Application of CD4TrmCXCR6 T cell as detection target cell in preparation of product for predicting immunotherapy curative effect of NSCLC brain metastatic tumor

Abstract

The invention provides an application of a CD4TrmCXCR6T cell as a detection target cell in preparation of a product for predicting the curative effect of NSCLC brain metastasis tumor receiving immunotherapy. The CD4TrmCXCR6T cell is a biomarker with response to prediction of NSCLC brain metastasis tumor receiving immunotherapy. According to the invention, a key immune cell subset, namely CD4TrmCXCR6T cells, is screened out, and the CD4TrmCXCR6T cells, which are used as positive predictive factors of ICI intracranial tumor response, show high functions and transcriptome similarity in cerebrospinal fluid and brain metastatic tumor environments. On the basis, key factors for predicting the immunotherapy curative effect of NSCLC brain metastatic tumor are identified; cD4TrmCXCR6T cell positive is a predictive index of the curative effect of NSCLC brain metastatic tumor receiving immunotherapy, and has great clinical significance and transformation value.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biological medicine, and particularly relates to application of CD4_Trm_CXCR6 T cells as detection target cells in preparation of a product for predicting the efficacy of immunotherapy for NSCLC brain metastases. BACKGROUND

[0002] The incidence of brain metastases in advanced non-small-cell lung cancers (NSCLC) is increasing, and the prognosis is poor, so new treatment strategies are urgently needed. For advanced NSCLC patients without driver genes, immune checkpoint inhibitors have become the standard treatment regimen. For patients with concomitant brain metastases, multiple retrospective studies or phase II studies have shown that immunotherapy has preliminary efficacy, with an intracranial objective response rate of 16.4%-52.5%. Immune monotherapy or combination therapy has become an important treatment option for brain metastases (BrM). However, due to the high heterogeneity of brain metastases in response to immunotherapy, only a small number of patients can achieve complete remission, and how to improve the clinical efficacy of brain metastases still faces major challenges, which highlights the urgency of finding biomarkers for predicting the efficacy of immunotherapy.

[0003] Brain metastases have specific heterogeneity, and their genomic and transcriptomic characteristics differ from those of lung cancer primary tumors, and extracranial tumor tissues have limited value in predicting intracranial response to systemic therapy. The risk and difficulty of tissue biopsy of brain metastases make cerebrospinal fluid an important liquid biopsy specimen for evaluating the biological characteristics of brain metastases. Our previous research and the results of other teams have confirmed that cerebrospinal fluid can be used to reveal the genetic characteristics and cellular composition of intracranial tumors in NSCLC. In addition, multiple studies have also used cerebrospinal fluid to reflect the tumor immune cell infiltration and microenvironment characteristics of brain metastases, but these studies mainly focus on brain metastases of solid tumors, and the sample size of lung cancer is very small.

[0004] Although the immune cells in the cerebrospinal fluid of patients with meningeal metastases can indicate the relevant characteristics of immune therapy response, due to the functional and phenotypic heterogeneity of immune cells between meningeal metastases and brain metastases, these results are not representative of brain metastases. Recent studies have shown that immune cell cytokines in cerebrospinal fluid can reflect the response of brain metastases patients to immunotherapy, suggesting the potential of cerebrospinal fluid as a surrogate specimen for intracranial tumor immunotherapy efficacy research. However, this study has a limited sample size and only focuses on cytokines, and is unable to deeply analyze the cellular behavior dynamics of NSCLC brain metastases in response to immunotherapy.

[0005] Therefore, there are still many unsolved mysteries about the response mechanism of brain metastases to immunotherapy and the application of cerebrospinal fluid as a liquid biopsy substitute. In addition, the biological consistency of cerebrospinal fluid and brain metastases, as well as the temporal variation characteristics of cerebrospinal fluid during immunotherapy are still unclear. SUMMARY

[0006] In view of the deficiencies in the prior art, the purpose of the present application is to provide the application of CD4_Trm_CXCR6 T cells as detection target cells in the preparation of products for predicting the efficacy of immunotherapy for NSCLC brain metastases. The present application finds that the detection results of CD4_Trm_CXCR6 T cells are predictive indicators of the efficacy of immunotherapy for NSCLC brain metastases, which has great clinical significance and transformation value.

[0007] To achieve the purpose of the present application, the present application adopts the following technical solutions:

[0008] In the first aspect, the present application provides the application of CD4_Trm_CXCR6 T cells as detection target cells in the preparation of products for predicting the efficacy of immunotherapy for NSCLC brain metastases. The CD4_Trm_CXCR6 T cells are biomarkers for predicting the response of NSCLC brain metastases to immunotherapy.

[0009] Preferably, any of the following characteristics of the CD4_Trm_CXCR6 T cell subpopulation in cerebrospinal fluid or brain metastases is a predictive indicator of the efficacy of immunotherapy for NSCLC brain metastases:

[0010] (1) The proportion of CD4_Trm_CXCR6 T cell subpopulation in cerebrospinal fluid in CD4 + T cell number is greater than 10%;

[0011] (2) The characteristic protein function score of CD4_Trm_CXCR6 T cells in cerebrospinal fluid is greater than 0.6;

[0012] (3) The characteristic protein or gene expression result of CD4_Trm_CXCR6 T cells in brain metastases is: CD3 + , PD1 + , CD4 + and CXCR6 + .

[0013] In the present application, our research provides a dynamic high-resolution single-cell map of cerebrospinal fluid and BrM cells of non-small cell lung cancer combined with BrM subjects during ICI treatment. Notably, we found a key immune cell subgroup, CD4_Trm_CXCR6 T cells, as a positive predictor of ICI intracranial tumor response, showing high functional and transcriptome similarity in both CSF and BrM tumor microenvironment. CXCR6 can be used as a specific marker for CD4+PD-1+ T cells associated with ICI response.

[0014] Preferably, the proportion of CD4_Trm_CXCR6 T cell subgroup in the cerebrospinal fluid is detected by single-cell transcriptome sequencing.

[0015] Preferably, the gene signature of CD4_Trm_CXCR6 T cells in single-cell transcriptome sequencing is CXCR6, CXCL13, IFN-gama, CTLA4, PRDM1 and HAVCR2, and the cell subgroup is defined and the proportion of CD4_Trm_CXCR6 T cells in CD4 + T cells is calculated.

[0016] Preferably, the characteristic protein function score of CD4_Trm_CXCR6 T cells in the cerebrospinal fluid is detected by proteomics.

[0017] Preferably, the characteristic proteins of CD4_Trm_CXCR6 T cells in proteomics detection include CD3D, CD2, CD5, CD4, CD6, CD28, HMGB2, SLAMF6, NFATC2, NFATC3, CD44, WNK1, RGS1, PD-1, TIM-3, EOMES, PRDM1, IFN-gamma, BLC(CXCL13), IL-21, GZMH, CST7, SKAP1, HSP40, P2RY10 and SH3KBP1.

[0018] Preferably, the characteristic protein or gene expression result of CD4_Trm_CXCR6 T cells of the brain metastasis tumor is detected by multiplex immunofluorescence and / or single-cell transcriptome.

[0019] In the present application, the characteristic protein expression is directly detected by multiplex immunofluorescence, or the transcriptome result corresponding to the characteristic protein is detected by single-cell transcriptome.

[0020] In a second aspect, the present application provides the use of a method for detecting CD4_Trm_CXCR6 T cells in the construction of a product for predicting the efficacy of immunotherapy for NSCLC brain metastasis, wherein the method for detecting CD4_Trm_CXCR6 T cells comprises:

[0021] obtaining a cerebrospinal fluid sample or a brain metastasis tissue sample from the subject before the subject receives the immunotherapy;

[0022] detecting the proportion of CD4_Trm_CXCR6 T cell subpopulation in CD4 + T cells in the cerebrospinal fluid by single-cell transcriptome sequencing, or detecting the characteristic protein score of CD4_Trm_CXCR6 T cells in the cerebrospinal fluid by proteomics, or detecting the characteristic protein or gene expression of CD4_Trm_CXCR6 T cells in the brain metastasis by multiplex immunofluorescence and / or single-cell transcriptome sequencing;

[0023] Based on the comparison of the cell subpopulation before the treatment between the immunotherapy response group and the progression group, the judgment standard for predicting the effective response of NSCLC brain metastasis to immunotherapy is:

[0024] the proportion of CD4_Trm_CXCR6 T cells in CD4 + T cells is greater than 10%;

[0025] or the characteristic protein score of CD4_Trm_CXCR6 T cells is greater than 0.6;

[0026] or the characteristic protein expression of CD4_Trm_CXCR6 T cells is: CD3 + , PD1 + , CD4 + , and CXCR6 + .

[0027] Preferably, the gene characteristics of the CD4_Trm_CXCR6 T cells in the single-cell transcriptome sequencing are: CXCR6, CXCL13, IFN-gama, CTLA4, PRDM1, and HAVCR2, according to which the cell subpopulation is defined and the proportion of CD4_Trm_CXCR6 T cells in CD4 + T cells is calculated.

[0028] Preferably, the characteristic proteins of the CD4_Trm_CXCR6 T cells in the proteomics detection include: CD3D, CD2, CD5, CD4, CD6, CD28, HMGB2, SLAMF6, NFATC2, NFATC3, CD44, WNK1, RGS1, PD-1, TIM-3, EOMES, PRDM1, IFN-gamma, BLC(CXCL13), IL-21, GZMH, CST7, SKAP1, HSP40, P2RY10, and SH3KBP1.

[0029] In a third aspect, the present application provides a system for predicting the efficacy of immunotherapy for NSCLC brain metastases, comprising:

[0030] a sample acquisition unit for acquiring a cerebrospinal fluid sample or a brain metastasis tissue sample from a subject before immunotherapy;

[0031] a cell detection unit for detecting the proportion of CD4_Trm_CXCR6 T cell subpopulation in CD4 + T cells in the cerebrospinal fluid by single-cell transcriptome sequencing, or detecting the characteristic protein score of CD4_Trm_CXCR6 T cells in the cerebrospinal fluid by proteomics, or detecting the characteristic protein or gene expression of CD4_Trm_CXCR6 T cells in the brain metastasis by multiplex immunofluorescence and / or single-cell transcriptome sequencing;

[0032] a data analysis unit for determining the therapeutic efficacy based on the comparison of the cell subpopulation before treatment between the immunotherapy response group and the progression group; the criteria for predicting the effective response of NSCLC brain metastases to immunotherapy are:

[0033] the proportion of CD4_Trm_CXCR6 T cells in CD4 + T cells is greater than 10%;

[0034] or the characteristic protein score of CD4_Trm_CXCR6 T cells is greater than 0.6;

[0035] or the characteristic protein or gene expression of CD4_Trm_CXCR6 T cells is: CD3 + , PD1 + , CD4 + and CXCR6 + .

[0036] Preferably, the gene characteristics of CD4_Trm_CXCR6 T cells in single-cell transcriptome sequencing are: CXCR6, CXCL13, IFN-gama, CTLA4, PRDM1 and HAVCR2, based on which the cell subpopulation is defined and the proportion of CD4_Trm_CXCR6 T cells in CD4 + T cells is calculated.

[0037] Preferably, the characteristic proteins of the CD4_Trm_CXCR6 T cells in the proteomics detection include: CD3D, CD2, CD5, CD4, CD6, CD28, HMGB2, SLAMF6, NFATC2, NFATC3, CD44, WNK1, RGS1, PD-1, TIM-3, EOMES, PRDM1, IFN-gamma, BLC (CXCL13), IL-21, GZMH, CST7, SKAP1, HSP40, P2RY10, and SH3KBP1.

[0038] In the present application, the key of the CD4_Trm_CXCR6 T cell subpopulation to ICI response lies in that it is closely related to lymphocyte activation and aggregation in both CSF and BrM, and CD4+PD-1+CXCR6+T cells can interact with classical dendritic cells (cDCs) to enhance antigen presentation and inflammatory activation.

[0039] Compared with the prior art, the present application has the following beneficial effects:

[0040] (1) Precise subject screening: Before the application of immunotherapy in non-small cell lung cancer brain metastasis subjects, the efficacy thereof can be predicted in advance, the response of the drug can be predicted, ineffective treatment can be avoided, and more precise treatment can be achieved.

[0041] (2) Joint strategy guidance: For subjects whose efficacy is predicted to be poor in advance, the step-up treatment can be used in advance to enhance the efficacy of the drug.

[0042] (3) Liquid biopsy is safer and easier: The use of cerebrospinal fluid liquid biopsy can reflect the efficacy prediction of brain metastasis, and has the advantages of simplicity and ease.

[0043] (4) Economic benefits: The early prediction of the efficacy of immunotherapy can help to develop a more cost-effective and reasonable treatment plan for the subjects. BRIEF DESCRIPTION OF DRAWINGS

[0044] Figure 1 The population setting of the exploration set and the validation set of the present application.

[0045] Figure 2 The whole picture of the immune cells in the cerebrospinal fluid of the non-small cell lung cancer brain metastasis patients before and after the application of immunotherapy.

[0046] Figure 3 The single cell transcriptome data result.

[0047] Figure 4 The proteomics verification result.

[0048] Figure 5Figure 6 is the result of verifying CD4_Trm_CXCR6 T cells by cerebrospinal fluid flow cytometry.

[0049] Figure 6 Figure 7 is the result of detecting CD4_Trm_CXCR6 T cells in brain metastasis tissue samples by multiplex immunofluorescence.

[0050] Figure 7 Figure 8 is the result of verifying CD4_Trm_CXCR6 T cells in brain metastasis and cerebrospinal fluid.

[0051] Figure 8 Figure 9 is a schematic diagram of CD4_Trm_CXCR6 T cells and lymphocyte activation and enhanced antigen presentation.

[0052] Figure 9 Figure 10 is a schematic diagram of an immune response with CD4_Trm_CXCR6 T cells as the core. DETAILED DESCRIPTION

[0053] The technical solutions of the present application will be further described below through specific embodiments. Those skilled in the art should understand that the embodiments are only used to help understand the present application and should not be regarded as specific limitations on the present application.

[0054] Unless otherwise specified in the embodiments, the techniques or conditions are performed according to the techniques or conditions described in the literature in the art or according to the product instructions. Unless otherwise specified, the reagents or instruments used are conventional products that can be commercially available through regular channels.

[0055] Glossary in the present application:

[0056] ICI: immune checkpoint inhibitor (ICI).

[0057] BrM: brain metastasis (BrM).

[0058] CSF: cerebrospinal fluid (CSF).

[0059] NSCLC: non-small-cell lung cancer (NSCLC).

[0060] CR: confirmed responder (CR).

[0061] PR: potential responder (PR).

[0062] NR: non-responder group (NR).

[0063] Example 1

[0064] Exploring the predictive indicators of immunotherapy efficacy in NSCLC patients with brain metastases

[0065] The immunotherapy in this example mainly refers to PD-1 inhibitors and PD-L1 inhibitors, including pembrolizumab, nivolumab, camrelizumab and sugemalimab.

[0066] 1. The population setting process of the exploration set is shown in Figure 1 .

[0067] Figure 1 Methodology A is the methodology of the exploration set and the validation set. As shown in the figure, we included NSCLC patients with brain metastases who planned to receive ICI treatment, and collected dynamic cerebrospinal fluid samples (n=20) before and during immunotherapy through lumbar puncture for cytological examination and single-cell transcriptome sequencing (scRNA-seq) analysis; At the same time, we collected BrM tumor tissues (n=4), and integrated the BrM tumor scRNA-seq dataset (n=16) from external databases.

[0068] Figure 1 Methodology B is the basic information and grouping of the exploration set. As shown in the figure, the patients were divided into three groups according to the efficacy: confirmed responder group (CR): including patients who did not receive brain radiotherapy but showed effective response. Potential responder group (PR): including patients who received brain radiotherapy and responded, and paired samples before and after ICI treatment were collected. Non-responder group (NR): including patients who did not respond or had disease progression after treatment, and paired samples before and after ICI treatment were collected. Another set of samples from patients who did not receive ICI treatment were used as background control.

[0069] Figure 1 Methodology C is the classic image of head MRI of the confirmed responder group (CR) and the non-responder group (NR). As shown in the figure, the brain metastases in the confirmed responder group (CR) decreased after treatment, and the brain metastases in the non-responder group (NR) increased after treatment.

[0070] 2. Predictive indicator screening

[0071] 2.1 Detection and analysis method of single-cell transcriptome sequencing (scRNA-seq)

[0072] 2.1.1 Detection steps

[0073] Qualified patients underwent lumbar puncture, and cerebrospinal fluid was collected. After centrifugation at 300g for 5 minutes, the cells were separated, resuspended, and stored in a new buffer. Brain metastasis tissue: after being placed in MACS tissue preservation solution, digested with collagenase I / IV and DNase I, passed through a 70-μm filter, red blood cells were lysed, washed with 0.04% BSA-PBS, and filtered through a 35-μm filter. After evaluating the cell activity of cerebrospinal fluid cells and brain metastasis cells using the BD Rhapsody Scanner (BD single cell library preparation system), the library was constructed by the BD single cell analysis system, and sequenced on the Illumina NextSeq platform. Single-cell transcriptome sequencing data of BrM tumor tissue (n=4) was obtained.

[0074] 2.1.2 Data integration

[0075] The single-cell transcriptome sequencing data of the collected BrM tumor tissue (n=4) was integrated with the BrM tumor scRNA-seq data set (n=16) from external databases. After integration, scRNA-seq data processing and analysis were performed.

[0076] 2.1.3 Data analysis through the NovelBrain cloud platform

[0077] Integration analysis: The Seurat package normalized the data, selected high-variable genes, corrected batch effects through "anchor points", and regressed sequencing depth, gene number, and mitochondrial read effects. Based on the top 2000 high-variable genes, PCA dimensionality reduction was performed, and the top 30 principal components were used for t-SNE / UMAP clustering (Louvain algorithm). Cell annotation: cell subgroups were defined according to differential genes (Log2FC>0.25, p<0.05, min.pct>0.10) and known marker genes, and double-labeled cells were excluded.

[0078] 2.2 Single-cell transcriptome identification of cell subgroups

[0079] The identified cell subgroups are shown in Figure 2 A-C, Figure 2 Panorama of cerebrospinal fluid immune cells before and after immunotherapy for non-small cell lung cancer patients with brain metastasis.

[0080] Figure 2 A is the t-SNE (t-distributed random neighbor embedding) visualization of cerebrospinal fluid immune cell scRNA-seq data. From the figure, it can be seen that CSF immune cells can be divided into 10 major subgroups, with CD4 + T cells, CD8 + T cells, monocytes, macrophages, regulatory T cells (Tregs), and classical dendritic cells (cDCs) being the main ones.

[0081] Figure 2 Figure 2B is a visualization plot of t-SNE of CSF immune cell scRNA-seq data, from which it can be seen that, in sample group division, the color reflects cell density, and the CR group has increased macrophage density, while the NR group has increased CD4 + T cell density.

[0082] Figure 2 Figure 2C is a plot of subdivided subgroups of each subgroup, from which it can be seen that, from left to right, T-sne shows the subdivided subtypes of CD4 + T cells, CD8 + T cells, NK cells, and B cells, and myeloid cells.

[0083] 2.3 Single-cell transcriptome data suggests that CD4_Trm_CXCR6 T cells are associated with immunotherapy response

[0084] Figure 3 Figure 3 is a single-cell transcriptome data result showing that CD4_Trm_CXCR6 T cells are associated with NSCLC brain metastasis immune response.

[0085] Figure 3 Figure 2A is a plot of subdivided subgroups of CD4+ T cells, from which it can be seen that the gene characteristics of CD4_Trm_CXCR6 T cells are: CXCR6, CXCL13, IFN-gama, CTLA4, PRDM1, and HAVCR2.

[0086] Figure 3 Figure 2B is a plot of comparison of each subdivided subgroup of CD4 + T cells among the four groups, from which it can be seen that, compared with the non-response group, the proportion of CD4_Trm_CXCR6 T cells in the confirmed response group is significantly increased; that is, the proportion of CD4_Trm_CXCR6 T cells / CD4 + T cells greater than 10% indicates an effective response to immunotherapy.

[0087] Figure 3 Figure 2C is a plot of density of each subdivided subgroup of CD4 + T cells, from which it can be seen that, compared with the non-response group, the density of CD4_Trm_CXCR6 T cells in the confirmed response group is increased, further confirming that CD4_Trm_CXCR6 T cells are associated with immunotherapy response.

[0088] Example 2

[0089] The predictive marker CD4_Trm_CXCR6 T cells screened in Example 1 were validated in the validation set.

[0090] 1. Population setting of the validation set

[0091] From Figure 1 The validation set population includes:

[0092] 1) Cerebrospinal fluid samples before and after immunotherapy were used for proteomics analysis, and the predictive effect of CD4_Trm_CXCR6 T cells on immunotherapy efficacy was verified by protein function score (N = 31).

[0093] 2) Flow cytometry was used to verify CD4_Trm_CXCR6 T cells in cerebrospinal fluid (N = 8).

[0094] 3) Brain metastasis tissue samples before and after immunotherapy were used for multiple immunofluorescence to verify the predictive effect of CD4_Trm_CXCR6 T cells on immunotherapy efficacy (N = 25).

[0095] 2. Marker validation experiment

[0096] 2.1. Proteomics verification

[0097] 2.1.1 Proteomics data generation and analysis:

[0098] Cerebrospinal fluid (n = 31) from NSCLC brain metastasis patients before and after immunotherapy was used for proteomics analysis. RayBiotech human antibody chip was used to detect 8000 proteins in the cerebrospinal fluid of brain metastasis patients. Mapix 8.1 software was used to analyze the signal, and the positive control point was standardized. For CD4_Trm_CXCR6 characteristic genes, differential proteins were screened and functional scores between groups were calculated.

[0099] The score was calculated according to the expression amount of functional proteins, and deconvolution analysis was performed to calculate the enrichment score of CD4_Trm_CXCR6 by applying relative ranking enrichment to protein array data.

[0100] 2.1.2 Proteomics analysis results

[0101] Figure 4 The proteomics verification results show that CD4_Trm_CXCR6 T cells are related to the immune response of NSCLC brain metastasis.

[0102] Figure 4Figure 6A shows the expression of signature proteins of CD4_Trm_CXCR6 T cells in the four groups. As can be seen from the figure, the characteristic proteins in the confirmed response group (CR) are significantly increased, including CD3D, CD2, CD5, CD4, CD6, CD28, HMGB2, SLAMF6, NFATC2, NFATC3, CD44, WNK1, RGS1, PD-1, TIM-3, EOMES, PRDM1, IFN-gamma, BLC (CXCL13), IL-21, GZMH, CST7, SKAP1, HSP40, P2RY10 and SH3KBP1.

[0103] Figure 4 Figure 6B shows the functional score of characteristic proteins of CD4_Trm_CXCR6 T cells in the four groups. As can be seen from the figure, the functional score of CD4_Trm_CXCR6 T cell proteins in the confirmed response group (CR) is significantly increased, i.e. the score is greater than 0.6, indicating an effective response of immunotherapy.

[0104] 2.2 Flow cytometry verification

[0105] 2.2.1 Verification of CD4_Trm_CXCR6 T cells in cerebrospinal fluid by flow cytometry in another cohort.

[0106] CSF from NSCLC patients with central nervous system metastasis (n=8) was collected to verify CD4_Trm_CXCR6 T cells by flow cytometry. After stimulation of cerebrospinal fluid cells, surface antibody labeling and intracellular factor staining were used, and BD FACSymphony TM S6 detection was used, and FlowJo V10.4 was used for analysis.

[0107] 2.2.2 Flow verification results.

[0108] Figure 5 Results of flow cytometry verification of CD4_Trm_CXCR6 T cells in cerebrospinal fluid.

[0109] Figure 5 Figure 6A shows the expression of signature proteins of CD4_Trm_CXCR6 T cells in the four groups. As can be seen from the figure, the characteristic proteins in the confirmed response group (CR) are significantly increased, including CD3D, CD2, CD5, CD4, CD6, CD28, HMGB2, SLAMF6, NFATC2, NFATC3, CD44, WNK1, RGS1, PD-1, TIM-3, EOMES, PRDM1, IFN-gamma, BLC (CXCL13), IL-21, GZMH, CST7, SKAP1, HSP40, P2RY10 and SH3KBP1. + + +

[0110] Figure 5 ​​​PD-1 and IFN-γ expression in CD4_Trm_CXCR6 T cells. As can be seen from the figure, PD-1 and IFN-γ in CD4_Trm_CXCR6 T cells are significantly increased.

[0111] Flow cytometry independently verified the presence of CD4_Trm_CXCR6 cell populations in the cerebrospinal fluid of NSCLC patients with brain metastases. These cells showed up-regulated expression of PD-1 (encoded by PDCD1), IFN-γ and other cytokines and exhaustion-related markers, further confirming the findings of single-cell RNA sequencing data.

[0112] 2.3 Verification based on multiplex immunofluorescence

[0113] 2.3.1 Multiplex immunofluorescence of brain metastases confirms the predictive role of CD4_Trm_CXCR6 T cell subsets for immunotherapy.

[0114] Brain metastasis tissues (n=25) from NSCLC patients with brain metastases before and after immunotherapy were used for multiplex immunofluorescence co-localization verification. PANO7-plex IHC kit was used for multiplex staining of brain metastasis paraffin sections (CXCR6, CD3, CD4, etc.). Mantra system scanning, QuPath software analysis of whole slide fluorescence images.

[0115] 2.3.2 Multiplex immunohistochemistry / immunofluorescence verification results.

[0116] Figure 6 The results of multiplex immunofluorescence detection of CD4_Trm_CXCR6 T cells in brain metastasis tissues were used to verify their role in predicting the efficacy of immunotherapy.

[0117] Figure 6 A is the classic multiplex immunofluorescence picture of each treatment group. As can be seen from the figure, the multiplex immunofluorescence antibody characteristics of CD4_Trm_CXCR6 T cells in brain metastasis tissues are: CD3 + , PD1 + , CD4 + and CXCR6 + . From the comparison of the classic images, it can be seen that the density of CD4_Trm_CXCR6 T cells increases in the confirmed response group (CR).

[0118] Figure 6Figure 6B shows the comparison of the proportion of cells expressing different antibodies in the confirmed response group (CR) and the non-response group (NR). As can be seen from the figure, the CD4_Trm_CXCR6 T cells in the confirmed response group (CR) brain metastasis tumor tissue significantly increased, while the other PD-1, CD4, CD8, CD3 labeled cell subgroups did not show significant differences. This indicates that the CD4_Trm_CXCR6 T cell subgroup has the potential to become a biomarker for predicting the efficacy of immunotherapy for brain metastasis. This finding is consistent with the results of the study of cerebrospinal fluid in the confirmed response group.

[0119] 2.4 Validation based on public datasets

[0120] 2.4.1 Public dataset collection and analysis

[0121] The scRNA-seq datasets of NSCLC brain metastasis (GSE131907, GSE143423, GSE186344) were obtained from the GEO database and analyzed uniformly after quality control. We integrated all brain metastasis data and analyzed the correlation of CD4_Trm_CXCR6 T cells in cerebrospinal fluid and brain metastasis.

[0122] 2.3.2 Analysis results

[0123] After integration, we found that CD4_Tex_CXCR6 cells in brain metastasis (BrM) and CD4_Trm_CXCR6 cells in cerebrospinal fluid (CSF) were highly similar in transcriptomics, characterized by high expression of PDCD1, CXCL13, CXCR6, and PRDM1, etc.

[0124] Figure 7 The validation results of CD4_Trm_CXCR6 T cells in brain metastasis and cerebrospinal fluid showed that CD4_Trm_CXCR6 T cells in brain metastasis and cerebrospinal fluid had high similarity.

[0125] Figure 7 Figure 6A shows the whole picture of CD4 T cells integrated with brain metastasis and cerebrospinal fluid. As can be seen from the figure, the comparison of CD4 T cells in brain metastasis and cerebrospinal fluid showed high overlap. +

[0126] Figure 7 Figure 6B shows the expression of CD4_Trm_CXCR6 T cells in brain metastasis (tumor, left) and cerebrospinal fluid (CSF, right). As can be seen from the figure, CD4_Tex_CXCR6 T in brain metastasis and CD4_Trm_CXCR6 T in cerebrospinal fluid significantly overlapped in the single cell dimension reduction space.

[0127] Figure 7 ​CD4_Trm_CXCR6 T cells in brain metastasis and cerebrospinal fluid. From the figure, it can be seen that CD4_Tex_CXCR6 T cells in brain metastasis have a high correlation with the overall gene expression profile of CD4_Trm_CXCR6 T cells in cerebrospinal fluid.

[0128] Figure 7 D-E is the pathway interaction network of CD4_Trm_CXCR6 T cells in brain metastasis. From the figure, it can be seen that, similar to CD4_Trm_CXCR6 T cells in cerebrospinal fluid, the signal pathway with the highest relative intensity in the signal sent by CD4_Tex_CXCR6 cells in brain metastasis is mediated by CD6 molecules. In addition, CD4_Tex_CXCR6 cells in brain metastasis also exhibit a strong CD40 outward signal, which is also up-regulated in CD4 + T cells in the immune response group of cerebrospinal fluid.

[0129] In summary, these findings highlight the high similarity in function and transcriptome of CD4_Trm_CXCR6 T cell subpopulations associated with immunotherapy response in cerebrospinal fluid and brain metastasis.

[0130] Example 3

[0131] The predictive marker CD4_Trm_CXCR6 T cells of Example 1 and Example 2 functionally elucidate the key of their immune response.

[0132] Figure 8 Schematic diagram of CD4_Trm_CXCR6 T cells and lymphocyte activation and enhancement of antigen presentation.

[0133] Figure 9 Schematic diagram of immune response with CD4_Trm_CXCR6 T cells as the core.

[0134] Figure 8 The figure shows that CD4_Trm_CXCR6 T cells promote lymphocyte activation and aggregation (left side) and activate antigen presenting cells to enhance antigen presentation and inflammatory activation (right side). From the figure, it can be seen that CD4_Trm_CXCR6 T cells in cerebrospinal fluid and brain metastasis are closely related to lymphocyte activation and aggregation on the one hand, and interact with antigen presenting cells such as classical dendritic cells (cDCs) to enhance antigen presentation and inflammatory activation on the other hand.

[0135] Figure 9The CD4_Trm_CXCR6 T cell is the core of the immune response, and the CD4_Trm_CXCR6 T cell subpopulation is the key to the ICI response, which is closely related to the activation of lymphocytes and antigen-presenting cells and inflammatory activation.

[0136] The applicant states that the above is only a specific embodiment of the present application, but the protection scope of the present application is not limited thereto, and those skilled in the art should understand that any changes or replacements within the technical scope disclosed by the present application can be easily thought of by those skilled in the art, and all fall within the protection scope and disclosure scope of the present application.

Claims

1. The use of CD4_Trm_CXCR6 T cells as detection target cells in the preparation of a product for predicting the efficacy of immunotherapy for NSCLC brain metastases, characterized in that: The CD4_Trm_CXCR6 T cells are biomarkers for predicting the response of NSCLC brain metastases to immunotherapy.

2. The use according to claim 1, characterized in that Any of the following characteristics of the CD4_Trm_CXCR6 T cell subset in cerebrospinal fluid is a predictor of efficacy of immunotherapy for NSCLC brain metastases: (1) The CD4_Trm_CXCR6 T cell subset in cerebrospinal fluid is + The proportion of T cells in the population is greater than 10%; (2) the characteristic protein function score of CD4_Trm_CXCR6 T cells in cerebrospinal fluid was greater than 0.6; (3) The characteristic protein or gene expression results of CD4_Trm_CXCR6 T cells in brain metastases are: CD3 + PD1 + 、CD4 + and CXCR6 + .

3. The use according to claim 2, characterized in that The proportion of CD4_Trm_CXCR6 T cell subsets in the cerebrospinal fluid was detected by single-cell transcriptome sequencing; Preferably, the gene features of the CD4_Trm_CXCR6 T cells in single-cell transcriptome sequencing are: CXCR6, CXCL13, IFN-gama, CTLA4, PRDM1 and HAVCR2, based on which the cell subpopulations are defined and the CD4_Trm_CXCR6 T cells in the CD4 + The proportion of T cells.

4. The use according to claim 2 or 3, characterized in that The characteristic protein function score of CD4_Trm_CXCR6 T cells in the cerebrospinal fluid is obtained by proteomics detection; Preferably, the characteristic proteins of the CD4_Trm_CXCR6 T cells in proteomic detection include: CD3D, CD2, CD5, CD4, CD6, CD28, HMGB2, SLAMF6, NFATC2, NFATC3, CD44, WNK1, RGS1, PD-1, TIM-3, EOMES, PRDM1, IFN-gamma, BLC, IL-21, GZMH, CST7, SKAP1, HSP40, P2RY10 and SH3KBP1.

5. The use according to any one of claims 2 to 4, characterized in that The characteristic protein or gene expression results of CD4_Trm_CXCR6 T cells of the brain metastasis tumor are obtained by multiple immunofluorescence and / or single cell transcriptome detection.

6. Application of a method for detecting CD4_Trm_CXCR6 T cells in constructing a product for predicting the efficacy of immunotherapy for NSCLC brain metastases, characterized in that: The method for detecting CD4_Trm_CXCR6 T cells comprises: Obtain cerebrospinal fluid samples or brain metastasis tissue samples from subjects before they receive immunotherapy; Single-cell transcriptome sequencing was used to detect the CD4_Trm_CXCR6 T cell subset in cerebrospinal fluid. + The proportion of T cells in the total number of cells, or the characteristic protein score of CD4_Trm_CXCR6 T cells in cerebrospinal fluid detected by proteomics, or the characteristic protein or gene expression of CD4_Trm_CXCR6 T cells in brain metastases detected by multiplex immunofluorescence and / or single-cell transcriptomics; The efficacy of treatment is determined by comparing the cell subpopulations in the immunotherapy response group and the progression group before treatment. The criteria for predicting effective response to immunotherapy in NSCLC brain metastases are: CD4_Trm_CXCR6 T cells in CD4 + The proportion of T cells is greater than 10%; or CD4_Trm_CXCR6 T cell signature protein score greater than 0.6; The characteristic protein or gene expression results of CD4_Trm_CXCR6 T cells are: CD3 + PD1 + 、CD4 + and CXCR6 + .

7. The use according to claim 6, characterized in that The gene features of the CD4_Trm_CXCR6 T cells in single-cell transcriptome sequencing are: CXCR6, CXCL13, IFN-gama, CTLA4, PRDM1 and HAVCR2, based on which the cell subsets were defined and the CD4_Trm_CXCR6 T cells in CD4 + The proportion of T cells; Preferably, the characteristic proteins of the CD4_Trm_CXCR6 T cells in proteomic detection include: CD3D, CD2, CD5, CD4, CD6, CD28, HMGB2, SLAMF6, NFATC2, NFATC3, CD44, WNK1, RGS1, PD-1, TIM-3, EOMES, PRDM1, IFN-gamma, BLC, IL-21, GZMH, CST7, SKAP1, HSP40, P2RY10 and SH3KBP1.

8. A system for predicting the efficacy of immunotherapy for NSCLC brain metastases, characterized in that: The system comprises: A sample acquisition unit, used to obtain cerebrospinal fluid samples or brain metastasis tissue samples from subjects before they receive immunotherapy; The cell detection unit detects the CD4_Trm_CXCR6 T cell subsets in cerebrospinal fluid by single-cell transcriptome sequencing. + The proportion of T cells in the total number of cells, or the characteristic protein score of CD4_Trm_CXCR6 T cells in cerebrospinal fluid detected by proteomics, or the characteristic protein or gene expression of CD4_Trm_CXCR6 T cells in brain metastases detected by multiplex immunofluorescence and / or single-cell transcriptomics; The data analysis unit determines treatment efficacy based on the comparison of cell subpopulations in the immunotherapy response group and the progression group before treatment. The criteria for predicting effective response to immunotherapy for NSCLC brain metastases are: CD4_Trm_CXCR6 T cells in CD4 + The proportion of T cells is greater than 10%; or CD4_Trm_CXCR6 T cell signature protein score greater than 0.6; The characteristic protein or gene expression results of CD4_Trm_CXCR6 T cells are: CD3 + PD1 + 、CD4 + and CXCR6 + .

9. The system for predicting the efficacy of immunotherapy for NSCLC brain metastases according to claim 8, characterized in that: The gene features of the CD4_Trm_CXCR6 T cells in single-cell transcriptome sequencing are: CXCR6, CXCL13, IFN-gama, CTLA4, PRDM1 and HAVCR2, based on which the cell subsets were defined and the CD4_Trm_CXCR6 T cells in CD4 + The proportion of T cells.

10. The system for predicting the efficacy of immunotherapy for NSCLC brain metastases according to claim 8 or 9, characterized in that: The characteristic proteins of the CD4_Trm_CXCR6 T cells in proteomic detection include: CD3D, CD2, CD5, CD4, CD6, CD28, HMGB2, SLAMF6, NFATC2, NFATC3, CD44, WNK1, RGS1, PD-1, TIM-3, EOMES, PRDM1, IFN-gamma, BLC, IL-21, GZMH, CST7, SKAP1, HSP40, P2RY10 and SH3KBP1.

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