Roles of the H-2Db and H-Kb genes in resistance to persistent Theiler’s murine encephalomyelitis virus infection of the central nervous system

Arièle Azoulay-Caylab,1, Sylvie Syan1, Michel Brahic1 and Jean-François Bureau1

Unité des Virus Lents, CNRS URA 1930, Institut Pasteur, 28 rue du Dr Roux, 75724 Paris Cedex 15, France1

Author for correspondence: Michel Brahic. Fax +33 1 40 61 31 67. e-mail mbrahic{at}pasteur.fr


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Theiler’s murine encephalomyelitis virus, a member of the Picornaviridae family, persists in the spinal cord of susceptible strains of mice. Resistant strains of mice, such as the H-2b strain, clear the virus infection after an acute encephalomyelitis. The H-2D locus, but not the H-2K locus, has a major effect on this resistance, although both loci code for MHC class I molecules with similar general properties. For the present work, we rendered susceptible H-2q FVB/N mice transgenic for either the H-2Dbgene, the H-2Kb gene or a chimeric H-2Db/Kb gene in which the exons encoding the peptide-binding groove of the H-2Kb gene have been replaced by those of the H-2Dbgene. Mice transgenic for either the H-2Dbgene or the chimeric H-2Db/Kb gene were significantly more resistant to persistent virus infection than mice transgenic for the H-2Kb gene, suggesting that the difference in the effects of the H-2Dbgene and the H-2Kb gene are due to the nature of the peptides presented by these class I molecules.


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The DA strain of Theiler’s murine encephalomyelitis virus (TMEV), a member of the Picornaviridae family, is responsible for a biphasic disease of the murine CNS. The first phase is an acute encephalomyelitis, which takes place during the first 2 weeks after intracerebral inoculation. This is followed, only in genetically susceptible animals, by a persistent infection of the white matter of the spinal cord, resulting in inflammation and chronic primary demyelination. This natural disease is one of the best animal model systems available for multiple sclerosis (Dal Canto & Lipton, 1977 ; Monteyne et al., 1997 ). Susceptibility to the second phase of disease varies greatly among inbred strains of mice (Lipton & Dal Canto, 1979 ; Lipton & Melvold, 1984 ). Several genetic loci implicated in susceptibility to virus persistence, demyelination and clinical disease have been identified (Brahic & Bureau, 1998 ). A locus with a major effect on demyelination was first located in the H-2D region of the MHC (Clatch et al., 1985 ; Lipton & Melvold, 1984 ; Rodriguez & David, 1985 ; Rodriguez et al., 1986 ). Resistance was dominant over susceptibility(Patick et al., 1990 ). Studying susceptibility to persistent TMEV infection in 17 inbred strains of mice, we later showed that the b haplotype was associated with resistance, the q haplotype was associated with full susceptibility, and the k, d,and s haplotypes were associated with intermediate susceptibility. The resistance that was brought by the b haplotype was dominant. The locus controlling virus persistence was mapped to the H-2D region of the MHC (Bureau et al., 1992 ). It is most likely that the H-2 loci that control the susceptibility to persistent TMEV infection and susceptibility to demyelination are the same (Bureau et al., 1992 ; Patick et al., 1990 ; Rodriguez et al., 1986 ).

Several observations suggest that the susceptibility gene present in the H-2D region is an MHC class I H-2D gene (Azoulay-Cayla et al., 1994 , 2000 ; Fiette et al., 1993 ; Lin et al., 1997 ; Pullen et al., 1993 ; Rodriguez et al., 1986 , 1993 ). The role of the H-2Db gene in resistance was formally demonstrated by showing that (H-2q) FVB/N mice, made transgenic for the H-2Db gene, are resistant to virus persistence (Azoulay-Cayla et al., 1994 ; Rodriguez & David, 1995 ) and that mutations in the H-2Dbgene reduce or delay virus clearance (Lipton et al., 1995 ).

The highly polymorphic exons 2 and 3 of class I genes code for the peptide-binding groove of the class I molecule. The polymorphism of this groove explains the large repertoire of peptides that can be presented to CD8+ T cells. The class I genes of the mouse are located in two sub-regions of the MHC, H-2K and H-2D/L. To date, no difference in the ability to present peptides has been demonstrated between the families of H-2K and H-2D/L proteins. In spite of this, resistance to both TMEV persistence and virus-induced demyelination is linked to the H-2D locus, and not to the H-2K locus, in strains with b, d and k H-2 haplotypes (Rodriguez et al., 1986 ). Class I genes are not expressed in cells of a normal CNS: expression is induced during infection. Interestingly, the levels of expression of the H-2D and H-2K molecules in the CNS following inoculation with TMEV are different (Altintas et al., 1993 ). Therefore, the exclusive role of the H-2D locus in virus clearance could be due either to particularities of gene expression in the CNS or to the nature of the viral peptides presented.

To examine if the peptide-binding groove of the H-2Db molecule bore all the determinants for virus clearance, we constructed transgenic FVB/N mice expressing a chimeric H-2Kb gene in which exons 2 and 3 were replaced by the corresponding exons of the H-2Db gene.

The D1 line is an FVB/N mouse transgenic for the H-2Dbgene and is resistant to persistent TMEV infection (Azoulay-Cayla et al., 1994 ). Two lines of FVB/N mice transgenic for the H-2Kb gene, named K1 and K2, and three lines of FVB/N mice transgenic for a chimeric H-2Db/Kb gene, named D/K1, D/K2 and D/K3, were constructed for the present study. To construct the chimeric gene, a BssHII–SpeI fragment containing exons 2 and 3 of the H-2Dbgene was exchanged with the corresponding fragment of the H-2Kb gene. All transgenes were genomic DNA segments that included the authentic class I promoter (Allen et al., 1984 ).

The level of expression of each transgenic class I molecule was determined and compared to that of the H-2Db and H-2Kb proteins of C57BL/6 mice by FACS analysis on splenic T cells from two animals of each line. FVB/N and H-2Db-/- mice were used as controls. The monoclonal antibodies KH95 and 5F1, which are specific for H-2Db and H-2Kb molecules, were used at saturating concentrations (Hasenkrug et al., 1987 ; Pérarnau et al., 1999 ; Sherman & Randolph, 1981 ). The D1, D/K1, D/K2 and D/K3 transgenic lines expressed 31, 26·5, 49 and 34%, respectively, of the level of the H-2Db molecules measured for C57BL/6 mice. As expected, no H-2Db expression was detected in either FVB/N or H-2Db-/-mice. For the K1 line, the expression of the H-2Kb molecule was 68% of that measured for C57BL/6 mice. H-2Kb molecules were not detected for FVB/N mice. In summary, all transgenes were expressed at similar levels. The fact that we studied heterozygous transgenic mice might explain that the transgenes were expressed at lower levels than the H-2Db and the H-2Kb genes in C57BL/6 mice.

The chimeric H-2Db/Kb molecule possesses the peptide-binding groove of the H-2Db molecule within the context of an H-2Kb molecule. It was important to determine if the anti-TMEV CTLs of H-2Db/Kb transgenic mice recognized peptide VP2122–130, an immunodominant H-2Db-restricted epitope (Dethlefs et al., 1997 ). D/K2 and C57BL/6 mice were inoculated intraperitoneally with 106 p.f.u. of TMEV and the cytotoxicity of splenocytes was measured using H-2b C57SV cells infected with TMEV or loaded with the VP2122–130 peptide as targets. As shown in Fig. 1, splenocytes from D/K2 mice lysed uninfected target cells loaded with the VP2122–130 peptide. Therefore, D/K2 transgenic mice raise TMEV-specific CTLs that recognize the same H-2Db-restricted VP2 epitope as the CTLs of C57BL/6 mice.



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Fig. 1. TMEV-specific CTL responses in C57BL/6 and FVB/N mice transgenic for the H-2Db/Kb chimeric gene are shown. Splenocytes from C57BL/6 and D/K2 mice were tested in a 51Cr-release assay against different target cells. The target cells were (H-2b) C57SV fibroblasts infected with TMEV ({blacksquare}), loaded with the VP2122–130 peptide ({bullet}) or uninfected ({triangleup}). Error bars show SEM for an experiment performed in triplicate. When no bar is shown, the SEM was negligible.

 
To examine if FVB/N mice transgenic for either the H-2Db gene or the H-2Db/Kb gene were more resistant to persistent TMEV infection than FVB/N mice transgenic for the H-2Kb gene, virus loads in the CNS of different transgenic lines were measured at various times post-inoculation (p.i.) by using a dot-blot assay, as described previously (Bureau et al., 1992 ) (Table 1 and Fig. 2). Pooled results for mice with the same transgene are shown in Fig. 2. At 45 days p.i. (Fig. 2C), virus loads in the spinal cord were lower for FVB/N mice transgenic for the H-2Db gene than those for FVB/N mice transgenic for the H-2Kb gene (P=0·0433). For both lines, virus loads were significantly lower than those for control FVB/N mice. FVB/N mice transgenic for the H-2Db/Kb chimeric gene were as resistant to persistent TMEV infection as FVB/N mice transgenic for the H-2Db gene (P=0·9850). However, FVB/N mice transgenic for the H-2Db/Kb chimeric gene were significantly more resistant than FVB/N mice transgenic for the H-2Kb gene (P=0·0145). These results indicate that the peptide-binding groove of the H-2Db molecule is an important determinant of resistance to persistent TMEV infection.


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Table 1. Persistent TMEV infection in the CNS at 6, 21 and 45 days p.i.

 


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Fig. 2. The amount of viral RNA in the central nervous system at 6 (A), 21 (B) and 45 (C) days p.i. is shown. The highest RNA dilution that gave a positive hybridization signal in a dot-blot assay was used as a measure of the amount of viral RNA. Data from mice bearing the same transgene have been pooled. Means of virus load were compared using the Anova and Scheffe tests. Open bars, spinal cord; shaded bars, brain; n, number of animals per group.

 
The virus load of all of the transgenic animals was lower than that of parental FVB/N mice. This could indicate the existence of an H-2Kb-restricted anti-TMEV response that was not detected in the H-2b strains. It could also be related to the fact that transgenic mice bear an extra H-2 haplotype.

No correlation was observed between the level of expression of the various transgenes and the phenotype of the corresponding mouse line. Although expression was studied on splenocytes and not on virus-containing CNS cells, the lack of correlation suggests that the levels of expression of the transgenes were above a threshold that ensured full function.

In order to look for qualitative differences between the mouse lines, we examined the spinal cord of animals for both histopathological lesions and the presence of viral antigens 45 days p.i. Out of 31 FVB/N mice examined, 30 showed numerous inflammatory lesions of the white matter with large numbers of cells positive for viral antigen (data not shown). In seven out of the nine H-2Db transgenic mice and in 19 out of the 20 H-2Db/Kb transgenic mice examined, there was a mild inflammation occurring predominantly in the white matter, but occasionally in the grey matter, of the spinal cord. No cells positive for viral antigen were found in nine H-2Db transgenic mice or in 18 out of the 20 H-2Db/Kb transgenic mice examined. A small number of cells positive for viral antigen (fewer than five per longitudinal section) were found in two H-2Db/Kb transgenic mice. Of the eight H-2Kb transgenic mice examined, six mice had conspicuous inflammation in the white matter of the spinal cord with many cells containing viral antigens. Therefore, these data were congruent with those obtained by measuring the amount of viral RNA in the spinal cord. The levels of both inflammation and viral antigen were lower in H-2Db and H-2Db/Kb transgenic mice than those in H-2Kb transgenic mice. The lesions in the latter were similar to those of susceptible FVB/N animals, although they were less extensive.

To shed light upon the mechanism by which the transgenes H-2Db and H-2Db/Kb reduce virus load, we studied virus infection in the H-2Db and H-2Db/Kb transgenic lines and in FVB/N controls both during acute encephalomyelitis (6 days p.i.) and at the beginning of chronic infection (21 days p.i.). At 6 days p.i., viral RNA levels in both the brain and the spinal cord were the same for the three lines of mice examined (Fig. 2A). Histopathologically, all transgenic and non-transgenic FVB/N mice showed the same pattern of infection and inflammation. Infected cells were mainly neurons of the hippocampus, the temporal cortex and the anterior horns of the spinal cord. At 21 days p.i., H-2Dband H-2Db/Kb transgenic mice were each infected with low levels of TMEV (Fig. 2B). Neither inflammation nor viral antigens were present in the brains of either control mice or H-2Dband H-2Db/Kbtransgenic mice. Mild inflammation and a small number of cells positive for viral antigen were found in the spinal cord of H-2Dband H-2Db/Kbtransgenic mice. In contrast, extensive inflammation and many cells containing viral antigen were observed in FVB/N mice. These results showed that resistance to persistent TMEV infection of FVB/N mice transgenic for either the H-2Db gene or the H-2Db/Kb gene corresponded to the ability to clear the infection after an early acute encephalomyelitis.

Two hypotheses can be formulated regarding the association of resistance with the H-2D and not the H-2K locus. Either this is due to chance and to the small number of H-2 alleles that were studied. Accordingly, resistance will also be linked to the H-2K locus when more alleles are examined. Or it is due to an intrinsic property of the groove of the H-2D/L family of class I molecules which makes them more efficient at presenting TMEV epitopes.

In summary, our results confirm that the H-2Db gene is essential for virus clearance, whereas the H-2Kb gene has a more modest role. Also, these data strongly suggest that the peptide-binding groove of the H-2Db molecule contains all of the biological functions necessary to eliminate TMEV from the CNS.


   Acknowledgments
 
We thank C. Babinet and P. Marchand of the transgenesis facility of the Institut Pasteur for their help and M. Gau for preparing the manuscript. A.A.-C. was supported by a fellowship from the Fondation pour la Recherche Médicale and the Association pour la Recherche sur la Sclérose en Plaques. This research was supported by grants from the Institut Pasteur Foundation, the Centre National de la Recherche Scientifique, the National Multiple Sclerosis Society, USA, and the Association pour la Recherche sur la Sclérose en Plaques.


   Footnotes
 
b Present address: Fédération de Neurologie, Hôpital de la Salpêtrière, 75651 Paris Cedex 13, France.


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Received 9 October 2000; accepted 15 January 2001.