Research On Lymphocyte Differentiation And Development Based On Multiomics And Bioinformatics

Lymphocytes are white blood cells with important immune function,which are very important for the body to resist infection,clear senescent cells and prevent tumorgenesis.Therefore,the study of lymphocytes has always been a hot spot in life science research.In lymphocytes,NK cells and T cells play a role in killing and immune regulation in innate immunity and adaptive immunity,respectively.At the same time,the good effect of immunotherapy based on NK cells and T cells in disease treatment makes the research of NK cells and T cells more and more important.The research on the development of NK cells and T cells can help us precisely understand the development process of NK cells and T cells,which is of great significance to understand the composition of the immune system,the development of diseases when the immune system is dysfunctional,and the treatment of diseases.The imperfection of previous technologies severely hindered research on NK cells and T cell development.With the development and improvement of a variety of omics sequencing technologies,we can study the development of NK cells and T cells more comprehensively.However,the current development process of NK cells and thymic T cells is not very clear.Understanding the development process of NK cells and thymic T cells can provide new insights for the study of lymphocyte development,and promote the development of immunotherapy based on NK cells and T cells.The integration of multiple omics technologies is essential to improve our understanding of human diseases and biology.Therefore,we use microarray,scRNA-seq,ATAC-seq,scATAC-seq and other genomics technologies,integrating transcriptomics and epigenomics and other bioinformatics methods,hope to study the transcriptional regulatory network of NK cells and thymic T cells during the development process,discover important transcription factors that regulate the transcriptional process,and reveal the important biological problems that have not revealed in the development process.This thesis is mainly divided into the following two parts.In the first part of the work,we first collected samples at different stages of NK cell differentiation for ATAC-seq library construction and sequencing.We obtained high-quality ATAC-seq data after rigorous quality control,and used these data to reveal the chromatin accessibility landscape in the process of NK cell differentiation.We discovered 6401 peaks with differentially-accessed chromatin sites across the genome,and identified two stages of accessible chromatin sites by unsupervised hierarchical clustering.Some known genes that regulate the development of NK cells also enriched in the corresponding stages of NK cells development,such as STAT5A in the early stage,and TBX21 in the late stage.From the functional enrichment analysis of these differential regulatory elements,we found that most of the sites opened in the early stage were enriched in phosphorylation and other functions related to maintaining the normal activities of the body,while the sites opened in the late stage participated in the construction of the immune system,etc.Immune response-related processes,which indicated that these regulatory elements more accessible in the late stage were accompanied by the expression of genes to regulate the development of NK cells.Subsequently,based on these epigenetic regulatory elements,we enriched transcription factors regulating NK cell differentiation using Homer and genomica.Using these enriched transcription factors and their gene expression levels,we constructed the transcriptional regulatory network during the differentiation of NK cells and described the dynamic changes of the transcriptional regulatory network within the different stages of NK cell development.Finally,we found that besides the known transcription factors,FOSL2 and EGR2 are significantly enriched during the differentiation of NK cells,and the genes regulated by FOSL2 and EGR2 are enriched with functions related to immune response,suggesting that FOSL2 and EGR2 can regulate the differentiation of NK cells.Subsequently,we proved that FOSL2 and EGR2 can indeed affect the differentiation and maturation of NK cells by knockdown experiments.In the second part of our work,we first used flow cytometry to sort CD45-positive lymphocytes from the mouse thymus,then constructed a library with 10X Genomics and sequenced to obtain single cell RNA-seq data.The data includes 1986 cells,and an average of 1784 genes per cell in average are detected.By clustering the single cell RNA-seq data,we obtained a total of 15 cell clusters corresponding to the different stages of T cell development,such as CD4/CD8 double negative,CD4/CD8 double positive,CD4 single positive and CD8 single positive,as well as some antigen-presenting cells and non-traditional lymphocytes.The development process of thymus T cells was described by pseudotime analysis.Secondly,by integrating single-cell RNA-seq and single-cell ATAC-seq data,we revealed the transcription factors that regulate the development of T cells.In addition to the enrichment of known TFs,we also found some new transcription factors are enriched.Based on these enriched transcription factors and their regulating differentially expressed genes,we constructed a transcriptional regulatory network for the development of thymic T cells.Moreover,we found that Ly6d and CD2 can serve as new markers to partition DP subtypes,then we developed a flow cytometry gating strategy to partition these DP subtypes at the protein level.Through this clustering,we also found some unknown phenomena in the development of thymic T cells:for example,(1)the unique proliferation behavior of DP thymocytes:DPbla cells differentiated into DPre during the cell cycle and tended to exit the cell cycle after division.The unique division mechanism of DP thymocytes may lead to a decline in the DP population during thymic involution.(2)In addition to thymic epithelial cells,thymocytes can also be regarded as antigen presenting cells for T cell development.We found that there was immunological synapses(ISs)between thymocytes by immunofluorescence,and we proved that thymocytes can serve as APCs for CD8-associated thymic selection.Finally,we downloaded and analyzed the single-cell data of human thymocytes.Through cross-species comparative analysis,we revealed the differences in transcription levels between humans and mice.Combined with ChIP-seq data,we found that Gatal may regulate the transcription distinctions between these species.