Article

Itk controls the spatiotemporal organization of T cell activation

Department of Immunology, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA.
Science Signaling (Impact Factor: 7.65). 10/2011; 4(193):ra66. DOI: 10.1126/scisignal.2001821
Source: PubMed

ABSTRACT During T cell activation by antigen-presenting cells (APCs), the diverse spatiotemporal organization of components of T cell signaling pathways modulates the efficiency of activation. Here, we found that loss of the tyrosine kinase interleukin-2 (IL-2)-inducible T cell kinase (Itk) in mice altered the spatiotemporal distributions of 14 of 16 sensors of T cell signaling molecules in the region of the interface between the T cell and the APC, which reduced the segregation of signaling intermediates into distinct spatiotemporal patterns. Activation of the Rho family guanosine triphosphatase Cdc42 at the center of the cell-cell interface was impaired, although the total cellular amount of active Cdc42 remained intact. The defect in Cdc42 localization resulted in impaired actin accumulation at the T cell-APC interface in Itk-deficient T cells. Reconstitution of cells with active Cdc42 that was specifically directed to the center of the interface restored actin accumulation in Itk-deficient T cells. Itk also controlled the central localization of the guanine nucleotide exchange factor SLAT [Switch-associated protein 70 (SWAP-70)-like adaptor of T cells], which may contribute to the activation of Cdc42 at the center of the interface. Together, these data illustrate how control of the spatiotemporal organization of T cell signaling controls critical aspects of T cell function.

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    • "Kinase dead ITK failed to disrupt cell contact and polarisation to APC [9] and studies have demonstrated that ITK plays role in actin-polymerisation in response to TCR-CD3 activation, which is independent of its kinase activity [6], [10]. Indeed, so important is the scaffold function of ITK that knocking it out results in the spatiotemporal disruption of 14 of the 16 components of the TCR signalling complex at the interface with the APC [11]. "
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    ABSTRACT: Interleukin-2 inducible tyrosine kinase (ITK) is expressed in T cells and plays a critical role in signalling through the T cell receptor. Evidence, mainly from knockout mice, has suggested that ITK plays a particularly important function in Th2 cells and this has prompted significant efforts to discover ITK inhibitors for the treatment of allergic disease. However, ITK is known to have functions outside of its kinase domain and in general kinase knockouts are often not good models for the behaviour of small molecule inhibitors. Consequently we have developed a transgenic mouse where the wild type Itk allele has been replaced by a kinase dead Itk allele containing an inactivating K390R point mutation (Itk-KD mice). We have characterised the immune phenotype of these naive mice and their responses to airway inflammation. Unlike Itk knockout (Itk-/-) mice, T-cells from Itk-KD mice can polymerise actin in response to CD3 activation. The lymph nodes from Itk-KD mice showed more prominent germinal centres than wild type mice and serum antibody levels were significantly abnormal. Unlike the Itk-/-, γδ T cells in the spleens of the Itk-KD mice had an impaired ability to secrete Th2 cytokines in response to anti-CD3 stimulation whilst the expression of ICOS was not significantly different to wild type. However ICOS expression is markedly increased on αβCD3+ cells from the spleens of naïve Itk-KD compared to WT mice. The Itk-KD mice were largely protected from inflammatory symptoms in an Ovalbumin model of airway inflammation. Consequently, our studies have revealed many similarities but some differences between Itk-/-and Itk-KD transgenic mice. The abnormal antibody response and enhanced ICOS expression on CD3+ cells has implications for the consideration of ITK as a therapeutic target.
    PLoS ONE 09/2014; 9(9):e107490. DOI:10.1371/journal.pone.0107490 · 3.23 Impact Factor
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    • "In addition, live fluorescent microscopy showed that viral particles utilize cytoplasmic dynein and the microtubule network to migrate towards the nucleus during early HIV infection (McDonald et al., 2002). ITK has been demonstrated to coordinate spatiotemporal organization of signaling molecules in response to T cell activation and mediate events such as actin polymerization and capping upon T cell receptor engagement and CXCR4 chemokine receptor signaling (Dombroski et al., 2005; Readinger et al., 2008; Singleton et al., 2011). We posit that it is the ability of ITK to coordinate cytoskeleton organization which mediates HIV assembly and release. "
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    ABSTRACT: Interleukin 2-inducible T cell kinase (ITK) influences T cell signaling by coordinating actin polymerization and polarization as well as recruitment of kinases and adapter proteins. ITK regulates multiple steps of HIV-1 replication, including virion assembly and release. Fluorescent microscopy was used to examine the functional interactions between ITK and HIV-1 Gag during viral particle release. ITK and Gag colocalized at the plasma membrane and were concentrated at sites of F-actin accumulation and membrane lipid rafts in HIV-1 infected T cells. There was polarized staining of ITK, Gag, and actin towards sites of T cell conjugates. Small molecule inhibitors of ITK disrupted F-actin capping, perturbed Gag-ITK colocalization, inhibited virus like particle release, and reduced HIV replication in primary human CD4+ T cells. These data provide insight as to how ITK influences HIV-1 replication and suggest that targeting host factors that regulate HIV-1 egress provides an innovative strategy for controlling HIV infection.
    Virology 12/2012; 436(1). DOI:10.1016/j.virol.2012.11.015 · 3.28 Impact Factor
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    • "An alternative approach is to target the expression of downstream signaling proteins that do not directly impact on early TCR signaling events. The loss of the tyrosine kinase interleukin-2 (IL2)-inducible T cell kinase (Itk) resulted in unusual spatial organization of the immunological synapse with a mislocalization of the Rho GTPase CDC42 and a concomitant loss of actin accumulation at the synapse (Singleton et al., 2011). These findings illustrate that a downstream signaling molecule can influence the synapse organization of upstream signaling proteins, suggesting that the non-linearity of signaling pathways is interlinked with protein trafficking and membrane compartmentalization at multiple stages. "
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    ABSTRACT: Engagement of the T cell antigen receptor (TCR) triggers signaling pathways that lead to T cell selection, differentiation and clonal expansion. Superimposed onto the biochemical network is a spatial organization that describes individual receptor molecules, dimers, oligomers and higher order structures. Here we discuss recent findings and new concepts that may regulate TCR organization in naïve and memory T cells. A key question that has emerged is how antigen-TCR interactions encode spatial information to direct T cell activation and differentiation. Single molecule super-resolution microscopy may become an important tool in decoding receptor organization at the molecular level.
    Frontiers in Immunology 11/2012; 3:352. DOI:10.3389/fimmu.2012.00352
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