Calcium and Cytotoxic T Lymphocytes

描述（由申请人提供）：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物杀死病毒感染的细胞和癌细胞，并在移植组织的免疫反应中发挥重要作用；不恰当的杀戮会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）以及用作生物武器的病毒性疾病（如天花）非常重要。它对理解和治疗癌症和自身免疫性疾病也很重要。最后，抑制CTL功能的能力对于器官移植的成功至关重要。ctl用于杀伤的主要机制之一是穿孔素途径，这涉及到细胞外释放成孔肽和水解酶，这些酶包含在专门的裂解颗粒中，进入与靶标形成的密切相关区域。众所周知，颗粒胞吐绝对需要通过质膜内流引起的细胞内钙的增加。然而，钙在颗粒胞吐中的具体作用尚不清楚，钙依赖步骤的数量尚不清楚，并且尚未确定钙依赖的分子。本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外分泌，或者是否需要在微域中增加高于胞质钙的量。2)研究颗粒重定向和重定向非依赖性胞吐对钙的依赖性。3)确定免疫突触的形成是否依赖于钙，并作为颗粒重定向的缓慢步骤。4)研究钙依赖性磷酸酶钙调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粒胞吐中的作用的理解。

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.