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The Tec kinase ITK optimizes differential NFAT, NF-κB, and Erk1/2 signaling to regulate graded gene induction in mouse CD8+ T cells [RNA-seq]

ABSTRACT: The Tec kinase ITK optimizes differential NFAT, NF-κB, and Erk1/2 signaling to regulate graded gene induction in mouse CD8+ T cells [RNA-seq]

PROVIDER: PRJNA704176 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR13766623_1.fastq.gz	Fastqsanger.gz
	SRR13766623_2.fastq.gz	Fastqsanger.gz
	SRR13766624_1.fastq.gz	Fastqsanger.gz
	SRR13766624_2.fastq.gz	Fastqsanger.gz
	SRR13766625_1.fastq.gz	Fastqsanger.gz

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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.

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The Tec kinase ITK optimizes differential NFAT, NF-κB, and Erk1/2 signaling to regulate graded gene induction in mouse CD8+ T cells [RNA-seq]

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