Project description:CD8+ DCs play key role in CD8+ T cell priming, however, the underlying signaling mechansim is unclear. We used a data-driven network-based systems biology approach and identified Hippo signaling kinases as key selective modualtors in CD8+ DCs. We focused on Mst1/Stk4 to further investigate the novel function of Hippo signaling in CD8+ DCs. All transcriptional profies were evalated by microarray. 1) We used microarrays of total DCs in previous (GSE98481) and current studies to reverse engineer a DC-specific signaling interactome (DCI). 2) We integrated DCI with profiles of sorted CD8+ with CD8- DCs to identify signaling drivers of CD8+ DCs. 3) We profiled and compared Mst1/2-KO with WT cells from total, CD8+ and CD8- DCs to understand the mechanism of Mst1 in CD8+ DCs.
Project description:In order to explore the regulatory mechanism of MST1 kinase on Tfh differentiation and function, we sorted Tfh cells in the spleen of WT and Mst1 KO mice 8 days after intraperitoneal sensitization, and extracted total RNA from the collected cells as samples for sequencing analysis.
Project description:To comprehensively understand how dendritic cells (DCs) are reprogrammed by lung fibroblasts- and their derived COX-2/PGE2, we employed lung fibroblasts isolated from WT or Ptgs2-/- mice, and collect their conditioned medium (CM) to stimulate the ex vivo cultured bone marrow (BM)-derived DCs (BM-DCs), with the PGE2 treatment as a control. After the treatment, BM-DCs were harvested for RNA extraction and the transcriptional profiles were analyzed by RNA sequencing (RNA-seq).
Project description:Analysis of the specific transcriptional changes on DCs provided by direct pattern recogition receptor (PRR) or IFNAR signaling that are required for DC maturation after poly IC stimulation. Results provide important information about the intricate differentiation process of DC maturation and the importance of type I IFNs for DC immunogenicity. WT/IFNA-/- or WT/PRR-/- mixed-chimera mice were injected with 50 ug Poly-IC i.p. in vivo 4 and 14 hr later, wild type and KO CD11chi CD3- DX5- B220- DCs were FACS sorted based on CD45.2 expression. Total RNA was isolated and expression profile was compared between unstimulated and activated WT and KO DCs.
Project description:In comparison to murine dendritic cells (DCs), less is known about the function of human DCs in tissues. Here, we analyzed, using lung tissues from humans and humanized mice, the role of human CD1c+ and CD141+ DCs in determining the type of CD8+ T cell immunity to live-attenuated influenza virus (LAIV) vaccine. We found that both lung DC subsets acquired influenza antigens in vivo and expanded specific cytotoxic CD8+ T cells in vitro. However, lung-tissue-resident CD1c+ DCs but not CD141+ DCs were able to drive CD103 expression on CD8+ T cells and promote CD8+ T cell accumulation in lung epithelia in vitro and in vivo. CD1c+ DCs induction of CD103 expression was dependent on membrane-bound TGF-?1. Thus, CD1c+ and CD141+ DCs generate CD8+ T cells with different properties, and CD1c+ DCs specialize in the regulation of mucosal CD8+ T cells. Total RNA were isolated from purified human CD1c+ (BDCA1+) and CD141+ (BDCA3+) mDCs sorted from different tissues, including human blood, spleen and lungs of humanized mice, and human lungs. Eighteen samples in total were analyzed from different donors and tissues.
Project description:To understand the functional relationship between brain dendritic cells (brain DCs) and other myeloid cells, we compared the gene expression profile of m/chDCs to that of bone marrow monocytes, brain microglia and classical spleen CD8+ and CD8- DCs. In order to obtain enough brain DCs for mRNA extraction, we expanded brain DCs with in vivo Flt3L treatment before purification. This study includes data from FACs Aria-purified brain DCs, bone marrow monocytes, brain microglia and classical spleen CD8+ and CD8- DCs. The genearray was performed to compare expression of cell surface endocytic receptor, cytokine receptors, TLRs, transcription factors, molecules for antigen processing and presentation. Each Series consists of 3 individuall samples
Project description:2 types of dendritic cells (DCs) can be generated in vitro in the presence of Flt3-L: CD4+ equivalent CD24- DCs and CD8+ equivalent CD24+ DCs. miR-142-/- mice show a severe defect in the generation of CD4+ equivalent CD24- DCs. To understand the underlying mechanism, RNA expression was analyzed by Affymetrix microarray from the 2 in vitro subtypes of DCs derived from miR-142+/+ and miR-142-/- bone marrow cells. We used microarrays to detail the global programme of gene expression in the presence or absence of miR-142 in in vitro derived DCs. Bone marrow cells from miR-142+/+ and miR-142-/- C57Bl/6 mice were isolated and incubated in the presence of Flt3-L for 8 days. in vitro derived wt and ko dendritic cells were devided into CD4+ and CD8+ equivalent DCs by FACS and sorted with a FACS-Aria. RNA was isolated and gene expression was investigated