FYF-ITK mutant nucleofected cells display defective PLCγ1 phosphorylation.

<p><i>(A)</i>, Thymocytes isolated from ITK^−/− mice and nucleofected with cDNA constructs encoding GFP-tagged WT-, FYF-, or K390R-ITK fusion proteins, or mock-nucleofected were stimulated or not with anti-mouse CD3ε antibodies and then analyzed by flow cytometry using Alexa 647-conjugated anti-PLCγ1 pY783 antibodies as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0045158#s4" target="_blank">Materials and Methods</a> section. Results are displayed as cell number versus fluorescence intensity. In each panel the grey dotted line histograms represent mock-nucleofected cells that were not stimulated as negative controls for setting an electronic gate (vertical line) for calculation of positive cells. Histograms of non-stimulated, nucleofected or mock-nucleofected cells were similar. The black dotted line histograms represent K390R-ITK nucleofected cells that were stimulated. The solid black line histograms represent WT-ITK nucleofected (left panel) and FYF-ITK mutant nucleofected (right panel) cells that were stimulated. The table inset lists anti-PLCγ1 MFI of the displayed stimulated cell histograms. <i>(B),</i> Average (±SEM) percentage of pY783 positive cells of five replicate experiments (except K390R; two replicate experiments) performed and analyzed as in panel A, and normalized as percentage of the respective WT control (average±SEM 39.6±8.7%), as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0045158#s4" target="_blank">Materials and Methods</a> section. <i>(C)</i> Average (±SEM) anti-PLCγ1 MFI of same experiments normalized as in panel B. WT control average MFI (±SEM) 616±130. The percentages of Y783 phosphorylation and MFI in FYF-mutant and K390R-ITK nucleofected cells are significantly lower from WT-ITK at p<0.05 determined by the student’s t test. No significant differences from Mock transfected controls. The expression of the GFP-ITK constructs was comparable in all experimental groups. Average (±SEM) GFP fluorescence MFI was 1508±48 for WT-ITK, 1590±63 for FYF-ITK mutant, and 1274±104 for K390R-ITK.</p

TCR-induced localization and conformational changes of ITK at the T cell-APC contact site.

<p>Jurkat cells that had been transfected with CFP/YFP chimeric constructs of WT- or FYF- ITK were incubated with SEE-pretreated Raji cells (labeled with Cy5 ester) and then fixed on slides and analyzed by epifluorescence microscopy for ITK localization and FRET as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0045158#s4" target="_blank">Materials and Methods</a> section. <i>(A),</i> representative example of conjugate between Jurkat transfected with WT-ITK (yellow) and SEE-pretreated Raji cell (red) displaying ITK localization under no-stimulation (3 min at 4°C) conditions. <i>(B),</i> the Jurkat cell in (A) presented as a pseudocolored intensity map with look-up table. <i>(C)</i>, the same cell analyzed for FRET and presented as a pseudocolored intensity map of E_app (%) with look-up table. <i>(D)</i>, representative example of conjugate between Jurkat transfected with WT-ITK and SEE-pretreated Raji displaying ITK localization under stimulation (3 min at 37°C) conditions. <i>(E)</i>, the Jurkat cell in (D) presented as a pseudocolored intensity map with look-up table. <i>(F)</i>, the same cell analyzed for FRET and presented as a pseudocolored intensity map of E_app (%) with look-up table. <i>(G)</i>, representative example of conjugate between Jurkat transfected with FYF-ITK and SEE-pretreated Raji displaying ITK localization under stimulation (3 min at 37°C) conditions. <i>(H)</i>, the Jurkat cell in (G) presented as a pseudocolored intensity map with look-up table. <i>(I)</i>, the same cell analyzed for FRET and presented as a pseudocolored intensity map of E_app (%) with look-up table. Images of FYF-ITK transfected Jurkat under non-stimulation conditions are identical to the image shown in panel A (data not shown). White stars in the intensity map panels represent the relative position of Raji cells.</p

TCR-induced changes in E_app at the T cell-APC contact site.

<p><i>(A)</i>, Jurkat T cells transfected with WT-ITK were incubated with SEE-pretreated Raji cells and E_app at the center and periphery (average of right and left sides) of the contact site was assessed as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0045158#s4" target="_blank">Materials and Methods</a> section. Red and black dots represent the E_app at the periphery and center of the same conjugate, respectively. The values displayed are those of 55 representative conjugates out of 114 total. Differences in E_app at the periphery and center of the contact of each individual conjugate are significant (p<0.0001, paired student’s t test). <i>(B),</i> Jurkat cells transfected with FYF-ITK and treated as in (A). The values of 55 conjugates are displayed. Differences in E_app at the periphery and center of the contact of each individual conjugate are not significant (p = 0.5973, paired student’s t test). <i>(C),</i> Jurkat cells transfected with pYC control (construct containing only the two fluorescent proteins separated by a short linker) and treated as in (A). The values of 21 conjugates are displayed. Differences in E_app at the periphery and center of the contact of each individual conjugate are not significant (p = 0.6366, paired student’s t test).</p

FYF-ITK mutant is deficient in its ability to become phosphorylated upon TCR stimulation.

<p><i>(A)</i>, Jurkat cells transfected with cDNA constructs encoding CFP/YFP chimeric WT-, F26S- and FYF-ITK mutants were stimulated with anti-CD3ε (+) or isotype control (-) antibodies, lysed, and ITK (both transfected and endogenous) immuno-precipitated (IP) with anti-ITK antibodies. The immune complexes were resolved by SDS-PAGE, proteins transferred onto PVDF membranes, and immuno-blotted (IB) sequentially with anti-phosphotyrosine and anti-ITK antibodies as indicated. The upper sets of panels represent transfected and the lower sets endogenous ITK. Signals were developed by chemiluminescence. <i>(B)</i>, Bands from three replicate experiments (including the one displayed in panel A) performed as in (A) were quantified using ImageJ software and displayed as the percentage of transfected WT-ITK phosphorylation calculated as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0045158#s4" target="_blank">materials and methods</a> section. The * denotes that the difference between FYF-mutant and WT or F26S is statistically significant at p<0.05 determined by the student’s t test.</p

TCR-induced association of FYF-ITK mutant with SLP-76 and LAT is intact.

<p><i>(A),</i> Jurkat cells transfected with the indicated CFP/YFP chimeric ITK mutant constructs were stimulated with anti-CD3ε or isotype control antibodies, lysed, and immunoprecipitated (IP) with anti-SLP-76 antibodies. Immune complexes were resolved by PAGE, proteins transferred onto PVDF membranes, and immunoblotted (IB) sequentially with anti-ITK and anti-SLP-76 antibodies (loading control) as indicated. <i>(B)</i>, lysates of cells similarly transfected, stimulated, and lysed were immunoprecipitated with anti-LAT antibodies and immune complexes resolved and sequentially immunoblotted with anti-ITK and anti-LAT antibodies. Bands were visualized by chemiluminescence as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0045158#s4" target="_blank">Materials and Methods</a> section. Results are representative of three replicate experiments with the exception of F26S that represents a single experiment.</p

Cumulative localization data of ITK at the T cell-APC contact site.

<p>Conjugates between Raji and Jurkat cells transfected with WT-ITK, FYF-ITK, or pYC (construct containing only the two fluorescent proteins separated by a short linker) were treated as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0045158#pone-0045158-g006" target="_blank">Figure 6</a> (A, D, G) and ITK localization was assessed as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0045158#s4" target="_blank">Materials and Methods</a>. Results are displayed as the average Localization Index (±SEM) for each transfectant calculated as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0045158#s4" target="_blank">Materials and Methods</a> section. WT/NS denotes non-stimulation conditions as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0045158#pone-0045158-g006" target="_blank">Figure 6A</a>. The number of conjugates analyzed in each group are WT, N = 129; FYF, N = 55; pYC, N = 21; WT/NS, N = 10. The * indicates p<0.05 between WT and the rest of the groups determined by the student’s t test. FYF-ITK is also significantly different from pYC at p<0.05.</p

FYF-mutant ITK fails to bind PIP₃.

<p>Whole cell lysates (WCL) obtained from HEK-293T cells transfected with untagged WT- or FYF-ITK mutant were resolved by SDS-PAGE and analyzed by immunoblotting with anti-ITK antibodies (left panels). An aliquot of each lysate was incubated with PIP₃-coated beads followed by washing, elution, and SDS-PAGE/immunoblot analysis for ITK content (right panels). Results are representative of three independent experiments.</p

Deficient IL-4 production by FYF-ITK mutant nucleofected T cells.

<p><i>(A),</i> Culture supernatants of murine <i>ITK</i>^−/− thymocytes that had been nucleofected with the indicated GFP-ITK cDNA constructs or mock nucleofected (M) and stimulated under Th₂ skewing conditions (as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0045158#s4" target="_blank">Materials and Methods</a>) were assessed for their IL-4 content by ELISA. Results are displayed as the averages (±SEM) of three replicate experiments. Non-stimulated culture supernatants of WT-ITK nucleofected cells contained an average of 69 pg IL-4 per ml. The WT-ITK values are significantly different from either K390R or FYF at p<0.05 as determined by the student’s t test. The expression of the GFP-ITK constructs was comparable in all experimental groups. Average GFP-MFI was 1509 for WT-ITK, 1655 for FYF-ITK, and 1325 for K390R-ITK. <i>(B),</i> Anti-ITK immune complexes from lysates of HEK-293T cells that had been transfected with the indicated untagged ITK constructs and then subjected to <i>in vitro</i> kinase assay were resolved by SDS-PAGE followed by sequential immunoblotting with anti-pY and anti-ITK antibodies. Data are representative of two replicate experiments.</p

Different molecular interactions in models M1–M7 produce different temporal profiles of PIP₃ binding to Itk.

<p>(<b>A</b>) Kinetics of PIP₃ association of Itk for fixed initial PIP₃ 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>_p), peak width (<i>τ</i>_w), and peak value or amplitude (<i>A</i>). The dimensionless asymmetry ratio <i>R</i> = <i>τ</i>_w/<i>τ</i>_p 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 PIP₃. 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.

<p>(A) Immunoblots showing Y₇₈₃-phosphorylated (upper panels) and total (lower panels) PLCγ1 protein amounts in <i>RAG2^−/−MHC^−/− 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_KL 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 (Itk⁰ = 140 molecules) and PIP₃ concentrations (PIP₃⁰ = 530 molecules), representing high-affinity OVA stimulation for <i>τ</i>_p = 2 min and <i>A</i> (shown as <i>A</i>_avg) = 40 molecules. Note we use <i>A</i> to represent the amplitude <i>A</i>^expt in experiments measuring fold change in Itk phosphorylation (see the main text for further details). The vertical orange bar indicates R<i>^expt</i> for OVA. Color legend in (D). (D) The color map shows which model is most robust (has the lowest D_KL) as <i>R^expt</i> and <i>A</i> (shown as <i>A</i>_avg) are varied for the same parameters as in (C). The color legend is depicted on the right.</p