Calcium and Cytotoxic T Lymphocytes

描述（由申请方提供）：本提案的长期目标是了解细胞内钙信号在细胞毒性T淋巴细胞（CTL）功能中的作用。这些免疫系统的关键效应子杀死病毒感染的细胞和癌细胞，并在对移植组织的免疫反应中发挥重要作用;不适当的杀伤可导致自身免疫性疾病，如狼疮，某些形式的糖尿病和关节炎。因此，了解CTL功能对于预防和治疗自然发生的病毒性疾病（如艾滋病和流感）以及病毒性疾病（如用作生物武器的天花）非常重要。它对于理解和治疗癌症和自身免疫性疾病也很重要。最后，抑制CTL功能的能力对于成功的器官移植至关重要。 CTL用于杀伤的主要机制之一是穿孔素途径，其涉及将特化裂解颗粒中所含的孔形成肽和水解酶胞吐释放到与靶标形成的紧密并置区域中。颗粒胞吐作用是绝对已知的，需要增加细胞内钙引起的跨质膜流入。然而，钙在颗粒胞吐中的具体作用尚不清楚，钙依赖性步骤的数量尚不清楚，并且尚未鉴定出赋予钙依赖性的分子。该提案的具体目标将使用一系列技术，包括我们开发的新型荧光成像方法，以：1）确定大量胞质钙的增加是否足以支持颗粒胞吐，或者是否需要微区中高于胞质钙的增加。 2)研究颗粒重定向和非重定向依赖性胞吐的钙依赖性。3)确定免疫突触的形成是否是钙依赖性的，并作为颗粒重定向的一个缓慢步骤。 4)研究钙依赖性磷酸酶钙调神经磷酸酶在颗粒胞吐中的作用。这些研究将大大加深我们对钙内流在溶胞颗粒胞吐中作用的理解。

DESCRIPTION (provided by applicant): The long-term goal of this proposal is to understand the role of intracellular calcium signals in the function of cytotoxic T lymphocytes (CTLs). These critical effectors of the immune system kill virus-infected cells and cancer cells and play a major role in the immune response to transplanted tissues; inappropriate killing can cause autoimmune diseases such as Lupus, certain forms of diabetes, and arthritis. Understanding CTL function is therefore important for preventing and treating naturally occurring viral diseases such as AIDS and influenza, and viral diseases such as smallpox used as biological weapons. It is also important for understanding and treating cancers and autoimmune diseases. Finally, the ability to suppress CTL function is vital for successful organ transplantation. One of the main mechanisms CTLs use to kill is the perforin pathway, which involves the exocytotic release of pore-forming peptides and hydrolytic enzymes contained in specialized lytic granules into an area of close apposition formed with the target. Granule exocytosis is known absolutely to require increased intracellular calcium caused by influx across the plasma membrane. However, the specific role(s) of calcium in granule exocytosis are unknown, the number of calcium-dependent steps is unclear, and molecules that confer calcium-dependence have not been identified. The specific aims of this proposal will use a battery of techniques, including novel fluorescence imaging methodologies we have developed, to: 1) determine whether bulk cytosolic calcium increases are sufficient to support granule exocytosis, or whether higher-than-cytosolic calcium increases in microdomains are required. 2) Investigate the calcium dependence of granule reorientation and of reorientation-independent exocytosis. 3) Determine whether immunological synapse formation is calcium dependent, and acts as a slow step in granule reorientation. 4) Investigate the role of the calcium-dependent phosphatase calcineurin in granule exocytosis. These studies will significantly further our understanding of the role of calcium influx in lytic granule exocytosis.