Exploiting Single-cell Omics Analysis To Study The Immune Atlas Of Atherosclerosis And Liver Cancers

The development of research in the biology and medicine areas extensively strengthens our understanding of the generation,progression,and intervention of different diseases,however,it is still a long way to overcome various diseases.The limitation of detection techniques also restricts the deeper inspections of the underlying mechanisms during disease occurrence.Cells are the basic elements of biology activities,the signal transduction across cells,the migration,differentiation,and proliferation of cells all significantly affect the normal operations of biological systems.Multiple single-cell detection methods that focus on single-cell genomic,epigenomic,transcriptomic,and metabolomic were developed to delineate the dynamic cell responses during diseases comprehensively.All these technical advances significantly enhance our understanding of the cellular functional differences in different disease statuses and the cellular responses in different environmental stimuli.Based on a variety of different single-cell omics technologies,in this thesis,we comprehensively elaborated the technical details of different methods,integrated and developed the single-cell data analysis methods,and applied these technologies to solve the biomedical problems related to basic science and clinical translation.It specifically includes:(1)Targeting the gadolinium isotope contamination problem in the single-cell mass cytometry system,this study developed a calibration method to remove the contamination signal;(2)Profiling the local immune landscape of carotid plaques of atherosclerosis patients at the single-cell level,this study uncovered the pro-inflammatory roles of plaque tissue-specific PD-1 positive T cell subset and elaborated a mechanism for targeting this cell subset to reverse the progression of human atherosclerosis;(3)Comparing the local tissue microenvironment of liver cancer patients with or without the infection of hepatitis B virus,this study revealed a specific T cell subset associated with hepatitis B virus infection,and the potential molecular regulatory mechanism of hepatitis B virus-induced hepatocellular carcinoma.The main innovative results in this thesis are as follows:1.In-depth characterization of the Gadolinium contamination signal in mass cytometry data and developed an algorithm for removing contamination signals.When applying single-cell mass cytometry for profiling single cells from differentpatient’s tissue samples,the single-cell data of some patients have severely signal co-expression between Gadolinium(Gd)-labeled signal channels.By comparing the clinical information of these patients,this study revealed that these patients had undertaken MRI scanning before the surgery and were administered Gadolinium-based contrast agents(GBCAs)to enhance the imaging quality.Thus,the residuals of Gd isotopes in tissue cells from GBCAs and the mixing of these Gd isotopes with the cellular antibody-conjugated Gd isotopes led to the signal contamination in the single-cell mass cytometry system.In this study,we deeply characterized the intensity and characteristics of the Gd contamination signals and proposed a new method to remove the Gd contamination signal in single-cell mass cytometry data.This study also designed additional experiments for proving the ability of this method in removing contamination signals and retaining the real single-cell biomarker signals.This research will improve the data quality and further application of single-cell mass cytometry system.2.In-depth characterization of immune atlas of carotid plaques in atherosclerosis patients,and uncovered critical immune subsets for restraining or reversing the disease progression of human atherosclerosis.Atherosclerosis is a chronic cardiovascular disease,and with the progress of the disease,the probability of acute cardiovascular and cerebrovascular incidents increases,which seriously threatens the safety of patients’lives.Previous studies have pointed out that atherosclerosis is a type of autoimmune disease and is accompanied by excessive local inflammation.By inhibiting the inflammatory response,the risk of cardiovascular and cerebrovascular incidents reduced slightly.Based on that,this study applied multiple single-cell detection methods to explore the immune microenvironment in the carotid plaques of atherosclerotic patients,for identifying the local inflammation-related cell subsets and functional characteristics.This study revealed the PD-1~+T cells in carotid plaques can still be activated and pro-inflammatory,and dissimilar with the tumoral exhausted T cells.Using the T cell receptors information,this identified the directional differentiation of PD-1~+T cells in plaque tissues.Based on the expression of PD-L1 is much lower in the plaque microenvironment compared with the tumor microenvironment,this study innovatively proposed a new therapeutic method to utilize the anti-PD-1 monoclonal antibodies captured by FcγRI receptors to replace the PD-L1 for restraining the pro-inflammatory roles of PD-1~+T cells in plaques and potentially further regulate the progression of human atherosclerosis.3.Characterizing HBV infection-related immune atlas in tissue samples of HCC patients,this study proposed the potential mechanism of HBV-infection induced HCC.Hepatitis B virus(HBV)infection is a key risk factor to induce hepatocellular carcinoma(HCC).Although lots of research have clarified their associations,the underlying mechanisms and intervention methods are still unclear.In this background,this study profiled the immune landscapes of the tumor tissues and tumor borders of HCC patients and the tissues of benign hepatic hemangioma patients by single-cell mass cytometry.By comparing the immune signatures between different tissue sites,this study revealed the tumor-specific immune suppression characteristics,and we comprehensively annotated the associations between HCC clinical staging,disease prognosis,liver damge induced by HBV infection with the different immune signatures.The subsequent comparisons of the tissue immune landscapes between HBV-infected and non-infected HCC patients revealed that the HBV infection has a more significant impact on the immune components in the tumor border.This study revealed PD-1~+CD103~+tissue-resident memory CD8~+T cells were expanded in the tumor border of HBV-infected patients.The following validation assays validated these T cells are HBV antigen-specific and compared with the PD-1~+CD103~+CD8~+T cells in HBV non-infected tumor border,these T cells have the weaker cytotoxic phenotypes.The enrichment of these T cells in the tumor border of HBV-infected patients potentially altered the immune microenvironment of normal liver tissues under HBV infection,which potentially contribute to hepatocarcinogenesis.