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.
