Bongsu Kang

The Majority of Infiltrating CD8+ T Cells in the Central Nervous System of Susceptible SJL/J Mice Infected with Theiler's Virus Are Virus Specific and Fully Functional

ABSTRACT Theilers virus infection of the central nervous system (CNS) induces an immune-mediated demyelinating disease in susceptible mouse strains, such as SJL/J, and serves as a relevant infectious model for human multiple sclerosis. It has been previously suggested that susceptible SJL/J mice do not mount an efficient cytotoxic T-lymphocyte (CTL) response to the virus. In addition, genetic studies have shown that resistance to Theilers virus-induced demyelinating disease is linked to the H-2D major histocompatibility complex class I locus, suggesting that a compromised CTL response may contribute to the susceptibility of SJL/J mice. Here we show that SJL/J mice do, in fact, generate a CD8+ T-cell response in the CNS that is directed against one dominant (VP3159-166) and two subdominant (VP111-20 and VP3173-181) capsid protein epitopes. These virus-specific CD8+ T cells produce gamma interferon (IFN-γ) and lyse target cells in the presence of the epitope peptides, indicating that these CNS-infiltrating CD8+ T cells are fully functional effector cells. Intracellular IFN-γ staining analysis indicates that greater than 50% of CNS-infiltrating CD8+ T cells are specific for these viral epitopes at 7 days postinfection. Therefore, the susceptibility of SJL/J mice is not due to the lack of an early functional Theilers murine encephalomyelitis virus-specific CTL response. Interestingly, T-cell responses to all three epitopes are restricted by the H-2Ks molecule, and this skewed class I restriction may be associated with susceptibility to demyelinating disease.

Differential Virus Replication, Cytokine Production, and Antigen-Presenting Function by Microglia from Susceptible and Resistant Mice Infected with Theiler's Virus

ABSTRACT Infection with Theilers murine encephalomyelitis virus (TMEV) in the central nervous system (CNS) causes an immune system-mediated demyelinating disease similar to human multiple sclerosis in susceptible but not resistant strains of mice. To understand the underlying mechanisms of differential susceptibility, we analyzed viral replication, cytokine production, and costimulatory molecule expression levels in microglia and macrophages in the CNS of virus-infected resistant C57BL/6 (B6) and susceptible SJL/J (SJL) mice. Our results indicated that message levels of TMEV, tumor necrosis factor alpha, beta interferon, and interleukin-6 were consistently higher in microglia from virus-infected SJL mice than in those from B6 mice. However, the levels of costimulatory molecule expression, as well as the ability to stimulate allogeneic T cells, were significantly lower in TMEV-infected SJL mice than in B6 mice. In addition, microglia from uninfected naïve mice displayed differential viral replication, T-cell stimulation, and cytokine production, similar to those of microglia from infected mice. These results strongly suggest that different levels of intrinsic susceptibility to TMEV infection, cytokine production, and T-cell activation ability by microglia contribute to the levels of viral persistence and antiviral T-cell responses in the CNS, which are critical for the differential susceptibility to TMEV-induced demyelinating disease between SJL and B6 mice.

Pathogenesis of virus-induced immune-mediated demyelination

Theilers murine encephalomyelitis virus-induced demyelinating disease has been extensively studied as an attractive infectiousmodel for human multiple sclerosis. Virus-specific inflammatory Th1 cell responses followed by autoimmune responses to myelin antigens play a crucial role in the pathogenic processes leading to demyelination. Antibody and cytotoxic T cells (CTL) responses to virus appears to be primarily protective from demyelinating disease. Although the role of Th1 and CTL responses in the induction of demyelinating disease is controversial, assessment of cytokine sproduced locally in the central nervous system (CNS) during the course of disease and the effects of altered inflammatory cytokine levels strongly support the importance of Th1 responses in this virus-induced demyelinating disease. Induction of various chemokines and cytokines in different glial and antigen presenting cells upon viral infection appears to be an important initiation mechanism for inflammatory Th1 responses in the CNS. Coupled with the initial inflammatory responses, viral persistence in the CNS may be acritical factor for sustaining inflammatory responses and consequent immune-mediated demyelinating disease.

Initial capsid-specific CD4+ T cell responses protect against Theiler's murine encephalomyelitisvirus-induced demyelinating disease

Central nervous system (CNS) infection by Theilers murine encephalomyelitis virus (TMEV) causes an immune‐mediated demyelinating disease similar to human multiple sclerosis in susceptible mice. To understand the pathogenic mechanisms, we analyzed the level, specificity, and function of CD4+ Th cells in susceptible SJL/J and resistant C57BL/6 mice. Compared to resistant mice, susceptible mice have three‐ to fourfold higher levels of overall CNS‐infiltrating CD4+ T cells during acute infection. CD4+ T cells in the CNS of both strains display various activation markers and produce high levels of IFN‐γ upon stimulation with anti‐CD3 antibody. However, susceptible mice display significantly fewer (tenfold) IFN‐γ‐producing Th1 cells specific for viral capsid epitopes as compared to resistant mice. Furthermore, preimmunization with capsid‐epitope peptides significantly increased capsid‐specific CD4+ T cells in the CNS during the early stages of viral infection and delayed the development of demyelinating disease in SJL/J mice. This suggests a protective role of capsid‐reactive Th cells during early viral infection. Therefore, a low level of the protective Th1 response to viral capsid proteins, in conjunction with Th1 responses to unknown epitopes may delay viral clearance in susceptible mice leading to pathogenesis of demyelination during acute infection, as compared to resistant mice.

Gender bias in Theiler's virus-induced demyelinating disease correlates with the level of antiviral immune responses

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.

Clonal Expansion of Infiltrating T Cells in the Spinal Cords of SJL/J Mice Infected with Theiler’s Virus

Intracerebral infection of susceptible mice with Theiler’s murine encephalomyelitis virus results in immune-mediated inflammatory demyelination in the white matter and consequent clinical symptoms. This system has been utilized as an important virus model for human multiple sclerosis. Although the potential involvement of virus-specific Th cells has been studied extensively, very little is known about the nature of T cells infiltrating the CNS during viral infection and their role in the development of demyelinating disease. In this study, the clonal nature of T cells in the spinal cord during the disease course was analyzed using size spectratyping and sequencing of the TCR β-chain CDR3 region. These studies clearly indicate that T cells are clonally expanded in the CNS after viral infection, although the overall TCR repertoire appears to be diverse. The clonal expansion appears to be Ag-driven in that it includes Th cells specific for known viral epitopes. Interestingly, such restricted accumulation of T cells was not detectable in the infiltrates of mice with proteolipid protein peptide-induced experimental autoimmune encephalomyelitis. The initial T cell repertoire (7–9 days postinfection) seems to be more diverse than that observed in the later stage (65 days) of virally induced demyelination, despite the more restricted utilization of Vβ subfamilies. These results strongly suggest continuous stimulation and clonal expansion of virus-specific T cells in the CNS of Theiler’s murine encephalomyelitis virus-infected mice during the entire course of demyelinating disease.

Theiler's Virus Infection Induces a Predominant Pathogenic CD4+ T Cell Response to RNA Polymerase in Susceptible SJL/J Mice

ABSTRACT Theilers murine encephalomyelitis virus (TMEV)-induced immune-mediated demyelinating disease in susceptible mouse strains has been extensively investigated as a relevant model for human multiple sclerosis. Previous investigations of antiviral T-cell responses focus on immune responses to viral capsid proteins, while virtually nothing is reported on immune responses to nonstructural proteins. In this study, we have identified noncapsid regions recognized by CD4+ T cells from TMEV-infected mice using an overlapping peptide library. Interestingly, a greater number of CD4+ T cells recognizing an epitope (3D21-36) of the 3D viral RNA polymerase, in contrast to capsid epitopes, were detected in the CNS of TMEV-infected SJL mice, whereas only a minor population of CD4+ T cells from infected C57BL/6 mice recognized this region. The effects of preimmunization and tolerization with these epitopes on the development of demyelinating disease indicated that capsid-specific CD4+ T cells are protective during the early stages of viral infection, whereas 3D21-36-specific CD4+ T cells exacerbate disease development. Therefore, protective versus pathogenic CD4+ T-cell responses directed to TMEV appear to be epitope dependent, and the differences in CD4+ T-cell responses to these epitopes between susceptible and resistant mice may play an important role in the resistance or susceptibility to virally induced demyelinating disease.

Functional maturation of proteolipid protein139–151-specific Th1 cells in the central nervous system in experimental autoimmune encephalomyelitis

Experimental autoimmune encephalomyelitis (EAE) is a widely adopted animal model system for studying human multiple sclerosis that affects the central nervous system (CNS). To understand the underlying pathogenic mechanisms of the autoimmune T cell response, localization, enumeration and characterization of autoreactive T cells are essential. We assessed encephalitogenic proteolipid protein epitope (PLP(139-151))-specific T cells in the periphery and CNS of SJL/J mice using MHC class II I-As multimers during both pre-clinical and clinical phases of PLP-induced EAE in conjunction with T cell function. Our results strongly suggest that PLP(139-151)-specific CD4+ T cells first expand primarily in the CNS-draining cervical lymph nodes and then migrate to the CNS. In the CNS, these PLP-specific CD4+ T cells accumulate, become activated and differentiate into effector cells that produce IFN-gamma in response to the self-peptide.

Type I interferon signals control Theiler's virus infection site, cellular infiltration and T cell stimulation in the CNS

Theilers murine encephalomyelitis virus (TMEV) establishes a persistent infection in the central nervous system (CNS). To examine the role of type I interferon (IFN-I)-mediated signals in TMEV infection, mice lacking a subunit of the type I IFN receptor (IFN-IR KO mice) were utilized. In contrast to wild type mice, IFN-IR KO mice developed rapid fatal encephalitis accompanied with greater viral load and infiltration of immune cells to the CNS. The proportion of virus-specific CD4(+) and CD8(+) T cell responses in the CNS was significantly lower in IFN-IR KO mice during the early stage of infection. Levels of IFN-γ and IL-17 produced by isolated primed CD4(+) T cells in response to DCs from TMEV-infected IFN-IR KO mice were also lower than those stimulated by DCs from TMEV-infected wild type control mice. The less efficient stimulation of virus-specific T cells by virus-infected antigen-presenting cells is attributable in part to the low level expression of activation markers on TMEV-infected cells from IFN-IR KO mice. However, due to high levels of cellular infiltration and viral loads in the CNS, the overall numbers of virus-specific T cells are higher in IFN-IR KO mice during the later stage of viral infection. These results suggest that IFN-I-mediated signals play important roles in controlling cellular infiltration to the CNS and shaping local T cell immune responses.

Effects of the major histocompatibility complex loci and T-cell receptor beta-chain repertoire on Theiler's virus-induced demyelinating disease

We have investigated the potential effects of H‐2 and T‐cell receptor (TCR) Vβ family genes on induction of T‐cell immunity and susceptibility to virally induced demyelinating disease by using BALB.S (H‐2KsAsDs) and BALB.S3R (H‐2KsAsDd/Ld) mice. These parameters were compared with those of highly susceptible SJL/J (H‐2KsAsDs) mice that contain only one‐half of TCR Vβ family genes compared with the above‐mentioned strains. Our results demonstrate that BALB.S but not BALB.S3R mice are susceptible similar to SJL/J mice. Although the level of CD4+ T‐cell infiltration to the CNS was elevated in susceptible mice, virus‐specific immune responses restricted with H‐2s were similar in these mice. No preferential use of Vβ families associated with differences in the major histocompatibility complex (MHC) components was apparent. However, the pattern and sequence of CDR3 distribution shows T‐cell clonal accumulation in the CNS associated with the H‐2 components. Further anti‐CD8 antibody treatment of resistant BALB.S3R mice abrogated resistance to demyelinating disease, indicating that CD8+ T cells restricted with H‐2Dd/Ld are most likely to exert resistance in BALB.S3R mice. These studies indicated that TCR Vβ and MHC class II genes are the secondary to a particular MHC class I gene expression in susceptibility to virally induced demyelinating disease.