Expression data of CD117+ splenocytes from C57BL/6 WT and RasGRP4 KO mice upon LPS stimulation
ABSTRACT: RasGRP4 is required for CD117+ DCs to optimally induce certain NK cell-dependent immune responses in vivo and in vitro in response to LPSRasGRP4 expressed in DCs played an important role on NK cell IFN-γ secretion . We used microarrays to detail the global programme of gene expression underlying cellularisation and identified distinct classes of up-regulated or down-regulated genes between the two DCs from WT and RasGRP4 mice respectively upon LPS stimulation. CD117+splenocytes were extracted from WT and RasGRP4 KO mice spleens aged 9-12 weeks and plated in 6-well plates with 1ug/mL of LPS for 0h,6h,12h,24h and 48h repectively for RNA extraction and hybridization on Affymetrix microarrays. Time course.
Project description:RasGRP4 is required for CD117+ DCs to optimally induce certain NK cell-dependent immune responses in vivo and in vitro in response to LPSRasGRP4 expressed in DCs played an important role on NK cell IFN-γ secretion . We used microarrays to detail the global programme of gene expression underlying cellularisation and identified distinct classes of up-regulated or down-regulated genes between the two DCs from WT and RasGRP4 mice respectively upon LPS stimulation. Overall design: CD117+splenocytes were extracted from WT and RasGRP4 KO mice spleens aged 9-12 weeks and plated in 6-well plates with 1ug/mL of LPS for 0h,6h,12h,24h and 48h repectively for RNA extraction and hybridization on Affymetrix microarrays. Time course.
Project description:Zbtb46 represses G-CSFR and LifR in cDCs Zbtb46 does not significantly affect gene expression in erythroid progenitors WT, Het, and KO cells were sorted from BM or spleen and analyzed. Pre MegE cells were sorted as CD117+ CD150+ CD105-CD41-CD16/32-. Pre CFU-E cells were sorted as CD117+ CD150+ CD105lo CD41- CD16/32-. CFU-E cells were sorted as CD117+ CD150- CD105+ Cd41- CD16/32-. Splenic CD4+ DCs were sorted as B220- CD11c+ MHCII+ CD8- CD172+ CD11b+ CD4+.
Project description:It is known that NK cells are a heterogeneous population of functionally distinct NK cell subsets. Here we report on different genomic, phenotypic and functional properties of four murine NK cell subsets distinguished by CD117 (c-kit), CD27 and CD11b expression. Gene expression was measured in NK cell subsets freshly sorted from murine C57Bl/6 splenocytes. Two to three different batches were analysed.
Project description:Mast cells are indispensable for LPS-induced septic hypothermia, in which TNF-α plays an essential role to initiate sepsis. Tec family non-receptor tyrosine kinases ITK and BTK regulate mast cell-derived TNF-α in response to allergic antigen, but their role in LPS-induced TNF-α production by mast cells and related pathology is unclear. We sought to investigate the role(s) of ITK and BTK in mast cell response in septic condition. We found that the absence of ITK and BTK leads to enhanced TNF-α production by bone marrow-derived mast cells (BMMC). Itk-/-Btk-/- mast cells exhibit hyperactive preformed and LPS-induced TNF-α production, along with enhanced expression of other related genes such as NF-κB targeted genes, compared to WT cells. Bone marrow cells from 8-week old WT, Itk-/-, Btk-/- and Itk-/-Btk-/- (double knockout: DKO) C57Bl/6 mice were cultured in murine Interleukin-3/Stem cell factor (IL-3/SCF) supplemented medium for 5 weeks to derive mast cells. WT, Itk-/-, Btk-/- and DKO bone marrow-derived mast cells (BMMC) were factor starved in medium without IL-3/SCF for 12 hours, followed by treatment with PBS (control) or 100 ng/ml LPS for 1 hour. Triplicates of each group were subjected to mouse whole genome genechip microarray analysis. Replicates were randomized on different chips to avoid systematic error.
Project description:Cancer-induced tolerance mostly involves myeloid suppressor cells, regulatory T cells and immunosuppressive cytokines, which all subvert adaptive immune responses against tumor cells. Here, we show that a subset of innate effectors, c-kit expressing NK cells (Kit+ NK), can participate in tumor-induced tolerance by compromising the NK cell arm of tumor immunosurveillance. IL-18 produced by tumor cells can convert Kit- into Kit+ NK cells that overexpress B7-H1/PD-L1 molecules. Upon tumor inoculation, Kit+ NK cells rapidly develop in lymphoid organs in a IL-18R/MyD88 dependent manner and directly kill Kit- NK cells in a B7-H1/PD-1-dependent manner, thereby promoting the progression of NK-controlled cancers. Our data suggest that, in a tumoral context, IL-18 subverts antitumor NK cell functions. Systemic neutralization of IL-18 by IL-18-binding protein may improve the NK-mediated immunosurveillance. Keywords: cell type comparison Overall design: Treatment Protocol: Two subpopulations of NK cells are sorted from mouse spleens with a FACS Vantage instrument (BD Bioscience). NK kit+ are defined as CD3-NK1.1+CD117+ cells and NK kit- as CD3-NK1.1+CD117- cells. Splenocytes from C57Bl/6 mice were sorted in two steps. First, they were enriched in NK cells (NK1.1+/CD3-) by a magnetic separation using NK cell Isolation Kit (Miltenyi Biotec). Second, enriched NK cells were stained with FITC-conjugated anti-CD117 (c-kit) and PE-conjugated anti-NK1.1 monoclonal antibodies to allow a sorting of NK kit+ and NK kit- cells. Antibodies were purchased from Pharmingen, San Diego, CA or eBioscience, San Diego, CA. The purity of NK cells was around 98% after cell sorting. Characteristics: CD3-NK1.1+CD117+ NK cells are compared to CD3-NK1.1+CD117- NK cells from spleen. Cell sorts and comparison are performed three times. Strain: C57Bl/6. Gender: female. Age: 7 weeks. C57Bl/6 mice were obtained from C.River Laboratories (L’Arbresle, France) and the Centre d’Elevage Janvier (Le Genest-st-Isle, France). Biomaterial provided: Material provided from laboratory of Prof. Laurence Zitvogel, Immunology, INSERM U805, Instiut Gustave Roussy, Villejuif, France.
Project description:We applied Illumina massively parallel signature sequencing to identify miRNomes in hematopoietic stem cells, bone morrow-derived immature DCs, mature DCs, and IL-10 and NO-producing regulatory DCs.The miRNomes of these DC subsets will contribute to investigate the significance of miRNAs in DC immunobiology. Examination of the miRNome in hematopoietic stem cells, bone morrow-derived immature DCs, mature DCs, and IL-10 and NO-producing regulatory DCs. All four mouse cell types.
Project description:Dendritic cells (DCs) are a complex group of cells which play a critical role in vertebrate immunity. They are subdivided into conventional DC (cDC) subsets (CD11b and CD8alpha in mouse) and plasmacytoid DCs (pDCs). Natural killer cells are innate lymphocytes involved in the recognition and killing of abnormal self cells, including virally infected cells or tumor cells. DCs and NK cells are activated very early upon viral infections and regulate one another. However, the global responses of DC and NK cells early after viral infection in vivo and their molecular regulation are not entirely characterized. The goal of this experiment was to use global gene expression profiling to assess the global genetic reprogramming of DC and NK cells during a viral infection in vivo, as compared to B lymphocytes, and to investigate the underlying molecular mechanisms This study includes data from cell sort purified DCs, NK cells and B cells isolated from the spleen of MCMV-infected mice. 2 independent replicates were made for each cell type except B cells. The control dataset for cells isolated from uninfected control animals has been previously published and is available in the GEO database as GSE9810. The complete dataset representing: (1) the infected Samples and (2) the uninfected control Samples from Series GSE9810 (re-processed using RMA), is linked below as a supplementary file. Comparison of the gene expression programs of wild-type spleen leukocyte subsets, including plasmacytoid DCs, CD8alpha conventional DCs, CD11b conventional DCs and NK cells, isolated from MCMV-infected versus control animals.
Project description:To further distinguish the different subset of DCs, changes in gene expression were analyzed using Agilent mouse gene expression 4×44,000 microarrays. BALB/C mouse bone marrow–derived imDCs and maDCs were generated from bone marrow progenitors. Bone marrow hematopoietic progenitor cells cocultured with Sca-1+Lin-CD117- MSCs could differentiate into a novel IL-10-dependent regDC subset-sDCs. The gene expression differentiations of the four groups (imDCs, maDCs, sDCs and neutralizing antibody-IL-10-treated sDCs) were analyzed by GeneChip microarray. The formed four groups of cells (imDCs, maDCs, sDCs and neutralizing antibody-IL-10-treated sDCs) were purified respectively, and then gene expression in them was measured.
Project description:DCs treated with PTX (PTX-DC) is able to induce EAE like PTX as adjuvant whereas neither LPS nor DCs treated with LPS (LPS-DC) fails to induce EAE. We want to identify genes that are responsible for EAE induction in DCs and genes that are able to toloerize EAE in DCs through the microarray. Bone marrow derived dendritic cells are either unstimulated or stimulated with LPS and PTX for 24h respectively. Cells are harveseted for RNA extraction and hybridization on Affymetrix microarrays.
Project description:Toll-like receptor (TLR)-induced maturation of dendritic cells (DCs) leads to the production of proinflammatory cytokines as well as the upregulation of various molecules involved in T cell activation. These are believed to be the critical events that account for the induction of the adaptive immune response. Here, we have examined the role of micro-RNA-155 (miR-155) in DC function and the induction of immunity. Using a model where the transfer of self-antigen-pulsed, TLR-matured DCs can induce a functional CD8 T cell response and autoimmunity, we find that DCs lacking miR-155 have an impaired ability to break immune tolerance. Importantly, transfer of self- antigen-pulsed DCs overexpressing miR-155 was sufficient to break tolerance in the absence of TLR stimuli. Although these unstimulated DCs induced T cell function in vivo, there was no evidence for the upregulation of costimulatory ligands or cytokine secretion. Further analysis showed that miR-155 influenced the level of the phosphatase SHIP1 in DCs, and that the lack of SHIP1 in DCs was sufficient to break T cell tolerance in vivo, again in the absence of TLR induced DC maturation. Our study demonstrates that the overexpression of miR-155 in DCs is a critical event that is alone sufficient to break self tolerance and promote a CD8-mediated autoimmune response in vivo. This process is independent of the induction of conventional DC maturation markers, indicating that miR-155 regulation of SHIP represents a unique axis that regulates DC function in vivo. Dendritic cell culture: Bone marrow was flushed from tibias and femurs of mice with HBSS. Bone marrow cells were cultured at 2x106 cells/ml in 10 ml non-tissue culture treated dishes in RPMI 1640 containing 10% LPS-free FBS, Penicillin:streptomycin glutamine 2-mercaptoethanol (cRPMI), with 40ng/ml murine GM-CSF (Peprotech). On day 3 of cultures 10ml of fresh media was added also containing 40ng/ml GM-CSF. On days 6 and 8, 10ml of media was removed and centrifuged to collect cells, which were resuspended in 10ml fresh media containing GM-CSF and were added back to dishes. Non-adherent cells were isolated on day 9. DCs were plated in 24-well plates at 2x106 cells/ml in 1ml of cRPMI with or without the class B CpG ODN1826 (ACGT DNA Technologies corporation, Toronto, ON, Canada) at 10μM final concentration overnight. Array analysis of mi-RNA expression: BMDCs from WT mice were either stimulated with CpG or left in media alone overnight. Whole RNA was isolated using mirVana miRNA isolation kit following the manufacturers instructions. RNA samples were labeled and hybridized to Agilent mouse miRNA 8x15K arrays (Agilent). Data was generated from 8 individual samples (4 unstimulated and 4 CpG-treated).