CD8+ T cells in acute and chronic demyelination

JHM病毒(JHMV)感染导致伴有原发性脱髓鞘的急性脑脊髓炎。在急性疾病期间，中枢神经系统(CNS)会产生强烈但部分无效的免疫反应。无法产生无菌免疫导致持续的中枢神经系统感染和慢性持续脱髓鞘，许多病理特征类似于多发性硬化症。CD8+T细胞是病毒清除的主要效应细胞。二 单独的CD8+T细胞效应器功能参与病毒清除。细胞毒性T淋巴细胞(CTL)介导的细胞溶解是病毒从星形胶质细胞和小胶质细胞清除的主要效应因子，而不是少突胶质细胞。相比之下，病毒在少突胶质细胞中的复制由干扰素-伽马(干扰素-伽马)控制。第一个目的是通过分析JHMV在新构建的转基因(TG)小鼠中的致病机制来检验干扰素-γ在预防病毒持续和慢性脱髓鞘方面的作用。在正常宿主的情况下，只有特定类型的中枢神经系统细胞不能对干扰素-γ做出反应，这为研究病毒复制的抑制和干扰素-γ免疫介导的中枢神经系统内的相互作用提供了一种新的工具。第二个目的是探讨少突胶质细胞对穿孔素介导的细胞溶解耐药的机制。这种不能改变少突胶质细胞中病毒复制的能力导致了病毒的持久性和慢性脱髓鞘。通过一种新的FACS方法从成人中枢神经系统中分离纯化少突胶质细胞、星形胶质细胞和小胶质细胞。CTL与这些靶点之间的相互作用将在体内和体内通过共聚焦显微镜进行检测。T细胞活化、穿孔素、颗粒酶B和颗粒酶B受体的定位将决定耐药机制。纯化的细胞还将在CTL与靶细胞相互作用前后检测抗凋亡基因(Bcl2)的表达。这种方法解决了一种可能性，而不是穿孔素传递或激活的缺陷，而是通过抑制细胞内信号事件来防止少突胶质细胞死亡。第三个目的是针对CTL介导的效应功能在竞争病毒清除之前在中枢神经系统内丢失的观察结果。这种效应器功能的丧失导致CTL无法完全消除JHMV。中枢神经系统细胞溶解活性的丧失将与干扰素-GammaR2受体的调节有关。这种受体最近被证明可以调节CTL的细胞溶解活性。此外，在急性JHMV感染期间，通过将来自中枢神经系统的CTL与体内丧失细胞溶解活性的中枢神经系统来源的细胞混合，研究了在急性JHMV感染期间诱导T细胞调节/抑制群的情况。这项建议侧重于急性感染期间中枢神经系统内免疫反应的调节。我们的假设是，导致CD8+T细胞保护性免疫反应不能完全清除CNS病毒的机制是病毒持续存在和慢性脱髓鞘的关键。

JHM virus (JHMV) infection results in an acute encephalomyelitis accompanied by primary demyelination. During acute disease a vigorous, but partially ineffective, immune response occurs within the central nervous system (CNS). The inability to effect sterilizing immunity results in persistent CNS infection and chronic ongoing demyelination with many pathological hallmarks similar to multiple sclerosis. CD8+ T cells are the predominant effectors of virus clearance. Two separate CD8+ T cell effector functions participate in virus clearance. Cytotoxic T lymphocyte (CTL) mediated cytolysis is the major effector of virus clearance from astrocytes and microglia, but not oligodendroglia. By contrast, virus replication in oligodendroglia is controlled by interferon gamma (IFN-gamma). The first Aim examines the role of IFN-gamma in preventing viral persistence and chronic demyelination by analysis of JHMV pathogenesis in newly constructed transgenic (TG) mice which express a decoy IFN-gammaR1 specifically in either oligodendroglia or astrocytes. The inability of only a specific CNS cell type to respond to IFN-gamma, within the context of an otherwise normal host, provides a novel tool to examine both inhibition of virus replication and the IFN-gamma immune mediated interactions within the CNS. The second Aim address the mechanism of oligodendroglial resistance to perforin mediated cytolysis. This inability to alter virus replication in oligodendroglia contributes to viral persistence and chronic demyelination. Oligodendroglia, astrocytes and microglia will be purified from the adult CNS by a newly develop FACS approach. Interactions between CTL and these targets will be examined in and in vivo by confocal microscopy. T cell activation, localization of perforin, Granzyme B and the Granzyme B receptor will define the mechanism of resistance. Purified cells will also be examined for expression of anti-apoptotic genes (Bcl-2) before and after CTL-target interaction. This approach addresses the possibility rather than a defect in delivery or activation of perforin, oligodendroglial death is prevented via inhibition of intracellular signaling events. The third Aim addresses the observation that CTL mediated effector functions within the CNS are lost prior to the compete virus clearance. This loss of effector function contributes to the inability of CTL to completely eliminate JHMV. Loss of CNS cytolytic activity will be correlated with regulation of the IFN-gammaR2 receptor. This receptor has recently been shown to regulate CTL cytolytic activity. In addition, induction of a T cell regulatory/suppressor population during acute JHMV infection is examined by mixing CTL derived from the CNS at the time of maximal expression of ex vivo cytolysis with cells derived from the CNS following in vivo loss of cytolytic activity. This proposal focuses on regulation of the immune response within the CNS during acute infection. Our hypothesis is that the mechanism which contribute to the inability of the CD8+ T cell protective immune response to completely eliminate CNS virus are critical to viral persistence and chronic demyelination.