Joan M. Goverman - US grants

9407222 Goverman This is a Research Planning Grant. The long-term objective is to investigate mechanisms in the immune system of establishing tolerance to self antigens. During the planning period of this award, experiments will be carried out to address one possible mechanism for inducing tolerance to the MBP121-140 epitope (amino acids 121-140 of mouse myelin basic protein, a predominant protein in the myelin sheath which has been used extensively as a model system for antibody production to self antigen). The proposed experiments involve the molecular characterization of the T cell receptors (TCRs) expressed on MBP121-140-specific T cells from both "shiverer" mutant mice, which are unable to express endogenous MBP (where the cells are not tolerized) as well as wild-type mice (where tolerance results in a minor response to these residues). Comparing the repertoires of these two different types of mice will permit the testing of the hypothesis that affinity of the T-cell receptor for its ligand is critical in inducing tolerance to this epitope. In this hypothesis, high affinity TCRs which mature in the presence of antigen become tolerized. The minor response observed in wild-type mice could then arise from a small number of cells that were not tolerized simply because they escaped the stochastic process of interaction with antigen, reflecting "leakiness" of tolerance mechanisms. The model predicts that a repertoire of TCRs would be found in shiverer mice that are not found in non shivering litter mates. All possible MBP121-140-specific TCRs would be present in shiverers because of the lack of ligand to tolerize high affinity receptors. However, only receptors representing low-affinity clones would be found in non-shivering mice that express MBP if the high affinity clones are deleted or unresponsive. These studies will lay the basis for an investigation of the in vivo mechanisms for establishing tolerance to MBP. %%% In its response to foreign p athogens, the immune system must be able to discriminate between foreign and self molecules. The recognition of "foreign" antigens is accomplished by lymphocytes. These cells undergo a developmental program in which a large variety of clones of lymphocytes express antigen receptors on their surfaces with different specificities. During lymphocyte maturation in the thymus, a selection process occurs in which those cells bearing receptors which recognize and bind to self-antigens are either destroyed (negative selection) or somehow induced to be non-reactive toward their antigens (anergy). This process is called tolerance. Without tolerance, the immune system would result in self-destruction of the organism. The goal of this project is to understand the molecular mechanism whereby this self-non self discrimination is achieved, using a well established mouse system. ***

DESCRIPTION (Adapted from the Investigator's abstract): Experimental allergic encephalomyelitis (EAE), an animal model of multiple sclerosis, is induced by generating T-cell mediated immunity to central nervous system antigens. The primary autoantigen in EAE in B10.PL mice is myelin basic protein (MBP). The immune response to MBP is predominantly directed toward residues MBP 1-11, with minor responses detected to MBP 31-50 and MBP 121-140. The basis for the dominance of MBP 1-11 and the extent to which tolerance mechanisms influence immunodominance are not understood. The role played by subdominant epitopes in autoimmune disease is also not well characterized. The extent to which subdominant epitopes participate in determinant spreading in the later stages of autoimmune disease may depend on the mechanisms responsible for their subdominance. Preliminary experiments show that endogenous expression of MBP significantly influences immunodominance of particular epitopes. In mice lacking endogenous MBP (MBP-/-), the strongest immune response is now directed toward MBP 121-140, with MBP 1-11 responses comparable to those observed in wild-type mice. Thus, wild-type mice induce tolerance in most MBP 121-140 specific T-cells while MBP 1-11 specific T-cells escape tolerance. The proposed research investigates the mechanisms underlying this differential tolerance. The investigators' hypothesis is that interactions between MBP 121-140/I-Au complexes and T-cells are of sufficient avidity to induce tolerance by clonal deletion, while interactions between MBP 1-11/I-Au complexes and T-cells are lower avidity and less able to induce tolerance. To test this hypothesis, kinetic experiments to compare both the binding affinities of MBP 1-11 and 121-140 for I-Au MHC protein as well as the effective affinities of MBP 1-11 and 121-140 specific T-cells for their respective ligands will be performed. Two possibilities for incomplete tolerance to MBP 121-140 will be addressed using both kinetic experiments and a comparison of T-cell receptor repertoires in MBP 121-140 specific T-cells from MBP+/+ ("wild-type") and MBP -/- mice: 1) the minor response in wild-type mice consists of clones that escaped tolerance induction because they express lower affinity T-cell receptors than those found in MBP -/- mice, or 2) some MBP 121-140 specific T-cells in wild type mice randomly escape tolerance mechanisms and are few in number but still highly reactive to antigen. T-cell receptor transgenic models will also be developed to define the mechanisms and the site of tolerance induction to MBP 121-140.

DESCRIPTION (Adapted from the Applicant's Abstract): Multiple sclerosis (MS) is a neurodegenerative demyelinating, disease of unknown etiology. MS plaques contain both CD4+ and CD8+ T-cells and susceptibility is linked to immune response genes, suggesting that MS occurs when autoreactive T-cells enter the central nervous system (CNS) and attack cells expressing myelin antigens. Myelin basic protein (MBP) is one likely target: for these T-cells because it is an abundant protein in the myelin sheath. Auto‑reactive CD4+ T-cells specific for MBP have been extensively studied in an animal model of MS, experimental allergic encephalomyelitis (EAE). EAE is induced by activating CD4+ T-cells through immunization of animals with MBP. Although this model demonstrates that MBP‑specific T-cells are present in the periphery of healthy animals, the investigators have shown that the repertoire of MBP‑specific T-cells in the periphery is strongly shaped by induction of immune tolerance to MBP in vivo. Some MBP‑specific T-cells are more prevalent in the periphery because they escape tolerance while other MBP‑specific T-cells are tightly regulated by tolerance. The mechanisms that induce tolerance to MBP, and how these mechanisms influence autoimmune disease, are unknown and are the focus of this application. In Aim 1, they will use newly developed TCR transgenic mouse models specific for highly tolerogenic MHC class II‑associated epitopes of MBP to define tolerance mechanisms and their impact on disease susceptibility. In Aims 2 and 3, they will investigate a new area: tolerance and autoimmune potential of MBP‑specific CD8+ cytotoxic T-cells (CTLs). Our recent data show that MBP‑specific CTLs are generated in vivo and regulated by tolerance induction. In this proposal, they show that these MBP‑specific CTLs induce a novel CNS autoimmune disease that differs from EAE and demonstrates new parallels to symptoms of MS. They will investigate mechanisms of tolerance that operate on these CTLs as well as their effector mechanisms and target cells within the CNS.

DESCRIPTION (provided by applicant): This application focuses on events that cause a breakdown in tolerance to antigens expressed in the central nervous system (CNS). Loss of tolerance to CNS antigens is believed to be a critical step leading to the development of multiple sclerosis (MS). Epidemiological studies have suggested that MS is triggered by exposure of genetically susceptible individuals to a pathogen(s) early in life. To study how tolerance to CNS antigens is broken, we established a transgenic mouse model in which all T cells express a transgenic T cell receptor specific for the CNS antigen myelin basic protein (MBP). These transgenic mice develop CNS autoimmune disease spontaneously, and the incidence of spontaneous disease increases with increasing microbial exposure in the environment. We will use this transgenic model to investigate how infection results in a breakdown of tolerance to CNS antigens using a novel method of disease induction. Preliminary data show that autoimmunity is induced in this transgenic model by infection with Streptococcus pneumonias and Group B Streptococcus (GBS) but not by several other bacterial strains. We also show that the MBP-specific TCR does not appear to cross-react with bacterial antigens. Additional preliminary data demonstrate that endogenous MBP is normally processed and presented by peripheral antigen-presenting cells (APCs), resulting in T cell tolerance in some cases and T cell ignorance in other cases. Based on these observations, we propose to test a new hypothesis that bacterial infection triggers autoimmune disease by increasing or altering the presentation of endogenous MBP epitopes in the periphery resulting in a loss of T cell ignorance. We will utilize this unique experimental system in combination with several transgenic, knock-out and knock-in mouse models bred onto the appropriate genetic background to test this hypothesis.

DESCRIPTION (provided by the applicant): Multiple sclerosis (MS) is an inflammatory, demyelinating disease of the central nervous system that is believed to result from erroneous activation of self-reactive T cells specific for myelin antigens. Experimental allergic encephalomyelitis (EAE) is an animal model for MS that is induced by immunization with myelin antigens. In the EAE-susceptible B10.PL strain, research has focused on T cells specific for the immunodominant epitope of myelin basic protein (MBP), AcMBP1-11. Our previous studies showed that the immunodominance of AcMBP1-11 is the product of immune tolerance induced by endogenous expression of MBP. We demonstrated using MBP-deficient mice that MBP121-150 is the most immunogenic region of MBP in the absence of tolerance. MBP121-150-specific T cells are subdominant in wild-type mice because central tolerance mechanisms eliminate most, but not all, of these T cells from the periphery. Using a MBP121-150-specific T cell receptor transgenic mouse model, we showed that the MBP121-150-specific T cells that escape central tolerance are pathogenic because they can be specifically triggered to induce EAE. New data presented here show that transgenic MBP121-150-specific T cells that reside in the periphery are prevented from causing spontaneous disease by regulatory T cells. Adoptive transfer of transgenic, naive MBP121-150-specific T cells into T cell deficient mice results in rapid and severe autoimmune disease. This autoimmunity is completely prevented by introducing CD4+ T cells. Surprisingly, the regulatory T cells do not inhibit expansion of the MBP-specific T cells in vivo after transfer or their migration to the brain. This proposal focuses on defining the phenotype (Aim 1) and mechanisms of action (Aim 2) of the regulatory T cells. We will test the hypothesis that regulatory T cells alter the cytokine milieu at sites of antigen presentation to inhibit activation of Th1 inflammatory T cells. Understanding how regulatory T cells prevent MBP-specific T cells from mediating disease is important for understanding the pathogenesis of MS and may provide insights into new therapeutic strategies.

DESCRIPTION (provided by applicant): Multiple sclerosis (MS) is an inflammatory, demyelinating disease of the central nervous system (CNS) that is believed to have an autoimmune etiology. It is the most common neurological disease in young adults. The pathogenesis of MS is not well understood and this has limited the ability to develop effective therapies. Experimental allergic encephalomyelitis (EAE), a widely used animal model for MS, has provided many insights into the activity of myelin-specific CD4+ T cells that can mediate CNS autoimmune disease. However, the clinical signs and pathology seen in MS patients is very heterogeneous and only a subset of the characteristics of MS patients is reproduced in classical CD4+ T cell-mediated EAE models. This observation suggests that new models are needed to investigate the diverse mechanisms contributing to this disease. Our laboratory developed a new EAE model based on the activity of myelin basic protein (MBP)- specific CDS+ T cells. Transfer of activated MBP-specific T cells induces autoimmune disease that recapitulates some of the clinical signs and pathology seen in MS patients that are not typically seen in classic EAE. We generated T cell receptor transgenic models of these CDS+ T cells and found that an unusual form of tolerance allows CDS+ T cells expressing a high affinity T cell receptor for MBP to escape tolerance and populate the peripheral repertoire. Interestingly, this tolerance can be broken by viral infection. In this application, we will investigate the molecular mechanisms underlying both the CDS+ T cell tolerance and the loss of tolerance due to infection in this model. We will also identify the CNS cells that present the MHC class l-associated MBP epitope to the CDS+ T cells and the consequences of interaction between the CDS+ T cells and CNS cells during disease. Finally, we will determine if the CDS+ and CD4+ T cells subsets utilize different tissue homing molecules to infiltrate the CNS and compare the CNS damage mediated by the CDS+ T cells to the pathology mediated by myelin-specific CD4+ T cells that induce EAE in the same mouse strain. The over-arching hypothesis guiding these studies is that myelin-specific CDS+ T cells differ from CD4+ T cells in the mechanisms used to escape tolerance, in their recognition of targets cells in the CNS and in the pathologic consequences of antigen recognition in the CNS. Testing this hypothesis will determine whether there are unique aspects of CNS autoimmune disease mediated by myelin-specific CDS+ T cells, a subject with significant clinical relevance to human disease.

DESCRIPTION (provided by applicant): Project Summary Multiple sclerosis (MS) is a neuroinflammatory disease of the central nervous system. The diverse clinical signs seen in MS patients reflect the wide distribution of inflammatory infiltrates, demyelinating plaques and axonal damage in the white matter tracks of the brain and spinal cord. Experimental allergic encephalomyelitis (EAE) is an animal model of MS that is induced by stimulating T cell-mediated immunity to myelin antigens. EAE has many similarities to MS, including the presence of inflammatory infiltrates and demyelination in the white matter. Unlike MS, however, the lesions are restricted predominantly to the spinal cord with significantly less inflammation seen in the brain. Thus, most rodent EAE models are not amenable to investigate mechanisms for targeting inflammation to the brain as well as the spinal cord. We have developed a unique model of EAE that allows us to determine the basis for different patterns of inflammation in the CNS. C3HeB/Fej x C3H.SW F1 mice generate T cells specific for three epitopes of myelin oligodendrocyte glycoprotein (MOG): MOG97-114, MOG79-90 and MOG35-55. Adoptive transfer of MOG97-114-specific T cells induces inflammation predominantly in the brain and not the spinal cord, while transfer of MOG79-90 and MOG35-55-specific T cells induces inflammation localized in the spinal cord and not the brain. T cells specific for all three epitopes generate IL-17+ and IFN-3+ cells, however, the IL-17:IFN-3 ratio is significantly higher for MOG97-114-specific T cells. By manipulating Th17:Th1 ratios for each specificity, we demonstrate that the localization of inflammatory cells in the brain versus the spinal cord is regulated by the ratio, and not the absolute number or epitope specificity, of myelin-specific Th17 and Th1 cells. Interestingly, MOG97-114 specific T cells also exhibit a higher functional avidity for their antigen compared to either MOG79-90 or MOG35-55-specific T cells. We propose to test the hypotheses that 1) T cell functional avidity for antigen determines the Th17:Th1 ratio in the responding population, 2) Th17 and Th1 cells differ in their ability to either migrate to, survive in, and/or proliferate in the brain versus the spinal cord and 3) resident cells within the brain and spinal cord differ in their response to infiltrating T cell populations biased toward Th17 or Th1.Project Narrative In multiple sclerosis, inflammatory lesions are typically disseminated in the white matter of the brain and frequently the spinal cord; however, lesions in most rodent models of MS predominate in the spinal cord with little brain inflammation. We developed a unique mouse model in which mechanisms mediated by different types of pathogenic T cells will be defined that regulates brain versus spinal cord inflammation. A better understanding of how, where and why different T cell subsets initiate and sustain inflammation in the central nervous system is critical to predict the efficacy and consequences of manipulating the activity of these T cells in MS therapies.

DESCRIPTION (provided by applicant): This application seeks continued support for a highly productive pre-doctoral training program in Basic and Cancer Immunology at the University of Washington. Administrative responsibility for this training grant will be based in the Department of Immunology. A cohesive and interactive group of training faculty, whose research addresses a broad range of areas in basic and cancer- related immunology, serve as faculty mentors. Trainees will be selected from an outstanding group of graduate students, including those recruited through the Departments and Programs in Immunology, Microbiology, and Molecular and Cellular Biology. The Training Grant Supervisory Committee, composed of the program director, two associate directors and three other members of the training faculty, evaluates applications, selects students for support, and reviews trainee progress. Ongoing, program-specific efforts seek to enhance further the recruitment of students from under-represented minorities. The training program will include an integrated curriculum of formal course work that builds in complexity, providing first a general foundation in biology and basic immunology, including courses in molecular and cellular biology, advanced immunology, and current issues in immunology, and then an in-depth consideration of cancer and cancer-related conditions in an immunological context. The formal course work will be supplemented by research- in-progress and visiting speaker seminars, biannual external review by outstanding visiting scientists, and, most importantly, by the performance of original research in the laboratories of our faculty. Student research projects address unanswered questions relevant to fundamental immunological processes and the translation of immunological principles into improved management of cancer and cancer-related conditions. Students present their work at national and international meetings, and when the work is mature publish their work in high quality journals. Through this training program, students will fulfill University requirements for receipt of the Ph.D. degree in their home graduate program. The goal of this training program is the selection and rigorous training of an outstanding group of young scientists, who will contribute disproportionately to the conduct of important research in cancer-related immunology.

DESCRIPTION (provided by applicant): This program seeks renewal of a program for post-doctoral training of physician and non-physician scientists in basic and translational immunology side-by-side in the rich, shared environment of the Department of Immunology at the University of Washington. The ultimate goal of the program is to develop the next generation of investigators, who will make important contributions to our understanding of fundamental immunology and of normal and abnormal immune function relevant to human immunological and infectious diseases. The physician and non-physician post-docs in our program complement each other: The physician investigators bring to the program unique perspectives derived from direct interaction with patients and laboratory research experience of varying degrees. The non-physician, Ph.D. trainees bring to the program a rich background of didactic and laboratory research experience, but limited familiarity with the potential clinical ramifications of their work. The key quality sought in all trainees is a clear commitment and outstanding potential for research.

DESCRIPTION (provided by applicant): There has been enormous growth in the past few years in our understanding of pathogenic mechanisms in autoimmunity, and a new recognition of the similarities and important differences between different autoimmune diseases. Examples of rapidly moving areas of research in autoimmunity are the identification and characterization of new subsets of pathogenic T cells that secrete unique inflammatory mediators and novel insights into the interface between genetic susceptibility, environmental stress and the microbiome. These findings have emerged largely from the work of basic scientists, and this FASEB summer conference on Autoimmunity has historically focused on discussion of new findings in basic research in autoimmunity. However, we believe that it is crucial for basic scientists to establish and maintain connections to scientists who are working to translate mechanisms into therapeutic targets. For example, the unexpected efficacy of therapeutic B cell depletion in some autoimmune diseases has prompted intense investigation by basic scientists into the role of B cells in pathogenic pathways. Therefore, while maintaining a focus on cutting edge basic research in autoimmunity, new sessions will be included in these conferences in which translational scientists are invited to discuss new strategies for treating autoimmune diseases with biologics, and current data on the successes and failures of manipulating the immune system in humans with autoimmune disease. We expect that increased emphasis on therapeutic treatment of autoimmune disease will attract both basic and industry scientists, and will stimulate strong interest among both junior and established investigators. The size and setting of this meeting are ideal to promote the open exchange of data and cross-fertilization of ideas that will stimulate new hypotheses and directions in autoimmunity research. PUBLIC HEALTH RELEVANCE: Autoimmune diseases are debilitating, chronic diseases resulting in significant decrease in quality of life. The incidence of autoimmunity has risen over the past decade such that collectively these disease exert a significant economic toll on society due to loss of livelihood and cost of health care. The goal of the FASEB Summer Conferences on Autoimmunity is to provide a forum for the exchange of data, ideas and hypotheses between basic and applied scientists in order to catalyze the development of new treatments for autoimmune diseases.

DESCRIPTION (provided by applicant): Multiple sclerosis (MS) is a devastating, demyelinating disease of the central nervous system (CNS) and is the leading cause of neurological disability in young adults. Self-reactive myelin-specific T cells are believed to initiate MS and to play a prominent pathogenic role throughout the course of disease. Developing therapies for MS has been challenging, in part because the heterogeneity seen in inflammatory infiltrates, lesions and clinical course suggest that the relative contribution of specific pathogenic mechanisms may vary among individual patients. This heterogeneity may reflect individual variation in the types of effector cells recruited to the CNS that induce tissue damage via distinct mechanisms. An additional level of complexity arises from the fact that the inflammatory milieu within the tissue is dynamic and will reflect the relative abundance of specific T cells subsets with different activities. Thus, a better understanding of how the activit of distinct T cell subsets in the same microenvironment influence each other and how their interactions with CNS resident cells act in concert to promulgate inflammation and induce tissue damage, is essential to develop effective therapeutic intervention. The role of CD4+ myelin-specific T cells has been extensively studied in MS and the animal model experimental autoimmune encephalomyelitis (EAE). Two CD4+ effector T cell subsets, IFN-? producing Th1 cells and IL-17-producing Th17 cells, have been implicated in the pathogenesis of MS, and these subsets induce different inflammatory patterns within the central nervous system (CNS) in EAE. The role of CD8+ myelin-specific T cells has not been well-studied, even though a large body of data implicates a role for CD8+ T cells in MS. Few models have been developed to study the role of CD8+ T cells in MS, therefore little is known about how this T cell subset influences the disease process. This application proposes to use newly developed tools to investigate the mechanisms by which CD8+ myelin-specific T cells influence EAE initiated by CD4+ T cells. Our fundamental hypothesis is that myelin-specific CD8+ T cells are recruited to the CNS during disease initiated by CD4+ T cells, and that the simultaneous, but distinct, activities of CD4+ and CD8+ T cells determine the pattern of lesion formation, tissue damage and clinical signs. Three aims are proposed to test this hypothesis: 1. DETERMINE HOW RECRUITMENT OF MYELIN-SPECIFIC CD8+ T CELLS MODIFIES THE COURSE OF ACUTE AND CHRONIC EAE INDUCED BY CD4+ T CELLS. 2: DEFINE THE EFFECTOR FUNCTIONS ACQUIRED BY CD8+ T CELLS RECRUITED TO THE CNS DURING CD4 T CELL-INITIATED EAE. 3: DETERMINE THE INFLUENCE OF MYELIN-SPECIFIC CD8+ T CELLS ON CNS AUTOIMMUNITY INDUCED BY TH1 VERSUS TH17 CELLS.

Project Summary/Abstract This renewal application builds on the strength of our world class Immunology faculty for scientific training at the interface of innate and adaptive immunity to continue our Immunology training program. We request support each year for five students in our predoctoral training program as well as two postdoctoral trainees. This training program is based in the Department of Immunology at the University of Washington, with Drs. Michael Gale, Jr. and Joan Goverman serving as multi-Principal Investigators and Program Co-Directors. Our training faculty include specialists in immune system development, infectious disease immunology, autoimmunity, allergy, cancer immunology, immunodeficiencies, innate immunity, vaccinology, and immunotherapeutics. Our program features predoctoral student training that combines research, coursework, and scientific learning in a rigorous training program to teach our students critical thinking, scientific approach, effective communication, and science ethics. Our new postdoctoral training program is designed to encompass a focus on research excellence, career exploration, teaching, training in grant writing and ethics, mentoring skills, and leadership. Predoctoral trainees are selected from our own Immunology graduate program, the University of Washington Molecular and Cellular Biology graduate program, and from the Medical Scientist Training Program; postdoctoral trainees will be selected from applicants within the labs of our training faculty. Training takes place at the University of Washington and our partner institutions including the Benaroya Research Institute, the Center for Infectious Disease Research, the Fred Hutchinson Cancer Research Center, Seattle Children's Research Institute and the Institute for Systems Biology. Our trainees benefit from the rich, diverse, and interactive Immunology community at the University of Washington and our partner institutions. We provide trainees the opportunity to attend specific national meetings annually, exposure to different career paths, formal evaluation of trainees' scientific presentations and progress by a committee composed of members of our training faculty, and dedicated interactions with selected Immunology seminar speakers. An External Advisory Committee provides ongoing evaluation of the training program.