miR-20a regulates cytokine production.

<p>Human naïve CD4⁺ T cells were transfected with plasmids encoding either miR-20a or miR-control (miR-Ctrl). 16 h after transfection, cells were stimulated with plate-bound CD3 and CD28. 48 h after stimulation, supernatants were collected and cytokines were measured by ELISA. Graphs in <b>(A)</b> show relative cytokine levels from miR-20a overexpressing cells compared to miR-control. Graphs in <b>(B)</b> show the absolute values of cytokine concentration, in pg/mL, from individual experiments. Data expressed as arbitrary units ± SEM of at least 4 independent experiments. Significat <i>P</i> values were done by using Student’s <i>t</i> Test. (*, <i>P</i> < 0.05; **, <i>P</i> < 0.01; ***, <i>P</i> < 0.001).</p

miR-20a inhibits early T-cell activation events.

<p>Human naïve CD4⁺ T cells were transfected with either miR-20a or miR- control expression plasmids. <b>(A)</b> 16h after transfection, cells were stimulated with immobilized CD3xCD28 mAbs for 6 hours and stained with an APC-conjugated CD69 mAb. Surface expression of CD69 was measured by flow cytometry. One representative experiment of 4 is shown. Graph in (<b>B</b>) represents relative mean fluorescent intensity (MFI) of CD69 expression of miR-20a overexpressing cells compared to miR-control. <b>(C)</b> Alexa Fluor 700 labelled cells were either left unstimulated or stimulated with platebound CD3xCD28 antibodies for 60 hours. Proliferation of cells was then analysed by flow cytometry by measuring the dilution of the fluorochrome by gating on GFP+ cells. Data represent arbitrary units ± SEM of at least 3 independent experiments. Significat <i>P</i> values were done by using Student’s <i>t</i> Test. (***, <i>P</i> < 0.001).</p

Expression of miR-20a and miR-20a precursors in human naïve CD4⁺ T cells.

<p>Human naïve CD4⁺ T cells were stimulated with plate-bound CD3 and CD28 mAbs for the indicated time periods. Quantification of <b>(A)</b> miR-20a and <b>(B)</b> miR-20a precursors (both pri- and pre-miRNAs) was performed using RT qPCR. <b>(C)</b> Naïve CD4⁺ T cells were also preincubated with DMSO, U0126 (10 μM), IKK VII (200 nM) and EGTA (10 mM) for 30 minutes. Subsequently, samples were either left unstimulated or stimulated with plate-bound CD3 for 4 hours at 37°C. Expression of miR-20a precursors was quantified using RT qPCR. <b>(D)</b> Quantification of miR-20a level upon overexpression. Human naïve CD4⁺ T cells were transfected with plasmids encoding either miR-20a or miR-control (miR-Ctrl). Cells were either left unstimulated or stimulated for 2 hours with CD3xCD28 mAbs. Quantification of miR-20a in both resting and activated cells was performed using real-time PCR. Unless otherwise mentioned, all the data was normalized to either resting cells or unstimulated control transfected cells. Data represent arbitrary units ± SEM of at least 3 (A, C, D) and 2 (B) independent experiments. Significant P values were done by using Student’s t Test. (*, P < 0.05; **, P < 0.01; ***, P < 0.001).</p

miR-20a does not affect the expression of signaling molecules and CD3 and CD28 receptors.

<p><b>(A)</b> Lysates from T cells transfected with either miR-20a or miR-control were prepared and analyzed by immunoblotting using the indicated Abs. (B) CD4⁺ T cells overexpressing either miR-20a or miR-control cells were stained with antibodies against CD3 and CD28 and analyzed by flow cytometry. Histograms overlay show the expression of CD3 (left) and CD28 (right) in miR-20a versus miR-control transfected cells.</p

miR-20a inhibits TCR-mediated signaling.

<p>Human naïve CD4⁺ T cells were transfected with plasmids encoding either miR-20a or miR-control (miR-Ctrl) and cultered for 16 hours. <b>(A)</b> Cells were stimulated with microbeads coated with CD3 and CD28 mAbs (iAbs) for the indicated time periods. Subsequently, lysates were analyzed by immunoblotting using the indicated Abs. Bands in <b>(A)</b> were quantified using the ImageQuant software and values were normalized to the corresponding β-actin signal. Graphs in <b>(B)</b> show the phosphorylation levels of the indicated molecules as arbitrary units ± SEM of at least 4 independent experiments. <b>(C)</b> CD4⁺ T cells were incubated with Indo-1AM, stimulated with CD3 and CD28 mAbs, and Ca⁺⁺ flux was measured by flow cytometry. Ionomycin is used to induce maximum Ca⁺⁺ flux. Graph in <b>(D)</b> shows quantification of Ca⁺⁺ flux expressed as arbitrary units ± SEM of at least 3 independent experiments. <b>(E)</b> CD4⁺ T cells were stimulated with iAbs for the indicated time periods. Subsequently, cell lysates were prepared and CD3ζ immunoprecipitations were analyzed by immunoblotting using the indicated Abs. Bands in <b>(E)</b> were quantified as described above. Graph in <b>(F)</b> shows the levels of CD3ζ-associated Zap-70 as arbitrary units ± SEM of 2 independent experiments. Significat <i>P</i> values were done by using Student’s <i>t</i> Test. (*, <i>P</i> < 0.05; **, <i>P</i> < 0.01; ***, <i>P</i> < 0.001).</p

Inhibition of miR-20a slightly increases TCR-mediated signaling.

<p>Human naïve CD4⁺ T cells were transfected with miR-20a or scramble decoys. <b>(A)</b> Quantification of miR-20a expression in resting and in stimulated cells upon stimulation with plate-bound CD3 and CD28 mAbs for 45 min was performed using RT qPCR. <b>(B)</b> 16 hours after transfection, cells were stimulated with microbeads coated with CD3 and CD28 mAbs for the indicated time periods. Subsequently, lysates were analyzed by immunoblotting using the indicated Abs. Bands in <b>(B)</b> were quantified using the ImageQuant software and values were normalized to the corresponding β-actin signal. Graphs in <b>(C)</b> show the phosphorylation levels of the indicated molecules as arbitrary units ± SEM of 3 independent experiments. Significat P values were calculated by using Student’s t Test. (*, P < 0.05).</p

Flow cytometry analysis of γH2AX-stained DNA double-strand breaks.

<p>Mean γH2AX intensity was assessed in PBMCs immediately, 1 h and 20 h after indicated exposure conditions. (A) Representative overlay histogram of γH2AX-intensity 1 h after indicated exposure (black line) and of corresponding control (gray line). (B) Difference of mean fluorescence intensity (MFI) of γH2AX and IgG-isotype control staining from 16 independent experiments at three different time points after exposure as mean ± SEM (***: P ≤ 0.001; ns: P > 0.05).</p

Downregulation of SIT results in an enhanced TCR-mediated signaling in primary human T cells.

<p>A) Primary human T cells were transfected with control or SIT-specific siRNA and 72 h later were stimulated with non-crosslinked CD3 (clone OKT3) mAb for the indicated times. Cell lysates were analyzed by immunoblotting using phosphospecific antibodies for ZAP-70, PLCγ-1, and Akt. Immunoblots verifying SIT downregulation are shown. B) Phosphorylated bands were quantified using the ImageQuant software and values were normalized to the corresponding β-actin signal. Graphs show the phosphorylation levels of ZAP-70, PLCγ-1, and Akt shown as of arbitrary units ± SEM of 2, 4, and 6 independent experiments, respectively. Statistical significance in B) * p<0.05 and **p<0.01.</p

Analysis of DNA Double-Strand Breaks and Cytotoxicity after 7 Tesla Magnetic Resonance Imaging of Isolated Human Lymphocytes

<div><p>The global use of magnetic resonance imaging (MRI) is constantly growing and the field strengths increasing. Yet, only little data about harmful biological effects caused by MRI exposure are available and published research analyzing the impact of MRI on DNA integrity reported controversial results. This in vitro study aimed to investigate the genotoxic and cytotoxic potential of 7 T ultra-high-field MRI on isolated human peripheral blood mononuclear cells. Hence, unstimulated mononuclear blood cells were exposed to 7 T static magnetic field alone or in combination with maximum permissible imaging gradients and radiofrequency pulses as well as to ionizing radiation during computed tomography and γ-ray exposure. DNA double-strand breaks were quantified by flow cytometry and automated microscopy analysis of immunofluorescence stained γH2AX. Cytotoxicity was studied by CellTiter-Blue viability assay and [³H]-thymidine proliferation assay. Exposure of unstimulated mononuclear blood cells to 7 T static magnetic field alone or combined with varying gradient magnetic fields and pulsed radiofrequency fields did not induce DNA double-strand breaks, whereas irradiation with X- and γ-rays led to a dose-dependent induction of γH2AX foci. The viability assay revealed a time- and dose-dependent decrease in metabolic activity only among samples exposed to γ-radiation. Further, there was no evidence for altered proliferation response after cells were exposed to 7 T MRI or low doses of ionizing radiation (≤ 0.2 Gy). These findings confirm the acceptance of MRI as a safe non-invasive diagnostic imaging tool, but whether MRI can induce other types of DNA lesions or DNA double-strand breaks during altered conditions still needs to be investigated.</p></div

T-cell subsets in lymph nodes.

<p>% of lymph node cells ± SEM. n = number of examined mice.</p