Njenga, M. Kariuki

Abstract We addressed the contributions of persistent virus infection and immune cells to the pathogenesis of Theiler's virus-induced demyelination, a model for human multiple sclerosis. We developed a model involving the transfer of spleen cells into immunodeficient C.B-17-scid (SCID) mice, which normally die of overwhelming virus encephalitis without demyelination when infected with Theiler's virus. Adoptive transfer of nonimmune spleen cells from BALB/c mice into SCID mice resulted in the survival of all mice. However, these mice developed extensive demyelination and virus Ag/RNA persistence in the spinal cord white matter. The most demyelination was observed when mice received an intermediate number of spleen cells (1.8-7.5 x 10(6)), whereas too few cells (0.5 x 10(6)) did not ameliorate the SCID phenotype, and too many cells (30 x 10(6)) resulted in almost complete viral clearance with minimal demyelination. Adoptive transfer of spleen cells depleted of either CD4+ or CD8+ T cells produced vacuolar demyelination associated with virus persistence. In contrast, reconstitution with both CD4+ and CD8+ T cells produced less severe demyelination and partial clearance of virus. These experiments support the hypothesis that demyelination is the result of a balance between persistent virus infection and immune injury mediated by either CD4+ or CD8+ T cells.

Infection of susceptible strains of mice with Daniel's (DA) strains of Theiler's murine encephalomyelitis virus (DAV) results in virus persistence in the central nervous system (CNS) white matter and chronic demyelination similar to that observed in multiple sclerosis. We investigated whether persistence is due to the immune system more efficiently clearing DAV from gray than from white matter of the CNS. Severe combined immunodeficient (SCID) and immunocompetent C.B-17 mice were infected with DAV to determine the kinetics, temporal distribution, and tropism of the virus in CNS. In early disease (6 h to 7 days postinfection), DAV replicated with similar kinetics in the brains and spinal cords of SCID and immunocompetent mice and in gray and white matter. DAV RNA was localized within 48 h in CNS cells of all phenotypes, including neurons, oligodendrocytes, astrocytes, and macrophages/microglia. In late disease (13 to 17 days postinfection), SCID mice became moribund and permitted higher DAV replication in both gray and white matter. In contrast, immunocompetent mice cleared virus from the gray matter but showed replication in the white matter of their brains and spinal cords. Reconstitution of SCID mice with nonimmune splenocytes or anti-DAV antibodies after establishment of infection demonstrated that both cellular and humoral immune responses decreased virus from the gray matter; however, the cellular responses were more effective. SCID mice reconstituted with splenocytes depleted of CD4+ or CD8+ T lymphocytes cleared virus from the gray matter but allowed replication in the white matter. These studies demonstrate that both neurons and glia are infected early following DAV infection but that virus persistence in the white matter is due to preferential clearance of virus from the gray matter by the immune system.

Mice with targeted disruption of the A beta gene of major histocompatibility complex class II molecules (Abo) were used to investigate the role of class II gene products in resistance or susceptibility to virus-induced chronic demyelination in the central nervous system (CNS). Class-II-deficient mice from the resistant H-2b [H-2b(Abo)] and nonmutant H-2b backgrounds were infected with Theiler's murine encephalomyelitis virus intracerebrally and examined for CNS virus persistence, demyelination, and neurologic clinical signs. Virus titers measured by plaque assays showed that 8 of 10 normally resistant nonmutant H-2b mice had cleared the virus within 21 days, whereas the other 2 mice had low titers. In contrast, all class II-deficient Abo mice had high virus titers for up to 90 days after infection (4.30 log10 PFU per g of CNS tissue). Virus antigens and RNA were localized to the brains (cortex, hippocampus, thalamus, and brain stem) and spinal cords of Abo mice. Colocalization identified persistent Theiler's murine encephalomyelitis virus in oligodendrocytes and astrocytes but not in macrophages. There was demyelination in 11 of 23 and 6 of 9 Abo mice 45 and 90 days after virus infection, respectively, whereas no demyelination was observed in infected nonmutant H-2b mice. Demyelinating lesions in Abo mice showed virus-specific CD8+ T cells and macrophages but no CD4+ T cells. Spasticity and paralysis were observed in chronically infected Abo mice but not in the nonmutant H-2b mice. These findings demonstrate that class II gene products are required for virus clearance from the CNS but not for demyelination and neurologic disease.

ABSTRACTC57BL/6 mice mount a cytotoxic T-lymphocyte (CTL) response against the Daniel’s strain of Theiler’s murine encephalomyelitis virus (TMEV) 7 days after infection and do not develop persistent infection or the demyelinating syndrome similar to multiple sclerosis seen in susceptible mice. The TMEV capsid peptide VP2121–130sensitizesH-2Db+target cells for killing by central-nervous-system-infiltrating lymphocytes (CNS-ILs) isolated from C57BL/6 mice infected intracranially. Db:VP2121–130peptide tetramers were used to stain CD8+CNS-ILs, revealing that 50 to 63% of these cells bear receptors specific for VP2121–130presented in the context of Db. No T cells bearing this specificity were found in the cervical lymph nodes or spleens of TMEV-infected mice.H-2bmice lacking CD4, class II, gamma interferon, or CD28 expression are susceptible to persistent virus infection but surprisingly still generate high frequencies of CD8+, Db:VP2121–130-specific T cells. However, CD4-negative mice generate a lower frequency of Db:VP2121–130-specific T cells than do class II negative or normalH-2banimals. Resistant tumor necrosis factor alpha receptor I knockout mice also generate a high frequency of CD8+CNS-ILs specific for Db:VP2121–130. Furthermore, normally susceptible FVB mice that express a Dbtransgene generate Db:VP2121–130-specific CD8+CNS-ILs at a frequency similar to that of C57BL/6 mice. These results demonstrate that VP2121–130presented in the context of Dbis an immunodominant epitope in TMEV infection and that the frequency of the VP2121–130-specific CTLs appears to be independent of several key inflammatory mediators and genetic background but is regulated in part by the expression of CD4.