Multiple sclerosis is an immune-mediated disease of the CNS and shows a sex-biased distribution in which 60–75% of all cases are female. A mouse model of multiple sclerosis, Theiler’s murine encephalomyelitis virus (TMEV)-induced demyelinating disease, also displays a gender bias. However, in the C57L/J strain of mice, males are susceptible to disease whereas females are completely resistant. In this study we determined the gender differences in the TMEV-specific immune response, which may be responsible for the gender bias in clinical disease. Our data clearly demonstrate that female C57L/J mice induce significantly higher levels of TMEV-specific neutralizing Ab as well as a stronger peripheral T cell response throughout the course of viral infection. In contrast, male mice have a higher level of TMEV-specific CD4+ and CD8+ T cell infiltration into the CNS as well as viral persistence. These results suggest that a higher level of the initial antiviral immune response in female mice may be able to effectively clear virus from the periphery and CNS and therefore prevent further disease manifestations. Male mice in contrast do not mount as effective an immune response, thereby allowing for eventual viral persistence in the CNS and continuous T cell expansion leading to clinical symptoms.

Multiple sclerosis (MS)3 is an immune-mediated disease of the CNS. A large number of infiltrating immune cells such as T cells and macrophages are found in the brain and spinal cord of patients with MS and are thought to be responsible for the demyelination of neurons found primarily in the white matter of the CNS (1, 2). Although the cause of MS has not been elucidated, epidemiological studies suggest a viral etiology for this disease (3, 4). When the first cases of MS were discovered it was noted that women were affected much more than men. Current studies have shown that as with many autoimmune diseases, MS shows a strong gender bias. In fact, 60–75% of all MS cases are female (5).

Theiler’s murine encephalomyelitis virus (TMEV) is a single- stranded picornavirus. When injected intracerebrally into susceptible strains of mice, the virus causes a disease that is clinically and histopathologically very similar to human MS (6, 7). Various immune parameters associated with this disease parallel those of human MS. Myelin breakdown is directly related to the clinical symptoms including gait spasticity and urinary incontinence. Strong autoimmunity to myelin Ags is induced following the initial demyelination by virus-specific T cells, and the incidence and severity of disease are associated with the gender of mice (8, 9, 10, 11, 12). Thus, TMEV provides an excellent model for studying MS.

Experimental autoimmune encephalomyelitis (EAE), an autoimmune mouse model for MS, shows a gender bias very similar to human MS with female SJL/J mice being much more susceptible than male SJL/J mice (13, 14, 15, 16). Studies with TMEV thus far have not yielded such definitive results. An early study using C57L/J mice showed that male mice are susceptible to TMEV-induced demyelinating disease (IDD), whereas females of this strain are completely resistant (10). A later study using SJL/J mice showed an opposite phenomena with female mice being more susceptible than males (17). In contrast, a recent study showed that male mice on the SJL/J background are more susceptible than females (18). However, the difference in clinical disease between sexes in the SJL/J strain is modest at best and is confounded by the fact that both females and males are highly susceptible. Studies with other virus-induced disease models such as HSV type I, coxsackievirus, and vesicular stomatitis virus have shown that male mice are significantly more susceptible to infection than female mice (19, 20, 21). However, it is not yet clear what underlying mechanisms are involved in gender-dependent susceptibility to autoimmune diseases and virally induced diseases.

We therefore sought to find a mouse strain that clearly shows a gender bias to TMEV-IDD to study the immune mechanisms involved. In the C57L/J strain of mice, males are both clinically (10) and histologically susceptible to TMEV-IDD, whereas females of this strain are completely resistant. Our analyses of the immune response to TMEV in male and female mice in this strain reveal striking differences. Female mice mount a much stronger humoral immune response compared with male mice. The enhanced Ab production in female mice is accompanied by an overall increase in the anti-TMEV splenic CD4+ T cell response. Conversely, male mice have an overall increase in the number of TMEV-specific CD4+ and CD8+ T cells in the CNS. The prolonged virus-specific immune response in the CNS of male mice may be due to prolonged viral persistence, and is likely a key contributing factor to the severe demyelination observed in male mice at late times after infection. These results demonstrate for the first time the underlying immune response differences involved in gender bias to TMEV-IDD and may be crucial in understanding the female bias in MS.

Four- to 6-wk-old male and female C57L/J mice were purchased from The Jackson Laboratory. All mice were housed at the Center for Comparative Medicine Facility at Northwestern University (Chicago, IL).

The BeAn strain of TMEV was propagated and titered in baby hamster kidney (BHK) cells grown in DMEM supplemented with 7.5% donor calf serum. The EL4 (H-2b) cell line was obtained from American Type Culture Collection and was maintained in RPMI 1640 supplemented with 10% FCS, glutamine/pyruvate, and antibiotics.

The rapid multiple peptide synthesis system (RaMPS; DuPont) was used to generate all synthetic peptides. The 2 mM peptide stocks were dissolved in 8% DMSO in PBS. Abs used for flow cytometry were purchased from BD Pharmingen.

Approximately 30 μl (1 × 106 PFU) of TMEV BeAn were injected into the right cerebral hemisphere of 5- to 7-wk-old mice. Sevofluorane was used to anesthetize mice before intracerebral injection. Clinical symptoms of disease were assessed weekly on the following grading scale: grade 0 = no clinical signs; grade 1 = mild waddling gait; grade 2 = moderate waddling gait and hind limb paralysis; grade 3 = severe hind limb paralysis. Data are represented as a percentage of mice affected with a score of 1 or higher.

Epon-embedded brains and spinal cords from male and female C57L/J mice were analyzed as previously described (22). Briefly, mice were anesthetized and perfused with 0.1% cold glutaraldehyde in PBS. Spinal cords were cut into 1-mm segments, postfixed in 1% OsO4, and embedded in epon. Sections were cut at 1-μm thickness, stained with toluidine blue, and analyzed by microscopy. Ten different sections of the lumbar region of the spinal cord of individual mice (two mice per experiment group) were graded for demyelination. A representative sample is shown.

Brain and spinal cord were removed from mice after perfusion with HBSS. After homogenization through a steel screen, the resultant homogenate was used to perform a standard plaque assay on BHK-21 monolayers (23). After methanol fixation, 0.1% crystal violet was used to visualize plaques on the monolayer. For Ab neutralization assays, serum collected from infected mice was incubated with TMEV for 1 h on ice, and serial dilutions were plated on BHK-21 monolayers in triplicate and incubated at 33°C for 4 days. Plaques were visualized as stated. The percentage of neutralization is calculated based on reduction of plaque numbers from untreated virus.

TMEV-specific Abs were measured at 8, 20, 40, 45, and 70 days after viral infection by ELISA. Plates (96-well) were coated with purified UV-irradiated TMEV and then incubated with pooled sera. Abs were detected with alkaline phosphatase-conjugated secondary Abs (Promega) and developed with p-nitrophenyl phosphate. Colorimetric readings were made at 405 nm. Ig subclasses were determined by using alkaline phosphate-conjugated isotype-specific secondary goat Abs (Promega).

ELISPOT plates (Millipore) were precoated with 1–5 μg/ml anti-IFN-γ Ab in 0.05 M carbonate buffer, pH 9.6, and then blocked with sterile PBS containing 1% BSA. Plates were incubated for 18 h with 2 × 104 CNS mononuclear cells plus 1 × 106 irradiated (3000 rad) syngeneic spleen cells or 1 × 106 splenocytes in 200 μl of HL-1 medium (BioWhittaker) at 37°C under 5% CO2 in the presence of 2 μM peptide. After washing, plates were incubated with biotin-conjugated anti-IFN-γ Ab (Endogen) overnight, followed with streptavidin-HRP for 3 h. Spots were developed using 3-amino-9-ethyl-carbazole (Sigma-Aldrich) in 0.05 M sodium acetate buffer (24).

Spleen cells (1 × 106 cell/well) were cultured in 96-well flat-bottom microculture plates in RPMI 1640 containing 0.5% syngeneic mouse serum and 5 × 10−5 M 2-ME. Triplicate cultures were stimulated with different TMEV capsid peptides (2 μM) for 72 h, pulsed with 1 μCi of [3H]TdR and then harvested 18 h later. Measurements of [3H]TdR uptake by the cells were determined in a scintillation counter and expressed as net CPM ± SEM after subtraction of the background count with PBS.

A total of 30 ml of sterile HBSS was perfused through the left ventricle. Brain and spinal cord were removed and forced through steel mesh and incubated at 37°C for 45 min in 250 μg/ml collagenase type 4 (Worthington Biochemical). A continuous 100% Percoll gradient (Amersham Biosciences) was used to enrich CNS infiltrating lymphocytes after centrifugation at 27,000 × g for 30 min, the bottom one-third of the gradient contains infiltrating mononuclear cells.

Freshly isolated CNS infiltrating lymphocytes were incubated in the presence of virus peptide or control and GolgiPlug in 96-well round-bottom plates for 6 h at 37°C. Fc receptors were then blocked using 50 μl of 2.4G2 hybridoma (American Type Culture Collection) supernatant by incubating at 4°C for 30 min. Allophycocyanin-conjugated anti-CD4 (clone L3T4) or anti-CD8 (clone Ly-2) Ab was then added and cells were incubated at 4°C for an additional 30 min. Following two washes, intracellular staining for IFN-γ staining was performed according to manufacturer’s protocol (BD Pharmingen) using PE-labeled rat monoclonal anti-IFN-γ. Cells were analyzed on a BD Biosciences FACSCalibur flow cytometer. Live cells were gated based on light scatter properties.

EL4 target cells were incubated with viral peptides for 2 h at 37°C and labeled with 51Cr (50 μCi per target) for 2 h. Cells were then washed three times with RPMI 1640 then resuspended at 3 × 104 cells/ml in RPMI 1640 supplemented with 5% FCS. 51Cr-labeled target cells were added to a 96-well round-bottom plate at 100 μl/well. CNS effector cells were added to the target cells immediately after isolation at varying concentrations and incubated for 6 h at 37°C. Supernatants were harvested and mean radioactivity values were calculated from duplicate wells. Percentage of specific lysis was calculated according to the formula: ((experimental 51Cr release − spontaneous 51Cr release)/(maximum 51Cr release with 1% Triton X-100 − spontaneous 51Cr release)) × 100%. Spontaneous lysis was 11 and 16% at days 8 and 21, respectively.

Previously, we observed that male mice of the C57L/J strain are more susceptible to TMEV-IDD than female mice (10). To confirm and extend such a gender-biased susceptibility to TMEV-IDD in this strain, male and female C57L/J mice were infected intracerebrally with 106 PFU TMEV and were monitored for clinical symptoms of disease. Male mice first began showing clinical signs of disease at 40 days postinfection and progressed until ∼105 days postinfection at the time of termination (Fig. 1,A). Strikingly, female mice never showed any clinical signs of disease up to 105 days postinfection. Histological examination of spinal cords from these mice at 63 days postinfection clearly shows inflammatory cell infiltrate in the meninges and demyelinating lesions extending deep into the anterior column in clinically affected male mice but no pathology at all in female mice infected with TMEV (Fig. 1,B). This pathology is even more severe at 115 days postinfection in male mice, at which time the inflammatory infiltrate extends from the leptomeninges into the white matter with very severe demyelination (Fig. 1,B). These histologic results are well correlated with enhanced disease susceptibility in male mice described earlier (Fig. 1 A). This gender dependent susceptibility to TMEV-IDD in the C57L/J strain of mice provides a good model for the study of gender bias in the immune response to viral infection.

FIGURE 1.

Clinical and histologic susceptibility of male and female mice infected with TMEV. A, Male (n = 19) and female (n = 12) mice were infected intracerebrally with 1 × 106 PFU TMEV and were monitored for clinical disease based on a 4 point scale (1 = mild waddling gate, 2 = severe waddling gate and partial hind limb paralysis, 3 = total hind limb paralysis, 4 = forelimb and hind limb paralysis). Percentage of total mice with a clinical score of ≤1 is depicted. B, One-micron thick, epon embedded section from the spinal cord of female (left) and male (right) mice at 63 (top) and 115 (bottom) days (D) postinfection stained with toluidine blue.

FIGURE 1.

Clinical and histologic susceptibility of male and female mice infected with TMEV. A, Male (n = 19) and female (n = 12) mice were infected intracerebrally with 1 × 106 PFU TMEV and were monitored for clinical disease based on a 4 point scale (1 = mild waddling gate, 2 = severe waddling gate and partial hind limb paralysis, 3 = total hind limb paralysis, 4 = forelimb and hind limb paralysis). Percentage of total mice with a clinical score of ≤1 is depicted. B, One-micron thick, epon embedded section from the spinal cord of female (left) and male (right) mice at 63 (top) and 115 (bottom) days (D) postinfection stained with toluidine blue.

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Previous studies from our laboratory and others have demonstrated that one of the major differences between resistant C57BL/6 mice and susceptible SJL/J mice is the ability of TMEV to persistently infect the CNS (23, 25, 26, 27). C57BL/6 mice rapidly eliminate the virus from the CNS within 2–4 wk after infection, protecting against pathology and clinical symptoms (23). Conversely, SJL mice are unable to clear the virus from the CNS leading to a persistent infection and clinical disease (25, 26, 27). To determine whether this would also be true for resistant female and susceptible male mice within the same C57L/J strain, viral plaque assays were performed to test this possibility. Brain and spinal cord homogenates from male and female C57L/J mice were obtained on days 7, 21, and 41 postinfection and were assayed for the ability to form lysis plaques on BHK monolayers. At 7 days postinfection in both male and female mice, the virus was nearly entirely localized in the brain and not the spinal cord (Fig. 2) with no significant difference between sexes. However, at day 21 postinfection, significantly more infectious virus (p < 0.005) was recovered from the spinal cord of male mice compared with that recovered from the spinal cord of female mice, although a higher level was found in the brains of female mice. This finding suggests that the virus migrates from the brain to the spinal cord in male mice whereas this shift is not as evident in female mice. At 41 days postinfection, the brain and spinal cord of female mice contained very little virus whereas male mice displayed a low, but significant, level of virus persistence in the brain and spinal cord. At 70 days postinfection there remained a low albeit elevated level of TMEV persistence in spinal cords from male but not female mice (data not shown). These results indicate that the CNS of male mice becomes persistently infected with TMEV whereas the CNS of female mice becomes virtually free of virus. This chronic viral persistence at late time points after infection in male mice likely leads to the increase in immune-mediated demyelination resulting in clinical and histopathological signs of disease.

FIGURE 2.

Comparison of viral persistence in the brain and spinal cord of male and female C57L/J mice at 7, 21, and 41 days post-TMEV infection. Brains and spinal cords from five mice were pooled, and measurement of plaque formation on BHK monolayers was performed in triplicate. The level of virus persistence is expressed as the number of plaques (PFU) per mouse CNS (mean ± SD) at each time point. There is no significant difference between male and female mice at 7 days postinfection in either the brain or spinal cord. The difference between male and female mice is highly significant at 21 days both in the brains and spinal cords (p < 0.005) and remains significant at 41 days in both the brain (p < 0.005) and spinal cord (p < 0.05). Representative data of two to three separate experiments. Student’s t test was used for statistical analysis between replicates of three.

FIGURE 2.

Comparison of viral persistence in the brain and spinal cord of male and female C57L/J mice at 7, 21, and 41 days post-TMEV infection. Brains and spinal cords from five mice were pooled, and measurement of plaque formation on BHK monolayers was performed in triplicate. The level of virus persistence is expressed as the number of plaques (PFU) per mouse CNS (mean ± SD) at each time point. There is no significant difference between male and female mice at 7 days postinfection in either the brain or spinal cord. The difference between male and female mice is highly significant at 21 days both in the brains and spinal cords (p < 0.005) and remains significant at 41 days in both the brain (p < 0.005) and spinal cord (p < 0.05). Representative data of two to three separate experiments. Student’s t test was used for statistical analysis between replicates of three.

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Several studies have previously indicated the importance of antiviral Ab production in the protection against TMEV-IDD (28, 29, 30). In addition, it is well documented in a variety of systems that females, both mice and humans, produce higher levels of Ab in response to pathogens and vaccination (31, 32, 33, 34). To investigate whether female mice produce higher levels of TMEV-specific Ab, the relative level of virus-specific serum IgG was determined. At 8 days postinfection, the level of TMEV-specific IgG is ∼2-fold higher in female mice compared with male mice (Fig. 3 A). This difference between male and female mice becomes far greater (>10-fold) at later times and reaches highly significant levels (p < 0.005) at 70 days postinfection. These data are consistent with an earlier study in SJL/J mice that showed increased specific Ab production in female mice compared with male mice (18).

FIGURE 3.

Assessment of TMEV-specific Ab production. A, Pooled sera from five male and five female mice were collected at 8, 20, 40, and 70 days after TMEV infection and analyzed by ELISA for total TMEV-specific IgG production. Sera from naive animals were used as controls. B, Anti-IgG1 and anti-IgG2a secondary Abs were used to determine the level of TMEV-specific IgG isotype production at 20 and 45 days after TMEV infection. C, Ab neutralization assays were performed at 8, 21, 41, and 70 days following infection by incubating serum from infected male and female mice (five each pooled) with virus for 1 h on ice and then plated on BHK monolayers. Percentage of neutralization is compared with virus alone control. ∗, p < 0.05, ∗∗, p < 0.005 (Student’s t test between replicates of three). Data are representation of three separate experiments.

FIGURE 3.

Assessment of TMEV-specific Ab production. A, Pooled sera from five male and five female mice were collected at 8, 20, 40, and 70 days after TMEV infection and analyzed by ELISA for total TMEV-specific IgG production. Sera from naive animals were used as controls. B, Anti-IgG1 and anti-IgG2a secondary Abs were used to determine the level of TMEV-specific IgG isotype production at 20 and 45 days after TMEV infection. C, Ab neutralization assays were performed at 8, 21, 41, and 70 days following infection by incubating serum from infected male and female mice (five each pooled) with virus for 1 h on ice and then plated on BHK monolayers. Percentage of neutralization is compared with virus alone control. ∗, p < 0.05, ∗∗, p < 0.005 (Student’s t test between replicates of three). Data are representation of three separate experiments.

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To determine whether there is a difference in IgG isotypes being produced, reflecting a Th1/Th2 shift, we compared the level of virus-specific IgG1 (Th2 subclass) and IgG2a (Th1 subclass) produced in response to TMEV in female and male mice. Both male and female mice produce higher levels of IgG2a compared with IgG1. However female mice show much higher levels of both IgG1 and IgG2a (Fig. 3 B), reflecting an overall increase in Ab production in female mice and not a Th1/Th2 shift.

To examine whether the increase in TMEV-specific IgG reflects enhanced viral neutralization, Ab neutralization assays were performed. At days 7, 21, 41, and 71 postinfection, serum from female mice neutralized virus significantly more effectively than serum from male mice (Fig. 3 C). Taken together, these results suggest that enhanced Ab production in female mice is likely an important factor in protection from clinical disease.

The peripheral CD4+ T cell response to TMEV is known to be critical for Ab production, aiding in the CD8+ T cell response as well as controlling viral persistence (35, 36, 37). Recently our laboratory identified several H-2b class II-restricted TMEV epitopes (38). To determine whether there is a difference in the virus-specific CD4+ T cell response in the periphery of male and female mice, the proliferative response and IFN-γ production by splenic T cells was examined after in vitro restimulation with UV-irradiated TMEV and the H-2b-restricted CD4+ T cell epitopes of TMEV capsid proteins, VP2201 and VP421. Splenocytes from female mice at both early (8 days) and late (50 days) times after infection proliferate more vigorously to UV-irradiated TMEV as well as to the different CD4+ T cell epitopes, compared with splenocytes from male mice (Fig. 4)

FIGURE 4.

IFN-γ and proliferation responses of splenocytes from male and female C57L/J mice. IFN-γ ELISPOT and proliferation assays were performed on splenocytes taken from five male and five female C57L/J mice at 8 and 50 days post-TMEV infection. The level of proliferation from 1 × 106 spleen cells was determined by [3H]TdR uptake assays after incubation with peptide or PBS for 96 h. IFN-γ-producing cells from 1 × 106 spleen cells were enumerated by ELISPOT assay after 18 h incubation with (VP2201–220 and VP421–40) or 0.3 μg of UV-irradiated TMEV 2 μM 20-mer peptides. Values given are CPM or spots with background (PBS stimulated) subtracted. ∗, p < 0.05, ∗∗, p < 0.005 (Student’s t test).

FIGURE 4.

IFN-γ and proliferation responses of splenocytes from male and female C57L/J mice. IFN-γ ELISPOT and proliferation assays were performed on splenocytes taken from five male and five female C57L/J mice at 8 and 50 days post-TMEV infection. The level of proliferation from 1 × 106 spleen cells was determined by [3H]TdR uptake assays after incubation with peptide or PBS for 96 h. IFN-γ-producing cells from 1 × 106 spleen cells were enumerated by ELISPOT assay after 18 h incubation with (VP2201–220 and VP421–40) or 0.3 μg of UV-irradiated TMEV 2 μM 20-mer peptides. Values given are CPM or spots with background (PBS stimulated) subtracted. ∗, p < 0.05, ∗∗, p < 0.005 (Student’s t test).

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IFN-γ has been shown to be essential for controlling TMEV infection and protecting the host from the development of disease (39). The level of early IFN-γ production by T cells in the spleen may affect whether the host will be able to clear the virus or will succumb to the chronic infection. At 8 days postinfection there are more virus specific IFN-γ-producing CD4+ (p < 0.005) cells in the spleen of female mice compared with male mice (Fig. 4). The stronger peripheral antiviral CD4+ T cell response in female mice is likely a key contributing factor to the heightened Ab production. At 50 days postinfection there is no virus-specific CD4+ T cell response detectable in the spleen of either female or male mice. It has been shown previously that females overall possess a greater number of CD4+ T cells (40). This greater number of CD4+ T cells (Th1 and Th2) with antiviral specificity in female mice may result in the increased Ab production compared with male mice.

Most studies analyzing the TMEV-specific CD8+ T cell responses have focused on CNS-infiltrating T cells. In contrast, the majority of previous studies analyzing CD4+ T cell responses to TMEV were primarily aimed at examining this response in the periphery. However, it is conceivable that CD4+ T cell responses in the periphery may reflect different roles from those in the CNS, the site of immune-mediated demyelination. To analyze T cell responses, we examined the potential differences in the overall levels of CD4+ and CD8+ T cells infiltrating the CNS in male and female mice infected with TMEV. The levels of CD4+ and CD8+ T cell populations in the CNS were assessed by flow cytometry using anti-CD4 PE and anti-CD8 allophycocyanin mAbs. The level of T cell infiltration was highest (55%) at 7 day postinfection and gradually decreased later (21 and 41 days postinfection) in both sexes (Fig. 5). However, the CD4:CD8 ratios of infiltrating T cells maintained at 1:3 throughout except in the CNS of male mice at a later time point (41 day), which was a 1:2 ratio due to a relative increase in the CNS-infiltrating CD4+ T cell population. Taken together, these data indicate no significant difference in the percentage or ratio of CD4+ or CD8+ T cells infiltrating the CNS of male and female mice at any time tested. It should be noted however that these data represent the total levels of CD4+ and CD8+ T cell infiltration without taking into account the Ag specificity of these cells.

FIGURE 5.

CD4+ and CD8+ T cell infiltration in the CNS of male and female mice. At days 8, 21, and 41 isolated infiltrating lymphocytes from TMEV-infected male (n = 5) and female (n = 5) C57L/J mice were stained for the presence of CD4 and CD8 and analyzed by flow cytometry. Numbers (inset) represent percentage of gated lymphocytes that are CD4+ or CD8+. Data are the representation of two to three experiments.

FIGURE 5.

CD4+ and CD8+ T cell infiltration in the CNS of male and female mice. At days 8, 21, and 41 isolated infiltrating lymphocytes from TMEV-infected male (n = 5) and female (n = 5) C57L/J mice were stained for the presence of CD4 and CD8 and analyzed by flow cytometry. Numbers (inset) represent percentage of gated lymphocytes that are CD4+ or CD8+. Data are the representation of two to three experiments.

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To determine the Ag specificity of CD4+ T cells in the CNS of male and female mice, ICS for IFN-γ was performed after stimulation with recently identified viral epitopes (VP2201 and VP421). Early after infection (day 7) the percentage of virus-specific IFN-γ-producing cells in the CNS of male mice is slightly higher (Fig. 6,A). At what appears to be the peak of the immune response in C57L/J mice (day 21), the percentage of virus-specific CD4+ T cells reaches similar levels. However, after the immune response has begun to contract (day 40), there are two times as many IFN-γ-producing CD4+ T cells in the CNS of male mice compared with female mice (Fig. 6,A). Due to the sensitive nature of the ICS assay, it is likely that we are detecting cells making only small amounts of IFN-γ as positive. To determine whether a difference exists in the number of cells that secrete a physiologically relevant amount of IFN-γ we used a secretion-based ELISPOT assay. Consistent with ICS results, we find that male mice have significantly (p < 0.005) more IFN-γ-producing CD4+ T cells within the CNS compared with female mice at 8 days following TMEV infection (Fig. 6 B). At 21 days postinfection there is no difference in the number of CD4+ T cells specific for either of the two Th epitopes. By 50 days after infection when the immune response in female mice begins to subside due to viral clearance, the CD4+ T cell response to TMEV remains high in male mice evidenced by higher numbers of VP2201- and VP421-specific T cells in male as compared with female mice. These experiments clearly indicate an overall higher virus-specific inflammatory CD4+ T cell response within the CNS of male mice at both early and late time points after infection. The higher level of inflammatory CD4+ T cell infiltrate in male mice compared with female mice may contribute to differential susceptibility to CNS inflammation and demyelination.

FIGURE 6.

TMEV-specific IFN-γ-producing CD4+ T cells in the CNS of male and female mice. A, Isolated infiltrating lymphocytes from male (n = 5) and female (n = 5) C57L/J mice were restimulated in culture with the CD4+-specific peptides, VP2201–220, VP421–40, and VP3171–190 and stained for CD4 and intracellular IFN-γ. Cells were then analyzed by flow cytometry. Live cells were gated by forward and side light scatter and the resulting plots show the percentage of CD4+ T cells that are IFN-γ-positive. Data are representative of two to three experiments. B, IFN-γ-producing cells from 1 × 106 infiltrating mononuclear cells (MNC) from TMEV-infected male and female C57L/J mice were measured by ELISPOT after 18 h incubation with VP2201–220 or VP421–40 at 8, 21, and 50 days postinfection in duplicate. ∗, p < 0.05, ∗∗, p < 0.005 (Student’s t test).

FIGURE 6.

TMEV-specific IFN-γ-producing CD4+ T cells in the CNS of male and female mice. A, Isolated infiltrating lymphocytes from male (n = 5) and female (n = 5) C57L/J mice were restimulated in culture with the CD4+-specific peptides, VP2201–220, VP421–40, and VP3171–190 and stained for CD4 and intracellular IFN-γ. Cells were then analyzed by flow cytometry. Live cells were gated by forward and side light scatter and the resulting plots show the percentage of CD4+ T cells that are IFN-γ-positive. Data are representative of two to three experiments. B, IFN-γ-producing cells from 1 × 106 infiltrating mononuclear cells (MNC) from TMEV-infected male and female C57L/J mice were measured by ELISPOT after 18 h incubation with VP2201–220 or VP421–40 at 8, 21, and 50 days postinfection in duplicate. ∗, p < 0.05, ∗∗, p < 0.005 (Student’s t test).

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The Ag specificity of the infiltrating CD8+ T cell population was also addressed by ICS for IFN-γ after stimulation with the predominant CD8+ T cell epitope VP2121–130. Beginning at 7 days after infection, male mice showed ∼13% higher numbers of virus-specific CD8+ T cells in the CNS compared with female mice (Fig. 7,A). The virus-specific cell increase in male mice is maintained at 21 days postinfection. By 41 days when overall infiltration declines, the percentage of CD8+ T cells specific for VP2121–130 is still 17% higher in male mice (46 vs 29%). To confirm and extend these results, IFN-γ ELISPOT assays were performed after stimulation with VP2121–130. A significantly higher number of VP2121–130-specific CD8+ T cells (p < 0.05) in the CNS of male compared with female mice was similarly observed at 8, 21, and 50 days after infection (Fig. 7 B).

FIGURE 7.

TMEV-specific IFN-γ-producing CD8+ T cells in the CNS of male and female mice. A, Isolated infiltrating lymphocytes from male (n = 5) and female (n = 5) C57L/J mice were restimulated in culture with the predominant CD8+ T cell epitope VP2121–130 and stained for CD8 and intracellular IFN-γ. These cells were analyzed by flow cytometry. Live cells were gated by forward and side light scatter and the resulting plots represent the percentage of CD8+ T cells that are IFN-γ-positive. Representation of two to three experiments. B, IFN-γ ELISPOT analysis of CNS infiltrating lymphocytes in female and male C57L/J mice at 8, 21, and 50 days postinfection was performed after 18 h stimulation with VP2121–130. C, Infiltrating lymphocytes from female and male C57L/J mice were cultured with VP2121–130 and 51Cr-loaded EL4 target cells for 6 h. The amount of 51Cr release was used as a measure of lysis. ∗, p < 0.05, ∗∗, p < 0.005 (Student’s t test).

FIGURE 7.

TMEV-specific IFN-γ-producing CD8+ T cells in the CNS of male and female mice. A, Isolated infiltrating lymphocytes from male (n = 5) and female (n = 5) C57L/J mice were restimulated in culture with the predominant CD8+ T cell epitope VP2121–130 and stained for CD8 and intracellular IFN-γ. These cells were analyzed by flow cytometry. Live cells were gated by forward and side light scatter and the resulting plots represent the percentage of CD8+ T cells that are IFN-γ-positive. Representation of two to three experiments. B, IFN-γ ELISPOT analysis of CNS infiltrating lymphocytes in female and male C57L/J mice at 8, 21, and 50 days postinfection was performed after 18 h stimulation with VP2121–130. C, Infiltrating lymphocytes from female and male C57L/J mice were cultured with VP2121–130 and 51Cr-loaded EL4 target cells for 6 h. The amount of 51Cr release was used as a measure of lysis. ∗, p < 0.05, ∗∗, p < 0.005 (Student’s t test).

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The cytolytic effector function of CD8+ T cells does not necessarily correlate to cytokine production by T cells specific for the same epitope. It is therefore important to assess lytic function of these cells to fully understand their role in the protection/pathogenesis of TMEV. To determine the potential differences in cytolytic efficiency between male and female mice, 51Cr release assays were performed at 8 and 21 days following infection with TMEV. At 8 days postinfection, there is no difference in the level of virus-specific lysis of VP2121–130-loaded EL4 target cells (Fig. 7,C). However, at 21 days postinfection, CD8+ T cells from male mice resulted in significantly more target cell lysis compared with female mice (p < 0.005). The superior TMEV-specific CD8+ T cell function, in both cytokine production and target cell lysis, appears to reflect higher virus-specific CD8+ T cell numbers in the CNS of male mice. There is elevated viral persistence in the spinal cords of male mice compared with female mice at late times after infection (Fig. 2). This increase in the amount of replicative virus may lead to the availability of a higher level of viral Ags in male mice leading to a prolonged immune response in the CNS of these mice. The elevated inflammatory CD4+ response combined with a prolonged inflammatory and lytic CD8+ T cell response to viral Ags in male mice may not be sufficient for viral clearance and likely leads to the onset of clinical symptoms and the development of demyelination.

It is clear from our current studies that the clinical signs of disease in male mice are proportional to the inflammation and demyelination found in the spinal cords of these mice (Fig. 1). As the disease progresses in male mice the extent of demyelination also increases. Female mice, on the contrary, did not develop clinical signs of disease and did not display detectable inflammation or demyelination within their CNS for up to 115 days postinfection. Susceptibility to TMEV-IDD is known to be dependent on the ability of TMEV to persist within the CNS of infected mice (23, 25, 26). Corresponding to the clinical and histological manifestations of disease, a higher level of viral replication was observed within the CNS of male mice compared with female mice (Fig. 2). The factors that contribute to increased viral replication and hence more severe disease in male mice are not understood. It is conceivable that the cells of the CNS in male mice are more permissive to viral infection and subsequent replication compared with female mice. In addition, the immune response to virus in female mice may be more effective in eliminating virus from the CNS thus affording protection from disease.

In our studies, resistant female C57L/J mice produce significantly higher levels of TMEV-specific IgG compared with male mice. This enhanced Ab production by female mice correlates to an increase in serum virus neutralization in females (Fig. 3). This is consistent with previous reports that associate TMEV-specific Ab production with protection from clinical disease (29, 30, 41). In addition, it has been shown that female mice in general produce significantly more specific Ab than males in response to various infections and immunizations (18, 31, 32). This increase in virus-specific Ab production in female mice may be a key factor in their resistance to TMEV.

In addition, female C57L/J mice displayed a more pronounced CD4+ T cell response in the periphery (spleen) measured by both TMEV-specific proliferation and IFN-γ secretion (Fig. 4). This observation is consistent with previous reports showing that bacillus Calmette-Guérin-sensitized female mice mount a stronger Th1 response in response to purified protein derivative challenge compared with male mice (42). CD4+ T cell help in the periphery is important in aiding the humoral immune response following infection. Therefore, the more pronounced CD4+ T cell response in female mice is consistent with the higher level of TMEV-specific serum IgG compared with male mice. In addition, the early Th1 response observed in the periphery of female mice infected with TMEV may be important in establishing an effective immune response that can then control early viral replication limiting chronic infection. However, the enhanced CD4+ T cell response in female mice although beneficial in controlling virus infection may be detrimental in an autoimmune disease such as EAE. In fact, females of several strains of mice including SJL, New Zealand White, and ASW appear to be more susceptible than males to EAE induced with proteolipid proteins, and myelin oligodendrocyte glycoprotein (MOG), myelin basic protein, and MOG92–106, respectively (43). C57L/J mice are highly susceptible to MOG35–55-induced EAE (44), however like C57BL/6 mice (45) no significant difference in susceptibility exists between males and females (our unpublished data). In contrast, male B10.PL and PL/J mice are more susceptible than females to EAE induced with myelin basic protein and MOG92–106, respectively (43). Therefore, gender-biased susceptibility to EAE may be dependent not only on the level of immune response but also the genetic background of mice and/or the encephalitogenic peptide used.

Although female C57L/J mice have a stronger anti-TMEV immune response in the periphery (Fig. 4), male mice have a greater number of TMEV-specific IFN-γ-producing CD4+ T cells in the CNS during the initial stage of the TMEV immune response and also at a chronic time point (Fig. 6). These results support the proposed pathogenic nature of these Th1 cells in the CNS (46, 47). In addition, Th1 responses to predominant viral epitopes have been reported to promote disease progression in TMEV-infected mice (48, 49, 50). Thus, these CNS infiltrating inflammatory CD4+ T cells may be a major effector population with respect to demyelination and subsequent disease progression leading to clinical disease in male but not female C57L/J mice.

The role of the CD8+ T cell response in TMEV-IDD has been a controversial issue. Studies have previously shown that mice deficient for different molecules involved in CD8+ T cell effector function as well as in MHC class I Ag presentation display enhanced susceptibility to disease (22, 51, 52, 53, 54). In contrast, other studies maintain a pathogenic role for CD8+ T cells based on the lack of clinical signs of disease after infection of mice lacking molecules involved in CD8+ T cell function (55, 56). Nevertheless, resistance to TMEV has been mapped to the MHC class I locus (57, 58) strongly suggesting CD8+ T cells are important to the outcome of disease. We observe no difference in the overall level of CD8+ T cell infiltration between male and female mice (Fig. 5). However, there is an increase in the level of virus-specific IFN-γ-producing CD8+ cells in the CNS of male mice at all times tested after infection (Fig. 7). There are also more TMEV-specific CD8+ T cells in the CNS of male mice capable of lysing epitope-loaded target cells compared with female mice (Fig. 7,C). We propose that the higher level of CD8+ T cell responses in male mice is due to enhanced virus persistence in the CNS of male mice compared with female mice. We have shown that there is significantly more virus present within the CNS of male mice at 21 and 41 days after infection (Fig. 2), and this increase in virus persistence may lead to continued viral Ag presentation in male mice and therefore a prolonged immune response. Although the CD8+ T cell response is thought to be protective at early times after infection, a prolonged inflammatory and lytic response by CD8+ T cells within the CNS of male mice may lead to the perpetuation of an inflammatory environment or direct damage to CNS cells leading to demyelination and clinical signs of disease. Alternatively, local expansion of virus-specific CD8+ T cells in the CNS of male mice may be greater than in female mice and this T cell population may also be involved in the pathogenesis of demyelination.

In this report we clearly identified differences in the adaptive immune response to TMEV between susceptible male and resistant female C57L/J mice. We hypothesize from these experiments that female mice are able to control virus infection more efficiently leading to decreased viral persistence. This protection may be attributable in part to robust early peripheral T cell responses and a strong antiviral humoral immune response. Male mice on the contrary are inefficient in controlling virus replication within the CNS leading to the establishment of a persistent infection and consequently the development of clinical and histopathologic disease. An effective early peripheral antiviral T cell response may be important in controlling viremia in TMEV-infected mice helping to protect mice from chronic infection and clinical disease. In contrast, a prolonged inflammatory T cell response in the CNS may be pathogenic. This dichotomous role for TMEV-specific T cells is well illustrated by the different levels of immune responses to TMEV in male and female C57L/J mice. The underlying factors that contribute to this difference in the TMEV specific immune response are not yet clearly understood. However preliminary experiments show that treating male mice with the female sex hormone estradiol renders these mice resistant to the clinical symptoms of TMEV (our unpublished observation), suggesting a role for estradiol as an immune modulator.

The authors have no financial conflict of interest.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1

This work was supported by the National Multiple Sclerosis Society Grant RG3126-A4 and the U.S. Public Health Service Grants NS 28752 and NS 33008.

3

Abbreviations used in this paper: MS, multiple sclerosis; TMEV, Theiler’s murine encephalomyelitis virus; IDD, induced demyelinating disease; ICS, intracellular cytokine staining; EAE, experimental autoimmune encephalomyelitis; MOG, myelin oligodendrocyte glycoprotein; BHK, baby hamster kidney.

1
Johnson, R. T..
1975
. The possible viral etiology of multiple sclerosis.
Adv. Neurol.
13
:
1
.-46.
2
McFarlin, D. E., H. F. McFarland.
1982
. Multiple sclerosis.
N. Engl. J. Med.
307
:
1183
.-1188.
3
Allen, I., B. Brankin.
1993
. Pathogenesis of multiple sclerosis: the immune diathesis and the role of viruses.
J. Neuropathol. Exp. Neurol.
52
:
95
.-105.
4
Dhib-Jalbut, S., D. E. McFarlin.
1990
. Immunology of multiple sclerosis.
Ann. Allergy
64
:
433
.-444.
5
Whitacre, C. C..
2001
. Sex differences in autoimmune disease.
Nat. Immunol.
2
:
777
.-780.
6
Lipton, H. L..
1975
. Theiler’s virus infection in mice: an unusual biphasic disease process leading to demyelination.
Infect. Immun.
11
:
1147
.-1155.
7
Lehrich, J. R., B. G. Arnason, F. H. Hochberg.
1976
. Demyelinative myelopathy in mice induced by the DA virus.
J. Neurol. Sci.
29
:
149
.-160.
8
Lipton, H. L., A. Friedmann.
1980
. Purification of Theiler’s murine encephalomyelitis virus and analysis of the structural virion polypeptides: correlation of the polypeptide profile with virulence.
J. Virol.
33
:
1165
.-1172.
9
Dal Canto, M. C., B. S. Kim, S. D. Miller, R. W. Melvold.
1996
. Theiler’s murine encephalomyelitis virus (TMEV)-induced demyelination: a model for human multiple sclerosis.
Methods
10
:
453
.-461.
10
Kappel, C. A., R. W. Melvold, B. S. Kim.
1990
. Influence of sex on susceptibility in the Theiler’s murine encephalomyelitis virus model for multiple sclerosis.
J. Neuroimmunol.
29
:
15
.-19.
11
Gran, B., B. Hemmer, R. Martin.
1999
. Molecular mimicry and multiple sclerosis: a possible role for degenerate T cell recognition in the induction of autoimmune responses.
J. Neural Transm.
55
:(Suppl.):
19
.-31.
12
Olson, J. K., T. N. Eagar, S. D. Miller.
2002
. Functional activation of myelin-specific T cells by virus-induced molecular mimicry.
J. Immunol.
169
:
2719
.-2726.
13
Cua, D. J., D. R. Hinton, S. A. Stohlman.
1995
. Self-antigen-induced Th2 responses in experimental allergic encephalomyelitis (EAE)-resistant mice: Th2-mediated suppression of autoimmune disease.
J. Immunol.
155
:
4052
.-4059.
14
Voskuhl, R. R., H. Pitchekian-Halabi, A. MacKenzie-Graham, H. F. McFarland, C. S. Raine.
1996
. Gender differences in autoimmune demyelination in the mouse: implications for multiple sclerosis.
Ann. Neurol.
39
:
724
.-733.
15
Dalal, M., S. Kim, R. R. Voskuhl.
1997
. Testosterone therapy ameliorates experimental autoimmune encephalomyelitis and induces a T helper 2 bias in the autoantigen-specific T lymphocyte response.
J. Immunol.
159
:
3
.-6.
16
Bebo, B. F., Jr, J. C. Schuster, A. A. Vandenbark, H. Offner.
1998
. Gender differences in experimental autoimmune encephalomyelitis develop during the induction of the immune response to encephalitogenic peptides.
J. Neurosci. Res.
52
:
420
.-426.
17
Hill, K. E., M. Pigmans, R. S. Fujinami, J. W. Rose.
1998
. Gender variations in early Theiler’s virus induced demyelinating disease: differential susceptibility and effects of IL-4, IL-10 and combined IL-4 with IL-10.
J. Neuroimmunol.
85
:
44
.-51.
18
Alley, J., S. Khasabov, D. Simone, A. Beitz, M. Rodriguez, M. K. Njenga.
2003
. More severe neurologic deficits in SJL/J male than female mice following Theiler’s virus-induced CNS demyelination.
Exp. Neurol.
180
:
14
.-24.
19
Han, X., P. Lundberg, B. Tanamachi, H. Openshaw, J. Longmate, E. Cantin.
2001
. Gender influences herpes simplex virus type 1 infection in normal and γ interferon-mutant mice.
J. Virol.
75
:
3048
.-3052.
20
Huber, S. A., J. Kupperman, M. K. Newell.
1999
. Hormonal regulation of CD4+ T-cell responses in coxsackievirus B3-induced myocarditis in mice.
J. Virol.
73
:
4689
.-4695.
21
Barna, M., T. Komatsu, Z. Bi, C. S. Reiss.
1996
. Sex differences in susceptibility to viral infection of the central nervous system.
J. Neuroimmunol.
67
:
31
.-39.
22
Pullen, L. C., S. D. Miller, M. C. Dal Canto, B. S. Kim.
1993
. Class I-deficient resistant mice intracerebrally inoculated with Theiler’s virus show an increased T cell response to viral antigens and susceptibility to demyelination.
Eur. J. Immunol.
23
:
2287
.-2293.
23
Pullen, L. C., S. H. Park, S. D. Miller, M. C. Dal Canto, B. S. Kim.
1995
. Treatment with bacterial LPS renders genetically resistant C57BL/6 mice susceptible to Theiler’s virus-induced demyelinating disease.
J. Immunol.
155
:
4497
.-4503.
24
Targoni, O. S., P. V. Lehmann.
1998
. Endogenous myelin basic protein inactivates the high avidity T cell repertoire.
J. Exp. Med.
187
:
2055
.-2063.
25
Rodriguez, M., J. Leibowitz, P. Lampert.
1983
. Persistent infection of oligodendrocytes in Theiler’s virus induced encephalomyelitis.
Ann. Neurol.
13
:
426
.-433.
26
Lipton, H. L., J. Kratochvil, P. Sethi, M. C. Dal Canto.
1984
. Theiler’s virus antigen detected in mouse spinal cord 2 1/2 years after infection.
Neurology
34
:
1117
.-1119.
27
Bureau, J. F., X. Montagutelli, F. Bihl, S. Lefebvre, J. L. Guenet, M. Brahic.
1993
. Mapping loci influencing the persistence of Theiler’s virus in the murine central nervous system.
Nat. Genet.
5
:
87
.-91.
28
Inoue, A., Y. K. Choe, B. S. Kim.
1994
. Analysis of antibody responses to predominant linear epitopes of Theiler’s murine encephalomyelitis virus.
J. Virol.
68
:
3324
.-3333.
29
Roos, R. P., S. Stein, M. Routbort, A. Senkowski, T. Bodwell, R. Wollmann.
1989
. Theiler’s murine encephalomyelitis virus neutralization escape mutants have a change in disease phenotype.
J. Virol.
63
:
4469
.-4473.
30
Zurbriggen, A., R. S. Fujinami.
1989
. A neutralization-resistant Theiler’s virus variant produces an altered disease pattern in the mouse central nervous system.
J. Virol.
63
:
1505
.-1513.
31
Eidinger, D., T. J. Garrett.
1972
. Studies of the regulatory effects of the sex hormones on antibody formation and stem cell differentiation.
J. Exp. Med.
136
:
1098
.-1116.
32
Weinstein, Y., S. Ran, S. Segal.
1984
. Sex-associated differences in the regulation of immune responses controlled by the MHC of the mouse.
J. Immunol.
132
:
656
.-661.
33
Michaels, R. M., K. D. Rogers.
1971
. A sex difference in immunologic responsiveness.
Pediatrics
47
:
120
.-123.
34
Vranckx, R., L. Muylle, J. Cole, R. Moldenhaser, M. E. Peetermans.
1986
. HBV vaccinations in medical and paramedical staff: the impact of age on immunization results.
Vox Sang.
50
:
220
.-222.
35
Murray, P. D., K. D. Pavelko, J. Leibowitz, X. Lin, M. Rodriguez.
1998
. CD4+ and CD8+ T cells make discrete contributions to demyelination and neurologic disease in a viral model of multiple sclerosis.
J. Virol.
72
:
7320
.-7329.
36
Njenga, M. K., K. D. Pavelko, J. Baisch, X. Lin, C. David, J. Leibowitz, M. Rodriguez.
1996
. Theiler’s virus persistence and demyelination in major histocompatibility complex class II-deficient mice.
J. Virol.
70
:
1729
.-1737.
37
Karls, K. A., P. W. Denton, R. W. Melvold.
2002
. Susceptibility to Theiler’s murine encephalomyelitis virus-induced demyelinating disease in BALB/cAnNCr mice is related to absence of a CD4+ T-cell subset.
Mult. Scler.
8
:
469
.-474.
38
Kang, B., H. K. Kang, B. S. Kim.
2005
. Identification of capsid epitopes of Theiler’s virus recognized by CNS-infiltrating CD4+ T cells from virus-infected C57BL/6 mice.
Virus Res.
108
:
57
.-61.
39
Rodriguez, M., K. Pavelko, R. L. Coffman.
1995
. Gamma interferon is critical for resistance to Theiler’s virus-induced demyelination.
J. Virol.
69
:
7286
.-7290.
40
Amadori, A., R. Zamarchi, G. De Silvestro, G. Forza, G. Cavatton, G. A. Danieli, M. Clementi, L. Chieco-Bianchi.
1995
. Genetic control of the CD4/CD8 T-cell ratio in humans.
Nat. Med.
1
:
1279
.-1283.
41
Yahikozawa, H., A. Inoue, C. S. Koh, Y. K. Choe, B. S. Kim.
1997
. Major linear antibody epitopes and capsid proteins differentially induce protective immunity against Theiler’s virus-induced demyelinating disease.
J. Virol.
71
:
3105
.-3113.
42
Huygen, K., K. Palfliet.
1984
. Strain variation in interferon γ production of BCG-sensitized mice challenged with PPD II: importance of one major autosomal locus and additional sexual influences.
Cell. Immunol.
85
:
75
.-81.
43
Papenfuss, T. L., C. J. Rogers, I. Gienapp, M. Yurrita, M. McClain, N. Damico, J. Valo, F. Song, C. C. Whitacre.
2004
. Sex differences in experimental autoimmune encephalomyelitis in multiple murine strains.
J. Neuroimmunol.
150
:
59
.-69.
44
Lindsey, J. W..
1996
. Characteristics of initial and reinduced experimental autoimmune encephalomyelitis.
Immunogenetics
44
:
292
.-297.
45
Okuda, Y., M. Okuda, C. C. Bernard.
2002
. Gender does not influence the susceptibility of C57BL/6 mice to develop chronic experimental autoimmune encephalomyelitis induced by myelin oligodendrocyte glycoprotein.
Immunol. Lett.
81
:
25
.-29.
46
Clatch, R. J., D. C. Pevear, E. Rozhon, R. P. Roos, S. D. Miller, H. L. Lipton.
1987
. Characterization and specificity of humoral immune responses to Theiler’s murine encephalomyelitis virus capsid proteins.
J. Gen. Virol.
68
:(Pt. 12):
3191
.-3196.
47
Clatch, R. J., H. L. Lipton, S. D. Miller.
1987
. Class II-restricted T cell responses in Theiler’s murine encephalomyelitis virus (TMEV)-induced demyelinating disease. II. Survey of host immune responses and central nervous system virus titers in inbred mouse strains.
Microb. Pathog.
3
:
327
.-337.
48
Yauch, R. L., B. S. Kim.
1994
. A predominant viral epitope recognized by T cells from the periphery and demyelinating lesions of SJL/J mice infected with Theiler’s virus is located within VP1(233–244).
J. Immunol.
153
:
4508
.-4519.
49
Yauch, R. L., K. Kerekes, K. Saujani, B. S. Kim.
1995
. 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.
J. Virol.
69
:
7315
.-7318.
50
Yauch, R. L., J. P. Palma, H. Yahikozawa, C.-S. Koh, B. S. Kim.
1998
. Role of individual T cell epitopes of Theiler’s virus in the pathogenesis of demyelination correlates with the ability to induce a Th1 response.
J. Virol.
72
:
6169
.-6174.
51
Fiette, L., C. Aubert, M. Brahic, C. P. Rossi.
1993
. Theiler’s virus infection of β2-microglobulin-deficient mice.
J. Virol.
67
:
589
.-592.
52
Rodriguez, M., A. K. Patick, L. R. Pease.
1990
. Abrogation of resistance to Theiler’s virus-induced demyelination in C57BL mice by total body irradiation.
J. Neuroimmunol.
26
:
189
.-199.
53
Palma, J. P., H.-G. Lee, M. Mohindru, B.-S. Kang, M. Dal Canto, S. D. Miller, B. S. Kim.
2001
. Enhanced susceptibility to Theiler’s virus-induced demyelinating disease in perforin-deficient mice.
J. Neuroimmunol.
116
:
125
.-135.
54
Rosenthal, A., R. S. Fujinami, P. W. Lampert.
1986
. Mechanism of Theiler’s virus-induced demyelination in nude mice.
Lab. Invest.
54
:
515
.-522.
55
Murray, P. D., D. B. McGavern, X. Lin, M. K. Njenga, J. Leibowitz, L. R. Pease, M. Rodriguez.
1998
. Perforin-dependent neurologic injury in a viral model of multiple sclerosis.
J. Neurosci.
18
:
7306
.-7314.
56
Rivera-Quinones, C., D. McGavern, J. D. Schmelzer, S. F. Hunter, P. A. Low, M. Rodriguez.
1998
. Absence of neurological deficits following extensive demyelination in a class I-deficient murine model of multiple sclerosis.
Nat. Med.
4
:
187
.-193.
57
Lipton, H. L., R. Melvold.
1984
. Genetic analysis of susceptibility to Theiler’s virus-induced demyelinating disease in mice.
J. Immunol.
132
:
1821
.-1825.
58
Rodriguez, M., C. S. David.
1985
. Demyelination induced by Theiler’s virus: influence of the H-2 haplotype.
J. Immunol.
135
:
2145
.-2148.