Inhibition of Diacylglycerol kinase alpha restores TCR-induced diacylglycerol signaling and restimulation-induced cell death in XLP1 T lymphocytes

X\u2010linked lymphoproliferative disease (XLP1) is a rare primary immunodeficiency associated with an unconstrained, life\u2010threatening expansion of CD8+ T cells following Epstein\u2010Barr virus (EBV) infection. XLP1 is caused by mutations in SAP, an adaptor protein that mediates signaling through the SLAM family receptors. SAP\u2010deficient cells exhibit impaired T cell receptor (TCR) restimulation\u2010 induced cell death (RICD), a key physiological process in maintaining lymphocyte homeostasis. We previously showed that SAP has a critical role in TCR\u2010induced inhibition of diacylglycerol kinase alpha (DGK\u3b1), which phosphorylates diacylglycerol (DAG) to phosphatidic acid, acting as a major regulator of T cell signaling. Here, we show that in SAP\u2010deficient Jurkat cells, DGK\u3b1 activity impairs immune synapse (IS) formation, MTOC reorientation and affects the integrity of DAG gradient. By taking advantage of confocal live\u2010cell imaging, we found that RNAi\u2010mediated silencing or pharmacological inhibition of DGK\u3b1 in SAP\u2010deficient cells rescues both immune synapse formation and MTOC repositioning, and the subsequent DAG accumulation at the IS. Inhibition/silencing of DGK\u3b1 was also sufficient to restore PKC\uf0e8\uf020and RasGRP1 recruitment at the immune synapse, as well as ERK1/2 activation and IL\u20102 production/IL\u20102 receptor membrane exposure in SAP knockdown primary T cells. Notably, DGK\u3b1 blockade specifically reestablished RICD in both SAP\u2010 silenced cells and XLP1 patients\u2019 T cells by restoring RSK\u2010mediated induction and phosphorylation of the pro\u2010apoptotic genes Nur77 and Nor1. Furthermore, in vivo inhibition of DGK\u3b1 prevented aberrant CD8+ T cell expansion, TNF\uf0e1\uf020and IFN\uf0e3\uf020production as well as tissue infiltration in Lymphocytic Choriomeningitis Virus\u2010infected SAP KO mice, a mouse model of XLP1. Altogether, these data demonstrates that DGK\u3b1 inhibition, by increasing localized DAG signaling at the IS, restores RICD in SAP deficient cells. In vivo, this limits the CD8+ T cell expansion and tissue damage that characterize XLP1. Collectively, these data highlight the key role for the SAP\u2010mediated DGK\u3b1 inhibition in regulating T lymphocyte homeostasis and highlight DGK\uf0e1\uf020as an attractive therapeutic target in XLP1 patients

Diacylglycerol Kinases: Shaping Diacylglycerol and Phosphatidic Acid Gradients to Control Cell Polarity

Diacylglycerol kinases (DGKs) terminate diacylglycerol (DAG) signaling and promote phosphatidic acid (PA) production. Isoform specific regulation of DGKs activity and localization allows DGKs to shape the DAG and PA gradients. The capacity of DGKs to constrain the areas of DAG signaling is exemplified by their role in defining the contact interface between T cells and antigen presenting cells: the immune synapse. Upon T cell receptor engagement, both DGK α and ζ metabolize DAG at the immune synapse thus constraining DAG signaling. Interestingly, their activity and localization are not fully redundant because DGKζ activity metabolizes the bulk of DAG in the cell, whereas DGKα limits the DAG signaling area localizing specifically at the periphery of the immune synapse. When DGKs terminate DAG signaling, the local PA production defines a new signaling domain, where PA recruits and activates a second wave of effector proteins. The best-characterized example is the role of DGKs in protrusion elongation and cell migration. Indeed, upon growth factor stimulation, several DGK isoforms, such as α, ζ, and γ, are recruited and activated at the plasma membrane. Here, local PA production controls cell migration by finely modulating cytoskeletal remodeling and integrin recycling. Interestingly, DGK-produced PA also controls the localization and activity of key players in cell polarity such as aPKC, Par3, and integrin β1. Thus, T cell polarization and directional migration may be just two instances of the general contribution of DGKs to the definition of cell polarity by local specification of membrane identity signaling

Wiskott-Aldrich syndrome protein interacts and inhibits diacylglycerol kinase alpha promoting IL-2 induction

Background: Phosphorylation of diacylglycerol by diacylglycerol-kinases represents a major inhibitory event constraining T cell activation upon antigen engagement. Efficient TCR signalling requires the inhibition of the alpha isoform of diacylglycerol kinase, DGKα, by an unidentified signalling pathway triggered by the protein adaptor SAP. We previously demonstrated that, in SAP absence, excessive DGKα activity makes the T cells resistant to restimulation-induced cell death (RICD), an apoptotic program counteracting excessive T cell clonal expansion. Results: Herein, we report that the Wiskott-Aldrich syndrome protein (WASp) inhibits DGKα through a specific interaction of the DGKα recoverin homology domain with the WH1 domain of WASp. Indeed, WASp is necessary and sufficient for DGKα inhibition, and this WASp function is independent of ARP2/3 activity. The adaptor protein NCK-1 and the small G protein CDC42 connect WASp-mediated DGKα inhibition to SAP and the TCR signalosome. In primary human T cells, this new signalling pathway is necessary for a full response in terms of IL-2 production, while minimally affecting TCR signalling and restimulation-induced cell death. Conversely, in T cells made resistant to RICD by SAP silencing, the enhanced DAG signalling due to DGKα inhibition is sufficient to restore apoptosis sensitivity. Conclusion: We discover a novel signalling pathway where, upon strong TCR activation, the complex between WASp and DGKα blocks DGKα activity, allowing a full cytokine response

A fully photonics-based coherent radar system

The next generation of radar (radio detection and ranging) systems needs to be based on software-defined radio to adapt to variable environments, with higher carrier frequencies for smaller antennas and broadened bandwidth for increased resolution. Today's digital microwave components (synthesizers and analogue-to-digital converters) suffer from limited bandwidth with high noise at increasing frequencies, so that fully digital radar systems can work up to only a few gigahertz, and noisy analogue up- and downconversions are necessary for higher frequencies. In contrast, photonics provide high precision and ultrawide bandwidth, allowing both the flexible generation of extremely stable radio-frequency signals with arbitrary waveforms up to millimetre waves, and the detection of such signals and their precise direct digitization without downconversion. Until now, the photonics-based generation and detection of radio-frequency signals have been studied separately and have not been tested in a radar system. Here we present the development and the field trial results of a fully photonics-based coherent radar demonstrator carried out within the project PHODIR. The proposed architecture exploits a single pulsed laser for generating tunable radar signals and receiving their echoes, avoiding radio-frequency up- and downconversion and guaranteeing both the software-defined approach and high resolution. Its performance exceeds state-of-the-art electronics at carrier frequencies above two gigahertz, and the detection of non-cooperating aeroplanes confirms the effectiveness and expected precision of the system

Wiskott-Aldrich syndrome protein interacts and inhibits diacylglycerol kinase alpha promoting IL-2 induction

BackgroundPhosphorylation of diacylglycerol by diacylglycerol-kinases represents a major inhibitory event constraining T cell activation upon antigen engagement. Efficient TCR signalling requires the inhibition of the alpha isoform of diacylglycerol kinase, DGKα, by an unidentified signalling pathway triggered by the protein adaptor SAP. We previously demonstrated that, in SAP absence, excessive DGKα activity makes the T cells resistant to restimulation-induced cell death (RICD), an apoptotic program counteracting excessive T cell clonal expansion.ResultsHerein, we report that the Wiskott-Aldrich syndrome protein (WASp) inhibits DGKα through a specific interaction of the DGKα recoverin homology domain with the WH1 domain of WASp. Indeed, WASp is necessary and sufficient for DGKα inhibition, and this WASp function is independent of ARP2/3 activity. The adaptor protein NCK-1 and the small G protein CDC42 connect WASp-mediated DGKα inhibition to SAP and the TCR signalosome. In primary human T cells, this new signalling pathway is necessary for a full response in terms of IL-2 production, while minimally affecting TCR signalling and restimulation-induced cell death. Conversely, in T cells made resistant to RICD by SAP silencing, the enhanced DAG signalling due to DGKα inhibition is sufficient to restore apoptosis sensitivity.ConclusionWe discover a novel signalling pathway where, upon strong TCR activation, the complex between WASp and DGKα blocks DGKα activity, allowing a full cytokine response

B7h triggering inhibits the migration of tumor cell lines

Vascular endothelial cells (ECs) and several cancer cells express B7h, which is the ligand of the ICOS T cell costimulatory molecule. We have previously shown that B7h triggering via a soluble form of ICOS (ICOS-Fc) inhibits the adhesion of polymorphonuclear and tumor cell lines to HUVECs; thus, we suggested that ICOS-Fc may act as an anti-inflammatory and antitumor agent. Because cancer cell migration and angiogenesis are crucial for metastasis dissemination, the aim of this work was to evaluate the effect of ICOS-Fc on the migration of cancer cells and ECs. ICOS-Fc specifically inhibited the migration of HUVECs, human dermal lymphatic ECs, and the HT29, HCT116, PC-3, HepG2, JR8, and M14 tumor cell lines expressing high levels of B7h, whereas it was ineffective in the RPMI7932, PCF-2, LM, and BHT-101 cell lines expressing low levels of B7h. Furthermore, ICOS-Fc downmodulated hepatocyte growth factor facilitated the epithelial-to-mesenchymal transition in HepG2 cells. Moreover, ICOS-Fc downmodulated the phosphorylation of focal adhesion kinase and the expression of \u3b2-Pix in both HUVECs and tumor cell lines. Finally, treatment with ICOS-Fc inhibited the development of lung metastases upon injection of NOD-SCID-IL2R\u3b3null mice with CF-PAC1 cells, as well as C57BL/6 mice with B16-F10 cells. Therefore, the B7h-ICOS interaction may modulate the spread of cancer metastases, which suggests the novel use of ICOS-Fc as an immunomodulatory drug. However, in the B16-F10-metastasized lungs, ICOS-Fc also increased IL-17A/RORc and decreased IL-10/Foxp3 expression, which indicates that it also exerts positive effects on the antitumor immune response

The diacylglycerol kinase α/Atypical PKC/β1 integrin pathway in SDF-1α mammary carcinoma invasiveness

Diacylglycerol kinase α (DGKα), by phosphorylating diacylglycerol into phosphatidic acid, provides a key signal driving cell migration and matrix invasion. We previously demonstrated that in epithelial cells activation of DGKα activity promotes cytoskeletal remodeling and matrix invasion by recruiting atypical PKC at ruffling sites and by promoting RCP-mediated recycling of α5β1 integrin to the tip of pseudopods. In here we investigate the signaling pathway by which DGKα mediates SDF-1α-induced matrix invasion of MDA-MB-231 invasive breast carcinoma cells. Indeed we showed that, following SDF-1α stimulation, DGKα is activated and localized at cell protrusion, thus promoting their elongation and mediating SDF-1α induced MMP-9 metalloproteinase secretion and matrix invasion. Phosphatidic acid generated by DGKα promotes localization at cell protrusions of atypical PKCs which play an essential role downstream of DGKα by promoting Rac-mediated protrusion elongation and localized recruitment of β1 integrin and MMP-9. We finally demonstrate that activation of DGKα, atypical PKCs signaling and β1 integrin are all essential for MDA-MB-231 invasiveness. These data indicates the existence of a SDF-1α induced DGKα - atypical PKC - β1 integrin signaling pathway, which is essential for matrix invasion of carcinoma cells

Diacylglycerol Kinases: Shaping Diacylglycerol and Phosphatidic Acid Gradients to Control Cell Polarity

Diacylglycerol kinases (DGKs) terminate diacylglycerol (DAG) signaling and promote phosphatidic acid (PA) production. Isoform specific regulation of DGKs activity and localization allows DGKs to shape the DAG and PA gradients. The capacity of DGKs to constrain the areas of DAG signaling is exemplified by their role in defining the contact interface between T cells and antigen presenting cells: the immune synapse. Upon T cell receptor engagement, both DGK α and ζ metabolize DAG at the immune synapse thus constraining DAG signaling. Interestingly, their activity and localization are not fully redundant because DGKζ activity metabolizes the bulk of DAG in the cell, whereas DGKα limits the DAG signaling area localizing specifically at the periphery of the immune synapse.When DGKs terminate DAG signaling, the local PA production defines a new signaling domain, where PA recruits and activates a second wave of effector proteins. The best-characterized example is the role of DGKs in protrusion elongation and cell migration. Indeed, upon growth factor stimulation, several DGK isoforms, such as α, ζ, and γ, are recruited and activated at the plasma membrane. Here, local PA production controls cell migration by finely modulating cytoskeletal remodeling and integrin recycling. Interestingly, DGK-produced PA also controls the localization and activity of key players in cell polarity such as aPKC, Par3, and integrin β1. Thus, T cell polarization and directional migration may be just two instances of the general contribution of DGKs to the definition of cell polarity by local specification of membrane identity signaling