Role of chemokines in the T cell response to ocular toxoplasmosis

描述(申请人提供)：在正常情况下，免疫系统的细胞在很大程度上被排除在眼睛之外。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主要集中在防止T细胞向免疫特异体组织的运输。然而，这种治疗的一个不可预见的并发症是无法控制感染，包括眼弓形体病，这是全球感染性后葡萄膜炎的主要原因。持续的T细胞介导的免疫反应对于限制弓形虫的复制和对视网膜的破坏是必要的。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反应的因素仍然知之甚少。趋化因子是T细胞迁移到炎症部位的关键，我们的初步分析表明，视网膜中CXCL10转录本的水平增加，大多数迁移到这个部位的T细胞表达CXCR3(CXCL10的受体)。此外，给予CXCL10特异性阻断抗体会导致眼内T细胞数量减少，眼部病理改变相应增加。最后，该实验室使用多光子显微镜产生的初步数据显示，慢性感染弓形虫的小鼠眼睛中的T细胞表现出一系列迁移行为。这项建议将特别关注CXCL10在CD8+T细胞反应中的作用，这将在以下特定目标中进行测试：特异性AIM I。CXCL10和CXCR3在眼弓形体感染过程中是否影响CD8+T细胞的运输？为了研究CXCL10在T细胞转运到眼睛中的作用，将开发一种T细胞转移系统，在该系统中，激活的T细胞将被转移到慢性感染的小鼠身上，并使用流式细胞仪和免疫组织化学(IHC)进行追踪。为了验证T细胞转运到眼睛需要CXCR3和CXCL10的假设，将使用抗CXCL10抗体和CXCR3-/-T细胞。在有或没有趋化因子的情况下，细胞转移对寄生虫负荷的影响将使用实时聚合酶链式反应和免疫组化进行检测。特异性目的II：CXCL10和CXCR3是否影响眼弓形体感染时T细胞在视网膜中的迁移行为？由于趋化因子影响T细胞运动和功能的各个方面(20)，提出了多光子视网膜活体成像，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CXCR3或CXCL10的缺失如何影响T细胞的行为。将评估几个迁移参数，包括速度、曲折指数和运动系数。这项提案中概述的研究将确定CXCL10和CXCR3在T细胞介导的视网膜弓形虫控制中的作用。此外，我们预计，我们的结果将为该模型用于检测额外的细胞因子、趋化因子和免疫疗法的作用提供原则上的证据。 公共卫生相关性：弓形虫感染眼睛是致盲的原因之一。许多人感染了这种寄生虫，但由于免疫系统的强烈反应，他们不会患上眼病。这项提案将确定免疫系统如何防止寄生虫对眼睛造成破坏。

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.