Role of chemokines in the T cell response to ocular toxoplasmosis

描述（由申请人提供）：在正常条件下，免疫系统的细胞大部分被排除在眼睛之外。然而，在炎症条件下，自身反应性或病原体特异性T细胞可以进入视网膜，这可能是有害的。因此，针对几种自身免疫性疾病的药物开发集中在防止T细胞运输到免疫特权组织。然而，这种治疗的一个不可预见的并发症是无法控制感染，包括眼弓形虫病，这是全球感染性后葡萄膜炎的主要原因。持续的T细胞介导的免疫应答对于限制弓形虫复制和视网膜破坏是必要的。通过允许T细胞进入和/或影响感染后眼睛内的迁移行为来支持免疫应答的因素仍然知之甚少。趋化因子对于T细胞迁移到炎症部位至关重要，我们的初步分析表明视网膜中CXCL 10转录水平增加，并且迁移到该部位的大多数T细胞表达CXCR 3（CXCL 10的受体）。此外，CXCL 10特异性阻断抗体的施用导致眼中T细胞数量的减少和眼部病理学的相应增加。最后，该实验室使用多光子显微镜生成的初步数据显示，长期感染T.弓形虫表现出一系列迁移行为。该提案将专门关注CXCL 10对CD 8 + T细胞反应的作用，并将在以下特定目标中进行测试：特定目的I。CXCL 10和CXCR 3是否影响眼弓形虫病期间CD 8 + T细胞的运输？为了检查CXCL 10在T细胞运输到眼睛中的作用，将开发T细胞转移系统，其中将活化的T细胞转移到慢性感染的小鼠中，并使用流式细胞术和免疫组织化学（IHC）进行跟踪。为了检验CXCR 3和CXCL 10是T细胞运输至眼睛所必需的假设，将使用抗CXCL 10抗体和CXCR 3-/- T细胞。将使用实时PCR和IHC检查在存在或不存在趋化因子的情况下细胞转移对寄生虫负荷的影响。第二章. CXCL 10和CXCR 3是否影响弓形虫病患者视网膜中T细胞的迁移行为？由于趋化因子影响T细胞运动和功能的各个方面（20），因此提出了视网膜的多光子活体成像，使用荧光标记的抗原特异性T细胞和转基因寄生虫来评估CXCR 3或CXCL 10的缺乏如何影响T细胞的行为。几个参数的迁移将进行评估，包括，速度，蜿蜒指数，和运动系数。 本提案中概述的研究将确定CXCL 10和CXCR 3在T细胞介导的T细胞调控中的作用。视网膜中的弓形虫此外，我们设想，我们的研究结果将提供该模型的实用性的原则证明，用于检查额外的细胞因子，趋化因子和免疫治疗剂的作用。 公共卫生相关性：眼部弓形虫感染可导致失明。许多人感染了这种寄生虫，但由于免疫系统的强烈反应而不会患上眼疾。这项提案将确定免疫系统如何防止寄生虫造成眼睛的破坏。

DESCRIPTION (provided by applicant): Under normal conditions, cells of the immune system are largely excluded from the eye. However, under inflammatory conditions, autoreactive or pathogen-specific T cells can gain entry to the retina, which can be detrimental. Thus, drug development for several autoimmune conditions has focused on the prevention of T cell trafficking to immune-privileged tissues. However, one unforeseen complication of such treatment is the inability to control infections, including ocular toxoplasmosis, the leading cause of infectious posterior uveitis worldwide. An ongoing T cell-mediated immune response is necessary to limit Toxoplasma gondii replication and destruction of the retina. The factors that support the immune response by allowing T cell entry into and/or influencing migratory behavior within the eye following infection remain poorly understood. Chemokines are crucial for T cell migration to sites of inflammation and our preliminary analysis demonstrates increased levels of CXCL10 transcript in the retina and that the majority of T cells that migrated into this site express CXCR3 (the receptor for CXCL10). In addition, administration of CXCL10-specific blocking antibodies leads to a decrease in T cell number in the eye and a corresponding increase in ocular pathology. Finally, preliminary data generated in this laboratory using multi-photon microscopy shows that T cells in the eyes of mice chronically with infected T. gondii exhibit a range of migratory behaviors. This proposal will focus specifically on the role of the CXCL10 on the CD8+ T cell response, which will be tested in the following specific aims: SPECIFIC AIM I. Do CXCL10 and CXCR3 affect the trafficking of CD8+ T cells during ocular toxoplasmosis? In order to examine the role of CXCL10 in T cell trafficking to the eye, a T cell transfer system will be developed, where activated T cells will be transferred to chronically infected mice and tracked using flow cytometry and immunohistochemistry (IHC). To test the hypothesis that CXCR3 and CXCL10 are required for T cell trafficking to the eye, anti-CXCL10 antibodies and CXCR3-/- T cells will be used. The effect of cell transfer on parasite burden in the presence or absence of chemokine will be examined using real-time PCR and IHC. SPECIFIC AIM II. Do CXCL10 and CXCR3 influence T cell migratory behavior in the retina during ocular toxoplasmosis? As chemokines influence various aspects of T cell movement and function (20), multi-photon intravital imagining of the retina is proposed, using fluorescently-labeled antigen-specific T cells and transgenic parasites to assess how the absence of CXCR3 or CXCL10 influence the behavior of T cells. Several parameters of migration will be assessed, including, velocity, meandering index, and motility coefficient. The studies outlined in this proposal will define the role of CXCL10 and CXCR3 in T cell-mediated control of T. gondii in the retina. Moreover, we envisage that our results will provide proof of principle for the utility of this model for examining the role of additional cytokines, chemokines, and immunotherapeutics. PUBLIC HEALTH RELEVANCE: Toxoplasma gondii infection of the eye is a cause of blindness. Many people are infected with this parasite, but do not develop eye disease due to a strong response by the immune system. This proposal will identify how the immune system prevents the parasite from causing destruction of the eye.