Class II-restricted T cell responses in Theiler's murine encephalomyelitis virus (TMEV)-induced demyelinating disease. I. Cross-specificity among TMEV substrains and related picornaviruses, but not myelin proteins

Following intracerebral inoculation of Theiler's murine encephalomyelitis virus (TMEV), susceptible mouse strains develop a chronic demyelinating disease characterized by mononuclear cell-rich infiltrates in the central nervous system. Current evidence strongly supports an immune-mediated basis for myelin breakdown, with an effector role proposed for TMEV-specific, major histocompatibility class II-restricted delayed-type hypersensitivity, which temporally correlates with disease onset and remains chronically elevated in susceptible mice. This study examined the fine specificity of class II-restricted T cell responses in TMEV-infected mice to better define the relevant virus-encoded T cell determinant(s) responsible for triggering the demyelinating process, and to determine if class II-restricted neuroantigen-specific autoimmune responses could be detected in mice with TMEV-induced demyelination. The data clearly show that T cell responses in TMEV-infected mice are directed against determinants shared by closely related TMEV strains and are cross-reactive with related picornaviruses, such as encephalomyocarditis virus. In contrast, class II-restricted autoimmune responses against syngeneic mouse spinal cord homogenate and the two major protein components of myelin, myelin basic protein and proteolipid protein, are not demonstrable in susceptible SJL/J mice undergoing chronic TMEV-induced demyelinating disease, but are readily seen in SJL/J mice displaying chronic, relapsing experimental allergic encephalomyelitis. Cross-reactivity (or lack thereof), as determined by functional T cell analyses, was found to correlate with the extent of exact amino acid homology between the TMEV capsid proteins, the two neuroantigens, and related picornaviruses. The data thus do not support a major role for autoimmune responses against myelin proteins in TMEV-induced demyelinating disease, but are consistent with our previously proposed hypothesis that TMEV-specific T cell responses constitute a major effector mechanism of myelin breakdown.     

Hierarchy of effects of the MHC and T cell receptor beta-chain genes in susceptibility to Theiler's murine encephalomyelitis virus-induced demyelinating disease.

Intracerebral inoculation of susceptible mice with Theiler's murine encephalomyelitis virus induces a demyelinating disease that is similar to human multiple sclerosis. This murine model for human multiple sclerosis is apparently immune-mediated and the genes involved in the immune response influence the outcome of disease susceptibility as observed with human multiple sclerosis. These genes include the MHC and TCR genes. However, the functional relationships among these genes on the disease susceptibility has not yet been studied. In this study, we demonstrate that the effect of the H-2s genotype from susceptible SJL/J mice overrides the resistant effect of the BALB/c TCR beta-chain gene in CXJ recombinant-inbred and BALB.S congenic mice. These results strongly suggest the presence of a hierarchy of genes involved in the immune response in Theiler's murine encephalomyelitis virus-induced demyelinating disease.     

Identification of a locus on mouse chromosome 3 involved in differential susceptibility to Theiler's murine encephalomyelitis virus-induced demyelinating disease.

Theiler's virus-induced demyelinating disease results from a chronic infection in the white matter of the central nervous system and provides an excellent model for human multiple sclerosis. Like multiple sclerosis, there are genetic risk factors in disease development, including genes associated with the major histocompatibility complex and with those encoding the beta chain of the T-cell receptor. Comparisons of the susceptible DBA/2 and resistant C57BL/6 strains have indicated an important role for the H-2D locus and for a non-H-2 gene (not involving the beta chain of the T-cell receptor) in differential susceptibility. In the present report, analysis of recombinant-inbred strains (BXD) between the DBA/2 and C57BL/6 strains indicated that this non-H-2 locus is located at the centromeric end of chromosome 3 near (4 +/- 4 centimorgans) the carbonic anhydrase-2 (Car-2) enzyme locus.     

The predominant virus antigen burden is present in macrophages in Theiler's murine encephalomyelitis virus-induced demyelinating disease.

Theiler's murine encephalomyelitis virus (TMEV) produces a persistent central nervous system infection and chronic, inflammatory demyelinating disease in susceptible mice. TMEV antigen(s) and RNA genome have been detected in astrocytes, oligodendrocytes, and macrophages during persistence. Whether there is a predominant cell type in which TMEV persists has not been resolved. Since TMEV-induced demyelinating lesions are infiltrated with macrophages and a number of other persistent viruses show near-exclusive tropism for these phagocytic cells, we used two-color immunofluorescent staining with conventional and confocal microscopy to colocalize TMEV to cells that stain with monoclonal antibodies (MOMA-2) [unknown antigen], Mac-1 [CD11b], FA-11 [CD66], and 2F8 [scavenger receptor]) to macrophages in BeAn-infected SJL mice. A predominant virus antigen burden within macrophages infiltrating demyelinating lesions was seen. A dichotomy of cells staining for virus antigen(s) was found with infected cells containing either a large or small virus antigen load. Ninety percent of cells with a large virus antigen load were large phagocytes (20 to 50 microns) that were readily detected at low power (5x objective). Cells with smaller amounts of virus antigen(s) turned out to be either these same large phagocytic cells or much smaller cells, approximately equal to 10 microns in diameter. Forty percent of cells with a small virus antigen load were macrophages. The unidentified approximately equal to 10-microns cells that are virus antigen positive and macrophage negative in this study could still be macrophages, or they may be oligodendrocytes. The fact that virus was detected in the cytoplasm and not phagolysosomes of macrophages and the sheer mass of fluorescently stained virus proteins in some macrophages suggest that TMEV persists in these phagocytic cells by active virus replication.     

Importance of amino acid 101 within capsid protein VP1 for modulation of Theiler's virus-induced disease.

We constructed a Theiler's virus mutant designated DA3304, in which the amino acid at position 101 of VP1 was changed from a threonine to an alanine. Because of this single amino acid change, DA3304 could still produce a biphasic central nervous system disease similar to that produced by the wild-type DA virus. However, DA3304 was significantly attenuated in both the acute and the chronic phases and induced smaller demyelinating lesions than the wild-type DA virus. The data are most compatible with the attenuated phenotype in DA3304 being due to the change of binding efficiency between the virus and receptor resulting from the physical alteration at the mutation site.     

Variations in genetic control of susceptibility to Theiler's murine encephalomyelitis virus (TMEV)-induced demyelinating disease. I. Differences between susceptible SJL/J and resistant BALB/c strains map near the T cell beta-chain constant gene on chromosome 6

In some susceptible mouse strains, intracerebral (IC) inoculation of Theiler's murine encephalomyelitis virus (TMEV) results in a persistent infection leading to chronic demyelinating disease. Previous genetic analyses between susceptible SJL/J and resistant C57BL/6 mice indicated a role for multiple unlinked genes in the development of clinical and histopathological disease, including a major influence of the D region of the H-2 complex. In this study, genetic analysis of a different strain combination (susceptible SJL/J and resistant BALB/c) also demonstrates the involvement of multiple genes, but the H-2 genotype (H-2s and H-2d, respectively) does not appear to contribute significantly to susceptibility differences. In both segregation studies and recombinant-inbred (R-I) analysis, clinical and histopathological disease occurs in both H-2s homozygotes and H-2d homozygotes (as well as H-2s/H-2d heterozygotes), with the actual frequency related to the proportion of non-H-2 genome from the susceptible strain. There appear to be at least two non-H-2 genes involved in differential susceptibility of SJL/J and BALB/c to TMEV-induced disease. Analysis of R-I strains generated from BALB/c and SJL/J progenitors indicates linkage of at least one of these non-H-2 genes to those encoding the constant portion of the beta-chain of the T cell receptor on chromosome 6. Many genes may actually be involved, but each strain comparison defines a different subset of these loci--only those at which the two strains in question carry "functionally" different alleles. Thus, different strain comparisons may accent the roles of different genes in resistance to the same infectious organism or disease process. In addition to the genes identified thus far, there may be yet other genes contributing to development of TMEV-induced disease, but their recognition may require analysis of still other strain combinations.     

Expression and potential role of inducible nitric oxide synthase in the central nervous system of Theiler's murine encephalomyelitis virus-induced demyelinating disease.

Intracerebral inoculation of susceptible strains of mice with Theiler's murine encephalomyelitis virus (TMEV) results in immune-mediated demyelinating disease. We examined the pathogenic roles of nitric oxide (NO) and inducible NO synthase (iNOS) in TMEV-induced demyelinating disease (TMEV-IDD). The presence of iNOS was confirmed in the spinal cords of TMEV-infected mice using immunohistochemical staining with anti-iNOS antibody on day 0 (control) and days 15, 30, 60, and 120. Aminoguanidine (AG), a specific inhibitor of iNOS, was injected intraperitoneally (ip) on 1, 3, 5, 8, 10, and 12 days post-TMEV inoculation as induction phase or 15, 17, 19, 22, 24, and 26 days as effector phase. Control animals in each experiment received phosphate-buffered saline (PBS) ip at similar time intervals. Few iNOS-positive cells were observed in the spinal cords of naive SJL/J mice. In the early phase (day 15) of TMEV-IDD, an increase of iNOS-positive cells was detected in the leptomeninges and perivascular space of the spinal cords. The number of iNOS-positive cells was increased and reached its peak on day 60, when histology of the animals showed peak infiltration with inflammatory cells. The clinical course of TMEV-IDD on each day postintracerebral infection was significantly reduced in mice treated with AG in the effector phase, and there was no significant difference between mice treated with AG in induction phase versus those administered PBS. Thus, NO production via iNOS appears to be a pathogenic factor in the effector phase of TMEV-IDD.     

Identification of a major T-cell epitope within VP3 amino acid residues 24 to 37 of Theiler's virus in demyelination-susceptible SJL/J mice.

Intracerebral inoculation of susceptible strains of mice with Theiler's murine encephalomyelitis virus (TMEV) results in a chronic, immunologically mediated demyelinating disease that shares many features with human multiple sclerosis. CD4+ T lymphocytes play a critical role in the pathogenesis of virus-induced demyelinating disease. We have identified a region within amino acid residues 24 to 37 of the VP3 capsid protein of TMEV (VP3(24-37)) that is recognized by T lymphocytes from the demyelination-susceptible SJL/J strain of mice. The T-cell response to VP3(24-37) represents a predominant Th-cell response against the virus from either TMEV-immunized or TMEV-infected SJL/J mice, and viral epitopes VP1(233-250), VP2(74-86), and VP3(24-37) account for most of the Th-cell response to TMEV.     

Lymphocytes from SJL/J mice immunized with spinal cord respond selectively to a peptide of proteolipid protein and transfer relapsing demyelinating experimental autoimmune encephalomyelitis.

Relapsing experimental autoimmune encephalomyelitis (R-EAE) can be induced in SJL/J mice by immunization with spinal cord homogenate and adjuvant. The specific Ag(s) responsible for acute disease and subsequent relapses in this model is unknown. Myelin basic protein (BP), an encephalitogenic peptide of BP (BP 87-99), and proteolipid protein (PLP) can each induce R-EAE in SJL/J mice, and a peptide of PLP (PLP 139-151) has been reported to induce acute EAE. To determine the encephalitogens in cord-immunized mice with R-EAE, the in vitro proliferative responses of lymph node cells (LNC) and central nervous system mononuclear cells to BP, BP peptides, and PLP peptides were examined during acute EAE and during relapses. LNC responded only to PLP peptides 139-151 and 141-151 and did not respond to BP or its peptides during acute or chronic disease. Central nervous system mononuclear cells also preferentially responded to PLP 139-151 and 141-151 during acute and relapsing disease. A PLP 139-151 peptide-specific Th cell line was selected from LNC of cord-immunized donors. Five million peptide-specific line cells transferred severe relapsing demyelinating EAE to naive recipients. We conclude that PLP peptide 139-151 is the major encephalitogen for R-EAE in cord-immunized SJL/J mice. We demonstrate for the first time that Th cells specific for this peptide are sufficient to transfer relapsing demyelinating EAE. The predominance of a PLP immune response rather than a BP response in SJL/J mice suggests that genetic background may determine the predominant myelin Ag response in human demyelinating diseases such as multiple sclerosis.     

Differences in avidity and epitope recognition of CD8(+) T cells infiltrating the central nervous systems of SJL/J mice infected with BeAn and DA strains of Theiler's murine encephalomyelitis virus

Theiler's murine encephalomyelitis virus (TMEV) infection induces immune-mediated demyelinating disease in susceptible mouse strains and serves as a relevant infectious model for human multiple sclerosis. To investigate the pathogenic mechanisms, two strains of TMEV (DA and BeAn), capable of inducing chronic demyelination in the central nervous system (CNS), have primarily been used. Here, we have compared the T-cell responses induced after infection with DA and BeAn strains in highly susceptible SJL/J mice. CD4(+) T-cell responses to known epitopes induced by these two strains were virtually identical. However, the CD8(+) T-cell response induced following DA infection in susceptible SJL/J mice was unable to recognize two of three H-2K(s)-restricted epitope regions of BeAn, due to single-amino-acid substitutions. Interestingly, T cells specific for the H-2K(s)-restricted epitope (VP1(11-20)) recognized by both strains showed a drastic increase in frequency as well as avidity after infection with DA virus. These results strongly suggest that the level and avidity of virus-specific CD8(+) T cells infiltrating the CNS could be drastically different after infection with these two strains of TMEV and may differentially influence the pathogenic and/or protective outcome.