Project description:Analysis of Foxp3(+)epigenetics(-) T cells, Foxp3(-)epigenetics(+) T cells, and Foxp3(+)epigenetics(+) T cells. Results indicate regulatory T cell (Treg) ontogenesis requires two independent processes, expression of the transcription factor Foxp3 and establishment of Treg epigenetic programs induced by T cell receptor (TCR) stimulation. GFP+CD4+ and GFP-CD4+ splenocytes were sorted from DEREG and DEREG/Scurfy mice. These cells were activated with anti-CD3/CD28 antibodies, and then transduced with Foxp3-expressing retrovirus (pGCSamIN, NGFR marker). NGFR+ T cells sorted were subjected to microarray analysis (Affymetrix, mouse genome 430 2.0 array). To normalize the experimental conditions, Tregs (GFP+ T cells from DEREG) and Tconv (GFP- T cells from DEREG) were also activated and transduced with empty vector. Two replicates each.
Project description:As we clarified before, the FOXP3 gene is an X-linked tumor suppressor gene of both human and mouse. We also clarified that the ERBB2, SKP2 and p21 genes were transcriptionally under control of FOXP3 in human epithelial cells. In order to further clarify the FOXP3 down stream targets in human cancer cells, we conducted a microarray analysis of FOXP3-induced gene expression profiling. A human prostate cancer cell line, LNCaP, was transfected either with a FOXP3-eGFP expressing vector or an eGFP-expressing vector. After 48 hrs of cell culture, we isolated eGFP-positive LNCaP cells by FACS sorting, and then total RNA from those cells were extracted by Qiagen's RNeasy column and they were applied to Affymetrix Human U133 2.0 array according to the manufacture's protocol. We clarified as yet unknown FOXP3 target genes in human prostate epithelial cells by this analysis.
Project description:Subcutaneous tumors from mock transfected LLC or b8 transfected LLC were established on flanks of C57B/6 FoxP3-Ires GFP mice. Mice were treated with isotype control antibodies or anti-b8 and Tregs (CD4+ GFP+) were sorted, pooled and total RNA isolated for RNAseq.
Project description:Goals and objectives of this study: to identify genes of the Treg signature induced by consitutive expression of GARP or FOXP3 in antigen-specific Th cells with potential role for stabililization & maintenance of the regulatory program. Keywords: T-cell receptor stimulation, gene-regulation, comparative gene expression profiling, cell type comparison, human, regulatory T cells, FOXP3, GARP Human alloantigen-specific Treg cells (THU) and Th cells (CD4-39), established and described recently (Ocklenburg et al.. Lab Invest.2006; 86: 724-737), were sitmulated for 3 days with cognate antigen (EBV B cells) and IL2 as described (Ocklenburg et al.. Lab Invest.2006; 86: 724-737) and analyzed using human Affymetrix U133 2.0 in monoplicate. Th cells had been transduced with a retroviral vector containing human GARP (LRRC32) or FOXP3 and an IRES-driven GFP as marker or empty GFP control, sorted for GFP+, expanded as described recently (Ocklenburg et al.. Lab Invest.2006; 86: 724-737), and characterized functially, phenotypically, and genetically as described (WO/2007/113301). Abbreviations: Treg THU d3 = alloantigen-specific Treg cells; GFP d3 = GFP-transduced alloantigen-specific Th cells (CD4-39); FoxP3 d3 = FOXP-transduced alloantigen-specific Th cells (CD4-39); Garp d3 = GARP-transduced alloantigen-specific Th cells (CD4-39); all stimulated for 3 days (=d3) with EBV B cells and IL2.
Project description:miRNA expression profiling in highly purified murine CD4+ Tconv and Treg cells. FoxP3-GFP-hCre1a(high) reporter mice were used to separate both populations based on surface markers and presence or absence of GFP. Two-condition experiment, Tconv vs. Treg. Biological replicates: 1 Tconv, 1 Treg, purified from the same pooled mice. One replicate on 1 array.
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. This dataset shows expression data of resting and mitogen stimulated (PMA / ionomycin) retrovirally transduced Jurkat T cells either expressing FOXP3(M-NM-^T2) (J-FOXP3) or an empty vector control (J-GFP). Expression profile of resting and PMA/ionomycin stimulated J-GFP and J-FOXP3 cells was analyzed (one microarray per condition).
Project description:The colonic lamina propria contains a distinct population of Foxp3+ T regulatory cells (Tregs) that modulate responses to commensal microbes. Analysis of gene expression revealed that the transcriptome of colonic Tregs is distinct from splenic and other tissue Tregs. Rorγ and Helios in colonic Tregs mark distinct populations: Rorγ+Helios- or Rorγ-Helios+ Tregs. We uncovered an unanticipated role for Rorγ, a transcription factor generally considered to be antagonistic to Foxp3. Rorγ in colonic Tregs accounts for a small but specific part of the colon-specific Treg signature. (1) Total colonic and splenic Foxp3+ Treg comparison: Lymphocytes were isolated from colonic lamina propria and spleens of Foxp3-ires-GFP mice, where GFP reports Foxp3 expression. TCRb+CD4+GFP+ cells were double sorted into Trizol. (2) Colonic Rorγ+ and Rorγ- Treg comparison: Foxp3-ires-Thy1.1 reporter mice were crossed to Rorc-GFP reporter mice to generate mice that report both Foxp3 and Rorγ expression. Rorγ+Foxp3+ Tregs (TCRb+CD4+Thy1.1+GFP+) and Rorγ-Foxp3+ Tregs (TCRb+CD4+Thy1.1+GFP-) from colonic lamina propria were double sorted into Trizol.To reduce variability and increase cell number, cells from multiple mice were pooled for sorting and at least three replicates were generated for all groups. RNA from 1.5-3.0 x104 cells was amplified, labeled and hybridized to Affymetrix Mouse Gene 1.0 ST Arrays.
Project description:Purpose: The goals of this study were to identify preferential gene expression signatures that are unique to telogen skin resident Tregs relative to peripheral Tregs Methods: Tregs from telogen skin and SDLNs were purified by cell sorting (using the Treg GFP reporter mouse line Foxp3-DTR/GFP) to generate mRNA transcription profiles. Results: Transcriptional profiling revealed a unique Skin treg signature relative to SDLN Tregs Conclusion: Our study represents the first detailed analysis of the skin Treg transcriptome. mRNA profiles of skin and SDLN Tregs isolated from 6 week old Foxp3-DTR/GFP mice.
Project description:miRNA expression profiling in highly purified murine CD4+ Tconv and Treg cells. FoxP3-GFP-hCre1a(high) reporter mice were used to separate both populations based on surface markers and presence or absence of GFP.
Project description:Expression profiling of PC12 cells stably transfected with GFP, GFP-SH2-Bb, or GFP-SH2-Bb(R555E), treated with or without NGF for 6h Keywords: response to NGF