upregulation of GZMB expression. DETAILED DESCRIPTION

[0036] The present invention is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way.

[0037] The experimental methods in the following examples, unless otherwise specified, are all conventional methods, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial channels.

[0038] The flow cytometry in the following embodiments refers to the flow cytometry described in "Liang Zhihui, Zhu Huifen, Chen Jiuwu. Basic Principles and Practical Techniques of Flow Cytometry [M]. Huazhong University of Science and Technology Press, 2008." The detection of GPR56 and GZMB in the following embodiments 1-3 are all detected by flow cytometry. In the following embodiments, T cells expressing GPR56 or GPR56-positive T cells are referred to as GPR56+T cells or GPR56+ cells; T cells expressing GZMB are referred to as GZMB+T cells or GZMB+ cells. GPR56 in the following embodiments is G protein-coupled receptor 56, also known as ADGRG1 / adhesion G protein-coupled receptor G1, referred to as GPR56. Granzyme B in the following embodiments is granzyme B, referred to as GZMB.

[0039] Example 1 Application of GPR56 in Screening for Early Primary Sjögren's Syndrome

[0040] Previous studies have shown that GPR56+T cells have stronger cytotoxic function, suggesting that this group of cells may be upregulated in autoimmune diseases and downregulated in diseases such as tumors and chronic viral infections. To this end, in this example, the expression of GPR56 and GZMB in peripheral blood CD4+T cells of patients with primary Sjögren's syndrome was detected.

[0041] 1. The proportions of GPR56+ and GZMB+ in peripheral blood CD4+T cells of patients with primary Sjögren's syndrome were significantly increased.

[0042] Peripheral blood samples were collected from 48 pSS patients and 41 healthy controls, and the extracted peripheral blood mononuclear cells were stained by flow cytometry using flow cytometry fluorescent antibodies. The antibodies used included APC anti-human CD4 (purchased from Biolegend, catalog number 317416), APC-Cy7 anti-human CD8 (purchased from BD, catalog number 557834), PE anti-human GPR56 (purchased from biolegend, catalog number 358204), and PerCP-Cy5.5 anti-human GZMB (purchased from biolegend, catalog number 372211). To stain intracellular GZMB, FoxP3 / transcription Factor Staining Buffer Kit (catalog number TNB-0607-KIT) from Tonbo Biosciences was used for cell fixation and permeabilization to achieve the purpose of intracellular molecule staining. The BD FACSCanto flow cytometer produced by BD was used to analyze GPR56 in CD4+ and CD8+ T cells, and the difference in the proportion of GPR56+ T cells between pSS patients and healthy controls was compared. Figure 1 shown. Figure 1 The figure shows an analysis and comparison of the proportion of GPR56-positive cells (GPR56+ cells) in peripheral blood CD4 and CD8 cells between normal controls and newly diagnosed pSS patients. HC represents normal controls, and pSS represents patients with primary Sjögren's syndrome.

[0043] The results showed that the proportions of GPR56+ and GZMB+ cells in the peripheral blood CD4+T cells of pSS patients were significantly increased, while the difference between pSS and healthy controls in CD8 cells has not yet reached statistical significance.

[0044] 2. Detection of GPR56 can help screen for early pSS patients in healthy people

[0045] The ROC curve was used to analyze the role of GPR56-positive CD4 cells (GPR56+CD4 cells) in the screening of early pSS patients. Figure 2 As shown, Figure 2 The ROC curve analysis of the diagnostic value of the proportion of GPR56+CD4 cells in the CD4+T cell subset for screening early pSS patients. The area under the curve for diagnosing early pSS patients in the healthy population reached 0.7558, which has a good clinical application potential for assisting the diagnosis of early pSS.

[0046] The quantitative standard for the proportion of GPR56+ cells is the percentage of cells expressing GPR56 protein in all T cells. Flow cytometry is used for detection, and the proportion of cells expressing GPR56 is determined by gating based on the boundaries of positive and negative. Figure 2 The cut-off value (critical value) calculated by the maximum value of the Youden index (sensitivity + specificity - 1) is 9.2%, and the corresponding sensitivity is 62.5% and the specificity is 78.05%. This indicates that if the measured GPR56+ ratio in CD4+T cells is greater than 9.2%, it indicates that the patient may have early Sjögren's syndrome. Based on this, the flow cytometric detection of GPR56 as a simple cell surface staining method has important application significance for screening early pSS patients from healthy people.

[0047] Example 2 Application of GPR56 in Screening for Early Lung Adenocarcinoma (LUAD)

[0048] 1. The proportion of GPR56+ in peripheral blood T cells of patients with early lung adenocarcinoma (LUAD) is significantly reduced

[0049] In order to clarify the clinical value of GPR56 in the screening of early lung adenocarcinoma patients, this example included 32 early LUAD patients (all stage IA) and 31 healthy controls. The surface of the extracted peripheral blood mononuclear cells was stained using flow cytometry fluorescent antibodies. The antibodies used included PerCP-Cy5.5 anti-human CD4 (purchased from Biolegend, catalog number 357414), APC-Cy7 anti-human CD8 (purchased from BD, catalog number 557834), APC anti-human GPR56 (purchased from biolegend, catalog number 391905), PE anti-CCR7 (purchased from biolegend, catalog number 353203), PE-Cy7anti-human CD45RA (purchased from biolegend, catalog number 304126), and the BD FACSCanto flow cytometer produced by BD was used to analyze GPR56 in the CD4+ and CD8+ T cells. Figure 3As shown in A, the detection of GPR56 in peripheral blood T cells of these individuals found that the proportion of GPR56+ cells in CD4+T cells and CD8+T cells of early LUAD patients was significantly reduced. Previous studies have shown that GPR56 is significantly co-expressed with granzyme B and perforin, indicating that anti-tumor immunity in early LUAD patients is reduced. T cells were further divided into four subgroups by CCR7 and CD45RA (CD45RA+CCR7+, initial T cells Tn; CD45RA-CCR7+, central memory T cells TCM; CD45RA-CCR7- effector memory T cells TEM; CD45RA+CCR7-, effector memory T cells TEMRA that re-express CD45RA), and the expression of GPR56 in the four subgroups was detected. The results showed that compared with healthy controls, the expression level of GPR56 in CD4+Tn and CD4+TCM cells of patients was significantly increased, while the expression level of GPR56 in CD4+TEM cells of patients was significantly decreased ( Figure 3 B). Further analysis of CD8 cells revealed that the expression rate of GPR56 in CD8 TEMRA cells of patients was significantly reduced compared with that in healthy controls, but significantly increased in CD8 Tn cells and CD8 TEM cells ( Figure 3 C).

[0050] 2. The GPR56+T cell subset has important diagnostic significance for screening early LUAD patients from healthy people. In view of the significant changes in the percentage of GPR56 expression (the percentage of cells expressing GPR56 protein) in CD4 cells, CD8 cells and their four subgroups (initial T cells Tn, central memory T cells TCM, effector memory T cells TEM, and effector memory T cells TEMRA that re-express CD45RA) of early LUAD patients, this example uses the receiver operating characteristic curve to analyze the diagnostic effect of GPR56+ cell-related subgroups in screening early LUAD from healthy controls. The area under the curve (AUC) was calculated, and the cut-off value (critical value) was determined according to the maximum value of the Youden index (sensitivity + specificity - 1). The results showed that the diagnostic efficiency of the GPR56+CD8 cell subset in the diagnosis of early LUAD was higher than that of the GPR56+CD4T cell subset, and the GPR56+CD8 TEMRA subset had the best diagnostic effect, with an AUC of 0.7978 ( Figure 3 We also found that the area under the ROC curve of GPR56+CD4 TCM reached 0.7031, and the area under the ROC curve of GPR56+CD8 TEM reached 0.7238 (see Table 1). Figure 4and Table 1), and also has good potential diagnostic application value for early LUAD patients. The above results show that the following indicators are all useful for early screening of LUAD patients, including the proportion of GPR56+ in CD4 cells, the proportion of GPR56+ in CD8 cells, and the proportion of GPR56+ cells in the four subpopulations of CD4 cells and CD8 cells. Among these indicators, according to the largest area under the curve AUC, the proportion of GPR56+ cells in CD8+TEMRA is the best indicator for early screening of LUAD patients. Table 1 shows the cut-off values ​​of each indicator for screening of early LUAD from healthy people and the corresponding sensitivity and specificity. If the measured proportion of the patient-related subpopulation is lower (for the indicator showing "<") or higher (for the indicator showing ">") than the corresponding cut-off value, it indicates that the patient may have early LUAD. Accordingly, the flow cytometry detection of GPR56, as a simple cell surface staining method, has important application significance for screening early LUAD patients from healthy people.

[0051] Table 1. Relevant indicators of ROC curve analysis of the diagnostic value of the proportion of GPR56+ cells in T cell subsets for screening early LUAD patients

[0052] Cell Subpopulations Area under the curve cut-off value Sensitivity % Specificity% GRP56+%inCD4TEMRA 0.6361 <24.40 87.5 41.94 GRP56+%inCD4Tn 0.6653 >0.1500 46.88 87.1 GRP56+%inCD4TEM 0.6905 <5.500 65.63 70.97 GRP56+%inCD4TCM 0.7031 >0.1500 78.13 70.97 GRP56+%inCD4 0.6507 <4.450 68.75 61.29 GRP56+%inCD8TEMRA 0.7978 <74.00 84.38 72.73 GRP56+%inCD8Tn 0.7107 >12.20 56.25 83.87 GRP56+%inCD8TEM 0.7238 >16.00 78.13 74.19 GRP56+%inCD8TCM 0.5756 <18.85 50 74.19 GRP56+%inCD8 0.7006 <47.85 81.25 54.84

[0053] Example 3 Anti-GPR56 stimulation of GPR56+T cells can significantly upregulate the expression of T cell granzyme B. In this example, peripheral blood was collected from 4 healthy people, and PBMC cells were obtained by density gradient centrifugation. GPR56+CD4 T cells (phenotype CD3+CD4+GPR56+) were sorted from human peripheral blood using BD's BD FACSAriaII flow cytometry sorting system, and stimulated with anti-GPR56 (purchased from biolegend, product number 358202). The cells were stimulated with anti-GPR56 for 24 hours and 48 hours at 37°C and 5% carbon dioxide, and compared with the initially unstimulated cells. The expression level of GZMB in GPR56+CD4 T cells was observed, the percentage of GZMB+ cells and the median fluorescence intensity (MFI) of the GZMB+ cell subset were detected, and compared using a paired t-test. The results are shown in Figure 5As shown, each broken line represents individual data measured once in an independent experiment. After 24 and 48 hours of anti-GPR56 stimulation, the Gzmb+ ratio in circulating CD4+GPR56+T cells increased significantly, and the median fluorescence of the Gzmb+ subpopulation increased significantly. These results indicate that GPR56 stimulation can cause changes in the expression of downstream Gzmb. The same method of stimulating CD8+GPR56+T cells can also significantly upregulate the expression of intracellular granzyme B. The upregulated granzyme B expression proves that the use of GPR56 in vitro can artificially upregulate the killing function of T cells. * indicates p<0.05; ** indicates p<0.01; ns indicates not significant.

[0054] The present invention has been described in detail above. It will be apparent to those skilled in the art that the present invention may be implemented in a wide range under equivalent parameters, concentrations and conditions without departing from the spirit and scope of the present invention and without the need for unnecessary experimentation. Although the present invention provides specific embodiments, it should be understood that further improvements may be made to the present invention. In short, according to the principles of the present invention, this application intends to include any changes, uses or improvements to the present invention, including changes made by conventional techniques known in the art that depart from the scope disclosed in this application. Applications of some of the basic features may be made within the scope of the following appended claims.

Claims

1. The application of the substance for detecting GPR56 in any of the following: A1) Use in the diagnosis of primary Sjögren's syndrome or in the preparation of a product for the diagnosis of primary Sjögren's syndrome; A2) Use in screening for primary Sjögren's syndrome or in preparing a product for screening for primary Sjögren's syndrome; A3) Use in early diagnosis of lung adenocarcinoma or in the preparation of a product for early diagnosis of lung adenocarcinoma; A4) Use in screening for early lung adenocarcinoma or in preparing a product for screening for early lung adenocarcinoma; A5) Use in preparing a product for promoting the expression of granzyme B; A6) Application in the preparation of products that enhance the killing function of T cells.

2. The use according to claim 1, wherein the substance for detecting GPR56 is the following B1) or B2): B1) a reagent or an instrument for detecting the proportion of cells expressing GPR56 in T cells; B2) Reagents or instruments for detecting the content of GPR56 protein in T cells.

3. The use according to claim 2, wherein the T cells are CD4+T cells or CD8+T cells in peripheral blood.

4. The use according to claim 1, wherein the substance for detecting GPR56 comprises an antibody that binds to GPR56.

5. A kit, characterized in that: The kit comprises the substance for detecting GPR56 according to any one of claims 1-4. The kit according to claim 5 , wherein the test sample of the kit is peripheral blood.

7. Use of the kit according to claim 5 or 6 in any of the following: A1) Use in the diagnosis of primary Sjögren's syndrome or in the preparation of a product for the diagnosis of primary Sjögren's syndrome; A2) Use in screening for primary Sjögren's syndrome or in preparing a product for screening for primary Sjögren's syndrome; A3) Use in early diagnosis of lung adenocarcinoma or in the preparation of a product for early diagnosis of lung adenocarcinoma; A4) Use in screening for early lung adenocarcinoma or in preparing a product for screening for early lung adenocarcinoma; A5) Use in the preparation of a product for promoting the expression of granzyme B; A6) Application in the preparation of products that enhance the killing function of T cells.

8. Use of GPR56 as a target in the preparation of products that promote the expression of granzyme B or in the preparation of products that enhance the killing function of T cells.

9. A method for screening or diagnosing primary Sjögren's syndrome, characterized in that: The method comprises the following C1) or C2): C1) detecting the proportion of cells expressing GPR56 protein in CD4+T cells of the patient to be tested, and determining whether the patient to be tested is a patient with primary Sjögren's syndrome according to the proportion value; C2) detecting the content of GPR56 protein in CD4+T cells of the patient to be tested, and determining whether the patient to be tested is a patient with primary Sjögren's syndrome according to the content value.

10. A method for screening or diagnosing early lung adenocarcinoma, characterized in that: The method comprises the following D1) or D2): D1) detecting the proportion of cells expressing GPR56 protein in CD4+T cells of the patient to be tested, and determining whether the patient to be tested is an early lung adenocarcinoma patient according to the proportion value; D2) detecting the level of GPR56 protein in CD4+T cells of the patient to be tested, and determining whether the patient to be tested is an early lung adenocarcinoma patient based on the level.

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