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

Macropinocytosis Is the Principal Uptake Mechanism of Antigen-Presenting Cells for Allergen-Specific Virus-like Nanoparticles

Virus-like nanoparticles (VNP) are regarded as efficient vaccination platforms and have proven to be useful for the non-anaphylactogenic delivery of allergen-specific immunotherapy in preclinical models previously. Herein, we sought to determine the mode of VNP uptake by antigen presenting cells (APC). Accordingly, we screened a collection of substances known to inhibit different uptake pathways by APC. The human leukemia monocytic cell line THP-1 and the murine dendritic cell line DC 2.4 were examined for the uptake of fluorescently labelled VNP in the presence or absence of inhibitors. The inhibitory effect of candidate substances that blocked VNP uptake in APC lines was subsequently evaluated in studies with primary APC present in splenocyte and lung cell homogenates in vitro and upon intratracheal application of VNP in vivo. The uptake of allergen-specific VNP in vitro and in vivo was mainly observed by macrophages and CD103+ dendritic cells and was sensitive to inhibitors that block macropinocytosis, such as hyperosmolarity induced by sucrose or the polyphenol compound Rottlerin at low micromolar concentrations but not by other inhibitors. Also, T-cell proliferation induced by allergen-specific VNP was significantly reduced by both substances. In contrast, substances that stimulate macropinocytosis, such as Heparin and phorbol myristate acetate (PMA), increased VNP-uptake and may, thus, help modulate allergen-specific T-cell responses. We have identified macropinocytosis as the principal uptake mechanism of APC for allergen-specific VNP in vitro and in vivo, paving the way for further improvement of VNP-based therapies, especially those that can be used for tolerance induction in allergy, in the future

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

The corepressor NCOR1 regulates the survival of single-positive thymocytes

Nuclear receptor corepressor 1 (NCOR1) is a transcriptional regulator bridging repressive chromatin modifying enzymes with transcription factors. NCOR1 regulates many biological processes, however its role in T cells is not known. Here we show that Cd4-Cre-mediated deletion of NCOR1 (NCOR1 cKO(Cd4)) resulted in a reduction of peripheral T cell numbers due to a decrease in single-positive (SP) thymocytes. In contrast, double-positive (DP) thymocyte numbers were not affected in the absence of NCOR1. The reduction in SP cells was due to diminished survival of NCOR1-null postselection TCR beta(hi)CD69(+) and mature TCR beta(hi)CD69(-) thymocytes. NCOR1-null thymocytes expressed elevated levels of the pro-apoptotic factor BIM and showed a higher fraction of cleaved caspase 3-positive cells upon TCR stimulation ex vivo. However, staphylococcal enterotoxin B (SEB)-mediated deletion of V beta 8(+) CD4SP thymocytes was normal, suggesting that negative selection is not altered in the absence of NCOR1. Finally, transgenic expression of the pro-survival protein BCL2 restored the population of CD69(+) thymocytes in NCOR1 cKO(Cd4) mice to a similar percentage as observed in WT mice. Together, these data identify NCOR1 as a crucial regulator of the survival of SP thymocytes and revealed that NCOR1 is essential for the proper generation of the peripheral T cell pool

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