Vß8+ T cells protect from demyelinating disease in a viral model of multiple sclerosis

Kristen M. Drescher1,2,4, Sean L. Johnston2, William Hogancamp1, Gerald H. Nabozny2, Chella S. David2, Ilonna J. Rimm3, Peter J. Wettstein2 and Moses Rodriguez1,2

1 Departments of Neurology and
2 Immunology, Mayo Clinic/Foundation, Rochester, MN 55905, USA
3 Department of Pediatric Oncology, Dana Farber Cancer Institute/Children's Hospital, Boston,MA 02115, USA

Correspondence to: M. Rodriguez, Department of Immunology, Mayo Clinic/Foundation, Guggenheim 428, 200 First Street SW, Rochester, MN 55905, USA


    Abstract
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Previous studies illustrated the influence of T cell subsets on susceptibility or resistance to demyelination in the Theiler's murine encephalomyelitis virus (TMEV) model of multiple sclerosis. Genetic segregation analysis showed a correlation with disease phenotype in this model with particular Vß genes. In this study we investigated the contribution of specific Vß TCR to the pathogenesis of virus-induced demyelinating disease. Spectratype analysis of cells infiltrating the CNS early in infection demonstrated an over-representation of Vß8+ T cells in mice expressing a susceptible H-2 haplotype. We infected transgenic mice expressing the Vß8.2 TCR directed against a non-TMEV antigen and found an increase in demyelinating disease in mice of either susceptible or resistant background compared with littermate controls. In addition, depletion studies with an anti-Vß8-specific antibody in both susceptible (B10.Q) and resistant (C57BL/6) mice resulted in increased demyelination. TCR analysis of VP2-specific cytotoxic T cell clones from mice with a resistant genotype identified only the Vß8.1 TCR, suggesting that limited T cell diversity is critical to TMEV clearance. Together, these results support a protective role for Vß8+ T cells in virus-induced demyelinating disease.

Keywords: demyelination, Theiler's murine encephalomyelitis virus, Theiler's virus


    Introduction
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Intracerebral infection (i.c.) of mice with Theiler's murine encephalomyelitis virus (TMEV) results in a biphasic disease in susceptible mouse strains. Susceptible mice experience acute encephalitis followed by chronic viral persistence and demyelination, while genetically resistant mice experience only the acute encephalitic phase of the disease and then clear the virus without further pathology (1). Several loci have been identified which influence TMEV-induced demyelination. The D region of the H-2 locus plays a critical role in determining susceptibility or resistance to TMEV-induced demyelination (25). Mice on a C57BL background with f, p, q, r, s or v haplotypes are susceptible, while mice with b, d or k haplotypes are resistant (3). Mice with other backgrounds have not been as well studied, although it has been shown that insertion of the Db (4) or Dd (5) gene into susceptible mice confers resistance to otherwise susceptible strains. Other genes implicated in influencing resistance/susceptibility to demyelination include an area on chromosome 3 near the Car-2 gene (6), an area on chromosome 6 near the Tcrb complex (7), a locus on chromosome 10 near the IFN-{gamma} gene (8), an area on chromosome 18 near the Mbp gene (8), and, most recently, a segment of chromosome 14 located between the Gnrh and Glud genes (9).

A common theme in Theiler's virus immunology is that components of the immune system may be either pathogenic or protective depending on the genetic background of the host. This paradox is illustrated by T cell depletion studies. Depletion of either CD4+ or CD8+ T cells in TMEV-infected mice of susceptible background results in less severe demyelination (1012). In contrast, depletion of T cell subsets in resistant mice results in induction of demyelination and virus persistence (10,12,13). Therefore, both MHC class I- and II-restricted cells participate in the pathogenesis of disease observed following TMEV infection. ß2-Microglobulin-deficient mice which have a deficiency in both MHC class I and CD8+ T cells develop large demyelinated lesions without significant neurological deficits (14,15). Ab° mice which express reduced levels of MHC class II and CD4+ T cells develop both demyelination and severe clinical disease (16,17). The mice in these studies were of a genetically resistant background, suggesting that both class I and class II participate in resistance to demyelination.

Several studies have tried to dissect the precise contribution of particular TCR genes to susceptibility or resistance to Theiler's virus-induced demyelination. Genetic segregation analysis determined that mice with large deletions in their Vß genes had significantly more demyelination than mice with intact Vß genes (18,19). Genetic analysis using congenic mice with specific deletions in their Vß genes allowed investigators the opportunity to evaluate the relative contributions of specific Vß gene deletions in mice susceptible and resistant to TMEV-induced demyelination. Mice with resistant H-2 genotypes were unaffected by large deletions in their TCR Vß genes, demonstrating that the remaining Vß repertoire was sufficient to protect mice from TMEV-induced damage. Mice of susceptible genotype with large deletions in their TCR Vß genes developed severe demyelination along with virus persistence. Of interest, these mice did not die of overwhelming acute neuronal encephalitis as is seen in Scid (20) and Rag–/– mice (21), indicating that the remaining T cell population in these mice was sufficient to protect from acute disease but nevertheless facilitated progression to chronic demyelinating disease.

The results of the genetic segregation analysis studies support a role for specific TCR genes in resistance/susceptibility to Theiler's virus-induced demyelination (18,22). To further understand the role of particular Vß genes in this model of multiple sclerosis (MS) we used spectratype analysis to identify the TCR Vß genes in the CNS of susceptible B10.Q mice after infection with TMEV and found that Vß8+ T cells were over-represented. We investigated the influence of one of these genes (Vß8.2) in genetically susceptible (B10.Q) and resistant (C57BL/6J) mice depleted of Vß8.2+ T cells by antibody treatment, as well as in mice of the same genotype expressing the Vß8.2 transgene. Spectratype analysis of cytotoxic T cell clones from the CNS of resistant H-2b mice stimulated in vitro identified only Vß8.1+ clones. Our findings support a role for Vß8+ T cells in protection from TMEV-induced demyelination in both B10.Q and C57BL/6J mice.


    Methods
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Virus
Daniel's strain of Theiler's virus was used for all experiments. The passage history of this virus has been described previously (1).

Mice
C57BL/6J and B10.Q mice were purchased from Jackson Laboratories (Bar Harbor, ME). Transgenic mice used in this study were bred in the Mayo Clinic Immunogenetics Mouse Facility (Rochester, MN). Transgenic C57L (H-2b) mice bearing a rearranged Vß8.2 TCR cloned from the T cell hybridoma DO-11.10 (ovalbumin specific) were prepared as previously described (23). In some experiments, transgenic mice were backcrossed to B10.Q (H-2q) animals. Animals were typed by flow cytometry for haplotype and the presence of the transgene. The antibodies used for this screening were MKDG (q specific), AF6/120 (b specific) and F23.1 (24).

Flow cytometric analysis
Mice were tail-bled and lymphocytes were isolated over a Ficoll-Hypaque gradient (Pharmacia, Piscataway, NJ). After centrifugation, cells were incubated with antibody on ice, and then washed. The cells were then incubated with a secondary FITC-conjugated F(ab)'2 fragment specific for mouse IgG (Accurate Chemicals, Westbury NY).

Infection, harvesting and morphology of the CNS
At 4–6 weeks of age, mice were infected i.c. with 2x105 p.f.u. of TMEV in a total volume of 10 µl. Forty-five days after infection, mice were perfused with Trump's fixative via intra-cardiac puncture. The 45 day time-point was chosen because it has been shown previously to distinguish between mice susceptible and resistant to TMEV-induced demyelination (13,25,26). Spinal cord blocks (1 mm) were embedded in glycol methacrylate and stained with a modified erichrome stain with a cresyl violet counterstain (27). Detailed morphologic analysis was performed on 10–15 coronal spinal cord sections from each animal. A total of 2951 spinal cord quadrants was studied. Each quadrant from every third spinal cord block from each animal was scored for the presence or absence of gray matter disease, meningeal inflammation and demyelination without knowledge of genotype or treatment. The score was expressed as a percent of the total number of quadrants with a particular abnormality. A maximum pathologic score of 100 indicated that there was disease in every quadrant of every spinal cord block from a particular mouse (28).

In vivo T cell depletion
One day prior to i.c. infection, mice were i.p. injected with 100 µg of F23.1, an antibody specific for Vß8. Mice were tail bled on day 3 post-infection and FACS analysis performed as described above to ensure that depletion was successful. FACS analysis was also performed upon termination of the experiment to confirm the effectiveness of clonal deletion. Control mice were injected with 100 µg of mouse IgG (Sigma, St Louis, MO).

Spectratyping
Spectratyping was performed using the methodology published by Johnston et al. (29). Brains and spinal cords were removed at 7 days post-infection. Lymphocytes were isolated over a Percoll (Pharmacia) gradient by centrifugation at 27,000 g for 30 min as previously described (30). RNA was extracted from the CNS-infiltrating lymphocytes (CNS-IL) using the Total RNA extraction kit (5'-3', Boulder, CO) as per the manufacturer's protocol. Twenty-four Vß-specific RT-PCR were performed using 10 ng total RNA per reaction, a Cß region-specific primer and previously published Vß region-specific primers (29). Following further amplification with a second nested Cß primer, the amplicons underwent single-stranded primer extension using a third nested Cß region primer end-labeled with 6-carboxyfluorescein. The resulting products were run on a 4.75% polyacrylamide/6 M urea denaturing gel. Product length was determined using the Prism Genescan software package (version 2.0.1; Applied Biosystems, Foster City, CA). Data analysis was performed using the Genotyper 1.1 software package (ABI).

Immunoperoxidase staining
Sections (10 µm thick) were cut and fixed in chilled acetone. Sections were stained with a mouse-anti-mouse Vß8.1, 8.2-specific mAb (clone MR5-2; PharMingen, San Diego, CA). In some cases, paraffin-embedded sections of spinal cords were deparaffinized in xylene and rehydrated through an ethanol series (100, 95, 90, 75% and PBS) prior to staining with a polyclonal rabbit anti-TMEV sera. The avidin–biotin immunoperoxidase technique was used (Vector, Burlingame, CA), as described previously (31). Slides were developed with Hanker Yates reagent (Polysciences, Warrington, PA), using hydrogen peroxide as the substrate. Slides were lightly counterstained with Mayer's hematoxylin.

Cytotoxic T lymphocyte (CTL) assay
C57SV cells transfected with the genes encoding the capsid proteins VP2, VP3 or VP4 were used as targets as previously described (30). In addition, cells expressing all three capsid proteins (LP) were used. Untransfected C57SV cells were used as a negative control. On the day of assay, target cells were labeled with 200 µCi of 51Cr-labeled sodium chromate (Amersham Life Sciences, Arlington Heights, IL), washed with RPMI and resuspended to 2x104/ml in RPMI with 5% FCS. The target cell suspensions (100 µl) were placed in 96-well round-bottom microtiter plates (Nalgene Nunc International, Naperville, IL). CNS-IL from Theiler's virus-infected mice (day 7 post-infection) were used as effector cells in this assay. The CNS-IL were resuspended to 2x106/ml in RPMI with 5% FCS and 2-fold serial dilutions were made to provide E:T ratios of 100:1 to 6.25:1. The effector cell suspensions (100 µl) were added in triplicate to the targets, resulting in final E:T ratios of 100:1, 50:1, 25:1, 12.5:1 and 6.25:1. Six wells of targets also received medium alone or 10% Triton X-100 (Sigma) to determine spontaneous release and maximum release of chromium from targets respectively. Plates were incubated for 5 h at 37°C in 5% CO2. Supernatants were harvested with Skatron supernatant collection system (Skatron, Sterling, VA) and assayed in a {gamma}-counter (Gamma 5500; Beckman Instruments, Irvine, CA) for the amount of radioactivity. Mean values were calculated from triplicate wells and results were expressed as percent specific lysis according to the formula [(experimental c.p.m. – spontaneous c.p.m.)/(maximum c.p.m. – spontaneous c.p.m.)]x100%.

Statistics
Data was analyzed using either the Student's t-test for normally distributed data or the Mann–Whitney rank-sum test for data that were not normally distributed.


    Results
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Spectratype analysis of CNS-IL from B10.Q mice demonstrates an over-representation of Vß8.1 and Vß8.2 TCR genes at day 7 post-infection with TMEV
Genetic segregation analysis has suggested that mice with large deletions in the Vß complex have increased levels of demyelination compared to mice with intact Vß genes (18,22). Previous work from our laboratory attempted to identify specific Vß genes involved in TMEV-induced pathology using RT-PCR analysis of the CNS-IL. While no specific TCR Vß genes were determined to be prominent, a caveat to the study was the relatively non-quantitative nature of the assay (32). More recently the technique of spectratyping was developed to more precisely identify Vß gene transcripts present in defined anatomical sites. This technique involves cDNA synthesis with a Cß gene primer, PCR amplification with specific Vß gene and Cß gene primers, followed by electrophoresis and subsequent identification. Spectratyping has been utilized successfully in identifying Vß gene usage in tumors (33,34), graft rejection (29,35) and TMEV (36). In this study, susceptible B10.Q mice were i.c. infected with Theiler's virus (Daniel's strain). At 7 days post-infection mice were sacrificed and CNS-IL were isolated (30). Total RNA was extracted from the CNS-IL, followed by cDNA synthesis and PCR amplification of Vß genes using specific primers. Spectratype analysis revealed restricted diversity in the number of Vß genes expressed by the CNS-IL of the B10.Q mice and a bias toward Vß8 gene usage in three of four of the mice examined with preferential expression of the Vß8.1 and Vß8.2 genes (Fig. 1Go). There was reduced Vß gene diversity in the remaining mouse, but the restriction towards Vß8 gene usage was not as striking as in the other mice. To determine whether Vß8+ T cells infiltrate the lesions of mice susceptible to TMEV-induced demyelinating disease, immunohistochemical staining was performed on spinal cord sections of mice showing virus persistence. Lesions were examined at day 45 post-infection, a time when well-formed infiltrated lesions are apparent in the white matter of the spinal cord. Abundant Vß8.1, 2+ cells were observed in the brains (Fig. 2AGo) and spinal cord lesions (Fig. 2BGo) of TMEV-infected B10.Q mice. No Vß8.1, 2+ cells were found in the CNS of non-infected B10.Q mice.



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Fig. 1. Spectratype analysis using Vß-specific primers from RNA of CNS-IL from B10.Q mice at day 7 post-infection with Theiler's virus. A predominance of Vß8+ TCR was identified in these mice. Total RNA was isolated and reverse transcribed using a Cß region-specific primer. Sequential PCR reactions were then performed using Vß-specific primers and a nested Cß primer, followed by primer extension with a 6-carboxyfluorescein-labeled nested Cß primer. Products were run on a polyacrylamide denaturing gel and analyzed as described in Methods.

 


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Fig. 2. Expression of Vß8+ cells in the brain (A) and spinal cord (B and C) of B10.Q mice after infection with Theiler's virus. Positively staining cells were observed in the demyelinated lesions of B10.Q mice. Frozen sections were stained using the immunoperoxidase technique with a mAb specific to Vß8.1, 8.2 (A and B) or a control antibody (C). The dark reaction product indicates positive staining. No positively staining cells were observed in the CNS of uninfected B10.Q mice.

 
A Vß8.2 transgene exacerbates the extent of demyelination in the spinal cords of Theiler's virus-infected H-2b mice
Previous studies utilizing mice bearing the Vß8.2 TCR transgene directed against ovalbumin have demonstrated an altered pattern of susceptibility to various pathological conditions, including collagen-induced arthritis (37), experimental autoimmune thyroiditis (38) and graft versus host disease (39). Prior data have demonstrated that >92% of the CD8+ T cells and 77% of CD4+ T cells express the Vß8.2 TCR in these transgenic mice, while only 14 and 9% of the CD8+ and CD4+ T cells express Vß8.2 in non-transgenic mice (37). To determine the effect of the Vß8.2 TCR in our model system, Vß8.2 transgenic mice on a background resistant to TMEV (H-2b) were infected i.c. with Theiler's virus. In addition, Vß8.2 transgene-positive mice were also backcrossed to B10.Q mice (background susceptible to demyelination) and screened by flow cytometry for both H-2q and the transgene. Mice homozygous for both q and the transgene were subsequently infected with TMEV. At 45 days post-infection, spinal cords were removed, embedded in glycol methacrylate, and examined for the presence or absence of gray matter disease, meningeal inflammation and demyelination. Transgene-positive B10.Q mice had increased levels of demyelination compared with the B10.Q transgene-negative littermate controls (Fig. 3A and BGo). The increased demyelination levels approached significance (P = 0.056; Table 1Go). In mice on a background resistant to demyelination, expression of the Vß8.2 TCR transgene resulted in statistically significant increased levels of demyelination compared to the non-transgenic control mice (P < 0.003; Table 1Go; Fig. 3C and DGo respectively). The extent of demyelination in transgene positive C57BL/6 was similar to the extent of demyelination in transgene negative normally susceptible B10.Q mice.



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Fig. 3. Demyelination in the spinal cord of Vß8.2 transgenic mice at day 45 after infection with Theiler's virus. Note the increased size of the demyelinated lesions in the spinal cords of a B10.Q transgene-positive mouse (A) as compared to the B10.Q transgene-negative littermate (B). Note marked presence of demyelination in Vß8.2 transgene-positive C57BL/6 mouse of normally resistant genotype (C) as compared to absence of demyelination in a C57BL/6 transgene-negative mouse (D). Spinal cord sections were embedded in glycol methacrylate plastic and stained with a modified erichrome/cresyl violet stain.

 

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Table 1. Presence of the Vß8.2 transgene exacerbates the extent of demyelination in the spinal cord of H-2b mice following Theiler's virus infection
 
Depletion of Vß8+ T cells enhances TMEV-induced demyelination in mice of resistant genotype
In experimental autoimmune encephalomyelitis (EAE), the autoimmune model of MS, Vß8.2+ T cells have been demonstrated to be encephalitogenic. Furthermore, EAE cannot be induced in rats depleted of Vß8.2+ T cells (40). To determine whether Vß8+ T cells participate in protection or pathogenesis in TMEV infection, mice susceptible (B10.Q) or resistant (C57BL/6J) to TMEV-induced demyelination were i.p. injected with either the F23.1 antibody (specific for Vß8+ T cells) or a control IgG. One day later mice were i.c. injected with TMEV. At day 3 post-infection, depletion was confirmed by flow cytometric analysis of peripheral blood lymphocytes. Depletion was considered successful if <1% of the T cells were positive when stained with the F23.1 antibody (Fig. 4A and BGo). Flow cytometry of the peripheral blood lymphocytes was also performed at the end of the experiment to ensure the effectiveness of depletion. Only those mice with >99% of the T cells negative for Vß8 were involved in the analysis. Depletion of F23.1+ cells resulted in an increased, but not statistically significant level of demyelination in susceptible B10.Q mice at day 45 post-infection (Table 2Go). However, depletion of Vß8+ T cells from resistant C57BL/6 mice resulted in statistically significant increased levels of demyelination (Table 2Go and Fig. 4DGo) compared with control-treated mice (Fig. 4CGo). All of the mice expressing a resistant haplotype converted to the demyelinating phenotype following depletion of Vß8+ T cells. The extent of demyelination in resistant mice depleted of Vß8.2 was similar to that observed in normal susceptible B10.Q mice. There was a 15-fold increase of demyelinating pathology in H-2b mice depleted of Vß8.2+ T cells as compared to non-depleted infected mice. In addition, TMEV antigens were detected in the white matter of infected C57BL/6 mice depleted of Vß8+ T cells (Fig. 4FGo), while no TMEV antigens were detected in control-treated infected C57BL/6 mice (Fig. 4EGo). Together, these data indicate that Vß8+ T cells play an integral role in protecting mice on a resistant background from virus-induced demyelination. Because the extent of gray matter disease was not affected by anti-Vß8 antibody treatment, and because the mice did not succumb to encephalitis and death during the acute disease, this indicates that cells other than Vß8+ T cells contribute to protection from acute encephalitis.



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Fig. 4. Flow cytometric analysis of peripheral blood lymphocytes of C57BL/6 mice following in vivo treatment with F23.1 mAb. Mice were i.p. injected with either a control IgG (A) or F23.1 (B). Double staining was performed as indicated in Methods using antibodies to Thy-1.2 (x-axis) and F23.1 (y-axis). At day 45 after infection with Theiler's virus, spinal cords were removed for morphometric analysis. Normal resistant C57BL/6 mice treated with the control IgG had no inflammation or demyelination (C). C57BL/6 mice depleted of Vß8+ T cells had prominent levels of demyelination and meningeal inflammation (D). Spinal cords sections were embedded in glycol methacrylate plastic and stained with a modified erichrome/cresyl violet stain. No virus antigen was detected in transgene-negative littermate controls (E). TMEV antigen was detected in the spinal cord of Vß8.2 transgenic mice on a C57BL/6 background (F). TMEV antigen staining was performed on paraffin-embedded sections with a rabbit polyclonal antisera using the ABC immunoperoxidase technique.

 

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Table 2. Vß 8+ T cells protect H-2b mice from spinal cord demyelination following Theiler's virus infection
 
TMEV-specific CTL cell clones are Vß8.1+
Previous data from our laboratory and others have demonstrated that cytotoxic T cell responses to TMEV are critical to resistance to demyelination. In addition, H-2b CTL are directed against an immunodominant VP2121–130 peptide in the context of H-2Db (41,42). This is the only CTL epitope that has been identified in H-2b mice following TMEV infection. One hypothesis regarding the critical role of Vß8+ T cells in H-2b mice is that these T cells recognize the VP2121–130 peptide. To test this hypothesis, CNS-IL were isolated from C57BL/6 mice at day 7 post-infection and re-stimulated in vitro with VP2-transfected fibroblasts. Spectratype analysis of two individual VP2-specific CTL clones revealed exclusive Vß8.1 usage (Fig. 5A–DGo). These data would suggest that the Vß8.1 TCR is critical to the development of the protective CTL response observed in H-2b mice.



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Fig. 5. Spectratype analysis of two VP2-stimulated CTL clones from H-2b mice. Only Vß8.1+ TCR were identified (A and B). Total RNA was isolated and reverse transcribed using a Cß region-specific primer. Sequential PCR reactions were then performed using Vß-specific primers and a nested Cß primer, followed by primer extension with a 6-carboxyfluorescein-labeled nested Cß primer. Products were run on a polyacrylamide denaturing gel and analyzed as described in Methods. Cytotoxicity assays using the clones in (A) and (B) as effector cells demonstrate that these clones are VP2-specific as they only recognize and kill C57SV target cells which are transfected and express the immunodominant peptide contained in the VP2 capsid protein (C and D respectively). No CTL epitopes have been mapped to the VP3 or VP4 capsid proteins. LP transfectants express the entire left-hand side of the TMEV genome which contains VP2, VP3 and VP4. No cytotoxicity was detected in the untransfected C57SV cells.

 

    Discussion
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
The concept that a particular Vß gene may be associated with susceptibility or resistance to disease has been well documented (37,39,43,44) The correlation between Vß8 family members and altered susceptibility to disease has been documented in autoimmune disease [i.e. EAE (40,45)and influenza (44)]. This has particular relevance to potential therapeutic strategies for human disease, since depletion of a particular Vß T cell population may hold promise for clinical improvement.

Despite the generation of a diverse T cell response in the CNS of TMEV-infected mice, predominant expression of Vß8.1+ and Vß8.2+ T cells was detected using spectratyping in susceptible B10.Q mice during the acute phase of the disease. This finding is significant, as previous studies in the autoimmune model of MS (EAE) have demonstrated that Vß8+ T cells are the predominant T cell population responsible for disease induction. In vivo depletion of Vß8.2+ T cells prevents EAE in otherwise susceptible animals (40). In addition, Vß8.2+ T cells have been shown to be pathogenic in experimental autoimmune uveitis (47), experimental autoimmune neuritis (48) and experimental autoimmune thyroiditis (38). Therefore, it is a reasonable hypothesis that Vß8+ T cell populations may contribute to the pathogenesis of virus-induced demyelination in susceptible strains of mice. In resistant mice TCR analysis of VP2-specific cytotoxic T cell clones identified only a single TCR, Vß8.1, suggesting that limited T cell diversity is important for TMEV clearance.

To analyze the influence of a skewed TCR repertoire in the TMEV model, we infected mice expressing the Vß8.2 TCR transgene with Theiler's virus. Because of the skewed T cell repertoire, it was postulated that mice of a resistant background would not clear early virus infection and would develop persistence. It was unknown however was whether the mice would succumb to acute neuronal infection as observed in Scid mice (49) or whether they would survive the acute disease to develop demyelination. Both H-2b and H-2q transgene-positive mice experienced demyelination following infection with TMEV, but only the H-2b Vß8.2 TCR transgenic mice experienced significantly more demyelination than their non-transgenic littermate controls. One interpretation of these results is that Vß8.2+ T cells, if not regulated by other T cell populations, may participate in the generation of TMEV-induced demyelination if virus is allowed to persist in the CNS. However, because only 77% of the CD4+ cells express the transgene, it is more likely that demyelination is the result of an insufficient number of T cells directed against viral peptide of a non-Vß8.2 specificity. This situation would be analogous to experiments from our lab demonstrating that adoptive transfer of an insufficient number of splenocytes into a Scid mouse (on a background resistant to demyelination) results in demyelination and virus persistence instead of protection from disease (20). An additional interpretation of these results is that epitope spreading is enhanced in the Vß8.2 transgene-positive mice. Studies by Miller et al. have demonstrated that by day 52 post-infection TMEV-infected mice develop a vigorous immune response against PLP139–151 that is partially responsible for the chronic demyelination observed in this model (53). Autoreactive T cells to this epitope have been demonstrated in these Vß8.2+ mice in the EAE model of demyelination (54).

To better study the influence of this TCR on disease progression in the TMEV model of demyelinating disease, we performed in vivo depletion studies in strains susceptible (B10.Q) and resistant (C57BL/6) to Theiler's virus-induced demyelination. The mice depleted of Vß8+ T cells did not succumb to early encephalitis observed in Scid (49), Rag–/– (21) or MHC class I/class II double knockout mice (50). Therefore, the remaining T cell, B cell, NK cell and macrophage populations are sufficient to protect against the neuronal disease. Normally resistant mice experienced significant levels of demyelination following depletion of Vß8+ T cells. The levels of disease present in the mice on a background resistant to TMEV-induced demyelination following depletion were similar to the levels seen in undepleted susceptible strains of mice. These data suggest that in the TMEV model of MS Vß8+ T cells are important for ameliorating demyelinating disease, as opposed to being pathogenic as has been proposed for EAE (40).

The results reported here contrast with studies by Mussette et al. using TMEV-infected SJL/J mice (36). In their study, no selective expansion of Vß populations was observed, despite similar technical approaches. These disparate results could be related to subtle differences in the virus inoculum, the time-points after infection examined or the method of RT-PCR. In the study by Mussette, later times (days 21 and 45) after infection were examined in SJL/J mice (36) in contrast to the 7 day time-point in our experiments. It may be that there is an increase in non-specific lymphocytes infiltrating demyelinated lesions at later times after infection, such that the predominance of Vß8 is not apparent. However, the most likely explanation for the differences observed between the two studies are the differences in the backgrounds of the mice. Almost all that is known regarding the immunogenetics of TMEV-induced disease is based on the C57BL background which was used for our experiments (3,1517,25,51,52). Minimal studies have been performed on the genetics of demyelinating disease in SJL/J mice.

In the in vivo depletion studies presented here, data suggests that Vß8+ T cells are important in protecting resistant strains of mice from demyelination. In contrast Vß8.2+ cells may be critical for induction of autoimmune-mediated demyelination. These results illustrate the potentially complex interactions that must be considered when evaluating the potential of TCR-based strategies for the treatment of human disease when the inciting antigen is unknown.


    Acknowledgments
 
K. M. D. is a fellow of the National Multiple Sclerosis Society. These experiments were supported by a grant from the Department of Neurology (Mayo Foundation), and grants R01NS24180 and R01NS32129 from the National Institutes of Health.


    Abbreviations
 
EAE experimental autoimmune encephalomyelitis
CNS-IL CNS-infiltrating lymphocytes
CTL cytotoxic T lymphocyte
i.c. intracerebral infection
MS multiple sclerosis
TMEV Theiler's murine encephalomyelitis virus

    Notes
 
4 Present address: Department of Medical Microbiology and Immunology, Creighton University, Omaha, NE 68178, USA Back

Transmitting editor: L. Steinman

Received 18 June 1999, accepted 5 November 1999.


    References
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

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  35. Dietrich, P. Y., Caignard, A., Lim, A., Chung, V., Pico, J. L., Pannetier, C., Kourilsky, P., Hercend, T., Even, J. and Triebel, F. 1994. In vivo T-cell clonal amplification at time of acute graft-versus-host disease. Blood 84:2815.[Abstract/Free Full Text]
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  53. Miller, S. D., Vanderlugt, C. L., Smith Begolka, W., Pao, W., Yauch, R. L., Neville, K. L., Katz-Levy, Y., Carrizosa, A. and Kim, B. S. 1997. Persistent infection with Theiler's virus leads to CNS autoimmunity via epitope spreading. Nat. Med. 3:1133.[ISI][Medline]
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