Project description:We profiled hematopoietic, lymphoid and peripheral fetal organs to systematically assess the heterogeneity of antigen receptors in immune cell populations across human tissues during development. Single-cell suspensions were obtained from fresh tissue. Cells were either DAPI-CD45+ or DAPI-CD45- FACS-isolated cells, or unsorted.
Project description:We profiled hematopoietic, lymphoid and peripheral fetal organs to systematically assess the heterogeneity of immune cell populations across human tissues during development. Single-cell suspensions were obtained from fresh tissue. Cells were either DAPI-CD45+ or DAPI-CD45- FACS-isolated cells, or unsorted.
Project description:Using single-cell RNAseq (scRNAseq) and paired VDJ analysis, we create the first comprehensive cell atlas of the healthy developing, paediatric and adult human gut, including 347,980 cells from up to 10 distinct anatomical sites. We use this data to trace the cellular composition of the gut throughout life, define novel cell markers and cell-cell interactions. We find four neuronal cell populations in the developing enteric nervous system, with expression patterns indicative of irritable bowel syndrome and Hirschsprung’s disease, and identify key cell players and communication networks initiating lymphoid structure formation in early human development.
Project description:Using single-cell RNAseq (scRNAseq) and paired VDJ analysis, we create the first comprehensive cell atlas of the healthy developing, paediatric and adult human gut, including 347,980 cells from up to 10 distinct anatomical sites. We use this data to trace the cellular composition of the gut throughout life, define novel cell markers and cell-cell interactions. We find four neuronal cell populations in the developing enteric nervous system, with expression patterns indicative of irritable bowel syndrome and Hirschsprung’s disease, and identify key cell players and communication networks initiating lymphoid structure formation in early human development.
Project description:For high-throughput sequencing and quantification of immunoglobulin repertoires, most methodologies utilise RNA. However, output varies enormously between recombined genes due to different promoter strengths and differential activation of lymphocyte subsets, precluding quantitation of recombinants on a per cell basis. To date, DNA-based approaches have used V gene primer cocktails, with substantial inherent biases. Here we describe VDJ-seq, which accurately quantitates immunoglobulin diversity at the DNA level in an unbiased manner. This is accomplished with a single primer extension step using biotinylated J gene primers. By addition of unique molecular identifiers (UMI) before primer extension, we reliably remove duplicate sequences and correct for sequencing and PCR errors. Furthermore, VDJ-seq captures productive and non-productive VDJ and DJ recombination events on a per cell basis. Library preparation takes 3 days, with 2 days of sequencing, and 1 day of data processing and analysis.
Project description:Porcine cytomegalovirus (PCMV) is a member of the genus Cytomegalovirus, subfamily Betaherpesvirinae, and family Herpesvirus. PCMV is a major immunosuppressive virus that mainly suppress the immune function of T lymphocytes and macrophages. PCMV is widely distributed all over the world, but there are not significantly different serotypes found. Moreover, the molecular mechanisms of host anti-PCMV infection and the molecular immunosuppressive mechanisms of PCMV is still not well charaterized. To understand the PCMV potential impact on the function of immune organs, we performed microarray assay to analyze the transcriptome of porcine immune organs after PCMV infection. We identified 5582 differential expression genes by PCMV infection in microarray. There are 2161 upregulated genes and 3421 down-regulated by PCMV infection genes compare to the uninfected control group. We confirmed 13 differentially expressed immune-related genes by quantitative real-time RT-PCR (qPCR). Gene ontology, gene interaction networks and KEGG pathway analysis uncovered the differentially expressed genes interaction regulatory network. These findings indicated that PCMV regulates multiple functional pathways, including immune system process, cellular process, metabolic process, networks of cytokine-cytokine receptor interaction, TGF-beta signaling pathway, lymphocytes receptor signaling pathway and TNF signaling pathway. Our study is the first comprehensive attempt to explore the host transcriptional response to PCMV infection in porcine immune systems. It provided new insights into the immunosuppressive molecular mechanisms and pathogenesis of PCMV. This previously unrecognized endogenous antiviral mechanism has implications for development of host-directed strategies to the prevention and treatment of immunosuppressive viral diseases. 2 samples were analysed. PCMV infected porcine immune organs; control porcine organs. piglets were divided into two groups of five pigs each, and maintained under controlled temperature and humidity. Each pig in the first group was inoculated with 5 ml of 109 PFU/ml PCMV SC strain by intramuscular and intranasal injection, and the other five pigs were injected with 5 ml of RPMI-1640 nutrient solution (Thermo Fisher Scientific, Waltham, UK) as the control. The sera, lymph nodes, spleens, and thymuses of the infected and control pigs were collected at 14 dpi