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）从感觉神经元中的潜伏状态重新激活并顺行转运至角膜似乎是复发性疱疹性角膜炎（角膜致盲性疾病）的主要原因。最近的证据，从我们的实验室和其他人强烈支持的免疫监视作用，CD 8 + T细胞在维持HSV-1在潜伏状态的感觉神经元，并证明IFN-？被用来阻止一些但不是所有神经元的再激活。对潜伏感染组织内影响记忆反应的因素了解较少。该提案的目标有两个方面：具体目标1将确定和表征CD 8 + T细胞用于抑制IFN-？难治性神经元中HSV-1再活化的效应机制;和特异性目标2将鉴定和表征负责在潜伏感染的TG中维持HSV特异性记忆CD 8+群体的因素。在具体目标1中，我们将证实初步数据强烈暗示CD 8 + T细胞溶解颗粒，特别是颗粒酶B（GrB）组分在控制HSV-1潜伏期，并检验使用溶解颗粒不会导致神经元破坏的假设。我们将比较在存在和不存在GrB和半胱天冬酶抑制剂的情况下，来自野生型、穿孔素缺陷（Pfn-/-）和GrB-/-小鼠的HSV特异性CD 8 + T细胞在体内和离体TG培养物中阻断HSV-1再活化的能力;并使用真实的时间活细胞荧光成像来直接确定CD 8 + T细胞是否向神经元中释放溶解颗粒，以及这种相互作用是否导致半胱天冬酶系统的激活和神经元的凋亡。我们还将测试的假设，即HSV-1的调节蛋白ICP 4，通过预测的GrB切割位点，与半胱天冬酶竞争作为GrB底物，并通过切割导致在GrB抑制HSV-1的再激活没有神经元的破坏失活。在特定目标2中，我们将产生骨髓嵌合体小鼠，其含有野生型CD 8 + T细胞和负责记忆性CD 8 + T细胞的稳态增殖和存活的细胞因子受体缺陷的那些细胞，并用缺乏免疫显性表位gB 498 -505的重组病毒感染它们，以比较维持HSV特异性CD 8 + T细胞的需要。在潜伏感染的TG中的记忆群体，表达或不表达同源抗原。我们还将测试潜伏病毒对CD 4 + T细胞帮助产生功能性CD 8 + T细胞记忆的需求的影响，并确定在TG中维持CD 8+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CD 8 + T细胞记忆的影响，这是疫苗设计中经常被忽视的重要因素。 公共卫生相关性。单纯疱疹病毒性角膜病的复发性发作导致进行性角膜瘢痕形成，并且是世界范围内失明的重要原因。这种疾病的复发性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。该补助金的目标是提供宿主免疫系统用于防止病毒从潜伏状态重新激活的机制的理解，因为这些信息将需要优化预防复发性疱疹疾病所需的宿主免疫反应。

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.