Application of gd t cells and immune checkpoint molecules in preparation of kit for evaluating efficacy of sLE treatment

Abstract

The application discloses application of gamma delta T cells and immune checkpoint molecules in preparation of a kit for evaluating SLE curative effect, and belongs to the technical field of biology. The application is blank in the prior art, and the expression levels of specific subgroups (TCRVd1, TCRVd2) of gamma delta T cells in peripheral blood of SLE patients are comprehensively detected from the gene and protein levels through mutual verification of flow cytometry, quantitative PCR technology and high-throughput liquid-phase protein chip analysis technology, the expression characteristics of immune checkpoint molecules PD-1 and TIGIT on the gamma delta T cell subgroup are combined, and the co-expression detection strategy of Foxp3 is combined, so that the precise evaluation of the clinical treatment effect of SLE patients is realized, and the disease states of SLE patients in the initial stage, refractory / recurrence and remission stage can be effectively evaluated. The application fills the blank in the prior art that the gamma delta T cell subgroup is combined with the immune checkpoint molecule for SLE curative effect evaluation, meanwhile, a standardized combined detection kit is constructed, the detection specificity is high, the result has strong correlation with clinical outcome, important laboratory basis can be provided for individualized treatment and prognosis judgment of SLE, and the application has a good clinical application prospect.

Description

Technical Field

[0001] This invention relates to the field of biological technology, and in particular to the application of γδT cells and immune checkpoint molecules in the preparation of kits for evaluating the efficacy of SLE treatment, specifically the application of a standardized kit for the joint detection of γδT cell subsets and immune checkpoint molecules in the evaluation of SLE treatment efficacy. Background Technology

[0002] Systemic lupus erythematosus (SLE) is a systemic autoimmune disease that predominantly affects women of childbearing age (15-45 years). Its core clinical features include multi-system and multi-organ involvement, recurrent relapses and remissions, and the presence of large amounts of autoantibodies. Without timely and effective treatment, it can lead to irreversible damage to affected organs and seriously endanger the patient's life. The core pathogenesis of SLE lies in the imbalance of the body's immune homeostasis, abnormal activation of T and B lymphocytes, and complement system disorders. T-cell immunodeficiency is closely related to the occurrence, development, and refractory relapse of the disease. Therefore, exploring precise diagnostic and treatment methods from an immunological perspective has significant theoretical and practical value for improving patient prognosis.

[0003] In existing technologies, it has been confirmed that γδT cells participate in the immunopathological process of SLE, and studies have found that elevated PD-1 expression and TIGIT deficiency in γδT cells of SLE patients increase the risk of SLE in mice. Flow cytometry, quantitative PCR, and high-throughput liquid protein chip analysis are also mature existing technologies when used individually. However, these technologies have not been effectively integrated or specifically developed, resulting in many technical bottlenecks in the clinical efficacy evaluation of SLE. The significant shortcomings of existing technologies are as follows: 1. Lack of combined efficacy assessment indicators and multi-technology verification system for γδT cell subsets and immune checkpoint molecules: Current technologies have only found that γδT cells are involved in SLE immunopathology, PD-1 expression is increased in γδT cells of SLE patients, and TIGIT deficiency increases the risk of SLE in mice. However, they have not combined specific γδT cell subsets (TCRVδ1, TCRVδ2) with immune checkpoint molecules such as PD-1 and TIGIT as specific indicators for SLE efficacy assessment. They have also not established a mutual verification system for flow cytometry, quantitative PCR technology and high-throughput liquid phase protein chip analysis technology. Therefore, it is impossible to comprehensively and accurately reflect the immune function repair status of patients after treatment from the gene, protein and cell function levels, and it is also difficult to distinguish the differences in immune characteristics at different stages of the disease.

[0004] 2. Insufficient Specificity and Targeting of Traditional Assessment Indicators: Current clinical practice mainly relies on complete blood count, lipid profile, immunoglobulins, complement (C3, C4), and autoantibody profiles (anti-dsDNA antibodies, ANA, etc.) as assessment criteria. However, these indicators can only reflect the overall immune and metabolic status of the body and cannot accurately characterize the repair status of cellular immune function. For example, a decrease in complement C3 and C4 levels only indicates that immune complexes have activated the complement system, but it is difficult to distinguish whether the patient is in the initial stage, refractory / relapsed stage, or remission stage; the correlation between anti-dsDNA antibody seroconversion and clinical efficacy lacks specificity, cannot effectively predict the patient's relapse risk, and cannot meet the needs of individualized treatment guidance.

[0005] 3. Existing gene testing kits are limited to diagnosis and cannot achieve efficacy assessment and multi-level verification: Most publicly available SLE-related detection technologies focus on disease diagnosis, such as diagnostic kits based on CYP51A1 gene transcription levels (CN116004795B) and diagnostic kits based on C10orf143 gene expression (CN118497330A). Both determine whether there is disease by the expression difference of a single gene, but such kits have obvious shortcomings: On the one hand, the relative expression level of the CYP51A1 gene is not significantly correlated with SLE disease activity and cannot reflect the changes in the condition before and after treatment; on the other hand, the C10orf143 gene can only achieve qualitative diagnosis of the disease, and cannot distinguish the disease stage or assess the regulatory effect of treatment on immune function. Moreover, it is not combined with multiple technologies for verification and is completely unsuitable for efficacy monitoring and prognosis.

[0006] 4. Lack of standardized combined detection kits and clear flow cytometry detection logic for SLE efficacy assessment: Flow cytometry detection of PD-1 / TIGIT molecules and γδT cell subsets are both mature existing technologies. However, existing technologies have not addressed the clinical needs of SLE efficacy assessment by clarifying the standardized logic of flow cytometry detection (from CD45+CD3+T cell delineation to specific subset analysis). The above detection technologies have not been integrated and developed, and there is a lack of standardized combined detection kits for γδT cell subsets and immune checkpoint molecules. At the same time, the detection strategy for co-expression of Foxp3 with PD-1 and TIGIT is not clear, making it impossible to achieve specific detection of γδT cell functional subsets. It is also difficult to form a unified and scalable SLE efficacy assessment detection system, resulting in the inability to routinely carry out related tests in clinical practice.

[0007] In summary, while existing technologies have confirmed the correlation between γδT cells, PD-1, TIGIT, and SLE, and possess the individual application basis for flow cytometry, quantitative PCR, and high-throughput liquid protein chip analysis, they have not combined γδT cell subsets with immune checkpoint molecules as indicators for SLE efficacy assessment. Furthermore, they have not established a multi-technology mutual validation system, developed standardized combined detection kits for SLE efficacy assessment, or defined flow cytometry detection logic. Additionally, they have not clarified the detection strategy for co-expression of Foxp3 with the aforementioned molecules, nor the stratified statistical methods for different disease states. This results in a lack of specific detection methods for cellular immune function, disease stage differentiation, and treatment efficacy quantification. Therefore, there is an urgent need to develop a precise and specific SLE efficacy assessment technology and standardized kits to address clinical needs. Summary of the Invention

[0008] To address the shortcomings of existing technologies, the purpose of this invention is to provide the application of γδT cells and their immune checkpoint molecules in the preparation of SLE efficacy assessment kits, filling the gap in existing technologies that lack the combined use of γδT cell subsets and immune checkpoint molecules for SLE efficacy assessment. This invention establishes a mutual validation system of flow cytometry, quantitative PCR, and high-throughput liquid protein chip analysis technologies, clarifies standardized flow cytometry detection logic and co-expression detection strategies for Foxp3, PD-1, and TIGIT, and develops a standardized combined detection kit for SLE efficacy assessment. This enables accurate evaluation of treatment effects in SLE patients, providing laboratory evidence for individualized SLE treatment and prognosis. The technical content of this invention is as follows: The purpose of this invention is to provide an application of γδT cells and immune checkpoint molecules in the preparation of a kit for evaluating the efficacy of SLE treatment. The core innovation lies in combining γδT cell subsets with immune checkpoint molecules as specific indicators for evaluating the efficacy of SLE treatment. The results are mutually verified by flow cytometry, quantitative PCR, and high-throughput liquid protein chip analysis to ensure the reliability of the test results at the gene and protein levels. The γδT cells include TCRVδ1 and TCRVδ2 subsets, and the immune checkpoint molecules include at least one of PD-1, Tim-3, and TIGIT.

[0009] Preferably, the kit defines a standardized flow cytometry detection logic: first, CD45+CD3+ T cell populations are labeled with anti-CD45 and anti-CD3 antibodies; then, CD3+TCRγδ+ T cell subsets, CD3+TCRVδ1+ T cell subsets, and CD3+TCRVδ2+ T cell subsets are further labeled; then, the expression of immune checkpoint molecules such as PD-1 and TIGIT, as well as Foxp3, on each of the above subsets is detected; simultaneously, the expression levels of secreted cytokines IL-17A and INF-γ are detected; among which, the TCRVδ1 T cell subset mainly expresses IL-17A, and the TCRVδ2 T cell subset mainly expresses INF-γ, forming a targeted functional subset detection system.

[0010] Preferably, the kit employs a co-expression detection strategy of Foxp3 with PD-1 and TIGIT to detect the expression ratio of at least one of the following cell subsets in the peripheral blood of SLE patients: PD-1+Foxp3+TCRγδ+ T cell subset, PD-1+Foxp3+TCRVδ1+ T cell subset, PD-1+Foxp3+TCRVδ2+ T cell subset, TIGIT+TCRVδ1+ T cell subset, and TIGIT+TCRVδ2+ T cell subset. As a core marker of regulatory T cells, the co-expression characteristics of Foxp3 with PD-1 and TIGIT on γδ T cell subsets can accurately reflect the cellular immune status of SLE patients, providing a more specific cellular immune indicator for the evaluation of SLE treatment efficacy.

[0011] Preferably, the kit also integrates quantitative PCR technology, high-throughput liquid phase protein chip technology, and ELISA: Quantitative PCR technology is used to detect the expression levels of relevant genes in peripheral blood mononuclear cells of SLE patients, including the TCR Vγ I-III subfamily and immune function-related molecules (PD-1, Foxp3, TIGIT, IL-17A, INF-γ, etc.), consistent with the cell subpopulation ratios and functional molecule expression trends detected by flow cytometry, validating the results at the gene level (real-time quantitative PCR); high-throughput liquid phase protein chip technology is used to detect the protein expression levels of various cytokines and immune checkpoint molecules such as IFN-γ, IL-1α, IL-6, and IL-10 in serum; ELISA is used to detect the protein expression level of IL-17A in serum, mutually validating the results at the protein level with those of flow cytometry and quantitative PCR, comprehensively supporting the assessment of the immune status of SLE patients; the kit can assess the immune status of SLE patients based on the expression characteristics of core indicators. Patients' clinical status (initial stage, refractory / relapsed stage, or remission stage) was stratified for assessment. Combined with clinical staging information, it was found that the expression ratio of PD-1+Foxp3+TCR Vδ1+γδT cell subsets and TIGIT+TCR Vδ1+γδT cell subsets in peripheral blood tended to be significantly higher in patients with refractory / relapsed stage than in patients with remission stage, providing a standardized assessment direction for subsequent large-sample stratified studies.

[0012] Preferably, the kit is a standardized combined detection kit developed for SLE efficacy assessment, containing supporting reagents for flow cytometry, quantitative PCR, and high-throughput liquid protein chip analysis: the flow cytometry detection section contains fluorescently labeled monoclonal antibodies, including at least one of anti-CD45 antibody, anti-CD3 antibody, anti-TCRγδ antibody, anti-TCRVδ1 antibody, anti-TCRVδ2 antibody, anti-PD-1 antibody, anti-TIGIT antibody, anti-Foxp3 antibody, anti-IL-17A antibody, and anti-INF-γ antibody, and each antibody is labeled with a different fluorophore to achieve specific labeling and accurate detection of γδT cell functional subsets; the quantitative PCR detection section contains RNA extraction reagents, reverse transcription reagents, PCR amplification reagents, and specific primers; the high-throughput liquid protein chip analysis section contains detection microspheres, detection antibodies, standards, and washing solutions, etc.

[0013] Preferably, the fluorophore includes at least three of PE, FITC, and APC-CY7, wherein the anti-CD45 antibody is labeled with APC-CY7, the anti-CD3 antibody is labeled with PE, and the anti-TCRγδ antibody is labeled with FITC. This labeling combination can effectively distinguish γδT cells from other immune cells, reduce background interference, and ensure the accuracy and repeatability of standardized detection.

[0014] Preferably, the kit further comprises at least one of sample processing reagents, cell fixation / permeabilization buffer, washing solution, and PBS resuspension, forming a complete standardized detection system. The sample processing reagents are used to isolate peripheral blood mononuclear cells, and the fixation / permeabilization buffer is used to achieve intracellular Foxp3 fluorescent labeling, meeting the standardization and operability requirements of routine clinical testing. The efficacy assessment includes determining the recovery status of immune function in SLE patients after treatment and predicting the risk of refractory / relapse in SLE patients. When patients are treated, the expression ratio of the PD-1+Foxp3+TCRVδ1+γδT cell subset in peripheral blood is significantly reduced, and quantitative PCR and high-throughput liquid phase protein chip analysis verify that the expression levels of related genes and proteins are simultaneously improved, indicating good immune function recovery and a low risk of refractory / relapse.

[0015] Compared with the prior art, the present invention can achieve the following beneficial effects: 1. Establishment of a novel efficacy evaluation index and multi-technology verification system combining γδT cell subsets and immune checkpoint molecules: This invention addresses a gap in existing technologies by combining specific γδT cell subsets (TCRVδ1, TCRVδ2) with immune checkpoint molecules PD-1 and TIGIT as specific cellular immune indicators for SLE efficacy evaluation. Simultaneously, it integrates flow cytometry, quantitative PCR, and high-throughput liquid protein chip analysis to mutually verify the efficacy at the gene, protein, and other levels. This fills the gap in existing technologies that only study the correlation between the aforementioned molecules / cells and SLE individually, without combined application and multi-technology verification, enriching the technical system for SLE efficacy evaluation and making the test results more reliable.

[0016] 2. Standardized flow cytometry detection logic and subpopulation functional specificity were clarified for the first time: This invention clarifies for the first time the standardized flow cytometry detection logic of "CD45+CD3+ T cells → CD3+TCRγδ+ T subpopulation → CD3+TCRVδ1+, CD3+TCRVδ2+ T cell subpopulation → expression of immune checkpoint molecules and cytokines on the subpopulation", and specifically reveals the functional characteristics of TCRVδ1 subpopulation mainly expressing IL-17A and TCRVδ2 subpopulation mainly expressing INF-γ, realizing the precise location and detection of γδT cell functional subpopulations, which is more targeted and logical than traditional flow cytometry detection.

[0017] 3. A standardized multi-technology combined detection kit for SLE efficacy assessment has been developed: Based on existing mature detection technologies, this invention integrates and develops a standardized combined detection kit that incorporates flow cytometry, quantitative PCR, and high-throughput liquid phase protein chip analysis technologies to meet the clinical needs of SLE efficacy assessment. It is equipped with dedicated reagents for sample processing, cell labeling, nucleic acid extraction, and protein detection, forming a unified and scalable detection system. This solves the problem that existing technologies lacked standardized kits and could not be routinely implemented in clinical practice. Furthermore, the detection operation is simple and rapid, requires only a small sample volume (peripheral blood only), and has low requirements for clinical sample processing, making it suitable for clinical testing applications in medical institutions at all levels.

[0018] 4. Precisely distinguishes disease stages and enables stratified assessment: The kit of this invention uses the combined detection results of γδT cell subsets and immune checkpoint molecules as the core indicators. Combined with multi-technology validation data, it is highly correlated with the clinical status of SLE patients (new onset, refractory / relapse, remission). Even without separate stratified statistical detection of the three disease states in 23 SLE patients, the characteristics of different disease stages can still be defined by the expression range of the core indicators. At the same time, it provides a standardized method for subsequent stratified statistical analysis. It can not only realize the immediate assessment of the treatment effect of SLE patients, but also accurately distinguish different stages of the disease, effectively predict the risk of refractory / relapse of patients, and provide important basis for clinicians to adjust individualized treatment plans. This helps to reduce the relapse rate of SLE patients and improve patient prognosis.

[0019] 5. Complete clinical information and correlation analysis of test results: This invention integrates the basic clinical information (gender, age), disease status (initial stage, refractory / relapse stage, remission stage) and various laboratory test data of SLE patients, and establishes a correlation analysis system between test indicators and clinical status, filling the gap in the existing technology that lacks integrated analysis of clinical information and test results, making efficacy evaluation more clinically practical. Attached Figure Description

[0020] The accompanying drawings, which are included to provide a further understanding of the invention and form part of this invention, illustrate exemplary embodiments of the invention and are used to explain the invention, but do not constitute an undue limitation of the invention. In the drawings:

[0021] Figure 1 The difference in the proportion of peripheral blood γδT cells and their subsets (Vδ1, Vδ2) between healthy controls and SLE patients; Figure 2 The expression differences of Foxp3 and PD-1 on peripheral blood γδT cells and their subsets between healthy controls and SLE patients; Figure 3Differences in the proportion of PD-1+Foxp3+γδT cells and their subsets in peripheral blood between healthy controls and SLE patients; Figure 4 Differences in the secretion of IL-17A and INF-γ by peripheral blood γδT cells and their subsets between healthy controls and SLE patients; Figure 5 Differences in TIGIT expression on peripheral blood γδT cells and their subsets between healthy controls and SLE patients; Figure 6 Gene expression levels of immune function-related molecules in the peripheral blood of SLE patients; Figure 7 Expression levels of TCR VγI~III subfamily genes in peripheral blood of SLE patients; Figure 8 High-throughput liquid phase protein chip analysis of the expression of relevant indicators in peripheral blood serum of SLE patients; Figure 9 ELISA method was used to detect the expression of relevant markers in peripheral blood serum of SLE patients; Figure 10 Correlation analysis of the expression ratio of functional subsets of peripheral blood γδ T cells in patients with SLE. Detailed Implementation

[0022] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below in conjunction with specific embodiments and corresponding drawings. Obviously, the described embodiments are only a part of the embodiments of this invention, and not all of them. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention.

[0023] The technical solutions provided by the various embodiments of the present invention will be described in detail below with reference to the accompanying drawings.

[0024] Reference Figures 1-10 This invention provides the application of γδT cells and immune checkpoint molecules in the preparation of kits for evaluating the efficacy of SLE. The core of this invention is to combine γδT cell subsets and immune checkpoint molecules as indicators for evaluating the efficacy of SLE, establish a mutual verification system of flow cytometry, quantitative PCR and high-throughput liquid protein chip analysis, clarify the standardized flow cytometry detection logic and the co-expression detection strategy of Foxp3 with PD-1 and TIGIT, and develop a standardized joint detection kit.

[0025] Clinical sample information and test grouping This study included 67 participants, including 41 SLE patients and 26 healthy controls. Table 1 shows a comparison of blood routine indicators between the healthy control group and the SLE patients. Table 1 Comparison of blood routine indicators between normal group and SLE patients All SLE patients met the 1997 revised diagnostic criteria for lupus by the American College of Rheumatology (ACR). The healthy controls were healthy individuals who underwent physical examinations. The baseline data of gender and age were comparable between the two groups.

[0026] Example 1: Composition of the Standardized Kit for Efficacy Assessment of Systemic Lupus Erythematosus A standardized multi-technology combined detection kit for SLE efficacy assessment is presented, and its components are shown in Table 2 below. The components of the kit form a complete detection system, ensuring the standardization, repeatability, and operability of the detection. Table 2 Composition of the SLE efficacy assessment kit Example 2: Detection method of the standardized reagent kit of the present invention The standardized reagent kit developed above for SLE efficacy assessment was used for multi-technology combined detection. The specific steps are as follows, and standardized operating procedures were established for all steps to ensure the accuracy and repeatability of the test results: (1) Collection and processing of clinical blood samples Peripheral blood samples were collected from SLE patients, and peripheral blood mononuclear cells were extracted using density gradient centrifugation with Ficoll separation solution. Informed consent was obtained from patients, and all samples were taken from fasting morning venous blood, anticoagulated with heparin. This part of the research protocol has been approved by the institution's ethics committee.

[0027] (2) RNA extraction and reverse transcription to synthesize cDNA Following the instructions, RNA was extracted from the collected peripheral blood mononuclear cells using the Trizol RNA extraction kit, and the first strand of cDNA was synthesized by reverse transcription using random primers and a reverse transcriptase kit. The quality of the synthesized cDNA was then determined by RT-PCR detection of the β2 microglobulin gene.

[0028] (3) Real-time quantitative PCR detection of TCR Vγ gene (SYBR green I staining method) Three Vγ (Vγ I~Vγ III) specific upstream primers were designed based on the TCR γ chain cDNA sequence, and a downstream primer Cγ was designed in the common region of Cγ1 and Cγ2. β2M was used as an internal reference gene. A negative control (without template) was included in all sample testing to exclude false positive results. Calculation of relative mRNA expression level: 2 -△Ct×100%, calculate the relative mRNA expression level difference of each subfamily of the TCRVγ gene in each sample, where ΔCt = Ct (target gene) - Ct (internal reference gene), as shown. Figure 6 The gene expression levels of immune-related molecules in the peripheral blood of SLE patients are shown. It can be seen that the gene expression levels of PD-1, TIGIT, IL-17A, and Foxp3 are significantly increased, while the gene expression level of IFN-γ is significantly decreased. Overall, this indicates an immune dysregulation state characterized by enhanced immunosuppression, increased pro-inflammatory factors, and decreased anti-inflammatory factors. The gene expression levels of these immune-related molecules are closely related to the cellular immune homeostasis of SLE patients and may affect their clinical outcome. Figure 7 The expression pattern of TCR VγI~III subfamily genes in peripheral blood PBMCs of SLE patients showed that: TCRVγI>TCR VγIII>TCR VγII, which is different from the expression pattern of healthy individuals. This suggests that SLE patients have a higher level of TCR VγI subfamily gene expression, and the imbalance of TCR VγI~III subfamily gene expression may be related to the disease state of SLE patients.

[0029] (4) Flow cytometry was used to detect the expression of γδ T cell functional subsets. Using flow cytometry antibodies such as PE-labeled anti-CD3 monoclonal antibody, FITC-labeled TCR γ / δ monoclonal antibody, PE-labeled FoxP3 monoclonal antibody, PE-labeled TIGIT monoclonal antibody, and PE-labeled PD-1 monoclonal antibody, the expression of surface molecules and functional subsets of γδ T cells in peripheral blood of SLE patients was detected by flow cytometry.

[0030] 1) Process the peripheral blood sample to be tested to form a single-cell suspension; specifically, process the peripheral blood sample to be tested according to conventional methods to separate the lymphocyte suspension. 2) Add monoclonal antibodies labeled with different fluorescence to the single-cell suspension obtained in step (1): anti-CD45 antibody, anti-CD3 antibody, anti-TCR γδ antibody, anti-PD-1 antibody, and isotype control antibody of the anti-PD-1 antibody. Mix gently and incubate in the dark. 3) After washing the cells, add fixation / permeabilization buffer, incubate in the dark, wash the cells again, mix thoroughly, and centrifuge to remove the supernatant; 4) Wash the cells, resuspend them in permeabilization buffer, and incubate in the dark; 5) Centrifuge to remove the supernatant, add fluorescently labeled anti-Foxp3 antibody and incubate in the dark; 6) After washing the cells, resuspend them in PBS and analyze them using flow cytometry to obtain data on the fluorescently labeled γδ T cell subsets. The results are as follows: Figure 1-5 As shown: Figure 1 Results: The proportion of total γδ T cells (CD3+TCRγδ+) in the peripheral blood of SLE patients was significantly lower than that in healthy controls (P=0.0001), mainly due to a significant decrease in the Vδ2 subset (CD3+TCRVδ2+) (P=0.0002), while there was no significant difference in the proportion of the Vδ1 subset (CD3+TCRVδ1+) (P=0.1579). Figure 2 Results: The expression ratios of Foxp3 (regulatory T cell marker) and PD-1 (immune checkpoint inhibitory molecule) on γδT cells and their Vδ1 and Vδ2 subsets were significantly higher in SLE patients than in healthy controls (P < 0.01), with the largest increase in the proportion of Foxp3+ and PD-1+ in the Vδ2 subset. Figure 3 Results: The proportion of PD-1+Foxp3+ double-positive cells in γδT cells and its Vδ1 subset was significantly higher in SLE patients than in healthy controls (P=0.0002, P=0.0011), suggesting that this subset has functional suppression. Figure 4 Results: The IL-17A secretion level of γδT cells and its Vδ1 subset was significantly increased in SLE patients (P=0.0027, P=0.0011), while the IFN-γ secretion level of the Vδ2 subset was significantly decreased (P=0.0022). Combined with the functional characteristics of "Vδ1 subset mainly secreting IL-17A and Vδ2 subset mainly secreting IFN-γ", it suggests that γδT cells in SLE patients have an immune disorder state with enhanced expression of pro-inflammatory factor (IL-17A) and decreased IFN-γ-mediated immune surveillance and killing function. Figure 5 Results: The proportion of TIGIT+γδ T cell subsets in γδ T cells and their Vδ1 and Vδ2 subsets was significantly higher in SLE patients than in healthy controls (P < 0.001), with the Vδ2 subset showing the most significant increase (0.36% vs 4.40%). This suggests that TIGIT, as a negative co-stimulatory molecule, may inhibit γδ T cell activation or response in SLE patients and play a major role in γδ T cell immune tolerance.

[0031] like Figure 10The correlation analysis of the expression ratios of peripheral blood γδ T cell functional subsets in SLE patients showed a correlation between the expression ratios of PD-1+, Foxp3+, and PD1+Foxp3+ subsets in γδ T cells across different groups. However, no significant correlation was found between the expression ratios of PD1+ γδ T cells, Foxp3+ γδ T cells, and PD1+Foxp3+ γδ T cells in healthy control cells. Furthermore, a positive correlation was found between the expression ratios of Foxp3+ γδ T cells and PD1+Foxp3+ γδ T cells in SLE patients, indicating a significantly increased proportion of Foxp3+ γδ T cells, consistent with the high expression of PD1+Foxp3+ γδ T cells. This suggests that SLE patients exhibit an immunosuppressive phenotype in γδ T cells.

[0032] (5) High-throughput liquid phase protein chip analysis of serum related markers in SLE patients.

[0033] 1) Reagent preparation Wash Buffer (10X-1X): Dilute with ddH2O at a ratio of 9:1.

[0034] Beads (50X-1X): Vortex the beads for 30 seconds. Take 100µL from each 50X Beads tube and add 1X Wash Buffer to a final volume of 5mL. Mix well. (If the kit provides 1X Beads, skip this step.) This example uses one bottle of 1X Beads.

[0035] Detection Antibody (50X-1X): Take 60ul from each tube of 50X Detection Antibody, add Detection Antibody Diluent to a final volume of 3mL, and mix well.

[0036] 2) Standard reconstitution a) Take out the standard sample and centrifuge at 2000 xg for 10s; b) Add 50 µL of Universal Assay Buffer to each of the standard tubes; c) Gently mix for 30 seconds; d) Place on ice for 5-10 minutes; e) Mix the standards in one tube, add Universal Assay Buffer, and finally obtain 250µL of mixed standards. In this example, there are 6 bottles of standards. Five bottles need to be dissolved first, and then the dissolved standards from the five bottles are added to the sixth bottle.

[0037] 3) Serial dilution of standard (4 times) a) Use the 8-tube PCR kit provided to dilute the standards; b) Add 200µL of the mixed standard to the first tube as standard 1; c) Add 150 µL of Universal Assay Buffer to tubes 2-8 respectively; d) Take 50µL of the mixed standard from tube 1 and add it to tube 2. Mix by blowing up and down 10 times, trying to avoid the generation of air bubbles. e) Replace with a new pipette tip, transfer 50µL of the diluted standard from tube 2 to tube 3, and mix by pipetting up and down 10 times. Repeat this process to complete the serial dilution of the mixed standard.

[0038] f) Place on ice for later use 4) Washing microspheres a) Vortex microspheres for 30 seconds; b) Add 50 µL / bottle of premixed microspheres to each well of a 96-well plate.

[0039] c) Place the 96-well plate into the magnetic separation plate, ensuring the plate is firmly held in place. Allow the plate to stand for 2 minutes to allow the microspheres to sink to the bottom. Then quickly invert the magnetic plate to pour out the liquid from the plate. Do not remove the 96-well plate from the magnetic separation plate during this process;

[0040] d) Add 150µL of 1X Wash Buffer to each well, let stand for 30 seconds, then invert the magnetic plate and pour out the liquid from the well plate; e) With the plate in an inverted position, use a paper towel to absorb any residual liquid on the surface of the perforated plate.

[0041] 5) Add sample incubation a) For serum samples, add 25 µL of Universal Assay Buffer + 25 µL of standard or sample to each well; b) Add 25 µL of Universal Assay Buffer to the blank control; c) Seal the well plate, incubate with shaking at 500 rpm at room temperature for 30 min, and then let it stand overnight at 4 degrees Celsius. The next day, remove the plate and incubate with shaking at 500 rpm at room temperature for 30 min.

[0042] 6) Washing the board a) Place the 96-well plate in the magnetic separation plate and let it stand for 2 minutes; b) Gently remove the sealing film to avoid liquid splashing; c) Invert the orifice plate to remove the liquid; d) Add 150 µL of 1X Wash Buffer to each well, let stand for 30 seconds, then invert the plate to remove the liquid. Repeat this process for a total of 3 washes.

[0043] e) At the end of the final rinse, use paper towels to absorb any remaining liquid.

[0044] 7) Add detection antibodies a) Add 25 µL of 1X detection antibody mixture to each well; b) Use a new sealing membrane to seal the orifice plate; c) Remove the 96-well plate from the magnetic separator and place it in a plate shaker and shake at 500 rpm at room temperature for 30 min.

[0045] 8) Washing the board a) Place the 96-well plate in the magnetic separation plate and let it stand for 2 minutes; b) Gently remove the sealing film to avoid liquid splashing; c) Invert the orifice plate to remove the liquid; d) Add 150 µL of 1X Wash Buffer to each well, let stand for 30 s, then invert the plate to remove the liquid. Repeat this washing process 3 times. e) At the end of the final rinse, use paper towels to absorb any remaining liquid.

[0046] 9) Add SA-PE a) Add 50 µL of SA-PE to each well; b) Seal the orifice plate with a new sealing membrane; c) Remove the 96-well plate from the magnetic separator and place it in a plate shaker and shake at 500 rpm at room temperature for 30 min.

[0047] 10) Washing the board a) Place the 96-well plate in the magnetic separation plate and let it stand for 2 minutes; b) Gently remove the sealing film to avoid liquid splashing; c) Invert the orifice plate to remove the liquid; d) Add 150µL of 1X Wash Buffer to each well, let stand for 30s, then invert the plate to remove the liquid. Repeat this washing process 3 times. e) At the end of the final rinse, use paper towels to absorb any remaining liquid.

[0048] 11) On-machine testing a) Add 120 µL of Reading Buffer to each well; b) Seal the orifice plate with a new sealing membrane; c) Remove the 96-well plate from the magnetic separator and place it in a plate shaker and shake at 500 rpm for 5 minutes at room temperature; d) Gently remove the sealing film and insert it into the Luminex 200 instrument to take the reading.

[0049] 12) Analyze the data A standard curve was fitted using a five-parameter nonlinear regression method, and the concentration values ​​were calculated. The results are as follows: Figure 8 The high-throughput liquid phase protein chip analysis of peripheral blood serum related indicators in SLE patients showed that the expression levels of IL-1α, IL-10, and IL-6 were significantly increased, while the expression levels of IFN-γ and IL-2 were significantly decreased. The expression level of IL-4 showed no significant difference, indicating an overall immune disorder state with an imbalance between pro-inflammatory and anti-inflammatory factors.

[0050] (6) ELISA method was used to detect the expression of serum related indicators in SLE patients.

[0051] 1. Reagent preparation 1) Wash Buffer: Dilute the concentrate (20x) with deionized water at a ratio of 1:20.

[0052] 2) Serial dilution of standards: a. IL-17A: Reconstitute the lyophilized standard with 1000 µl of diluent. Gently rotate the vial or shake slightly, then let the reconstituted standard stand at room temperature for 10 minutes to ensure complete dissolution of the dry powder. Gently shake to ensure uniform mixing of the standard (concentration 1000 pg / ml). Take 7 clean sample tubes and label them as: S1, S2, S3, S4, S5, S6, S7, and S8. Add 20 μL of the reconstituted standard working solution and 980 μL of diluent to sample tube S1 and mix well. Add 150 μL of diluent to each of sample tubes S2-S8. Transfer 150 μL of the standard from tube S1 to tube S2 and mix well. Then transfer 150 μL of the mixed standard from tube S2 to tube S3 and mix well. Repeat the above steps to continuously dilute up to tube S8. This creates a standard curve, using the diluent as a blank control. (Concentrations: 20, 10, 5, 2.5, 1.25, 0.625, 0.3125, 0.156, 0 pg / ml).

[0053] b. TGF-β: Reconstitute the lyophilized standard with 750 µl of diluent. Gently rotate the vial or shake slightly, then let the reconstituted standard stand at room temperature for 10 minutes to ensure complete dissolution of the dry powder. Gently shake to ensure homogeneous mixing of the standard (concentration 1000 pg / ml). Take 7 clean sample tubes and label them as: S1, S2, S3, S4, S5, S6, and S7. Add 300 μL of the reconstituted standard working solution to sample tube S1, and add 150 μL of diluent to sample tubes S2 through S7 respectively. Transfer 150 μL of the standard from sample tube S1 to tube S2 and mix well. Then transfer 150 μL of the mixed standard from tube S2 to tube S3 and mix well. Repeat the above steps to continuously dilute to tube S7. This creates a standard curve, with the diluent serving as a blank control. (Concentrations: 1000, 500, 250, 125, 62.5, 31.25, 15.625, 0 pg / ml).

[0054] 3) Antibody working solution: According to the required amount in this example, IL-17A and TGF-β are diluted 50x with the antibody diluent to concentrate the antibody to the working concentration. Prepare and use immediately.

[0055] 4) HRP working solution: a. To prepare the working concentration of TGF-β, dilute 50x concentrated HRP with HRP diluent according to the dosage required in this embodiment. Prepare and use immediately.

[0056] b. According to the required dosage in this embodiment, IL-17A is diluted with HRP diluent to 50x concentrated high-sensitivity HRP to the working concentration, and should be prepared and used immediately.

[0057] 2. Sample processing 1) Final dilution factor of IL-17A sample: 2x 2) Final dilution factor of TGF-β sample: 14 times (The centrifuged sample was activated according to the instructions. During the activation process, the sample was diluted 7 times. According to the preliminary experimental results, it needs to be diluted another 2 times). 3. Operating Procedures 1) Prepare all reagents, samples and standards.

[0058] 2) Add 300μl Wash Buffer (1x) to each well and wash once.

[0059] 3) Add 100 μL of diluted sample or diluted standard to the corresponding well. Add 100 μL of diluent to the blank well. Incubate with shaking for 120 minutes at room temperature.

[0060] 4) Add 300µL Wash Buffer (1x) to each well and wash three times.

[0061] 5) Add 1× detection antibody working solution (100ul / well) to each well, cover with sealing film, and incubate with shaking at room temperature for 60 minutes.

[0062] 6) Add 300µL Wash Buffer (1x) to each well and wash three times.

[0063] 7) Add 1×HRP working solution or 1×high-sensitivity HRP working solution (100ul / well) to each well, gently shake to mix, cover with sealing film, and incubate at room temperature for 30 minutes with shaking.

[0064] 8) Add 300µL Wash Buffer (1x) to each well and wash six times.

[0065] 9) Add 100µL of TMB substrate solution to each well, cover with a sealing film, and incubate at room temperature in the dark with shaking for 10-20 minutes.

[0066] 10) Add 100 μl of stop solution to each well to terminate the reaction.

[0067] 11) Read the 450nm OD value using a microplate reader. The result is as follows: Figure 9The ELISA method shown indicates that the expression levels of relevant indicators in the peripheral blood serum of SLE patients are significantly elevated. It can be seen that the expression levels of TGF-β and IL-17A in the peripheral blood serum of SLE patients are also significantly elevated, reflecting the immune dysregulation characteristics of the imbalance between the secretion of pro-inflammatory and anti-inflammatory factors in SLE patients, and that SLE patients are in a state of low immune function.

[0068] The above description is merely an embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principle of the present invention should be included within the scope of the claims of the present invention.

Claims

1. The application of reagents for detecting γδT cells and immune checkpoint molecules in the preparation of kits for evaluating the efficacy of SLE treatment, characterized in that, The γδT cells include the TCRVδ1 and TCRVδ2 subsets, and the immune checkpoint molecules include PD-1 and TIGIT. The combination of the γδT cell subsets and immune checkpoint molecules is used as a specific indicator for evaluating the efficacy of SLE treatment. The kit contains fluorescently labeled monoclonal antibodies, which are composed of anti-CD45 antibody, anti-CD3 antibody, anti-TCRγδ antibody, anti-TCRVδ1 antibody, anti-TCRVδ2 antibody, anti-PD-1 antibody, anti-TIGIT antibody, anti-Foxp3 antibody, anti-IL-17A antibody, and anti-INF-γ antibody.

2. The application according to claim 1, characterized in that, The kit employs a co-expression detection strategy for Foxp3, PD-1, and TIGIT to detect the expression ratios of PD-1+Foxp3+TCRγδ+T, PD-1+Foxp3+TCRVδ1+T cell subsets, PD-1+Foxp3+TCRVδ2+T cell subsets, TIGIT+TCRVδ1+T cell subsets, and TIGIT+TCRVδ2+T cell subsets in the peripheral blood of SLE patients.

3. The application according to claim 1, characterized in that, The kit is also used to detect the expression levels of cytokines INF-γ and IL-17A secreted by γδT cells in the peripheral blood of SLE patients, and the combined detection results of γδT cell subsets and immune checkpoint molecules are used in synergistic ways to evaluate the efficacy of SLE treatment.

4. The application according to claim 1, characterized in that, The kit also includes at least one of the following: sample processing reagents, cell fixation or permeabilization buffer, washing solution, PBS resuspension solution, RNA extraction reagent, reverse transcription reagent, PCR amplification reagent, and high-throughput liquid phase protein chip analysis reagent, forming a standardized SLE efficacy assessment and detection system.

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