Visualizing immune responses to Toxoplasma gondii

描述(申请人提供)：细胞内原生动物寄生虫弓形虫是一种广泛传播的病原体，可感染包括人类在内的许多物种，导致免疫受损个体的出生缺陷和神经系统疾病。免疫活性宿主的感染通常是无症状的，因为强健的I型免疫反应可以控制急性感染，并允许建立慢性期。在慢性期，寄生虫持续存在于脑和肌肉组织内的包囊中。使用小鼠感染模型的研究为免疫反应如何控制感染提供了相当大的洞察力。然而，在我们对寄生虫如何与免疫系统相互作用的理解上仍然存在很大差距。寄生虫是如何操纵抗原提呈细胞功能的？在启动过程中，哪些类型的抗原提呈细胞将弓形虫抗原呈现给T细胞？CDS T细胞和已知对免疫保护至关重要的细胞因子IFNy实际上是如何控制寄生虫的生长的？被侵袭的靶细胞CTL裂解后寄生虫的命运如何？在感染的慢性期，CDS T细胞在防止寄生虫再次出现方面起什么作用？通过直接可视化CDS T细胞在保护免受感染过程中的作用，我们希望对这些问题有更深入的了解。在这项建议中，我描述了使用双光子激光扫描显微镜对组织内荧光标记的CDS T细胞、寄生虫和抗原提呈细胞进行实时成像的实验。成像方法将与常规的流式细胞术和免疫反应的功能分析相补充。在目标1中，我们将研究寄生虫感染细胞启动幼稚CDS的细胞动力学。在目标2，我们将研究干扰素-γ的产生和效应CDS T细胞的CTL杀伤。在目标3中，我们将通过检测慢性感染小鼠大脑中T细胞和寄生虫的行为来研究CDS T细胞在慢性感染中的作用。这些研究应该为我们的免疫系统如何保护我们免受弓形虫的潜在致命感染提供洞察力，弓形虫是NIAID B类优先病原体。

DESCRIPTION (provided by the applicant): The intracellular protozoan parasite Toxoplasma gondii is a wide-spread pathogen that can infect many species including humans, leading to birth defects and neurological disease in immunocompromised individuals. Infection of immunocompetent hosts is generally asymptomatic due to a robust type I immune response that controls acute infection and allow establishment of a chronic phase. During the chronic phase the parasite persists in cysts within brain and muscle tissues. Studies using the mouse model of infection have provided considerable insight into how immune responses control infection. Nevertheless, large gaps remain in our understanding of how the parasite interacts with the immune system. How does the parasite manipulate antigen presenting cell function? What types of antigen presenting cells display T. gondii antigens to T cells during priming? How do CDS T cells and the cytokine IFNy, which are known to be crucial for immune protection, actually control the growth of the parasite? What is the fate for parasites following CTL lysis of an invaded target cell? What is the role of CDS T cells in preventing the re-emergence of the parasite during the chronic phase of infection? By directly visualizing CDS T cells in the process of protecting from infection, we hope to gain insight into these questions. In this proposal, I describe experiments using Two Photon Laser Scanning Microscopy to perform real-time imaging of fluorescently labeled CDS T cells, parasites, and antigen presenting cells within tissues. The imaging approach will be complemented by conventional flow cytometric and functional assays of immune responses. In Aim 1, we will investigate the cellular dynamics of priming of naive CDS by parasite infected cells. In Aim 2, we will investigate INF-y production and CTL killing by effector CDS T cells. In Aim 3, we will examine the role of CDS T cells during chronic infection by examining the behavior of T cells and parasites in the brains of chronically infected mice. These studies should provide insights into how our immune systems protect us from potentially lethal infection with Toxoplasma, an NIAID Category B Priority Pathogen.