Imaging mass spectrometry flow-based biomarker for superficial lymph node tuberculosis and screening method, detection reagent and application thereof

Abstract

This invention belongs to the fields of biomedical detection and immunology, specifically relating to a biomarker for superficial lymph node tuberculosis based on imaging mass cytometry, along with its screening method, detection reagents, and applications. It reveals for the first time the dynamic expression patterns of the immune checkpoint molecule VISTA in different pathological stages (ST0-ST4) of superficial lymph node tuberculosis. It provides a method for screening the biomarker based on imaging mass cytometry (IMC), as well as detection reagents and their application in the preparation of products for monitoring the course of superficial lymph node tuberculosis, prognostic assessment, or efficacy evaluation. Through the combined analysis of high-dimensional single cells and spatial information, it achieves precise analysis of the lymph node tissue microenvironment at different pathological stages, possessing significant clinical application value.

Description

Technical Field

[0001] This invention belongs to the field of biomedical detection and immunology technology, specifically relating to a biomarker for superficial lymph node tuberculosis based on imaging mass cytometry, its screening method, detection reagents, and applications. Background Technology

[0002] Superficial lymph node tuberculosis is one of the most common extrapulmonary tuberculosis types, with a complex pathological process. Clinically, it is classified into nodular (ST1), infiltrative (ST2), abscessive (ST3), and ulcerative fistula (ST4) types. Clinical studies have shown that effective intervention in the ST1 and ST2 stages can prevent or reduce lymph node calcification and secondary necrosis, achieving clinical cure. However, once the disease progresses to the ST3 or ST4 stages, the local immune environment undergoes irreversible changes, making clinical management extremely difficult and often requiring surgical intervention. Therefore, identifying biomarkers that can accurately reflect pathological progression in the early stages of the disease and guide treatment decisions is crucial.

[0003] Traditional immunohistochemistry and immunofluorescence techniques are limited by the number of detection channels, making it difficult to perform high-throughput, multi-parameter simultaneous analysis of complex tissue microenvironments at the single-cell level. Imaging Mass Cytometry (IMC) combines the high-dimensional detection capabilities of CyTOF with the spatial information resolution capabilities of tissue imaging, enabling in-depth analysis of the tissue microenvironment at the subcellular level and providing a powerful tool for studying the composition, spatial structure, and interactions of immune cells.

[0004] VISTA (V-domain Ig suppressor of T cell activation) is a negative costimulatory molecule (immune checkpoint) that plays a crucial role in regulating T cell activation. However, the dynamic expression pattern of VISTA in different pathological stages (ST0-ST4) of superficial lymph node tuberculosis and its potential as a biomarker have not yet been clearly reported. Summary of the Invention

[0005] The purpose of this invention is to provide a biomarker for superficial lymph node tuberculosis based on imaging mass cytometry, as well as its screening method, detection reagents, and applications.

[0006] To address the problems existing in the prior art, the present invention adopts the following technical solution: A biomarker for superficial lymph node tuberculosis based on imaging mass cytometry, wherein the biomarker is VISTA protein.

[0007] The aforementioned imaging mass cytometry-based biomarkers for superficial lymph node tuberculosis exhibit the following characteristics in the expression levels of the VISTA protein at different pathological stages of superficial lymph node tuberculosis: (1) The expression level is highest in the ST0 phase, i.e., in normal control tissue adjacent to the lesion; (2) Expression levels begin to decrease in ST1 (nodular type) and ST2 (infiltrative type); (3) The expression level was significantly reduced in ST3 (abscess type) and ST4 (ulcer fistula type), showing a statistically significant difference compared with ST0.

[0008] A method for screening biomarkers for superficial lymph node tuberculosis based on imaging mass cytometry includes the following steps: (1) Collect formalin-fixed paraffin-embedded tissue samples from patients with superficial lymph node tuberculosis at different pathological stages (ST0, ST1, ST2, ST3, ST4); (2) Design and prepare a set of antibodies conjugated to metal isotope markers, the antibodies including at least anti-VISTA antibody, and stain the tissue sample sections using imaging mass cytometry. (3) Image data were acquired using an imaging mass spectrometry flow cytometry system to obtain multi-channel images. After image preprocessing and cell segmentation, protein expression data at the single-cell level were extracted. (4) Use dimensionality reduction algorithm to perform cell population visualization analysis, identify and distinguish different cell types, analyze the expression level of VISTA in each cell population at different pathological stages, and screen out biomarkers whose expression level is significantly correlated with the disease stage.

[0009] In the above screening method, the dimensionality reduction algorithm in step (4) is t-distributed random neighborhood embedding (t-SNE).

[0010] In the above screening method, the cell types mentioned in step (4) include CD4+ T cells, CD8+ T cells and M1 macrophages.

[0011] A detection reagent for a biomarker of superficial lymph node tuberculosis based on imaging mass cytometry, the detection reagent comprising an antibody that specifically binds to the VISTA protein.

[0012] In the above-mentioned detection reagent, the antibody is coupled to a detectable marker; preferably, the detectable marker is a metal isotope marker.

[0013] The above-mentioned detection reagents also include at least one selected from anti-CD4 antibody, anti-CD8 antibody, anti-CD68 antibody, anti-FOXP3 antibody, and anti-PD1 antibody, each antibody being conjugated to a different detectable marker.

[0014] A detection kit for biomarkers of superficial lymph node tuberculosis based on imaging mass cytometry, comprising: First antibody: Anti-human VISTA monoclonal antibody conjugated to metal isotope 169Tm, working concentration 1:200; Second antibody: Anti-human CD8 monoclonal antibody conjugated with the metal isotope 146Nd, working concentration 1:400; Third antibody: anti-human CD4 monoclonal antibody conjugated with metal isotope 149Sm, working concentration 1:400; Fourth antibody: Anti-human CD68 monoclonal antibody conjugated with the metal isotope 175Lu, working concentration 1:300; Nuclear dyes: intercalating agents containing 191Ir and 193Ir; Other necessary reagents: antigen retrieval buffer, antibody dilution solution, and washing buffer.

[0015] The above-mentioned test reagents are used in the preparation of products for monitoring the course of superficial lymph node tuberculosis, prognostic assessment, or efficacy evaluation.

[0016] The monitoring of superficial lymph node tuberculosis includes dynamically detecting changes in the expression of VISTA in lymph node tissue before and after treatment. If the expression level of VISTA is significantly lower than that of ST0 stage, it indicates that the disease has entered the middle and late stages.

[0017] The prognostic assessment of superficial lymph node tuberculosis includes detecting the VISTA expression level in the patient's lesion tissue before treatment. If the VISTA expression level rises after treatment, it indicates that the treatment is effective.

[0018] The prognostic assessment of superficial lymph node tuberculosis also includes: detecting the expression level of VISTA in CD8+ T cells in the lesion tissue of the patient before treatment, with the expression level of VISTA in normal tissue adjacent to the ST0 stage lesion as a reference. If VISTA is highly expressed, it indicates that the patient responds well to early intervention treatment. If the VISTA expression level drops to a level comparable to that of ST3 or ST4 stage, it indicates that the patient responds poorly to conventional treatment.

[0019] A method for screening anti-tuberculosis immunomodulatory drugs, based on imaging mass cytometry, includes the following steps: (1) Using the detection reagent of any one of claims 6-9 to stain superficial lymph node tuberculosis tissue models or lymph node tissue sections of patients before and after treatment with candidate drugs; (2) Image data were acquired using an imaging mass cytometry system, and VISTA protein expression data at the single-cell level were extracted; (3) By comparing the changes in the expression level of VISTA in specific cell populations before and after treatment with the control group, candidate drugs that can upregulate VISTA expression or restore its function can be screened.

[0020] In the above method, the specific cell population includes CD8+ T cells, CD4+ T cells, and M1 macrophages.

[0021] Compared with the prior art, the present invention has the following beneficial effects: 1. This invention reveals for the first time the dynamic expression pattern of VISTA in different pathological stages (ST0-ST4) of superficial lymph node tuberculosis, namely, a gradual decrease in expression from high in the ST0 stage to significantly low expression in the ST3 / ST4 stage. This discovery provides new insights into the immune dysregulation mechanism in the progression of tuberculosis.

[0022] 2. The expression level of VISTA is highly correlated with the severity of the disease (ST stage). In particular, the downregulation of its expression can serve as a key indicator for the disease to shift from a controllable stage (ST1 / ST2) to a refractory stage (ST3 / ST4), and has important diagnostic and prognostic value.

[0023] 3. The imaging mass cytometry (IMC) screening method employed in this invention enables high-throughput, multi-parameter analysis of VISTA expression in multiple cell types simultaneously at both single-cell and spatial levels. This method overcomes the shortcomings of traditional immunohistochemistry techniques, such as low throughput and inaccurate quantification, providing a reliable platform for the precise screening of biomarkers related to the tissue microenvironment.

[0024] 4. VISTA biomarker-based detection reagents can not only be used for clinical auxiliary diagnosis and efficacy monitoring, but also provide a theoretical basis and target for developing novel immunotherapy strategies for advanced tuberculosis (such as combined immune checkpoint inhibitors). Attached Figure Description

[0025] To more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings used in the following description of the embodiments will be briefly introduced. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0026] Figure 1 A: The expression distribution of VISTA on CD8+ T cells is shown by t-SNE analysis; Figure 1 B: Distribution of VISTA expression on CD8+ T cells in different stages of lymph node tuberculosis using t-SNE analysis; Figure 1C: The bar chart shows the statistical analysis of VISTA expression levels on CD8+ T cells at five different stages (ST0, ST1, ST2, ST3, ST4); Figure 1 D: The expression distribution of VISTA on CD4+ T cells is shown by t-SNE analysis; Figure 1 E: Distribution of VISTA expression on CD4+ T cells in different stages of lymph node tuberculosis using t-SNE analysis; Figure 1 F: The bar chart shows the statistical analysis of VISTA expression levels on CD4+ T cells at five different stages (ST0, ST1, ST2, ST3, ST4); Figure 2 A: The distribution of VISTA expression on M1 macrophages at different stages of lymph node tuberculosis was shown by t-SNE analysis; Figure 2 B: The bar chart shows the statistical analysis of VISTA expression levels on M1 macrophages at five different stages (ST0, ST1, ST2, ST3, ST4). Detailed Implementation

[0027] To make the above-mentioned objects, features and advantages of the present invention more apparent and understandable, the specific embodiments of the present invention will be described in detail below with reference to the examples in the specification.

[0028] Many specific details are set forth in the following description in order to provide a full understanding of the invention. However, the invention may also be practiced in other ways different from those described herein, and those skilled in the art can make similar extensions without departing from the spirit of the invention. Therefore, the invention is not limited to the specific embodiments disclosed below.

[0029] Secondly, the term "one embodiment" or "embodiment" as used herein refers to a specific feature, structure, or characteristic that may be included in at least one implementation of the present invention. The phrase "in one embodiment" appearing in different places in this specification does not necessarily refer to the same embodiment, nor is it a single or selective embodiment that is mutually exclusive with other embodiments.

[0030] Unless otherwise specified, all raw materials used in the examples are commercially available.

[0031] Example 1 This embodiment provides a biomarker for superficial lymph node tuberculosis based on imaging mass cytometry, the biomarker being the VISTA protein. Its expression levels at different pathological stages of superficial lymph node tuberculosis exhibit the following characteristics: 1. The highest expression level was observed in the ST0 phase (normal control tissue adjacent to the lesion); 2. Expression levels begin to decrease in ST1 (nodular type) and ST2 (infiltrative type); 3. The expression level was significantly reduced in ST3 (abscess type) and ST4 (ulcerative fistula type), showing a statistically significant difference compared with ST0.

[0032] This biomarker is used in the development of products for monitoring the course of superficial lymph node tuberculosis, assessing prognosis, or determining treatment efficacy. Specific applications include: dynamically monitoring changes in VISTA expression in lymph node tissues before and after treatment; if VISTA expression levels are significantly lower than in ST0 stage, it indicates the disease has progressed to an intermediate or advanced stage; detecting VISTA expression levels in lesion tissues before treatment; if VISTA expression levels rise after treatment, it indicates effective treatment; and detecting VISTA expression levels in CD8+ T cells in lesion tissues before treatment, using the expression levels of VISTA in normal tissues adjacent to ST0 stage lesions as a reference; high VISTA expression indicates a good response to early intervention, while a decrease in VISTA expression levels to levels comparable to ST3 or ST4 stage indicates a poor response to conventional treatment.

[0033] This embodiment provides a method for screening biomarkers for superficial lymph node tuberculosis based on imaging mass cytometry, specifically including the following steps: Formalin-fixed paraffin-embedded tissue samples were collected from patients with superficial lymph node tuberculosis at different pathological stages (ST0, ST1, ST2, ST3, and ST4). Specifically, formalin-fixed paraffin-embedded (FFPE) tissue samples were collected from patients diagnosed with superficial lymph node tuberculosis at the Department of Pathology, Nanjing Integrated Traditional Chinese and Western Medicine Hospital, affiliated with Nanjing University of Chinese Medicine. Based on clinical and pathological characteristics, the samples were divided into five groups: ST0 (perilesion control lymph nodes), ST1 (nodular type), ST2 (infiltrative type), ST3 (abscess type), and ST4 (ulcerative fistula type). The tissues were prepared into 4 μm thick FFPE sections.

[0034] Design and prepare a set of antibodies conjugated to metal isotope markers, said antibodies including at least anti-VISTA antibody, and stain the tissue sample sections using imaging mass cytometry: Specifically, a combination of multiple antibodies was designed to characterize immune and non-immune cells in lymph nodes. Key antibodies included anti-VISTA antibody, anti-CD4 antibody, anti-CD8 antibody, anti-FOXP3 antibody, anti-PD1 antibody, and anti-CD68 antibody. All antibodies were conjugated with metal isotope markers (such as lanthanides) of different atomic mass numbers. The conjugation process was strictly carried out according to the protocol provided by Standard BioTools, and the optimal dilution of each antibody was optimized through pre-experiments to ensure strong staining signal specificity, high signal-to-noise ratio, and no crosstalk between channels. Subsequently, IMC staining was performed: (1) Dewaxing and repair: FFPE sections were dewaxed sequentially with xylene and graded ethanol, and antigen repair was induced by heat. (2) ROI selection: Based on the H&E staining results of adjacent sections, under the guidance of pathologists, regions of interest (ROIs) containing complete pathological features of the lymph node cortex and medulla were selected on each tissue section. (3) Antibody incubation: The conjugated antibodies were mixed into a staining working solution and incubated with the tissue sections at 4°C overnight. (4) DNA staining: The cell nuclei were stained with PBS solution containing intercalator-Iridium and incubated at room temperature for 30 minutes.

[0035] Image data was acquired using an imaging mass cytometry system to obtain multi-channel images. After image preprocessing and cell segmentation, protein expression data at the single-cell level were extracted. Specifically, after staining, cleaning, and drying, the slides were placed in the Hyperion imaging system (Fluidigm) for laser ablation. The aerosol generated by ablation was carried into the plasma by the carrier gas. After the isotope tags were ionized, they were detected by mass spectrometry to obtain the original MCD file, i.e., the multi-channel image. Data processing and analysis: (1) Image preprocessing: The protein expression pattern was observed using MCD Viewer software, and the original data was exported as a multi-channel image in OME-TIFF format. (2) Cell segmentation: The image was imported into ilastik software for pixel classification to generate a probability map. Then, in CellProfiler software, the watershed algorithm was used to identify individual cell nuclei based on nuclear staining (Ir) signals and to define cell boundaries outward to generate single-cell data. (3) Extraction of protein expression data at the single-cell level: The expression intensity of all markers in each single cell was extracted using histoCAT software. The Harmony algorithm was used to eliminate batch effects.

[0036] Cell population visualization analysis was performed using dimensionality reduction algorithms to identify and differentiate different cell types. The expression levels of VISTA in each cell population were analyzed at different pathological stages, and biomarkers showing significant correlations between expression levels and disease stages were screened. Specifically, the Rphenograph algorithm was used to perform unsupervised clustering of cells, and heatmaps were generated based on the median expression of biomarkers to identify and distinguish different cell types. In this embodiment, the identified cell types included CD4+ T cells, CD8+ T cells, and M1 macrophages. Cell population visualization analysis was performed using the t-distributed random neighborhood embedding (t-SNE) dimensionality reduction algorithm to analyze the expression level of VISTA in each cell population at different pathological stages, and biomarkers whose expression levels were significantly correlated with disease stages were screened.

[0037] The key findings are as follows: pass Figure 1 A to Figure 1 F and Figure 2 A to Figure 2 Analysis of the imaging mass cytometry data shown in Figure B indicates that: (a) Expression of VISTA in CD8+ T cells ( Figure 1 A-1C) Figure 1 Figure A shows the overall expression distribution of VISTA on CD8+ T cells obtained through t-SNE dimensionality reduction analysis. Each point in the figure represents a cell, and the color intensity indicates the expression intensity of VISTA. It can be seen that in the CD8+ T cell population of all samples, there are obvious regions with high VISTA expression (bright color) and low expression (dark color), suggesting that VISTA expression is heterogeneous.

[0038] Figure 1 B further performed t-SNE analysis on the samples according to pathological stages (ST0 to ST4) to show the expression distribution of VISTA on CD8+ T cells in each stage of lymph node tuberculosis. It can be seen that in ST0 stage (adjacent control), the CD8+ T cell population generally shows high brightness (high expression); as the disease progresses to ST1 and ST2 stages, the high expression area gradually decreases; by ST3 and ST4 stages, the CD8+ T cell population almost completely turns dark (low expression). This image clearly shows that the expression of VISTA on CD8+ T cells is negatively correlated with disease stage.

[0039] like Figure 1 A and Figure 1 As shown in Figure B, VISTA expression in CD8+ T cells exhibited a clear phase specificity: high expression in ST0 phase and low expression in ST3 / ST4 phase.

[0040] Figure 1C is a bar chart that quantitatively analyzed the expression level of VISTA on CD8+ T cells in five phases. The results showed that the expression level was highest in ST0 phase; it decreased sequentially in ST1 and ST2 phases; and the expression level was lowest in ST3 and ST4 phases, with statistically significant differences compared to ST0 phase (p<0.001). This bar chart validated the above observations of t-SNE.

[0041] like Figure 1 Statistical analysis of C showed that VISTA was most highly expressed on CD8+ T cells in ST0 phase, then gradually decreased in ST1 and ST2 phases, and reached its lowest level in ST3 and ST4 phases, with statistically significant differences (p<0.001).

[0042] (II) Expression of VISTA in CD4+ T cells ( Figure 1 D-1F) Figure 1 D shows the overall t-SNE expression distribution of VISTA on CD4+ T cells, which also reveals the heterogeneity of VISTA expression.

[0043] Figure 1 E shows the expression distribution of VISTA on CD4+ T cells in different stages of lymph node tuberculosis. The variation pattern is consistent with that of CD8+ T cells: high expression in ST0 stage, gradually downregulated in ST1 and ST2 stages, and almost no expression in ST3 and ST4 stages.

[0044] Figure 1 The bar chart of F quantitatively confirmed the above trend: the expression level of VISTA on CD4+ T cells was highest in ST0 phase and lowest in ST3 and ST4 phases, and the difference was statistically significant.

[0045] like Figure 1 D to Figure 1 As shown in F, in CD4+ T cells, VISTA expression also showed a consistent downregulation trend, with the highest expression in ST0 phase and the lowest expression in ST3 / ST4 phase.

[0046] (III) Expression of VISTA in M1 macrophages ( Figure 2 A-2B) Figure 2 A shows the t-SNE distribution of VISTA on M1 macrophages at different stages of lymph node tuberculosis. It can be seen that in M1 macrophages, VISTA also exhibits a characteristic downregulation trend of high expression in ST0 phase and low expression in ST3 / ST4 phase.

[0047] Figure 2 The bar chart in section B quantitatively analyzed the expression level of VISTA in M1 macrophages at five stages, further confirming that the downregulation trend was statistically significant.

[0048] In summary, the expression of VISTA in CD8+ T cells, CD4+ T cells, and M1 macrophages was significantly negatively correlated with the disease stage of superficial lymph node tuberculosis, specifically high expression in the ST0 stage and significant decrease in the intermediate and late stages (ST3 / ST4). These results demonstrate that the downregulation of VISTA expression is a key molecular event in the transition of superficial lymph node tuberculosis from the early reversible stage to the late refractory stage, confirming that VISTA can serve as an effective biomarker for assessing disease progression and treatment response.

[0049] This embodiment provides a detection reagent for biomarkers of superficial lymph node tuberculosis based on imaging mass cytometry. The reagent comprises an antibody that specifically binds to the VISTA protein, which is conjugated to a detectable metal isotope marker. The reagent also includes at least one antibody selected from anti-CD4, anti-CD8, anti-CD68, anti-FOXP3, and anti-PD1, each antibody being conjugated to a different detectable marker. This detection reagent is used in the preparation of products for monitoring the course of superficial lymph node tuberculosis, assessing prognosis, or determining treatment efficacy.

[0050] This embodiment provides a detection kit for biomarkers of superficial lymph node tuberculosis based on imaging mass cytometry, comprising: a first antibody: an anti-human VISTA monoclonal antibody conjugated to the metal isotope 169Tm, with a working concentration of 1:200; a second antibody: an anti-human CD8 monoclonal antibody conjugated to the metal isotope 146Nd, with a working concentration of 1:400; a third antibody: an anti-human CD4 monoclonal antibody conjugated to the metal isotope 149Sm, with a working concentration of 1:400; a fourth antibody: an anti-human CD68 monoclonal antibody conjugated to the metal isotope 175Lu, with a working concentration of 1:300; a nuclear dye: an intercalating agent containing 191Ir and 193Ir; and other necessary reagents: antigen retrieval buffer, antibody dilution solution, and washing buffer.

[0051] A method for screening anti-tuberculosis immunomodulatory drugs, based on imaging mass cytometry, includes the following steps: (1) The above-mentioned detection reagents (i.e., antibodies that specifically bind to VISTA protein and their conjugates with metal isotope markers, which may selectively contain antibodies such as anti-CD4 and anti-CD8) were used to stain superficial lymph node tuberculosis tissue models or lymph node tissue sections of patients before and after treatment with candidate drugs. (2) Image data were acquired using an imaging mass cytometry system, and VISTA protein expression data at the single-cell level were extracted; (3) By comparing the changes in VISTA expression levels in specific cell populations before and after treatment with those in the treatment group and the control group, candidate drugs that can upregulate VISTA expression or restore its function are screened. The specific cell populations include CD8+ T cells, CD4+ T cells, and M1 macrophages.

[0052] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.

Claims

1. A biomarker for superficial lymph node tuberculosis based on imaging mass cytometry flow, characterized by, The biomarker is the VISTA protein.

2. The imaging mass spectrometry flow based biomarker for superficial lymph node tuberculosis as claimed in claim 1, wherein, The expression levels of the VISTA protein at different pathological stages of superficial lymph node tuberculosis exhibit the following characteristics: (1) The expression level is highest in the ST0 phase, i.e., in normal control tissue adjacent to the lesion; (2) Expression levels begin to decrease in ST1 (nodular type) and ST2 (infiltrative type); (3) The expression level was significantly reduced in ST3 (abscess type) and ST4 (ulcer fistula type), showing a statistically significant difference compared with ST0.

3. A method for screening biomarkers for superficial lymph node tuberculosis based on imaging mass cytometry flow according to claim 1 or 2, characterized in that, Includes the following steps: (1) Collect formalin-fixed paraffin-embedded tissue samples from patients with superficial lymph node tuberculosis at different pathological stages (ST0, ST1, ST2, ST3, ST4); (2) Design and prepare a set of antibodies conjugated to metal isotope markers, the antibodies including at least anti-VISTA antibody, and stain the tissue sample sections using imaging mass cytometry. (3) Image data were acquired using an imaging mass cytometry system to obtain multi-channel images. After image preprocessing and cell segmentation, protein expression data at the single-cell level were extracted. (4) Use dimensionality reduction algorithm to perform cell population visualization analysis, identify and distinguish different cell types, analyze the expression level of VISTA in each cell population at different pathological stages, and screen out biomarkers whose expression level is significantly correlated with the disease stage.

4. The method for screening biomarkers for superficial lymph node tuberculosis based on imaging mass cytometry according to claim 3, characterized in that, The dimensionality reduction algorithm in step (4) is t-distributed random neighborhood embedding; the cell types in step (4) include CD4+ T cells, CD8+ T cells and M1 macrophages.

5. A detection reagent for biomarkers of superficial lymph node tuberculosis based on imaging mass cytometry according to claim 1 or 2, characterized in that, The detection reagent contains an antibody that specifically binds to the VISTA protein; the antibody is conjugated to a detectable marker; the detectable marker is a metal isotope marker; the detection reagent also includes at least one selected from anti-CD4 antibody, anti-CD8 antibody, anti-CD68 antibody, anti-FOXP3 antibody, and anti-PD1 antibody, each antibody being conjugated to a different detectable marker.

6. A kit for biomarker detection for superficial lymph node tuberculosis based on imaging mass cytometry flow according to claim 5, characterized in that, Include: First antibody: Anti-human VISTA monoclonal antibody conjugated to metal isotope 169Tm, working concentration 1:200; Second antibody: Anti-human CD8 monoclonal antibody conjugated with the metal isotope 146Nd, working concentration 1:400; Third antibody: anti-human CD4 monoclonal antibody conjugated with metal isotope 149Sm, working concentration 1:400; Fourth antibody: Anti-human CD68 monoclonal antibody conjugated with the metal isotope 175Lu, working concentration 1:300; Nuclear dyes: intercalating agents containing 191Ir and 193Ir; Other necessary reagents: antigen retrieval buffer, antibody dilution solution, and washing buffer.

7. The application of the detection reagent for biomarkers of superficial lymph node tuberculosis based on imaging mass cytometry according to any one of claims 5 or 6 in the preparation of products for monitoring the course of superficial lymph node tuberculosis, prognostic assessment or efficacy judgment.

8. The application of the imaging mass cytometry-based biomarker for superficial lymph node tuberculosis according to claim 1 or 2 in the preparation of products for monitoring the course of superficial lymph node tuberculosis, prognostic assessment, or efficacy judgment; wherein the monitoring of the course of superficial lymph node tuberculosis includes dynamically detecting the expression changes of VISTA in lymph node tissues before and after treatment, and if the VISTA expression level is significantly lower than the ST0 stage level, it indicates that the disease has entered the middle and late stages.

9. The use of biomarkers based on imaging mass spectrometry flow for superficial lymph node tuberculosis according to claim 8 for the manufacture of a product for superficial lymph node tuberculosis course monitoring, prognosis evaluation or efficacy judgment, characterized by, The prognostic assessment of superficial lymph node tuberculosis includes detecting the VISTA expression level in the patient's lesion tissue before treatment. If the VISTA expression level rises after treatment, it indicates that the treatment is effective. The prognostic assessment of superficial lymph node tuberculosis includes: detecting the expression level of VISTA in CD8+ T cells in the lesion tissue of the patient before treatment, with the expression level of VISTA in normal tissue adjacent to the ST0 stage lesion as a reference. If VISTA is highly expressed, it indicates that the patient responds well to early intervention treatment. If the expression level of VISTA drops to a level comparable to that of ST3 or ST4 stage, it indicates that the patient responds poorly to conventional treatment.

10. A method for screening immunomodulatory drugs against tuberculosis as claimed in claim 5 wherein, Based on imaging mass cytometry, the process includes the following steps: (1) The detection reagent described above is used to stain superficial lymph node tuberculosis tissue models or lymph node tissue sections of patients before and after treatment with candidate drugs; (2) Image data were acquired using an imaging mass cytometry system, and VISTA protein expression data at the single-cell level were extracted; (3) By comparing the changes in the expression level of VISTA in specific cell populations before and after treatment with the control group, candidate drugs that can upregulate VISTA expression or restore its function can be screened.

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