Stephen Miller - US grants

We propose to continue our investigation of the inductive events and suppressor T cell/T cell factor (Ts/TsF)-mediated regulation of delayed-type hypersensitivity (DTH) responses to 2,4-dinitrofluorobenzene (DNFB). As precise analysis of the induction and effector mechanisms of complex inductive and regulatory circuits is possible only via the use of cells and factors from cloned T cell lines and hybridomas, particular emphasis will be given to the isolation and characterization of clonal sources of regulatory Ts/TsF and clonal sources of potential targets of these regulatory cells. Studies on the inductive events of DTH will focus on further characterization, on both the population and clonal levels, of the hapten-specific, MHC Class II-restricted T cell subsets involved in this process [helper T cells for DTH (Thdh), proliferative T cells (Tprlf), and precursor DTH effector cells (pTDH)]. Studies on the inductive and effector events of antigen-specific Ts circuits induced by the intravenous injection of syngeneic DNP-modified spleen cells (DNP-SP) will focus on several areas. The role of L3T4+ Ts-inducer (Ts-i) cells in the Ts circuit will be probed using in vivo injection of monoclonal GK1.5 antibody (directed against the MHC Class II T cell receptor-associated L3T4a determinant) and by examining the suppressive mechanisms of our recently described L3T4+ Ts-i clones. The mechanisms and cellular targets of efferent (elicitation phase) and afferent (induction phase)-acting Ts-1 and Ts-2 effector cells will be investigated using both polyclonal and clonal sources of these cells and their targets. Lastly, major emphasis will be placed on investigation of the biochemistry, receptor genes, and molecular mechanisms of effector suppression using a monoclonal, DNP-specific, efferent-acting TsF (Clone 26.10.2) as a probe. Studies are proposed wherein genetic and molecular mechanisms of the divergent pathways by which this monoclonal TsF appears to mediate suppression will be examined - a) directly and specifically on DTH-effector TDH cells; b) indirectly via triggering of a nonspecific factor from antigen-primed Ts-auxiliary cells; and, c) via induction of second-order Ts. We feel that the past experience of our laboratory in the study of DTH immunoregulation and our recently developed technology for producing clonal sources of both DTH effector and Ts cells make these goals attainable. Understanding of Ts/TsF regulation of DTH responses gained by these studies should prove valuable for our future ability to manipulate the immune system for both the prevention and treatment of neoplasia and human immunoregulatory disease.

Experimental autoimmune encephalomyelitis (EAE) is a T cell-mediated inflammatory demyelinating disease of the CNS that serves as a model for human multiple sclerosis (MS). In the SJL/J mice, a relapsing-remitting form of EAE (R-EAE) is induced following active immunization with proteolipid protein (PLP), myelin basic protein (MBP), or the immunodominant epitopes on these molecules (PLP139-151 or MBP84-104) or following the adoptive transfer of peptide-specific Th1 cells. Based on the relapsing-remitting course of the disease, along with our finding that disease progression (relapses) in these peptide-induced R- EAE models are due primarily to the recruitment of T cell responses against non-crossreactive endogenous myelin epitopes on the same or different myelin proteins (intramolecular or intermolecular epitope spreading), we hypothesize that disease remission results from specific form(s) of immunoregulation. Specific Aim 1 of current proposal will build on our productive studies from the previous funding period to further delineate the role of several overlapping immunoregulatory mechanisms which appear to function in ameliorating CNS responses to the disease initiating epitopes leading to initiation of disease remission. These include: a switch of CNS cytokines from pro-inflammatory to anti-inflammatory (i.e., a Th1/Th2 switch) and/or the possible activation of antigen- or TcR-specific regulatory T cell populations in response to the disease-initiating T cells. Secondly, we will continue to elucidate the mechanisms responsible for downregulation of disease induction and progression following extrinsic induction of antigen-specific peripheral tolerance induced by the i.v. injection of protein/peptide-pulsed, ethylene carbodiimide (ECDI)-fixed antigen presenting cells (Ag-SP). In comparison to tolerance induced by the i.v. or oral administration of soluble peptide, tolerance induced by Ag-SP was shown to be highly effective for prevention and treatment of R-EAE, and a powerful tool for identification of the specificity of pathologic epitopes at various stages of the relapsing-remitting disease process. Specific Aim 2 will further test the hypothesis that unresponsiveness induced by the i.v. injection of Ag-SP is primarily mediated by clonal anergy/deletion of encephalitogenic Th1 cells. The effects of tolerance at varying times during the disease process on the T cell repertoire (using both immunoscope analysis and in vivo tracking of SJL Thy 1.1 congenic and SJL PLP139-151-specific TcR transgenic T cell populations), activation state, CNS homing properties, and cytokine expression patterns of effector Th1 cells will be determined. In addition, in vitro experiments utilizing encephalitogenic Th1 clones and in vivo experiments using TcR transgenic mouse systems will be employed to directly assess the relative contributions of clonal anergy vs. deletion to the unresponsive state. These studies should enhance our understanding of both intrinsic mechanisms of spontaneous disease remission, and continue to delineate the cellular and molecular mechanisms of a highly efficient extrinsic method of inducing peripheral immune tolerance proven effective for the treatment of pre-existing autoimmune disorders.

Multiple sclerosis is a neurological disease of major economic and social importance in which a viral etiology is still strongly suspected. Because of the historical importance of experimental animal models to understanding human diseases,investigation of multiple sclerosis models can be expected to lead to a clearer insight into the pathogenesis of this disease. Of the few available experimental animal models of virus-induced demyelination, TMEV infection in mice is possibly the most relevant to multiple sclerosis. The prospect is that continued studies of this model will lead to innovative approaches which may ultimately link a specific virus(es) with multiple sclerosis. It is clear that a multidisciplinary approach is needed to answer relevant questions about the molecular pathogenesis of TMEV infection. This application is a renewal of the program project which has been expanded to include six separate projects and two cores. Project 1 involves continuing studies of T cell immunity in the TMEV model of demyelination. Using in vivo-derived T cells and in vitro-propagated T cell clones and hybrids, the effector phenotype and epitope-specificity of the TMEV-specific T cell repertoire will be analyzed. In Project 2, the immunogenetic control of TMEV-induced demyelinating disease will be further investigated. Specific approaches include continued identification and analysis of MHC and non-MHC genes involved, a search for active protective mechanisms in resistant animals and patterns of genetically controlled responses to viral capsid proteins. Project 3 involves the use of X-ray crystallographic methods to determine the atomic structures of TMEV and the virus co-crystallized with neutralizing antibodies. In Project 4 it is proposed that resistant mouse strains preferentially develop immune responses less destructive and more helpful to the host. Thus, the immune resistance to TMEV-induced demyelinating disease will be pursued. In Project 5 mutations of TMEV surface residues will test whether amino acids in the putative viral receptor attachment site (pit) are involved in binding to the cell receptor. Also, by deleting the two major VP1 loops, we hope to produce a viable TMEV that is susceptible to the "WIN" antiviral compounds. Project 6 involves the use of recombinant DNA technology to express individual TMEV proteins to determine function and immunogenicity.

Multiple sclerosis (MS) is a neurological disease of major economic and social importance in which a viral etiology is still strongly suspected. Because of the historical importance of experimental animal models to understanding human diseases, investigation of multiple sclerosis models can be expected to lead to a clearer insight into the pathogenesis of this disease. Of the few available experimental animal models of virus-induced demyelination, Theiler's murine encephalomyelitis virus (TMEV) infection in mice is possibly the most relevant to MS. The prospect is that continued studies of this model will lead to innovative approaches which may ultimately link a specific virus(es) with MS and possibly provide approaches for the treatment of the disease. It is clear that a multidisciplinary approach is needed to answer relevant questions about the molecular pathogenesis of TMEV-induced demyelinating disease. Project 1 involves continuing studies of T cell immunity in the TMEV model. Using in vivo-derived polyclonal T cell populations and in vitro-propagated T cell clones and hybrids, effector phenotype, epitope specificity, T cell receptor usage, and immunopathologic potential of TMEV- specific peripheral and CNS-infiltrating CD4+ T cells subsets (i.e., Th1 and Th2 cells) and their soluble lymphokine products will be assessed in both susceptible and resistant mouse strains. In addition, the effects of specific (tolerance) and nonspecific (monoclonal antibody depletion) immunoregulation on the course of demyelination will be studied. Project 5 will further investigate the immunogenetic control of TMEV-induced demyelinating disease by continued identification and analysis of MHC and non-MHC genes associated with susceptibility and examine the role of active suppression in genetically resistant animals. Project 7 will continue experiments on introducing point mutations into TMEV surface residues to test whether amino acids in the putative viral receptor attachment site (pit) are involved in binding to the cellular receptor.

Proteolipid protein (PLP) is the major protein component of CNS myelin and appears to be a major target of immune responses both in murine models of experimental autoimmune encephalomyelitis (EAE) and perhaps in human multiple sclerosis (MS). A chronic-relapsing form of EAE (R-EAE) is induced in inbred SJL/J mice following either active immunization with intact PLP or the major encephalitogenic determinant of the PLP molecule which encompasses amino acids 139-151 (PLP139-151(s)), or the adoptive transfer of T cell lines/clones specific for the PLP139-151(s) epitope. PLP-induced R-EAE follows a relapsing-remitting course of paralysis and is characterized histologically by perivascular mononuclear cell-rich infiltrates of the white matter of the central nervous system (CNS) and areas of acute and chronic demyelination. The well-understood genetics of the murine host and the similarities in both clinical course and histopathology of murine R-EAE and MS make it an ideal animal model for the study of immunopathogenic and immunoregulatory aspects of MS. We propose to examine the neuroantigen specificity, effector phenotype, T cell receptor usage, and lymphokine-producing profile of both peripheral and CNS-infiltrating neuroantigen-specific T cell-mediated immune (CMI) responses throughout the relapsing clinical course of PLP-induced R-EAE to determine if epitopes different than that which induced the initial paralytic episode are targeted during clinical relapses. We also propose to examine the conditions and mechanisms by which the induction and/or expression of PLP-induced R-EAE can be specifically downregulated (following the induction of neuroantigen-specific immunological tolerance). In addition, aspects of the molecular pathogenesis of demyelination will be addressed by defining and comparing encephalitogenic and tolerogenic PLP epitopes, and determining the effects of in vivo and in vitro tolerance induction on the ability of encephalitogenic PLP139-151(S)-specific T cell clones to produce particular lymphokines and/or subsequently mediate clinical disease. These studies should lead to a better understanding of the fine specificity, immunopathologic role, and immunoregulation of PLP-specific T cell-mediated immune responses which may be applicable to the understanding and treatment of human MS.

Multiple sclerosis (MS) is a neurological disease of major economic and social importance in which a viral etiology is still strongly suspected. Because of the historical importance of experimental animal models to understanding human diseases, investigation of multiple sclerosis models can be expected to lead to a clearer insight into the pathogenesis of this disease. Of the few available experimental animal models of virus-induced demyelination, Theiler's murine encephalomyelitis virus (TMEV) infection in mice is possibly the most relevant to MS. The prospect is that continued studies of this model will lead to innovative approaches which may ultimately link a specific virus(es) with MS and possibly provide approaches for the treatment of the disease. It is clear that a multidisciplinary approach is needed to answer relevant questions about the molecular pathogenesis of TMEV-induced demyelinating disease. Project 1 involves continuing studies of T cell immunity in the TMEV model. Using in vivo-derived polyclonal T cell populations and in vitro-propagated T cell clones and hybrids, effector phenotype, epitope specificity, T cell receptor usage, and immunopathologic potential of TMEV- specific peripheral and CNS-infiltrating CD4+ T cells subsets (i.e., Th1 and Th2 cells) and their soluble lymphokine products will be assessed in both susceptible and resistant mouse strains. In addition, the effects of specific (tolerance) and nonspecific (monoclonal antibody depletion) immunoregulation on the course of demyelination will be studied. Project 5 will further investigate the immunogenetic control of TMEV-induced demyelinating disease by continued identification and analysis of MHC and non-MHC genes associated with susceptibility and examine the role of active suppression in genetically resistant animals. Project 7 will continue experiments on introducing point mutations into TMEV surface residues to test whether amino acids in the putative viral receptor attachment site (pit) are involved in binding to the cellular receptor.

This proposal is a request for new funding of an institutional pre- and postdoctoral NRSA Training Program in Immunology and Molecular Pathogenesis at Northwestern University Medical School. The training program will be run in conjunction with the Immunology and Molecular Pathogenesis graduate track, a component of the highly successful Integrated Graduate Program in the Life Sciences (IGP). The IGP will be the mechanism for recruitment of a pool of highly qualified graduate students from which predoctoral candidates will be selected. The training program will also be supported by the Northwestern Interdepartmental Immunobiology Center. The training program will stress the interactive nature of immunology and molecular pathogenesis, two traditionally related disciplines. Successful research in immunology and molecular pathogenesis requires consultation and collaboration among colleagues, therefore this program includes nineteen trainers with primary appointments in both basic science (Microbiology-Immunology, Pathology and Pharmacology) and clinical departments (Medicine and Neurology) at the Medical School Campus all of whom currently hold peer-reviewed funding. The program also includes five training consultants who are highly funded medical experts in various disciplines related to immunology and/or molecular pathogenesis who will not serve as trainers, but will serve in various capacities to lend a clinical perspective to the training process. The training program therefore has the effect of bringing together a cadre of highly productive, well-funded researchers experienced in pre- and postdoctoral training, who can impart both basic and clinical perspectives to the trainees. The result will be a more productive research environment, both for the pre- and postdoctoral students and for the many research projects funded by grants from the NIH and other federal and private agencies. The training grant also proposes to foster trainee interactions with scientists at other institutions through trainee travel to national meetings (to present and receive critiques on their research and to develop contacts with other scientists) and through visits of prominent scientists to Northwestern Medical School as trainee-invited speakers. The training program also serves to focus the activities at the University aimed at educating students in the ethics of science and at recruiting minorities to graduate and post-graduate studies in immunology and molecular pathogenesis. The program requests six predoctoral slots and two postdoctoral slots per year to be phased in over the first two years of the program, thus permitting three predoctoral trainees and one postdoctoral trainee to be appointed each year for two years terms. Predoctoral students will be appointed at the end of the second year after they have completed their coursework and qualifying exams and have identified a research advisor, while postdoctoral candidates will be appointed at the outset of their training. The proposed program will give training in immunology and molecular pathogenesis at Northwestern the constancy it needs to maintain its momentum and continued growth, allowing the continued supply of highly trained young investigators with primary interests in the basic mechanisms of the disease process.

Experimental autoimmune encephalomyelitis (EAE) is a T cell- mediated inflammatory demyelinating disease of the CNS that serves as a model for human multiple sclerosis. In the SJL/J mice, a relapsing-remitting form of EAE (R-EAE) is induced following active immunization with proteolipid protein (PLP), myelin basic protein (MBP), or the immunodominant epitopes on these molecules (PLP139-151 or MBP84-104) or following adoptive transfer of peptide- specific Th1 cells. Based on the relapsing-remitting course of the disease, along with the finding that Date Released: 05/15/1997 disease progression (relapses) in these peptide induced R-EAE models are due primarily to the recruitment of T cell responses against non- crossreactive endogenous myelin epitopes on the same or different myelin proteins (intermolecular or intramolecular epitope spreading), it is hypothesized that disease remission results from specific form(s) of immunoregulation. Specific Aim 1 will continue studies from the previous funding period to test several non-mutually exculsive hypotheses by which responses to disease initiating epitopes may be down-regulated leading to disease remission: 1) potential switching of CNS cytokines from pro-inflammatory to anti-inflammatory; 2)CTLA- 4-mediated anergy and/or Fas/FasL-mediated apoptosis of disease initiating Th1 cells; 3) activation of antigen- or TCR-specific regulatory T cell populations in response to disease-initiating T cells. Specific Aim 2 will elucidate the mechanisms responsible for downregulation of disease following extrinsic induction of antigen-specific peripheral tolerance induced by the i.v. injection of protein/peptide-pulsed, ethylene carbodiimide (ECDI)-fixed antigen presenting cells (Ag-SP). Specific Aim 2 will further test the hypothesis that unresponsiveness induced by the i.v. injection of Ag-SP is primarily mediated by clonal anergy or encephalitogenic Th1 cells. The effects of tolerance at varying times during the disease process on the activation state, CNS homing properties, and cytokine expression patterns of effector Th1 cells will be determined. In addition, in vitro experiments utilizing encephalitogenic Th1 clones and in vivo experiments using TCR transgenic mouse systems will be employed to directly assess the relative contributions of clonal anergy vs. deletion to the unresponsive state. These studies would enhance our understanding of both intrinsic mechanisms of spontaneous disease remission, an delineate the molecular mechanisms of a highly efficient extrinsic method of inducing peripheral immune tolerance proven effective for the treatment of pre-existing autoimmune disorders.

Theiler~s murine encephalomyelitis virus (TMEV)-inducted demyelinting disease is one of the most relevant of the few available experimental animal modesl of virus-induced demyelination. TMEV are natural mouse pathogens. Intracerebral inoculation of susceptible mouse strains with the BeAn strain of TMEV results in a chronic, progressive, CNS inflammatory demyelinating disorder which is related to life-long persistent CNS virus infection and characterized by spasti hind limb paralysis. Our previous studies have characterized the effector nature, phenotype, repertoire, and specificity of the T cells involved in initiating CNS demyelination and regulating the disease process using antigen-specific tolerance. These studies clearly demonstrate that the demyelination is initiated by virus-specific Th1 cells targeting viral epitopes persistently presented in the CNS. TMEV-induced demyelination is considered a highly relevant animal model for multiple sclerosis (MS) since both diseases are characterized by progressive demyelinating lesions with accompanying mononuclear cell infiltrates in which CD4 plus T cells and activated macrophages predominate. MS is generally considered to involve an autoimmune pathology, but epidemiological evidence strongly suggests a viral trigger. Our preliminary evidence shows that responses to the immunodominant, encephalitogenic CD4 plus T cell epitope on myelin proteolipid protein (PLP139-151) arise in mice 2- 4 weeks after the onset of TMEV-induced demyelination. This proposal will test the hypothesis that the chronic stages of TMEV-induced demyelinationare mediated by CD4 plus T cell responses to both TMEV and to PLP139-151 and perhaps other self myelin epitopes activated secondary to initial myelin damage. The specificity, receptor repertoire, and relative contributions of virus- and myelin-specific T cell and antibody responses to the immunopathologic process at varying stagesof the disease process will be assessed by analyzing both peripheral and CNS- infiltrating T cells. The mechanisms (molecular mimicry vs. Epitope spreading) by which anti-myelin epitope responses arise in these mice will be tested. In addition, we will continue or studies of the efficacy, specificity, and molecular mechanisms by which peripheral tolerance and antagonists of B7/Cd28-mediated costimulation inhibit clinical and histologic disease progression. These studies should define the mechanism(s) by which anti-self responses arise during persistent CNS virus infection and determine their functional contribution to chronic disease pathology. In addition, our studies on specific immunoregulation of TMEV-induced demyelinating disease may be applicable to the understanding the etiology of MS and other chronic (auto) immune diseases and for design of specific treatment strategies.

Theiler's murine encephalomyelitis virus (TMEV)-induced demyelinating disease is one of the most relevant of the few available experimental animal models of virus-induced demyelination. TMEV are natural mouse pathogens. Intracerebral inoculation of susceptible mouse strains with the BeAn strain of TMEV results in a chronic, progressive, CNS inflammatory demyelinating disorder which is related to life-long persistent CNS virus infection and characterized by spastic hind limb paralysis. Our previous studies have characterized the effector nature, phenotype, repertoire, and specificity of the T cells involved in initiating CNS demyelination and regulating the disease process using antigen-specific tolerance. These studies clearly demonstrate that the demyelination is initiated by virus- specific Th1 cells targeting viral epitopes persistently presented in the CNS. TMEV-induced demyelination is considered a highly relevant animal model for multiple sclerosis (MS) since both diseases are characterized by progressive demyelinating lesions with accompanying mononuclear cell infiltrates in which CD4+ T cells and activated macrophages predominate. MS is generally considered to involve an autoimmune pathology, but epidemiological evidence strongly suggests a viral trigger. Our preliminary evidence shows that responses to the immunodominant, encephalitogenic CD4+ T cell epitope on myelin proteolipid protein (PLP 139-1 51) arise in mice 2-4 weeks after the onset of TMEV-induced demyelination. This proposal will test the hypothesis that the chronic stages of TMEV-induced demyelination are mediated by CD4+ T cell responses to both TMEV and to PLP139-151 and perhaps other self myelin epitopes activated secondary to initial myelin damage. The specificity, receptor repertoire, and relative contributions of virus- and myelin-specific T cell and antibody responses to the immunopathologic process at varying stages of the disease process will be assessed by analyzing both peripheral and CNS-infiltrating T cells. The mechanisms (molecular mimicry vs. epitope spreading) by which anti-myelin epitope responses arise in these mice will be tested. In addition, we will continue our studies of the efficacy, specificity, and molecular mechanisms by which peripheral tolerance and antagonists of B7/CD28-mediated costimulation inhibit clinical and histologic disease progression. These studies should define the mechanism(s) by which anti-self responses arise during persistent CNS virus infection and determine their functional contribution to chronic disease pathology. In addition, our studies on specific immunoregulation of TMEV-induced demyelinating disease may be applicable to the understanding the etiology of MS and other chronic (auto)immune diseases and for design of specific treatment strategies.

Multiple sclerosis (MS) is a neurological disease of major socioeconomic importance in which a viral etiology is strongly suspected. Because of the historical importance of experimental animal models to understanding human disease, investigation of MS models can be expected to lead to clearer insights into the pathogenesis of this human disease. Of the few available experimental animal models of virus-induced demyelination, Theiler's murine encephalomyelitis virus (TMEV) infection in mice is possibly the most relevant to MS. Clearly, a multidisciplinary approach is needed to answer relevant questions about the molecular pathogenesis of TMEV-induced demyelinating disease. This application represents a request for the renewal of a highly successful collaborative research effort which has been in effect for the past 11 years. This Program Grant is a unique in that it represents the only multidisciplinary approach to the study of the molecular pathogenesis of TMEV infection and of its utility as a model of MS. Funding is requested for the continuation of Projects 1 (Dr. Miller), and 3 (Dr. Lipton), and for the inclusion a new project directed jointly by Drs. Kim and Melvold (Project 2). Project 1 will determine the mechanism(s) by which myelin-specific T cell responses arise during persistent CNS TMEV infection, ascertain their functional contribution to chronic disease pathology, and define the specificity and mechanisms of specific immunoregulation of TMEV-induced demyelinating disease using peripheral tolerance and antagonists of B7/CD28 costimulation. Project 2 will examine the functional contribution of CD8+ cytotoxic T cells in disease pathogenesis and resistance in susceptible and resistant mouse strains. Project 3 will employ molecular and biochemical approaches to identify the host cellular receptor for TMEV and determine its tissue distribution and function. In addition, this project will investigate the mechanisms of persistence of TMEV virions as related to their ability to induce apoptosis in murine cells. Important information relative to the nature, specificity (anti-viral and anti-self), and immunoregulation of immune responses involved in resistance/susceptibility to TMEV-induced demyelination and to the viral and host genetic elements involved in disease resistance/susceptibility, cellular susceptibility to virus infection, and virus persistence should be forthcoming. These studies should add to our understanding of the etiology and immunopathologic mechanisms of tissue injury in MS and other chronic (auto)immune diseases, aid in design of specific treatment strategies, and hopefully lead to innovative approaches which may ultimately link a specific virus(es) with MS.

DESCRIPTION (Adapted from the Applicant's Abstract): Activation of naive T-cells requires TCR occupancy plus CD28-mediated co-stimulatory signals delivered by ligation of B7-1 and B7-2 on antigen presenting cells (APCs). In contrast, B7 ligation of CTLA-4 expressed on activated T-cells delivers a potent negative regulatory signal and may be involved in tolerance induction. These studies during the previous application period have clearly shown that strategies targeting these interactions have potent positive and negative regulatory activity on the initiation and progression of both acute and relapsing-remitting forms of experimental autoimmune encephalomyelitis (EAE), a CD4+ T-cell-mediated autoimmune disease which serves as a model for multiple sclerosis. The hypothesis under test in this renewal application is that the B7-CD28 co-stimulatory interactions play a critical role in positively regulating the activation and effector functions of autoreactive T-cells, while B7-CTLA-4 co-stimulatory interactions negatively regulate autoimmune disease and are required for the induction/maintenance of immune tolerance. Building on their preliminary data describing a new EAE model in NOD mice induced by priming with proteolipid protein peptide, PLP56-70, Aim 1 will employ a multifaceted approach, utilizing NOD mice with targeted deletion of B7-1, B7-2, B7-1/B7-2, or CD28 in comparison with antibody blocking studies, to further delineate the roles of the individual co-stimulatory receptors and ligands in the induction and effector phases of EAE and in activating myelin peptide-specific T-cell responses. This aim will also examine the individual roles of B7-1 and B7-2 in induction of cell surface activation and homing antigens, proliferation, patterns of cytokine/chemokine mRNA and protein expression, and the ability to activate wildtype T-cells and Th1 clones for adoptive transfer of R-EAE. In addition, they will explore the mechanistic basis behind the resistance of CD28 knockout mice to EAE induction. Aim 2 will expand their published studies examining the effects of modifying co-stimulatory signals in animals with a pre-existing autoimmune disease employing the PLP139-151-induced R-EAE model in SJL and (SJL x B10.PL)F1 mice in which relapses are due to the activation of encephalitogenic T-cells specific for endogenous; myelin epitopes induced by epitope spreading. Treatment of mice with intact anti-B7-1 mAb following the initial clinical episode results in a significant increase in relapse incidence and exacerbation of disease severity. However, similar treatment with the F(ab) fragments of anti-B7-1 mAb blocked epitope spreading, and significantly ameliorated CNS histopathology and prevented clinical relapses. The cellular targets and molecular mechanisms of intact anti-B7-1-mediated R-EAE exacerbation and anti-B7-1 F(ab) fragment-induced protection from disease relapses will be assessed by examining the Th1/Th2 phenotype and functional responses of peripheral and CNS-resident APCs and of T-cells specific for both the initiating and relapse-associated myelin epitopes. Aim 3 will explore the role of CTLA-4 in negatively regulating R-EAE pathogenesis and epitope spreading, and determine the role of B7-1, B7-2 and CTLA-4 in the induction and maintenance of peripheral tolerance induced by the i.v. injection peptide-pulsed, ECDI-fixed APCs. These studies should enhance our understanding of the role of co-stimulatory molecules in disease initiation and regulation of epitope spreading in chronic autoimmunity and provide vital information relative to the potential targeting of co-stimulatory molecules for treatment of pre-existing immune-mediated disorders.

DESCRIPTION (Adapted from the Investigator's Abstract): The mechanism(s) underlying the initiation and progression of autoimmune diseases are not well understood. Clinical and epidemiologic evidence supports a role for viral infections. Postulated mechanisms include: molecular' mimicry (activation of autoreactive T cells secondarily by viral epitopes shared or cross-reactive with self antigens); epitope spreading (de novo activation of autoreactive T cells by sequestered antigens released secondary to self tissue destruction); and superantigens (non-specific stimulation of autoreactive T cells bearing particular VB receptors). Evidence for a viral etiology is particularly strong for multiple sclerosis (MS), a human CD4+ T cell mediated demyelinating disease associated with anti-myelin responses. The investigators have used the Theiler's murine encephalomyelitis virus (TMEV)-induced demyelinating disease model of MS to examine mechanisms of initiation and progression of virus-induced autoimmunity. TMEV is a natural mouse pathogen which establishes a life-long persistent CNS infection. Demyelination following infection of SJL mice with the wildtype BeAn strain of TMEV is initiated by virus-specific CD4+ Thl cells targeting virus persisting in CNS macrophages/microglia. Autoreactivity to the immunodominant myelin proteolipid protein epitope, PLP139-151, arises via epitope spreading two months post infection and plays a pathologic role in disease progression. To test the ability of molecular mimicry to initiate CNS demyelination, they engineered TMEV expressing PLP139-151 in the viral leader (PLP139-BeAn). SJL mice infected with PLP1 39-BeAn develop a severe, rapid-onset clinical demyelinating disease characterized by early activation of PLP1 39-151-specific T cells. They will test the hypothesis that CNS autoimmunity can be initiated by molecular mimicry induced by infection with a neurotropic virus. Aim 1 will further characterize PLP139-BeAn-induced disease by determining the pathologic role of PLP139-151-specific T cells (using tolerance and adoptive transfer) and the precursor frequency and temporal appearance of CD4+ and CD8+ TMEV- and myelin epitope-specific responses. Aim 2 will directly test the molecular mimicry hypothesis by assessing activation of autoreactive T cells1and clinical disease following infection with TMEV encoding molecular mimics of PLP139-151 naturally expressed by mouse hepatitis virus and H. influenzae. Relevant to human MS, Aim 3 will assess activation of autoreactive T cells and clinical disease induced by infection with TMEV encoding molecular mimics of the MS-associated MBP85 99 epitope naturally expressed by HSV, EBV, influenza A virus and Reovirus type 3 in HLA-DR2 transgenic mice. These studies should further our understanding of the potential role of molecular mimicry in MS etiology by defining activation requirements for autoreactive T cells primed by infection with a virus expressing molecular mimics of defined murine and human encephalitogenic myelin peptides and assessing their pathologic capacity.

Description (provided by applicant) Theiler's murine encephalomyelitis virus (TMEV)-induced demyelinating disease (TMEV-IDD) is the most relevant of the few available experimental animal models of virus-induced demyelination. MS is generally considered to involve an autoimmune pathology, but epidemiological evidence strongly suggests a viral trigger. TMEV are natural mouse pathogens. Intracerebral inoculation of susceptible mouse strains with the BeAn strain of TMEV results in a chronic, progressive, CNS inflammatory demyelinating disorder which is related to life-long persistent CNS virus infection and characterized by spastic hind limb paralysis. TMEV-IDD is considered a highly relevant animal model for multiple sclerosis (MS) since both diseases are characterized by progressive demyelinating lesions with accompanying mononuclear cell infiltrates in which CD4+ T cells and activated microglia/macrophages predominate. Our previous studies have shown that demyelination is initiated by virus specific CD4+ T cells targeting viral epitopes presented in the CNS by microglia/macrophages persistently infected with TMEV. Chronic disease is characterized by the induction of autoimmune responses to a variety of encephalitogenic myelin epitopes which arise via epitope spreading and which appear to play a pathologic role in chronic disease. We will continue to test the overall hypothesis that de novo priming of auto reactive T cells specific for endogenous myelin epitopes occurs both in the CNS and the peripheral lymphoid system and that these autoimmune responses play a major pathologic role in chronic disease progression. Aim 1 will use a variety of approaches to precisely quantitate the temporal appearance of Th1/Th2 cells in peripheral lymphoid organs, peripheral blood and the CNS specific for a panel of encephalitogenic myelin epitopes (using ELISPOT analysis, intracellular cytokine staining, and MHC class II peptide tetramers), determine the T cell receptor (TCR) repertoire (using immunoscope) of these T cells, and assess their encephalitogenic potential (using tolerance and adoptive transfer). Aim 2 will utilize a TCR transgenic T cell transfer system to analyze the temporal appearance and anatomic location of activation of naive myelin-specific T cells stimulated by endogenous myelin epitopes released during acute clinical disease. Aim 3 will determine the relative functional ability of virus-infected peripheral (dendritic cells, macrophages, and B cells) and CNS-resident (astrocytes, microglia, and infiltrating macrophages) APC populations derived from primary cultures and isolated from mice with ongoing TMEV-induced demyelinating disease to produce innate immune cytokines and to process and present TMEV and encephalitogenic myelin epitopes on PLP, MBP, MOG to a panel of myelin epitope-specific naive T cells and activated Th1/Th2 clones. These studies should define the mechanism(s) by which myelin epitope-specific autoimmune responses arise during persistent CNS virus infection and determine their functional contribution to chronic disease. In addition, studies using epitope-specific tolerance to treat chronic TMEV-IDD are applicable for the future design of antigenspecific treatment strategies for MS.

DESCRIPTION (provided by applicant): Multiple sclerosis (MS) is an immune-mediated central nervous system (CNS) disease characterized by perivascular CD4 + T cell and mononuclear cell infiltration with subsequent primary demyelination of axonal tracks leading to progressive paralysis. MS is generally considered to be an autoimmune disease involving autoreactive T cell responses to MBP, PLP, and/or myelin-oligodendrocyte glycoprotein (MOG), however a clear cut cause-effect relationship between myelin reactivity and disease pathology has yet to be demonstrated. Although MS is generally considered to involve an autoimmune pathology, little is known regarding its etiology and there are limited therapeutic strategies available to specifically inhibit and prevent ongoing disease. We propose to investigate intrinsic regulatory mechanisms that influence both the susceptibility to and progression of MS using two mouse models of experimental autoimmune encephalomyelitis (EAE) that either follow a relapsingremitting (R-EAE) or chronic (C-EAE) disease course. CD4+CD25 + regulatory T cells (TR) are potent inhibitors of CD4 + T cell responses that are activated in an antigen-specific manner via TCR cross-linking and have been shown to mediate protection against the initiation of several spontaneous autoimmune disorders, such as diabetes and autoimmune gastritis. Our preliminary data clearly demonstrate that supplementation of TR significantly protects against the initiation of EAE by inhibiting the proliferation and effector function of both naive and previously-activated encephalitogenic CD4 + T cells, while depletion or inactivation of these cells leads to exacerbated disease symptoms. We have also shown that TR-mediated disease inhibition is associated with an increased frequency of myelin peptide-specific protective Th2 cells within the peripheral lymphoid organs and significantly decreased numbers of mononuclear cells infiltrating the CNS. The experiments in this proposal will test the hypothesis that CD4+CD25 + TR play a critical role in conferring resistance to EAE susceptibility and in regulating disease progression by inducing antigen-specific unresponsiveness to disease-relevant self-myelin peptides. The proposed experiments will elucidate the cellular and molecular mechanisms by which TR inhibit the functional activity of encephalitogenic effector Thl cells in acute and relapsing EAE, thus blocking the initiation/progression of clinical demyelinating disease. Three specific aims are proposed: AIM 1 will determine the role of CD4+CD25 + TR in modulating the initiation, progression, and recovery phases of R-EAE and C-EAE. AIM 2 will determine the contribution of CD4+CD25 + TR to age-, gender-, and strain-associated susceptibility to EAE. AIM 3 will elucidate the mechanisms by which CD4+CD25 + TR influence encephalitogenic T cell activation, expansion, trafficking, and/or effector function. These studies should further our understanding of the role of TR in regulating both the induction and progression of CNS autoimmunity and provide vital information relative to optimizing strategies designed to enhance intrinsic regulatory mechanisms for the prevention and treatment of human autoimmune diseases.

DESCRIPTION (provided by applicant): Theiler's murine encephalomyelitis virus (TMEV)-induced demyelinating disease (TMEV-IDD) is widely considered the most relevant animal model of virus-induced autoimmune-mediated demyelinating disease. MS is believed to involve an autoimmune pathology, but epidemiological evidence strongly suggests a viral trigger. TMEV are natural mouse pathogens and intracerebral inoculation of susceptible SJL mice with the BeAn strain of TMEV results in a chronic-progressive, inflammatory immune-mediated demyelinating disorder which is related to life-long persistent CNS virus infection and characterized by spastic hind limb paralysis. In contrast, resistant C57BL/6 mice make a potent CTL response and rapidly clear the infection. Like MS, TMEV-IDD is characterized by progressive demyelination with accompanying mononuclear cell infiltrates dominated by CD4+ T cells and activated APCs (microglia/macrophages/DCs). Our previous studies have shown that demyelination is initiated by virus-specific CD4+ T cells targeting viral epitopes presented by persistently infected CNS- resident APCs. Chronic demyelination is mediated by induction of autoimmune responses to a variety of endogenous encephalitogenic myelin epitopes which arise via epitope spreading. This application proposes continue our productive studies funded by an NIH PPG for the past 21 years. Based on extensive new preliminary data, we will examine the role of innate immune regulatory and innate immune stimulatory mechanisms involved in regulating susceptibility/resistance to TMEV-IDD at both the level of immune- mediated virus clearance to acute infection and regulation of autoimmune processes during chronic demyelination. Employing depletion, supplementation and genetic approaches, Aim 1 will test the hypothesis that activation of CD4+CD25+Foxp3+ Tregs plays a major role in regulating susceptibility to TMEV-IDD by inhibiting development of virus-specific CD4, CD8, and antibody responses during acute infection leading to establishment of CNS virus persistence and hence eventual development of chronic autoimmunity. Aim 2 will test the hypothesis that the pro-inflammatory effects of both myelin epitope-specific autoreactive Th1 (IFN-3) and Th17 (IL-17) cells are critical for mediating demyelinating immunopathology during the chronic autoimmune phase of TMEV-IDD. Aim 3, will test the hypothesis that peripherally-derived, CNS-resident DCs play a critical role in driving the activation of epitope spreading to myelin-specific CD4+ Th1/17 cells responsible for CNS pathology in chronic TMEV-IDD. Innate immune responses (expression of cytokines;chemokines;and antigen presentation molecules);and functional ability of CNS-derived DCs, MUs and microglia from TMEV-infected mice to activate T cell proliferation and differentiation of naove CD4 and memory CD4+ (Th1, Th2 and Th17) and CD8+ T cells by will be determined. These studies will provide important information on the mechanisms underlying susceptibility/resistance in virus-induced demyelination and are applicable for the future design of treatment strategies for MS and other CNS inflammatory diseases. PUBLIC HEALTH RELEVANCE: Multiple sclerosis (MS) is an autoimmune paralytic disease caused by immune cell-mediated destruction of myelin-producing oligodendrocytes in the central nervous system. Certain forms of MS are suspected to occur as a secondary consequence a virus infection in genetically susceptible individuals. We will employ a mouse model of MS induced by infection with Theiler's murine encephalomyelitis virus to study the role of regulatory T cells in determining genetic susceptibility to disease in different strains of inbred mice, and to determine the role of dendritic cells, specialized antigen presenting cells of the innate immune system, both in inducing virus immunity in the acute stages of the disease and in inducing development of autoimmune T cells making destructive cytokines (IFN-3 and IL-17) in the chronic phase of disease.

DESCRIPTION (provided by applicant): Atherosclerosis is caused by a chronic inflammation of large arterial blood vessels superimposed upon by hyperlipidemias, as well as other risk factor. The processes involved in the initiation of atherosclerosis are still poorly understood, but there is incontrovertible evidence that autoimmunity, characterized by a T cell response to specific auto-antigens, plays a major role. Currently, one of the more promising methods for the prevention and treatment of autoimmune diseases is to induce antigen-specific T cell tolerance by intravenous treatment with antigen-coupled ethylene carbodiimide (ECDI)-fixed splenocytes. The fixation of splenocytes (or other donor cells) with ECDI in the presence of antigen results in the formation of peptide bonds between free amino and carboxyl groups, which bind the antigen to the cells, generating a antigen carrier system capable of inducing antigen- specific T cell tolerance. Current evidence indicates that tolerance induced by antigen-coupled cells is due, at least in part, to the up-regulation of CD4+CD25+FoxP3+ regulatory T cells (Tregs) that function in the suppression of pathogenic T cells. The goals of this proposal are to determine whether antigen-coupled cells can modulate the development of atherosclerotic lesions in experimental animals. Accordingly, the first objective of this proposal is to determine the effectiveness of tolerance induced by atherosclerosis-specific antigen-coupled cells in: a) preventing the formation of atherosclerotic lesions;b) reducing lesion size and severity, and/or c) causing lesion regression in two well-establish murine models of atherosclerosis: the low-density lipoprotein receptor deficient mouse (LDLR-/-) and the apolipoprotein E deficient mouse (apoE-/-). The second objective is to investigate the effect of tolerance on atherosclerosis antigen-specific T cell and antibody responses focusing on whether antigen-specific Tregs play a significant role in the prevention of atherosclerotic lesions in this tolerance model. PUBLIC HEALTH RELEVANCE: Atherosclerosis and its complications are a major public health problem, accounting for over half the annual mortality in the United States. This disease begins, at least in part, as a result of an autoimmune reaction in the major arterial blood vessels. In this application we propose to examine whether this autoimmune reaction can be neutralized thereby preventing or causing the regression of atherosclerosis in animal models of this disease.

? DESCRIPTION: The undesired destruction of healthy cells by the immune system results in the loss of tissue function and complicates strategies to restore tissue function. The current standard therapy for autoimmune disease involves generalized immunosuppression, which is in most cases is not clinically efficacious and leads to numerous undesired side effects. Dr. Stephen Miller, (co-PI) pioneered an approach in which splenocytes were cross-linked with specific auto antigens, and their delivery to the spleen induced tolerance specifically to the auto antigen. This approach was recently adapted for a clinical trial in multiple sclerosis (MS) patients, and was the first-in-man study to report the induction antigen specific tolerance. However, the use of cellular carriers for tolerance induction in the clinical arena is challenging due to the considerable ex-vivo laboratory manipulation that is required, which is expensive, increases the number of donor cells needed and introduces further opportunity for technical error. Our long-term goal is to develop a particle-based platform that can be an off-the-shelf product for induction of tolerance to specific antigens to inhibit the specific undesired immune response while not altering the remaining elements of the immune response. We have demonstrated that antigen-loaded particles delivered intravenously can induce tolerance for the prevention and treatment of experimental autoimmune encephalomyelitis (EAE), the mouse model of MS. With the goal of moving this technology toward the clinic, we propose to extend these studies to address fundamental questions about the particle design and their mechanisms of action, and also critical questions regarding the ability to target the variety of antigens and cell populations underlying disease. Specific Aim 1 will investigate the particle design parameters and identify the cellular mechanisms by which particles injected intravenously are able to modulate inflammation and induce antigen specific tolerance. Our results suggest the liver as a critical site involved in tolerance induction from the particles, which distinguishes it from the previous work with antigen-coupled splenocytes. We propose to investigate the particle composition and size to distinguish i) the impact of the carrier on immune cell polarization, ii) te efficacy of antigen presentation, and iii) in vivo trafficking of the particles. Specific Aim 2 wil determine the cellular and molecular mechanisms by which Ag-PLG tolerance is induced and maintained in naïve, activated, and memory T cells. We propose to test the ability to induce tolerance with particles encapsulating multiple peptides/proteins and to examine the separate and combined contributions of anergy and Tregs to the induction and maintenance of tolerance. Successful completion of these studies would identify particles that are novel, safe, efficient and clinically relevant tools to inhibit antigen-specific T-cells for therapy of autoimmune diseases. This innovative approach has far reaching implications for decreasing specific immune responses in applications such as autoimmune disease, rejection of transplanted cells, and allergies to food antigens or airborne particulates.

DESCRIPTION (provided by applicant): The long-term goal of the proposed preclinical studies is to develop a clinically applicable tolerogenic protocol for use in human islet allotransplantatin in T1D. The central component of our strategy is the delivery of antigens on leukocytes treated with the chemical cross linker 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (ECDI). Autoantigen-coupled splenocytes given IV prevent and treat autoimmunity in mice. In transplant models, ECDI-fixed donor splenocytes given IV on days -7 and +1 - as induce long-term donor-specific tolerance to islet allografts, and when combined with short-term rapamycin (RAPA), also to heart allografts in mice. A first-in-human clinical trial of autologous, peptide-coupled cels in multiple sclerosis (MS) recently established the clinical feasibility of this novel tolerogenic strategy. To test whether the profound tolerogenic efficacy of alloantigen delivery via ECDI-fixed cells (ADEC) will translate to islet transplantation in nonhuman primates (NHP), we will study the following specific aims: AIM #1: To manufacture ADEC products meeting prospectively defined release criteria for evaluation as tolerogens in islet allotransplantation in RM. AIM #2: T determine the efficacy of ADEC in inducing tolerance to islet allografts in RM with low and high memory alloreactivity transiently treated with RAPA, sTNFR, ¿-IL-6R, and LFA3-Ig. AIM #3: To examine the effects of the immunotherapeutic protocol on mechanisms underlying the induction, maintenance, and/or loss of donor-specific tolerance to islet allografts in RM. The innovation of this proposal lies expressly in the preemptive use of potent, yet safe, cellular immunotherapeutics as antigen-specific, negative vaccines. Our protocol targets innate, heterologous, and adaptive direct and indirect pathway immunity (and can be extended to target autoimmunity) and has, despite complete avoidance of generalized T and/or B cell depletion and costimulation blockade, a high potential for inducing durable tolerance to islet allografts in NHP. The proposed studies will provide novel insights into the role of ADEC and concomitant immunotherapy for tolerance induction to islet allografts, a critical step toward clinical translaton of this antigen-specific tolerance strategy. PUBLIC HEALTH RELEVANCE: Human islet allotransplantation restores insulin independence and near normoglycemia, protects from severe hypoglycemia and slows the progression of microvascular complications in those people with type 1 diabetes (T1D). The numerous undesirable side effects and the high costs of chronic immunosuppression limit the applicability of islet transplants. To overcome this limitation, the goal of this proposal is to develop in a pre clinical model of T1D (diabetic non-human primates) tolerogenic protocol(s) for use in human islet allotransplantation in T1D.

? DESCRIPTION (provided by applicant): Anticonvulsants are the standard treatment for epilepsy, but only control the symptoms without addressing the mechanisms of the disease. About 1/3 of patients develop drug-resistant epilepsies and only some are candidates for resective surgery as a final attempt to reduce seizure occurrence. Recently, our laboratory has pioneered the characterization of inflammatory cell infiltrates in surgically removed fresh brain samples in a search for novel therapies that may target the cause of epilepsy. Our data strongly indicate a role for immune cell activation in the epileptic brain irrespective of the particular etiology of epilepsy. We discovered significant brain infiltration of functionally activated lymphocytes in both epileptic patients and experimental animals. Additionally, steroids that are known for their anti-inflammatory properties have shown efficacy in a number of types of drug-resistant epilepsy. However, the profound immunosuppressive and other severe side effects of chronic steroid use have prevented widespread prescription of these drugs to otherwise treatable patients. The goal of this proposal is to use mouse models to design novel therapies to treat epilepsy using directed immunomodulatory approaches independent of broad-acting immunosuppressive agents. We propose to test the hypothesis that dampening ongoing inflammation in the brain could effectively reduce epileptogenic effects of early-life seizures and ultimately prevent epilepsy in the absence of systemic immunosuppression. This proposal has two aims. Specific Aim 1 will determine the efficacy and underlying mechanisms by which biodegradable nanoparticles formulated from the FDA-approved biopolymer poly(lactide-co- glycolide) (PLG) induce leukocyte sequestration in the spleen and reduction of brain inflammation to prevent the priming effect of early-life seizures. Specific Aim 2 is designed to assess the efficacy of autologous natural regulatory T cell (nTreg) infusion and the potential synergistic effect of PLG nanoparticle treatment in combination with exogenously introduced nTregs in the amelioration of neuroinflammation in murine models of epilepsy. Our previous work documented the success of using biodegradable nanoparticles to treat a variety of inflammatory immune-mediated diseases in animal models. Strikingly, our preliminary results indicate that treatment with PLG nanoparticles can improve seizure outcomes in our two-hit model of epileptogenesis. We propose to elucidate the underlying therapeutic mechanisms. Additionally, we propose to test the ability of a patient's nTregs to dampen immune responses in the epileptic brain alone or in combination with PLG nanoparticles. We have previously reported that supplementation of nTregs could significantly reduce disease severity in several animal models of multiple sclerosis by preventing brain inflammation. Our approaches that target inflammation and restrict brain infiltration by inflammatory leukocyte subsets in the absence of the complicating effects of steroids are highly novel and may be a major step forward for the translation of novel treatments for the root cause of epilepsy.

Although multiple sclerosis (MS) is the most common neurological disorder to afflict young adults, much remains unknown with regard to the etiology and pathogenesis of this disease. Epidemiologic evidence indicates that there are environmental, gender and genetic factors that influence disease incidence. Nevertheless, the triggering event that initiates the autoimmune response against the myelin sheath is unclear. MS usually begins as a remitting/relapsing inflammatory demyelinating disorder, but in most individuals the disease progresses to a chronic neurological condition that correlates with the accumulation of axonal damage. To further our understanding of this disorder, we have developed a new mouse model of inducible, widespread oligodendrocyte ablation by inducing expression of diptheria toxin A under control of the PLP promoter that results in extensive CNS demyelination in adult animals (DTA model). Strikingly, these animals display robust CNS remyelination that correlates with the recovery from the severe neurological symptoms that the mice display at the peak of disease. At the peak of the early disease course the blood brain barrier remains intact, T cells are not detected within the CNS and axons are preserved. Despite the robust early recovery that these animals display in response to oligodendrocyte (ODC) ablation, within 6 months they succumb to a severe inflammatory neurological condition supporting the `inside-out' model of MS pathogenesis. This late phase progressive disease is characterized by CNS accumulation of myelin-specific CD4+ T cells and widespread focal demyelination. We propose to utilize the DTA model to study fundamental aspects of adult-onset remyelination and demyelination. We will test the hypothesis that the initial CD4+ T cell response to ODC ablation is protective/regulatory, but eventual loss of myelin peptide-specific tolerance/regulation leads to the induction of CD4+ T cell-mediated late-onset disease. We will explore the role that both innate and adaptive immune responses play in development of chronic inflammatory demyelination. While our previously published work shows that there is an increase in the number and activation of CD11b+ cells in the CNS following initial ODC ablation, the question remains whether microglia or peripheral macrophages/DCs are the predominant antigen presenting cells that activate the later influx of pathogenic CD4+ T cells. We will also determine, similar to MS pathogenesis, why there is a lengthy lag time between the initial ODC ablation and the late-onset CD4+ T cell-mediated chronic demyelinating phase. To define the immune mechanisms underlying the transition to late-onset T cell-mediated demyelination, we will exploit strategies to increase the frequency and infiltration of myelin-specific effector and regulatory T cells into the CNS of the DTA mice during the initial disease phase. We will also elucidate the underlying immunopathologic T cell mechanism(s) driving late-onset immune- mediated demyelination. These studies will examine the exciting possibility that the initial ODC loss and demyelination trigger autoreactive myelin-specific T cell responses in a model of chronic progressive MS.    

PROJECT SUMMARY It is estimated that scleroderma affects about 300,000 Americans, predominantly women. It is a progressive and untreatable disease of unknown cause and high mortality. No therapy to date has been shown to significantly alter survival but only alleviate the pain. The pathogenesis of scleroderma is characterized by overt inflammation followed by progressive tissue damage and fibrosis secondary to excessive production of collagen and accumulation of myofibroblasts in lesional tissues causing organ failure. Recent genomic studies have revealed potential pathogenic role of inflammatory myeloid cells, including inflammatory monocytes (?IMs) and macrophages (M?s). We have shown for the first time accumulation of MARCO+ ?IMs and M?s within the lesional skin and lung of scleroderma patients and mice with bleomycin-induced scleroderma. MARCO is a type II glycoprotein scavenger receptor expressed by activated phagocytic antigen presenting cells, and is linked to alternate (M2) activation of M?s, resulting in pro-fibrotic activities. The function and pathological contribution of MARCO+ ?IMs and M?s and the therapeutic potential of targeting these cells in scleroderma have never been addressed. We pioneered an innovative strategy targeting ?IMs and M?s by leveraging the drug-like biological properties of carboxylated poly(lactic-co-glycolic acid) (PLGA) nanoparticles. We showed that negatively-charged 500 nM diameter PLGA particles are selectively recognized and bound by ?IMs and M?s via MARCO. PLGA-associated ?IMs/M?s undergo sequestration in the spleen and apoptosis, culminating in reduced immune pathology in bleomycin-treated mice. We hypothesize that PLGA nanoparticle can be used i) as a unique biological probe for illuminating the pathogenic roles of ?IMs and M?s in scleroderma, and to ii) treat inflammation-driven fibrotic diseases. We will test this hypothesis in 3 aims: Aim 1 will measure MARCO levels to determine their clinical significance using skin biopsies from scleroderma patients and healthy controls. Aim 2 will evaluate the efficacy of MARCO-targeting PLGA nanoparticle treatment on progression and alleviation of fibrosis formation in the bleomycin murine model of scleroderma. Aim 3 will delineate mechanisms underlying the deregulated fibrogenic nature of MARCO+ ?IMs and M?s in vitro. In summary, this application leverages exciting preliminary findings from the Miller and Varga labs implicating a pathologic role for MARCO+ ?IMs and M?s in the pathogenesis of scleroderma and showing the potential ability to regulate their pro-fibrotic function using a cutting-edge and translationally-relevant nanoparticle approach. These studies under the co-direction of two well-established PIs benefit from their complementary expertise in inflammation, fibrosis and scleroderma research. The results should significantly advance our understanding of scleroderma by illuminating the pro-fibrotic mechanism(s) of action of MARCO+ ?IMs and M?s, validating the feasibility of a novel therapeutic in preclinical scleroderma models, developing strategies for the identification of patients suitable for the MARCO-targeting therapy.

Multiple Sclerosis (MS) is an autoimmune disease of the CNS characterized by demyelination and neurodegeneration in response to perivascular T cell and mononuclear cell infiltration. Currently FDA-approved MS disease modifying therapies are global immunosuppressants mediating non-specific inhibition of T cell activation/function and/or trafficking. These drugs generally have limited efficacy and/or are associated with serious side effects. The Miller lab has recently demonstrated an effective means of ameliorating disease in the EAE mouse model of MS via tolerance induction in autoreactive T cells induced by the intravenous infusion of 500nM carboxylated poly(lactic-co-glycolic acid) (PLG) nanoparticles coupled with or encapsulating myelin peptides (Ag-PLG). Ag-PLG nanoparticles effectively reduce disease in relapsing-remitting (RR-EAE) and chronic-progressive (C-EAE) mouse models of experimental autoimmune encephalomyelitis (EAE) by tolerizing encephalitogenic Th1/17 cells. The Ag-PLG tolerance approach is currently undergoing phase 1 testing in celiac disease patients. Additionally, there are currently no FDA-approved therapies marketed for promoting remyelination. We have recently found that digoxin, an FDA-approved cardiac glycoside (Na+/K+ ATPase), and miconazole, an FDA-approved anti-fungal agent, promote oligodendrocyte differentiation and maturation and robustly induce remyelination with negligible side effects in two non-T cell-mediated demyelination models - cuprizone and DTA-induced mouse demyelinating models. As both autoimmunity and neurodegeneration underlie MS pathogenesis, effective disease modifying therapies need to both regulate the immune system and promote restoration of neuronal function, including remyelination. Thus, we will test the hypothesis that remyelination can be more efficiently induced in mice in which underlying autoimmune responses are specifically regulated. The proposed research will examine the effects of therapy with digoxin or miconazole to promote endogenous remyelination by stimulating oligodendrocyte progenitor cells (OPCs) alone or in combination with Ag-PLG tolerance-based immunotherapy in T cell-mediated demyelinating disease. The drugs will be tested in two autoimmune-mediated mouse EAE models of relapsing-remitting and chronic-progressive MS providing the ability to test myelin repair promoting strategies in the two major clinical disease types observed in MS patients. We hypothesize that since both autoimmunity and neurodegeneration underlie MS pathogenesis, effective disease modifying therapies need to both regulate the immune system and promote restoration of neuronal function by stimulating myelin repair. These studies will combine the complimentary expertise of the Miller lab and collaborators (Drs. Popko and Tesar) in tolerance-based immunotherapy and molecular aspects of myelination, and will hopefully pave the way for future clinical studies employing this combinatorial therapeutic approach in MS patients.

PROJECT SUMMARY/ABSTRACT: Neuromyelitis optica (NMO) is an autoimmune disease of the CNS characterized by T cell and antibody responses to the water channel protein Aquaporin 4 (AQP4) and myelin oligodendrocyte protein (MOG). NMO patients often suffer from blindness and paralysis as a result of chronic damage to the optic nerves and spinal cord. Current treatments such as high dose corticosteroids, plasmapheresis and rituximab are expensive, non- curative and fail to provide durable disease control. The Miller lab has developed a method using carboxylated biodegradable nanoparticles composed of the FDA-approved biopolymer polylactide-co-glycolide (PLG) to encapsulate proteins or peptides [PLG(Ag)] for induction of T cell tolerance for treatment of autoimmune models of Multiple Sclerosis (EAE) as well as other autoimmune and allergic diseases. Following i.v. infusion, PLG(Ag) NPs are taken up by tolerogenic antigen presenting cells (APCs) in the liver and splenic marginal zone via the MARCO scavenger receptor. These APCs present the encapsulated antigen and induce tolerance by several mechanisms such as anergy induction and the activation of various subsets of Ag-specific Treg cells. Significantly, PLG NPs encapsulating gliadin are presently undergoing phase 2 clinical testing in celiac disease patients. NMO is a prime candidate autoimmune disease which may benefit from this tolerogenic therapy due to substantial evidence that the disease is primarily driven by autoimmune responses to AQP4. Therefore, we propose to test the hypothesis that tolerogenic treatment with PLG nanoparticles encapsulating AQP4 and/or its immunodominant T cell epitopes will induce specific immunotherapy of NMO via induction of anergy and AQP4-specific Tregs. Aim 1 will establish mouse models of NMO by transfer of activated T cells from AQP4 KO mice primed with either recombinant AQP4 protein (rAQP4), yielding T cells specific for multiple AQP4 epitopes, or with T cells from KO mice primed with NMO-inducing I- Ab/I-As-restricted AQP4201-220 or I-Ab-restricted AQP4135?153 epitopes to wildtype B6 or SJL recipients. As NMO patients often display autoantibodies to MOG and PLP as well as to AQP4 due, we will assess if epitope spreading leads to T cell and antibody responses to these myelin proteins in NMO mice. Aim 2 will determine the effects of tolerance induced by PLG NPs encapsulating the immunodominant AQP4201-220 and/or AQP4135- 153 T cell epitopes in rAQP4 and AQP4 peptide-induced NMO mice. Aim 3 will determine the effects of tolerance induced by PLG NPs encapsulating rAQP4 on disease initiated by transfer of rAQP4 vs. AQP4 peptide-specific T cells due to the possibility that human NMO patients recognize multiple AQP4 determinants presented by different HLA class II molecules. Tolerance induction will be assessed by clinical scoring, evaluation of demyelination and optic neuritis, and by immune criteria (T cell proliferation and cytokine production and Ab responses). It is anticipated that successful demonstration of AQP4-specific tolerance will propel the rapid clinical testing of this novel immune tolerance inducing platform for the treatment of NMO.