Dataset Information

Transcriptional signatures of Itk-deficient CD3+, CD4+ and CD8+ T-cells.

ABSTRACT: 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 A (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.

SUBMITTER: Blomberg KE

PROVIDER: S-EPMC2689280 | biostudies-literature | 2009 May

REPOSITORIES: biostudies-literature

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Transcriptional signatures of Itk-deficient CD3+, CD4+ and CD8+ T-cells.

Blomberg K Emelie M KE Boucheron Nicole N Lindvall Jessica M JM Yu Liang L Raberger Julia J Berglöf Anna A Ellmeier Wilfried W Smith Ci Edvard CE

BMC genomics 20090518

<h4>Background</h4>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.<h4>Results</h4>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 ...[more]

PMID: 19450280

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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.

Project description:T-cell activation is an essential step of the immune response and relies on the tightly controlled orchestration of hundreds of genes/proteins, yet the cellular and molecular events underlying this complex process are not fully understood, especially at the genome-scale. Significantly, a comparative genome-scale transcriptional analysis of two T-cell subsets (CD4+ and CD8+) against each other and against the naturally mixed population (CD3+ cells) remains unexplored.Comparison of the microarray-based gene expression patterns between CD3+ T cells, and the CD4+ and CD8+ subsets revealed largely conserved, but not identical, transcriptional patterns. We employed a Gene-Ontology-driven transcriptional analysis coupled with protein abundance assays in order to identify novel T-cell activation genes and cell-type-specific genes associated with the immune response. We identified potential genes involved in the communication between the two subsets (including IL23A, NR4A2, CD83, PSMB2, -8, MIF, IFI16, TNFAIP1, POU2AF1, and OTUB1) and would-be effector-function-specific genes (XCL2, SLAMF7, TNFSF4, -5, -9, CSF3, CD48 and CD244). Chemokines induced during T-cell activation, but not previously identified in T cells, include CCL20, CXCL9, -10, -11 (in all three populations), and XCL2 (preferentially in CD8+ T cells). Increased expression of other unexpected cytokines (GPI, OSM and MIF) suggests their involvement in T-cell activation with their functions yet to be examined. Differential expression of many receptors, not previously reported in the context of T-cell activation, including CCR5, CCR7, IL1R2, IL1RAP, IL6R, TNFRSF25 and TNFRSF1A, suggests their role in this immune process. Several receptors involved in TCR activation (CD3D, CD3G, TRAT1, ITGAL, ITGB1, ITGB2, CD8A and B (CD8+ T-cell specific) along with LCK, ZAP70 and TYROBP were synchronously downregulated. Members of cell-surface receptors (HLA-Ds and KLRs), none previously identified in the context of T-cell activation, were also downregulated.This comparative genome-scale, transcriptional analysis of T-cell activation in the CD4+ and CD8+ subsets and the mixed CD3+ populations made possible the identification of many immune-response genes not previously identified in the context of T-cell activation. Significantly, it made possible to identify the temporal patterns of many previously known T-cell activation genes, and also identify genes implicated in effector functions of and communication between CD4+ and CD8+ T cells.

Dataset Information

Transcriptional signatures of Itk-deficient CD3+, CD4+ and CD8+ T-cells.

Publications

Transcriptional signatures of Itk-deficient CD3+, CD4+ and CD8+ T-cells.

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