Katherine L. Neville

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. Theilers 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.

Microglia are activated to become competent antigen presenting and effector cells in the inflammatory environment of the Theiler's virus model of multiple sclerosis

Theilers murine encephalomyelitis virus-induced demyelinating disease (TMEV-IDD) is a well-characterized murine model of the chronic-progressive form of human multiple sclerosis (MS) characterized by the activation of myelin-specific autoreactive CD4 Th1 cells via epitope spreading. To gain an understanding of the potential role of central nervous system (CNS)-resident cells in the presentation of endogenous myelin epitopes, we determined the individual antigen presentation and effector potential of resident microglia vs. infiltrating macrophages in the CNS of mice with ongoing TMEV-IDD by performing functional analysis of these populations separated to high purity by flow cytometric sorting based on their level of CD45 expression. Unlike microglia from nai;ve mice, peptide-pulsed CD45(lo) microglia isolated at the onset of clinical disease were as efficient as CNS-infiltrating CD45(hi) macrophages in activating proliferation and IFN-gamma production by myelin-peptide specific Th1 cells. In contrast, during the chronic stages of TMEV-IDD, CNS-infiltrating macrophages were more highly activated than the resident microglia as reflected both by higher expression of cell surface molecules associated with APC function and enhanced functional ability of spinal cord-infiltrating macrophages to stimulate T cell proliferation in vitro. Interestingly, both microglia and infiltrating macrophages expressed similar profiles of effector molecules such as IL-1, IL-6, IL-12 p40, TNF-alpha, and iNOS. Collectively, this is the first report comparing the antigen-presenting phenotype and function of microglia and infiltrating macrophages in a virus-induced model of CNS demyelination demonstrating that the resident microglia are capable APCs and may play an important role in antigen presentation at the onset of clinical disease and contribute to effector myelin destruction.

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 ∼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.

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.

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 β2M-deficient SJL mice have similar disease incidence rates to wild-type controls, however β2M-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. β2M-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.

Myelin-specific tolerance attenuates the progression of a virus-induced demyelinating disease: implications for the treatment of MS

Theilers murine encephalomyelitis virus-induced demyelinating disease (TMEV-IDD), a multiple sclerosis (MS) model, is a central nervous system (CNS) demyelinating disease characterized by early peripheral T cell responses to virus epitopes which spreads to myelin epitopes during chronic disease. We show that CD4(+) T cells isolated from the spinal cords of chronically infected SJL mice proliferate and secrete pro-inflammatory cytokines upon in vitro challenge with both TMEV epitopes and proteolipid protein (PLP(139-151)). Importantly, myelin-specific tolerance induced by intravenous administration of MP4, a fusion of the myelin proteins myelin basic protein (MBP) and PLP, to SJL mice with ongoing TMEV-IDD attenuated disease progression and resulted in significantly less demyelination and decreased inflammatory cell infiltration in the CNS. Paradoxically, peptide-specific splenic T cell proliferative and IFN-gamma responses were enhanced in the tolerized mice. Collectively, these results indicate that myelin-specific T cell responses contribute to chronic disease progression in this virus-induced model of MS, and suggest caution in the use of antigen-specific tolerance for treatment of ongoing autoimmune disease.

CD154 Blockade Results in Transient Reduction in Theiler's Murine Encephalomyelitis Virus-Induced Demyelinating Disease

ABSTRACT Transient CD154 blockade at the onset of Theilers murine encephalomyelitis virus-induced demyelinating disease ameliorated disease progression for 80 days, reduced immune cell infiltration, and transiently increased viral loads in the central nervous system. Peripheral antiviral and autoimmune T-cell responses were normal, and disease severity returned to control levels by day 120.

CD28 Costimulatory Blockade Exacerbates Disease Severity and Accelerates Epitope Spreading in a Virus-Induced Autoimmune Disease

ABSTRACT Theilers 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.