Search Results (9)

Linker for activation of T cells (LAT) is an essential adaptor protein in early T cell receptor (TCR) signaling that propagates multiple signaling pathways. However, how LAT spatial organization facilitates signal initiation and propagation after TCR triggering is not clear. To differentiate de novo assembly in the plasma membrane from pre-existing LAT vesicles and clusters, we developed imaging protocols and analyses to capture the organization and dynamics of single LAT molecules immediately after TCR engagement. We could observe individual LAT molecules in the plasma membrane that assembled into immobile signaling entities requiring LAT phosphorylation. This step-wise assembly process was temporally highly coordinated via the zeta-chain-associated protein kinase 70 (Zap70)-LAT-growth factor receptor-bound protein 2 (Grb2) pathway. While multiple spatial organization co-existed even within the plasma membrane, our data suggest that de novo plasma membrane assemblies facilitated signal propagation. Full article

Post-translational ubiquitination is an essential mechanism for the regulation of protein stability and function, which contributes to the regulation of the immune system. Cbl, an E3 ubiquitin ligase, is particularly well-characterized in the context of T and NK cell signaling, where it serves as a key regulator of receptor downstream signaling events and as a modulator of cell activation. Cbl promotes the proteasomal degradation of TCR/CD3 subunits as well as the protein kinases Fyn and Lck in T cells. Additionally, the scaffold protein linker for activation of T cells (LAT) is a universal target for Cbl-mediated ubiquitination and degradation in both T and NK cells. Recent findings suggest that CrkII-mediated ubiquitination and degradation of C3G by Cbl during early T cell activation may also be relevant to NK cell signaling. Given its role in modulating immune responses and its manageable impact on autoimmunity, Cbl is being investigated as a target for cancer immunotherapy. This review explores the ubiquitin ligase activity of Cbl and its implications for CAR T and NK cell immunotherapies. Full article

The positional cloning of single nucleotide polymorphisms (SNPs) of the neutrophil cytosolic factor 1 (Ncf1) gene, advocating that a low oxidative burst drives autoimmune disease, demands an understanding of the underlying molecular causes. A cellular target could be T cells, which have been shown to be regulated by reactive oxygen species (ROS). However, the pathways by which ROS mediate T cell signaling remain unclear. The adaptor molecule linker for activation of T cells (LAT) is essential for coupling T cell receptor-mediated antigen recognition to downstream responses, and it contains several cysteine residues that have previously been suggested to be involved in redox regulation. To address the possibility that ROS regulate T cell-dependent inflammation through LAT, we established a mouse strain with cysteine-to-serine mutations at positions 120 and 172 (LAT^SS). We found that redox regulation of LAT through C120 and C172 mediate its localization and phosphorylation. LAT^SS mice had reduced numbers of double-positive thymocytes and naïve peripheral T cells. Importantly, redox insensitivity of LAT enhanced T cell-dependent autoimmune inflammation in collagen-induced arthritis (CIA), a mouse model of rheumatoid arthritis (RA). This effect was reversed on an NCF1-mutated (NCF1^m1j), ROS-deficient, background. Overall, our data show that LAT is redox-regulated, acts to repress T cell activation, and is targeted by ROS induced by NCF1 in antigen-presenting cells (APCs). Full article

T lymphocytes are key players in adaptive immune responses through the recognition of peptide antigens through the T Cell Receptor (TCR). After TCR engagement, a signaling cascade is activated, leading to T cell activation, proliferation, and differentiation into effector cells. Delicate control of activation signals coupled to the TCR is needed to avoid uncontrolled immune responses involving T cells. It has been previously shown that mice deficient in the expression of the adaptor NTAL (Non-T cell activation linker), a molecule structurally and evolutionarily related to the transmembrane adaptor LAT (Linker for the Activation of T cells), develop an autoimmune syndrome characterized by the presence of autoantibodies and enlarged spleens. In the present work we intended to deepen investigation into the negative regulatory functions of the NTAL adaptor in T cells and its potential relationship with autoimmune disorders. For this purpose, in this work we used Jurkat cells as a T cell model, and we lentivirally transfected them to express the NTAL adaptor in order to analyze the effect on intracellular signals associated with the TCR. In addition, we analyzed the expression of NTAL in primary CD4+ T cells from healthy donors and Rheumatoid Arthritis (RA) patients. Our results showed that NTAL expression in Jurkat cells decreased calcium fluxes and PLC-γ1 activation upon stimulation through the TCR complex. Moreover, we showed that NTAL was also expressed in activated human CD4+ T cells, and that the increase of its expression was reduced in CD4+ T cells from RA patients. Our results, together with previous reports, suggest a relevant role for the NTAL adaptor as a negative regulator of early intracellular TCR signaling, with a potential implication in RA. Full article

Adaptor molecules play a crucial role in signal transduction in immune cells. Several adaptor molecules, such as the linker for the activation of T cells (LAT) and SH2 domain-containing leukocyte protein of 76 kDa (SLP-76), are essential for T cell development and activation following T cell receptor (TCR) aggregation, suggesting that adaptor molecules are good therapeutic targets for T cell-mediated immune disorders, such as autoimmune diseases and allergies. Signal-transducing adaptor protein (STAP)-2 is a member of the STAP family of adaptor proteins. STAP-2 functions as a scaffold for various intracellular proteins, including BRK, signal transducer, and activator of transcription (STAT)3, STAT5, and myeloid differentiation primary response protein (MyD88). In T cells, STAP-2 is involved in stromal cell-derived factor (SDF)-1α-induced migration, integrin-dependent cell adhesion, and Fas-mediated apoptosis. We previously reported the critical function of STAP-2 in TCR-mediated T cell activation and T cell-mediated autoimmune diseases. Here, we review how STAP-2 affects the pathogenesis of T cell-mediated inflammation and immune diseases in order to develop novel STAP-2-targeting therapeutic strategies. Full article

In this study, the effect of GBR fermented with the Pediococcus pentosaceus SP024 strain on IgE/Ag mediated passive cutaneous anaphylaxis (PCA) was investigated. Protocatechuic acid and trans-ferulic acid levels in GBR-SP024 increased more than those in unfermented GBR, respec-tively. The inhibitory activity of GBR-SP024 on β-hexosaminidase release and the level of proin-flammatory cytokine mRNA expression (tumor necrosis factor-α (TNF-α) and interleukin 4 (IL-4)) was observed in IgE/Ag-stimulated RBL-2H3 cells. Western blot analysis showed that GBR-SP024 significantly inhibited the phosphorylation of the linker for activation of T cell (LAT) and nuclear factor-κB (NF-κB) in IgE/Ag-stimulated RBL-2H3 cells. Further, we investigated the anti-allergic effect of GBR-SP024 using PCA murine model. The number of infiltrated immune cells and degranulated mast cells in GBR-SP024 treated dermis was lower than that in the GBR-treated mice. In addition, mRNA expression of 5-lipoxygenase (5-LOX) in the dermis of ear tissue declined in the GBR-SP024–treated group, compared to that in the GBR group. GBR-SP024 was also more effective than GBR at reducing the levels of IL-33 protein expression in IgE/Ag-stimulated BALB/c mice. Our study suggests the potential usage of GBR-SP024 as a dietary supplement or an adjuvant for treating IgE-dependent-allergic diseases. Full article

Peripheral immune regulation is critical for the maintenance of self-tolerance. Here we have investigated signaling processes that distinguish T cells with regulatory capability from effector T cells. The murine Tg4 T cell receptor recognizes a peptide derived from the self-antigen myelin basic protein. T cells from Tg4 T cell receptor transgenic mice can be used to generate effector T cells and three types of T cells with regulatory capability, inducible regulatory T cells, T cells tolerized by repeated in vivo antigenic peptide exposure or T cells treated with the tolerogenic drug UCB9608 (a phosphatidylinositol 4 kinase IIIβ inhibitor). We comparatively studied signaling in all of these T cells by activating them with the same antigen presenting cells presenting the same myelin basic protein peptide. Supramolecular signaling structures, as efficiently detected by large-scale live cell imaging, are critical mediators of T cell activation. The formation of a supramolecular signaling complex anchored by the adaptor protein linker for activation of T cells (LAT) was consistently terminated more rapidly in Tg4 T cells with regulatory capability. Such termination could be partially reversed by blocking the inhibitory receptors CTLA-4 and PD-1. Our work suggests that attenuation of proximal signaling may favor regulatory over effector function in T cells. Full article

Intracellular signaling through the T cell receptor (TCR) is essential for T cell development and function. Proper TCR signaling requires the sequential activities of Lck and ZAP-70 kinases, which result in the phosphorylation of tyrosine residues located in the CD3 ITAMs and the LAT adaptor, respectively. LAT, linker for the activation of T cells, is a transmembrane adaptor protein that acts as a scaffold coupling the early signals coming from the TCR with downstream signaling pathways leading to cellular responses. The leukemic T cell line Jurkat and its derivative mutants J.CaM1.6 (Lck deficient) and J.CaM2 (LAT deficient) have been widely used to study the first signaling events upon TCR triggering. In this work, we describe the loss of LAT adaptor expression found in a subline of J.CaM1.6 cells and analyze cis-elements responsible for the LAT expression defect. This new cell subline, which we have called J.CaM1.7, can re-express LAT adaptor after Protein Kinase C (PKC) activation, which suggests that activation-induced LAT expression is not affected in this new cell subline. Contrary to J.CaM1.6 cells, re-expression of Lck in J.CaM1.7 cells was not sufficient to recover TCR-associated signals, and both LAT and Lck had to be introduced to recover activatory intracellular signals triggered after CD3 crosslinking. Overall, our work shows that the new LAT negative J.CaM1.7 cell subline could represent a new model to study the functions of the tyrosine kinase Lck and the LAT adaptor in TCR signaling, and their mutual interaction, which seems to constitute an essential early signaling event associated with the TCR/CD3 complex. Full article

The spleen tyrosine kinase (Syk) is essential for immunoreceptor tyrosine-based activation motif (ITAM)-dependent platelet activation, and it is stimulated by Src-family kinase (SFK)-/Syk-mediated phosphorylation of Y352 (interdomain-B) and Y525/526 (kinase domain). Additional sites for Syk phosphorylation and protein interactions are known but remain elusive. Since Syk S297 phosphorylation (interdomain-B) was detected in platelets, we hypothesized that this phosphorylation site regulates Syk activity via protein kinase C (PKC)-and cyclic adenosine monophosphate (cAMP)-dependent pathways. ADP, the GPVI-agonist convulxin, and the GPIbα-agonist echicetin beads (EB) were used to stimulate human platelets with/without effectors. Platelet aggregation and intracellular messengers were analyzed, along with phosphoproteins, by immunoblotting using phosphosite-specific antibodies or phos-tags. ADP, convulxin, and EB upregulated Syk S297 phosphorylation, which was inhibited by iloprost (cAMP pathway). Convulxin-stimulated Syk S297 phosphorylation was stoichiometric, transient, abolished by the PKC inhibitor GF109203X, and mimicked by the PKC activator PDBu. Convulxin/EB stimulated Syk S297, Y352, and Y525/526 phosphorylation, which was inhibited by SFK and Syk inhibitors. GFX and iloprost inhibited convulxin/EB-induced Syk S297 phosphorylation but enhanced Syk tyrosine (Y352/Y525/526) and substrate (linker adaptor for T cells (LAT), phospholipase γ2 (PLC γ2)) phosphorylation. GFX enhanced convulxin/EB-increases of inositol monophosphate/Ca²⁺. ITAM-activated Syk stimulates PKC-dependent Syk S297 phosphorylation, which is reduced by SFK/Syk/PKC inhibition and cAMP. Inhibition of Syk S297 phosphorylation coincides with enhanced Syk activation, suggesting that S297 phosphorylation represents a mechanism for feedback inhibition in human platelets. Full article