Jeffrey A. Bluestone - US grants

CD8+ cytotoxic cells and CD4+ helper cells expressing an antigen receptor composed of the TcR alpha and beta chains constitute the major T cell subsets. Recently, we have used a monoclonal anti-murine CD3 antibody derived in our laboratory to identify a new T cell subset in the fetal and adult thymus of mice. These CD3+, CD8-, CD4- cells appear early in thymic ontogeny, are maintained in the mature thymus throughout adult life, and express a novel TcR composed of a CD3 complex associated with the TcR gamma gene product disulfide-linked to a fourth glycoprotein, termed TcR delta. Although similar TcR gamma delta/CD3-bearing T cells have been identified in humans, the physiological role of the TcR gamma delta/CD3 T cells is at present unknown. The major objective of this study is to determine the physiologic role of this subset of T cells. The repertoire of TcR alpha beta T cells is clearly skewed toward recognition of major histocompatibility complex (MHC) antigens. In contrast, initial studies of TcR gamma delta cells isolated from both mice and humans suggested that these cells functioned in a non-MHC- restricted fashion. In recent studies, we have generated an MHC- specific cytotoxic TcR gamma delta/CD3 cell line from athymic nu/nu mice. This TcR gamma delta-expressing CTL clearly recognizes a beta2-microglobulin-dependent class I molecule. Additional MHC-specific TcR gamma delta T cells from CD3+, CD8-, CD4- peripheral T cells isolated from athymic nu/nu and euthymic mice will be generated and their repertoires compared. Attempts will be made to generate additional antigen-specific TcR gamma delta T cells, identify the ligand for these cells and perform detailed analyses of the TcR gamma delta structure expressed on peripheral CD8-, CD4- T cells. The activation requirements of purified CD3+, CD8-, CD4- peripheral T cells will be studied using anti-TcR- and anti-non-TcR- specific monoclonal antibodies (mAb) to induce proliferation, lymphokine production, and cytolytic activity. The TcR gamma and TcR delta proteins as well as a novel TcR complex expressed on peripheral CD8-, CD4-T cells will be characterized in an attempt to determine the degree of heterogeneity of T cells expressing non-TcR alpha beta proteins. Finally, we will produce mAb against antigen-specific TcR gamma delta-bearing cells as a means of studying the TcR complex and for use as immunomodulating reagents in vivo.

Monoclonal antibody (mAb) treatment of organ transplant recipients has been a major development in the treatment of allograft rejection. The advantages of mAbs include their known specificity, consistent biological activity, and ease of administration. Of all mAbs clinically and experimentally tested in man, the most efficacious is the OKT3 mAb which recognizes the T cells receptor (TcR) complex on the surface of alloreactive cells. However, several limitations to the use of the OKT3 reagent persists. These include the significant adverse reactions following the initial dose, production by the recipient of antibodies to the OKT3 antibody, and the broad transient nature of the induced immunosuppression. Our laboratory has recently developed the first anti-murine CD3 mAb that reacts with all mouse T cells expressing a TcR complex. Initial studies in the laboratory suggest that treatment with the mAb in vivo has profound long term immunosuppressive effects including prolonged skin graft survival and depressed cellular immunity. It appears that the initial immunosuppression is a consequence of T cell depletion and receptor blockade. In addition, T cells that survive treatment may continue to be hyporesponsive. Thus, this hamster mAb provides the first anti- CD3 that can be used in a well-defined small animal model to provide a systematic evaluation of the use of anti-CD3 in vivo to suppress transplantation responses. Anti-CD3 antibodies not only function to suppress immune responses but can function as an antigen-non-specific mitogen capable of activating a variety of T cell functions. Thus, a major potential; of anti-CD3 treatment in vivo may lie in its ability to facilitate ongoing antigen-specific immune response, enhance bone marrow engraftment and augment immune potential of immunocompromised individuals. In this regard, preliminary findings suggest that in vivo treatment with low doses of anti-murine CD3 mAb results in a substantial activation of T cells including IL-2 receptor expression, secretion of lymphokines such as colony stimulating factor (CSF) and extramedullary hematopoiesis. Thus, these activating in vivo effects, previously not recognized in human studies, may provide a model system for analyzing the immunopotentiating effects of anti-CD3 mAb in vivo.

OKT3 is a murine monoclonal antibody directed against the CD3epsilon chain of the T cell receptor complex on human T-cells. OKT3 has been widely used over the past decade in transplantation to treat corticosteroid- resistant organ graft rejections, and more recently, as prophylaxis of rejection. This mAb induces an efficient and rapid immunosuppression, at least in part due to, the prevention of allorecognition as a result of modulation of the T cell receptor (TCR) from the surface of T lymphocytes and clearance of T cells from the circulation. However, there are several clinical and scientific issues that remain unresolved regarding anti-CD3 therapy. First, a variety of side effects are associated with OKT3 that limits its usefulness in other diseases such as autoimmunity and bone marrow transplantation. These limitations are, in large part, a result of the first dose toxicity as a consequence of T cell activation and the subsequent massive cytokine release. In addition, the therapy has been limited by the generation of a potent humoral antibody response against the murine mAb limiting the potential for retreatment. During the past 4 years, Dr. Bluestone's laboratory has systematically evaluated the use of anti-CD3 to suppress transplantation responses in vivo first in a well- defined, small animal model and then in the hu-SPL-SCID model in collaboration with Dr. Thistlethwaite. The toxic effects of the mAb were defined and largely eliminated, immunosuppressive agents such as deoxyspergualin and CTLA4Ig were used in conjunction with anti-CD3 to potentiate immunosuppression and block the humoral response, and genetically-engineered monoclonal anti-CD3 antibodies were developed to examine the role of T cell activation and Co-stimulation in immune suppression. However, many questions remain including: the relative efficacy of "activating" and "non-activating" anti-CD3 mAbs; the signal transducing ability of "non-activating" anti-CD3 mAbs and how it regulates T cell activation; the role of co-stimulatory in the activating and toleragenic effects of anti-CD3; and the role of other inhibitors of T cell signalling, such as cyclosporin A in the efficacy of anti-CD3 therapy. In order to answer these questions the following specific aims are proposed: 1. To study the in vivo effects of non-mitogenic anti-CD3 mAbs alone and in conjunction with donor antigen; 2. To determine the role of CD28/B7 interactions in anti-CD3-mediated immunosuppression; 3. To determine the molecular mechanism(s) controlling apoptosis and anergy in mice treated with mitogenic anti-CD3. The results of these studies will provide important insights into the effects of anti-CD3 and the CD28/B7 family of cell surface molecules in the regulation of transplant rejection that may translate into clinical application.

The overall goals of this Transplantation Program Project are to dissect the mechanisms which lead to allograft rejection and to facilitate the implementation of scientific insights gained in the laboratory as treatment modalities in the clinic. To attain these general goals, four complementary projects have been proposed: 1. A hamster mAb anti-murine CD3 mAb will be used to evaluate systematically the use of anti-CD3 to suppress transplantation responses in vivo in a well-defined, small animal model. The anti-CD3 and combined mAbs therapies will be exploited to more efficiently suppress transplantation responses in vivo. Other T cell subset-specific mAbs and antigens including staphylococcal toxins will be examined for their ability to selectively suppress T cell subsets which may play a predominant role in graft rejection. These studies will hopefully lead to the development of more sophisticated immunosuppressive regimens that selectively alters immunity towards the organ transplant. 2. The kinds of T cells involved in rejection of allografts which differ from the host in specific, defined regions of the MHC complex as well as the mechanisms by which these T cells cause graft rejection will be defined. Alloreactive T cells from unprimed spleen and lymph nodes, from the lymph nodes draining the site of a rejecting skin allograft, and from rejecting skin allografts will be characterized in terms of the array of lymphokines produced, the cell surface molecules expressed, and the specific epitopes with which these clones react. 3. The human/SCID mouse model will be developed to study the human cell interaction involved in graft rejection. This model will aid in the analyses of novel therapeutic approaches to overcome rejection of human allografts. 4. New modalities in man based on the basic research performed in the small animal and human/SCID mouse model systems will be directly analyzed at the humoral, cellular and molecular levels. Together, the four projects should provide information as to the mechanism of graft rejection and should help to develop new therapeutic modalities that will be directly implemented in the clinic.

Activation of immune responses against viral infections or tumors depends on a complex interaction of T calls, foreign antigen and antigen-presenting calls. In some cases, the immune response is suboptimal, therefore, efforts towards developing an ideal form of immunotherapy have focussed on enhancing one or more of these immune components. The approach to immunotherapy presented in this study differs from those previously examined in that an attempt will be made to enhance the immune response by augmenting the native ability of T cells to respond to antigen through T cell receptor (TCR)-CD3 complex without invoking alternate efferent or afferent immune mechanisms. It is known that monoclonal antibodies (mAb) recognizing certain T call surface molecules (i.e. TCR, CD3, CD2, etc.) can activate T cells in the absence of nominal antigen. The mAb mimics physiologic antigen and bypasses the antigen-specific recognition mechanism. Our laboratory has developed a monoclonal anti-murine CD3 antibody that reacts with all murine mouse T cells expressing a TCR complex. Although the anti-CD3 mAb functions to suppress immune responses to organ graft recipients as a consequence of T cell depletion and receptor blockade, our studies suggest that the monoclonal antibody can function in vivo as an antigen-independent mitogen capable of activating a variety of T cell functions. In this regard, we have demonstrated that in vivo treatment with low doses of anti-CD3 mAb results in a substantial activation of T cells including IL-2 receptor expression, secretion of lymphokines such as colony stimulating factors, IL-2, and interferon-gamma and rejection of malignant progressor tumors. The proposed studies are designed to: 1) Better understand the mechanism of activation of T calls by mAbs; 2) Develop insights into the mechanism by which anti-CD3 antibodies administered In vivo alter the immune response; 3) Evaluate the role of accessory cells in vivo using heterobifuncional reagents to specifically target the anti-CD3 antibodies to T cells; 4) Exploit the activation properties of anti-CD3 as well as other T cell reagents to modulate immune responses against tumors and viral infections; and 5) Utilize anti-CD3-mediated induction of lymphokines in promoting hematopoiesis.

The overall goal of the UCCRC program in Immunology and Cancer is to dissect the multiple closely interrelated factors that determine immune responses to conventional and tumor antigens. This program is closely related to the Committee on Immunology which has been highly successful in developing interactive research and educational efforts to enhance the scientific endeavors of the UCCRC and the University. One example of the interactive nature of this program is the ongoing USPHS-NCI Program Project: "Immunity and Cancer" H. Schreiber, Program Director. The overall goals of the UCCRC Program in Immunology and Cancer are: (1) to enhance the scientific interactions among the various immunology researchers interested in cancer; (2) to bring together individuals interested in the most basic elements of the immune response to coordinate efforts towards a better understanding of tumor immunology; (3) to develop organized educational programs for predoctoral and postdoctoral students in order to foster increased interest in cancer research and (4) to manipulate the immune system in the treatment of cancer. Ultimately, these activities will lead to developing preclinical models to test novel immunotherapies for cancer and clinical studies through collaborative efforts with the Developmental Therapeutics Program of the UCCRC.

germ free condition; biomedical facility; laboratory mouse; animal colony;

animal breeding; biomedical facility; genetic strain; genetically modified animals; animal colony; animal care; genetic models; laboratory mouse;

Initiation and maintenance of peripheral T cell tolerance relies on a complex set of events dependent on the responding T cell subpopulation, the antigen and the antigen presenting cells (APC), expression of co- stimulatory molecules and cytokines, and the genes expressed during the early signalling cascade following exposure to antigen. The overall goals of this Tolerance Program Project are to dissect the mechanisms which lead to peripheral T cell tolerance by examining the molecular, biochemical, and immunobiological consequences of T cell interactions with antigen presenting cells. To achieve these general goals, four complementary projects will be undertaken: 1. To study the early molecular events following T cell activation to determine the genetic elements that control the functional outcome of T cell receptor (TCR)/ligation. The molecular basis of T cell tolerance will be studied by analyzing the transcriptional regulation of the IL-2 gene following T cell activation and tolerance induction. In addition, novel genes, including a newly discovered bcl-2-like gene (bcl-x), will be studied for its role in T cell inactivation and antigen-induced cell death (apoptosis). 2. To dissect the role of cell surface adhesion molecules in regulating T cell tolerance. Functional activation of T cells depends not only on the interaction of the TCR with its ligand but, in addition, the signals transduced by so called co-stimulatory molecules present on T cells that bind to ligands expressed on APC. The role of one such co- stimulatory molecule/ligand interaction, CD28/B7, in the induction of peripheral anergy will be examined both in vitro and in vivo using a combination of mAbs, soluble receptors, and genetically-deficient mice. Other cell surface molecules such as CTLA4, HSA, and ICAM-1 will also be studied to determine their role in peripheral tolerance induction and maintenance. These studies will include analyses of both clonal, naive, and in vivo-derived T cells. 3. To determine the mechanism of T cell tolerance induction in distinct T cell subsets using different APC. A variety of T cell subsets exist. Activation of these individual subsets are dictated by the nature of the antigens recognized, characteristics of the APC, and cytokine milieu. Only the Th1 of T cells have been studied, in depth, with regard to anergy induction. Other T cell subsets including: Th2 cells; CD8+ IL-2 producing cells; and CD8+ IL-4 producing cells will be examined for tolerance induction using T cell clones derived against selected nominal antigens, and subsets will be compared for differences in expression of genetic and biochemical components that regulate T cell activation. 4. The in vivo basis for peripheral T cell tolerance will be examined using transplantation and transgenic mouse models. Individual antigen-presenting cells derived from epithelial tissue and pancreatic islets will be manipulated using gene therapy and transgenic mouse technology in order to express selective co-stimulatory molecules in these cell types. These animals will be examined in vivo for the initiation and maintenance of tolerance. T cells derived from these animals will be examined molecularly and biochemically to compare in vivo tolerized T cells inactivated in vitro. Members of this Program Project grant bring together in a unique set of reagents, model systems, and insights into the mechanisms of T cell anergy and tolerance. Together, these projects should provide information on the mechanism of peripheral T cell tolerance and help to develop new therapeutic modalities treating autoimmunity and graft rejection.

More than half a decade after their identification, the biological role of TCRgammadelta cells remains uncertain. While TCRgammadelta cells are clearly dominant in certain pathologic conditions, their lack of significant involvement in most classical T cell responses suggests that they perform a unique and special role in host immune defense. In fact, TCRgammadelta cells have several unique features that distinguish them from TCRalphabeta cells including: (a) preferential expression in epithelial tissue; (b) a broad repertoire skewed towards non-classical major histocompatibility complex (MHC) class I antigens, heat shock proteins, and non-processes antigens; and (c) substantial expansion during a variety of inflammatory diseases thus constituting a major arm of the immune system. The major objective of the previous grant was to dissect and functionally characterize this unique T cell subset. During this time, we identified TCRgammadelta cells showing that TCRgammadelta cells can be MHC-specific (classical class I-, class II-, and TL-specific) and nominal antigen- specific. The antigen-specific TCRgammadelta cells were used: 1) To demonstrate that the TCRgammadelta repertoire is diverse; 2) To create TCRgammadelta transgenic mice that establish positive and negative selection in TCRgammadelta development; 3) To produce multiple antisera and mABs that have been widely used to characterize TCRgammadelta subsets and examine the functional potential (i.e. lymphokine production and cytolytic activity) of this novel T cell population in normal and transgenic mice. During the next grant period, we will use the mABs, T cell clones and transgenic mice to define TCRgammadelta cell activation and intracellular signal transduction pathways, the physiologic processes that affect positive and negative selection, and the genetic elements that control antigen recognition by this eclectic population. Specifically, we will: 1) Study the precise antigen specificity and physiologic role of herpesvirus-specific and TL-specific TCRgammadelta cells. We will examine the role of antigen processing and the requirement for peptide- presentation in the specificity of these T cells; 2) Examine the biochemical and molecular events associated with TCRgammadelta maturation and selection. We will examine directly the TCR complex and early signal transduction pathways in developing TCRgammadelta cells and during antigen-driven activation; and 3) Determine the genetic elements that control TCRgammadelta repertoire development. We will identify the genes and antigens that control strain-specific repertoire differences; These studies will help to define the role of TCRgammadelta cells in immune responses.

Clonal deletion in the thymus of potentially autoreactive T cells remains the major mechanism of maintaining tolerance in the central lymphoid system. Since, under certain circumstances (autoimmunity, extrathymic T cell development and transplantation), the first T cell engagement with antigen occurs in the periphery, distinct mechanisms must be either inherent or introduced therapeutically to prevent T cell reactivity. The major goals of this proposal are: to study the T cell response to antigens expressed in the peripheral tissue; to manipulate the T cell/antigen-presenting cell interaction to promote T cell tolerance; to determine the underlying basis for T cell unresponsiveness in vivo; and to use gene therapy and transgenic mouse technology to manipulate T cell responses in vivo. In order to accomplish these goals, we will: 1) Study the role of co-stimulatory molecules in the induction of T cell tolerance following transplantation. These studies will use a number of unique reagents that regulate co-stimulatory molecules including: mAbs (anti- murine CTLA-4, anti-CD28, anti-B7 and anti-ICAM-1); CTLA4lg; CD28- deficient and CTLA-4-deficient mice; and B7/Islet beta cell and CTLA4lg/keratinocyte transgenic mice. 2) To develop novel gene therapy approaches to introduce inhibitors of co-stimulation directly into transplanted tissue as a means of blocking transplant recognition. In these studies, adenovirus-medicated gene transfer will be used to introduce the genes for co-stimulation antagonists, such as CTLA4lg, directly into the pancreatic islets. In addition, a transgenic mouse expressing CTLA4lg in keratinocytes will be used in transplantation experiments to study the effect of local immunosuppression on proximally and distally transplanted tissue. 3) To study peripheral T cell tolerance of intraepithelial lymphocytes (IELs) in T cell receptor transgenic mice exposed to nominal antigen expressed on "non- professional" antigen presenting cells. Although potentially autoreactive T cells are effectively deleted from the lymphoid tissue, T cells that develop extrathymically, such as IELs, are present, express normal levels of the TCR, but are functionally inactivated. However, following continuous antigen exposure, these cells undergo ligand-induced cell death (apoptosis) not unlike what is observed in the thymus. The present studies will examine the nature of the biochemical signalling defect(s) in these cells and determine whether the induced unresponsiveness is a result of similar genetic changes [i.e., induction of cell death genes [bcl-x] or transcription factors] as has been observed in vitro. In addition, we will use the CD28-deficient and CTLA4lg transgenic mice to determine the role of CD28/B7 in this peripheral tolerance model. Finally, we will use the adenovirus to introduce co-stimulatory molecules in an attempt to reverse tolerance as a model for autoimmune inflammatory bowel disease. Together these studies will provide insights into the basis of peripheral T cell tolerance in vivo and provide novel therapeutic approaches to tolerance induction.

The overall theme of this Program Project is to understand the mechanisms which regulate peripheral tolerance and to facilitate the implementation of scientific advances gained using in vitro model systems to the establishment of peripheral tolerance in vivo. Initiation and maintenance of peripheral T cell tolerance relies on a complex set of events dependent on the responding T cell subpopulation, the antigen and the antigen-presenting cells (APC), expression of co- stimulatory molecules and cytokines, and the genes expressed during the early signalling cascade following exposure to antigen. The overall goals are to dissect the mechanisms which lead to peripheral T cell tolerance by examining the molecular, biochemical, and immunobiological consequences of T cell interactions with antigen presenting cells. To achieve these general goals, three complementary projects will be undertaken: 1. To understand the interactions between receptor-mediated signal transduction pathways that regulate the functions of different T lymphocyte subsets. The Pis will further characterize the signaling block in anergic T cells to produce IL-2. They also will characterize the CD28-mediated signaling events that promote augmented cytokine gene expression and that prevent energy induction, focussing on serine/threonine kinase intermediates that may lead to activation of Jnk-1/Jnk-2. They will identify additional costimulatory molecules that regulate the growth of Th2 cells. 2. To define the molecular basis of for the activity of co-stimulatory molecules. The PI will continue to define the molecular basis of the opposing effects of CTLA-4 and CD28 on the immune response in collaboration with the other Program Project investigators. In addition, Dr. Thompson will study the role CD30, 4-1-BB, and OX40 as co-stimulatory molecules, emphasizing the role of the TRAF family of molecules in mediating their effects. He also will continue his studies of the role of co-stimulation in preventing apoptosis through the bcl-xl and Fas pathways. 3. To determine the mechanisms of tolerance induction and the individual roles of CD28/CTLA-4 and B7- 1/B7-2. The PI will determine the individual roles of B7-1 and B7-2 on donor and host tissues during the initiation and progression of transplant rejection. He also will further characterize the biological effects of CTLA4 engagement and blockade on exposure to antigen in vitro and in vivo, determining the importance of the stage of the immune response, the tissue transplanted, and the APC targeted. These projects should provide information on the mechanism of peripheral T cell tolerance and help develop new therapeutic approaches for autoimmunity and graft rejection.

Description (provided by applicant): The Flow Cytometry Core will meet the special needs of the Program Project members. The availability of quality sorting and analysis capabilities is essential for the success of the research efforts proposed in this Program Project Grant. All the program members will be active users of both the MoFlo cell sorting and LSR fluorescence analyzer in the studies dealing from: sorting of small numbers of cells based on either cell surface or transduced gene markers; and multiparameter analyses to detect biochemical and biological changes in rare populations of lymphocytes as well as whole populations transduced with various genes. In many cases, the studies proposed in the individual programs will require triple or even quadruple staining combined with state of the art cell sorting for cell surface and/or intracellular proteins. Thus, the FIRST SPECIFIC AIM of this Core is to provide access to a newly purchased high-speed cell sorter, MoFlo. The SECOND SPECIFIC AIM of this core is to provide access to the flow analyzers including the multiparameter LSR.

Human asthma is characterized by infiltration of eosinophils and other inflammatory cells into the airways. The pathological events that lead to eosinophilic airway inflammation are a direct result of activation of T lymphocytes. It is therefore important to understand the mechanisms of T cell activation in asthma, how external interventions might alter T cell activation, and what consequences on airway inflammation and function might follow. Activation of T cells is known to require two stimulatory signals. The first is provided by the interaction of antigen receptor with its appropriate antigen in the context of self MHC molecules. The second, non-cognate interaction, involves interaction between ligands on the T cell surface and on antigen presenting cells (called "T cell co- stimulation"). We have found that this non-cognate interaction between the CD28 family of molecules on T cells and B7 molecules on antigen presenting cells and other cells is involved in the pathogenesis of eosinophilic airway inflammation and hyperresponsiveness in S. mansoni soluble egg antigen (SEA) sensitized and challenged mice. Treatment with a soluble CD28/B7 interaction antagonist, CTLA4Ig, at the time of antigen challenge can suppress airway eosinophilia. The overall goal of this study is to elucidate the role of CD28/B7 co-stimulatory interactions in the pathophysiology of allergic asthma. In Specific Aim #1, we evaluate the individual roles of CD28, B7-1, B7-2, and CTLA4 in SEA-induced airway inflammation and hyperresponsiveness by inhibiting molecular function through administration of soluble antagonists or blocking monoclonal antibodies in vivo, in normal or genetically engineered "knockout" mice that lack individual molecules along the co-stimulatory pathway. To assess the influence of these interventions, we will measure airway constrictor responsiveness, assess airway inflammation histologically, quantify and characterize BAL fluid cells, and examine the proliferative and cytokine secretory responses of T lymphocytes from these animals after SEA or control challenges. Adoptive transfer experiments will further clarify the molecular mechanisms of the proposed interventions. In Specific Aim #2, we will use immunohistochemical analyses to determine the spatial and temporal evolution of T cell activation in relation to the sensitization/challenge process, and in relation to the development of airway inflammation and constrictor hyperresponsiveness. In Specific Aim #3, we will test whether modulating T-cell activation or secretion by local overexpression of B7-1, B7-2, CTLA4Ig, IFNg, or IL-4 within airways will prevent or enhance airway inflammation and hyperreactivity in S. mansoni sensitized and challenged mice in a fashion predicted by the results of Specific Aims #1 and #2. We will use replication-deficient adenoviral vectors encoding these immunoregulatory molecules to effect gene transfer and overexpression within airway epithelium. Together, these studies should elucidate the role of T cell costimulation in allergic airway inflammation and hyperresponsiveness. The knowledge gained will suggest potential new therapeutic interventions for asthma.

CD28 molecule; leukocyte activation /transformation; T lymphocyte; protein tyrosine phosphatase; phosphorylation; tissue /cell culture; immunoprecipitation; laboratory mouse; monoclonal antibody;

Recent technical advances have made pancreatic islet cell transplantation a feasible approach to the treatment of insulin- dependent diabetes mellitus (IDDM). However, the need for continuous, often debilitating, immunosuppressive drug therapy in transplant recipients makes the use of this therapy in diabetic patients most problematic. Thus, the development of long term donor-specific tolerance remains a primary goal of transplant biology. Activation of graft-specific pathogenic T-lymphocytes requires a first signal originating from the ligation of the TCR complex and an additional second co-stimulatory signal. However, novel immunotherapies successfully developed in small animal models to block costimulation often fail to induce the desired immunologic effects in large animals. It is critical to move these therapies forward towards the clinic using primate models that lack the confounding factors present in clinical transplantation. In order to accomplish this goal, the following specific aims are proposed. Specific Aim number 1. To determine the effect of blockade of the CD28/B7 co-stimulatory pathway on the survival of allogeneic islet cells transplanted into insulin- dependent Cynomolgus monkeys. In this study, we propose to administer CD28/B7 antagonists short-term in the absence of other immunosuppressive drugs, to prolong pancreatic islet allograft survival, prevent the development of donor specific antibodies and promote tolerance in a non-human primate model of islet transplantation; Specific Aim number 2. To integrate costimulation inhibition into novel drug combination treatment strategies in the non-human primate in order to establish consistent long term donor-specific tolerance. Antagonism of CD28 co-stimulation, even utilizing optimal reagents and dosing, may not completely inhibit T cell activation and differentiation. We hypothesize that additional treatments (anti-IL-2R, anti-ICAM- 1, 4-1BBFc) aimed at preventing the development of proinflammatory cell-mediated anti-donor responses will facilitate engraftment and induce a consistent donor specific tolerance in a non-human primate transplant model. Together the experiments proposed above will provide a detailed study of co- stimulation blockade in an islet transplant model in non-human primates. The immunologic, biologic and physiologic studies will afford us an opportunity to explore the basis of T cell activation and tolerance induction. The outcome of these studies will have important implications for islet cell transplantation for the treatment of IDDM.

DESCRIPTION (adapted from the applicant's abstract): OKT3 therapy for the treatment of organ graft rejection is complicated by severe first dose side effects caused by T cell activation-induced cytokine release in vivo. Moreover, OKT3 causes pan-immunosuppression that can lead to increased infections and cancer. The investigators have developed a novel Fc receptor (FcR) non-binding form of anti-murine CD3 mAb, 2C11-IgG3, which suppresses immune responses in the absence of first dose side effects. In vitro, 2C11-IgG3 has short-lived effects on naive T cells, but delivers a partial signal to activated T cells that results in clonal inactivation of Th1 cells, proliferation/ cytokine production by Th2 cells, and Th2 deviation of undifferentiated T cells. Biochemical analyses of the early activation events in both T cell subsets show an identical pattern of partial phosphorylation of T cell receptor (TCR) zeta and ZAP-70 similar to that observed in T cells treated with altered CD4 receptor blockade during T cell activation. These results suggest that this novel TCR antagonist can differentially alter the intracellular signals that regulate Th1 and Th2 development selectively on antigen-experienced T cells. The investigators hypothesize that unbalanced biochemical signaling, exemplified by this mAb, is a common mechanism to regulate T cell differentiation and tolerance induction in vivo. The proposed study will focus on several questions: 1) Do the biochemical and functional changes induced by 2C11-IgG3 in vitro also occur in vivo in the allogeneic islet transplant model? 2) Can the mechanisms that regulate this process be defined? 3) What are the minimal biochemical signaling events required for Th cell differentiation? To answer these questions, the applicant proposes the following specific aims: 1) Determine the in vivo effect of 2C11-IgG3 mAb on T cell signaling in the allogeneic islet transplant model. 2) Use biochemical analyses and retroviral gene transfer to define the proximal signaling events associated with clonal inactivation, cell death, and T helper subset differentiation induced by 2C11-IgG3. 3) Use biochemical analyses and retroviral gene transfer to define the distal signaling events associated with clonal inactivation, cell death, and T helper subset differentiation induced by 2C11-IgG3.

flow cytometry; biomedical equipment purchase;

DESCRIPTION (provided by applicant): The treatment and cure of autoimmunity remains of paramount importance. The challenges to developing successful therapies are broad, ranging from complex genetics, similarities and differences among target tissues, differential pathogenic mechanisms and an incomplete knowledge of the target antigens. We have shown that the Non-Obese Diabetes (NOD) mouse strain can be used as a mouse model of multiple autoimmune disorders (AID). These other autoimmune diseases were most apparent when regulatory T cells (Tregs) were eliminated and co-stimulatory pathways altered. For instance, NOD mice develop a spontaneous autoimmune disease of the peripheral nervous system, termed Spontaneous Autoimmune Peripheral Polyneuropathy (SAPP), in the absence of CD28 interaction with B7-2. In addition, we observed that in the complete absence of CD28 signals, NOD mice were deficient in Tregs and developed SAPP, sialadenitis, autoimmune thyroiditis and a newly appreciated autoimmune exocrine disease similar to that observed in "fulminant type 1 diabetes" described in Japanese and some Australian patients. Significantly, these various autoimmune diseases could use different pathogenic and co-stimulatory pathways and result from the recognition of distinct as well as potentially overlapping self-antigen specificities. These results have led to the conclusion that the NOD mouse represents a unique model for studying multi-organ autoimmunity. The combination of genetic propensity for autoimmunity and the tools that we have developed in this mouse strain will be exploited to address several key questions. Do unique and/or overlapping antigen specificities distinguish/link these diseases? Are the pathogenic pathways evident for one disease critical for the manifestation of others? Are there common co-stimulation pathways that control the susceptibility and progression of these distinct autoimmune diseases? The following aims are proposed to address these questions: Specific Aim #1: To generate tissue antigen-specific effector and regulatory T cell TCR Tg mice based on candidate antigens. Specific Aim #2: To generate tissue antigen-specific effector and regulatory T cell TCR Tg mice using T cell hybridomas and mimotopes. Specific Aim #3: To determine the effector and regulatory pathways and the role of co-stimulation in the distinct autoimmune diseases in NOD mice. Specific Aim #4: To develop green fluorescence protein (GFP)-specific systems to study autoimmunity in NOD mice. Together, the results of these studies will test the hypothesis that the phenotypic manifestation of multi- organ autoimmune diseases is regulated by a coalescence of common and tissue-specific pathways. Moreover these common and distinct pathways are critical for understanding of the immunopathology of these different autoimmune diseases and development of novel therapies.

DESCRIPTION (provided by applicant): The University of California at San Francisco Diabetes Center, an organized research unit at UCSF, has functioned for more than a half-century as a basic and clinical research enterprise at the forefront of diabetes research. Historically, the program has had an ongoing enrichment program, Pilot & Feasibility studies, strong basic science and clinical research interface. The goal of the Center is to support a highly interactive team involved either directly or indirectly in Type 1 and Type 2 diabetes research to advance the study and treatment of the disease. In this application, the UCSF Diabetes Center proposes to establish a Diabetes Research and Training Center (DRTC) that will support Core Laboratories, an Enrichment Program and Pilot and Feasibility studies. The Center will encompass a broad range of intellectual and research expertise from over 12 departments and organized research units and three UCSF campuses focused on basic research with an eye towards clinical application. The center will combine immunology, metabolic research, cell biology and genetics in the field of diabetes to develop unique approaches to understand and treat this devastating disease. Investigators of the DRTC are organized in the following programmatic areas: Cell Biology of Islets, Developmental Biology, Islet Transplantation and Immunology, Autoimmunity, Receptors and Signaling, and Obesity & Metabolism and Obesity, Complications of Diabetes. Seven Core facilities are designed to facilitate interdisciplinary investigations of these scientists ( Enrichment Core, Islet Production Facility Core, Microscopy & Cellular Imaging Core, Genomics & Bioinformatics Core, Mouse Genetics Core, Mouse Metabolism, and Human Metabolism Cores). A Pilot and Feasibility Grant Program serves to foster new initiatives in diabetes research primarily of junior faculty and those senior faculty from outside the diabetes focus area. An intensive academic enrichment program which organizes seminars and various symposia is designed to keep Center investigators abreast of the latest discoveries and to maintain the research program at this center at the forefront of biomedical science.

DESCRIPTION (Provided by applicant): The primary objective of the DRTC Mouse Models Core is to create a shared resource for the establishment, maintenance and experimentation on mouse models of type 1 and type 2 diabetes and related disorders to assist DRTC investigators in meeting their research objectives. Specific aims of the core are: 1.Produce transgenic mice in standard laboratory strains; 2. Produce transgenic mice in the NOD mouse background; 3. Produce transgenic mice carrying bacterial artificial chromosomes (BACs), in standard and NOD backgrounds; 4. Provide standardized ES cells and assistance with producing gene targeted clones; 5. Establish gene-targeting procedures for targeting the NOD chromosomes in embryonic stem (ES) stern cell lines derived from NOD x 129 F1 mice, facilitating the generation of gene knockout and knock-in mice that can be rapidly inbred to homozygosity in NOD; 6. Produce gene targeted mice via blastocyst injection of standard and NOD ES cell clones; and 7. Provide liaison to the Stanford University Gene Trap Resource, facilitating the screening by DTRC members for mouse insertional mutations with phenotypes relevant to types I and II diabetes. To meet these aims, the core will operate in a fashion similar to most institutional transgenic and targeted mutagenesis cores. This facility will, however, emphasize and specifically support diabetes-related projects. For example, the core will establish routine microinjection and gene-targeting procedures using zygotes and embryonic stem (ES) cell lines from the NOD (and potentially other) mouse strains that are of key significance for diabetes research. The Core will import and maintain relevant mouse strains and ES cell lines relevant in diabetes research and it will establish the capability to derive new ES lines to complement external sources. In addition, the Core will subscribe to a regional consortium that is producing new mutations in the mouse through 'gene-trap' insertion technologies, so as to identify new mutant mice showing developmental or physiological phenotypes relevant to research of the UCSF DRTC in types I and II diabetes. A major focus of the core will be on establishing the capability to genetically manipulate the non-obese diabetic (NOD) mouse via transgenic and gene targeting experiments.

DESCRIPTION (provided by applicant) The overall objective of the Microscopy and Cellular Imaging Core Laboratory is to consolidate, enhance and disseminate Diabetes Center resources and expertise in tissue and cell imaging technologies. Although extraordinarily powerful, many imaging techniques are sophisticated and difficult to learn and develop independently, and therefore lend themselves well to the cooperative structure of a core laboratory. In addition, because these technologies change rapidly, the core will provide an important information and training function by keeping participants abreast of the latest advances. The Core will provide the following: Access to specialized instrumentation, software, reagents, and information for histology and microscopy applications. Access will require oversight and maintenance of Core resources by staff. Standardized protocols will allow inter-study comparisons of data within the DRTC. The resources and standardized applications also will facilitate the entry of other laboratories at U.C.S.F. to diabetes research. Advice and training of DRTC members will be provided in application of imaging technologies. Development of new resources and technologies including new protocols and new equipment through common equipment grant applications. The Core will provide information, training, assistance and resources, but experiments and data collection will be performed by individual investigators. By enhancing the efforts of 22 funded investigators, including multiple members of all six Biomedical Research Programs and both basic and clinical investigators, the Core will accelerate progress towards the overall programmatic goals of the UCSF DRTC.

DESCRIPTION (provided by applicant): The major objectives of this core are to produce and provide, to the UCSF DRTC community, pancreatic tissues and purified islets from mouse, non-human primates (NHP), and human donor pancreas. Our focus is to provide each investigator the best possible pancreatic tissue and islets, ensuring the quality and reproducibility of these samples. We will focus on developing new techniques that aim to minimize islet cell death, maximize islet yields and optimize islet function. We will work closely with experts in the islet, b-cell field to identify factors that will contribute to us reaching our goals for better new islet isolation techniques. The Core will continue to develop assays to better assess the quality of a pancreatic islet preparation. Investigate more informative quality control assays, which will prospectively predict in vivo islet function rather than the limited prospective studies relied upon today. The Core will work with the basic scientists, which we provide pancreas tissue and islets, to help us take full advantage of new research in islet cell expansion and neogenesis into large-scale islet transplantation settings. The Core will also maintain the state-of-the-art human islet isolation facilities meeting all FDA's cGMP regulations for human islet isolation and transplantation. The Core will also maintain the NHP and rodent pancreatic islet isolation facilities. The Core will focus on taking the positive aspects to what has been developed in the human islet isolation laboratory and apply these techniques and assays to both the NHP and rodent facilities and vise versa. The Islet Production Facility Core will play a critical role in the experimental and clinical programs of the DRTC and our objective is to provide the best possible tissues and to aid in the advancement of islet and beta-cells research.

Description (provided by applicant): The DRTC Genomics Core unites the related resources and expertise of several unique genomics activities at UCSF. Although all of these activities already exist, by bringing them together the UCSF DRTC will enhance these activities, reduce duplication of effort, develop DRTC-specific resources, and increase access for DRTC members. Each facility has a full range of bioinformatics resources relevant to its particular technology. This Core works on the underlying principle that it will provide information, training, assistance and resources, but to get truly meaningful results, the individual investigators must be involved in each step of data production and analysis.

DESCRIPTION (provided by the applicant): The UCSF Diabetes Center Pilot & Feasibility (P&F) Program was established 18 months ago to encourage the pursuit of highly novel research for understanding and treating type 1 and type 2 diabetes. The program has been targeted towards all members of the UCSF community - the existing faculty of the Diabetes Center as well as other investigators from other departments and institutes at UCSF - in order to capitalize on the many and varied opportunities within the institution that have pointed relevance to diabetes. The P&F program implemented at UCSF provides investigators with two years of support at $25,000 per year to support conceptually innovative studies. As importantly, these awards are hoped to provide seed funding that will generate sufficient data to pursue additional research efforts through other funding mechanisms. The DRTC P&F Program will endeavor to: 1. facilitate and allow new investigators and junior faculty to establish research programs in diabetes; 2. facilitate and support established faculty wishing to transfer skills and research developments to the arena of diabetes research; 3. foster highly innovative directions in basic and clinical diabetes research, including clinical studies in minority and other underserved patient groups; and 4. foster new and innovative collaborations amongst DRTC UCSF Faculty. The Pilot & Feasibility Program committee is will be chaired by Dr. Douglas Hanahan and the Committee includes Drs. Gerold Grodsky, Michael German and Ira Goldfine. As a group this committee represents all the aspects of the Center to assist the director in the management of the program.

[unreadable] DESCRIPTION (provided by applicant): Recent advances in the understanding of T-cell activation have led to new therapeutic approaches in the treatment of immunological disorders. One attractive target of intervention has been the blockade of T cell-mediated co-stimulatory pathways, which result in more selective effects on only those T-cells that have encountered specific antigen. In fact, in some instances, CD28/B7 co-stimulatory pathway antagonists can induce antigen-specific tolerance that prevents the progression of autoimmune diseases and organ graft rejection. However, many other cell surface molecules exist on naive T cells that may play an important role in initiating immunity from the quiescent state. Moreover, it is clear that quiescent T cells can also function as regulatory T cells to promote immune tolerance by an as yet unknown mechanism suggesting that some of the cell surface proteins may provide functional activities beyond just determining whether cells effectively go into cell cycle. Notch, Serrate, and Delta are type 1 integral membrane proteins involved in cell-cell signaling crucial to normal cellular differentiation and in organogenesis. In the immune system, Notch signaling is critical for normal T cell development. Recently, a role for Notch signaling in mature T cells has been proposed based on the observation that engagement of Notch by Serrate could induce a profound state of unresponsiveness in peripheral T cells in vivo. Most interesting was the observation that these "unresponsive" T cells possessed regulatory abilities that on several levels appear similar to the CD25+ regulatory cells that are able to regulate autoimmunity. Our preliminary studies suggest that Notch-1 is an important regulator of T cell signaling in resting T cells and may be involved in tolerance. We hypothesize that Notch-1 plays a critical role in the intrinsic regulation of T cell responses by influencing the primary signaling pathways critical for activating naive T cells. This results in T cell inactivation or altered differentiation. Moreover, we hypothesize that manipulation of immune responses through Notch-1 can be used to alter autoimmunity and transplantation tolerance. In this application we propose to explore the mechanisms of Notch signaling in T cells and its application as follows: Specific Aim #1. To Identify the biochemical, cellular and genetic effects of Notch signaling in T cells; and Specific Aim #2. In vivo application of Notch signaling for the establishment of peripheral T cell tolerance. [unreadable] [unreadable]

CTLA-4 was first described as a negative regulatory molecule by our group in 1994. During the past 11 years the molecule has been extensively studied. The molecule has been shown to have an intrinsic effect on T cell activation by directly delivering negative signals to T cells to shut down activation and differentiation. In addition, there have been a number of studies suggesting an extrinsic role for CTLA-4 as the molecule has been proposed to be the effector molecule by which regulatory T cells suppress immunity. All of these "functions" have been complicated by the recent discovery that CTLA-4 can be expressed as a B7 ligand non-binding variant that controls T cell activation. In addition, polymorphisms in the CTLA-4 gene has been linked to Graves Disease and Type 1 Diabetes. Thus, in this renewal, we will continue to addreess the fundamental biology of CTLA-4. We propose to directly address both the intrinsic and extrinsic role of CTLA-4 in the regulation of initial T cell activation, ongoing autoimmune responses and maintenance of tolerance. The following specific aims are proposed: 1. To study the biochemical basis of B7 ligand-dependent and ligand-independent CTLA-4-mediated inhibition of T cell function;2. To study the intrinsic role of CTLA-4 in T cell development, regulation of tolerance induction and development of diabetes in NOD mice;and 3. To study the intrinsic role of CTLA-4 in T cell development, regulation of tolerance induction and development of diabetes in NOD mice. We will use a combination of novel mice and reagents, combined with TCR transgenic and bone marrow chimera models to identify the cellular and mechanistic basis for CTLA-4 regulation. The results of these studies will provide insights into the mechanisms of CTLA-4 regulation of immunity, and test the the role of CTLA-4 in lymphoproliferative/homeostasis versus immune activation and tolerance are mediated by distinct extrinsic versus intrinsic pathways. Importantly, the information learned from this study may have important implications for public health for several reasons. T cells play a central role in immunity and autoimmunity. As such, any means to modulate T cell activity may lead to novel therapeutic approaches to treat these disease. Moreover, the fundamental importance of CTLA-4 in the induction and maintenance of peripheral tolerance is central to our understanding of disease etiology and current therapeutic approaches.

immune tolerance /unresponsiveness

The Immune Tolerance Network is dedicated to the clinical evaluation of novel tolerance-inducing therapies that will [unreadable]re-educate[unreadable] the immune system to eliminate injurious immune responses and graft rejection. The ITN conducts clinical trials that include novel tolerance-inducing therapies for heart transplantation and the prevention of graft rejection. In addition, to understand the underlying mechanisms of action of the candidate therapies and to monitor tolerance, the ITN has established state of the art core laboratory facilities to conduct integrated mechanistic studies, and to develop and evaluate markers and assays to measure induction, maintenance, and loss of tolerance in humans.

Autoimmunity is reaching epidemic proportions with tens of millions of people suffering from diseases such as multiple sclerosis, rheumatoid arthritis, type 1 diabetes (T1D), systemic lupus erythematosus and others. In addition to its financial costs, the long term complications of these diseases can be devastating. A curative therapy is desperately needed. At present, efforts to prevent or reverse autoimmune diseases have been limited by the lack of safe and effective immunotherapies. With this challenge in mind, we believe the induction of immunological tolerance is a fundamental requirement for any effective therapy. Previous studies, including numerous published reports from our ACEs group and others, have focused on the use of regulatory T cells (Tregs) as one means of restoring tolerance in autoimmunity. This notion is based on the core principle that treatment with Tregs will lead to the induction of long-term tolerance. Most significantly, there is strong evidence that self-antigen-specific Tregs are most effective to treat a variety of autoimmune diseases. However, the identification and application of these cells have been compromised by the lack of effective isolation and expansion protocols for these low frequency cells. Thus, novel approaches are necessary to address this need. Specifically, this application proposes to develop novel approaches to generate a sufficient quantity of antigen-specific Tregs capable of restoring tolerance and averting autoimmunity. These studies will be performed as a collaborative effort between the Abbas and Bluestone labs where multiple mouse models have been established and be used to rapidly test theoretical cell manipulations. These efforts will inform and enhance the bulk research efforts in his proposal develoted to human Treg studies and the development of engenerred Tregs that can be used both to study the biology of Tregs and potentially be developed for clinical application. To address these key issues, the following specific aims are proposed. Aim 1.Develop engineered antigen-specific Tregs by introducing autoreactive T cell receptors (TCRs) and other therapeutic genes. Aim 2. Assess the mechanisms and safety of cellular therapy with engineered Tregs. Aim 3. To generate high numbers of autoreactive engineered Tregs capable of suppressing pathogenic autoreactive T cell responses.

The Immune Tolerance Network is dedicated to the clinical evaluation of novel tolerance-inducing therapies that will [unreadable]re-educate[unreadable] the immune system to eliminate injurious immune responses. The ITN is conducting clinical trials in autoimmune diseases such as rheumatoid arthritis. In addition, to understand the underlying mechanisms of action of the candidate therapies and to monitor tolerance, the ITN has established state of the art core laboratory facilities to conduct integrated mechanistic studies and clinical research to develop and evaluate markers and assays to measure induction, maintenance, and loss of tolerance in humans.

The Immune Tolerance Network is dedicated to the clinical evaluation of novel tolerance-inducing therapies that will [unreadable]re-educate[unreadable] the immune system to eliminate injurious immune responses and graft rejection. The ITN conducts clinical trials that include novel tolerance-inducing therapies for lung transplantation and the prevention of graft rejection. In addition, to understand the underlying mechanisms of action of the candidate therapies and to monitor tolerance, the ITN has established state of the art core laboratory facilities to conduct integrated mechanistic studies, and to develop and evaluate markers and assays to measure induction, maintenance, and loss of tolerance in humans.

The Immune Tolerance Network is dedicated to the clinical evaluation of novel tolerance-inducing therapies that will [unreadable]re-educate[unreadable] the immune system to eliminate injurious immune responses and graft rejection. The ITN conducts clinical trials that include novel tolerance-inducing therapies for liver transplantation, kidney transplantation, bone marrow transplantation, stem cell transplantation, pancreatic islet transplantationand the prevention of graft rejection. In addition, to understand the underlying mechanisms of action of the candidate therapies and to monitor tolerance, the ITN has established state of the art core laboratory facilities to conduct integrated mechanistic studies, and to develop and evaluate markers and assays to measure induction, maintenance, and loss of tolerance in humans.

DESCRIPTION (provided by applicant): Our overall goal is to understand how insulin-producing ?-cells are generated and to apply that knowledge to the production of ?-cells for patients with Diabetes. Our general approach in this application is to use human islets and human embryonic stem cells to model human ?-cell genesis and turn over. We will continue to use animal models to determine how ?-cell expansion technologies work in vivo -- how they interact with the immune system and impact metabolism -- in order to develop these ideas into practical and safe human therapies. Our Specific Aims explore three approaches to ?-cell genesis: neogenesis, proliferation, and reprogramming/transdifferentiation: Specific Aim 1: Translate results of regeneration screens to human ?-cells. Using zebrafish, we have identified small molecules that enhance ?-cell regeneration. We will validate these hits in human Islets and ES cells, explore their mechanisms of action, and test their activity in preclinical animal models. Specific Aim 2: Determine the efficacy of GPCR signaling in ?-cell genesis. We have established that GPCR signaling plays a critical role in two physiologic settings of ?-cell expansion: pregnancy and infancy. We will test the importance of these pathways in the neogenesis and turnover of human ?-cells. Specific Aim 3: Establish the role of the immune system in islet regeneration. Current models of islet regeneration all cause pancreatic damage and provoke an immune response. We will determine the role of these responses in islet regeneration and reprogramming in preparation for moving these technologies to human therapy. Specific Aim 4: Monitor and control ER stress during ?-cell genesis. We have developed technologies for monitoring and controlling the unfolded protein response (UPR) in living cells. We will utilize these technologies to determine the role of ER stress and the UPR during ?-cell genesis in human ES cells and live mice. PUBLIC HEALTH RELEVANCE: These studies are directed towards the application of basic knowledge of the mechanisms by which the insulin producing cells in the pancreas are generated to the clinical problem of how to produce more of these cells for patients with Diabetes.

Purpose: The DERC Islet Metabolism Core assists investigators who currently study or have plans to study pancreatic islet cell biology, transplantation and metabolic analysis. Components: 1. Islet Purification. Processing and preparation of human and mouse islets for use in islet transplantation, genomic studies, and cell biology studies. 2. Islet Functional Analysis. Real time biological and biochemical analysis of insulin production and other functional aspects of human and mouse islets to characterize islet activity under different conditions and to assess islet preparation quality for transplantation studies. 3. Mouse Metabolic Analysis. Analysis of the metabolic status of an intact mouse including measurements of fat content, the monitoring of glucose levels in response to exogenous glucose or insulin stimulation and the analysis of oxygen consumption and/or activity. Benefits to DERC Community: These highly specialized capabilities are beyond the reach of almost ail individual laboratories. The Islet Metabolism Core consolidates these unique capabilities and enables the application of these capabilities to research studies by DERC investigators. Standardized protocols ensure reproducibility in analyses conducted on different days. Standardization also enables individual laboratories to compare outcomes. Technology Development: There is an ongoing commitment to continue the prior improvements in islet isolation and analysis. A broader work-up of islet function standard for all preps will be combined with a Core-wide database to assist investigators in making cross-study inferences. Significant improvements in the metabolic analysis of intact mice are expected to represent another major effort in the next DERC cycle.

DESCRIPTION (provided by applicant): The long-term goal of this project is to develop a regulatory T cell (Treg)-based approach for the induction of donor-specific immunologic tolerance in liver transplant recipients. Liver transplantation can be life-saving therapy for live failure. However, the maintenance of the transplanted liver requires continuous immunosuppression to prevent rejection by the host immune system. Although ongoing refinement of immunosuppression regimens has substantially reduced the incidence of acute rejection after transplantation, long-term outcomes have stagnated partly due to morbidity and mortality associated with immunosuppression. Therefore, a main focus of research has been to promote tolerance to transplanted livers so that immunosuppression can be minimized or completely withdrawn. In the past decade, we have learned that tolerance in organ transplantation is linked to the development and persistence of Tregs. In multiple preclinical models, therapeutic administration of Tregs has proven efficacy in controlling allograft rejection and inducing donor-specific tolerance. Alloantigen-specific Treg are more effective and potentially safer than non-specific Treg by offering targeted therapy instead of indiscriminate regulation. A key point in Treg-based regimens in the transplant setting is that, because of the exceptionally high frequency of donor-reactive T cells, debulking of the host alloreactive repertoire and adjunct immunosuppression are needed to create a more favorable setting for Tregs to control alloimmunity and to ensure long-term graft tolerance. We aim to translate these basic and clinical findings into a practical and effective clinical protocol. We plan to test the ue of donor- specific Tregs in the context of a Treg-supportive immunosuppression regimen as an approach to induce liver transplant tolerance. As a first step, we propose to conduct an open-label, single center, phase I, dose escalation trial to determine the safety of administering a single escalating dose of donor-specific Tregs in liver transplant patients. We will perform mechanistic analyses of the study patients to assess the impact of the Treg therapy on recipient's immune reactivity to the donor. The R34 grant will allow the investigators to finalize all facets of the clinical trial protocol, Treg manufacturing, and concomitant mechanistic studies, along with securing Investigational New Drug and Institutional Review Board approvals for the trial. To our knowledge, this proposal represents the first application of Tregs in solid organ transplantation to induce graft tolerance. It is strongly aligned with NIAID's goal to evaluate approaches that include tolerogenic, anti-inflammatory, and immunomodulatory strategies to treat and prevent immune-mediated diseases. PUBLIC HEALTH RELEVANCE: Regulatory T cells (Tregs) have recently been shown to be critical for the development of natural and acquired immunologic tolerance in a variety of settings, suggesting that Treg administration may be useful for the therapeutic induction of immunologic tolerance. Organ transplant recipients continue to suffer from immunologic rejection as well as serious side effects from non-specific immunosuppressive medication. Our long-term objective is to develop Treg cell therapy to induce transplant tolerance so that immunosuppression can be minimized or withdrawn. Therefore, we propose to develop the first clinical trial in solid organ transplantation to test the safety of an immunosuppression regimen that favors the development of Tregs, in conjunction with an infusion of donor-specific Tregs in the liver transplant setting.

DESCRIPTION (provided by applicant): Our overall goal is to understand how insulin-producing ?-cells are generated and to apply that knowledge to the production of ?-cells for patients with Diabetes. Our general approach in this application is to use human islets and human embryonic stem cells to model human ?-cell genesis and turn over. We will continue to use animal models to determine how ?-cell expansion technologies work in vivo -- how they interact with the immune system and impact metabolism -- in order to develop these ideas into practical and safe human therapies. Our Specific Aims explore three approaches to ?-cell genesis: neogenesis, proliferation, and reprogramming/transdifferentiation: Specific Aim 1: Translate results of regeneration screens to human ?-cells. Using zebrafish, we have identified small molecules that enhance ?-cell regeneration. We will validate these hits in human Islets and ES cells, explore their mechanisms of action, and test their activity in preclinical animal models. Specific Aim 2: Determine the efficacy of GPCR signaling in ?-cell genesis. We have established that GPCR signaling plays a critical role in two physiologic settings of ?-cell expansion: pregnancy and infancy. We will test the importance of these pathways in the neogenesis and turnover of human ?-cells. Specific Aim 3: Establish the role of the immune system in islet regeneration. Current models of islet regeneration all cause pancreatic damage and provoke an immune response. We will determine the role of these responses in islet regeneration and reprogramming in preparation for moving these technologies to human therapy. Specific Aim 4: Monitor and control ER stress during ?-cell genesis. We have developed technologies for monitoring and controlling the unfolded protein response (UPR) in living cells. We will utilize these technologies to determine the role of ER stress and the UPR during ?-cell genesis in human ES cells and live mice. PUBLIC HEALTH RELEVANCE: These studies are directed towards the application of basic knowledge of the mechanisms by which the insulin producing cells in the pancreas are generated to the clinical problem of how to produce more of these cells for patients with Diabetes.

DESCRIPTION (provided by applicant): Type 1 Diabetes (T1D) is an autoimmune disease resulting in the destruction of pancreatic islet insulin- producing beta cells. T1D is mainly drive by autoreactive T cells but components of the innate immune system have been implicated as well, including monocytes, dendritic cells and natural killer (NK) cells. NK cells belong to the innate lymphoid cell (ILC) family, which consists in innate cells that share a lymphoid morphology and other properties. Novel populations of ILCs have been defined recently and ILCs are now subdivided in three main categories: ILC1 cells (NK cells); ILC2 cells (IL-13- and IL-5-producing GATA-3+ innate helper cells, and ILC3 cells (ROR?t+ ILCs, which include distinct subsets of IL-22- and/or IL-17-producing ILCs). Functionally, ILC2s and ILC3s are involved in protective immunity against infections but they can also induce or control chronic inflammation depending on the circumstances. Importantly, the role of ILCs in autoimmunity is largely unknown. Thus, in this proposal we will address the overall hypothesis that ILC populations play a critical role in the development and progression of autoimmune diabetes. We will take advantage of innovative mouse models that include reporter mice allowing the identification and deletion of ROR?t+ ILC3s and IL-5/13-producing ILC2 cells selectively. Most significantly, we observe that ILCs are the first cells found in the pancreas of T1D-susceptible non-obese diabetic (NOD) mice suggesting a potential role in disease initiation. Finally, we will address another key hypothesis, namely, that ILC2 cells, present in tissues, including the pancreas, are critically involved in the negative side effects of IL-2 therapies designed to promote regulatory T cells (Tregs). Recent studies to test the safety of IL-2 therapy in new-onset T1D patients have validated mouse work demonstrating increased numbers of Tregs..However, in some instances, the treatment resulted in transient ¿ cell dysfunction, increased Th17 cells and eosinophilia. Importantly, our preliminary studies suggest that ILC2s present in the pancreas of NOD mice respond to IL-2 treatment and may be responsible for some of these unwanted effects. In this R21 Revised application we propose the following specific aims to address these hypotheses. 1) To characterize the ILC2 and ILC3 subsets in the NOD mouse; 2) To determine the functional effect of ILC2s on the development and regulation of autoimmune diabetes; and 3) To examine the effect of IL-2 therapy on ILC2s and the role of ILC2s in the biological and clinical effect of IL-2 therapy. These studies will characterize novel ILC subsets i the pancreas during diabetes and identify their influence on disease. To our knowledge, this will be the first characterization of ILC2s and ROR?t+ ILC3s in autoimmune diabetes. The results of our study may shift the current T1D dogma from a T cell-centric paradigm to one that includes an involvement of ILCs. Additionally, our studies could have important clinical implications by identifying novel therapeutic targets in T1D and allowing better a prediction of off-target effecs of therapy directed at pathways shared by ILCs and T cells.

DESCRIPTION (provided by applicant): The long-term goal of this project is to develop a regulatory T cell (Treg)-based approach for the induction of donor-specific immunologic tolerance in liver transplant recipients. Liver transplantation can be life-saving therapy for live failure. However, the maintenance of the transplanted liver requires continuous immunosuppression to prevent rejection by the host immune system. Although ongoing refinement of immunosuppression regimens has substantially reduced the incidence of acute rejection after transplantation, long-term outcomes have stagnated partly due to morbidity and mortality associated with immunosuppression. Therefore, a main focus of research has been to promote tolerance to transplanted livers so that immunosuppression can be minimized or completely withdrawn. In the past decade, we have learned that tolerance in organ transplantation is linked to the development and persistence of Tregs. In multiple preclinical models, therapeutic administration of Tregs has proven efficacy in controlling allograft rejection and inducing donor-specific tolerance. Alloantigen-specific Treg are more effective and potentially safer than non-specific Treg by offering targeted therapy instead of indiscriminate regulation. A key point in Treg-based regimens in the transplant setting is that, because of the exceptionally high frequency of donor-reactive T cells, debulking of the host alloreactive repertoire and adjunct immunosuppression are needed to create a more favorable setting for Tregs to control alloimmunity and to ensure long-term graft tolerance. We aim to translate these basic and clinical findings into a practical and effective clinical protocol. We plan to test the ue of donor- alloantigen-reactive Tregs in the context of a Treg-supportive immunosuppression regimen as an approach to induce liver transplant tolerance. As a first step, we propose to conduct an open-label, two-center, phase I, dose escalation trial to determine the safety of administering a single escalating dose of donor-specific Tregs in liver transplant patients. We will perform mechanistic analyses of the study patients to assess the impact of the Treg therapy on recipient's immune reactivity to the donor. We have finalized the clinical trial protocol, Treg manufacturing process, and detailed plan for mechanistic studies. FDA has reviewed the Investigational New Drug (IND) application for this trial and the IND is currently open. This proposal represents the first application of donor alloantigen-reactiveTregs in solid organ transplantation to induce graft tolerance. It is strongly aligned with NIAID's goal to evaluate approaches that include tolerogenic, anti-inflammatory, and immunomodulatory strategies to treat and prevent immune-mediated diseases.

Project Summary/Abstract Type 1 Diabetes (T1D) is caused by pathogenic autoreactive T cells that recognize and destroy the islet tissue. However, the presence of T cells reactive to self-antigens is not sufficient for disease to occur as autoreactive T cells can be found in healthy control subjects; thus various means are available that control unwanted responses. One of the most important mechanisms is the activity of regulatory T cells (Tregs), which arise both in thymus and in the peripheral immune system as a consequence of exposure to antigens. Defects in Treg numbers, phenotype, and/or function have been described in T1D and other autoimmune diseases. Because immunosuppression by Tregs specific for a limited number of Ags is dominant and can efficiently thwart a polyclonal autoreactive response, Tregs are an attractive target for antigen-specific tolerogenic therapies. However, the specificity and repertoire of Tregs recognizing self-Ags in humans and mice and whether they are different in individuals with autoimmunity is unknown. Recent studies have uncovered unique aspects of self-peptides presentation to autoreactive T cells by MHC class II alleles predisposing to autoimmunity. These peculiarities potentially circumvent negative selection of autoreactive T cells in the thymus and lead to autoreactivity in the target tissue. Given the well-known skewing of the tTreg repertoire towards self-reactivity, biochemical complexities may affect the development and repertoire of Tregs and their ability to recognize self-Ags in the targeted tissues ? suggesting that the Ag specificity of local Tregs will be instrumental in controlling autoimmunity in the tissue. Thus, identifying the Ag specificity of Tregs, especially at the site of inflammation, is critical to improve our understanding of Treg deficiencies in T1D. In this project, we will address this question by examining the overall hypothesis that the biochemical intricacies that promote effector immunity may paradoxically alter Treg efficacy and contribute to the overall failure of Tregs to control autoreactive Tconv cells in autoimmune diseases such as T1D. We propose the following Specific Aims to address this question: 1: Characterization of Treg specificity against known I-Ag7-restricted autoantigens 2: Antigen focused approach to define the specificity of Tregs in T1D patients. 3: Identification of novel ligands for Tregs in NOD mice and T1D patients. It is recognized that altered Treg immunoregulation is an inherent factor in autoimmunity but defining specific Treg defects in T1D patients has been challenging. Thus, defining the specificity and repertoire of Tregs and their reactive TCRs would be an important step towards the discovery of selective Treg defects in T1D and greatly improve our understanding of the immunopathology of disease. Moreover, it would pave the way for therapeutic opportunities aimed at restoring proper Treg immunoregulation in pancreatic islets.

? DESCRIPTION (provided by applicant): Although stem cell-based therapies can potentially be used to treat numerous prevalent diseases, their successful clinical translation requires overcoming the major roadblock of immune rejection. In addition, in autoimmune diseases such as Type 1 diabetes (T1D), where a breakdown in immune tolerance leads to the immune-mediated destruction of pancreatic insulin-producing cells, the underlying autoimmunity needs to be altered to allow successful engraftment without immunosuppression. Developing approaches to manipulate immune tolerance in the context of autoimmunity is thus essential for improving methods to cure and treat human autoimmune diseases. Within the immune system, the thymus plays a critical role in establishing central immune tolerance through the education of developing T cells. The thymus enforces tolerance through the deletion of T cells that recognize self-antigens and the production of potent regulatory T cells (Tregs) that can suppress immune responses. Given that self-identity is encoded by thymic epithelial cells (TECs), an appealing approach to manipulating immune tolerance in the context of stem cell therapies would be to reprogram the immune system through the generation of stem cell-derived TECs. Recently, our group has developed a novel method to differentiate human pluripotent stem cells (hPSCs) into thymic epithelial progenitors (TEPs) that mature into functional thymic tissue upon transplantation into immunodeficient mice. Importantly, these hPSC-derived TEPs acquire characteristics of mature TECs that allow generation of functional T cells capable of mounting allogeneic immune responses as well as formation of Tregs that maintain immune tolerance through crucial inhibition of self-reactive T cells. This unique and highly innovative tool will now be used to establish novel humanized murine models that more closely mimic in vivo human immune responses. Humanized mice transplanted with hPSC-derived TEPs will be used to study engraftment of another stem cell derivative (pancreatic beta cells). Additionally, a model of T1D autoimmunity will be developed through the generation of islet-specific autoreactive T cells within the thymus. Indeed, by altering expression of antigens such as insulin in hPSC-derived TEPs, we will have an opportunity to modify the immune repertoire as well as potentially impact the development of Tregs specific to islets. This will allw us to examine the early stages of T1D in an animal model using human immune cells and targets. Finally, these humanized mouse models will be used to study the impact of manipulation of thymic function and Treg administration that have the potential to alter allogeneic and autoimmune responses to stem cell-derived grafts. Taken together, our proposed studies will provide unique tools to enable improved studies of stem cell derivatives transplantation and modeling of autoimmune disorders such as T1D. Furthermore, our work will lay the foundation for the potential that thymic immune tolerance can be manipulated to improve engraftment of stem cell derivatives in the context of autoimmunity.

ABSTRACT In type I diabetes, pancreatic islets are destroyed by an autoimmune reaction. Thus, a general strategic goal for preventing islet loss is to locally suppress this autoimmune response, while avoiding systemic immune suppression. FoxP3-expressing T regulatory cells (Tregs) are immune cells that play a central role in local tolerance, and therefore could serve as a potential platform for a cell therapy to prevent islet loss. Tregs have many attractive attributes as a suppressive cell therapy: they can exert dominant immunosuppressive actions, through cell-cell interactions or production of suppressive cytokines; they can in principle be long lived; and tolerance induced by Tregs can persist through infectious tolerance by conferring tolerogenic properties to neighboring cells. In mouse models, single infusion of Tregs can prevent and reverse diabetes indefinitely. The encouraging preliminary data in mouse models have let to the development of ongoing clinical trials of Treg cell therapies in humans. Nonetheless, Treg cell therapy presents several key challenges: it is hard to specifically target them to the islets and avoid systemic suppression, they are hard to expand in sufficient numbers (especially relative to conventional effector T cells), and their cell fate can be changed, especially in particular inflammatory microenvironments. In the past few years, however, there have been remarkable advances in using conventional T cells for cancer therapy (e.g. CAR T cells), including an explosion of new synthetic biology tools that can be used to precisely target T cells to disease tissues, to control their fate and proliferation, and to even give them the capability to locally deliver non-natural therapeutic payloads. Here our goal is to bring these new tools to bear on the problem of engineering improved islet-specific therapeutic suppressive cells. Our aims are to: 1) engineer natural Tregs with improved targeting and recognition of islets 2) develop tools to selectively expand therapeutic Tregs in vivo and enhance their stability 3) design synthetic suppressor cells that disarm effectors, dampen inflammation and promote islet repair These approaches wed cutting-edge synthetic biology with multiple strategies for developing therapeutic suppressor cells. We will establish proof-of-concept data using mouse model of autoimmune diabetes and apply them to test engineered human Tregs targeted to human islets using a humanized mouse model of autoimmune islet inflammation. Successful completion of this study will provide preclinical data for future implementation of these strategies in clinical trials.