Redox regulation of intestinal T cells

描述（由申请人提供）：T细胞受体信号转导的调节可以通过外源性细胞因子、细胞间接触和内源性代谢物介导，对抗原刺激产生专门的功能反应。尽管肠道存在敌对的免疫邻域，粘膜固有层T细胞（LPT）仍然对来自“安全”腔内蛋白的抗原反应低下；但它们仍能对病原体产生强有力的免疫反应。因此，我们建议研究调节粘膜T细胞低反应性维持的机制，以及使这种通常静止的粘膜T细胞在需要时做出反应的环境信号。T细胞受体（TCR）发出的信号转导对所有功能反应都至关重要，如增殖、免疫介质（如Fas配体、受体和粘附蛋白）的表面表达、分化、活化诱导的细胞死亡和细胞因子基因表达。在本提案中，我们将重点关注来自氧不完全还原的活性氧（ROS），如过氧化氢，作为第二信使，因此是TCR发出的信号转导的内源性调节剂。我们最近报道，LPT对TCR交联反应能力的减弱，通过弱增殖来衡量，反映了它们启动TCR信号转导能力的显著丧失。为了理解LPT信号传导能力的下降，我们研究了与TCR的a链和¿链直接相关的酶途径，重点研究了与TCR/CD3复合物相关的酪氨酸激酶和磷酸酶之间的潜在不平衡。由于质膜很容易渗透过氧化氢，外源性低浓度、无毒的H2O2刺激酪氨酸磷酸化模式，反映TCR参与。与TCR交联所观察到的类似，h2o2诱导的LPT中酪氨酸磷酸化明显减弱。因此，我们假设并报道了低反应性LPT的氧化还原状态倾向于增加还原能力。此外，克罗恩病衍生的LPT（炎症性肠病的一种形式）对TCR结扎(例如，增殖、IFN-？炎症T细胞中的氧化还原状态是氧化的，这使得TCR参与后能够进行强烈的信号转导。与我们的发现一致，最近有报道称，在正常肠道中，固有层巨噬细胞不分泌半胱氨酸，而半胱氨酸是LPT调节其胞质氧化还原电位所必需的。这些结果支持以下中心假设：粘膜T细胞的细胞内还原能力调节了细胞产生细胞内活性氧的能力，改变了T细胞受体在正常肠粘膜中启动免疫反应的阈值。这一假设将在以下两个具体目标中得到解决：目标1。定义tcr活化LPT中生成的活性氧在合成和/或稳定性方面的内在缺陷。目标2。确定细胞质氧化还原电位设定LPT反应的阈值。

DESCRIPTION (provided by applicant): Modulation of signal transduction emanating from the T cell receptor can be mediated by exogenous cytokines, cell-cell contact, and endogenous metabolites, yielding a specialized functional response to antigenic stimulation. Despite the hostile immunological neighborhood of the intestinal tract, mucosal lamina propria T cells (LPT) remain hyporesponsive to antigens derived from "safe" luminal proteins; yet retain their capacity to mount a vigorous immune response to pathogens. We therefore propose to investigate the mechanisms regulating the maintenance of hyporesponsiveness in mucosal T cells and the environmental signals that enable this normally quiescent mucosal T cell to respond when needed. Signal transduction emanating from the T cell receptor (TCR) is critical for all functional responses, such as proliferation, surface expression of immune mediators such as Fas Ligand, receptors, and adhesion proteins, differentiation, activation induced cell death, and cytokine gene expression. In this proposal we will focus on reactive oxygen species (ROS) derived from incomplete reduction of oxygen, such as hydrogen peroxide, acting as second messengers and thus endogenous modulators of signal transduction emanating from the TCR. We recently reported that the diminished capability of LPT to respond to TCR cross-linking, as measured by weak proliferation, is mirrored by a dramatic loss in their ability to initiate signal transduction from the TCR. To understand this decrease in LPT signaling capacity we investigated the enzymatic pathway directly linked to the a and ¿ chains of the TCR, focusing on a potential imbalance between tyrosine kinases and phosphatases associated with the TCR/CD3 complex. Since the plasma membrane is readily permeable to hydrogen peroxide, exogenous administration of low, non-toxic concentrations of H2O2 stimulates a tyrosine phosphorylation pattern that reflects TCR engagement. Similar to what is observed with TCR cross-linking, H2O2-induced tyrosine phosphorylation in LPT is significantly muted. We therefore hypothesized and then reported that the redox status of the hyporesponsive LPT is tilted toward increased reductive capability. In addition, the Crohn's disease-derived (a form of inflammatory bowel disease) LPT responds robustly to TCR ligation (e.g., proliferation, IFN-? production), and the redox status in this inflamed T cell is oxidative, which enables strong signal transduction after TCR engagement. Consistent with our findings it was recently reported that in the normal intestine, lamina propria macrophages do not secrete cysteine, which is required by the LPT to regulate their cytosolic redox potential. These results support the following central hypothesis: The intracellular reducing capacity of the mucosal T cell, which modulates that cell's ability to generate intracellular reactive oxygen species, alters the threshold at which engagement of the T cell receptor initiates an immune response in the normal intestinal mucosa. This hypothesis will be addressed in the following two specific aims: Aim 1. Define the intrinsic defect in the synthesis and/or stability of reactive oxygen species generated in TCR-activated LPT. Aim 2. Establish that cytosolic redox potential sets the threshold for an LPT response. RELEVANCE: To protect us from infections we have a complex system of white blood cells, the principal of these is called the T lymphocyte, which makes strategic decisions for host defense. T cells isolated from the colon or small bowel normally respond very weakly when compared to T cells isolated from blood, while T cells isolated from inflamed intestinal areas in patients with an infection or intestinal cramping and bowel disease due to chronic inflammation respond with still yet another distinct character. This proposal will define those intrinsic properties of the mucosal T cell that enables it to toggle between these responsive and unresponsive states. We recently identified that a chemical balance (called the redox potential) within the T cell regulates whether it can respond weakly or strongly to the outside world. We also showed that by switching the balance within a mucosal T cell to the alternate type this treated intestinal T cell now behaves more like a normal blood-derived T cell. This project will define the exact molecules in the T cell that causes this imbalance. We can then target this molecule for 'correction' and in the future determine whether this type of treatment is an effective therapy for mucosal infections, vaccine development, or mucosal autoimmune-like diseases.