| Esophageal cancer is a high-frequency malignant tumor of the digestive tract in the world,ranking 8th in the incidence of malignant tumors in the world and 6th in the mortality rate.The incidence of esophageal cancer in high-risk areas can reach 500 times that in low-risk areas,showing obvious regional differences.Among them,China is a country with a high incidence of esophageal cancer.Its morbidity and death rank first in the world,which seriously endangers people’s health.Therefore,finding new prevention and treatment methods for esophageal cancer is an important subject in the field of esophageal cancer research in China.In recent years,checkpoint therapy has made encouraging progress in a variety of malignant tumors,and has become a landmark progress in the field of human malignant tumors following surgery,chemotherapy and radiotherapy.The latest phase Ⅱ multicenter clinical trial showed that immune checkpoint inhibitors can effectively improve the survival rate of patients with advanced esophageal cancer.Recent studies have shown that the composition and interaction of Tumor-infiltrating lymphocytes(TILs)and the expression of checkpoint molecules in immune cells are closely related to the effect of immunotherapy.It is suggested that elucidating the key immune cell subsets of tumor invasion and the interaction mechanism with tumor microenvironment is the key to effective immunotherapy intervention.TRM(Tissue resident memory T cells,TRM)cells are a newly discovered group of memory T cells characterized by high expression of CD 103 and CD69 molecules and specific colonization in non-lymphatic peripheral tissues.TRM is considered the first line of defense in peripheral tissues such as the mucosa,skin,digestive tract,and respiratory tract immune response.Existing research shows that TRM plays an important role in viral infections,allergic dermatitis,and various inflammatory diseases.The study of mouse infection model showed that the ability of TRM cells to clear local tissue pathogens was significantly stronger than that of peripheral blood circulating T cells.It is suggested that TRM cells play a very important role in maintaining the local immune homeostasis of tissues.However,research on the role of TRM in the human tumor microenvironment has just begun.Recently,only a few studies based on the analysis of immunohistochemistry or gene expression and prognosis have shown that the number of tumor infiltrating TRM is related to the prognosis of patients with lung cancer and melanoma.Therefore,this study intends to rely on the advantages of clinical biological sample resources and take human esophageal cancer tissue-specific memory T.cells as the entry point to try to find out the biological characteristics of esophageal cancer infiltrating TRM so as to provide new ideas for finding new esophageal cancer prevention methods.PART ONE Composition of TILs Cell Subsets in Esophageal CancerBackgroundEsophageal cancer is a high-frequency malignant tumor of the digestive tract worldwide.China is a country with a high incidence of esophageal cancer.Its morbidity and mortality rank first in the world,which seriously endangers the health of our people.In recent years,checkpoint therapy has made encouraging progress in a variety of malignant tumors,and has become a landmark progress in the field of human malignant tumors following surgery,chemotherapy and radiotherapy.The latest phase Ⅱ multicenter clinical trial showed that immune checkpoint inhibitors can effectively improve the survival rate of patients with advanced esophageal cancer.Recent studies have shown that the composition and interaction of Tumor-infiltrating lymphocytes(TILs)and the expression of checkpoint molecules in immune cells are closely related to the effect of immunotherapy.TRM(Tissue resident memory T cells,TRRM)cells are a newly discovered group of memory T cells characterized by high expression of CD 103 and CD69 molecules and specific colonization in non-lymphatic peripheral tissues.TRRM is considered the first line of defense in peripheral tissues such as the mucosa,skin,digestive tract,and respiratory tract immuune response.Existing research shows that TRM plays an important role in viral infections,allergic deRMatitis,and various inflammatory diseases.TRM(Tissue resident memory T cells,TRM)cells are a newly discovered group of memory T cells characterized by high expression of CD 103 and CD69 molecules and specific colonization in non-lymphatic peripheral tissues.TRM is considered the first line of defense in peripheral tissues such as the mucosa,skin,digestive tract,and respiratory tract immune response.Existing research shows that TRM plays an important role in viral infections,allergic dermatitis,and various inflammatory diseases.The study of mouse infection model showed that the ability of TRM cells to clear local tissue pathogens was significantly stronger than that of peripheral blood circulating T cells.It is suggested that TRM cells play a very important role in maintaining the local immune homeostasis of tissues.However,research on the role of TRM in the human tumor microenvironment has just begun.Recently,only a few studies based on the analysis of immunohistochemistry or gene expression and prognosis have shown that the number of tumor infiltrating TRM is related to the prognosis of patients with lung cancer and melanoma.SPAD(Spanning-tree Progression Analysis of Density-normalized Events,SPADE)analysis of esophageal cancer and paired normal tissue infiltrating T cells through single-cell analysis platform Cytobank found that CD8+TRM cells(CD8+CD103+CD69+TRM cells)content is significantly higher than normal tissues.Through traditional gated analysis,we also confirmed that human CD8+TRM cells are specifically expressed in esophageal cancer tissue and paired normal esophageal tissue,and the content of CD8+TRM cells in human esophageal cancer tissue Significantly higher than normal tissue.The above preliminary research results suggest that the abnormal increase of CD8+TRM cells is a very important feature of T cell composition changes in the inflammatory microenvironment of human esophageal cancer.Therefore,further exploring the role of CD8+TRM cells in the immune microenvironment of human esophageal cancer and its potential mechanisms involved in tumor immune response and regulation can provide a new immunotherapy strategy for targeting key cells that interfere with the immune microenvironment of esophageal cancer.ObjectiveIdentify key TILs cell subpopulations that are abnormally elevated or decreased in esophageal cancer tissues.Thus,the position of CD8+TRM cells in TILs cells of esophageal cancer tissue was clarified.MethodsThis part of the research work will be informed by the hospital ethics committee and the patient’s informed consent.After the patient is informed of the research related matters and signed the informed consent,Firstly,fresh tumor tissues and relatively normal esophageal tissues of patients with esophageal cancer surgery were collected,and single cell suspensions were prepared by primary tissue infiltration lymphocyte separation method.Then multi-color fluorescent antibody labeling and high-throughput multi-color flow cytometry were used to detect the expression of TILs cells and their subpopulations in esophageal cancer and matched normal tissues,and to identify key TILs cell subpopulations that were abnormally increased or decreased in esophageal cancer tissues.Thus,the position of CD8+TRM cells in TILs cells of esophageal cancer tissue was clarified.It lays the foundation for the next step of isolating and identifying the distribution and characteristics of esophageal cancer CD8+TRM cells in esophageal cancer tissues.Results(1)SPADE analysis of CD3+T cells in human esophageal cancer and paired normal tissues revealed that TRM cells in human esophageal cancer tissues increased significantly in proportion and quantity compared with normal tissues.(2)Flow gated analysis showed that TRM cells in human esophageal cancer tissues was significantly higher in proportion and quantity than matched normal tissues.ConclusionsCD8+TRM cells were significantly enriched in human esophageal cancer tissues.PART TWO Study on the Subpopulation,Distribution,Phenotype and Biological Characteristics of CD8+TRMCells Infiltrated in Esophageal CancerBackgroundAt present,TRM cells are considered to have the following characteristics:(1)have a long-term self-renewal ability;(2)are abundantly expressed in immune barrier tissues such as epithelial mucosa;(3)identify microbial products,cytokines,danger signal molecules and stress-related ligand(4)rapid secretion of a variety of inflammatory factors.Classical infection immunology studies show that after initial infection,mouse CD8+TRM cells in vaginal mucosa can quickly identify pathogens and generate specific immune memory;When exposed to the same antigen again,it secretes a large number of cytokines to promote humoral immune response,DC cell maturation and natural killer cell activation.It is suggested that TRM cells are widely involved in the regulation of various immune responses in the body.But its role in tumorigenesis and development is unclear.Our previous research found that CD8+TRM cells were significantly increased in human esophageal cancer tissue.In addition,previous studies have shown that CD8+TRM cells can initiate innate and adaptive immunity in chronic inflammation models,and can regulate a variety of immune responses.From this we speculated that CD8+TRM cells are one of the key factors for inflammatory immune disorders and tumor progression in human esophageal cancer.ObjectiveThis part of the research focuses on the detection of subpopulations and tissue distribution of CD8+TRM cells in a single cell suspension of esophageal cancer and its paired normal tissues by using a variety of CD8+TRM cell-related cell surface-labeled antibodies and multicolor flow detection analysis technology And other phenotypic characteristics.To elucidate the tissue distribution,differentiation-related phenotype,and immune checkpoint molecule expression characteristics of esophageal cancer tissue infiltrating CD8+TRM cells.Therefore,the basic biological characteristics such as differentiation phenotype,immune checkpoint molecule expression,and tissue tropism were clarified In order to further clarify its regulatory mechanism in esophageal cancer-related inflammatory tissue remodeling and further polarization.Methods(1)Tissue distribution characteristics of esophageal cancer infiltrating CD8+TRM cells:surgically remove freshly paired esophageal cancer and normal tissues,prepare a single cell suspension in a sterile environment,and then perform multicolor flow cytometry(CD45,CD3,CD8,CD 103,CD69),to analyze the proportion and number of CD8+TRM cells in different tissues.(2)Identification of differentiation-related phenotypes:Multi-color flow cytometry(CD45,CD3,CD8,CD 103,CD69,CD45RA,CD45RO)was used to analyze the differentiation-related phenotypic characteristics of tumors and paired normal tissues infiltrating CD8+TRM cells.(3)Immune checkpoint molecular detection:Through multi-color flow cytometry(CD45,CD3,CD8,CD103,CD69,PD-1,Tim-3),analysis of tumor and paired normal tissue infiltration CD8+TRM cell immune checkpoint molecular expression characteristics.Results(1)Esophageal cancer tumor infiltrating T cells are mainly CD4 and CD8 cells.Therefore,further analysis of CD8+TRM cells and CD4+TRM cells:in human esophageal cancer paired tissues showed that CD8+TRM cells and CD4+TRM cells were higher expressed in normal esophageal tissues and tumors.However,statistical analysis showed that the proportion and absolute number of CD8+TRM cells in human esophageal cancer tissues were significantly higher than those in matched normal tissues(P<0.0001).On the contrary,although the number of CD4+TRM is also increasing in esophageal cancer tissues,the proportion of CD4+TRM in human esophageal cancer tissues is decreasing compared with that of normal esophageal tissues.(2)Our research on human esophageal cancer and paired normal tissue CD3+TRM cells showed that human esophageal cancer infiltrated CD3+TRM cells was significantly higher than the normal paired tissue.Further analysis showed that human esophageal cancer and paired normal esophageal tissue TRM cells were mainly CD8+TRM cells and CD4+TRM cells,and only a small subset of TRM cells were γδT cells and iNKT cells.More importantly,our parallel comparison of TRM cell subsets in tumor tissues found that human esophageal cancer infiltrating CD8+TRM cells had an absolute advantage in proportion and number over the remaining TRM cell subsets.(3)Studies based on a mouse chronic autoimmune disease model have shown that CD8+TRM cells that persist in diseased tissues show an immune exhaustion and can continuously recruit proinflammatory CD4+Th cells and macrophage cell.It is suggested that CD8+TRM cells undergo immune exhaustion in the presence of chronic inflammation and further promote local inflammation.Our research shows that human esophageal cancer and paired normal esophageal TRM cells are mainly effector memory T cells(TEM)and central memory T cells(TCM).Further research shows that the expression of PD-1 in CD8+TRM cells of human esophageal cancer tissue was significantly higher than that of normal esophageal tissue.We also confirmed by viSNE(visual stochastic network embedding,viSNE)analysis that PD-1 expression of CD8+TRM cells in human esophageal cancer tissues was significantly higher than that in normal esophageal tissues.It is suggested that certain factors in the inflammatory immune microenvironment of human esophageal cancer promote CD8+TRM cell immune exhaustion.The persistence of chronic inflammation in the tumor microenvironment has been recognized to be closely related to the imbalance of tumor immune regulation.Therefore,we have reason to believe that the CD8+TRM in human esophageal cancer tissues is significantly increased and the phenomenon of immune exhaustion is closely related to the homeostasis of tumor inflammatory microenvironment.Conclusions(1)CD8+TRM cells predominate in human esophageal cancer tissue infiltrating TRM cells.(2)Human esophageal cancer infiltrating CD8+TRM cells show an immune exhaustion phenotype.PART THREE Correlation between esophageal cancer infiltrating TRM cells and clinicopathological features and prognosisBackgroundThe above research proves that CD8+TRM cells dominate the human esophageal cancer infiltrating TRM cells.By analyzing the correlation between the number of infiltrating CD8+TRM cells and clinical features and prognosis of esophageal cancer,Provide new ideas for targeted intervention of key cells(molecules)in immune regulation related to esophageal cancer,It also provides a potential possibility for clinical application of CD8+TRM cells as predictive indicators of esophageal cancer prognosis and targeted intervention for CD8+TRM cell-mediated immune suppression in esophageal cancer,which has clinical transformation significance.ObjectiveThis part of the research work will focus on the detection of the number of CD8+TRM cells in tumor tissues of 122 patients with esophageal cancer by immunohistochemical staining using esophageal cancer tissue chips or paraffin sections from previous patients,and analyze the CD8+TRM cells with clinical data Correlation between cell number and tumor TNM stage and clinicopathological characteristics.In order to clarify the correlation between human CD8+TRM cells and the clinicopathological characteristics and prognosis of esophageal cancer.Methods(1)The number of CD8+TRM cells in tumor tissues of 122 patients with esophageal cancer was detected by immunohistochemical staining using esophageal cancer tissue chips or paraffin sections from previous patients.(2)Correlation analysis of esophageal cancer infiltrating CD8+TRM cells and clinicopathological characteristics of patients:collect clinical data of patients,analyze the correlation between the number of CD8+TRM cells and tumor TNM stage and other clinical pathological characteristics.(3)Correlation analysis of esophageal cancer infiltrating CD8+TRM cells and patients’ prognosis:The relationship between the number of CD8+TRM cells and the patient’s prognosis was analyzed by immunohistochemical staining of esophageal cancer tissue chips from previous patients.Results(1)The overall survival of patients with increased CD8+TRM cells is higher than that of patients with esophageal cancer with reduced CD8+TRM cells,(log-rank P<0.05)(2)The disease-free survival of patients with an increased number of CD8+TRM cells was higher than that of patients with esophageal cancer with a reduced number of CD8+TRM cells,(log-rank P<0.05)Conclusions(1)The prognosis of esophageal cancer patients with high expression of CD8+TRM cells is good.CD8+TRM cells can be used as a biological indicator to judge the prognosis of esophageal cancer patients.(2)CD8+TRM cells can be used as predictive indicators to screen patients with esophageal cancer who have received clinical trials of immunotherapy. |
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