Cooperation of CD8+ T cells and phagocytes to eliminate Toxoplasma cysts

描述(申请人提供)：弓形虫建立了一种重要的慢性感染，能够在艾滋病患者和其他免疫功能受损的人中引起危及生命的弓形体脑炎。持续感染的基础是组织囊肿，在宿主的生命中，它在很大程度上保持静止，但可以重新激活并导致疾病。这一阶段的寄生虫不受目前任何药物治疗的影响，通常被认为是不可接触的。然而，我们最近的研究表明，当免疫T细胞被转移到已经形成大量弓形虫包囊的感染免疫缺陷动物中时，免疫T细胞能够从感染宿主的脑中清除包囊。这种T细胞介导的免疫过程与组织囊周围单个核细胞的聚集有关，从形态上讲，主要是小胶质细胞和巨噬细胞。由于累积的吞噬细胞穿透囊内，这些细胞似乎是主要的效应细胞，在免疫T细胞启动这一过程后，破坏囊并将其从脑中清除。CD8+免疫T细胞具有启动这种抗囊变免疫过程的强大活性，而T细胞的保护活性需要穿孔素。总目标 本研究的目的是确定穿孔素介导的CD8+免疫T细胞活性与吞噬细胞协同清除脑内弓形虫包囊的分子机制。在第一个目标中，我们将确定CD8+免疫T细胞是否在大脑中积累并附着到含囊肿的细胞上。我们还将在体外确定CD8+T细胞是否以穿孔素依赖的方式溶解囊性细胞，以及CD8+T细胞的穿孔素介导的活性是否诱导脑囊肿周围的吞噬细胞聚集。在第二个目标中，我们将确定CD8+T细胞识别含囊细胞的关键分子MHC I类分子(S)。然后，我们将通过筛选预测的缓体表位多肽和/或缓体cDNA文库，确定鉴定出的MHC I类分子呈现的弓形虫包囊表位(S)，以供CD8+T细胞识别。第三个目的是确定吞噬细胞在CD8+T细胞启动免疫过程后清除弓形虫包囊的机制。我们将利用缺乏每种机制所需分子的小鼠，研究诱导型一氧化氮合酶和自噬在杀死缓殖子和消除包囊方面的作用。从这三个特定目的的研究中产生的信息将为开发一种新的方法(S)提供必要的基础，该方法激活CD8+T细胞和吞噬细胞以消除可重新激活并引起弓形虫脑炎的弓形虫包囊。

DESCRIPTION (provided by applicant): Toxoplasma gondii establishes an important chronic infection capable of causing life-threatening toxoplasmic encephalitis in AIDS patients and other immunocompromised individuals. The basis of persistence of the infection is the tissue cyst, which remains largely quiescent for the life of the host, but can reactivate and cause disease. This stage of the parasite is not affected by any of the current drug treatments and it has been generally regarded as untouchable. However, our recent studies revealed that the immune T cells are able to eliminate T. gondii cysts from the brains of infected hosts when the T cells are transferred into infected immunodeficient animals that have already developed large numbers of the cysts. This T cell-mediated immune process is associated with accumulation of mononuclear cells, primarily microglia and macrophages morphologically, around tissue cysts. Since the accumulated phagocytes penetrate within the cyst, these cells appear to be the main effector cells that destroy the cysts and eliminate them from the brain after initiation of this process by immune T cells. CD8+ immune T cells possess a potent activity to initiate this anti-cyst immune process, and the protective activity of the T cells requires perforin. The overall goal of this research project is to determine the molecular mechanisms by which perforin-mediated activity of CD8+ immune T cells cooperates with the phagocytes to remove T. gondii cysts from the brain. In the first aim, we will determine if CD8+ immune T cells accumulate and attach to the cyst-containing cells in the brain. We will also determine if the CD8+ T cells lyse cyst-containing cells in a perforin-dependent manner in vitro and whether the perforin- mediated activity of CD8+ T cells induces the accumulation of phagocytes around the cysts in the brain. In the second aim, we will determine the MHC class I molecule(s) critical for recognition of cyst-containing cells by CD8+ T cells. We will then determine the T. gondii cyst epitope(s) presented by the identified MHC class I molecule for recognition by the CD8+ T cells by screening predicted bradyzoite epitope peptides and/or bradyzoite cDNA library. The third aim is to determine the mechanisms by which phagocytes eliminate T. gondii cysts after initiation of the immune process by CD8+ T cells. We will examine the roles of inducible nitric oxide synthase and autophagy in killing of bradyzoites and elimination of cysts using mice lacking a molecule required for each mechanism. The information generated from the studies in these three specific aims all together will provide the essential basis for developing a novel method(s) to activate CD8+ T cells and phagocytes to eliminate T. gondii cysts that can reactivate and cause toxoplasmic encephalitis.