Calcium and Cytotoxic T Lymphocytes

描述(由申请人提供)：这项建议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者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主要作用；不适当的杀伤可能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的功能对于预防和治疗艾滋病和流感等自然发生的病毒疾病以及用作生物武器的天花等病毒疾病非常重要。它对了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，它涉及到特殊裂解颗粒中的造孔肽和水解酶的胞外释放，进入与靶标形成的紧密结合的区域。众所周知，颗粒胞吐绝对需要细胞内钙离子通过质膜内流而增加。然而，钙在颗粒胞吐中的具体作用(S)尚不清楚，钙依赖步骤的数量也不清楚，赋予钙依赖的分子也尚未确定。这项提案的具体目的将使用一系列技术，包括我们开发的新的荧光成像方法，以：1)确定大量胞浆钙是否足以支持颗粒胞吐，或者是否需要在微域中高于胞浆钙的升高。2)研究颗粒重定位和非重定位胞吐对钙的依赖性。3)确定免疫突触的形成是否是钙依赖的，并作为颗粒重定向的缓慢步骤。4)探讨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血颗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.