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

Theiler's 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 Theiler's 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 (VP3(159-166)) and two subdominant (VP1(11-20) and VP3(173-181)) capsid protein epitopes. These virus-specific CD8(+) T cells produce gamma interferon (IFN-gamma) 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-gamma 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 Theiler's murine encephalomyelitis virus-specific CTL response. Interestingly, T-cell responses to all three epitopes are restricted by the H-2K(s) molecule, and this skewed class I restriction may be associated with susceptibility to demyelinating disease.     

Differences in avidity and epitope recognition of CD8(+) T cells infiltrating the central nervous systems of SJL/J mice infected with BeAn and DA strains of Theiler's murine encephalomyelitis virus

Theiler's murine encephalomyelitis virus (TMEV) infection induces immune-mediated demyelinating disease in susceptible mouse strains and serves as a relevant infectious model for human multiple sclerosis. To investigate the pathogenic mechanisms, two strains of TMEV (DA and BeAn), capable of inducing chronic demyelination in the central nervous system (CNS), have primarily been used. Here, we have compared the T-cell responses induced after infection with DA and BeAn strains in highly susceptible SJL/J mice. CD4(+) T-cell responses to known epitopes induced by these two strains were virtually identical. However, the CD8(+) T-cell response induced following DA infection in susceptible SJL/J mice was unable to recognize two of three H-2K(s)-restricted epitope regions of BeAn, due to single-amino-acid substitutions. Interestingly, T cells specific for the H-2K(s)-restricted epitope (VP1(11-20)) recognized by both strains showed a drastic increase in frequency as well as avidity after infection with DA virus. These results strongly suggest that the level and avidity of virus-specific CD8(+) T cells infiltrating the CNS could be drastically different after infection with these two strains of TMEV and may differentially influence the pathogenic and/or protective outcome.     

Viral load increases in SJL/J mice persistently infected by Theiler's virus after inactivation of the beta(2)m gene.

We show that inactivating the beta(2)m gene increases the viral load of SJL/J mice persistently infected by Theiler's virus. Together with previous results, this shows that the characteristics of Tmevp1, a locus which controls the amount of viral RNA that persists in the central nervous system, are those of an H-2 class I gene.     

Opsonization of tumor-reactive T cells in SJL/J mice bearing syngeneic tumors

Summary The role of antigen-reactive cell opsonization (ARCO) in a syngeneic tumor system and its effect on tumor progression was investigated. Thus, anti-tumor reactive T cells were prepared in vivo by immunization of normal SJL/J mice with mitomycin C-inactivated tumor cells of the syngeneic transplantable reticulum cell sarcoma (RCS) line LA-6. Dividing cells were subsequently labeled by injecting iodo-2-deoxyuridine (¹²⁵IUdR) into the same animals 3 days later. Antigen-reactive cells (*ARC) present in the radiolabeled, nylon wool-fractionated spleen cell population taken from these mice on day 4 and injected IV into syngeneic SJL/J mice bearing LA-6 tumors were diverted to the liver and away from the spleen. The effect was maximal by 8 days following inoculation of tumor cells, and was specific inasmuch as ¹²⁵IUdR-labeled cells prepared by immunization with allogeneic spleen or tumor cells which were not opsonized in day-8 LA-6 tumor-bearing mice. Opsonization of *ARC in day-8 LA-6 tumor-bearing mice was completely abrogated by either prior injection of heat-aggregated immunoglobulin into the mice or preincubation of the *ARC in solubilized tumor antigen before injection into tumor-bearing mice, demonstrating the involvement of Fc receptors in the host and antigen-specific receptors on the *ARC, respectively, in the opsonizing process. When anti-LA-6 reactive T cells were incubated in serum from LA-6 tumor-bearing mice and then injected IV into normal syngeneic SJL/J mice, a similar liver diversion was observed. Serum from cyclophosphamide-pretreated mice injected with LA-6 or serum from mice given mitomycin C-inactivated LA-6 cells did not cause opsonization of tumor-reactive T cells, while a mixture of these two sera did have some *ARC opsonizing activity. Further experiments with SJL/J mice bearing spontaneous RCS tumor indicate that tumor-reactive T cells are also opsonized in these mice. The above studies and others suggested that ARCO may play an important role in vivo in the survival of tumors. Abbreviations used in this paper are: ARC, antigen-reactive cells; ARC, radiolabeled antigen-reactive cells; ARCO, antigen-reactive cell opsonization; LA-6 tumor line derived in our laboratory; L.I., localization index; PEG, polyethylene glycol; RCS, reticulum cell sarcoma; STA, soluble tumor antigen; TBS, tumor-bearer serum     

Minus-strand RNA synthesis in the spinal cords of mice persistently infected with Theiler''s virus.

Theiler's virus, a murine picornavirus, causes a chronic neurological disease characterized by primary demyelination in SJL/J mice. The lesions are very reminiscent of those of multiple sclerosis. Theiler's virus persists in oligodendrocytes and to a lesser extent in astrocytes and macrophages throughout the disease. Viral RNA and capsid protein syntheses are minimal in these cells. This restriction could play a central role in the mechanism of virus persistence. By quantitating plus- and minus-strand RNAs in infected central nervous system cells, we showed that RNA replication was blocked at the level of minus-strand RNA synthesis.     

Restricted virus replication in the spinal cords of nude mice infected with a Theiler''s virus variant.

The Daniels strain of Theiler's murine encephalomyelitis produces a chronic disease which is an animal model for human demyelinating disorders. Previously, we selected a neutralization-resistant virus variant producing an altered and diminished central nervous system disease in immunocompetent mice which was evident during the later stage of infection (after 4 weeks) (A. Zurbriggen and R. S. Fujinami, J. Virol. 63:1505-1513, 1989). The exact epitope determining neurovirulence was precisely mapped to a capsid protein, VP-1, and represents a neutralizing region (A. Zurbriggen, J. M. Hogle, and R. S. Fujinami, J. Exp. Med. 170:2037-2049, 1989). Here, we present experiments with immunoincompetent animals to determine viral replication, spread, and targeting to the central nervous system in the absence of detectable antibodies or functional T cells. Nude mice were infected orally, and the virus was monitored by plaque assay, immunohistochemistry, and in situ hybridization. Early during the infection (1 week), the variant virus induced an acute disease comparable to that induced by the wild-type virus in these nude mice. Alterations in tropism in the central nervous system were not apparent when wild-type parental Daniels strain virus was compared with the variant virus. Moreover, variant virus replicated in tissue culture (BHK-21 cells) to similarly high titers in a time course identical to that of the wild-type virus (A. Zurbriggen and R. S. Fujinami, J. Virol. 63:1505-1513, 1989). However, replication of the variant virus versus the wild-type virus within the spinal cord of athymic nude mice infected per os was substantially restricted by 6 weeks postinfection. Therefore, the reduced neurovirulence in the later stage (6 weeks) of the disease is most likely due to a diminished growth rate or spread of the variant virus in the central nervous system rather than to marked differences in viral tropism.     

Surgical transplantation of mouse neural stem cells into the spinal cords of mice infected with neurotropic mouse hepatitis virus

Mice infected with the neurotropic JHM strain of mouse hepatitis virus (MHV) develop pathological and clinical outcomes similar to patients with the demyelinating disease Multiple Sclerosis (MS). We have shown that transplantation of NSCs into the spinal cords of sick mice results in a significant improvement in both remyelination and in clinical outcome. Cell replacement therapies for the treatment of chronic neurologic diseases are now a reality and in vivo models are vital in understanding the interactions between the engrafted cells and host tissue microenvironment. This presentation provides an adapted method for transplanting cells into the spinal cord of JHMV-infected mice. In brief, we provide a procedure for i) preparation of NSCs prior to transplant, ii) pre-operative care of mice, iii) exposure of the spinal cord via laminectomy, iv) stereotactic injection of NSCs, and iv) post-operative care.     

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.

Intracerebral inoculation of susceptible strains of mice with Theiler's murine encephalomyelitis virus (TMEV) results in a chronic, immunologically mediated demyelinating disease that shares many features with human multiple sclerosis. CD4+ T lymphocytes play a critical role in the pathogenesis of virus-induced demyelinating disease. We have identified a region within amino acid residues 24 to 37 of the VP3 capsid protein of TMEV (VP3(24-37)) that is recognized by T lymphocytes from the demyelination-susceptible SJL/J strain of mice. The T-cell response to VP3(24-37) represents a predominant Th-cell response against the virus from either TMEV-immunized or TMEV-infected SJL/J mice, and viral epitopes VP1(233-250), VP2(74-86), and VP3(24-37) account for most of the Th-cell response to TMEV.     

Preferential induction of protective T cell responses to Theiler's virus in resistant (C57BL/6 x SJL)F1 mice

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-2(s)) but not in strains such as C57BL/6 (H-2(b)). In addition, it has been shown that (C57BL/6 × SJL/J)F1 mice (F1 mice), which carry both resistant and susceptible MHC haplotypes (H-2(b/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-2(b)-restricted CD8(+) T cell responses than of the H-2(s)-restricted CD8(+) 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.     

Clonal expansion of superantigen-reactive T cells is resistant to FK506 in mice with AIDS.

Subcutaneous injection of FK506 (10 mg/kg of body weight) completely blocked the clonal expansion of staphylococcal enterotoxin A (SEA)-reactive T cells in healthy (control) mice after SEA injection but did not disturb it in mice with murine AIDS (MAIDS) caused by infection with LP-BM5 murine leukemia virus. MAIDS mice are characterized by utilization of a FK506-insensitive pathway for clonal expansion of superantigen-reactive T cells.     

Characterization of the molecules on SJL/J lymphomas which stimulate syngeneic T cells

I-A antigens isolated from SJL reticulum cell sarcoma (RCS) cells show greater heterogeneity with respect to charge and size of the A alpha chains than do I-A antigens isolated from normal SJL spleen cells. The relevance of these structural changes in RCS I-A to the previously shown syngeneic T cell stimulatory properties of RCS was investigated. It was shown that RCS cells expressed acidic forms of the A alpha chain on their cell surface which were not present on SJL spleen cells, peritoneal cells, or dendritic cells. The only normal cells which showed A alpha chain heterogeneity approaching that of RCS cells were LPS-induced B cell blasts. Treatment with tunicamycin completely abolished the RCS A alpha chain heterogeneity, whereas exposure to neuraminidase removed the charge heterogeneity, but not the size heterogeneity. Parallel studies of the syngeneic T cell proliferative response to RCS showed that tunicamycin abolished, while neuraminidase enhanced, the ability of RCS cells to stimulate syngeneic T cells. It is suggested that polysaccharide chains on RCS I-A molecules are particularly important for the biologic properties of these lymphoma cells. The possibility that highly glycosylated I-A antigens on normal B cell blasts are also involved in the stimulation of syngeneic T cells is discussed.     

Cytotoxic T cells in the peritoneal cavity of mice infected with ectromelia virus.

Peritoneal exudate cells from mice infected with ectromelia virus were cytotoxic for virus-infected target cells as measured in a 51Cr release assay. Cytotoxic activity seemed to be T cell-dependent as it was largely abolished by treatment with anti-theta serum and complement but was not impaired by macrophage depletion. The kinetics of development of cytotoxicity in the peritoneal cavity lagged behind spleen cytotoxicity by 1-2 days. Peak activity in peritoneal cells was present about 6 days after intravenous infection with virus. These studies suggest that macrophages present in the free peritoneal cell populations of ectromelia-infected mice are not cytotoxic for virus-infected target cells. The effect of macrophages in virus clearance is therefore likely to be due to phagocytic rather than cytotoxic effects.     

Properties of reticulum cell sarcomas in SJL/J mice

While T cells from SJL and from F₁ hybrids of SJL that do not express I-E antigens give strong proliferative responses to RCS, T cells from F₁ hybrids expressing surface I-E do not. The nature of the stimulating antigen on the RCS cell surface was examined using monoclonal antibodies. Complete inhibition of the T-cell proliferative response was obtained with antibodies to I-A antigens, whereas antibodies to I-E antigens did not inhibit at all. This inhibition was mediated via an effect of the antibodies on the stimulating cells. Biochemical characterization of immunoprecipitated ¹²⁵I- and 's S-labeled RCS antigens was performed using two-dimensional gel electrophoresis. Using this technique, I-A antigens were readily detected. However, neither Ia.7-specific antibodies nor antibodies specific for E_α : E _β complexes precipitated any E alpha or E beta chains. Comparison of I-A antigens from RCS and normal SJL spleen cells revealed minor mobility differences in the gels, possibly due to differences in glycosylation, the significance of which needs to be further evaluated. Examination of RNA extracted from RCS, using E alpha and A alpha cDNA probes showed that RCS cells do not transcribe the E alpha gene as has been shown previously for normal H-2 ^s cells. Furthermore, DNA from RCS cells showed a defect in the E alpha gene similar to that known to exist in normal H-2 ^s cells. Our findings exclude the presence of E alpha on RCS cells and suggest a major role for I-A, either alone or in conjunction with another as yet unidentified cell surface antigen, in the stimulation of T cells.     

B lymphocyte colony-forming cells in the SJL/J mouse.

Mercatoethanol-induced B lymphocyte cloning in semi-solid agar has been used to study lymphocyte colony formation by cells from the SJL/J mouse thymus. From the 3rd month of life, the SJL/J mouse thymus. From the 3rd month of life, the SJL thymus develops an increasing frequency of cells forming B lymphocyte colonies in agar. The peak frequency in 6- to 12-month-old mice was one colony per 1000 to 2000 cultured thymus cells. In contrast, 10 to 100 times lower frequencies were found in the thymus of five other inbred mouse strains. The rise in B lymphocyte colony-forming cells correlated well with the age-related rise in Ig-positive cells and approximately 50% of the colony cells reacted with anti-micron-serum indicating the B lymphocyte nature of the colony cells. Colony-forming cells from the thymus showed higher sensitivity than colony-forming spleen cells to cortisol and irradiation. Cell transfer experiments and thymus grafting suggested that the increased frequency of colony-forming cells in the thymus is caused by development of special thymus-seeking B lymphocytes in ageing SJL/J mice. Finally, B lymphocyte colony-forming cells were found to be more frequent in the thymus, spleen, and lymph nodes from healthy aged mice than in lymphoid organs from mice with spontaneous reticulum cell tumors.     

Characterization of T cell lines and clones from SJL/J and (BALB/c x SJL/J)F1 mice specific for myelin basic protein

Lines of thymus-derived lymphocytes reactive against bovine myelin basic protein (BP) were established in vitro from SJL/J mice. These lines are stable in long-term culture and mediate inflammatory central nervous system (CNS) lesions and a low incidence of clinical experimental allergic encephalomyelitis (EAE) when injected into recipient SJL/J mice. The line cells proliferate in response to BP of bovine, rat, or mouse origin. Clones were derived from these lines, and the characteristics of these clones were analyzed. The clones express Thy-1, Ly-1, and L3T4 antigens and are negative for Ly-T2. The clones all proliferate in response to bovine BP, with different clones showing varying degrees of cross-reactivity between bovine, rat, and mouse BP. The proliferative response is MHC-restricted; antigen-presenting cells from I-As strains are required. Compatible with their phenotype as helper cells, some of the clones will provide help to primed B cells stimulating antibody production in an in vitro assay. When injected into recipients pretreated with pertussis and irradiation, clones that showed proliferation to mouse BP induced the development of inflammatory lesions in the CNS, with mortality of 28% of the recipients. T cell lines were also established in (BALB/c x SJL/J)F1 mice. In contrast to the homozygous SJL/J lines, these lines were highly encephalitogenic, inducing a high incidence of clinical and histologic EAE when injected in vivo.     

Age-associated parallel increase of Foxp3CD4 regulatory and CD44CD4 memory T cells in SJL/J mice

Effector/memory T cells (Tem) are required to maintain successful immunity, while regulatory T cells (Treg) are required to prevent excessive/uncontrolled inflammation and/or autoimmunity. Although both Tem and Treg cells are increased during aging, the relationship between the increased proportion of Foxp3⁺ Treg cells and CD44⁺ Tem cells with aging is not clearly understood. We found in this report that Foxp3⁺ Treg cells are increased in parallel with CD44⁺ Tem cells in SJL/J mice with aging, and that all Foxp3⁺ Treg cells are of CD44⁺ Tem phenotype, suggesting that the increased Foxp3⁺ Treg cells originated from the expanded pool of CD44⁺ Tem cells with aging. Our in vitro kinetic studies further suggested that Foxp3⁺ Treg cells are converted through the CD44⁺ stage. Furthermore, we observed that although the balance between Foxp3⁺ Treg and CD44⁺Foxp3⁻ Tem cells remained with aging, the aged mice have higher ratios of both Tem and Treg cells vs. naïve T cells resulting in the “shrunken” naïve T cell pools. Our results suggest that an age-associated imbalance of T cell repertoire is a mechanism that contributes to spontaneous occurrence of Hodgkin’s-like lymphoma in aged SJL/J mice.     

Epitope specificity of autoreactive T and B cells associated with experimental autoimmune encephalomyelitis and optic neuritis induced by oligodendrocyte-specific protein in SJL/J mice

The encephalitogenic potential of oligodendrocyte-specific protein (OSP) in mice, its specific localization in the intralamellar tight junctions in CNS myelin, and the detection of autoreactivity against OSP in multiple sclerosis (MS) strongly suggest the relevance of autoreactivity against OSP in the pathogenesis of MS. In this study, we have characterized the autoimmune T and B cells that are associated with clinicopathological manifestations of OSP-induced MS-like disease in mice by using recombinant soluble mouse OSP (smOSP) and synthetic overlapping peptides spanning smOSP. SJL/J mice immunized with smOSP developed chronic relapsing clinical experimental autoimmune encephalomyelitis accompanied with intense perivascular and parenchymal inflammatory infiltrates, widespread demyelination, axonal loss, and remarkable optic neuritis. The smOSP-primed lymph node cells reacted predominantly against OSP55-80 and to a lesser extent also to OSP22-46 and OSP179-207. Unexpectedly, in vitro selection with smOSP resulted in pathogenic smOSP-specific CD4+ T cells that reacted equally well against OSP55-80, OSP22-46, OSP45-66, and OSP179-207. Fine analysis of the anti-OSP autoimmunity revealed that the disease is primarily associated with CD4+ T cells directed against the major (OSP55-80) and the minor (OSP179-207) encephalitogenic regions that were further delineated, both in vitro and in vivo, to OSP55-66 and OSP194-207, respectively. In contrast, the OSP-induced Abs were predominantly directed against OSP22-46; these Abs were mostly of IgG1 isotype, but high levels of IgG2a and IgG2b and significant levels of IgE were also observed. The reactivity of pathogenic T cells to two encephalitogenic regions, OSP55-80 and OSP179-207, and their diverse TCRVbeta gene repertoire may impose difficulties for epitope-directed or TCR-targeting approaches to immune-specific modulation of OSP-related pathogenesis.     

Role of the immune response in Sindbis virus-induced paralysis of SJL/J mice

The pathologic role of the specific immune and inflammatory responses to viral infections of the CNS was investigated by using mice which are susceptible (SJL/J) and resistant (C57Bl6 and BALB/c) to the development of experimental autoimmune encephalomyelitis (EAE). Intracerebral inoculation of 10(4) PFU of Sindbis virus (SV) into 6- to 8-wk-old SJL/J mice resulted in a severe and sometimes fatal encephalomyelitis. A mild to severe hind leg paralysis was observed around days 6 to 7 postinfection (pi) which closely resembled EAE stages and persisted for up to 8 wk pi. Immunosuppression with cyclophosphamide on day 4 alleviated the severity of this disease. Significant perivascular and parenchymal infiltration was present in the brains and spinal cords of SV-infected SJL/J mice for up to 1 mo. This apparent immunopathologic reaction was found to be a characteristic of SJL/J mice, because infection of 6- to 8-wk-old BALB/c and C57Bl6 mice with SV did not cause paralytic disease. These mice also exhibited a significantly milder cellular infiltrate which was mostly resolved on day 12 to 14 pi. Titers of virus in the brain and spinal cords of mice were comparable with clearance by day 7 pi. SV-specific lymphoproliferation and serum antibody responses were also comparable in all mice. SV-infected SJL/J mice developed antibodies to myelin components as demonstrated in Western blots and responded to myelin basic protein by lymphoproliferation. Lymph node cells from these mice, after in vitro challenge with myelin basic protein, transferred a mild EAE-like disease to naive recipients and potentiated subclinical EAE into a severe disease.     

Zinc aspartate suppresses T cell activation in vitro and relapsing experimental autoimmune encephalomyelitis in SJL/J mice

Zinc is an essential trace element with a critical role in normal growth and development and in immune homeostasis. Zinc deficiency impairs both the innate and the adaptive immune system and can be normalized by zinc supplementation. On the other end of the spectrum, high dosages of zinc diminish immune cell functions similar to zinc deficiency. Here, we investigated the influence of zinc aspartate on proliferation and cytokine production of stimulated human T cells and mouse splenocytes in vitro. Furthermore, the effect of zinc aspartate was examined in mice with experimental autoimmune encephalomyelitis (EAE), an animal model of Multiple Sclerosis (MS) with a Th1/Th17 T cell-mediated immunopathogenesis. Zinc aspartate suppressed proliferation as well as IL-2, IL-10 and IL-17 production in stimulated human T cells and mouse splenocytes. Importantly, administration of a medium range dose of 30 μg/day zinc aspartate [1.5 mg/kg body weight (BW)] in a therapeutic manner led to a significant reduction of the clinical severity of the EAE during the first relapse of the disease. A lower zinc aspartate dose (6 μg/day, 0.3 mg/kg BW) had no significant therapeutic effect on the severity of the EAE, while administration of higher zinc aspartate amounts (120 μg/day, 6 mg/kg BW) led to more severe disease. Taken together, our data suggest that zinc aspartate can modulate activation, proliferation and cytokine production of effector T cells in vitro and in vivo and that activated autoreactive T cells may be potential therapeutic targets of tightly controlled zinc supplementation in autoimmune diseases like MS.