An immunoreactive signature of monocyte-derived dendritic cells from patients with Down syndrome
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ABSTRACT: Monocyte-derived dendritic cells (MoDCs) were obtained from two controls and two Down syndrome patients. A microarray-based transcriptomic analysis was performed on the Clariom D Assay, Human platform (ThermoFisher Scientific).
Project description:Monocytes infiltrating tumors acquire various states that distinctly impact cancer treatment. Here, we show that resistance of tumors to radiotherapy (RT) is controlled by the accumulation of monocyte-derived dendritic cells (moDCs). These moDCs are characterized by the expression of CD301b and have a superior capacity to generate regulatory T cells (Tregs). Accordingly, moDC depletion limits Treg generation and improves the therapeutic outcome of RT. Mechanistically, we demonstrate that granulocyte-macrophage colony-stimulating factor (GM-CSF) derived from radioresistant tumor cells following RT is necessary for the accumulation of moDCs. Our results unravel the immunosuppressive function of moDCs and identify GM-CSF as an immunotherapeutic target during RT.
Project description:Persistent activation of canonical and non-canonical NF-κB pathways is a hallmark of the malignant Hodgkin and Reed-Sternberg cells in classical Hodgkin lymphoma (cHL). We identified lymphotoxin alpha (LTA), which is secreted by the cHL cell line L-1236 in high concentrations. This cytokine contributes to the constitutive activation of the canonical and non-canonical NF-κB pathways. L-1236 cells were purchased from the DSMZ (Braunschweig, Germany) and cultured in RPMI (Gibco) with 10% heat-inactivated fetal calf serum (FCS; Gibco). Cells were transducted with the lentiCRIPSPR v2 vector containing gRNAs which target the second (g2) and the fourth exon (g3) of LTA. Following single cell clonal selection, L-1236 control cells (v2) and two LTA knockout (KO) clones g2_1 and g3_4 were cultured in normal culture medium for 72 h. The RNA was extracted using the RNeasy kit (Qiagen) according to manufacturer’s instructions. Preparation of cDNA, fragmentation and labeling was performed with the GeneChIPTM WT PLUS reagent kit (ThermoFisher Scientific). Samples were hybridized to the human Clariom™ S Assay (ThermoFisher Scientific).
Project description:miRNA expression profiling of human monocyte-derived dendritic cells (moDCs) during maturation. Immature, 4h and 16h LPS-activated moDCs were used.
Project description:miRNA expression profiling of human monocyte-derived dendritic cells (moDCs) during maturation. Immature, 4h and 16h LPS-activated moDCs were used. In this study were analysed 2 biological samples of RNA extracted from 4h-post LPS stimulation moDCs and 2 bfrom 16h-post LPS stimulation. The RNA from 0h-post LPS stimulation were used as reference sample. Were also performed dye-swaps of each biological sample as technical replicates.
Project description:Aim of the experiment: To examine changes in gene expression in HT1080 fibroblasts following treatment with purified recombinant wild-type typhoid toxin (TOX) from Salmonella Typhi relative to a mutant H160Q variant lacking DNase activity (TOX-H160Q). Experimental workflow: HT1080 were seeded one day before intoxication into T75 tissue culture flasks for a 30% confluency in DMEM supplemented with 10% FBS and PenStrep. The next day, TOX (5 ng/ml) or negative control TOX-H160Q (5 ng/ml) was incubated for 2h with cells followed by three washes with PBS and a 48h chase in DMEM supplemented with 10% FBS and PenStrep. After 48 h, cells were collected by trypsinisation. RNA was isolated and samples analysed using human Clariom S assay (ThermoFisher Scientific, 902927). Analysis was performed with Transcriptome Analysis Console 4.0 software (Applied Biosystems, Thermo Fisher Scientific).
Project description:Purpose: To compare the cortical neuroonal differentiation capacity of clonal isogenic hESC lines with different levels of alpha-synuclein (aSyn) expression. Methods 1: Shef4 hESCs was used as a parental line to create an allelic series of clonal transgenic hESC lines expressing a human SNCA (encoding aSyn) contruct. Clonal transgenic lines with high (S8, S37) and low (S9, S34) aSyn expression were established and characterized. Methods 2: hESCs were cultured on Laminin-521 (BioLamina) in StemMACS iPS-Brew XF self-renewal media (Miltenyi) prior to lifting for isolation of total RNA. Methods 3: Cortical neuronal progenitor cells were differentiated from hESCs on Laminin-111 coated (Biolamina) 24-well plates at an initial plating density of 80,000 cells/cm2. Differentiation commenced in neural induction media (NIM) consisting of 50% DMEM/F12 (ThermoFisher Scientific), 50% Neurobasal Media (ThermoFisher Scientific), B27 supplement with Retinoic Acid (ThermoFisher Scientific), N2 supplement (ThermoFisher Scientific) and 2 mM L-Glutamine (ThermoFisher Scientific), 10μM SB431542 (Tocris) and 100 nM LDN-193189 (Miltenyl Biotec). From day 4 onwards, the base media was changed to 50% NIM, 25% DMEM/F12 and 25% Neurobasal Media. SB431542 (10 μM) and LDN-193189 (100 nM) were present for the first 12 days of differentiation. Cells were lifted and re-plated at day 12 and day 17 with Collagenase Type IV (Life Technologies). At day 25, differentiated cells were lifted for total RNA isolation.
Project description:To ditinguish different immune response and investigate the rule of proper recognition of fungi, a transcriptional analysis on moDCs exposed to a pathogenic and a harmless fungi was performed. Monocyte-derived dendritic cells were treated with Candida albicans, two different forms of Saccaromyces cerevisiae (whole organism and spore ascum) or untreated.
Project description:Primary human monocytes were isolated and differentiated into moDCs. These moDCs were infected with different Zika virus (ZIKV) strains (namely DN-1, DN-2 and H/PF/2013) and YF17D (as compared to non-infected controls, \\"mock\\") to better understand the gene expression changes induced by these different viruses. moDCs were harvested at 24 hours post-infection and microarray performed with the Affymetrix GeneChip Human Gene 2.0 ST Array.
Project description:Affymetrix Human Gene 2.0 ST microarray (ThermoFisher Scientific, Waltham, MA, USA) was used to select differentially expressed genes.