Abul K. Abbas - US grants

The objective of this project is to examine the mechanisms of generation of memory CD4 T cells, and the functional and biochemical characteristics of memory cells. The experimental system for studying CD4 memory uses adoptive transfer of primed T cells expressing a transgenic TCR (DO.11.10) into normal recipients, in which the antigen-specific cells can be followed quantitatively and purified. The specific aims of the project are the following: 1. Stimuli for the generation and maintenance of memory cells: The influence of priming conditions on the generation of memory cells will be defined, in order to establish correlations between the initial T cell expansion and differentiation and the size of the memory pool that develops subsequently. Using knockout mice lacking molecules involved in T cell activation and homeostasis (e.g., CD28, CD40L, IL-2, FasL), we will examine the role of these molecules in the development of memory cells. 2. Functional responses of memory T cells: Long-lived memory DO.11 T cells will be isolated from transfer recipients and analyzed for ex vivo responses to antigen, costimulation, and cytokines. The recall responses of these memory cells to immunogenic and tolerogenic forms of antigen will be defined. 3. Gene expression and biochemical alterations in memory T cells: We will examine the activation of selected genes and signaling pathways in memory cells to identify biochemical changes that may contribute to the survival and functional attributes of these cells. We will focus on pro- and anti-apoptotic proteins, cytokines and cytokine receptors, and intracellular biochemical intermediates that may account for the relative costimulator independence and tolerance resistance of memory cells. The physiologic importance of genes expressed in memory cells will be examined by introducing these genes into T cells by retrovirus-mediated transfer, and examining effects on memory cell generation. Thus, this proposal addresses basic issues of immunologic memory, which is fundamental to effective vaccination. Defining the stimuli and genes that control memory T cell development should lead to more rational strategies for vaccine development than the largely empirical approaches that are in common use.

DESCRIPTION (Provided by the Applicant): The objectives of this continuing project are to study the biology and biochemical regulation of a pathway of apoptosis in CD4 T lymphocytes that is mediated by engagement of the Fas death receptor. The importance of this form of apoptosis (called activation-induced cell death [AICD]) in the maintenance of self-tolerance is illustrated by the autoimmune diseases that develop in mice and humans with inherited defects in Fas or Fas ligand (FasL). The specific aims of the project are the following. 1. Biology of Fas-mediated AICD. In this aim we will examine the hypothesis that AICD is induced by repeated T-cell activation, and define the conditions of antigen exposure that trigger this death pathway. We will use homogeneous T-cells from T-cell receptor (TCR) transgenic mice to test if and when AICD is triggered by immunization with antigen in adjuvant, infection with viruses expressing the antigen, and exposure to the antigen expressed as a systemic self protein. We will determine the factors that influence the choice between T-cell proliferation and apoptosis, including the concentration of antigen, the frequency and persistence of antigen exposure, the frequency of responding T-cells, and the amounts of IL-2 produced by the T-cells. We will define the effects of defective AICD on the survival of T-cells, the functional interactions between antigen-specific B- and T-cells, and the development of autoimmunity. We will also examine the consequences of disrupting Fas signals in T-cells by expressing the Fas antagonist, FLIP, or a dominant negative mutant of the adapter protein, FADD, by retrovirus-mediated gene transfer. 2. Regulation of Fas-mediated AICD. In this aim we will define the biochemical correlates of T-cell sensitivity to AICD induced by different types of antigen exposure in vitro and in vivo, focusing on the expression of the pro-apoptotic molecule FasL, the anti-apoptotic molecule FLIP, caspases, and other possible inhibitors of death pathways. We will examine the mechanisms by which IL-2 paradoxically potentiates Fas-mediated death, emphasizing the role of Stat5 signals on the transcriptional activation of FasL and inhibition of FLIP. We will also initiate studies to define the regulation of AICD by other cytokines, specifically IFN-g, and the biochemical mechanisms and functional implications of this regulation. The studies in this project will tell us how an important control mechanism for immune responses and autoreactivity is regulated, and may provide clues about manipulating this pathway therapeutically.

DESCRIPTION (provided by applicant): The objective of this Program Project is to understand the mechanisms of peripheral tolerance and why tolerance fails in systemic and tissue-specific autoimmune diseases. Common themes in the program are the concept that proximal signals in T cells influence the choice between tolerance and autoimmunity, and the use of genetic disruptions in mice that result in breakdown of immune tolerance. The proposed studies rely on combined functional and biochemical analyses, with a strong emphasis on in vivo model systems. The four component projects are highly complementary in terms of scientific themes, expertise, and tools and technologies. 1. Mechanisms of T cell tolerance vs. autoimmunity in vivo. (P.I.: A. Abbas). The goals of this project are to analyze the roles of two mechanisms of tolerance, anergy and deletion, in tolerance to transgene-encoded systemic and tissue-restricted secreted and membrane forms of "self" antigens. We will define the consequences of T cell encounter with self-antigens, the mechanisms of anergy induction in vivo, and the roles of CTLA-4, Fas, cytokines (IL-2, TGF-beta), and infections in determining the choice between tolerance and autoimmunity. 2. CTLA-4 based tolerance in autoimmune diabetes (P.I.: J. Bluestone). The goals of this project are to examine the role of CTLA-4 in T cell tolerance and suppression in vivo, and the molecular basis of CTLA-4 mediated T cell regulation. In addition, novel membrane-bound single chain Fvs and dimeric complexes will be developed to selectively ligate CTLA-4 in close proximity to TCR engagement, and tested for their ability to induce tolerance and treat autoimmune disease. 3. Regulation of CD45 signaling in tolerance and autoimmunity (P.I. A. Weiss). The goals of this project are to exploit a newly discovered model of autoimmunity caused by a genetic disruption that prevents CD45 dimerization, as a model for defects in signaling receptors that interfere with self-tolerance. The cell lineages responsible for autoimmunity and the role of antigen will be defined, and the molecular basis of CD45 regulation of lymphocyte responses and self-tolerance will be examined. 4. Src tyrosine kinases and autoimmunity (P.I.: A. DeFranco). The goals of this project are to examine the mechanisms by which deletion of two kinases important in lymphocyte responses, Lyn and Fyn, leads to systemic autoimmunity. The cells involved in autoimmunity will be defined, and the molecular mechanisms by which these kinases contribute to lymphocyte regulation and self- tolerance will be examined. The two scientific cores (A. Animals, and B. Flow cytometry) will support all four projects, and develop new mouse strains and analytical techniques for the proposed studies. Thus, the information gained from this Program Project will answer fundamental questions about the choice between self-tolerance and autoimmunity and may provide new strategies for inducing tolerance.

DESCRIPTION (provided by applicant): The goal of this project is to explore the mechanisms underlying the well-recognized link between immune deficiencies and autoimmunity. We have developed a transgenic mouse model in which CD4 helper T-cells specific for ovalbumin (Ova) become tolerant (functionally unresponsive) when they encounter a secreted form of Ova (sOva) produced as a self antigen in lymphocyte-sufficient hosts. In striking contrast, if the same T-cells see the circulating Ova in a lymphopenic (RAG-/-) host, tolerance fails, resulting in a severe, systemic autoimmune reaction that is usually acutely lethal. Our hypothesis is that multiple classes of host (endogenous) lymphocytes help to maintain self-tolerance by several mechanisms, including: competition for antigen and other stimuli, antigen presentation under tolerogenic conditions, and production of regulatory cytokines. Using this model, we will address the following specific aims. 1. Regulatory functions of endogenous lymphocytes: We will determine which types of host lymphocytes function to maintain tolerance by two approaches. First, mice expressing the sOva will be depleted of selected lymphocytes (CD4 or CDS T cells, B cells, gamma-delta cells, NK-T cells). Ova-specific T-cells will be transferred into these hosts and followed for tolerance or autoimmune reactions. Second, lymphopenic hosts expressing the sOva will be reconstituted with different lymphocyte populations, and the responses of Ova-specific T cells will again be examined. 2. Mechanisms by which endogenous lymphocytes maintain tolerance: To determine how endogenous lymphocytes help to maintain self-tolerance and prevent the activation of self-reactive T-cells. We will first define the types of responses of these T-cells that correlate with tolerance vs pathologic autoimmunity. We will then inhibit or eliminate selected molecules (such as cytokines) from the self-reactive T-cells or the host, or selected non-lymphoid cell types from the host, to define how endogenous lymphocytes control the balance between tolerance and autoimmunity. 3. Imaging tolerance and autoimmunity: We will use conventional immunohistochemistry and 2-photon confocal microscopy to define the migration of self-reactive T-cells in a self antigen-expressing host, in the presence and absence of endogenous lymphocytes. These studies will tell us if the anatomy of antigen recognition and competition with host lymphocytes influence the maintenance or failure of tolerance. This project will provide valuable information about why immune deficiencies are associated with autoimmunity, and may give insights into the general mechanisms of failure of tolerance and development of systemic autoimmunity.

The overall objective of this project is to define the cellular and molecular mechanisms of CD4 T-cell tolerance to a systemic self-antigen. The studies will test the hypotheses that cell-intrinsic tolerance in T-cells results from a combination of proximal signaling blocks and imbalanced cytokine production, and when the cell-intrinsic mechanisms of anergy and deletion fail, prolonged self-antigen recognition without other stimuli may lead to progressive development of regulatory T-cells. The studies will rely on a well-established transgenic model of systemic T-cell tolerance which has some unique strengths, notably that it is amenable to biochemical and molecular analyses of cells that have encountered self-antigen, and it is the only system in which effector and regulatory T-cells are generated sequentially from a monoclonal T-cell population in response to self-antigen recognition in peripheral tissues. The following specific aims will be addressed: 1. Mechanisms of cell-intrinsic tolerance (anergy and deletion) induced by a systemic self-antigen. These studies will define signaling blocks in T-cells rendered anergic by recognition of systemic self-antigen in vivo, using a novel multiplex phosphoprotein array and other techniques, and examine the roles of known T-cell regulators (CTLA-4, Fas, Bim) in systemic tolerance. In addition, the studies will explore the novel idea that imbalanced production of IFN-y without IL-2 contributes to T-cell tolerance, and define the mechanisms underlying this unexpected role of IFN-y as a tolerance-inducing cytokine. 2. Induction and functions of peripherally generated regulatory T-cells (Treg). Using a model of sequential development of effector T-cells and Treg in response to recognition of systemic antigen, these studies will define the lineage relationships between these two cell populations and the roles of cytokines in controlling the balance between effector and regulatory cells. A model of parent to F1 graft-vs-host reaction will be used to examine the development of effector and regulatory T-cells in situations of polyclonal T-cell reactivity. Thus, this project uses a defined experimental system and a variety of precise analytical methods to study fundamental mechanisms of peripheral T-cell tolerance, its induction and maintenance, and its regulation by external signals. There are numerous close interactions between this project and studies of CTLA-4 and Treg, signaling pathways, interactions of central and peripheral tolerance mechanisms. The results are not only of biological significance, but will also provide valuable leads for strategies to induce tolerance as a therapeutic modality.

DESCRIPTION (provided by applicant): The goal of this project is to explore the mechanisms underlying the well-recognized link between immune deficiencies and autoimmunity. We have developed a transgenic mouse model in which CD4 helper T-cells specific for ovalbumin (Ova) become tolerant (functionally unresponsive) when they encounter a secreted form of Ova (sOva) produced as a self antigen in lymphocyte-sufficient hosts. In striking contrast, if the same T-cells see the circulating Ova in a lymphopenic (RAG-/-) host, tolerance fails, resulting in a severe, systemic autoimmune reaction that is usually acutely lethal. Our hypothesis is that multiple classes of host (endogenous) lymphocytes help to maintain self-tolerance by several mechanisms, including: competition for antigen and other stimuli, antigen presentation under tolerogenic conditions, and production of regulatory cytokines. Using this model, we will address the following specific aims. 1. Regulatory functions of endogenous lymphocytes: We will determine which types of host lymphocytes function to maintain tolerance by two approaches. First, mice expressing the sOva will be depleted of selected lymphocytes (CD4 or CDS T cells, B cells, gamma-delta cells, NK-T cells). Ova-specific T-cells will be transferred into these hosts and followed for tolerance or autoimmune reactions. Second, lymphopenic hosts expressing the sOva will be reconstituted with different lymphocyte populations, and the responses of Ova-specific T cells will again be examined. 2. Mechanisms by which endogenous lymphocytes maintain tolerance: To determine how endogenous lymphocytes help to maintain self-tolerance and prevent the activation of self-reactive T-cells. We will first define the types of responses of these T-cells that correlate with tolerance vs pathologic autoimmunity. We will then inhibit or eliminate selected molecules (such as cytokines) from the self-reactive T-cells or the host, or selected non-lymphoid cell types from the host, to define how endogenous lymphocytes control the balance between tolerance and autoimmunity. 3. Imaging tolerance and autoimmunity: We will use conventional immunohistochemistry and 2-photon confocal microscopy to define the migration of self-reactive T-cells in a self antigen-expressing host, in the presence and absence of endogenous lymphocytes. These studies will tell us if the anatomy of antigen recognition and competition with host lymphocytes influence the maintenance or failure of tolerance. This project will provide valuable information about why immune deficiencies are associated with autoimmunity, and may give insights into the general mechanisms of failure of tolerance and development of systemic autoimmunity.

DESCRIPTION (provided by applicant): The objectives of this PPG are to understand what controls the choice between tolerance and autoimmunity. Using established experimental models, the component projects will explore the molecular pathways and genetic modifiers of central and peripheral T-cell tolerance to self-antigens. Project 1. Mechanisms of peripheral CD4 T-cell tolerance (PI: A. Abbas). The aims of this project are to define: 1. signaling abnormalities and the roles of known immune regulators and cytokines (IFN-y, IL-2) in T cell anergy and deletion induced by a transgene-encoded systemic secreted protein, and 2. the mechanisms that control the relative development of effector and regulatory T-cells specific for the systemic self-antigen. Project 2. CTLA-4 function in tolerance and autoimmunity (PI: J. Bluestone). This project will explore the hypothesis that the ligand-binding and ligand-independent forms of the inhibitory receptor CTLA-4 play distinct roles at different stages of T-cell responses. The specific aims are to study: 1. the molecular mechanisms of action of the two forms of CTLA-4, 2. the roles of the two forms of CTLA-4 in cell-intrinsic tolerance, and 3. their roles in preventing lymphoproliferation and maintaining homeostasis. Project 3. Regulation of CD45 signaling in tolerance and autoimmunity (PI: A. Weiss). This project will focus on CD45 as a paradigm for the role of tyrosine phosphatase signaling in maintaining self-tolerance. The specific aims are to study: 1. the influence of an inactivating CD45 wedge mutation on T- and B-cell tolerance, 2. the roles of myeloid cell abnormalities caused by the mutation, 3. genetic modifiers of the autoimmune phenotype, and 4. molecular basis of the regulatory function of CD45, specifically the wedge. Project 4. Control of autoimmunity by central tolerance (PI: M. Anderson). Building on the seminal discovery of the role of the AIRE protein in thymic negative selection, this project will explore: 1. genetic modifiers of autoimmunity caused by AIRE deficiency, 2. interactions between central tolerance and peripheral regulatory mechanisms, and 3. AIRE-mediated self-antigen expression as a therapeutic strategy for autoimmunity. Cores. Core A (animals) will create new genetically modified mouse strains, maintain strains shared by all projects, and assist with breeding and genotyping for genetic studies. Core B (flow cytometry) will provide state-of-the-art multi-parameter analyses and high-speed cell sorting for all projects. Thus, this PPG brings together four highly complementary, interactive and productive investigators to address fundamental questions about the biology and mechanisms of T-cell tolerance and autoimmunity. The proposed studies will lay the foundation for developing new immunological therapies for immune mediated inflammatory diseases. PROJECT 1: Mechanisms of peripheral CD4 T-cell tolerance (Abbas, A.) PROJECT 1 DESCRIPTION (provided by applicant): The overall objective of this project is to define the cellular and molecular mechanisms of CD4 T-cell tolerance to a systemic self-antigen. The studies will test the hypotheses that cell-intrinsic tolerance in T-cells results from a combination of proximal signaling blocks and imbalanced cytokine production, and when the cell-intrinsic mechanisms of anergy and deletion fail, prolonged self-antigen recognition without other stimuli may lead to progressive development of regulatory T-cells. The studies will rely on a well-established transgenic model of systemic T-cell tolerance which has some unique strengths, notably that it is amenable to biochemical and molecular analyses of cells that have encountered self-antigen, and it is the only system in which effector and regulatory T-cells are generated sequentially from a monoclonal T-cell population in response to self-antigen recognition in peripheral tissues. The following specific aims will be addressed: 1. Mechanisms of cell-intrinsic tolerance (anergy and deletion) induced by a systemic self-antigen. These studies will define signaling blocks in T-cells rendered anergic by recognition of systemic self-antigen in vivo, using a novel multiplex phosphoprotein array and other techniques, and examine the roles of known T-cell regulators (CTLA-4, Fas, Bim) in systemic tolerance. In addition, the studies will explore the novel idea that imbalanced production of IFN-y without IL-2 contributes to T-cell tolerance, and define the mechanisms underlying this unexpected role of IFN-y as a tolerance-inducing cytokine. 2. Induction and functions of peripherally generated regulatory T-cells (Treg). Using a model of sequential development of effector T-cells and Treg in response to recognition of systemic antigen, these studies will define the lineage relationships between these two cell populations and the roles of cytokines in controlling the balance between effector and regulatory cells. A model of parent to F1 graft-vs-host reaction will be used to examine the development of effector and regulatory T-cells in situations of polyclonal T-cell reactivity. Thus, this project uses a defined experimental system and a variety of precise analytical methods to study fundamental mechanisms of peripheral T-cell tolerance, its induction and maintenance, and its regulation by external signals. There are numerous close interactions between this project and project 2 (studies of CTLA-4 and Treg), project 3 (signaling pathways), and project 4 (interactions of central and peripheral tolerance mechanisms). The results are not only of biological significance, but will also provide valuable leads for strategies to induce tolerance as a therapeutic modality.

This application is being submitted on behalf of the Organizing Committee of the International[unreadable] Congress of Immunology (ICI) to request partial support for the 13th Congress, in August 2007.[unreadable] The 13th International Congress of Immunology (www.immunorio2007.org.br) in Rio de Janeiro,[unreadable] Brazil, sponsored by the International Union of Immunological Societies (IUIS), Latin American[unreadable] Association of Immunology (ALAI) and Brazilian Society of Immunology (SBI), is being planned[unreadable] to attain the highest scientific level, with the participation of the most renowned immunologists[unreadable] from all over the world, as a unique scientific learning and networking opportunity for basic and[unreadable] clinical researchers, physicians, and academics involved in multi-disciplinary immunology. This[unreadable] is the first time the ICI is being held in Latin Amrica, and the Organizing Committee feels[unreadable] strongly that it must provide a realistic overview of problems related to immunology issues in[unreadable] developing countries. Equally importantly, this is a ?golden age? of Immunology, driven by the[unreadable] development of novel therapies and the application of new technologies to the study of disease.[unreadable] In light of these developments, the scientific platform of the Congress has been created as a[unreadable] highly interactive forum to present and debate new ideas, treatments, and cutting edge research[unreadable] within immunology and related subjects. It is also a unique opportunity to identify new strategies[unreadable] and solutions in major areas such as Autoimmmunity, Allergy, Cancer and Transplantation, and[unreadable] Immunotherapy/Vaccination, as well as a wide range of topics in basic Immunology. We are[unreadable] also committed to providing equitable access and the chance to participate in the 13th[unreadable] International Congress of Immunology to students and young researchers from developing[unreadable] countries and different continents such as Southeast Asia, Africa, and Latin America. The[unreadable] support from this grant will be invaluable for providing travel opportunities for young scientists[unreadable] from the entire world, who often do not have access to funds for foreign conferences.

DESCRIPTION (provided by applicant): Interleukin-2 (IL-2) is the prototypic T-cell growth factor but elimination of IL-2 results in severe immune dysregulation and systemic autoimmunity because the obligatory function of this cytokine is to maintain CD4+ CD25+ FoxP3+ regulatory T-lymphocytes (Treg). Defining the role of IL-2 in the maintenance of Treg, and how effector and memory cells are generated in the absence of IL-2, is fundamental for understanding the biology of this cytokine and the pathogenesis of diseases caused by its absence (and more generally, by the absence of Treg), and also for developing optimal strategies for using this cytokine and its antagonists as therapeutic immune response modifiers. We will address these issues along the following specific aims. 1. What mechanisms underlie the obligatory role of IL-2 in the maintenance of functional Treg? To test the hypothesis that the obligatory functions of IL-2 are to prevent apoptotic death of Treg and to promote expression of genes involved in Treg function, we will define the phenotypic and functional characterisitics of Treg that develop in the periphery and the thymus in the absence of IL-2 signals. We will use the genetic approach of deleting Bim, the sensor of cytokine deprivation, to generate viable Treg in the absence of IL-2. We will define the functions of these cells in vitro and in vivo, and analyze the biochemical pathways that are defective in these cells compared to FoxP3+ cells that develop in the presence of IL-2. Using retroviral gene transduction, we will examine the biochemical signals that link IL-2 to Treg survival and function, particularly Akt and Stat5. We will ask if different amounts and duration of IL-2 signal differentially affect effector/memory cells vs Treg, in order to optimize strategies for therapeutic use of IL-2 and its antagonists. 2. What is the role of IL-2 in effector and memory responses, and how do these responses develop in the absence of IL-2? To test the hypothesis that IL-2 functions to provide activated T-cells the capacity to overcome Treg control, and responses become IL-2-independent in the absence of Treg, we will compare the activation of normal and CD25-deficient T-cells in normal recipients and in IL-2/CD25xCD28 double-knockout Treg-deficient mice we have developed that have a full lymphoid system. We will also define the biochemical correlates of T-cell activation in the presence and absence of Treg and IL-2. We will examine the early events after T-cell recognition of antigen in the absence of Treg, using in vivo imaging methods. 3. What is the basis of the immunological disease associated with IL-2 deficiency? The autoimmune disease of IL-2-knockout BALB/c mice is characterized by rapid-onset hemolytic anemia and lymphoproliferation, and is associated with markedly enhanced IFN-g production (Th1 responses) and activation of dendritic cells (DCs). We will define the mechanism of the enhanced T-cell cytokine response and its contribution to the autoimmune disease, and explore the possibility that DCs are spontaneously activated b5cause of the absence of endogenous Treg. Thus, this project addresses fundamental issues of the functions of IL-2 and how its dual role in activating effector/memory responses and Treg is orchestrated. These studies will shed light on the mechanisms of immune regulation and tolerance, and may help to optimize protocols for the therapeutic uses of IL-2 and its antagonists.7. PROJECT NARRATIVE: Interleukin-2 (IL-2) is a T-cell growth factor that has been used therapeutically to boost immune responses, and IL-2 antagonists have been used as immunosuppressive agents to prevent rejection of allografts and treat graft-vs-host disease. Recent results have shown that IL-2 has a dual biological role it stimulates immune responses by promoting the development of effector and memory cells, and it limits the same responses by virtue of its essential role in the maintenance of functional regulatory T cells (Treg). If we are to use IL-2 or its antagonists as therapeutic agents, and understand its role in immune regulation, we must elucidate how its dual functions are orchestrated. This project uses transgenic and gene knockout mouse models to define why IL-2 is required for Treg survival and function, and how effector and memory responses can be induced in its absence. We will also analyze the mechanisms of the autoimmune disease caused by the absence of IL-2, and determine if different amounts and duration of IL-2 administration can be used to differentially activate effector cells and Treg.

The objective of this project is to define the plasticity and stability of regulatory T cells (Tregs) and the possibility of converting effector cells to functional Tregs, as a prelude to cellular therapy. Our hypothesis is that activated T cells go through a phase of transient Foxp3 expression that is a critical decision point, and stable FoxpS expression is a key determinant of Treg stability. Our approach is to exploit mouse models to address these questions in vivo and in vitro and to develop predictive phenotypic and molecular markers for Treg stability, and use this information to define the stability of human Tregs and to genetically manipulate human T cells in order to program their differentiation pathway. The following Specific Aims are proposed. 1. Functional and phenotypic plasticity of regulatory T cells (Tregs) and effector T cells (TefO. In this Aim, we will generate antigen-specific natural (thymic) and adaptive (peripheral) Tregs using TCR transgenic mice expressing fluorescent reporters for FoxpS and key cytokines (IFN-v, IL-17 and IL-10) and different forms of antigen exposure. Purified Tregs will be transferred into normal recipients and exposed to antigen with and without inflammatory stimuli, or transferred into recipients with active autoimmune reactions. The cells will be followed for FoxpS and cytokine expression and surface phenotype by staining, and unctional responses after sorting. Tregs will be isolated from healthy volunteers and patients with autoimmune disease (initially T1D) and followed in vitro for conversion to effectors in the presence of activating and inflammatory stimuli. A novel transgenic mouse strain that expresses a lineage marker of past roxp3 expression will be used to examine the fates of these cells. The conversion of effector cells into Tregs be examined using the same approaches. 2.'Biochemical basis of Treg stability and conversion. T cells will be examined under conditions of stability and conversion for changes in surface phenotype, expression of Treg-specific genes, and methylation of the :oxp3 locus. The functional consequence of transient FoxpS expression will be analyzed in mouse and human cells using shRNA knockdowns and knockout mice. Attempts will be made to maintain the stability of regs by expressing selected genes in human Tregs.

Project Summary/Abstract FOCIS 2009 - FOCIS 2013 Understanding function and regulation of the immune system is critical for many diseases, including autoimmune diseases, cancer, allergy/asthma, infectious diseases, immune deficiency, and transplant rejection. To rapidly exploit scientific advances in this area, two major barriers need to be overcome: First, the translation of basic findings into clinically and medically relevant contexts, and second, the exchange of relevant findings in immunological sciences which unify these diverse diseases through multidisciplinary approaches which cut across traditional clinical boundaries. FOCIS, the Federation of Clinical Immunology Societies, is the leading international organization which exists to meet these needs. Linking more than 20 major professional societies representing 40,000 professionals with this common vision is a service that will ensure progress towards the goal of improving human health through immunology. The FOCIS Annual Meeting provides a scientific forum that fosters a cross-disciplinary approach required to understand and treat immune-based diseases. The evolution of the immunology field is a reflection of the shared pathophysiology of many diseases, including: autoimmune diseases, cancer, allergy/asthma, infectious diseases, immune deficiency and transplant rejection. While clinicians necessarily remain linked to diseases associated with their own specialty, the immunological sciences underlying recent advances in diagnosing and treating these diverse diseases are multidisciplinary and cut across standard clinical boundaries. The ultimate goal of the FOCIS annual meeting is to create a better understanding of the shared pathophysiological underpinnings of clinical immunology and the new therapeutic approaches suggested by these novel relationships, including the increasingly widespread use of biologics in therapy. The FOCIS Annual Meeting offers investigators and clinicians an opportunity to understand and translate breakthroughs in basic science and clinical applications from a variety of different fields. In addition to highlighting the best science, the FOCIS Annual Meeting is an incubator for developing scientists and practitioners alike to meet with one another along with representatives of relevant biotechnology and pharmaceutical industry. Each day includes poster and oral abstract presentations to give young investigators the chance to have their work presented and critiqued. FOCIS takes a specific interest in the development of young investigators and provide travel awards to them to supplement their costs to attend the meeting. By sharing discoveries with one another, delegates acquire new ideas for research and applied science from disciplines to which they would otherwise not be exposed at disease- or organ-specific meetings. The FOCIS Annual Meeting is an ideal forum for new investigators to learn about the different translational approaches that are bridging the basic immunology laboratories within clinical practice. In addition to encouraging the participation of new investigators, FOCIS recognizes the importance of participation from a wide range of demographics. Every effort is made to include a diverse representation within the FOCIS Organizing Committee and the Annual Meeting's scientific program. The success of all eight of the previous FOCIS meetings attests to this effort. Support is now requested to support the 2009- 2013 FOCIS meetings, specifically the educational programs and travel fellowships, which will continue this valuable effort.