Project description:the molecular mechanisms behind the turnover of medullry thymic epithelial cells remain unclear. We performeds single cell RNA-seq of TECs from mice 15 days after depleting cytokine RANKL and CD40L signaling.
Project description:C57BL/6 mouse lymph node stromal cells treated with anti-CD40L were isolated for scRNA-seq, more than 2x104 cells were run on the 10X Chromium Controller (10X Genomics) to partition single cells with uniquely barcoded beads and processed for sequencing library preparation using the Chromium Single Cell 3’ Reagent Kit. cDNA libraries were sequenced on a NovaSeq 6000 sequencing system. 4 x 103 cells per sample were captured on the 10X Chromium chip. 5-10 x 104 reads/cell were obtained with characterization of 2-3 x 103 transcripts/cell.
Project description:RANKL (receptor acrivator of NFkB ligand) is a member of TNF superfamily cytokines. In the gastrointestinal tract, RANKL is expressed in the stromal cells of Peyer's patches, and involved in the development of the specialized intestinal epithelial cells, called M cells. To identify the genes involved in M-cell development, we treated BALB/c mice with recombinant GST-RANKL. After RANKL-treatment, epithelial cells were isolated from small intestine, and used for microarray analysis. Total of 15 samples were analyzed. We generated the Excel sheet for comparing gene expression.
Project description:To identify the microRNAs that are involved in osteoclastogenesis, microRNA expression profiles in mouse bone marrow macrophages (BMMs) stimulated with RANKL (BMOc) were compared with that of control untreated BMMs. These results provide insights into the mechanisms to regulate osteoclastogenesis and bone resorption activities in osteoclasts by microRNA. BMMs were cultured with 20 ng/ml M-CSF in the presence or absence of 50 ng/ml RANKL for 24 hours. Cells were collected for total RNA isolation, and were subjected to microRNA array analysis.
Project description:Atherosclerosis, the underlying vascular cause of cardiovascular disease, has a strong inflammatory component. The co-stimulatory CD40-CD40 ligand (CD40L) signaling axis is a pivotal regulator of immune responses in atherosclerosis. However, therapeutic long-term inhibition of CD40L will severely compromise the immune system making it a non-viable treatment option. To circumvent this issue, cell-specific inhibition may present a better approach to target the CD40-CD40L axis. Therefore, we generated T cell and platelet-specific knockout mice for CD40L and apolipoprotein E, which were aged for 28 weeks to study their effects on immune status and atherosclerosis. Here, we show that T cell specific deficiency in CD40L signaling reduced plaque progression through hampered Th1 polarization as well as reduced antigen-dependent proliferation and oxLDL IgM production. DC-specific CD40 deficient mice displayed a similar phenotype. Platelet-specific CD40L deficiency, however, failed to decrease atherosclerosis, but ameliorated atherothrombosis. Together, our results illuminate the divergent cell-specific mechanisms of CD40-CD40L signaling in atherosclerosis, which may lead to advances in targeted therapies.
Project description:RANKL (receptor acrivator of NFkB ligand) is a member of TNF superfamily cytokines. In the gastrointestinal tract, RANKL is expressed in the stromal cells of Peyer's patches, and involved in the development of the specialized intestinal epithelial cells, called M cells. To identify the genes involved in M-cell development, we treated BALB/c mice with recombinant GST-RANKL. After RANKL-treatment, epithelial cells were isolated from small intestine, and used for microarray analysis.
Project description:To assess the nature of CD8+CD40L+ memory Tcells, we compared the gene expression to CD8+CD40L- and CD4+ counterparts, and found similarities in expression of genes encoding cytokines PBMCs were isolated from five healthy donors. The cells were stimulated with PMA/Ionomycin for 6 h, and subsequently sorted into the CD4+CD45RA-CD40L+, CD8+CD45RA-CD40L-, CD8+CD45RA-CD40L+ populations by FACS-sort
Project description:To assess the nature of CD8+CD40L+ memory Tcells, we compared the gene expression to CD8+CD40L- and CD4+ counterparts, and found similarities in expression of genes encoding cytokines. PBMCs were isolated from five healthy donors. The cells were stimulated with PMA/Ionomycin for 6 h, and subsequently sorted into the CD4+CD45RA-CD40L+, CD8+CD45RA-CD40L-, CD8+CD45RA-CD40L+ populations by FACS-sort.
Project description:To assess the nature of CD8+CD40L+ memory Tcells, we compared the gene expression to CD8+CD40L- and CD4+ counterparts, and found similarities in expression of genes encoding cytokines