CD8-deficient SJL mice display enhanced susceptibility to Theiler's virus infection and increased demyelinating pathology.

Theiler's murine encephalomyelitis virus (TMEV) infection of the central nervous system (CNS) induces a chronic, progressive demyelinating disease in susceptible mouse strains characterized by inflammatory mononuclear infiltrates and spastic hind limb paralysis. Our lab has previously demonstrated a critical role for TMEV- and myelin-specific CD4(+) T cells in initiating and perpetuating this pathology. It has however, also been shown that the MHC class I loci are associated with susceptibility/resistance to TMEV infection and persistence. For this reason, we investigated the contribution of CD8(+) T cells to the TMEV-induced demyelinating pathology in the highly susceptible SJL/J mouse strain. Here we show that beta2M-deficient SJL mice have similar disease incidence rates to wild-type controls, however beta2M-deficient mice demonstrated earlier onset of clinical disease, elevated in vitro responses to TMEV and myelin proteolipid (PLP) epitopes, and significantly higher levels of CNS demyelination and macrophage infiltration at 50 days post-infection. beta2M-deficient mice also displayed a significant elevation in persisting viral titers, as well as an increase in macrophage-derived pro-inflammatory cytokine mRNA expression in the spinal cord at this same time point. Taken together, these results indicate that CD8(+) T cells are not required for clinical or histologic disease initiation or progression in TMEV-infected SJL mice. Rather, these data stress the critical role of CD4(+) T cells in this capacity and further emphasize the potential for CD8(+) T cells to contribute to protection from TMEV-induced demyelination.

Virus-induced autoimmunity: epitope spreading to myelin autoepitopes in Theiler's virus infection of the central nervous system.

Epidemiological studies indicate that host immunogenetics and history of infection, particularly by viruses, may be a necessary cofactor for the induction of a variety of autoimmune diseases. To date, however, there is no clear-cut evidence, either in experimental animal models or in human autoimmune disease, that supports either molecular mimicry (Wucherpfennig and Strominger, 1995; Fujinami and Oldstone, 1985) or a role for superantigens (Scherer et al., 1993) in the initiation of T cell-mediated autoimmunity. In contrast, the current data provide compelling evidence in support of a major role for epitope spreading in the induction of myelin-specific autoimmunity in mice persistently infected with TMEV. It is significant that two picornaviruses closely related to TMEV, coxsackievirus (Rose and Hill, 1996) and encephalomyocarditis virus (EMCV) (Kyu et al., 1992), have been similarly shown to persist (either the viral RNA or the infectious virus) in their target organs and have been associated with the development of chronic autoimmune diseases, including myocarditis and diabetes. Thus, inflammatory responses induced by viruses that trigger proinflammatory Th1 responses, and have the ability to persist in genetically susceptible hosts, may lead to chronic organ-specific autoimmune disease via epitope spreading. Epitope spreading has important implications for the design of antigen-specific therapies for the potential treatment of MS and other autoimmune diseases. This process indicates that autoimmune diseases are evolving entities and that the specificity of the effector autoantigen-specific T cells varies during the chronic disease process. Our experiments employing tolerance in R-EAE clearly indicate that antigen-specific treatment of ongoing disease is possible for preventing disease relapses, provided the proper relapse-associated epitope is targeted (Vanderlugt et al., 1999). However, the ability to identify relapse-associated epitopes in humans will be a difficult task because immunodominance will vary in every individual. The use of costimulatory antagonists that can induce anergy without requiring prior knowledge of the exact epitopes (Miller et al., 1995b), or the use of therapies that induce bystander suppression (Nicholson et al., 1997; Brocke et al., 1996), may thus be more practical current alternative therapies for the treatment of human autoimmune disease.

Temporal development of autoreactive Th1 responses and endogenous presentation of self myelin epitopes by central nervous system-resident APCs in Theiler's virus-infected mice.

Theiler's murine encephalomyelitis virus (TMEV)-induced demyelinating disease is a chronic-progressive, immune-mediated CNS demyelinating disease and a relevant model of multiple sclerosis. Myelin destruction is initiated by TMEV-specific CD4(+) T cells targeting persistently infected CNS-resident APCs leading to activation of myelin epitope-specific CD4(+) T cells via epitope spreading. We examined the temporal development of virus- and myelin-specific T cell responses and acquisition of virus and myelin epitopes by CNS-resident APCs during the chronic disease course. CD4(+) T cell responses to virus epitopes arise within 1 wk after infection and persist over a >300-day period. In contrast, myelin-specific T cell responses are first apparent approximately 50-60 days postinfection, appear in an ordered progression associated with their relative encephalitogenic dominance, and also persist. Consistent with disease initiation by virus-specific CD4(+) T cells, CNS mononuclear cells from TMEV-infected SJL mice endogenously process and present virus epitopes throughout the disease course, while myelin epitopes are presented only after initiation of myelin damage (>50-60 days postinfection). Activated F4/80(+) APCs expressing high levels of MHC class II and B7 costimulatory molecules and ingested myelin debris chronically accumulate in the CNS. These results suggest a process of autoimmune induction in which virus-specific T cell-mediated bystander myelin destruction leads to the recruitment and activation of infiltrating and CNS-resident APCs that process and present endogenous myelin epitopes to autoreactive T cells in a hierarchical order.

CD28 costimulatory blockade exacerbates disease severity and accelerates epitope spreading in a virus-induced autoimmune disease.

Theiler's murine encephalomyelitis virus (TMEV) is a natural mouse pathogen which causes a lifelong persistent infection of the central nervous system (CNS) accompanied by T-cell-mediated myelin destruction leading to chronic, progressive hind limb paralysis. TMEV-induced demyelinating disease (TMEV-IDD) is considered to be a highly relevant animal model for the human autoimmune disease multiple sclerosis (MS), which is thought to be initiated as a secondary consequence of a virus infection. Although TMEV-IDD is initiated by virus-specific CD4(+) T cells targeting CNS-persistent virus, CD4(+) T-cell responses against self myelin protein epitopes activated via epitope spreading contribute to chronic disease pathogenesis. We thus examined the ability of antibodies directed against B7 costimulatory molecules to regulate this chronic virus-induced immunopathologic process. Contrary to previous studies showing that blockade of B7-CD28 costimulatory interactions inhibit the initiation of experimental autoimmune encephalomyelitis, treatment of SJL mice at the time of TMEV infection with murine CTLA-4 immunoglobulin or a combination of anti-B7-1 and anti-B7-2 antibodies significantly enhanced clinical disease severity. Costimulatory blockade inhibited early TMEV-specific T-cell and antibody responses critical in clearing peripheral virus infection. The inhibition of virus-specific immune responses led to significantly increased CNS viral titers resulting in increased damage to myelin-producing oligodendrocytes. Following clearance of the costimulatory antagonists, epitope spreading to myelin epitopes was accelerated as a result of the increased availability of myelin epitopes leading to a more severe chronic disease course. Our results raise concern about the potential use of B7-CD28 costimulatory blockade to treat human autoimmune diseases potentially associated with acute or persistent virus infections.

Pathologic role and temporal appearance of newly emerging autoepitopes in relapsing experimental autoimmune encephalomyelitis.

Relapsing experimental autoimmune encephalomyelitis (R-EAE) is a CD4+ T cell-mediated demyelinating disease model for multiple sclerosis. Myelin destruction during the initial relapsing phase of R-EAE in SJL mice initiated by immunization with the proteolipid protein (PLP) epitope PLP139-151 is associated with activation of T cells specific for the endogenous, non-cross-reactive PLP178-191 epitope (intramolecular epitope spreading), while relapses in R-EAE induced with the myelin basic protein (MBP) epitope MBP84-104 are associated with PLP139-151-specific responses (intermolecular epitope spreading). Here, we demonstrate that T cells specific for endogenous myelin epitopes play the major pathologic role in mediating clinical relapses. T cells specific for relapse-associated epitopes can serially transfer disease to naive recipients and are demonstrable in the CNS of mice with chronic R-EAE. More importantly, induction of myelin-specific tolerance to relapse-associated epitopes, by i.v. injection of ethylene carbodiimide-fixed peptide-pulsed APCs, either before disease initiation or during remission from acute disease effectively blocks the expression of the initial disease relapse. Further, blockade of B7-1-mediated costimulation with anti-B7-1 F(ab) during disease remission from acute PLP139-151-induced disease prevents clinical relapses by inhibiting activation of PLP178-191-specific T cells. The protective effects of anti-B7-1 F(ab) treatment are long-lasting and highly effective even when administered following the initial relapsing episode wherein spreading to a MBP epitope (MBP84-104) is inhibited. Collectively, these data indicate that epitope spreading is B7-1 dependent, plays a major pathologic role in disease progression, and follows a hierarchical order associated with the relative encephalitogenic dominance of the myelin epitopes (PLP139-151 > PLP178-191 > MBP84-104).

Endogenous presentation of self myelin epitopes by CNS-resident APCs in Theiler's virus-infected mice.

The mechanisms underlying the initiation of virus-induced autoimmune disease are not well understood. Theiler's murine encephalomyelitis virus-induced demyelinating disease (TMEV-IDD), a mouse model of multiple sclerosis, is initiated by TMEV-specific CD4(+) T cells targeting virally infected central nervous system-resident (CNS-resident) antigen-presenting cells (APCs), leading to chronic activation of myelin epitope-specific CD4(+) T cells via epitope spreading. Here we show that F4/80(+), I-A(s+), CD45(+) macrophages/microglia isolated from the CNS of TMEV-infected SJL mice have the ability to endogenously process and present virus epitopes at both acute and chronic stages of the disease. Relevant to the initiation of virus-induced autoimmune disease, only CNS APCs isolated from TMEV-infected mice with preexisting myelin damage, not those isolated from naive mice or mice with acute disease, were able to endogenously present a variety of proteolipid protein epitopes to specific Th1 lines. These results offer a mechanism by which localized virus-induced, T cell-mediated inflammatory myelin destruction leads to the recruitment/activation of CNS-resident APCs that can process and present endogenous self epitopes to autoantigen-specific T cells, and thus provide a mechanistic basis by which epitope spreading occurs.

The functional significance of epitope spreading and its regulation by co-stimulatory molecules.

Epitope spreading is a process whereby epitopes distinct from and non-cross-reactive with an inducing epitope become major targets of an ongoing immune response. This phenomenon has been defined in experimental and natural situations as a consequence of acute or persistent infection and secondary to chronic tissue destruction that occurs during progressive autoimmune disease. We have investigated the functional significance of this process in the chronic stages of both autoimmune and virus-induced central nervous system (CNS) demyelinating disease models in the SJL/J mouse. During the relapsing-remitting course of experimental autoimmune encephalomyelitis (R-EAE) induced with defined encephalitogenic myelin peptides, CD4+ T cells specific for endogenous epitopes on both the initiating myelin protein (intramolecular epitope spreading) and distinct myelin proteins (intermolecular epitope spreading) are primed secondary to myelin destruction during acute disease and play a major functional role in mediating disease relapses. Similarly, epitope spreading to endogenous myelin epitopes appears to play a major functional role in the chronic-progressive course of Theiler's murine encephalomyelitis virus-induced demyelinating disease (TMEV-IDD), a virus-induced CD4+ T-cell-mediated immunopathology. In TMEV-IDD, myelin destruction is initiated by virus-specific CD4+ T cells which target virus epitopes persisting in CNS-derived antigen-presenting cells. However, the chronic stage of this progressive disease is associated with the activation of CD4+ T cells specific for multiple myelin epitopes. In both models, the temporal course of T-cell activation occurs in a hierarchical order of epitope dominance, spreading first to the most immunodominant epitope and progressing to lesser immunodominant epitopes. In addition, epitope spreading in R-EAE is regulated predominantly by CD28/B7-1 co-stimulatory interactions, as antagonism of B7-1-mediated co-stimulation using anti-B7-1 F(ab) fragments is an effective ameliorative therapy for ongoing disease. The process of epitope spreading has obvious important implications for the design of antigen-specific therapies for the treatment of autoimmune disease since these therapies will have to identify and target endogenous self epitopes associated with chronic tissue destruction.

Persistent infection with Theiler's virus leads to CNS autoimmunity via epitope spreading.

Multiple sclerosis (MS) is a T cell-mediated autoimmune demyelinating disease, which may be initiated by a virus infection. Theiler's murine encephalomyelitis virus (TMEV), a natural mouse pathogen, is a picornavirus that induces a chronic, CD4+ T cell-mediated demyelinating disease with a clinical course and histopathology similar to that of chronic progressive MS (ref. 3). Demyelination in TMEV-infected mice is initiated by a mononuclear inflammatory response mediated by virus-specific CD4+ T cells targeting virus, which chronically persists in the CNS (ref. 4-6). We show that beginning 3-4 weeks after disease onset, T-cell responses to multiple myelin autoepitopes arise in an ordered progression and may play a pathologic role in chronic disease. Kinetic and functional studies show that T-cell responses to the immunodominant myelin proteolipid protein epitope (PLP139-151) did not arise because of cross-reactivity between TMEV and self epitopes (that is, molecular mimicry), but because of de novo priming of self-reactive T cells to sequestered autoantigens released secondary to virus-specific T cell-mediated demyelination (that is, epitope spreading). Epitope spreading is an important alternate mechanism to explain the etiology of virus-induced organ-specific autoimmune diseases.

Level of corticosterone replacement determines body weight gain in adrenalectomized rats with VMH lesions.

Adrenalectomized female rats with lesions of the ventromedial hypothalamus or sham lesions were given SC implants of wax pellets or a fused mixture of corticosterone-cholesterol (40, 75, or 130% by weight). In animals with sham lesions, high dosages of corticosterone proved to be catabolic (r = -0.61 between plasma corticosterone and weight change). In marked contrast, animals with VMH lesions displayed substantial weight gains at all circulating levels of corticosterone, with a significant positive correlation (r = +0.48) between these two variables. It is concluded that: a) damage to the basomedial hypothalamus alters an organism's response to corticosterone at both ends of the dose-response curve, and b) both Type I and Type II corticosterone receptors in the brain play a role in hypothalamic obesity.

Hyperphagia and obesity in female rats with temporal lobe lesions.

Damage to the temporal lobes in cats, dogs, and primates has long been known to result in hyperphagia and obesity, but research into the role of this area of the brain in feeding behavior has largely been neglected because of an inability to produce similar results in rats. The present study reports hyperphagia and obesity in female rats with small electrolytic lesions centered in the posterodorsal amygdala. Daily food intake more than doubled in the first few days after surgery and mean weight gain was more than four times that observed in animals with sham lesions during the first 26 days. The rats with lesions were not hyperresponsive to a switch in diets (lab chow to high-fat, and back). In all animals that gained abnormal amounts of weight, the posterior extent of the lesions extended through the amygdalohippocampal area into the ventral hippocampal formation. The results suggest that the temporal lobe is an important extrahypothalamic site for the regulation of food intake in rodents.

Abnormal weight gain in rats with amygdaloid lesions.

Marked weight gain was observed in female rats given small electrolytic lesions in the dorsal posterior portion of the amygdala. With a standard lab pellet diet, weight gains typically ranged between 20-30 g during the first 3 postoperative days, and between 60-100 g over the first 20 days. Rats with sham lesions generally gained only 5-15 g in 20 days. The results are consistent with much older studies that reported obesity in cats, dogs, and primates with lesions of the amygdala.