Transcription profiling of mouse Itk-deficient CD3+, CD4+ and CD8+ T-cells
Ontology highlight
ABSTRACT:
ORGANISM(S): Mus musculus
SUBMITTER: Emelie Blomberg
PROVIDER: E-GEOD-12466 | biostudies-arrayexpress |
REPOSITORIES: biostudies-arrayexpress
Similar Datasets
Project description:The Tec-family kinase Itk plays an important role during T-cell activation and function, and controls also conventional versus innate-like T-cell development. We have characterized the transcriptome of Itk-deficient CD3+ T-cells, including CD4+ and CD8+ subsets, using Affymetrix microarrays. The largest difference between Itk-/- and Wt CD3+ T-cells was found in unstimulated cells, e.g. for killer cell lectin-like receptors. Compared to anti-CD3-stimulation, anti-CD3/CD28 significantly decreased the number of transcripts suggesting that the CD28 co-stimulatory pathway is mainly independent of Itk. The signatures of CD4+ and CD8+ T-cell subsets identified a greater differential expression than in total CD3+ cells. Cyclosporin (CsA)-treatment had a stronger effect on transcriptional regulation than Itk-deficiency, suggesting that only a fraction of TCR-mediated calcineurin/NFAT-activation is dependent on Itk. Bioinformatic analysis of NFAT-sites of the group of transcripts similarly regulated by Itk-deficiency and CsA-treatment, followed by chromatin-immunoprecipitation, revealed NFATc1-binding to the Bub1, IL7R, Ctla2a, Ctla2b, and Schlafen1 genes. Finally, to identify transcripts that are regulated by Tec-family kinases in general, we compared the expression profile of Itk-deficient T-cells with that of Btk-deficient B-cells and a common set of transcripts was found. Taken together, our study provides a general overview about the global transcriptional changes in the absence of Itk. Experiment Overall Design: CD3+ T-cells from pooled suspensions of spleen and lymph nodes of Wt and Itk knockout mice on C57BL/6 background were isolated after negative depletion. Unstimulated as well as stimulated T-cells were studied. Stimulations were done with anti-CD3 (1 mg/ml) with or without anti-CD28 (3 mg/ml) in the presence or absence of CsA (1 mg/ml) for 24 hrs. For each stimulus, at least duplicate samples were used.
2009-05-29 | E-GEOD-12464 | biostudies-arrayexpress
Project description:The Tec-family kinase Itk plays an important role during T-cell activation and function, and controls also conventional versus innate-like T-cell development. We have characterized the transcriptome of Itk-deficient CD3+ T-cells, including CD4+ and CD8+ subsets, using Affymetrix microarrays. The largest difference between Itk-/- and Wt CD3+ T-cells was found in unstimulated cells, e.g. for killer cell lectin-like receptors. Compared to anti-CD3-stimulation, anti-CD3/CD28 significantly decreased the number of transcripts suggesting that the CD28 co-stimulatory pathway is mainly independent of Itk. The signatures of CD4+ and CD8+ T-cell subsets identified a greater differential expression than in total CD3+ cells. Cyclosporin (CsA)-treatment had a stronger effect on transcriptional regulation than Itk-deficiency, suggesting that only a fraction of TCR-mediated calcineurin/NFAT-activation is dependent on Itk. Bioinformatic analysis of NFAT-sites of the group of transcripts similarly regulated by Itk-deficiency and CsA-treatment, followed by chromatin-immunoprecipitation, revealed NFATc1-binding to the Bub1, IL7R, Ctla2a, Ctla2b, and Schlafen1 genes. Finally, to identify transcripts that are regulated by Tec-family kinases in general, we compared the expression profile of Itk-deficient T-cells with that of Btk-deficient B-cells and a common set of transcripts was found. Taken together, our study provides a general overview about the global transcriptional changes in the absence of Itk. Experiment Overall Design: CD3+ CD4+ and CD8+ T-cells from pooled suspensions of spleen and lymph nodes of Wt and Itk knockout mice on C57BL/6 background were isolated after negative depletion. Unstimulated as well as stimulated T-cells were studied. Stimulations were done with anti-CD3 (1 mg/ml) for 24 hrs. For the CD4+ T-cells we collected triplicates from the Itk knockout mice and duplicates from the Wt group. For the CD8+ T-cells, we got duplicates from Itk knockout , while we obtained a single sample from Wt owing to the low cell yield for resting Wt CD8+ T-cells. After CD3-stimulation we got a single sample from the CD8+ subset of both Wt and Itk knockout, while for the CD4+ subsets we collected duplicates.
2009-05-29 | E-GEOD-12465 | biostudies-arrayexpress
Project description:The Tec-family kinase Itk plays an important role during T-cell activation and function, and controls also conventional versus innate-like T-cell development. We have characterized the transcriptome of Itk-deficient CD3+ T-cells, including CD4+ and CD8+ subsets, using Affymetrix microarrays. The largest difference between Itk-/- and Wt CD3+ T-cells was found in unstimulated cells, e.g. for killer cell lectin-like receptors. Compared to anti-CD3-stimulation, anti-CD3/CD28 significantly decreased the number of transcripts suggesting that the CD28 co-stimulatory pathway is mainly independent of Itk. The signatures of CD4+ and CD8+ T-cell subsets identified a greater differential expression than in total CD3+ cells. Cyclosporin (CsA)-treatment had a stronger effect on transcriptional regulation than Itk-deficiency, suggesting that only a fraction of TCR-mediated calcineurin/NFAT-activation is dependent on Itk. Bioinformatic analysis of NFAT-sites of the group of transcripts similarly regulated by Itk-deficiency and CsA-treatment, followed by chromatin-immunoprecipitation, revealed NFATc1-binding to the Bub1, IL7R, Ctla2a, Ctla2b, and Schlafen1 genes. Finally, to identify transcripts that are regulated by Tec-family kinases in general, we compared the expression profile of Itk-deficient T-cells with that of Btk-deficient B-cells and a common set of transcripts was found. Taken together, our study provides a general overview about the global transcriptional changes in the absence of Itk.
2009-05-20 | GSE12464 | GEO
Project description:The Tec-family kinase Itk plays an important role during T-cell activation and function, and controls also conventional versus innate-like T-cell development. We have characterized the transcriptome of Itk-deficient CD3+ T-cells, including CD4+ and CD8+ subsets, using Affymetrix microarrays. The largest difference between Itk-/- and Wt CD3+ T-cells was found in unstimulated cells, e.g. for killer cell lectin-like receptors. Compared to anti-CD3-stimulation, anti-CD3/CD28 significantly decreased the number of transcripts suggesting that the CD28 co-stimulatory pathway is mainly independent of Itk. The signatures of CD4+ and CD8+ T-cell subsets identified a greater differential expression than in total CD3+ cells. Cyclosporin (CsA)-treatment had a stronger effect on transcriptional regulation than Itk-deficiency, suggesting that only a fraction of TCR-mediated calcineurin/NFAT-activation is dependent on Itk. Bioinformatic analysis of NFAT-sites of the group of transcripts similarly regulated by Itk-deficiency and CsA-treatment, followed by chromatin-immunoprecipitation, revealed NFATc1-binding to the Bub1, IL7R, Ctla2a, Ctla2b, and Schlafen1 genes. Finally, to identify transcripts that are regulated by Tec-family kinases in general, we compared the expression profile of Itk-deficient T-cells with that of Btk-deficient B-cells and a common set of transcripts was found. Taken together, our study provides a general overview about the global transcriptional changes in the absence of Itk.
2009-05-20 | GSE12465 | GEO
Project description:A microarray study was performed in unstimulated and TCR-stimulated CD4 + T cells and Treg in wild type and conditional Trim28 KO mice to identify genes that are regulated by Trim28. These experiments constitute a portion of the study described below: Paper Abstract: Peripheral T cell activation and differentiation into specialized effectors are regulated by TCR- and cytokine-mediated signals that induce clonal expansion and unique transcriptional factors. These processes may include active chromatin modification by nuclear factors. In search of such molecules, we found Trim28, a component of large nuclear chromatin-regulatory complex is tightly controlled upon TCR stimulation at the level of phosphorylation, and examined global impact of Trim28 loss in especially CD4+ T cells, by generating T cell-specific conditional Trim28 KO mice (CKO). CD4+ T cells from CKO mice showed defective IL-2 production and T cell proliferation associated with defective upregulation of cell-cycle associated proteins. Accordingly, young CKO showed T-lymphopenia. Surprisingly, Trim28 CKO mice eventually accumulated auto-reactive memory-phenotype T cells that produced inflammatory IL-17. CKO mice are also susceptible to induced auto-inflammatory disease with TH-17 dominant immune response. Loss of Trim28 showed aberrant accumulation of TH-17 and FoxP3+ T cells, two key T cells in inflammation vs. tolerance. We found CKO T cells showed a cell-extrinsic promotion of TH-17 and FoxP3+ T cell development by a mechanism involving overproduction of TGF-beta. Our study revealed unexpected roles of Trim28, a global chromatin regulator in both T cell activation and tolerance. Trim28 conditional KO mice and age-matched control mice were sacrificed, and neive CD4+ T cells (CD4+CD62+CD25-) and Treg (CD4+CD62+CD25+) were sorted. Stimulation of naive T cells was done with anti-CD3 and anti-CD28 for 13 hours. We collected quadruplicates for each group.
2012-04-28 | E-GEOD-32224 | biostudies-arrayexpress
Project description:Regulatory T cells (Tregs) play a critical role in controlling autoimmunity and limiting tissue destruction and inflammation. IL2-inducible T cell kinase (Itk) belongs to the Tec family of tyrosine kinases and is an important component of TCR-mediated signaling. Here we showed that either genetic ablation of Itk signaling, or inhibition of Itk signaling pathways resulted in increased frequency of “noncanonical” CD4+CD25−FoxP3+ Tregs (ncTregs), as well as of “canonical” CD4+CD25+FoxP3+ Tregs (canTregs). Using in vivo models, we showed that ncTregs can prevent the development of acute graft-versus-host disease (GVHD), in part by reducing conventional T cell proliferation, proinflammatory cytokine production, and tissue damage. This reduction in GVHD occurred without disruption of graft-versus-leukemia (GVL) effects. RNA sequencing revealed that a number of effector, cell adhesion, and migration molecules were upregulated in Itk-/- ncTregs. Furthermore, disrupting the SLP76: ITK interaction using a specific peptide inhibitor led to enhanced Treg development in both mouse cells and primary human cells. This peptide inhibitor also significantly reduced inflammatory cytokine production in primary GVHD patient samples and mouse T cells without causing cell death or apoptosis. We provide evidence that specifically targeting Itk signaling could be a therapeutic strategy to treat autoimmune disorders
2021-10-19 | GSE185327 | GEO
Project description:Transcriptional signatures of Itk-deficiency using CD3+ T-cells
| PRJNA114225 | ENA
Project description:We analyzed gene expression profiles of unstimulated Herpesvirus Saimiri (HVS) transformed T cells (CD4+) of patients harboring a homozygous R335W mutation in the IL-2 inducible T cell kinase (ITK) compared to healthy control HVS cells. We identified a set of 1927 Affymetrix probe sets showing significant misregulation in the ITK deficient cells (Welch's T-test, p<0.05). HVS T cell lines (ITK wt or ITK R335W, each CD4+) from three independent culturing time points were used for RNA extraction and hybridization on Affymetrix microarrays.
2011-12-20 | E-GEOD-28200 | biostudies-arrayexpress
Project description:Genomic aberrations are of predominant importance to the biology and clinical outcome of patients with chronic lymphocytic leukemia (CLL), and FISH-based genomic risk classifications are routinely used in clinical decision making in CLL. One of the known limitations of CLL FISH is the inability to comprehensively interrogate the CLL genome for genomic changes. In an effort at overcoming the existing limitations in CLL genome analysis, we have analyzed high-purity DNA isolated from FACS-sorted CD19+ cells and paired CD3+ or buccal cells from 255 CLL patients for acquired genomic copy number aberrations (aCNA) using ultra-high-density Affymetrix SNP 6.0 arrays. Overall, two or more subchromosomal aCNA were found in 39% (100/255) of all cases analyzed, while ≥3 subchromosomal aCNA were detected in 20% (50/255) of cases. Subsequently, we have correlated genomic lesion loads (genomic complexity) with the clinical outcome measures time to first therapy (TTFT) and overall survival (OS). Using multivariate analyses incorporating the most important prognostic factors in CLL together with SNP 6.0 array-based genomic lesion loads at various thresholds, we identify elevated CLL genomic complexity as an independent and powerful marker for the identification of CLL patients with aggressive disease and short survival. we have analyzed high-purity DNA isolated from FACS-sorted CD19+ cells and paired CD3+ or buccal cells from 255 CLL patients for acquired genomic copy number aberrations (aCNA) using ultra-high-density Affymetrix SNP 6.0 arrays
2011-11-29 | E-GEOD-30777 | biostudies-arrayexpress
Project description:Expanding the sequencing depth of the peptides showing a statistically significant quantitative change arising from a biological stimulation is critical. Here we demonstrate that optimization of LC gradient and analytical column construction can reveal over 30,000 unique peptides and 23,000 phosphopeptides at high confidence. The quantitative reproducibility of different analytical workflows was evaluated by comparing the phosphoproteome of CD3/4 stimulated and unstimulated T-cells as a model system. A fritless, 50 cm-long column packed with 1.9 µm particles operated with a standard pressure HPLC significantly improved the sequencing depth 51% and decreased the selected ion chromatogram peak spreading. Most importantly, under the optimal workflow we observed an improvement of 330% in detection of significantly changed phosphopeptides in the stimulated cells compared with the other workflows. The discovery power of the optimized column configuration was illustrated by identification of significantly altered phosphopeptides harboring novel sites from proteins previously established as important in T cell signaling including A-Raf, B-Raf, c-Myc, CARMA1, Fyn, ITK, LAT, NFAT1/2/3, PKCα, PLCγ1/2, RAF1, and SOS1. Taken together, our results revealed a simple column fabrication methodology that provides an inexpensive improvement for single-run LC-MS/MS analysis to optimize peptide sequencing depth, dynamic range, sensitivity, and label free quantitative reproducibility.
2018-10-26 | PXD005650 | Pride