Preferential Induction of Protective T Cell Responses to Theiler's Virus ...

JOURNAL OF VIROLOGY, Mar. 2011, p. 3033–3040 0022-538X/11/$12.00 doi:10.1128/JVI.02400-10 Copyright © 2011, American Society for Microbiology. All Rights Reserved.

Vol. 85, No. 6

Preferential Induction of Protective T Cell Responses to Theiler’s Virus in Resistant (C57BL/6 ⫻ SJL)F1 Mice䌤 Young-Hee Jin, Hyun Seok Kang, Mani Mohindru, and Byung S. Kim* Department of Microbiology-Immunology and Neuroscience Institute, Northwestern University Medical School, 303 East Chicago Avenue, Chicago, Illinois 60611 Received 17 November 2010/Accepted 16 December 2010

Infection of the central nervous system (CNS) with Theiler’s murine encephalomyelitis virus (TMEV) induces an immune-mediated demyelinating disease in susceptible mouse strains such as SJL/J (H-2s) but not in strains such as C57BL/6 (H-2b). In addition, it has been shown that (C57BL/6 ⴛ SJL/J)F1 mice (F1 mice), which carry both resistant and susceptible MHC haplotypes (H-2b/s), are resistant to both viral persistence and TMEV-induced demyelinating disease. In this study, we further analyzed the immune responses underlying the resistance of F1 mice. Our study shows that the resistance of F1 mice is associated with a higher level of the initial virus-specific H-2b-restricted CD8ⴙ T cell responses than of the H-2s-restricted CD8ⴙ T cell responses. In contrast, pathogenic Th17 responses to viral epitopes are lower in F1 mice than in susceptible SJL/J mice. Dominant effects of resistant genes expressed in antigen-presenting cells of F1 mice on regulation of viral replication and induction of protective T cell responses appear to play a crucial role in disease resistance. Although the F1 mice are resistant to disease, the level of viral RNA in the CNS was intermediate between those of SJL/J and C57BL/6 mice, indicating the presence of a threshold of viral expression for pathogenesis. Intracerebral infection of susceptible mice with Theiler’s murine encephalomyelitis virus (TMEV) induces a chronic, progressive demyelinating disease that is clinically and histopathologically similar to a form of human multiple sclerosis (MS) (25). In addition, the various immunological and genetic factors that affect disease outcome in TMEV-infected mice closely parallel those associated with the development of MS (23). Combined with a suspected viral etiology for MS (1, 13, 46), these similarities make TMEV-induced demyelinating disease (TMEV-IDD) an attractive and relevant infectious model for investigating this human demyelinating disease. Development of TMEV-IDD in highly susceptible SJL/J (H-2s) mice (SJL mice) is associated with chronic viral persistence in the central nervous system (CNS) (7, 26, 44), whereas resistant C57BL/6 (H-2b) mice (B6 mice) clear the virus within 2 to 4 weeks of infection (41). Thus, viral persistence appears to be a critical factor in the disease development. Interestingly, F1 mice crossed between resistant B6 and susceptible SJL mice [(C57BL/6 ⫻ SJL/J)F1 mice; herein referred to as F1 mice] are able to clear the virus and are relatively resistant to TMEVIDD, indicating that genes involved in the resistance are dominant genetic traits (8, 9, 27). Resistance to TMEV-IDD has been closely associated with the major histocompatibility complex (MHC) class I locus (27, 42), which suggests that class I-restricted CD8⫹ T cells are an important mediator of the protection and/or pathogenesis. It has been of great interest to define and characterize the class I-restricted CD8⫹ T cell responses in both resistant and susceptible mice. Resistant H-2b mice mount CD8⫹ T cell

responses to one highly dominant (VP2121-130) (6, 12, 19) and two minor (VP2165-173 and VP3110-120) viral epitopes of TMEV (29). Similarly, CNS-infiltrating CD8⫹ T cells in susceptible SJL mice recognize a dominant (VP3159-166) and two subdominant (VP3173-181 and VP111-20) epitopes (20). Interestingly, all of the epitopes for CD8⫹ T cells from resistant B6 mice are restricted by H-2Db, whereas CD8⫹ T cells from susceptible SJL mice recognize the epitopes in conjunction with H-2Ks. Despite the similar proportions of CNS-infiltrating CD8⫹ T cells that recognize TMEV epitopes in both resistant B6 and susceptible SJL mice, the overall number of CD8⫹ T cells in the CNS is significantly lower in virus-infected SJL mice than in resistant B6 mice (30). Therefore, the inefficient viral clearance in mice that are susceptible to TMEV-IDD may reflect an insufficient number of, rather than deficient function of, protective CD8⫹ T cells compared to those in the resistant mice. In contrast to the protective role of virus-specific CD8⫹ T cells, CD4⫹ T cell responses are considered to play a pivotal role in the pathogenesis of demyelinating disease (22, 38, 48). Our recent studies demonstrated that Th17 cells in particular play a critical pathogenic role; hence, the treatment of susceptible mice with anti-interleukin 17 (anti-IL-17) antibody renders them resistant to the disease (16). Therefore, it is likely that a balance between the protective virus-specific CD8⫹ T cell responses and pathogenic Th17 cell levels may determine the outcome of disease development. Despite extensive studies on immunological parameters of resistant and susceptible strains, the immune response levels, the CD4⫹ T cell types, and the distribution of MHC haplotype-restricted T cells in resistant F1 mice expressing both MHCs are unknown. For clinical application, it is important to understand how protective and pathogenic immune responses function in an environment where the expression of resistant and susceptible genes are mixed. In this study, we directly compared the TMEVspecific CD4⫹ and CD8⫹ T cell responses among the proto-

* Corresponding author. Mailing address: Department of Microbiology-Immunology, Northwestern University Medical School, 303 E. Chicago Ave., Chicago, IL 60611. Phone: (312) 503-8693. Fax: (312) 503-1339. E-mail: [email protected]. 䌤 Published ahead of print on 29 December 2010. 3033

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FIG. 1. Levels of viral loads and mononuclear cells in the CNS of TMEV-infected SJL, F1, and B6 mice at 10 and 45 days postinfection. Female SJL/J (SJL) and C57BL/6 (B6) mice were purchased from the Charles River Laboratories (Charles River, MA) through the National Cancer Institute (Frederick, MD). Female (C57BL/6 ⫻ SJL)F1 (F1) mice were purchase from the Jackson Laboratory. (A) Viral persistence levels in the brain (BR) and spinal cord (SC) of infected mice at 10, 45, and 80 days postinfection (DPI) were determined by quantitative PCR in SYBR green master mix using an iCycler (Bio-Rad, Hercules, CA). Data are values from a representative experiment from three independent experiments conducted with CNS pools from three mice per group. The values given are means ⫾ standard deviations of triplicate reactions. Statistically significant differences among the groups at a given time point are indicated with asterisks (***, P ⬍ 0.001). For multigroup comparisons, one-way analysis of variance (ANOVA) with the Tukey-Kramer multiple-comparison test was used. (B) Numbers in fluorescence-activated cell sorting (FACS) plots are percentages of CD4⫹ and CD8⫹ T cells and CD4/CD8 ratios in the CNS. (C) Overall numbers of mononuclear cell types in the CNS. Data are representative of three experiments using three mice per group. MP, macrophage. CNS-infiltrating mononuclear cells were enriched at the bottom one-third of a continuous 100% Percoll (Pharmacia, Piscataway, NJ) gradient.

typically susceptible SJL (H-2s), resistant B6 (H-2b), and their F1 (H-2b/s) mice. Our results revealed that virus-specific CD8⫹ T cell responses in F1 mice preferentially recognize the viral determinants of resistant parents. In addition, F1 mice induce lower levels of pathogenic Th17 responses than the susceptible parent mice. These results indicate that F1 mice acquire resistance to the demyelinating disease by preferentially selecting protective CD8⫹ T cell responses and nonpathogenic CD4⫹ T cell development from the resistant parents. Our studies further suggest that antigen-presenting cells (APCs) play a critical role in the reduced development of pathogenic Th17 cells in F1 mice. We believe that our results provide an important insight into the immunological mechanisms associated with mixed genetic contributions to resistance/susceptibility of the host to virus-induced chronic inflammatory diseases. Reduced viral load and CD4ⴙ T cell infiltration in the CNS of (B6 ⴛ SJL)F1 mice. It is known that (B6 ⫻ SJL)F1 mice carrying both H-2b and H-2s genes are relatively resistant to TMEV-induced demyelinating disease (9, 27). However, the underlying mechanisms for this resistance remain unclear. To further correlate the disease susceptibility with viral persistence levels, viral message levels in the CNS of mice at 10, 45, and 80 days postinfection were assessed by quantitative PCR (Fig. 1A). The results show that the brains and spinal cords of F1 mice carry significantly higher levels of viral message than resistant B6 parent mice, particularly during late stages (45 and 80 days) of infection. However, the viral levels of F1 mice were

significantly lower than those of susceptible SJL mice, suggesting that F1 mice are able to clear virus more efficiently. These results demonstrate that viral persistence levels are loosely associated with disease susceptibility and may not be an accurate indicator of the development of demyelinating disease. The viral level in F1 mice may border the threshold of pathogenesis, and thus, an extensive time period is required to develop a demyelinating disease. Since CNS-infiltrating T cell responses are most likely involved in the protection against and/or pathogenesis of demyelinating disease, we first compared the levels of CD4⫹ and CD8⫹ T cells accumulated in the CNS of infected mice (Fig. 1B). The proportion of CD4⫹ T cells in the CNS of F1 mice was consistently lower than that of susceptible SJL mice (17% versus 24%) but higher than that of resistant B6 mice (9%). Consequently, the ratio of CD4⫹ to CD8⫹ T cells in SJL mice was greater than 3 at the early stage of infection and remained higher at 47 days postinfection. However, the CD4⫹/CD8⫹ ratios of F1 mice were similar to those of B6 mice, which were approximately 2- to 3-fold lower than those of SJL mice during the course of viral infection. When the overall cellular infiltration to the CNS was considered, only CD4⫹ T cell levels were significantly higher (⬎2-fold) in SJL mice than in F1 and B6 mice at 10 days postinfection (Fig. 1C). The differences in the infiltration levels of CD8⫹ T cells, B cells, macrophages, dendritic cells, and NK cells were not noticeable. These results suggest that the relatively higher levels of CD4⫹ T cells in SJL

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FIG. 2. CD4⫹ T cell responses in SJL, F1, and B6 mice at 8 days after TMEV infection. (A) Proportions of IFN-␥-producing CD4⫹ cells in the CNS were determined by intracellular staining using phycoerythrin (PE)-labeled rat monoclonal anti-IFN-␥ (XMG1.2) antibody (Pharmingen, San Diego, CA) after stimulation with 2 ␮M SJL CD4 structural peptides (SJL SP), SJL CD4 nonstructural peptides (SJL NSP), or B6 CD4 peptides (B6 CD4). (B) IFN-␥-producing CD4⫹ cell numbers in the CNS after stimulation with the indicated epitope mixtures are shown. (C) mRNA expression levels of cytokines and transcription factors in the CNS at 10 days postinfection were analyzed by quantitative PCR. Data are expressed as fold induction of expression after normalization to the GAPDH (glyceraldehyde-3phosphate dehydrogenase) mRNA levels using the following primer sets: IFN-␥ (5⬘-ACTGGCAAAAGGATGGTGAC-3⬘ and 5⬘-TGAGC TCATTGAATGCTTGG-3⬘); T-bet (5⬘-CAACAACCCCTTTGCGA AAG-3⬘ and 5⬘-TCCCCCAAGCAGTTGACAGT-3⬘); IL-17A (5⬘-CT CCAGAAGGCCCTCAGACTAC-3⬘ and 5⬘-AGCTTTCCCTCCGCA TTGACACAG-3⬘); ROR-␥t (5⬘-CCGCTGAGAGGGCTTCAC-3⬘ and 5⬘-TGCAGGAGTAGGCCACATTACA-3⬘); and GAPDH (5⬘-A ACTTTGGCATTGTGGAAGGG-CTC-3⬘ and 5⬘-TGCCTGCTTCA CCACCTTGAT-3⬘). The values given are means ⫾ standard deviations of triplicate experiments. Statistically significant differences are indicated with asterisks (*, P ⬍ 0.05; **, P ⬍ 0.01; ***, P ⬍ 0.001). A representative result of three separate experiments using three mice per group is shown. The significance of the differences (two-tailed P value) between experimental groups was analyzed with an unpaired Student t test using the InStat program (GraphPAD Software, San Diego, CA). (D) Proliferative responses and cytokine levels (IFN-␥ and IL-17) of splenocytes (1 ⫻ 106 cells/well) from mice at 10 days postinfection following stimulation for 2 days with 2 ␮M I-As-restricted structural capsid peptides (SJL SP), I-As-restricted nonstructural peptides (SJL NSP), or I-Ab-restricted CD4⫹ T cell epitope peptides (B6 CD4). SJL SP included VP1233–250, VP274–86, and VP324–37; SJL NSP included 3D6–23, 3D21–36, and 3D412–430; B6 CD4 included VP2203–220 and VP425–38. Proliferation was measured by determining the [3H]thymidine ([3H]TdR) uptake by the cells in triplicate and expressed as net counts per minute (⌬cpm ⫾ SEM). Cytokine levels in

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mice may contribute to the development of demyelinating disease. Intermediate CD4ⴙ T cell responses to viral epitopes in F1 mice. To compare the overall levels of activated CD4⫹ T cells and their cytokine production levels in the CNS of TMEVinfected SJL, F1, and B6 mice, levels of gamma interferon (IFN-␥)-producing CD4⫹ T cells were assessed at 8 days postinfection following stimulation with I-As- and I-Ab-restricted epitopes. The responses in F1 mice to both of the I-A-restricted epitopes were intermediate to those of the SJL and B6 mice, respectively (Fig. 2A and B). In addition, it has recently been shown that Th17 cells play a critical role in the pathogenesis of TMEV-induced demyelinating disease (16). To assess the relative levels of Th1 and Th17 types in vivo at the inflammatory site, we also compared IFN-␥ and IL-17 mRNA levels, as well as the transcription factors for the cytokines (T box expressed in T cells [T-bet] and retinoic acid orphan receptor gamma T [ROR-␥t], respectively) expressed in the CNS of virus-infected F1, SJL, and B6 mice at 8 days postinfection (Fig. 2C). The results showed that there are little differences in the IFN-␥ and T-bet levels, indicating similar Th1 type responses among F1, SJL, and B6 mice. However, the levels of IL-17 and ROR-␥t representing Th17 responses in F1 mice were significantly lower than those of susceptible SJL mice but higher than those of resistant B6 mice. These results strongly suggest that the induction of intermediate levels of Th17 responses in F1 mice may dampen the pathogenesis of demyelinating disease in comparison to that in SJL mice yet may be able to exert higher pathogenic potential than what occurs in B6 mice, which leads to delayed disease development. Levels of peripheral CD4⫹ T cell responses of SJL, F1, and B6 mice infected with TMEV were also assessed (Fig. 2D). Interestingly, F1 mice showed significantly reduced levels of proliferation to both SJL and B6 epitopes compared to SJL and B6 mice, respectively. CD4⫹ T cells from F1 mice produced significantly lower levels of IL-17 in response to I-Asrestricted epitopes than SJL CD4⫹ T cells. However, the cytokine levels produced by F1 CD4⫹ T cells in response to I-Ab-restricted epitopes were significantly lower than those produced by B6 CD4⫹ T cells in response to the epitopes. Together, the overall IL-17 production in the periphery of F1 mice is lower than that in susceptible SJL mice (Fig. 2D), similar to what occurs in the CNS (Fig. 2C). In contrast, the overall level of protective IFN-␥ produced in the periphery of resistant B6 mice is greater than the levels in F1 and SJL mice (Fig. 2D). Thus, the intermediate cytokine levels produced by F1 CD4⫹ T cells may provide porous protection against developing a TMEV-induced demyelinating disorder. Preferential CD8ⴙ T cell responses to H-2b-restricted epitopes in the CNS of F1 mice. It has previously been shown that antiviral CD8⫹ T cell responses associated with H-2Db

the culture supernatants were assessed with an ELISA. The results of triplicate analyses of a single representative experiment from three separate experiments are shown. The values are means ⫾ standard deviations of results from triplicate cultures. Statistically significant differences are indicated with asterisks (*, P ⬍ 0.05; **, P ⬍ 0.01; ***, P ⬍ 0.001).

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FIG. 3. Comparison of CNS-infiltrating CD8⫹ T cell levels specific for viral epitopes among B6, F1, and SJL mice at 10 and 47 days after TMEV infection. (A) Levels of IFN-␥ producing CD8⫹ cells in the CNS were determined by intracellular staining after stimulation for 6 h with 2 ␮M CD8 epitope peptides. Numbers in the FACS plots represent percentages of IFN-␥-producing CD8⫹ cells of the total infiltrating CD8⫹ cells. (B) The numbers of IFN-␥-producing cells per mouse strain CNS (⫻104) in the absence (none) or presence of epitope peptides: VP2121–120 as the predominant B6 epitope, and VP3159–166, VP3173–181, and VP111–20 as the equivalent SJL epitopes. Statistically significant differences are indicated with asterisks (*, P ⬍ 0.05; **, P ⬍ 0.01; ***, P ⬍ 0.001). The data are results of three separate experiments using three mice per group.

can convert genetically susceptible mice to be resistant to TMEV-IDD (32). Therefore, it is conceivable that resistant F1 mice may preferentially develop antiviral CD8⫹ T cell responses associated with the resistant H-2Db haplotype of B6 mice. To determine this possibility, viral epitope peptides restricted with H-2Db and H-2Ks were used to detect virusspecific CD8⫹ T cell levels in the CNS of B6, SJL, and F1 mice at 10 and 47 days after TMEV infection (Fig. 3). The results indicate that the level of capsid-specific CD8⫹ T cell response to H-2Db-restricted epitopes in virus-infected F1 mice is equal to or greater than that of the resistant parental B6 mice. However, the levels of CD8⫹ T cell responses to H-2Ks-restricted epitopes in F1 mice are minimal (Fig. 3A). Therefore, the CD8⫹ T cell response in the CNS of F1 mice is rather similar to that of resistant B6 mice throughout the course of viral infection. Interestingly, higher levels of CD8⫹ T cell responses were induced in resistant B6 and F1 mice at the early stage of viral infection but lower levels were induced at a late stage in comparison to the responses of susceptible SJL mice (Fig. 3B). This difference may reflect viral persistence in the CNS of

mice, as the viral load is relatively higher in SJL mice. Nevertheless, the overall level of virus-specific CD8⫹ T cells and/or the preferential H-2Db-restricted CD8⫹ T cell response appears to correlate with resistance to TMEV-induced demyelination. The lower levels of H-2Ks-restricted CD8⫹ T cell responses than of H-2Db-restricted CD8⫹ T cell responses observed in F1 mice (Fig. 3) were unexpected. To examine the possibility that differential expression of H-2Db and/or H-2Ks in F1 mice contributes to the preferred H-2Db-restricted CD8⫹ T cell response, we examined the expression of H-2 class I and class II molecules (not shown). Our results indicate that the expression levels of both H-2Db and H-2Ks in the CNS of F1 mice after viral infection are similarly reduced compared to the levels of infected B6 and SJL mice, respectively. Thus, the expression pattern of MHC class I does not explain the preferred H-2Db-restricted CD8⫹ T cell responses in F1 mice. The expression levels of I-Ab and I-As molecules in F1 mice were not significantly reduced compared to those in parent mice (not shown). Similarly, the expression levels of costimulatory molecules (CD80, CD86, and CD40) on CNS

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FIG. 4. Viral load, cellular infiltration, and cytokine production in F1 versus SJL mice carrying VP2-specific TCR-Tg. (A) Virus levels in the CNS of SJL TCR-Tg and F1 TCR-Tg mice at 5, 8, and 21 days postinfection (DPI) were determined by quantitative PCR using the above (Fig. 2) and following primer sets: TMEV (VP1) (5⬘-TGACTAAGCAGGACTATGCCTTCC-3⬘ and 5⬘-CAACGAGCCACATATGCGGATTAC-3⬘) and GAPDH (5⬘-AACTTTGGCATTGTGGAAGGG-CTC-3⬘ and 5⬘-TGCCTGCTTCACCACCTTGAT-3⬘). mRNA levels are shown as fold induction after normalization with GAPDH mRNA levels. (B) Cytokine mRNA levels in the CNS of SJL TCR-Tg and F1 TCR-Tg mice were analyzed by quantitative PCR using the following additional primer sets: IL-6 (5⬘-AGTTGCCTTCTTGGGACTGA-3⬘ and 5⬘-TCCACGATTTC CCAGAGAAC-3⬘); TNF-␣ (5⬘-CTGTGAAGGGAATGGGTGTT-3⬘ and 5⬘-GGTCACTGTCCCAGCATCTT-3⬘). Data are expressed as fold induction after normalization to GAPDH mRNA levels. The results shown are for a representative experiment of three separate experiments using three mice per group. (C and D) Responses of splenic CD4⫹ T cells from SJL TCR-Tg and F1 TCR-Tg mice at 5 and 8 days postinfection to the cognate epitope. (C) Proliferative responses of T cells from F1 TCR-Tg and SJL TCR-Tg mice to 2 ␮M VP272-86 for 2 days. (D) ELISA determinations of cytokine levels (IFN-␥ and IL-17) in the above-described cultures. Statistically significant differences are indicated with asterisks (*, P ⬍ 0.05; **, P ⬍ 0.01; ***, P ⬍ 0.001). The data are from a representative experiment of three separate experiments using three mice per group.

macrophages and microglia did not correlate with the preferential H-2Db-restricted CD8⫹ T cell responses (not shown). Therefore, the level and/or affinity of H-2Db-restricted epitopes and H-2Ks-restricted epitopes may be haplotype dependent, and the differences may facilitate the induction of an H-2Db-restricted CD8⫹ T cell response in F1 mice. The viral load and IL-17 production level are lower in F1 VP2-specific TCR-Tg mice than in SJL mice. In order to further understand the role of a resistant B6 background gene in F1 mice, we have tested virus-specific T cell receptor transgene (TCR-Tg)-bearing CD4⫹ T cell responses in F1 and SJL mice (Fig. 4). For these experiments, we utilized F1 TCR-Tg and SJL TCR-Tg mice which express the transgenic TCR recognizing VP274-86, a major CD4⫹ T cell epitope restricted to I-As (16). Greater than 80% of the CD4⫹ T cells express VP2specific TCR in both TCR-Tg mice bearing I-As/b and those bearing I-As. Thus, the utilization of these TCR-Tg mice would reveal the potential contributions of the F1 and SJL backgrounds, including the differences in the expression of I-A molecules to the development of clonal CD4⫹ T cells specific for a viral epitope.

Similar to the loads in non-TCR-Tg mice (Fig. 1), viral loads in the CNS of F1 TCR-Tg mice were markedly lower than those of SJL TCR-Tg mice (Fig. 4A). In addition, the level of CD4⫹ T cells infiltrating the CNS was drastically reduced in the F1 mice throughout the viral infection. The reduced cellular infiltration accompanied reduced production of proinflammatory cytokines, such as IL-6, IL-17, IFN-␥, and tumor necrosis factor alpha (TNF-␣), in the CNS of infected F1 mice at the early stage (5 days postinfection) of viral infection (Fig. 4B). However, at the peak of the immune responses (8 days postinfection), only IL-6 and IL-17 levels, which are associated with Th17 responses, were significantly lower than those of SJL TCR-Tg mice. For these experiments, the levels of cytokine genes expressed in the CNS during viral infection were assessed without an external stimulation to mirror the status of activated cell types in the CNS. In vitro restimulation of these CNS cells with the corresponding epitope peptide showed a pattern similar to that in the infected mice, although the levels were much greater (not shown). Similarly, peripheral CD4⫹ T cell responses (proliferation and cytokine production) of F1 TCR-Tg mice to the cognate VP272-86 peptide were decreased

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compared to that of SJL TCR-Tg mice, except for the proliferation at 5 days postinfection (Fig. 4C and D). However, IFN-␥-producing Th1 levels remain lower in the periphery of F1 mice than in SJL mice at these time points of viral infection, in contrast to the results in the CNS (Fig. 4B). These results strongly suggest that the development of virus-specific pathogenic Th17 cells, including an identical I-As-restricted clonal CD4⫹ T cell response, is significantly reduced in resistant F1 mice compared to susceptible SJL mice. The reduction of Th17 cell development in F1 mice likely reflects the lower levels of viral load, which are associated with Th17 cell levels in infected mice (16). Th17 development is lower with virus-infected F1 APCs than with SJL APCs. We have previously shown that TMEV-infected APCs from resistant B6 mice induce low levels of Th17 development in vitro compared to those from susceptible SJL mice (16). To determine the contribution of antigen-presenting cells to the development of Th cell types, we stimulated CD4⫹ T cells isolated from naïve SJL VP2-specific-TCR-Tg mice with F1 and SJL macrophages isolated from virus-infected F1 and SJL mice or with macrophages infected with TMEV in vitro (Fig. 5A and B). The proliferation level was significantly higher in cultures stimulated with F1 macrophages infected either in vitro or in vivo than in those stimulated with SJL macrophages. Thus, macrophages from F1 mice appear to be more efficient in expanding the identical virus-specific Th cells than macrophages from SJL mice. The level of IFN-␥ production was higher in cultures stimulated with macrophages from virus-infected F1 mice (Fig. 5A), although the levels were similar when cultures were stimulated with macrophages infected in vitro (Fig. 5B). In contrast, the level of IL-17 produced in cultures stimulated with either in vitro- or in vivo-infected F1 macrophages was significantly lower than that produced in cultures stimulated with the respective SJL macrophages. To further determine whether the lower Th17 development by F1 macrophages reflects relative resistance to viral infection, we compared the levels of viral proteins (Fig. 5C) and messages (Fig. 5D) produced in SJL, F1, and B6 macrophages after infection with TMEV for 24 h. The viral protein-producing cells assessed by green fluorescent protein (GFP) production (21) showed a low viral infection (15%) in F1 cells similar to that in B6 macrophages (16%) but in contrast to a high viral infection (⬃40%) in SJL macrophages. Interestingly, however, the viral message level in F1 macrophages was higher than that in B6 macrophages, suggesting an intermediate permissiveness to the viral infection. Since F1 cells are less permissive to TMEV infection and consequently produce lower levels of proinflammatory cytokines, including IL-6 (not shown), similarly to B6 cells (20, 21), the lower level of Th17 development by F1 cells appears to reflect their relative resistance to the viral infection. These results suggest that a combination of low levels of viral loads and pathogenic Th17 cells and a high level of protective Th1 cells in the CNS of F1 mice compared to susceptible SJL mice leads to the resistance to pathogenesis in F1 mice. Earlier genetic studies with this infectious model indicated that the progeny F1 (H-2b/s) mice of resistant B6 (H-2b) and susceptible SJL (H-2s) mice are resistant to TMEV-induced demyelinating disease (27). In recent years, we have defined many immune responses, including innate cytokine responses

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FIG. 5. (A) Proliferation of CD4⫹ T cells (1 ⫻ 105 cells/well) from naïve SJL TCR-Tg mice upon stimulation with isolated peritoneal macrophages (2 ⫻ 104 cells/well) from TMEV-infected SJL and F1 mice at 8 days postinfection in the absence or presence of 2 ␮M VP272–86 for 3 days. Cytokine levels in the culture supernatants were determined using an ELISA. (B) Proliferation of CD4⫹ T cells (1 ⫻ 105 cells/well) from naïve SJL TCR-Tg mice upon stimulation with macrophages (2 ⫻ 104 cells/well) infected with TMEV (10 MOI for 24 h) in vitro in the presence of 2 ␮M VP272–86 for 3 days was assessed. Cytokine levels in the supernatants were determined using an ELISA. (C) Peritoneal macrophages isolated from SJL, F1, and B6 mice were infected with enhanced GFP-TMEV (MOI ⫽ 10) for 24 h, and then the proportion of GFP⫹ cells was assessed using flow cytometry in conjunction with CD11b. (D) The relative viral message levels in macrophages from TMEV-infected SJL, F1, and B6 mice and in isolated macrophages infected in vitro with TMEV for 24 h were determined using quantitative PCR.

to viral infection and CD4⫹ and CD8⫹ T cell responses in virus-infected resistant B6 and susceptible SJL mice (17, 18, 36, 37). Therefore, in this study, we further utilized these parameters, which differ between resistant B6 and susceptible SJL

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mice, to investigate the mechanisms of resistance to TMEVinduced demyelinating disease in F1 mice. Numerous previous studies have established that CD4⫹ T cell responses, Th1 cells in particular, play a pivotal pathogenic role in the development of demyelinating disease in susceptible SJL mice (22, 38, 47, 48). However, our recent studies demonstrated that Th17 cells play a critical pathogenic role, as the levels of Th17 cells in the CNS tightly correlate with the resistance/susceptibility of TMEV-induced demyelinating disease (16). In contrast, the role of Th1 cells remains somewhat unclear, as the treatment of mice with anti-IFN-␥ antibody exacerbates the disease development while infusion of recombinant IFN-␥ promotes it (40, 45). In addition, the presence of virusspecific Th1 cells prior to viral infection appears to be protective but also pathogenic during the infection (33, 48). Interestingly, the overall IFN-␥-producing CD4⫹ T cell responses in the CNS seem to be similar among TMEV-infected SJL, B6, and F1 mice (Fig. 4), but the level of IL-17-producing CD4⫹ T cell response in virus-infected SJL mice is higher than that in resistant B6 and F1 mice. IL-17 is shown to inhibit the apoptosis of virus-infected cells induced by viral infection and cytolytic T cell engagement via upregulation of survival molecules on the target cells (16). Therefore, the lower level of Th17 cells in F1 mice likely contributes to the resistance of F1 mice, rendering them free of disease for a prolonged time period. Unlike the potential involvement of CD4⫹ T cells in viral pathogenesis, CD8⫹ T cells are known to play an important role in clearing viruses from infected hosts (5, 15, 39, 43). Thus, protection from disease development caused by these pathogens may rely heavily on this effector cell population. Previous studies suggest that SJL mice and other susceptible strains do not mount strong TMEV-specific cytotoxic T lymphocyte responses in the CNS (11, 24, 30). In addition, it is conceivable that differences in MHC haplotype restriction between B6 and SJL mice may affect the level of effective viral clearance since the H-2D rather than the H-2K locus is associated with the resistance (3, 10, 28). Interestingly, virus-specific CD8⫹ T cells in B6 utilize H-2D-restricted recognition, as opposed to the H-2K-restricted recognition in SJL mice (20, 29). Since all the predominant antiviral CD8⫹ T cell responses have been identified previously (19, 20, 29), the direct comparison of these CD8⫹ T cell responses provides a credible estimation of the actual magnitude and kinetics of the overall class I-restricted T cell response to TMEV in these resistant and susceptible mouse strains. It is interesting to note that the number and epitope recognition of CNS-infiltrating CD8⫹ T cells from relatively resistant F1 (H-2b/s) mice are nearly identical to those from resistant B6 (H-2b) mice, in contrast to the poor recognition of H-2Ks-restricted epitopes (Fig. 3). These results indicate that CD8⫹ T cell responses restricted with H-2Db are strongly preferred compared to H-2Ks-restricted responses in F1 mice when presented by both class I molecules. The underlying mechanism for this haplotype-dependent differential CD8⫹ T cell response is currently unknown. It is conceivable that a higher expression of H-2Db than H-2Ds or H-2Ks in F1 mice may contribute to the preferred H-2Dbrestricted CD8⫹ T cell response (2). However, the expression level of H-2Db in the CNS of F1 mice after viral infection is significantly reduced compared to the level in B6 mice, simi-

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larly to the expression of H-2Ks in F1 mice compared to that in SJL mice (not shown). Therefore, the differences in the affinity of H-2Db- and H-2Ks-restricted epitopes may result in the preferred induction of H-2Db-restricted CD8⫹ T cell responses in F1 mice. Nevertheless, the overall levels of CNSinfiltrating CD8⫹ T cells in these virus-infected mice correlate well with the susceptibility to TMEV-induced demyelination. However, viral loads in resistant F1 mice remain relatively higher than those in B6 mice (Fig. 1). It is conceivable that the level of the virus-specific CD8⫹ T cell response alone may not be sufficient for effective viral clearance from the CNS. Since IL-17 produced by pathogenic Th17 cells promotes viral persistence (16) and the level of Th17 cells in F1 mice is higher than that in B6 mice, the unfavorable ratio of protective CD8⫹ T cells to pathogenic Th cells may result in higher viral persistence and eventual pathogenicity in F1 mice. The role of persisting virus-specific CD8⫹ T cells in the CNS is not yet clear. In some circumstances, CD8⫹ cytotoxic T lymphocytes may be pathogenic due to the destruction of virus-infected cells in an effort to clear the virus (4, 14, 31, 34, 35). Therefore, the persisting CD8⫹ T cell response in the CNS may reflect an immune response to continued viral loads, and these T cells at the late stage of infection may contribute to the pathogenesis of demyelinating disease. Further studies on the mechanisms involved in differentially protective versus pathogenic CD4⫹ and CD8⫹ T cell responses among genetically different mice, including F1 mice, will help to understand the relationship between T cell responses and persistent infections, which may lead to intervention in chronic infection-induced diseases. This work was supported by grants NS23349 and NS33008 from the USPHS and a grant (RG 4001-A6) from the National Multiple Sclerosis Society. REFERENCES 1. Allen, I., and B. Brankin. 1993. Pathogenesis of multiple sclerosis—the immune diathesis and the role of viruses. J. Neuropathol. Exp. Neurol. 52:95–105. 2. Altintas, A., Z. Cai, L. R. Pease, and M. Rodriguez. 1993. Differential expression of H-2K and H-2D in the central nervous system of mice infected with Theiler’s virus. J. Immunol. 151:2803–2812. 3. Azoulay-Cayla, A., S. Syan, M. Brahic, and J. F. Bureau. 2001. Roles of the H-2Db and H-2Kb genes in resistance to persistent Theiler’s murine encephalomyelitis virus infection of the central nervous system. J. Gen. Virol. 82:1043–1047. 4. Baenziger, J., H. Hengartner, R. Zinkernagel, and G. Cole. 1986. Induction or prevention of immunopathological disease by cloned cytotoxic T cell lines specific for lymphocytic choriomeningitis virus. Eur. J. Immunol. 16:387–393. 5. Begolka, W. S., et al. 2001. CD8-deficient SJL mice display enhanced susceptibility to Theiler’s virus infection and increased demyelinating pathology. J. Neurovirol. 7:409–420. 6. Borson, N. D., et al. 1997. Brain-infiltrating cytolytic T lymphocytes specific for Theiler’s virus recognize H2Db molecules complexed with a viral VP2 peptide lacking a consensus anchor residue. J. Virol. 71:5244–5250. 7. Bureau, J. F., et al. 1993. Mapping loci influencing the persistence of Theiler’s virus in the murine central nervous system. Nat. Genet. 5:87–91. 8. Bureau, J. F., et al. 1992. The interaction of two groups of murine genes determines the persistence of Theiler’s virus in the central nervous system. J. Virol. 66:4698–4704. 9. Clatch, R. J., H. L. Lipton, and S. D. Miller. 1986. Characterization of Theiler’s murine encephalomyelitis virus (TMEV)-specific delayed-type hypersensitivity responses in TMEV-induced demyelinating disease: correlation with clinical signs. J. Immunol. 136:920–927. 10. Clatch, R. J., R. W. Melvold, S. D. Miller, and H. L. Lipton. 1985. Theiler’s murine encephalomyelitis virus (TMEV)-induced demyelinating disease in mice is influenced by the H-2D region: correlation with TEMV-specific delayed-type hypersensitivity. J. Immunol. 135:1408–1414. 11. Dethlefs, S., M. Brahic, and E. L. Larsson-Sciard. 1997. An early, abundant cytotoxic T-lymphocyte response against Theiler’s virus is critical for preventing viral persistence. J. Virol. 71:8875–8878.

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