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

<p>Abstract</p> <p>Background</p> <p>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.</p> <p>Results</p> <p>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 <it>Bub1</it>, <it>IL7R, Ctla2a</it>, <it>Ctla2b</it>, and <it>Schlafen1 </it>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.</p> <p>Conclusion</p> <p>Taken together, our study provides a general overview about the global transcriptional changes in the absence of Itk.</p

The protein tyrosine kinase Tec regulates a CD44highCD62L- Th17 subset

The generation of Th17 cells has to be tightly controlled during an immune response. In this study, we report an increase in a CD44 2 effector/memory Th17 populations

The transcription factor MAZR preferentially acts as a transcriptional repressor in mast cells and plays a minor role in the regulation of effector functions in response to FcεRI stimulation.

Mast cells are key players in type I hypersensitivity reactions in humans and mice and their activity has to be tightly controlled. Previous studies implicated the transcription factor MAZR in the regulation of mast cell function. To study the role of MAZR in mast cells, we generated a conditional Mazr allele and crossed Mazr (F/F) mice with the Vav-iCre deleter strain, which is active in all hematopoietic cells. MAZR-null BM-derived mast cells (BMMC) were phenotypically indistinguishable from wild-type BMMCs, although the numbers of IL-3 generated Mazr (F/F) Vav-iCre BMMCs were reduced in comparison to Mazr (F/F) BMMCs, showing that MAZR is required for the efficient generation of BMMC in vitro. A gene expression analysis revealed that MAZR-deficiency resulted in the dysregulation of 128 genes, with more genes up- than down-regulated in the absence of MAZR, indicating that MAZR acts as a transcriptional repressor in mast cells. Among the up-regulated genes were the chemokines Ccl5, Cxcl10, Cxcl12, the chemokine receptor Ccr5 and the cytokine IL18, suggesting an immunoregulatory role for MAZR in mast cells. Enforced expression of MAZR in mature Mazr-deficient BMMCs rescued the altered expression pattern of some genes tested, suggesting direct regulation of these genes by MAZR. Upon FcεRI stimulation, Mazr expression was transiently down-regulated in BMMCs. However, early and late effector functions in response to FcεRI-mediated stimulation were not impaired in the absence of MAZR, with the exception of IL-6, which was slightly decreased. Taken together, out data indicate that MAZR preferentially acts as a transcriptional repressor in mast cells, however MAZR plays only a minor role in the transcriptional networks that regulate early and late effector functions in mast cells in response to FcεRI stimulation

Histone deacetylase 1 controls CD4+ T cell trafficking in autoinflammatory diseases.

CD4+ T cell trafficking is a fundamental property of adaptive immunity. In this study, we uncover a novel role for histone deacetylase 1 (HDAC1) in controlling effector CD4+ T cell migration, thereby providing mechanistic insight into why a T cell-specific deletion of HDAC1 protects against experimental autoimmune encephalomyelitis (EAE). HDAC1-deficient CD4+ T cells downregulated genes associated with leukocyte extravasation. In vitro, HDAC1-deficient CD4+ T cells displayed aberrant morphology and migration on surfaces coated with integrin LFA-1 ligand ICAM-1 and showed an impaired ability to arrest on and to migrate across a monolayer of primary mouse brain microvascular endothelial cells under physiological flow. Moreover, HDAC1 deficiency reduced homing of CD4+ T cells into the intestinal epithelium and lamina propria preventing weight-loss, crypt damage and intestinal inflammation in adoptive CD4+ T cell transfer colitis. This correlated with reduced expression levels of LFA-1 integrin chains CD11a and CD18 as well as of selectin ligands CD43, CD44 and CD162 on transferred circulating HDAC1-deficient CD4+ T cells. Our data reveal that HDAC1 controls T cell-mediated autoimmunity via the regulation of CD4+ T cell trafficking into the CNS and intestinal tissues

Reduced mast cell numbers <i>in vitro</i> but normal mast cell homeostasis <i>in vivo</i>.

<div><p>(A) Diagram showing the cumulative numbers of c-kit⁺FcεRI⁺<i>Mazr</i>^F/F and <i>Mazr</i>^<i>F/F</i><i>Vav-iCre</i> BMMC over the course of 5 weeks of culture. Cells were counted by CASY counter, then percentages of c-kit⁺FcεRI⁺ BMMC among PI-negative cells (= alive) was determined by flow cytometry. The summary of three independent experiments with a total of 6 independent cell batches is shown. Mean ± SEM is shown.</p> <p>(B) Number of PI-negative (= alive) <i>Mazr</i>^F/F and <i>Mazr</i>^<i>F/F</i><i>Vav-iCre</i> BMMC during 5 days of IL-3 starvation. The summary of 3 experiments is shown. Mean ± SEM is shown.</p> <p>(C) Toluidine blue staining of paraffin-embedded 5 µm ear sections of <i>Mazr</i>^F/F and <i>Mazr</i>^<i>F/F</i><i>Vav-iCre</i> mice showing mast cells in pink/purple color (examples indicated by arrowheads). Diagram at the right indicates mean ± SEM of mast cell number per field of view (fov) calculated over 10 individual sections per ear (n=4). Magnification 20x.</p> <p>(D) Percentage of EYFP⁺c-kit⁺FcεRI⁺ mast cells from peritoneal lavage of wild-type (Mazr^F/+Rosa26^+/EYFPMcpt5Cre) and mast cell-specific MAZR-null (Mazr^F/FRosa26^+/EYFPMcpt5Cre) mice (n=8 and 9, respectively).</p></div

MAZR is not essential for the differentiation of BM-derived mast cells.

<div><p>(A) Diagram shows qRTPCR analysis of <i>Mazr</i> expression in thymus, CD4⁺ and CD8⁺ T cells and in IgE-primed BM-derived mast cells (BMMC). Expression levels are normalized to <i>Hprt</i> expression and levels in thymocytes were set as 1 (100%). Columns represent a summary of three independent samples. Mean ± SEM is shown.</p> <p>(B) Histograms depict expression of cell surface markers on <i>Mazr</i>^F/F and <i>Mazr</i>^<i>F/F</i><i>Vav-iCre</i> BMMC (after 5 weeks of culture). Filled gray areas are isotype control stainings. Data are representative of three independent experiments.</p> <p>(C) Flow cytometric analysis showing up-regulation of FcεRI levels in <i>Mazr</i>^F/F and <i>Mazr</i>^<i>F/F</i><i>Vav-iCre</i> BMMC. Filled gray areas are isotype control stainings. The solid black line shows the levels of cell-surface bound IgE after 15 min incubation. The dotted line shows the levels of cell-surface bound IgE after overnight priming. Data are representative of three independent experiments. </p> <p>(D) Toluidine blue staining of 5 week-cultured <i>Mazr</i>^F/F and <i>Mazr</i>^<i>F/F</i><i>Vav-iCre</i> BMMC prepared by a cytospin. Magnification 20x.</p></div

Minor defects in early and late mast cell effector functions in the absence of MAZR.

<div><p>(A) Diagram shows qRTPCR analysis of <i>Mazr</i> expression in resting anti-TNP IgE-primed BMMCs and in BMMCs activated for the indicated time points with TNP. Expression levels are normalized to <i>Hprt</i> expression and levels in IgE-primed non-activated mast cells were set as 1 (100%). Data show summary of three samples analyzed. Mean ± SEM is shown.</p> <p>(B) Plasma histamine levels in a systemic anaphylaxis model are shown. <i>Mazr</i>^F/F and <i>Mazr</i>^<i>F/F</i><i>Vav-iCre</i> mice were primed (i.v.) with anti-TNP IgE, challenged 24 hours later by i.v. injection of TNP or PBS. Serum was collected 2 minutes post-injection and histamine levels were determined by ELISA, n=7.</p> <p>(C) Absorbance (OD) of Evans Blue dye extravasated in a passive cutaneous anaphylaxis model from the ears of <i>Mazr</i>^F/F and <i>Mazr</i>^<i>F/F</i><i>Vav-iCre</i> mice is shown. Mice were injected with PBS and anti-TNP IgE into left and right ear, respectively, and 24 hours later mice were challenged by i.v. injection with TNP/Evans Blue dye. Extravasation of Evans Blue dye in the ear was measured 4 hours later. Diagram shows summary of 9 mice. Mean ± SEM is shown.</p> <p>(D) Anti-TNP-IgE-primed <i>Mazr</i>^F/F and <i>Mazr</i>^<i>F/F</i><i>Vav-iCre</i> BMMCs were activated for 10 min with TNP or PMA/ionomycin. β-Hexosaminidase release levels of <i>Mazr</i>^<i>F/F</i> BMMCs were set to 1 (n=10).</p> <p>(E) Anti-TNP-IgE-primed <i>Mazr</i>^F/F and <i>Mazr</i>^<i>F/F</i><i>Vav-iCre</i> BMMCs were activated for 60 min with TNP. LTB₄ levels were determined by ELISA. Mean ± SEM is shown. (n=4).</p> <p>(F) Flow cytometric analysis of calcium flux in anti-TNP-IgE-primed <i>Mazr</i>^F/F and <i>Mazr</i>^<i>F/F</i><i>Vav-iCre</i> BMMCs that have been activated with TNP. Data are representative of 3 independent experiments. </p> <p>(G) Cytokine production of anti-TNP-IgE-primed <i>Mazr</i>^F/F and <i>Mazr</i>^<i>F/F</i><i>Vav-iCre</i> BMMCs that were activated by plate-bound TNP for 24 hours (at least 5 independent mast cell batches were analyzed). Cytokine production of <i>Mazr</i>^<i>F/F</i> BMMCs was set to 1.</p></div