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Lung Natural Helper cells

ABSTRACT: Natural Helper cells constitute a unique lineage of Th2-cytokine producting innate lymphocytes, here we characterize the gene expression profile of non-stimulated or PMA/ionomycin-stimulated Natural Helper cells from naive C57Bl/6 mouse lungs. We used microarrays to detail the gene expression profile of stimulated and unstimulated lung Natural Helper cells in mice. Lineage(-)Sca-1(+)c-Kit(-/low)CD127(+)CD25(+) Lung Natural Helper cells were purified from naive 6-8 week old B6 mice by FACS. RNA was extracted immediately from un-stimulated Natural Helper cells after FACS. Stimulated Natural Helper cells were cultured with PMA/ionomycin for 3 days followed by RNA extraction.

ORGANISM(S): Mus musculus

SUBMITTER: Timotheus Halim

PROVIDER: E-GEOD-36057 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Lung Natural Helper cells

Project description:Natural Helper cells constitute a unique lineage of Th2-cytokine producting innate lymphocytes, here we characterize the gene expression profile of non-stimulated or PMA/ionomycin-stimulated Natural Helper cells from naive C57Bl/6 mouse lungs. We used microarrays to detail the gene expression profile of stimulated and unstimulated lung Natural Helper cells in mice.

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Project description:Naive CD4 T helper cells were isolated using magnetic negative selection kit from 4 weeks old Balb\c mice. The cells were activated with anti CD3/CD28 antibodies in the presence of Th1 th2 skewing cytokines and antibodies. The cells were disconnected for resting conditions after 48hours and further proliferated for 7 days in the presence of IL2 and skewing cytokines. In the 7th day the cells were collected and re-stimulated for 1hr using PMA/ionomycin and RNA was extracted for transcriptomics.

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Project description:<p>We use next generation sequencing to investigate the different transcriptomes of closely related CD4+ T-cells from healthy human donors to elucidate the genetic programs that underlie their specialized immune functions. Six cell types were included: Regulatory T-cells (CD25hiCD127low/neg with >95% FOXP3+ purity), regulatory T-cells activated using PMA/ionomycin, CD25-CD45RA+ ('naive' helper T-cells), CD25-CD45RO+ ('memory' helper T-cells), activated Th17 cells (>98% IL17A+ purity) and activated IL17-CD4+ T-cells (called 'ThPI'). Poly-T capture beads were used to isolate mRNA from total RNA, and fragment sizes of ~200 were sequenced from both ends on Illumina's genome analyzer. We confirm many of the canonical signature genes of T-cell populations, but also discover new genes whose expression is limited to specific CD4 T-cell lineages, including long non-coding RNAs. Additionally, we find that genes encoded at loci linked to multiple human autoimmune diseases are enriched for preferential expression upon T-cell activation, suggesting that an aberrant response to T-cell activation is fundamental to pathogenesis.</p>

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Epigenetic analysis of TCR signaling [Dnase-Seq]

Project description:DNaseI-Sequencing of unstimulated and PMA/Ionomycin-stimulated human T-blast cells

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EZH2 rip-seq in mouse Th1 helper cells, resting and re-stimulated with pma/ionomycin

Project description:EZH2 rip-seq in mouse Th1 helper cells, resting and re-stimulated with pma/ionomycin

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Project description:Bulk RNA-sequencing of FACS isolated human fetal lung, intestine, and kidney endothelial cells

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FOXP3 binding sites in promoter regions of human Jurkat T cells

Project description:The transcription factor (TF) Forkhead Box P3 (FOXP3) is constitutively expressed in high levels in natural occurring CD4+CD25+ regulatory T cells (nTreg) and is not only the most accepted marker for that cell population, but is considered lineage determinative. Chromatin immunoprecipitation (ChIP) of transcription factors in combination with genomic tiling microarray analysis (ChIP-on-Chip) has been shown to be an appropriate tool to identify FOXP3 transcription factor binding sites (TFBS) on a genome-wide scale. In combination with microarray expression analysis the ChIP-on-Chip technique allows to identify direct FOXP3 target genes. ChIP-on-Chip analysis of human FOXP3M-NM-^T2 isoform expressed in resting and PMA / ionomycin stimulated Jurkat T cells revealed several thousand putative FOXP3 binding sites and importance of intronic regions for FOXP3 binding. Knowledge of general distribution patterns of FOXP3 TFBS in the human genome under resting and activated conditions contributes to a better understanding of this TF and its influence on direct target genes with importance for Treg cell phenotype and function. ChIP-DNA from FOXP3(M-NM-^T2) expressing Jurkat T cells under resting and PMA / ionomycin stimulated conditions from duplicate experiments was analyzed. FOXP3-specific tiling array data were analyzed in reference to an individual isotype control dataset (J-FOXP3 ChIP'd with FOXP3 antibody vs. J-FOXP3 ChIP'd with isotype control antibody). In total 8 tiling array analyses were performed (2x resting J-FOXP3 with FOXP3-IP, 2x resting J-FOXP3 with isotype-IP, 2x PMA/iono J-FOXP3 with FOXP3-IP, 2x PMA/iono J-FOXP3 with isotype-IP)

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Methylome of CD4+ T cell subsets

Project description:We compared the methylated and non-methylated regions in the genome of ex vivo-isolated naive CD4+ T cells, Th1 cells, Th17 cells and regulatory T cells by methyl-CpG binding domain protein sequencing (MBD-seq). Naive T cells and Th1 cells share more methylated regions than naive T cells and Th17 cells or Th1 and Th17 cells. However, analysis of the non-methylated regions revealed the highest similarity between Th1 and Th17 cells. Another aim was the analysis of the Th17 lineage on the basis of the methylome. We searched for regions absent in the methylome of Th17 but present in naive T cells, Th1 cells and regulatory T cells. Here, we identified differential methylation in the loci of Il17a, Chn2, Dpp4 and Dclk1. CD4+ T effector cells were prepared ex vivo, stimulated with PMA/Ionomycin, subjected to a comercially available cytokine secretion kit (IL-17A and IFNg), stained by adding fluorescence-labeled antibodies against CD3, CD4 and CD45RB and sorted by flow cytometry. We sorted naive CD4+ T cells (CD3+CD4+CD45RB_high), Th1 cells (CD3+CD4+CD45RB_low_IFNg+IL17A-), Th17 cells (CD3+CD4+CD45RB_low_IFNg-IL17A+) and regulatory T cells (CD3+CD4+CD25++).

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