11 research outputs found

    Effect of induction with the chemokine CCL4 on signaling molecules.

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    <p><b>A</b> Th1 lymphocytes from TCR<sup>tg</sup>CD152-deficient and TCR<sup>tg</sup>CD152-competent mice were analyzed for ERK phosphorylation on day 2 and day 5 of a recall response. Signaling via CCR5 was induced in serum-starved lymphocytes by treatment with 20 nM CCL4 for 5 (heavy black line) or 20 min (heavy grey line). Filled histograms indicate ERK phosphorylation before CCL4 application. <b>B</b> Migration assay in Th1 lymphocytes from TCR<sup>tg</sup>CD152-deficient and TCR<sup>tg</sup>CD152-competent mice on day 5 of a recall response. A portion of the lymphocytes was incubated with the PKCθ-specific inhibitor Rottlerin for 20 h. <b>C</b> Western blot detection of signaling proteins in lysates of TCR<sup>tg</sup>CD152-deficient and TCR<sup>tg</sup>CD152-competent Th1 lymphocytes on day 5 of a recall response. The cells were treated as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031391#pone-0031391-g002" target="_blank">Fig. 2 A</a>. <b>D</b> The band intensity of pGRK2 relative to GRK2 was quantified using Image Gauge 4.0. Representative data from at least two experiments are shown.</p

    T Cells of Infants Are Mature, but Hyporeactive Due to Limited Ca<sup>2+</sup> Influx

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    <div><p>CD4 T cells in human infants and adults differ in the initiation and strength of their responses. The molecular basis for these differences is not yet understood. To address this the principle key molecular events of TCR- and CD28-induced signaling in naive CD4 T cells, such as Ca<sup>2+</sup> influx, NFAT expression, phosphorylation and translocation into the nucleus, ERK activation and IL-2 response, were analyzed over at least the first 3 years of life. We report dramatically reduced IL-2 and TNFα responses in naive CD31<sup>+</sup> T cells during infancy. Looking at the obligatory Ca<sup>2+</sup> influx required to induce T cell activation and proliferation, we demonstrate characteristic patterns of impairment for each stage of infancy that are partly due to the differential usage of Ca<sup>2+</sup> stores. Consistent with those findings, translocation of NFATc2 is limited, but still dependent on Ca<sup>2+</sup> influx as demonstrated by sensitivity to cyclosporin A (CsA) treatment. Thus weak Ca<sup>2+</sup> influx functions as a catalyst for the implementation of restricted IL-2 response in T cells during infancy. Our studies also define limited mobilization of Ca<sup>2+</sup> ions as a characteristic property of T cells during infancy. This work adds to our understanding of infants’ poor T cell responsiveness against pathogens.</p></div

    Frequencies of naive CD31<sup>+</sup> T cells within the CD4<sup>+</sup>CD45RA<sup>+</sup> compartment remain constant over ages.

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    <p>(A) T cell subsets of CB or PBMCs analyzed by flow cytometry. Representative flow cytometry dot plots and histograms are shown with the percentage of CD4<sup>+</sup>CD45RA<sup>+</sup> T cell subsets for CB (top), infant (middle; 2.8 months), and adult (bottom) samples. (B) Frequencies of CD4<sup>+</sup> among lymphocytes, (C) of CD45RA<sup>+</sup> among CD4<sup>+</sup> T cells, and (D) CD31<sup>+</sup> among peripheral CD4<sup>+</sup>CD45<sup>+</sup> cells are shown (* <i>P<</i>0.05; two-sided ANOVA Tukey-Kramer post-hoc test). The mean value and standard deviations (SD) are indicated. Age of infants and children is indicated in months. n = number of individuals.</p

    CD152-enhanced migration is IFNγ independent.

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    <p>CD4<sup>+</sup> OVA-specific TCR<sup>tg</sup> splenocytes from CD152<sup>−/−</sup> and CD152<sup>+/+</sup> were stimulated under Th1 conditions (as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0005702#pone-0005702-g002" target="_blank">Fig. 2</a>) in the presence of 20 ng/ml recombinant IFN-γ or 10 µg/ml blocking anti-IFN-γ -antibodies. On day 6 after inducing a recall response cells were analyzed for migration towards CCL4 in chemotaxis assays. The data represent one out of 3 similar experiments.</p