Cytotoxic Lymphocytes and HSV-1 Corneal Lesions

描述(由申请人提供)： 单纯疱疹病毒1型(HSV-1)在感觉神经元潜伏状态的重新激活和向角膜的顺行运输似乎是复发的疱疹角膜炎的主要原因，疱疹角膜炎是一种致盲的角膜疾病。最近来自我们实验室和其他实验室的证据有力地支持了CD8+T细胞在维持HSV-1在感觉神经元潜伏状态中的免疫监视作用，并证明了干扰素？被用来阻断部分但不是所有神经元的重新激活。对潜伏感染组织中影响记忆反应的因素知之甚少。这项建议的目标有两个：特定目标1将识别和表征CD8+T细胞用来抑制对干扰素-1无效的神经元中HSV-1重新激活的效应机制；特定目标2将识别和表征负责在潜伏感染的TG中维持HSV特异性记忆CD8+群体的因素。在具体目标1中，我们将证实CD8+T细胞裂解颗粒，特别是成分颗粒酶B(GRB)在控制HSV-1潜伏期方面的初步数据，并检验使用裂解颗粒不会导致神经元破坏的假设。我们将比较来自野生型、穿孔素缺陷(PFN-/-)和GRB-/-小鼠的HSV特异性CD8+T细胞在GRB和caspase抑制剂存在和不存在的情况下阻止体内和体外TG培养中HSV-1重新激活的能力；并使用实时细胞荧光成像来直接确定CD8+T细胞是否向神经元释放裂解颗粒，以及这种相互作用是否导致caspase系统的激活和神经元的凋亡。我们还将测试这样的假设，即HSV-1调节蛋白ICP4通过预测的GRB裂解位点与作为GRB底物的半胱氨酸酶竞争，并被裂解失活，从而在不破坏神经元的情况下抑制HSV-1的重新激活。在特定目标2中，我们将产生含有野生型CD8+T细胞的骨髓嵌合体小鼠，以及那些负责稳态增殖和记忆性CD8+T细胞存活的细胞因子受体缺乏的小鼠，并用缺乏免疫优势表位gB498-505的重组病毒感染它们，以比较在潜伏感染的TG中维持HSV特异性CD8+记忆群的需求。我们还将测试潜伏病毒对产生功能性CD8+T细胞记忆的CD4+T细胞的需求的影响，并确定在TG中维持CD8+记忆群体是否需要从外周血中补充。这些研究将确定组织微环境对维持CD8+T细胞记忆的影响，这是疫苗设计中经常被忽视的一个重要因素。与公共卫生相关。反复发作的单纯疱疹病毒角膜疾病导致进行性角膜瘢痕形成，是全世界失明的重要原因。这种疾病的复发本质是由于病毒能够在感觉神经元中建立潜伏(静止)感染，然后周期性地重新激活并感染由这些神经元支配的组织。这笔赠款的目的是了解宿主免疫系统用来防止病毒从潜伏状态重新激活的机制，因为这些信息将是优化宿主免疫反应所必需的，以防止疱疹复发。

DESCRIPTION (provided by applicant): Reactivation of herpes simplex virus type 1 (HSV-1) from a latent state in sensory neurons and anterograde transport to the cornea appears to be a predominant cause of recurrent herpes keratitis, a blinding disease of the cornea. Recent evidence from our laboratory and others strongly support an immunosurveillance role for CD8+ T cells in maintaining HSV-1 in a latent state in sensory neurons, and demonstrate that IFN-? is employed to block reactivation in some, but not all neurons. Less well understood are the factors within latently infected tissue that influence the memory response. The goals of this proposal are two-fold: Specific Aim 1 will identify and characterize the effector mechanism used by CD8+ T cells to inhibit HSV-1 reactivation in neurons that are refractory to IFN-?; and Specific Aim 2 will identify and characterize the factors responsible for maintaining the HSV-specific memory CD8+ population in the latently infected TG. In Specific Aim 1 we will confirm preliminary data strongly implicating CD8+ T cell lytic granules and specifically the component granzyme B (GrB) in controlling HSV-1 latency, and test the hypothesis that the use of lytic granules does not lead to neuronal destruction. We will compare the capacity of HSV-specific CD8+ T cells from wild type, perforin deficient (Pfn-/-) and GrB-/- mice to block HSV-1 reactivation in vivo and in ex vivo TG cultures in the presence and absence of GrB and caspase inhibitors; and use real time live cell fluorescence imaging to directly determine if CD8+ T cells release lytic granules into neurons, and if this interaction leads to activation of the caspase system and apoptosis in neurons. We will also test the hypothesis that the HSV-1 regulatory protein ICP4, via predicted GrB cleavage sites, competes with caspases as GrB substrate and is inactivated by cleavage resulting in GrB inhibition of HSV-1 reactivation without neuronal destruction. In Specific Aim 2 we will generate bone marrow chimera mice containing wild type CD8+ T cells and those deficient in receptors for cytokines responsible for homeostatic proliferation and survival of memory CD8+ T cells, and infect them with recombinant virus lacking the immunodominant epitope gB498-505 to compare the requirements for maintaining an HSV-specific CD8+ memory population in latently infected TG that do or do not express cognate antigen. We will also test the effect of latent virus on the requirement for CD4+ T cell help in generating functional CD8+ T cell memory, and determine if maintaining a CD8+ memory population in the TG requires replenishment from the peripheral blood. These studies will define the effect of a tissue microenvironment on maintenance of CD8+ T cell memory, an important factor that is often overlooked in vaccine design. PUBLIC HEALTH RELEVANCE. Recurrent bouts of herpes simplex virus corneal disease result in progressive corneal scarring and represent an important cause of blindness world-wide. The recurrent nature of the disease is due to the ability of the virus to establish a latent (quiescent) infection in sensory neurons, and then periodically reactivate and infect the tissue innervated by those neurons. The goal of this grant is to provide an understanding of the mechanisms used by the host immune system to prevent the virus from reactivating from the latent state, as this information will be required to optimize the host immune response necessary to prevent recurrent herpetic disease.