Ali Naji - US grants

We intend to study factors which induce or amplify MHC antigen expression on parenchymal cells of allografts. Pancreatic islet cells should prove interesting for these studies, since passenger leukocytes with antigen presenting function can be deleted from them by tissue culture, and since islet cells ordinarily express only class I antigens, but can be induced by exposure to lymphokines (such as interferon) to express class II antigens and to increase their expression of class I antigens. Assessment of whether modulation of MHC antigens imparts to islets the potential to present alloantigens or nominal antigens may provide insight into intragraft immuno-regulating mechanisms. We will employ in vitro cytotoxicity assays to test the effect of hyperexpression of class I antigens on islet lysis. Islets induced to express class II neo-antigens will be assessed for antigen presenting capability in vitro and in islet transplant experiments. Islets obtained from a new line of transgenic mice in which beta cell class II expression has been induced by recombinant DNA techniques will be of particular interest. Islet transplant studies will attempt to determine whether Ia bearing beta cells can initiate rejection or autoimmunity. If parenchymal cells are themselves found capable of presenting antigen to T lymphocytes, then blocking MHC antigen induction might be a promising mode of therapeutic intervention before or during allograft rejection.

Rejection remains a potent threat to the long term success of organ transplants and significant morbidity is associated with even the best of modern immunosuppressive agents. The best hope for improving allograft survival, and particularly for preventing chronic rejection, is the induction of a state of donor-specific tolerance. The overall goal of this project is to explore the mechanism(s) of allograft tolerance which follows inoculation of the donor alloantigen directly into the thymus. Previous studies by ourselves and others have confirmed the efficacy of intrathymic inocula of endocrine or lymphohematopoietic cells to promote acceptance of subsequent extrathymic cellular or organ allografts in rodents. The first specific aim of this project is to examine whether deletion is the primary mechanism responsible for thymus-mediated tolerance. By utilizing several murine models with well-defined alloantigenic expressions, we will employ immune repertoire analysis of Vbeta-bearing T cells to explore clonal deletion as the basis for intrathymic tolerance. The second specific aim of this application is to assess the roles of the counterregulatory cytokine network as the mechanism of active clonotypic suppression responsible for the induction of intrathymic tolerance. Novel MHC knockout mice will be utilized to assess the role of the host lymphoid cells to promote specific cytokine profiles important in the induction and maintenance of tolerance following intrathymic inoculation with donor alloantigen. In specific aim 3 we will utilize a preclinical model of SCID mice reconstituted with human lymphohematopoietic and thymus grafts to simulate the human immune system. SCID-hu constructs will be used to assess the ability of the human thymus to nurture autografts and allografts of pancreatic islets and to reverse experimental diabetes. The impact of the residence of allogeneic islets in the human thymus on the peripheral T cell repertoire will be assessed as well as the possible induction of specific tolerance to extrathymic islets from the same donor. Intrathymic inocula of allogeneic lymphoid cells will also be tested as a tolerogen to promote donor-specific unresponsiveness to extrathymic islet allografts. Collectively, the present application proposes to delineate the mechanism(s) of acquired thymus-mediated immune tolerance and to utilize this information to optimize its potential utility in man.

The overall goal of this program project is to characterize the cellular and molecular basis of the immune pathogenesis of insulin-dependent diabetes mellitus (IDDM). The information obtained will be utilized to design a specific immune intervention strategy to prevent or treat IDDM. The program project involves a collaborative interaction between four departments. The grant is organized into four individual projects supported by three cores. The expertise of the project leaders bridge the disciplines of immunology, biochemistry, genetics, and pathology. The will use a wide range of techniques to characterize the immunologic and molecular perturbations, leading to the selective destruction of pancreatic beta cells. Project 1 will focus on the feasibility of islet- directed gene transfer to express several well characterized chemokines within islet allografts to promote local immunomodulation. Project 2 will investigate the intra-islet T-cell immunoregulatory events and the delineation of several putative autoimmune diabetes of NOD mice. Novel immune intervention strategies will be applied to treatment of diabetic NOD mice with islet transplantation. Project 3 will focus on the physical and metabolic characteristics of islet tissue to explore the technology of genetic engineering for modification of islet tissue prior to transplantation. Project 4 will focus efforts on characterizing the sphingomyelinase/ceramide and phospholipase A2 signal transduction pathways in mediating the effect of cytokine in the beta cell destruction. Core units for project administration, islet isolation, and vector preparation will provide necessary coordination and infrastructure for the program project. Collectively, this program project is an integrated and focused multidisciplinary effort to list the mechanisms that lead to the destruction of pancreatic beta cells in diabetes mellitus. The proposed studies are designed to elucidate the pathophysiology of IDDM and to utilize modern transplantation and molecular techniques to improve the treatment of the disease.

The pancreatic islet isolation and vector core (Core B) is an integral component of this program project. The purpose of this core is to assist the project leaders and their investigative personnel in three critical ways: 1) isolation of large numbers of highly purified pancreatic islets, 2) quality control of purity, quantity, and viability of islets needed for physiological and molecular studies as well as for successful transplantation in diabetic recipients and 3) construction and quality controlled delivery of adenoviral vectors containing the desired recombinant gene. The overlapping and synergistic need for both isolated islet and recombinant adenoviral vectors by the investigators of this program project prompted us to propose the implementation of Core B. The need for such a core is particularly poignant given the lack of a bona fide and easily accessible islet isolation core at the University of Pennsylvania. Additionally, given our 'in-house' expertise in construction of adenoviral vectors, we fell that the most time- and cost-efficient means of developing adenoviral vectors is the proposed 'in house' Core facility\ (Core B).

Insulin-dependent diabetes mellitus (IDDM) is mediated by a T lymphocyte- dependent autoimmune destruction of pancreatic b-cells. Although insulin therapy has increased the lifespan of type I diabetic patients, it has failed to influence the long-term complications of diabetes. The basis of these long-term complications is due to the failure of exogenous insulin therapy to achieve fine specificity of normal glucose homeostasis. The recent results of the DCCT that improved glycemic control reduces the progression of secondary complications of IDDM has provided impetus for the design of protocols to achieve euglycemia. In the recent past, much effort has been focused on identifying the autoantigens implicated in IDDM, with an ultimate goal of using them for immune intervention strategies to prevent IDDM during the preclinical stages. This method of study, although meritorious, does little to aid those patients already afflicted with established diabetes. For these patients, islet cell transplantation remains the most practical and most specific treatment to restore normoglycemia and thus, prevent the development of long-term complications of IDDM. A major drawback of the widespread application of islet transplantation for the treatment of diabetes has been the vulnerability of islet allografts to immunologic rejection. Recent advances in gene therapy provide hope for the molecular engineering of islets to promote donor- specific unresponsiveness. Both adenoviral and adenoassociated viral- mediated gene transfer offer the novel opportunity of transducing somatic, non-replicating cells with desirable genes. The overall objective of this project is to induce islet allograft tolerance. We propose to employ several well-characterized chemokines (i.e., uteroglobin, CTLA4Ig, vIL10, and TGFb1), utilizing the latest technologies of gene therapy to express these secretory immunomodulatory proteins within the islet cell allograft. These secretory cytokines have the capacity to immunomodulate the intraislet milieu and block several critical steps of the T cell activation cascade important in islet- directed alloimmune responses. Through the technique of islet-directed DNA virus-mediated gene therapy, we will introduce a "multiple hit" concept of local immunomodulation to promote islet allograft tolerance.

DESCRIPTION (adapted from applicant's abstract): Maternally transmitted immunoglobulins (Ig) are a major component of protective humoral immunity in neonates. The repertoire of these Ig specificities is a direct reflection of the immunologic experience of the mother. In the context of autoimmune disease, maternally derived Ig might contribute to the initiation of autoimmunity in genetically susceptible offspring. Indeed, a major hallmark of autoimmune diabetes in humans and NOD mice is the presence of a myriad of autoantibodies reactive to islet (beta) cell autoantigens. Detection of such islet-reactive antibodies has been utilized as a predictive marker for susceptibility to the development of autoimmune diabetes in genetically predisposed individuals. However, the exact contribution of such autoantibodies to the ontogeny of anti-islet autoimmunity remains to be elucidated. In this regard, maternal transmission of islet-reactive autoantibodies could serve to promote the initiation of (beta) cell inflammation in the susceptible child. The present proposal plans to determine the contribution of maternally derived diabetes associated Ig specificities in the development of diabetes in NOD offspring. The availability of genetically B cell deficient (uMT-/-) NOD mice provides a unique means of specifically eliminating maternal Ig from diabetes susceptible progeny. First, the applicant will assess whether maternal islet-reactive autoantibodies are transferred to NOD offspring. Next, the applicant will examine the impact of eliminating maternal antibodies on the evolution of islet-directed autoimmunity. Finally, the applicant will focus on determining the influence of the islet-reactive subset of maternally derived antibodies on diabetogenesis. In accomplishing these goals, it is hoped to gain insight into the contribution of diabetes associated autoantibodies to the initiation of islet inflammation. Such knowledge will permit the design of preventive measures aimed at reducing the risk of progression to diabetes in genetically susceptible children.

Insulin dependent diabetes mellitus (IDDM) in humans and NOD mice is an autoimmune disease caused by the selective destruction of pancreatic beta cells by autoreactive T lymphocytes. In addition to the well characterized loss of T-cell tolerance to islet autoantigens, the presence of anti-islet antibodies highlights a concomitant dysregulation of B-cell tolerance to these antigens. The requisite role of B-cells in diabetogenesis has recently been established in studies of B-cell deficient NOD mice which were found to be protected from insulitis and diabetes. Whether the role of B-cells in NOD diabetogenesis is mediated by the activated autoreactive B-cells and whether this role involves antigen presentation by B-cells in unknown. The investigator plans to study the mechanisms underlying the role of B-cells in NOD diabetogenesis. Thus, specific aim 1 will be focused on elucidating the contribution of MHC class I and class II mediated antigen presentation by B-cells to NOD diabetogenesis. In specific aim 2, studies are proposed to assess the contribution of the autoreactive subset of B- cells to NOD diabetogenesis by skewing the NOD B-cell repertoire away from diabetes associated Ig specificities. Specific aim 3 will address the importance of C3-mediated B-cell activation and antigen uptake in the development of anti-islet autoantibodies and diabetogenesis in NOD mice. In specific aim 4, the studies to be pursued aimed at elucidating the regulation of self-reactive B-cells in the NOD microenvironment. Insights into the mechanisms by which B-cells contribute to the etiology of autoimmune diabetes will provide the basis for the design of novel strategies for the prevention of IDDM.

Insulin dependent diabetes mellitus (IDDM) in humans and NOD mice is an autoimmune disease caused by the selective destruction of pancreatic beta cells by autoreactive T lymphocytes. In addition to the well characterize loss of T cell tolerance to islet autoantigens, the presence of anti-islet antibodies highlights a concomitant dysregulation of B cell tolerance to these antigens. The requisite role of B cells in diabetogenesis has recently been established in studies of B cell deficient NOD mice where were found to be protected from insulitis and diabetes. Whether the role of B cells in NOD diabetogenesis is mediated by the activated autoreactive B cells and whether this role involves antigen presentation by B cells is unknown. In the present proposal we plan to study the mechanisms underlying the role of B cells in NOD diabetogenesis. Thus, specific Aim 1 will be focused on elucidating the contribution of MHC class I- and class II-mediated antigen presentation by B cells to NOD diabetogenesis. In specific Aim 2 we propose studies to assess the contribution of the autoreactive subset of B cells to NOD diabetogenesis by skewing the NOD B cell repertoire away from diabetes associated Ig specificities. Specific aim 3 will address the importance of C3-mediated B cell activation and antigen uptake in the development of anti-islet autoantibodies and diabetogenesis in NOD mice. Finally, in specific aim 4 we will pursue studies aimed at elucidating the regulation of self-reactive B cells in the NOD microenvironment. Insights into the mechanisms by which B cells contribute to the etiology of autoimmune diabetes will provide the basis for the design of novel strategies for the prevention of IDDM.

Peripheral T cell tolerance mechanisms, unlike those operational in the thymus, target mature self reactive T cells. By virtue of acting on mature T cells, such mechanisms involved a tenuous balance between antigen mediated activation leading to effector function and deletion/anergy of mature autoreactive T cells. An important mechanism for the elimination of mature antigen reactive T cells involves a process of activation induced clonal deletion. The overall hypothesis tested in this proposal is that an imbalance in the homeostasis governing activation versus activation- induced clonal deletion of mature T cells contributes to autoimmunity. Using the NOD mouse as a paradigm of spontaneous T cell mediated autoimmune disease, our studies have demonstrated that mature NOD CD4+ T cells are characterized by a profound inertia in activation and resistance to activation induced death. Thus, in the present proposal we will first determine whether peripheral NOD CD4+ T cell activation is a stringent process requiring a higher threshold stimulus for triggering as compared to activation of non-autoimmune CD4+ T cells. We will then determine if in vivo activated aberrant activation properties of peripheral NOD CD4+ T cells result from intrinsic T cell characteristics or stem from unique properties of NOD APCs. An understanding of the mechanisms by which diabetogenic T cells abnormally persist in NOD mice can provide the basis for the design of novel strategies for the prevention of IDDM.

DESCRIPTION (Provided by applicant): The transplant program at the University of Pennsylvania (UP)has had a long-standing interest in the application of pancreatic islet transplantation in the treatment of diabetes mellitus. The program has continued basic investigations of the biological barriers to islet transplantation, with emphasis on the induction of immunological tolerance. Insights gained from experimental islet transplantation have led to recent successful applications of this therapy and achievement of euglycemia in patients with Type I diabetes mellitus. Based on these advances, a comprehensive islet transplantation program was established at the UP integrating a multidisciplinary team of investigative scientists and clinicians with recognized expertise in diabetes research and transplantation. Since the inception of the program in 1999, significant progress has been made in building its critical components: 1) construction of an Food and Drug Administrative (FDA)-compliant current good manufacturing practices (cGMP) facility devoted exclusively to islet processing; 2) recruitment and training of personnel qualified to perform islet isolation and assignment of dedicated transplant surgeons for on-site recovery of cadaveric pancreata; 3) establishment of a strong collaboration with the local organ procurement organization, which has led to a marked increase in the procurement of the human cadaveric pancreata; 4) distribution of isolated pancreatic islets to regional and national investigators engaged in diabetes research; and 5) development of a standardized islet quality index. In the present application, this strong infrastructure will serve as the mechanism for increasing the availability of human cadaveric pancreata suitable for preparation of isolated islets. This goal will be accomplished by procurement of pancreata from an expanded donor pool, including non-heart-beating organ donors. Another emphasis for the program will be the refinement of the islet quality index, based on physiological, biochemical, and morphological parameters, for correlation with the outcome of islet transplantation. With this strong platform in place, the program is poised to realize its goal of generating large quantities of highly purified and quality-controlled human islets for transplantation into Type I insulin-dependent diabetic patients.

DESCRIPTION (Provided by applicant): The transplant program at the University of Pennsylvania (UP)has had a long-standing interest in the application of pancreatic islet transplantation in the treatment of diabetes mellitus. The program has continued basic investigations of the biological barriers to islet transplantation, with emphasis on the induction of immunological tolerance. Insights gained from experimental islet transplantation have led to recent successful applications of this therapy and achievement of euglycemia in patients with Type I diabetes mellitus. Based on these advances, a comprehensive islet transplantation program was established at the UP integrating a multidisciplinary team of investigative scientists and clinicians with recognized expertise in diabetes research and transplantation. Since the inception of the program in 1999, significant progress has been made in building its critical components: 1) construction of an Food and Drug Administrative (FDA)-compliant current good manufacturing practices (cGMP) facility devoted exclusively to islet processing; 2) recruitment and training of personnel qualified to perform islet isolation and assignment of dedicated transplant surgeons for on-site recovery of cadaveric pancreata; 3) establishment of a strong collaboration with the local organ procurement organization, which has led to a marked increase in the procurement of the human cadaveric pancreata; 4) distribution of isolated pancreatic islets to regional and national investigators engaged in diabetes research; and 5) development of a standardized islet quality index. In the present application, this strong infrastructure will serve as the mechanism for increasing the availability of human cadaveric pancreata suitable for preparation of isolated islets. This goal will be accomplished by procurement of pancreata from an expanded donor pool, including non-heart-beating organ donors. Another emphasis for the program will be the refinement of the islet quality index, based on physiological, biochemical, and morphological parameters, for correlation with the outcome of islet transplantation. With this strong platform in place, the program is poised to realize its goal of generating large quantities of highly purified and quality-controlled human islets for transplantation into Type I insulin-dependent diabetic patients.

DESCRIPTION (provided by applicant): The Diabetes Control and Complications Trial (DCCT) established that the microvascular complications of diabetes can be prevented by maintaining near normal glycemic control in patients with type 1 diabetes (T1D). This seminal outcome has provided a strong impetus for developing effective tolerogenic strategies for a cell replacement via pancreas or isolated islet transplantation in T1D patients. The recent success of the "Edmonton protocol" was a major advance in the field of islet transplantation. However, despite initial achievement of an insulin independent state, a progressive loss of beta cell function culminates in the recurrence of diabetes in a majority of these recipients. Thus, the Edmonton approach, which targets the T lymphocyte compartment alone, seems insufficient in preventing immunological rejection and recurrent anti-beta cell autoimmunity. Despite the critical role of T lymphocytes in these two processes, it is established that a concomitant and specific B lymphocyte response against beta-cell derived allo- and auto-antigenic epitopes also occurs. In rodent studies we demonstrated that interruption of B lymphocyte function curtails the T cell mediated destruction of islet allografts. Thus, we hypothesize that an induction immunotherapy regimen which targets both the B- and T lymphocyte compartments, will promote immunological tolerance to islet allografts. Our preclinical studies test this premise in non-human primates (NHPs) and indicate that the combined use of anti-B and T-lymphocyte antibodies (i.e., Rituximab and Thymoglobulin) results in long-term islet allograft survival without the need for maintenance therapy with a calcineurin inhibitor (CNI) agent. In the present application we propose to determine the efficacy of combined B- and T- lymphocyte directed immunotherapy for promoting immunological tolerance to islet allografts in T1D patients. We will initiate a clinical trial which will: 1) build upon our experience with the "Edmonton protocol", by incorporating the B lymphocyte specific monoclonal antibody, Rituximab, into its induction regimen and 2) assess the efficacy of a combined induction regimen including Thymoglobulin and Rituximab followed by CNI-free maintenance monotherapy with Rapamycin. Importantly, the latter protocol, which parallels that used in our preclinical NHP studies, will permit the inclusion of T1D patients with microalbuminuria into islet transplantation trials. A series of prospective in vivo metabolic studies will be undertaken to specifically evaluate beta cell function and secretory capacity following transplantation that 1) includes Rituximab and 2) eliminates CNI agents. Our mechanistic studies are designed to test the hypothesis that Rituximab immunotherapy provides a "tolerogenic window" for the reconstituting B lymphocyte repertoire, during which allo- and auto-reactive clones with a transitional phenotype are subject to negative selection. Following islet cell transplantation we will: 1) monitor the development of alloantibodies to HLA antigens and autoantibodies to islet antigens, 2) analyze the immunoglobulin repertoire for clonal persistence and heterogeneity using antibody CDR3 spectratyping, 3) survey the cytokine profiles of autoantigen-specfic T cells reactive to islet beta-cells by ELISpot, and 4) perform immunophenotyping and functional assays of circulating lymphocytes to assess global alterations in lymphocyte differentiation and energy. Overall, the proposed studies will determine whether a balanced immunotherapy regimen targeting the B- and T- lymphocyte compartments promotes a state of immunological tolerance to islet allografts, while obviating the need for chronic immunosuppression.

DESCRIPTION (provided by applicant): Islet transplantation is the most specific therapy for restoration of euglycemia in patients with type 1 diabetes. Despite the recent success of clinical islet transplantation, achievement of long-term allograft survival remains an elusive goal even in the presence of chronic immunosuppression. Therefore, a major objective in advancing the field of islet transplantation is to develop novel immune intervention strategies capable of inducing permanent allograft survival while eliminating the chronic use of immunosuppressive agents. Presently the mainstay of immunosuppressive therapy for islet transplantation includes induction with T lymphocyte specific antibodies followed by maintenance with T cell specific pharmacological agents. This 'T-cell focused' approach is based on the well-established function of T lymphocytes as the effector population responsible for graft rejection. Notwithstanding the logic of this approach, both clinical and experimental data clearly indicate that T cell directed immunosuppression fails to promote immunological tolerance to islet allografts. Here, we suggest that a more balanced consideration of the components of the adaptive immune response to allografts provides a strong rationale for an integrated approach to immunosuppression: one including both the T- and B- lymphocyte compartments. It is well established that host B lymphocytes, in addition to the alloreactive T cell compartment, mount a robust immune response against donor alloantigens following transplantation. Despite this well-recognized feature of the alloimmune response, the B lymphocyte compartment has been largely neglected as a target of induction immunotherapy. Our preliminary exploratory studies have indicated that host B lymphocytes play a key role as antigen presenting cells in the pathogenesis of acute allograft rejection. Furthermore, our pilot studies in non-human primate recipients suggest that transient B lymphocyte depletion using an anti-CD20 mAb (i.e., Rituxan) at the induction phase promotes long-term islet allograft survival, while minimizing the need for chronic immunosuppression. These results have compelled us to propose a more balanced approach to induction immunotherapy by integrating both T- and B- lymphocyte specific antibodies (i.e., Thymoglobulin and Rituxan) into a tolerogenic regimen. In this project, we will determine the efficacy of such an induction immunotherapy regimen in a rigorous preclinical model and investigate its mechanistic basis in promoting islet allograft survival in non-human primates. Our overall contention is that interruption of T- and B- lymphocyte function during the induction phase represents a novel and potentially powerful means of promoting immunological tolerance to islet allografts.

[unreadable] DESCRIPTION (provided by applicant): The transplant program at the University of Pennsylvania has had a long standing interest in the application of pancreatic islet transplantation for treatment of type 1 diabetes mellitus. The comprehensive islet transplantation program at the University of Pennsylvania was established with the primary objective of uniting a group of multidisciplinary scientists and clinicians to focus their expertise on the development of novel strategies for successful islet transplantation. Since the inception of the program critical scientific and complimentary infrastructure milestones have been achieved that include: 1) construction of an FDA compliant cGMP facility devoted exclusively to islet processing, 2) recruitment and training of personnel qualified to perform islet isolation and assignment of dedicated transplant surgeons for on-site recovery of human pancreas, 3) establishment of a strong collaboration with the local organ procurement organization which has led to marked increase in the procurement of deceased donor human pancreas, 4) distribution of isolated pancreatic islets to regional and national scientist engaged in diabetes research 5) initiation of clinical islet transplantation in T1D subjects 6) establishment of a GCRC based program for metabolic evaluation of islet transplant recipients, 7) development of a standardized islet quality index based on the biochemical and physiological parameters, 8) development of innovative immunotherapy protocols for induction of immunological tolerance to islet allografts. [unreadable] [unreadable] In the present proposal this strong infrastructure will serve as the mechanism for further growth of our ICR program in the acquisition of human deceased donor pancreas which will be critical for production of clinical grade islets for transplantation. This goal will be accomplished by procurement of pancreas from an expanded donor pool including non-heart beating organ donors. In addition we will continue to recover and distribute islets from type 2 diabetic donors for physiologic and genomic investigations into the pathogenesis of type 2 diabetes. Importantly, the leading scientists with expertise in the islet biochemistry, functional genomics, proteomics and islet development and neogenesis will utilize state of the art bioassays to develop an islet quality index with predictive value in the decision making algorithm prior to transplantation of the islet preparation. The ICR program at Penn will continue to be an active member of the steering committee; utilizing the bioinformatics resources of the ABCC, we will share our clinical and basic research data with other ICR centers for collaborative investigations to advance the biology of islet processing and transplantation. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Islet transplantation is the most specific therapy for beta cell replacement and achievement of glucose homeostasis. However, despite a T cell-directed immunotherapy, majority of islet allografts, succumb to rejection~ typically co-incident with the production of donor specific IgG alloantibodies, which in addition to being poor prognostic clinical indicators are thought to exert a pathogenic role in vivo. Our preliminary studies in a murine model indicated a requisite role of B-cell antigen presentation in activation o alloreactive CD4 T lymphocytes. Therefore, our contention is that the induction of robust transplantation tolerance will require unresponsiveness at the level of both the B- and T-cell compartments. As such, we performed a preclinical trial of islet transplantation in Cynomolgus macaques utilizing an induction immunotherapy regimen, which included a CD20 specific B cell depleting agent. The results of this trial indicated that transient B cell depletion at the time of transplantation protects islet allografts from rejection for a significant length of time. However, donor-specific IgG alloantibodies were eventually produced in the majority of the recipients, coincident with the loss of islet graft function. Therefore, the main mechanistic proposition of th present application is that establishment of B cell tolerance is required for achievement of immunological tolerance to islet allografts. Our goal is to develop a clinically feasible, B cell-directed immunotherapeutic strategy based on the homeostatic mechanisms governing the development of B cell tolerance to self-antigens. To this end, we aim to induce donor-specific B cell tolerance by targeting the key regulatory pathway of B cell survival, life-span and selection: the TNF-related cytokine known as BLyS/BAFF. It was recently recognized that this cytokine regulates antigen mediated negative selection of newly emerging transitional B cells and, thereby, serves as the major micro-environmental cue responsible for shaping the mature B cell repertoire. Here, we hypothesize that limiting the availability of systemic BLyS/BAFF during B cell compartment reconstitution in the presence of an islet allograft will promote a sustained state of B cell tolerance. We will test this clinically pertinent concept in the setting of islet alo-transplantation in Cynomolgus monkeys. PUBLIC HEALTH RELEVANCE: The rejection of transplanted organs places a major burden on public health resources in the United States. Furthermore, the requirement for life-long immunosuppression to maintain transplant function not only poses a major cost burden on the health care system, but also imposes a dramatic degree of morbidity on the recipient. In this application, we propose a novel avenue of investigations into the possibility of inducing donor specific immunological tolerance to allografts by targeting recipient B-lymphocytes.

Penn Human Pancreas Procurement and Analysis Program Abstract Utilizing our existing infrastructure and scientific collaborations, we have assembled 6 cores with expertise ranging from pancreas procurement and islet isolation to data integration for a comprehensive and integrated Human Pancreas Procurement and Analysis Program based at the University of Pennsylvania. Core A will procure a spectrum of human pancreata and detailed donor medical history; perform high resolution HLA typing by next generation sequencing; isolate islets; and distribute islets and tissues to the other Cores for further analysis or processing. Core B will perform physiological phenotyping on the isolated islets. Core C will quantify and characterize memory T cell subsets by flow cytometry and single cell qPCR analysis; characterize suppressive activity of Tregs and the ability of related effector cells to be suppressed; B cell phenotyping; and generate chromatin accessibility maps of enhancers in pathogenic cell types. Core D will perform multiple advanced modalities for the molecular profiling of isolated islets including RNAseq and microRNAseq of sorted islet cell populations; mass cytometry for single cell quantification of more than 20 cell surface and intracellular markers; and single cell RNAseq. Core E will process tissues using multiple modalities that will allow for analysis using advanced technologies such as multiplexed immunoflourescent staining, combinatorial barcoded FISH (combFISH), whole slide imaging, and quantitative image analysis of protein markers and immune cell infiltrates. This Core will adapt 2-dimensional mass cytometry to pancreatic sections utilizing multiplexed ion beam imaging (MIBI) technology. This Core will also archive tissues as well as DNA and blood, and facilitate sample distribution to HPPAP approved researchers. Finally, Core F will assemble, annotate and maintain an open access database for the Program and its member-researchers, and collaborate with the HIRN in the sharing of data from both programs. The entire Program will be executed by an Administrative Core consisting of the PIs, with assistance from an Executive Committee consisting of the core leaders. The Administrative Core will interface with an external committee to review applications for HPPAP biosample use, and will collaborate with the HIRN. The Program will also interact with the HPPAP member/PANC DB user community to provide a richly annotated source of physiologic, genomic and immunologic data on the tissue-based landscape governing T1D.

Penn Human Pancreas Analysis Program ? T2D Abstract Building on our existing infrastructure and scientific collaborations, and the expertise gained during the first two years of the HPAP effort for Type 1 Diabetes, we have assembled five cores with expertise ranging from pancreas procurement and islet isolation to data integration. These Cores form a for a comprehensive and integrated Penn Human Pancreas Analysis Program ? T2D based entirely at the University of Pennsylvania. Core A will procure a spectrum of human pancreata and detailed donor medical history; perform high resolution HLA typing by next generation sequencing; isolate islets; and distribute islets and tissues to the other Cores for further analysis or processing. Core B will perform physiological phenotyping on the isolated islets. Core C will perform multiple advanced modalities for the molecular profiling of isolated islets including RNAseq, ATACseq and DNA methylome analysis of sorted islet cell populations; single cell ATACseq and RNAseq, and mass cytometry for single cell quantification of more than 20 cell surface and intracellular markers. Core D will process tissues using multiple modalities that will allow for analysis using advanced technologies such as multiplexed immunoflourescent staining, whole slide imaging, and imaging mass cytometry. This Core will also archive tissues, DNA and blood, as well as other T2D-relevant organs such as skeletal muscle, intestine, adipose tissue and liver for future use by other NIDDK-approved consortia. Finally, Core E will assemble, annotate and maintain an open-access database for the Program and its member-researchers, and collaborate with the HIRN in the sharing of data from both programs. The entire program will directed by an Executive Committee consisting of the core leaders and the PI, who will be the interface with HIRN and NIDDK leadership. HPAP-T2D will provide physiologic, genomic, genetic, and histological analysis of the pancreas in type 2 diabetes at unprecedented detail, share the rich data with researchers world-wide before publication, and thus enable breakthrough discoveries in our understanding of this disease that has reached epidemic levels world-wide.

The Human Pancreas Analysis Program for Type 1 Diabetes (HPAP-T1D) Abstract In 2016, the NIH NIDDK selected a multi-disciplinary team of investigators from three institutions (UPENN, Vanderbilt, University of Florida) to establish the pilot phase of the Human Pancreas Analysis Program (HPAP). Over the past three years, type 1 diabetes (T1D)-relevant tissues from more than 50 organ donors were profiled at the anatomic, physiologic, metabolic, immunologic, genomic and epigenomic levels. The resulting data were compiled and organized into the publicly accessible PANC-DB database and website. Here, we propose not only to continue, but to expand our efforts to apply and develop state-of-the-art technologies designed to phenotype and molecularly profile human tissues relevant to the etiology of T1D through a series of innovative efforts by six Cores. Core A (Pancreas Procurement and Islet Isolation) will procure/process pancreatic islets, pancreas and lymphoid organs, expand donor outreach (in collaboration with the well-established nPOD program) and increase the collection of non-pancreatic tissues. Core B (PhysiologicalPhenotyping)will provide a comprehensive metabolic profile and probe the key regulatory steps that govern hormone secretion from the major pancreatic endocrine cell types. Core C (Immunobiology) will develop an immune atlas of peripancreatic lymphoid populations, obtain transcriptomic profiles of the T1D- specific T cells, and perform immune repertoire profiling of B and T cells in association with single cell and antigen-specific cell approaches. Core D (Advanced Molecular Profiling) will perform RNAseq, ATACseq and DNA methylome analysis on sorted alpha-cell, beta-cell and exocrine cell population as well as scRNAseq and scATACseq and carry out whole genome sequencing. In addition, islet endocrine and major lymphocyte populations will be quantified precisely using flow CyTOF. Core E (Tissue Analysis & Biobanking) will analyze pancreatic tissue architecture and immune cell/epithelial cell interactions using multiple modalities including imaging mass cytometry, multi-spectral imaging and CODEX. Complete image data will be made available via PancreatlasTM and PANC-DB. Finally, Core F (PANC-DB, Data Analysis and Integration) will expand the PANC-DB resource by adding new features that will make the public web page even more useful, as well as add a Computational Biology and Data Science Unit for applying state-of-the-art analytical tools, allowing for the integration and visualization of generated datasets using different experimental modalities such as multi-spectral imaging and omics technologies. In addition, Core F will continue to expand its outreach activities, exemplified via the deposition of transcriptome and epigenome data into the Diabetes Epigenome Atlas (DGA). HPAP-T1D will be directed by an experienced, collaborative multi-PI team that confers weekly and will meet in-person on a biannual basis in coordination with NIDDK leadership to review the progress of the entire program.

Abstract Kidney transplant is the treatment of choice for patients with end-stage renal disease (ESRD), as it extends survival, improves quality of life and is highly cost-effective. However, for about a third of patients on the waitlist, pre-existing anti-HLA antibodies (i.e. allo-sensitization) presents a major barrier to successful transplant. HLA antibody responses are maintained by memory B cells (Bmem) and plasma cells (PC). Unfortunately, desensitization approaches have been largely ineffective due to incomplete depletion of allo-specific B cells and PCs. WE HYPOTHESIZE that stringent depletion of donor-specific B cells and PCs is required for a clinically significant reduction of allo-antibodies necessary to achieve successful kidney transplantation. We have shown that engineered T cell immunotherapies employing synthetic chimeric antigen receptors (CARs) can induce durable remission of B cell lineage and plasma cell malignancies. Two CAR-T cell therapies that target CD19 (CART-19) and B cell maturation antigen (CART-BCMA) result in depletion of malignant cells but also physiologic B cells and PCs. Importantly, we have shown that CART-BCMA and CART-19 can be safely administered together. Based on this experience, our GOAL is to leverage this innovative platform to target Bmem and PCs and promote reduction of preformed anti-HLA antibodies, thus providing a window of opportunity for transplantation. Specifically, we propose a single-arm proof-of-concept CLINICAL TRIAL that combines CART-19 with CART-BCMA as a novel desensitization measure in kidney transplant candidates with a cPRA ?99.9%. MECHANISTIC studies will evaluate the cellular, humoral and molecular immune correlates of CART-19 + CART-BCMA immunotherapy in highly sensitized kidney transplant candidates. These are focused on the CAR T cells, T- and B-cell immunity (both allo-specific and protective) and, in the event of successful transplantation, the allograft biology. An INFECTIOUS DISEASE STUDY proposal will evaluate vaccine response and immune function in our renal transplant candidates, including our study cohort. The multi-center team (Penn, NYU, MGH) brings together investigators with extensive experience in CART therapy, desensitization, and outstanding depth of laboratory expertise to carry out robust mechanistic studies.