Unknown,Transcriptomics,Genomics,Proteomics

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ATAC-seq examining chromatin accessibility of mouse splenic IL-10+ and IL-10-CD19+CD21hiCD24hi B cells

ABSTRACT: Chromatin profiling of mouse splenic ex-vivo IL-10+, IL-10-CD19+CD21hiCD23hiCD24hi B cells and IL-10- follicular B cells, after antigen induced arthritis. IL-10eGFP reporter (Vert-X) mice were used to sort IL-10+ and IL-10- subsets. ATAC-seq was used in conjunction with microarray data to understand the chromatin profiles and transcriptional landscape of mouse regulatory B cells, compared to other B cell subsets.

INSTRUMENT(S): Illumina HiSeq 4000

ORGANISM(S): Mus musculus

SUBMITTER: Claudia Mauri

PROVIDER: E-MTAB-8393 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Tumor-associated macrophages (TAM) represent an abundant cell population of the immune infiltrate in solid tumors and have been shown to orchestrate escape from immune surveillance. Macrophages display a very plastic phenotype which is recapitulated in vitro by classifying certain subsets according to exposure with defined, individual cytokines. The tumor-promoting M2 macrophages are polarized in vitro by differentiating human monocyte-derived macrophages with the T helper cell type 2 (Th2) response cytokines interleukin-4 and interleukin-13 or the immunosuppressive cytokine interleukin-10. Notably, only the latter macrophage subset undergoes apoptosis when treated with the colony stimulating factor 1 receptor (CSF1R) blocking antibody emactuzumab. However, under physiologic conditions the phenotype of TAM is shaped by a combination of cytokines. Hence, we evaluated if the addition of IL-10 to IL-4 or IL-13 differentiated macrophages is able to override IL-4/-13 mediated signaling and to restore susceptibility to emactuzumab. Though addition of IL-10 did not restore emactuzumab susceptibility, we surprisingly detected that only IL-4 differentiated macrophages sustained their specific marker expression while IL-10 skewed the IL-13 differentiated macrophage profile towards the IL-10 regulated phenotype. In-depth characterization by gene expression profiling revealed unique signatures of IL-4+IL-10 and IL-13+IL-10 differentiated macrophage subsets characterized by upregulation of the canonical NFÎºB signaling or Wnt/Î²-catenin signaling pathways, respectively. In silico-based analysis of a large cohort of cancer patients revealed distinct interleukin-4 or interleukin-13 overexpression patterns in a subset of patients with partial co-expression of IL-10 but almost absent IL-4/IL-13 co-expression. These patients may have less TAM depletion under therapy with CSF1R inhibitors. Five different macrophage subtypes were differentiated for six days with respect to different cytokines and transcriptomically profiled with three biological replicates derived from three different human, healthy volunteer donors

Project description:To determine characterize human B cells that express IL-10 on a molecular level, we compared the global gene expression of primary CD19pos B cells secreting IL-10 or not, upon activation with anti-CD40, IL-4 and CpG for 2 days. Human B cells from healthy donors were sorted according to CD19 expression by magnetic cell separation. The cells were stimulated at 2.5E06 cells/ml by activating anti-CD40 mAb (1 M-BM-5g/ml, clone 82111, RnD Systems), recombinant human IL-4 (10 ng/ml, Immunotools) and CpG2006 (3 M-BM-5g/ml) for 2 days following restimulation for 3 h with phorbol myristate acetate (PMA, 10 ng/ml) and ionomycin (1 M-BM-5g/ml). Staining of viable IL-10 secreting human CD19pos B cells was performed by the IL-10 capture assay according the manufacturers instructions (Miltenyi Biotec, n=2). Finally, the cells were sorted into IL-10posCD69pos and IL-10negCD69pos cells by flow cytometric cell sorting on a BD Aria II sorter (Becton Dickinson). Dead cells were excluded using propidium iodide. Total RNA was extracted using the RNeasy Mini kit (Qiagen). The integrity and amount of isolated RNA was assessed for each sample using an Agilent 2100 Bioanalyzer (Agilent, Waldbronn, Germany) and a NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies, Wilmington, DE). Double-stranded complementary RNA was synthesized from 1 M-BM-5g total RNA using Message AmpII Biotin (Ambion, USA). Fifteen micrograms of fragmented cRNA of each sample were hybridized to four HG-U133A plus 2.0 GeneChips (Affymetrix). Hybridization was performed in a Hybridization Oven 640, and chips were washed and stained in the Fluidics Station 400 (both Affymetrix). Finally, the arrays were scanned with a GeneChip Scanner 3000 using the GCOS software, version 1.4, both Affymetrix. All relevant GCOS data of quality checked microarrays were analyzed with High Performance Chip Data Analysis (HPCDA, unpublished), using the BioRetis database (www.bioretis-analysis.de), as described and validated previously.

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ATAC-seq examining chromatin accessibility of mouse splenic IL-10+ and IL-10-CD19+CD21hiCD24hi B cells

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