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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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 BssHIISpeI 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 VP2122130, 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 VP2122130 peptide as targets. As shown in Fig. 1
, splenocytes from D/K2 mice lysed uninfected target cells loaded with the VP2122130 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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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.
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Footnotes |
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References |
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Received 9 October 2000;
accepted 15 January 2001.