12 research outputs found

    Assay specificity and possible additional analyses.

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    <p>(A) ActCasp3 staining (left) and side scatter signal (right) in the DP gate with and without A2 tetramer stimulation for 24 h in the presence or absence of caspase inhibitor Z-VAD (OMe)-FMK. (B) Simultaneous stimulation of PD670-labeled and unlabeled cells in the same well with K<sup>b</sup>-Q7 tetramer for 24 h. ActCasp3 staining in the PD670 positive and negative DP gate with and without stimulation with tetramer for 24 h. (C) PD670 signal (X axis) in the DP gate with and without stimulation with A2, Q7, G4, and E1 tetramers for 24 h. (D) Vα2 (left) and CD69 (middle) staining in the DP gate with and without stimulation with A2, Q7, G4, and E1 tetramers for 24 h. Tetramer-PE signal (right) in ActCasp3<sup>+</sup> and ActCasp3<sup>−</sup> DP gate compared with that of non-treated B6 DP thymocytes. (E) Vα2 (left), CD69 (middle) and tetramer-PE (right) signals in the DP gate with and without stimulation with K<sup>b</sup>-A2 tetramer for 24 h in the presence or absence of caspase inhibitor Z-VAD (OMe)-FMK.</p

    Alternative gating strategies.

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    <p>(A) ActCasp3 (Y axis) and Vα2 staining (X axis) in the DP gate with and without stimulation with anti-CD3/28 beads or E1 and A2 H-2K<sup>b</sup> tetramers for 24 h. (B) ActCasp3 (Y axis) and side scatter signal (X axis) in the DP gate with and without stimulation with anti-CD3/28 beads or E1 and A2 tetramers for 24 h. (C) ActCasp3 (Y axis) and forward scatter signal (X axis) in the DP gate with and without stimulation with anti-CD3/28 beads or E1 and A2 tetramers for 24 h.</p

    Development of triple-transgenic cells in hosts lacking MHC class I and II yields TCR<sup>int</sup>, single α-chain thymocytes.

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    <p>(A) Bone marrow from B6 mice was used to reconstitute irradiated CD45.1 (top) or MHC<sup>o/o</sup> (bottom) hosts. After eight weeks, thymocytes were harvested and analyzed for expression of either α-chain in TCR<sup>lo</sup>, TCR<sup>int</sup>, and TCR<sup>hi</sup>, Vβ5-positive gates. (B) Bone marrow from triple-transgenic mice was used to reconstitute irradiated CD45.1 (top) or MHC<sup>o/o</sup> (bottom) hosts. After eight weeks, thymocytes were harvested and analyzed for expression of either α-chain in TCR<sup>lo</sup>, TCR<sup>int</sup>, and TCR<sup>hi</sup>, Vβ5-positive gates. FACS plots representative of 7-9 mice per group in three independent experiments.</p

    Detection of dual Vα expressing cells in subsets gated based on Vα2-positive or Vα3.2-positive populations.

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    <p>Cells positive for subunits indicated above each plot were analyzed for the expression of the other subunit. Gates were set based on total lymphocyte population (gray lines). Top panel represents detection of dual Vα expressing cells by surface staining, bottom panel by intracellular staining. Results are representative of six mice per genotype.</p

    Vβ5 transgene skews expression of Vα2 from CD4 to CD8 subset, but Vα3.2 skews to CD8s with or without Vβ5 transgene.

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    <p>(A) LN cells from B6 mice (WT), <i>Tcra</i><sup>−/−</sup> mice transgenic for Vα2 and Vα3.2 miniloci, with or without transgenic rearranged Vβ5, were gated for Vα2 or Vα3.2 expression to show the assortment of cells to CD4 or CD8 subpopulations. FACS plots representative of >10 mice per genotype. (B) shows the CD8∶CD4 ratio of peripheral T cells from mouse strains expressing all the potential combinations of transgenes used here: Vα2 and Vα3.2 miniloci individually or together, and rearranged Vβ5 transgene. Data represent 8–17 mice per genotype.</p

    Vα3.2-expressing T cells are more frequent than Vα2-expressing cells in dual α-chain minilocus mice.

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    <p>(A) Vα2, Vα3.2 and Vβ5 expression in lymph node T cells from B6 mice (WT), <i>Tcra</i><sup>−/−</sup> mice transgenic for Vα2 or Vα3.2 miniloci, <i>Tcra</i><sup>−/−</sup> mice transgenic for both Vα2 and Vα3.2 miniloci, and <i>Tcra</i><sup>−/−</sup> mice transgenic for Vα2 and Vα3.2 miniloci plus the rearranged Vβ5 gene. FACS plots representative of >10 mice per genotype. (B) Ratio of Vα3: Vα2-bearing T cells in CD4 and CD8 peripheral T cell subsets in the mice strains described in (A). Data represent >10 mice per genotype.</p

    Triple-transgenic thymocytes exhibit efficient allelic exclusion of TCR Vα chains.

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    <p>(A) Double-negative (DN) and CD4 and CD8 single positive (SP) thymocytes from B6 (top), Vα2 Vα3.2 (middle) and Vα2 Vα3.2 Vβ5 (bottom) mice were analyzed for surface expression of Vα2, Vα3.2 and Vβ5 in TCR<sup>int</sup> and TCR<sup>hi</sup> gate. (B) DN and CD4 and CD8 SP thymocytes from B6 (top), Vα2 Vα3.2 (middle) and Vα2 Vα3.2 Vβ5 (bottom) mice were analyzed for intracellular expression of Vα2, Vα3.2 and Vβ5 in TCR<sup>int</sup> and TCR<sup>hi</sup> gate. FACS plots representative of >10 mice per genotype and are derived from the same dataset as <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0114320#pone-0114320-g004" target="_blank">Fig. 4</a>.</p

    Different molecular interactions in models M1–M7 produce different temporal profiles of PIP<sub>3</sub> binding to Itk.

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    <p>(<b>A</b>) Kinetics of PIP<sub>3</sub> association of Itk for fixed initial PIP<sub>3</sub> and Itk concentrations (100 and 370 molecules, respectively) in models with feedbacks (M1–M4, and M7, left panel) and no feedbacks (M5–M6, right panel). (B) The shapes of the temporal profiles can be characterized by the parameters peak time (<i>τ</i><sub>p</sub>), peak width (<i>τ</i><sub>w</sub>), and peak value or amplitude (<i>A</i>). The dimensionless asymmetry ratio <i>R</i> = <i>τ</i><sub>w</sub>/<i>τ</i><sub>p</sub> quantifies how symmetric the shape of the time profile is. A larger R value indicates larger asymmetry. (C) Variations in R in models M1–M7 for different initial concentrations of Itk and PIP<sub>3</sub>. Color scales for R values are shown on the right of each panel.</p

    Experimentally measured PLCγ1activation kinetics in DP thymocytes stimulated with TCR ligands of different affinities and robustness of <i>in silico</i> models.

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    <p>(A) Immunoblots showing Y<sub>783</sub>-phosphorylated (upper panels) and total (lower panels) PLCγ1 protein amounts in <i>RAG2<sup>−/−</sup>MHC<sup>−/−</sup> OT1 TCR-transgenic</i> DP thymocytes stimulated for the indicated times with MHCI tetramers presenting the indicated altered peptide ligands (APL). (B) Phospho-PLCγ1 levels normalized to total PLCγ1 protein amounts plotted over time for the indicated APLs. Their TCR affinity decreases in the order OVA (black)>Q4R7 (red)>Q4H7 (blue)>G4 (green). Band intensities were quantified via scanning and analysis with <i>ImageJ</i> software. Representative of several independent experiments. (C) Variation of the Kulback-Leibler distance D<sub>KL</sub> with <i>R</i> for models M1–M3 (blue, red and black, respectively), M7 (yellow), and M4–M6 (orange, purple, and maroon, respectively) at high initial Itk (Itk<sup>0</sup> = 140 molecules) and PIP<sub>3</sub> concentrations (PIP<sub>3</sub><sup>0</sup> = 530 molecules), representing high-affinity OVA stimulation for <i>τ</i><sub>p</sub> = 2 min and <i>A</i> (shown as <i>A</i><sub>avg</sub>) = 40 molecules. Note we use <i>A</i> to represent the amplitude <i>A</i><sup>expt</sup> in experiments measuring fold change in Itk phosphorylation (see the main text for further details). The vertical orange bar indicates R<i><sup>expt</sup></i> for OVA. Color legend in (D). (D) The color map shows which model is most robust (has the lowest D<sub>KL</sub>) as <i>R<sup>expt</sup></i> and <i>A</i> (shown as <i>A</i><sub>avg</sub>) are varied for the same parameters as in (C). The color legend is depicted on the right.</p
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