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 (LATSS). We found that redox regulation of LAT through C120 and C172 mediate its localization and phosphorylation. LATSS 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 (NCF1m1j), 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).

Protein 4.1R, an 80 000 MW membrane skeleton protein, is a vital component of the red blood cell membrane cytoskeleton that stabilizes the spectrin-actin network and regulates membrane properties of deformability and mechanical stability. It has been shown that 4.1R is expressed in T cells, including CD8+ T cells, but its role in CD8+ T cells remains unclear. Here, we have explored the role of 4.1R in CD8+ T cells using 4.1R-/- mice. Our results showed that cell activation, proliferation and secretion levels of interleukin-2 and interferon-γ were significantly increased in 4.1R-/- CD8+ T cells. Furthermore, the phosphorylation levels of linker for activation of T cells (LAT) and its downstream signaling molecule extracellular signal-regulated kinase were enhanced in the absence of 4.1R. In vitro co-immunoprecipitation experiments showed a direct interaction between 4.1R and LAT. Moreover, 4.1R-/- CD8+ T cells and mice exhibited an enhanced T-cell-dependent immune response. These data enabled the identification of a negative regulation function for 4.1R in CD8+ T cells by a direct association between 4.1R and LAT, possibly through inhibiting phosphorylation of LAT and then modulating intracellular signal transduction.

In mast cells, induction of HSP70 expression during antigen stimulation has not been reported.
Mouse bone marrow-derived mast cells (BMMC) were stimulated with IgE/Ag or HSP70. Induction of HSP70 expression and signaling protein phosphorylation were evaluated by immunoblotting.
HSP70 expression is induced in BMMC at an early stage of IgE/Ag-dependent stimulation, some of which is released from the cells in a granule-associated form. Induction of HSP70 expression was also observed with an IgE/Ag-stimulated human basophilic cell line, indicating that the phenomenon is not restricted to mouse BMMC. The induction of HSP70 expression, and its release, followed a similar time course to that of degranulation. Released HSP70 seems to be responsible for degranulation and production of eicosanoids, at least in part, because a neutralizing anti-HSP70 antibody mitigated these activities and because exogenous HSP70 not only induced immediate degranulation followed by autocrine HSP70 expression but also enhanced degranulation in IgE/Ag-stimulated BMMC. Extracellular HSP70 was found to induce phosphorylation of linker for activation of T cells (LAT) and a series of downstream signaling molecules in BMMC. We further found that Fyn, Lyn, and spleen tyrosine kinase (Syk), which are known to concern LAT phosphorylation in IgE/Ag-stimulated BMMC, were not phosphorylated in HSP70-stimulated BMMC, whereas lymphocyte-specific protein tyrosine kinase (Lck) was phosphorylated.
FcεRI stimulation in BMMC and basophils induces HSP70 expression and its release. Extracellular HSP70 induces degranulation and mediator release via phosphorylation of LAT.

OBJECTIVE: Severe aplastic anemia (SAA) is a rare immune-regulated disease characterized by severe pancytopenia and bone marrow failure, caused by destruction of hematopoietic cells by the activated T lymphocytes. Linker for activation of T cells (LAT), a transmembrane adaptor protein, plays a key role in T-cell and mast cell functions. However, it remains unclear how LAT may change in patients with SAA. This study aims at understanding the role of lymphocyte LAT in SAA.
METHODS: The expression of LAT, related signaling molecules, and T-cell effector molecules was determined by flow cytometry. LAT mRNA was evaluated by quantitative real-time PCR. Cytokine production by cultured T cells was determined by ELISA.
RESULTS: Patients with SAA had an increased levels of LAT and both total phosphorylated LAT and of the related molecule (ZAP-70) in circulating T cells compared with normal controls. In patients with SAA, the expression of LAT was positively associated with the expression of perforin and granzyme B in CD8(+) T cells. Inhibition of LAT expression in T cells from patients with SAA decreased the activation of the CD4(+) and CD8(+) T-cell subsets. Overexpression of LAT in T cells from normal controls increased the activation of CD4(+) and CD8(+) T-cell subsets with increased apoptosis of K562 cells in coculture.
CONCLUSIONS: Our findings demonstrate that dysregulation of LAT expression and activation may contribute to over-function of T cells, imbalance of Th1/Th2 subsets and thus lead to hematopoiesis failure in SAA. Immunosuppressive therapy dramatically reduced the expression of LAT making it an attractive therapeutic target in SAA.

Complement is undeniably quintessential for innate immunity by detecting and eliminating infectious microorganisms. Recent work, however, highlights an equally profound impact of complement on the induction and regulation of a wide range of immune cells. In particular, the complement regulator CD46 emerges as a key sensor of immune activation and a vital modulator of adaptive immunity. In this review, we summarize the current knowledge of CD46-mediated signalling events and their functional consequences on immune-competent cells with a specific focus on those in CD4(+) T cells. We will also discuss the promises and challenges that potential therapeutic modulation of CD46 may hold and pose.

Recently, some endocannabinoids were reported to show anti-inflammatory and anti-allergic activities. In this respect, various arachidonoyl endocannabinoids were screened for the inhibition of allergic response in IgE-activated RBL-2H3 cells. Among arachidonoyl endocannabinoids with a low cytotoxicity, only NA-5HT remarkably inhibited the release of β-hexosaminidase (IC(50), 13.58 μM), a marker of degranulation, and tumor necrosis factor-α (IC(50), 12.52 μM), a pro-inflammatory cytokine, in IgE-activated RBL-2H3 cells. Additionally, NA-5HT markedly suppressed the formation of prostaglandin D(2) (PGD(2)) with IC(50) value of 1.27 μM and leukotriene B(4) (LTB(4)) with IC(50) value of 1.20 μM, and slightly LTC4. When effect of NA-5HT on early stage of FcεRI cascade was investigated, it significantly inhibited phosphorylation of Syk, but not Lyn. Furthermore, NA-5HT suppressed phosphorylation of PLCγ1/2 and PKCδ, related to degranulation process, as well as phosphorylation of LAT, ERK1/2, p38, JNK, Gab2, PI3K and Akt, implicated in the expression of pro-inflammatory cytokines. Relative to its effect on the late stage, NA-5HT slightly reduced phosphorylation of 5-lipoxygenase (5-LO) and cyclooxygenase-2 (COX-2). Additionally, NA-5HT significantly reduced the level of p40(phox), and partially inhibited the expression of p47(phox) and p67(phox). From these results, it is suggested that NA-5HT expresses anti-allergic action by suppressing the activation of Syk, LAT, p38, JNK, PI3K and Akt, as well as the expression of ERK1/2 and NADPH oxidase subunits. Further, a strong inhibition of PGD(2) or LTB(4) biosynthesis by NA-5HT may be an additional mechanism for its anti-allergic action. Such anti-allergic actions of NA-5HT may contribute to further information about its biological functions.

Genetic and biochemical studies have identified a large number of molecules involved in T cell signaling. They have provided us with a comprehensive understanding of protein-protein interactions and protein modifications that take place upon antigen recognition. Diffraction limited fluorescence microscopy has been used to study the distribution of signaling molecules on a cellular level. Specifically, the discovery of microclusters and the immunological synapse demonstrates that T cell signaling cascades utilizes spatial association and segregation. Recent advancements in live cell imaging have allowed us to visualize the spatio-temporal mechanisms of T cell signaling at nanometer scale resolution. This led to the discovery that proteins are organized in distinct membrane domains prior and during T cell activation. Evidently, plasma membrane structures and signaling molecule distributions at all length scales (molecular to cellular) are intrinsic to the mechanisms that govern signaling initiation, transduction, and inhibition. Here we provide an overview of possible plasma membrane models, molecular assemblies that have been described to date, how they can be visualized and how they might contribute to T cell signaling.