JDRF Autoimmunity

Center Abstracts

Cord Blood Therapies for Type 1 Diabetes

Mark Atkinson

of

University of Florida

Applicant: Atkinson, Mark A.

Composite: Cord Blood Therapies for Type 1 Diabetes

SCIENTIFIC ABSTRACT

Objective: Identifying the therapeutic potential of umbilical cord blood (UCB) for tolerance induction, and defining the role for two UCB constituents (i.e., Treg, mesenchyml stem cells (MSC)) in the process of imparting immune regulation.

Background/Rationale: We recently received FDA approval for a highly innovative pilot study aimed at the reversal of recent onset T1D utilizing autologus UCB transfusion in that study's protocol, initially approved for 14 individuals, subjects with recent onset T1D receive intravenous infusion of autologus UCB (obtained from banked material). The primary purpose of this effort is designed to address issues related to safety, whereas questions related to the metabolic and immunologic aspects are subject to farm more limited investigation. That trial (which is ongoing but nearing enrollment completion at the time of this application's submission) has, in fact, proven a success in that the therapy not only appears "safe" but in addition, a trend suggestive of a potential therapeutic benefit has been identified. At the same time, a clear need for improving and expanding this line of investigation exists in that our knowledge of the mechanisms underlying this form of intervention remains poor. Furthermore, recent advances in our understanding of immune regulation provided by Treg and MSC suggest additional, perhaps even improved opportunities for therapeutic intervention utilizing these cells, when derived from UCB, are warranted. Taking these notions together, we believe the studies proposed in this Center/Academic R&D application represent a novel opportunity to identify the therapeutic capacity of UCB and its cellular constituents to prevent/reverse T1D, induce tolerance, and understand the contributions of Treg and MSC to both the process of tolerance induction as well as the pathogenic defects which underly the natural history of T1D.

Description of Project: The overall purpose of this Center application is to test whether T1D can be prevented or reversed by transfusion of UCB or two populations of cells derived from UCB, Treg and MSC. In addition to evaluating therapeutic efficacy, the Center's Projects and Cores seeks to collectively improve our mechanistic understanding of the potential of UCB to modulate immune reactivities (in general) and induce tolerance in an autoimmune setting, with particular focus on the role for Treg and MSC in these processes. The successful completion of these studies could uncover an innovative method for curing T1D as well as for inducing tolerance, establish the foundation for additional/expanded investigations on the potential for this therapy to treat immune based-disorders, and expand our insights as to the role for Treg and MSC in the process of immune regulation in early childhood.

Anticipated Outcome: The successful completion of these studies will impact an extensive number of significant needs, includeing: 1) To uncover novel means for safe-yet-effective cell based therapies for tolerance induction and in this particula case, T1D prevention and reversal; 2) To understand the immunoregulatory potential of UCB, as well as the role for Treg and MSC in this process; 3) To identify novel assays of cellular immunity and immune tolerance in T1D; 4) To understand key differences in the regulatory capacity of UCB versus other tissue sources (i.e., peripheral blood, bone marrow) in early childhood.

Relevance to Type I Diabetes: The programs investigators believe that the information gleaned from these studies represent KEY underpinnings to uncovering a means to prevent or reverse type 1 diabetes. Applicant: Schatz, Desmond A.

Project 1: Autologus Umbilical Cord Therapies for Type 1 Diabetes

SCIENTIFIC ABSTRACT

Objective: Project 1 seeks to identify the therapeutic potential of autologus umbilical cord blood (UCB), or specialized immune cells derived from UCB in subjects with- or at increased-risk for T1D.

Background/Rationale: Rationale supporting such an effort finds its basis on multiple fronts. First, in 2005, we received FDA approval for a highly innovative pilot study whose goal involved analysis of the safety and feasibility of autologus UCB transfusion in those with T1D; an effort that was highly successful in terms of recruitment and suprizingly, subject availability. Second, we and others within this Center application have found multiple lines of evidence supporting a role for Treg and MSC in T1D (humans or animal models); efforts that support a potential role for such cells in regulating tolerance. Third, while multiple interventions have recently been approved for therapeutic testing in individuals’ with- or at increased-risk for T1D (many being part of NIH TrialNet or ITN), very few of these therapies are available for young individuals. This, combined with TrialNet’s recently re- initiating studies aimed at identifying individuals at increased risk for T1D, will result in a group of at-risk subjects for whom little to no therapeutic options seeking to delay or prevent the disease are available. Finally, we believe the studies proposed in this application would be greatly aided by the investigational resources and insights into the mechanisms underlying autoimmunity afforded by the network of JDRF Autoimmunity Centers.

Description of Project: The proposal will test the hypothesis that T1D can be prevented or the rate of metabolic loss slowed by transfusion with autologus umbilical cord or its cellular derived products. In addition to evaluating therapeutic efficacy, Project 1’s specific aims also seek to improve our mechanistic understanding of the potential of UCB or its cell derived products to modulate immune reactivities (in general) and induce tolerance in an autoimmune setting. These studies will also, through studies of UCB derived Treg and MSC from subjects with- or at increased-risk for T1D, allow for examination of potential defects in their activity leading to the development of T1D or respond to current therapies; questions that will not only be performed by Projects 1, 2 & 3, but in addition, by the very innovative Core B utilizing Humanized Mouse Models. Finally, Project 1 will find much scientific support and potentially, avenues for therapeutic enhancement by knowledge gains provided by Core C; one of the world’s leading academic centers for cell therapies.

Anticipated Outcome: The successful completion of these studies could uncover an innovative method for tolerance induction, establish the foundation for additional/expanded investigations on the potential for this therapy to treat immune based-disorders, and expand our insights as to the role for Treg and MSC in the process of immune regulation in early childhood.

Relevance to Type I Diabetes: The program investigators believe that the information gleaned from these studies represent KEY underpinnings to uncovering a means to prevent or reverse type 1 diabetes.

Applicant: Riley, James L.

Project 2: Expanded Umbilical Cord Blood Regulatory T Cells for the Treatment of T1D

SCIENTIFIC ABSTRACT

Objective:

The central theme of this application is that improved T cell culture systems can enable successful adoptive transfers of CB TREG cells by improving expansion, engraftment, persistence and function.

Background/Rationale:

Murine models have demonstrated expanded TREGS can prevent or reverse type 1 diabetes (T1D), but this has not yet been attempted in humans yet in part due to the complexity of peripheral blood (PB) which makes identifying and purifying TREGS in PB difficult. In contrast, umbilical cord blood (CB) T cells are mainly comprised of just two populations, naïve and TREG cells. Thus, CB TREGS are attractive for therapeutic applications because they are largely free of effector cells, easily purified and have their full replicative capacity intact. The availability of T1D patients who have frozen, autologous CB presents a tremendous opportunity to test whether ex vivo expanded Treg cells can prevent or reverse T1D disease progression in humans. Recent data from our laboratory and others indicates that human, CB derived TREG subsets have distinct growth requirements from peripheral TREG subsets and effector T cells, yet there are no clinically scalable and FDA compliant culture systems optimized for CB TREG. Our strategy is to develop novel artificial antigen presenting cells (aAPC) specifically engineered to propagate human CB TREGS with potent suppressor functions and to test the ability of these TREGS to prevent progression of T1D using the state of art humanized murine models (Core B). Our project is supported by extensive preliminary data as we have already constructed a large panel of aAPCs to expand human PB TREGS, established robust assays to measure TREG function and have created an efficient system to rapidly generate antigen specific T cells.

Description of Project:

The following two specific aims will test the central hypothesis that optimally expanded human CB TREG cell subsets can control T1D progression: (1) Define the optimal aAPC to expand CB TREGS for therapeutic use; (2) Determine whether antigen specific CB TREGS are more effective in controlling T1D than polyclonal TREG cells.

Anticipated Outcome:

Project 2 will export the optimal TREG aAPC technology to Core C for adaptation to large scale tissue culture under clinical grade GMP manufacturing conditions for clinical testing and eventually this technology will employed in a Phase I clinical trial (Project 1). Project 2 will work very closely with Core B to test the function of expanded TREGS in state of the art humanized murine models and in the later years of the grant, Project 2 will work closely with Project 3 to explore whether synergy exists between mesenchymal stem cells and TREGS in the treatment of T1D.

Relevance to Type I Diabetes:

The program investigators believe that the information gleaned from these studies represent KEY underpinnings to uncovering a means to prevent or reverse type 1 diabetes. Applicant: Sayegh, Mohamed H.

Project 3: Immunomodulatory Functions of Human MSCs in Type 1 Diabetes

SCIENTIFIC ABSTRACT

Objective:

The overall goal of Project 3 is to characterize human MSCs obtained from peripheral blood, cord blood and bone marrow, as well as to identify the immunomodulatory functions of these cells as they relate to the pathogenesis and natural history of T1D.

Background/Rationale:

While the developmental plasticity of mesenchymal stem cells (MSC) has resulted in their becoming the focus of a remarkable degree of scientific interest (given their potential application for replacing injured tissues), MSCs have also been noted for their capacity to provide profound immunomodulatory activities in vivo. As a result, MSC therapy has recently become the therapeutic focus for a number of clinical trials involving a wide variety of human disorders, including those autoimmune in nature. However, quite remarkalbly, that list of trials does not include autoimmune type 1 diabetes (T1D). Indeed, as of today, the role of MSCs in the pathogenesis of human T1D remains largely unexplored, and their therapeutic potential for attenuating this disease is virtually unknown.

Description of Project:

Project 3 (both alone and in combination with Core B – Humanized Mouse Models Core) should provide data allowing for an improved understanding of the mechanisms leading to the beneficial effects of autologous cord blood transfusion in T1D. In addition, through cooperative arrangements with Core C (Cell Separation and Expansion Core), Project 3 will work with Project 1 to establish a clinical trial seeking to test the efficacy of MSC therapy to prevent or reverse T1D.

Anticipated Outcome:

Our overall hypothesis is that MSCs, through their immunomodulatory activities, will provide an ideal cell-based therapy for the prevention or reversal of T1D. This hypothesis is largely but not exclusively based on our preliminary studies in the NOD mouse model of T1D utilizing bone marrow-derived MSCs. By extension, we would posit that cord blood MSCs possess immunomodulatory functions that have the capacity to control the autoimmune responses of autoreactive T cells in individuals with T1D who are subject to cellular transfusion. These immunomodulatory functions would involve a tri-cell interaction of MSCs, dendritic cells and regulatory T cells, resulting in a tipping of the balance towards regulation of the autoreactive T cell repertoire, preventing the autoimmune destruction of insulin-secreting beta cells.

Relevance to Type I Diabetes:

We believe that results of this research will provide vital mechanistic data to support the concept of translating cord blood therapies, including those involving MSCs, to individuals with T1D. The program investigators believe that the information gleaned from these studies represent KEY underpinnings to uncovering a means to prevent or reverse type 1 diabetes. Applicant: Atkinson, Mark A.

Core A: Administrative Core

SCIENTIFIC ABSTRACT

Objective:

The purpose of the Administrative Core is to serve as a central element for fiscal and scientific coordination for this Center award.

Background/Rationale:

While systems analysis has identified many elements resident to “successful” multi- investigator/multi-institutional research programs, one key aspect is that of effective leadership, supported by Administrative unit, to serve as a central element for communication as well as fiscal and scientific coordination.

Description of Project:

The four goals of this Administrative Core A in this Center application are as follows: 1) To coordinate the budgetary and fiscal aspects of the Program. The proposed Program involves direct cost disbursements to investigators of the three Projects (Project 1 – Umbilical Cord Blood; Project 2 – Cord Blood Regulatory T Cells; Project 3 – Cord Blood Mesenchyml Cells) and three Cores (Core A – Administration; Core B – Humanized Mouse; Core C – Cell Seperation and Expansion). Careful administrative oversight will be provided by Core A in order to provide JDRF staff and lay reviewers annual reports containing sound fiscal information as well as a straightforward indication of scientific progress. 2) To facilitate communication among investigators within the Center. This will take form through performance of many activites including regularly scheduled meetings between Program investigators, organization of Center retreats, training of Project Investigators by Program Cores, and more. 3) To work with an External Advisory Committee and ad hoc JDRF reviewers (both MSRC and LRC) to provide ongoing feedback with regard to the progress and success of the Center. In addition to being responsive to the annual external review, a Program Executive Committee (comprised of the Project and Core Pis) will meet on an ongoing basis and provide counsel to all Program Investigators regarding their program and recommendations for improvement. 4) To organize the collection of human materials, generate appropriate data sets, and facilitate communication of Program results.

Anticipated Outcome:

We believe Core A will represent a key component to an effective Program, and one that will provides both short- and long-term benefits in seeing the Center, as well as JDRF, meet their stated goal; providing a means to prevent and reverse type 1 diabetes.

Relevance to Type I Diabetes:

The programs investigators believe that the information gleaned from these studies represent KEY underpinnings to uncovering a means to prevent or reverse type 1 diabetes. Applicant: Greiner, Dale L.

Core B: The Humanized Mouse Core

SCIENTIFIC ABSTRACT

Objective:

The Overall Goal of the Humanized Mouse Core (Core B) is to establish humanized mouse models that will support studies within the Center seeking to identify the mechanisms by which autologous umbilical cord blood (UCB) infusion provides therapeutic benefit for individuals with type 1 diabetes (T1D).

Background/Rationale:

To accomplish this goal, the Core will model the Center’s clinical trials by performing experiments involving infusion of autologous UCB (Project 1), regulatory T cell (Treg) expanded from UCB (Project 2), or UCB derived mesenchymal stem cells (MSC; Project 3) into unique immunodeficient mice; animals that have been engrafted with an intact human immune system established from the same cord blood donor. The Humanized Mouse Core will be responsible for the establishment, characterization, maintenance, treatment, and manipulation of humanized mice for all studies posed by the Center’s investigators. Core B will also be responsible for developing and characterizing new stocks of genetically modified mice to meet the emerging needs of the Center, as well as to establish and validate new humanized mouse models as needed for specific goals of the Center’s projects.

Description of Project:

The specific objectives and responsibilities of the Humanized Mouse Core include: 1. Provide humanized mouse models amenable to studies of human immune responses in vivo; 2. Develop procedures wherein Humanized Mouse Models will recapitulate conditions similar to the therapeutic trials posed within the Center; and 3. Perform trafficking studies and immunomodulatory analyses of human cell populations that are generated by Projects within the Center involving humanized mice.

Anticipated Outcome:

Core B will provide a remarkably innovative and novel in vivo model for uncovering the mechanisms by which UCB therapies modulate immune responses and hopefully, lead to the prevention and reversal of T1D.

Relevance to Type I Diabetes:

The program investigators believe that the information gleaned from these studies represent KEY underpinnings to uncovering a means to prevent or reverse type 1 diabetes.

Applicant: Levine, Bruce L.

Core C: Cell Separation and Expansion Core

SCIENTIFIC ABSTRACT

Objective: The rationale for the Clinical Cell and Vaccine Production Facility (CVPF) to serve as the Cell Separation and Expansion Core.

Background/Rationale: Core C, is justified by the fact that experimental cell therapy requires focused scientific, technical and regulatory expertise and resources that are not available to most individual researchers. The mission of the CVPF is to enable the translation from research bench to bedside of novel cellular therapies. The first element in meeting this mission is scale-up and validation of cell processing isolation and culture procedures. The CVPF was the first to develop a method for efficient large- scale culture of purified CD4+ T cells from HIV+ study subjects for a clinical trial initiated in 1996. Since that time, the CVPF has developed clinical scale technology for the support of over 20 clinical trials in various diseases, including the world’s first lentiviral vector clinical trial. The second part of the CVPF mission is to perform cell processing of donor and patient cells to be reinfused as investigational cellular vaccines. This includes performing release testing (Quality Control) that addresses the FDA requirements for demonstrating safety, purity, potency and identity of the cellular vaccines. To date, the CVPF has produced 300 vaccines for 180 people enrolled in these trials. Finally, translation of research procedures for use in cell therapy clinical trials requires a detailed knowledge of current Good Manufacturing Practices (cGMP) and regulations enforced by the FDA.

Description of Project: The ultimate goal of Core C is to support the development of clinical grade umbilical cord derived regulatory T cells (Tregs) for use in patients with Type 1 diabetes. To meet this goal, Core C will support Center projects and develop methods needed for cord blood Treg cell separation and expansion. The first major aim of Core C is to develop and validate the best clinically compatible methods for the isolation and culture of Tregs from cord blood and provide these cells for the studies proposed in Project 2. Following pre-clinical validation and scale-up studies, Core C will isolate, expland, test and ship cord blood Tregs to the clinical site for the clinical trial proposed in Project 1. Leveraging expertise in mesenchymal progenitor cells, Core C will also collaborate with Project 3 in developing and validating clinically compatible methods for isolation and culture of these cells from bone marrow and cord blood. Finally, Core C will provide cGMP integrated clinical regulatory expertise in support of the investigational New Drug application to the FDA. The second major aim of the Core C is to establish a cGMP Master Cell Bank (MCB) and characterize a Working Cell Bank (WCB) for a second generation artificial Antigen Presenting Cell (aAPC) line obtained from Project 2. Following quality control and potency testing, this line will be able to be used in the expansion of cord blood Tregs for the cell therapy clinical trial described in Project 1.

Anticipated Outcome: This core represents a vital component to attempts seeking to utilize cell based therapies as a means to prevent or reverse type 1 diabetes.

Relevance to Type I Diabetes: The program investigators believe that the information gleaned from these studies represent KEY underpinnings to uncovering a means to prevent or reverse type 1 diabetes.

The JDRF Collaborative Center for Cell Therapy

Jeffrey Bluestone

of

UCSF

SCIENTIFIC ABSTRACT The JDRF Collaborative Center for Cell Therapy (J. Bluestone, UCSF)

Objective:

The mission of the JDRF COLLABORATIVE CENTER FOR CELL THERAPY at the University of California, San Francisco (UCSF), Columbia University (CU), La Jolla Institute for Allergy and Immunology (LIAI), Benaroya Institute (BI) and the University of Pennsylvania (UPenn) is to promote basic and clinical research that will lead to successful antigen-specific regulatory T cell therapy in patients with ongoing auto-aggressive Type 1 Diabetes.

Background/Rationale:

The approach is multi-faceted taking advantage of the novel tolerogenic and autoantigen-specific properties of regulatory T cells pioneered by these research groups. The insights into the generation, expansion and functional characterization of regulatory T cells, combined with superb tools for conducting a successful clinical program will allow early proof of principle phase I/II trials using antigen-specific Tregs developed under cGMP conditions.

Description of Project:

In an effort to take advantage of the current basic biology and move aggressively towards the first clinical trials in type 1 Diabetes, the following projects will be conducted: Project #1 (Bluestone) Expansion of Tregs for the treatment of autoimmune diabetes Project #2 (June) Second Generation APCs as stimuli of polyclonal and antigen-specific regulatory T cells Project #3. (Herold) Anti-CD3 plus antigen based induction of regulatory CD8+ T cells Project #4. (Ziegler) – Use of Foxp3 to induce and regulate antigen-specific Tregs

In addition, the Center will support two pilot projects aimed at developing new approaches to regulatory cell therapy. Project #5. (Von Herrath) – Ag mechanism by which CD11c+DX5+ cells protect from diabetes Project #6 (Buckner) – Generation of Ag-specific Tregs from CD4+CD25- T cells using MHC-peptide tetramer Finally, the center will support Cell Expansion Core; a Clinical Operations and Regulatory Affairs Core; and an Administrative Core. Together these projects will focus on rapid clinical application of the biology gleaned from the basic projects.

Anticipated Outcome:

We anticipate that using novel and innovative tools, we will achieve robust expansion and functional integrity of Tregs and other suppressor cell populations for use in the treatment of Type 1 Diabetes.

Relevance to Type I Diabetes:

Our studies are aimed at developing the wherewithal to expand regulatory cells from the limited sources available. Efficient expansion of functional Tregs in vitro would make relatively straightforward the routine therapeutic use of cell therapy for the treatment and prevention of Diabetes. It is believed that correct administration of novel immunotherapeutic agents will result in stable tolerance to leading to sustained insulin independence without associated procedural risks and drug toxicity.

SCIENTIFIC ABSTRACT Expansion of Tregs for the treatment of Autoimmune Diabetes (Project 1 J. Bluestone, UCSF)

Many studies have shown the enormous potential of regulatory T cells (Treg) to suppress pathologic immune responses in autoimmune diseases. Functional defects in Treg in mouse model systems of Type 1 Diabetes (T1D) and other autoimmune diseases suggest that abnormalities in the Treg of T1D patients may contribute to the pathogenesis of the disease. Recently an effective immunomodulatory ant-CD3 antibody, called hOKT3γ1 (Ala- Ala), has been developed in our laboratory. This mAb was shown to slow the progression of diabetes in new onset patients. There is growing data in the NOD mouse model that anti-CD3 therapy increases both the number and functional activity of Treg. Thus, we believe that there is great potential in using antigen-specific Tregs as therapeutics in this disease. Although Tregs represent only a small percentage of the CD4+ T cell subset, express a diverse repertoire and decrease in number and function over time correlating with disease progression, we have been able to select and expand antigen-specific Tregs to treat diabetes in NOD mice. Therefore, we proposed that an ability to expand Tregs in an antigen–specific manner, in combination with anti-CD3 therapy from patients with T1D. Success in this arena would both reduce the cell number needed and provide for greater degree of selectivity and presumably lower risk for this cell therapy. Finally, a number of antigenic peptides, restricted to known disease-related MHC class II molecules (many developed by our collaborators in Seattle) are available to use to expand antigen-specific Tregs in humans. Thus, we believe that the development of rapid and efficient expansion of antigen-specific Tregs has provided a new opportunity for cell therapy. Therefore, in Project #1 the major goal will be to develop a robust expansion of antigen-specific CD4+ Tregs using MHC-peptide dimercoated beads, anti-CD28 and high doses of IL-2. To accomplish this goal we propose to:

1. Characterize Treg expression/function in T1D patients versus patients treated with the immunomodulatory drug hOKT3γ1 (Ala-Ala), and normal subjects;

2. Expand and analyze antigen-specific Tregs from T1D patients;

3. Develop a method for expanding human CD4+ Tregs specific for putative autoantigens including GAD, IA-2, proinsulin and IGRP for use in recent onset type 1 diabetes.

SCIENTIFIC ABSTRACT Second generation APCs as Stimuli of Polyclonal and Antigen-Specific Regulatory T Cells (Project 2 C. June, UPENN)

The principle that adoptively transferred T lymphocytes have therapeutic promise for a variety of infections and cancers has been established. There are recent exciting advances in the biology of suppressor or regulatory T cells (TREG) that indicate their potential for the therapy of autoimmune disorders. Our long-term goal is to develop second generation forms of adoptive immunotherapy with TREG for type 1 diabetes (T1D). Project 2 will address the therapy of (T1D) by developing and testing TREG with potent suppressor functions. The central theme of this application is that improved T cell culture systems can enable successful adoptive transfers of T cells by improving engraftment, persistence and function. Recent data from our laboratory and others indicates that human TREG subsets have growth requirements distinct from effector T cells, yet there are no clinically scalable and FDA compliant culture systems optimized for TREG. Our strategy is to develop novel artificial antigen presenting cells (aAPC) specifically engineered to propagate human TREG with potent suppressor functions. Our laboratory has a long term interest in the development of aAPCs, as we previously pioneered the first robust aAPC for the culture of effector CD4 T cells. Our approach to expand polyclonal and antigen specific TREG for T1D will be based on the K562 aAPC line that is optimized for natural and adaptive subsets of human TREG. Our project is supported by extensive preliminary data as we have already constructed a library of over 20 lentiviral vectors to express various costimulatory molecules and cytokines in the aAPC in order to mimic the physiologic process used to differentiate and expand human TREG. The following three specific aims will test the central hypothesis that human TREG cell subsets have distinct costimulatory and cytokine requirements for long term growth and effector function by: (1) Developing enhanced cell-based aAPC for TREG adoptive immunotherapy; (2) Determining the replicative capacity of human TREG cell subsets from normal and autoimmune donors, and (3) Adapting cell-based aAPC for clinical use in phase I pilot trials to test polyclonal and antigen-specific TREG in the setting of T1D. Using antigenic targets developed in Project 1, Project 2 will interact with Project 1 in the development of antigen-specific TREG. Project 2 will then export the optimal TREG aAPC technology to Core A for adaptation to large scale tissue culture under clinical grade GMP manufacturing conditions for clinical testing as described in the Clinical Core. Finally, this project builds on long term collaboration between investigators in Projects 1 and 2 to develop new immunotherapies. In addition, the feasibility of Project 2 is enhanced by an experienced investigative team at the University of Pennsylvania that has worked together for more than a decade to develop aAPC, and that has a track record of taking these approaches to clinical trials.

Success in our proposed experiments will lead to the development of a new approach for inducing tolerance to islet allografts for human patients with type I diabetes.

SCIENTIFIC ABSTRACT Anti-CD3 based induction of regulatory CD8+ Tcells (Project 3 K. Herold, Columbia University)

Objective:

The objective of the proposed studies is to characterize, isolated and expand CD8+ antigen- specific T cells from patients with T1DM. We hypothesize that some of these cells, including those induced with anti-CD3 mAb have regulatory function. Our objective is to isolate these cells, characterize them, and expand the population so that they can be readministered to patients.

Background/Rationale:

In previous trials of the anti-CD3 monoclonal antibody, hOKT3γ1(Ala-Ala) in patients with new onset Type 1 diabetes and in studies in vitro, we found that the anti-CD3 mAb induces a subpopulation of CD8+ T cells with regulatory function. We have studied antigen specific CD8+ T cells, identified with Class I MHC tetramers, from patients with T1DM and have found that they have a phenotype that is different from conventional antigen specific T cells. We have also found that the proportion of these cells increases after treatment with anti-CD3 mAb. Our hypothesis is that anti-CD3 mAb induces a subpopulation of antigen specific regulatory T cells and that these cells may be used for adoptive immune therapy.

Description of Project:

We will isolate tetramer+ cells and determine their functional phenotype. We will test whether they lyse beta cells or alternatively have regulatory function. We will first determine the changes in the CD8+ antigen specific T cells over time after diagnosis of diabetes. We will study the phenotype of the cells ex vivo and compare gene expression in these cells to conventional antigen reactive cells and cells from normal subjects. We will also grow these cells in vitro using the anti-CD3 mAb or artificial antigen presenting cells including HLA-A2+ K562 cells and HLA-Ig/anti-CD28 coated beads. Finally, we will use the methods that are developed in the first 2 aims to grow the antigen specific T cells from patients treated with anti-CD3 mAb. We will test the function of these cells in order to differentiate their effector from regulatory function.

Anticipated Outcome:

We anticipate that before treatment, there will be cells with either regulatory or effector function. We also anticipate that the antigen specific CD8+ T cells in patients after treatment with anti-CD3 mAb will have regulatory function. Based on previous experiences with the use of agents such as the HLA-Ig-coated antigen presenting cells, we anticipate that we will be able to expand the cells at least 10,000 fold.

Relevance to Type I Diabetes:

These studies are based on our findings from successful treatment of patients with T1DM with anti-CD3 mAb. Our plan is to isolate the cells that, we postulate, mediate the clinical effects and to administer these cells to patients rather than systemic retreatment.. We postulate that this immune therapy will prevent the loss of insulin production that occurs as part of the natural history of Type 1 diabetes. Retention of insulin production would be expected to improve metabolic control of the disease.

SCIENTIFIC ABSTRACT Use of FoxP3 to Induce and Regulate Regulatory T Cells (Project 4 S. Ziegler, Benaroya Research Institute at Virginia Mason)

Objective:

The objective of this proposal is to determine whether T cells can be manipulated in such a way to convert otherwise pathogenic cells into regulatory one. We will attempt this through the use of the transcription factor FOXP3. FOXP3 has been found to be expressed primarily in regulatory T cells, and to be critical for their development and function. The studies in this proposal will determine whether FOXP3 function can be enhanced through the identification and characterization of interacting proteins, and whether ectopic expression of FOXP3 has therapeutic efficacy.

Background/Rationale:

Immunological tolerance to self-antigens is a tightly regulated process. Several lines of evidence demonstrate two fundamental mechanisms for controlling immunologic reactivity to self tissues: A primary mechanism for self tolerance is deletion of self-reactive cells in the thymus. However, this mechanism is not perfect and auto- reactive clones do escape into the periphery. Tolerance is maintained in the periphery through a variety of mechanisms including T cell anergy and T cell indifference or ignorance. Recently, another, more active, mechanism of tolerance induction has been identified that is controlled by a population of regulatory T cells that actively suppress the function of auto-reactive T cells. These T cells, known as regulatory T cells, have the ability to inhibit the development of autoimmunity when transferred into the appropriate host. However, the mechanism by which these cells perform their tasks is as yet unclear. Recent work has shown that the forkhead/winged-helix protein FoxP3 is expressed predominantly in regulatory T cells, and is both necessary and sufficient for their development and function.

Description of Project:

The finding that FoxP3 expression determines regulatory T cell fate, as well as the ability of ectopic FoxP3 expression to convert naIve T cells into regulatory T cells, suggests that FoxP3 may be useful in cell-based therapies for autoimmune diseases. As a first step, the experiments in this proposal are designed to test the feasibility of manipulating FoxP3 expression and function as a means of converting both normal and pathogenic T cells to regulatory T cell-like cells. We will use an in vitro cell culture system to determine whether introduction of FoxP3 into primary antigen-specific human T cells can convert these cells into antigen-specific regulatory T cells. We will also use an animal model of type I diabetes in the mouse to test the ability of ectopic FoxP3 expression to convert diabetogenic T cells into regulatory T cells. Several FoxP3-interacting proteins have been identified, and the ability of these proteins to enhance FoxP3 function will be assessed.

Anticipated Outcome:

For the first part of the proposal, we expect that the interacting proteins we have identified will be involved in regulating FOXP3 function in regulatory T cells. We also expect that manipulating their function, through alterations in expression levels or through mutagenesis, will also alter FOXP3 function. For the second part, we fully expect that introduction of FOXP3 into human CD4+ T cells will convert them to a regulatory phenotype. Also, in our humanized models of type I diabetes, we expect that introduction of FOXP3 into pathogenic T cells will convert them to a regulatory phenotype, and that their re-introduction will ameliorate disease.

Relevance to Type I Diabetes:

The ability to modify the function of diabetogenic T cells in such a way that they become therapeutic has potential implication in the treatment of type I diabetes. With the availability of reagents for isolating self-reactive T cells from patients, introduction of FOXP3, or manipulation of its function, may prove to be a viable therapeutic approach.

SCIENTIFIC ABSTRACT Ag mechanism by which CD11c+DX5+ cells protect from diabetes (Project 5 M. Von Herrath, La Jolla Institute for Allergy and Immunology)

One major issue for the clinical application of polyclonal Tregs in type 1 diabetes is the potential for systemic immune suppression. Therefore, suitable preclinical models have to be employed to test whether introduction of Tregs will affect the immune control and outcome of acute or chronic viral infections. Conversely, long term efficacy is likely to depend on persistence of the Tregs in the face of ongoing immunity to viruses, bacteria and other inflammatory responses that might “inactivate” Tregs through TLR or other innate immune pathways. Thus, additional studies are needed to determine how the presence of viral infections might affect activity and in vivo persistence of transferred Tregs. The experiments in this grant application are designed to answer these safety and efficacy questions. We will assess viral titers and duration of persistence, the amount of immunopathology, numbers of anti-viral T cells, and the direct function and reactivity of Tregs and their interaction with effector cells. These studies will enhance the confidence in future trials in terms of safety and long term efficacy and will also deepen our mechanistic understanding Treg function in the face of inevitable ‘real-life’ environments.

Aim 1: Effect of polyclonally expanded CD4+CD25+CD127lo/- (received from JB at UCSF) and antigen-induced IL-10+ Tregs on acute and chronic LCMV and flu infections. Viral clearance, CTL, immunity, immunopathology, and cytokine profiles will be assessed with and without Treg transfers.

Aim 2: Effect of viral infections (acute and chronic LCMV, flu and mCMV) on in vivo function of polyclonal CD4+CD25+CD127low/- Treg and antigen-induced IL-10+ Treg function (FoxP3 expression, ex vivo suppressive capacity, diabetes development or recurrence, cytokine production, use of FoxP3 and IL-10 reporter mice) – Treg activity and function will be assessed before, during and after viral infections.

Tools: JB (Jeff Bluestone UCSF) polyclonal Tregs, BDC 2.5 Tregs, LCMV CD4 SMARTA Tregs, FoxP3 and IL-10 reporter mice, LCMV and NOD diabetes models.

Infection models: Acute (Armstrong) and chronic (Clone 13) LCMV infections, acute flu infection, (chronic mCMV infection if needed).

SCIENTIFIC ABSTRACT Generation of Ag-specific Tregs from CD4+CD25- T cells using MHC-peptide tetramer (Project 6 J. Buckner, Benaroya Research Institute at Virginia Mason)

Objective:

To optimized the conditions under which antigen specific T regulatory cells can be generated de novo from CD4+CD25- T cells, isolated by use of MHC-peptide tetramers and expanded in vitro with the ultimate goal of utilizing this method of TR generation and expansion for islet specific therapy.

Background/Rationale:

CD4+CD25+ regulatory T cells (TR) are implicated in the pathogenesis of diabetes and recently the transfer of islet specific TR has been shown to be therapeutic in animal models of diabetes. TR can be isolated directly from the blood but are very rare thus limiting the number available for expansion. However an alternative to direct isolation from the blood is the generation of Tr from CD4+CD25- T cells. Our laboratory has demonstrated that CD4+CD25+FoxP3+ regulatory T cells can also be generated de novo as a result of activation of CD4+CD25- FoxP3- T cells. Further, our laboratory has shown that antigen specific TR can be generated de novo and isolated with HLA class II tetramers.

Description of Project:

Initial studies will utilize the foreign antigen HA and will focus on optimizing the in vitro culture conditions for TR generation and expansion. Once ideal conditions are identified using this model system we will proceed to studies utilizing islet specific antigens. The general approach will be to isolate CD4+CD25- T cells from subjects, activate with antigen, following a 10 day incubation the resulting CD4+CD25+ T etramer+ cells will be isolated, then expanded further in vitro. Culture conditions during the generation phase and expansion will be optimized. At each step in the process phenotypic characteristics and regulatory functional of the resulting cells will be examined.

Anticipated Outcome:

We expect to improve our ability to generate and isolate of tetramer positive TR and develop a method which will allow expansion of 50-100 fold of these regulatory cells.

Relevance to Type I Diabetes:

The ultimate goal of this work is to develop a cell- based method of immunotherapy, by generating and expanding islet specific TR in vitro.

SCIENTIFIC ABSTRACT Human Islet and Cellular Therapy Core at UCSF (Core A J. Bluestone, UCSF)

Experimental cell therapy requires focused scientific, technical and regulatory expertise and resources that are not available to or fundable by individual researchers. The purpose of the Cell Expansion Core A is to support the development of clinical grade Tregs for use in patients with T1D. The core will support the development of tools needed for cell expansion including: development of clinical grade antibody/tetramer coupled beads; K562 artificial APCs, standardized lots of human serum and media; viral, bacterial and endotoxin testing; development and application of standard operating procedures for individual cell batches and appropriate other supplies and core needs. The overall mission of the Clinical Cell and Vaccine Production Facility (CVPF) at the University of Pennsylvania and the Human Islet and Cell Therapy Facility (HICTF) at UCSF is to enable the translation from research bench to bedside of novel cellular and gene therapies. The Core will support scale-up and validation of large-scale cell processing procedures. The CVPF was the first to develop a closed system method for the efficient large-scale culture of purified CD4+ T cells from HIV+ study subjects for a clinical trial initiated. The CVPF has developed clinical scale technology for the support of over 20 cell and gene therapy clinical trials in various cancers and HIV, including the world’s first lentiviral vector clinical trial. The HICTF has developed a human islet transplant program and performed over 50 islet preparations. The CVPF and HICTF will process donor and patient cells that are to be reinfused to study subjects as investigational cellular therapies. This includes performing release testing (Quality Control) that addresses the requirements for demonstrating safety, purity, potency and identity of the cellular final products. All Quality Control testing and release is overseen by Quality Assurance Officers. To date, the CVPF has produced for infusion over 170 cellular products for over 100 people enrolled in these trials. The CVPF will also serve as a cell and tissue repository, complying with HIPAA guidelines, in support of the projects in this proposal. Finally, translation of research procedures for use in cell therapy clinical trials requires not only the use of clinical grade or clinically compatible reagents and materials, but also a detailed knowledge of Good Manufacturing Practices and regulations enforced by the FDA for all clinical trials. The Core staff provides regulatory support for the investigators in this proposal to translate their discoveries at the bench to clinical trials and to support FDA annual report submission and FDA inquiries.

SCIENTIFIC ABSTRACT Human Islet and Cellular Therapy Core at UCSF (Core A B. Levine, UPENN)

Objective:

The purpose of the Cell Expansion Core A is to support the development of clinical grade Tregs for use in patients with T1 D. Core A will serve as the translational 'bench to bedside' Good Manufacturing Practice resource in the two clinical trials of Tregs for treatment of T1 D.

Background/Rationale:

Experimental cell therapy requires focused scientific, technical and regulatory expertise and resources that are not available to or fundable by individual researchers.

Description of Project:

The core will support the development of tools needed for cell expansion including: development of clinical grade antibody/tetramer coupled beads; K562 artificial APCs, standardized lots of human serum and media; viral, bacterial and endotoxin testing; development and application of standard operating procedures for individual cell batches and appropriate other supplies and core needs. The overall mission of the Clinical Cell and Vaccine Production Facility (CVPF) at the University of Pennsylvania is to enable the translation from research bench to bedside of novel cellular and gene therapies. To fulfill the mission of the CVPF in supporting new clinical trials, the first element is the scale-up and validation of large-scale cell processing procedures. The CVPF was the first to develop a closed system method for the efficient large-scale culture of purified CD4+ T cells from HIV+ study subjects for a clinical trial initiated in 1996. Since that time, the CVPF has developed clinical scale technology for the support of over 20 cell and gene therapy clinical trials in various cancers and HIV, including the world's first lentiviral vector clinical trial. The second element of the CVPF's mission is to perform the cell processing of donor and patient cells that are to be reinfused to study subjects as investigational cellular therapies. This includes performing release testing (Quality Control) that addresses the requirements for demonstrating safety, purity, potency and identity of the cellular final products. All Quality Control testing and release is overseen by an outside Quality Assurance Officer. To date, the CVPF has produced for infusion over 170 cellular products for over 100 people enrolled in these trials. The CVPF will also serve as a cell and tissue repository, complying with HIPAA guidelines, in support of the projects in this proposal. Finally, translation of research procedures for use in cell therapy clinical trials requires not only the use of clinical grade or clinically compatible reagents and materials, but also a detailed knowledge of Good Manufacturing Practices and other regulations enforced by the FDA for all clinical trials. The CVPF staff provides Good Manufacturing Practice regulatory support for the investigators in this proposal to translate th'4!ir discoveries at the bench to clinical trials and to support ongoing requirements of FDA annual report submission"and FDA inquiries.

Anticipated Outcome:

The Core will first perform the clinical trial scale-up, validation and qualification necessary to support the first clinical trial of Tregs in T1 D. This includes qualification of a clinical grade method of Treg enrichment/separation f~om an apheresis product, optimization of clinical scale in vitro expansion, the development of a potency assay, and stability/shipping validation. A second trial will be performed with a next generation antigen presenting cells developed by Project #2.

Relevance to Type I Diabetes:

The proposed clinical trials are designed to test antigen-specific regulatory T cell therapy in patients with ongoing autoaggressive Type 1 diabetes.

SCIENTIFIC ABSTRACT Clinical Operations & Regulatory Affairs (CORA) (Core B June, UPENN & K. Herold, Columbia University)

Successful translation of experiment cell therapies for T1D from concept through to clinical study demands a complex skill set of specialized scientific, technical and regulatory expertise. This expertise must be accompanied by a dedicated infrastructure that meets or exceeds the rigorous requirements and regulatory guidelines necessary to ensure patient safety, particularly in early trials. The Office of Human Research and the Clinical Trials Units at the University of Pennsylvania in conjunction with the Clinical Operations Center at Columbia University are uniquely and ideally suited to perform the core functions in support of the translational and clinical research plans of the JDRF Collaborative Center for Cell Therapy. The Clinnical Operations and Regulatory Affairs (CORA) Core provides an experienced team of physician scientists, and regulatory specialists with expertise in complex clinical trials involving T1D and cell based therapies. In addition, extensive preliminary data has established Standard Operating Procedures to permit efficient and safe evaluation of new cell based therapies for T1D according to Good Clinical Practices, Included in the CORA Core are four functional components: Clinical Operations; Regulatory Affairs; Monitoring and Quality Assurance; and Data Management and Analysis. The Core will oversee production and clinical operations to ensure that the regulations and guidelines of the Food and Drug Administration (FDA), and local regulatory agencies are followed. The CORA core will prepare IND’s, revisions/amendments, reports and annual renewals for the IND. This information will also be prepared for use by the institutional IRB and GCRC’s as needed. In addition, the CORA Core has the responsibility of reporting to the Data Safety Monitoring Board. The CORA infrastructure will enable the Program to rapidly of reporting to the Data Safety Monitoring Board. The CORA infrastructure will enable the Program to rapidly translate scientific milestones to safe and efficient pilot clinical trials to test and evaluate Treg infusions in patients with T1D.

SCIENTIFIC ABSTRACT Administrative Core (Core C J. Bluestone, UCSF)

The mission of the JDRF Collaborative Center for Cell Therapy is to promote the basic and clinical research that will lead to successful regulatory cell therapy in patients with ongoing autoaggressive Type 1 diabetes. The approach will be multi-faceted taking advantage of the faculty at each institution with strong research projects in both basic and clinical immunology. Efforts will be focused on efforts towards identifying novel approaches to cell therapy coupled with the most effective immunotherapies pioneered at the Diabetes Center at UCSF, University of Pennsylvania and Columbia University. This endeavor underlines a new approach to the treatment of Type 1 Diabetes by bringing together committed groups of researchers with strong, complimentary, credentials in basic science, clinical research and the treatment of diabetes. The JDRF Collaborative Center for Cellular Therapy is part of the Diabetes Center at UCSF. The Diabetes Center is an organized research unit at University of California, San Francisco. This unit has functioned for more than a half-century as a basic and clinical research enterprise at the forefront of diabetes research. The Center supports various seminar series, early stage research, and a strong educational program. Special emphasis is placed on creating a collaborative basic and clinical research environment to promote interdisciplinary research, facilitate and encourage new research in Type 1 diabetes, provide state-of-the art facilities for research and continue to build a superb translational research program. This unit has interdisciplinary research, facilitate and encourage new research in Type 1 diabetes, provide state-of-the art facilities for research and continue to build a superb translational research program. The Administrative Core will organize the Center activities. The core will schedule monthly videoconferencing /quarterly face-to-face meetings to discuss ongoing research and clinical efforts, and organized the core and financial aspects of the Center research program. The Core will support a Pilot and Feasibility Program that will foster efforts in the cell therapy research arena. Finally, we will appoint an external advisory group that will meet once a year to discuss advances and progress in our efforts with very specific duties.

JDRF Center for Developing Immune Therapies for Type 1 Diabetes

Richard Flavell

of

Yale University

Applicant: FLAVELL, Richard A

Project 1 – A humanized mouse model of Type 1 diabetes

SCIENTIFIC ABSTRACT

Objective:

The objective of this project is to generate novel humanized mouse models that allow study of type 1 diabetes process and testing of therapies.

Background/Rationale:

Type 1 diabetes (T1D) results from the inactivation and subsequent destruction of the islets of Langerhans by autoreactive T cells. Despite the development of good mouse and rat models for this disease, what is really needed is a model for the human disease in which the human immune system destroys human islets. The purpose of this proposal is to develop a humanized mouse model that reflects the natural history of this disease in people.

Description of Project:

This project will generate transgenic mice which allow for engraftment of a human immune system and specifically investigate genes involved in diabetes.

Anticipated Outcome:

We anticipate generating new “fully humanized” transgene and knockin mice which can recapitulate human T1D in a mouse.

Relevance to Type I Diabetes:

The development of humanized animal models that allow study of disease process and testing of therapy is of major importance in developing strategies to translate knowledge in preclinical models to the ultimate prevention and cure of type 1 diabetes in humans.

Applicant: Wen, Li

Project 2: of JDRF Center for Developing Immune Therapies for Type 1 Diabetes

SCIENTIFIC ABSTRACT

Objective:

To generate human anti-GAD BCR knock-in mouse and investigate human anti-GAD T and B cells in humanized T1D animal model for developing immune therapies for type 1 diabetes.

Background/Rationale:

B cells are an important component in the development of T1D. Autoantibodies specific for islet cell proteins are regularly detected in patients with T1D and in individuals who have a high risk of developing the disease. Islet autoantibodies have been utilized as effective bio-markers for predicting the onset of disease in those high risk individuals. Although islet auto-antibodies are not believed to directly cause beta cell damage, they can cause beta cell damage by virtue of enhancing islet auto-antigen presentation to diabetogenic T cells. Although there is good evidence for the importance of B cells in the development of diabetes in the NOD mouse model, most of the data in human studies were obtained in vitro, and therefore, the role of B cells in human T1D still remains largely unknown due to the lack of an appropriate model system.

Description of Project:

In this project, we propose to generate a humanized anti-GAD B cell receptor transgenic knock-in mouse for investigating the role of a) islet specific auto-antibody in T1D development, b) the B cells expressing anti-GAD as antigen presenting cells in T1D development, c) the interaction of human anti-GAD B cells with human anti-GAD T cells in the disease development and d) other human immune molecules in diabetes development in an advanced humanized mouse model of T1D (generated by Project 1).

Anticipated Outcome:

We anticipate to generate human anti-GAD BCR knock-in mouse and to study anti-GAD B cell and T cell in advanced humanized mouse model of T1D in order to develop immune therapies for type 1 diabetes.

Relevance to Type I Diabetes:

Anti-GAD autoantibody has been one of the dominant islet autoantibodies for clinical prediction and diagnosis of T1D development. Individuals who either have high risk of developing the disease or have already diagnosed with T1D also showed T cell responses to GAD autoantigen. However, little is not known the in vivo role of anti-GAD B cell and its interation with anti-GAD T cell in vivo. Our study will provide valuable in vivo information for the above questions. This is highly relevant to T1D and the mission of JDRF as our study could potentially translate into clinical settings by developing immune therapies for T1D. Applicant: Sherwin, Robert

Project 3: Role of Reg, a New Beta Cell Autoantigen in Human T1DM

SCIENTIFIC ABSTRACT

Objective:

To investigate the relevance of the new islet autoantigen Reg in human T1D by using a mouse model transgenic for the disease predisposing human MHC DQ8 molecule and an islet-expressed member of the human Reg family of proteins.

Background/Rationale:

Reg proteins play a dual role in type 1 diabetes mellitus (T1DM). They act as autoantigens and as beta- cell trophic factors. It is therefore possible that Reg protein, locally released in an attempt to regenerate and protect beta cells, in turn activates autoreactive Reg specific T-cells promoting inflammation and release of more Reg. Thus a vicious cycle ensues converting a protective into an islet-destructive process. Our data show that the autoagressive immune response against RegII in NOD mice and humans with recent onset of T1DM is predominantly directed against its N-terminal fragment (Ntfr).

Description of Project:

We will concentrate on the immune response directed against the N-terminal portion of the Reg molecule and propose to carry our findings towards patients with T1DM by using two humanized mouse models. A mouse transgenic for the MHC DQ8 molecule will allow the development of reagents to block the interaction between autoagressive Ntfr specific T-cells and the DQ8 Ntfr (Reg) peptide complex on the antigen presenting cells. We will investigate if these reagents - single chain antibodies derived from phage display libraries recognizing DQ8 Ntfr peptide complexes – influence the Reg-directed autoimmune response in the DQ8 mice and test them on cells derived from human carriers of the DQ8 allele. In the second model mice will additionally express a human Reg molecule in their islets to study the DQ- restricted autoimmune response against a human Reg molecule. Furthermore these mice will be bred onto a background suitable for reconstitution with human haematopoietic stem cells to investigate if the reconstituted immune system recognizes the hReg transgene.

Anticipated Outcome:

We expect to answer the following questions thereby characterizing the potential role of Reg,a new beta cell autoantigen, in human T1DM: -Do HLA DQ8 transgenic mice show a spontaneous autoimmune response to the N-terminal fragment of RegII and the transgenic, islet-expressed human Reg member? -Does vaccination with Ntfr accelerate diabetes in the transgenic mice as it does in NOD mice? -Can either the spontaneous or the accelerated autoimmune response be blocked or attenuated by treatment with antibodies that recognize DQ8 Ntfr peptide complexes?

Relevance to Type I Diabetes:

The hypothesis to be addressed is that early insult to beta cells initiates a vicious circle involving attempted islet cell regeneration followed by enhanced autoimmunity. One of the targets of the autoimmune-response is a protein, namely Reg, that supports the regeneration and protection of islets. There is strong support for this model from the NOD mouse model, and understanding this process might significantly affect current approaches to therapies. For example, agents that may enhance beta cell regeneration may, in the end, accelerate their destruction since immune responses would be stimulated, and they are generally rapid and completely eliminate antigen.

Our preliminary data indicate that the autoimmune response against the N-terminal part of Reg may be an important contributor to the pathogenesis of T1DM not only in NOD mice, but also in human patients. We were able to expand NtfrII specific autoagressive T-cells in vivo to an extent that they became the dominating force in the pathogenesis of the disease by as few as two consecutive vaccinations with NtfrII, suggesting that the autoagressive NtfrII specific T-cells either exist in comparatively large numbers in NOD mice or that they are able to respond vigorously to stimulation with epitopes contained within this part of RegII. Our preliminary experiments furthermore demonstrated that the B-cell response in NOD mice preferentially targeted NtfrII and that autoantibodies against peptides within the N-terminal region of two human Reg members could be isolated from serum of diabetic patients but not from serum of normal controls.

In the current proposal we will test if antibodies with the capacity to block MHC peptide complexes that arise when T-cell epitopes of islet autoantigens are presented might serve as diagnostic and therapeutic tools in T1DM. We plan to evaluate these reagents in the context of our studies of the autoimmune response directed against the N-terminal region of the autoantigen RegII and human Reg proteins. However, the approach to isolate these reagents is not restricted to Reg, but could be applied to any (auto) antigen. It should be emphasized that the transfer of this novel approach for specific immune- intervention to mice expressing the human DQ8 molecule as proposed here could generate a reagent for the study of cells derived from human DQ8 patients with T1DM. A DQ8 Ntfr peptide complex recognizing antibody will at this stage only have diagnostic application in human DQ8+ patients. However, in the future this approach has significant therapeutic potential. In summary this application offers the potential to identify new immune therapeutic targets for human diabetes and to develop a highly specific, direct immune intervention for T1DM that has a reduced risk of unwanted side effects.

Applicant: Bothwell, Alfred

Project 4: Functional Analysis of Islet Specific Human T Cells

SCIENTIFIC ABSTRACT

Objective:

The role of CD4 and CD8 T cells as well as the function of regulatory T cells has been well studied in rodent models of T1DM, but there is very little functional data that has identified the role of human T cells in causing the disease. While there are common autoantigens in both mouse and human T1DM, the in vivo immune responses of the antigen specific cells may differ significantly. For example, the contributions of adhesion molecules, specific autoantigenic peptides, chemokines and cytokines and MHC molecules may not be the same in humans and mice. The goal of this project is to establish a model in which we can reconstruct and study mechanisms of cellular homing and activation of human diabetes antigen specific T cells and to define their functional properties.

Background/Rationale:

The role of islet specific T cells in the development of type 1 diabetes is well established. Considerable insight into molecules that can initiate and advance disease in rodents is available. However there are significant differences between important molecules that affect cell homing and lymphocyte activation. Thus, a humanized mouse model in which human antigen specific T cells can be manipulated and characterized is essential for basic understand of disease mechanisms that could lead to progress in translational objectives.

Description of Project:

The first aim is to characterize the immune interactions of immunodeficient mice reconstituted with human peripheral blood mononuclear cells (PBMC) or CD34+ progenitor cells with islets expressing human HLA- DR, DQ or HLA-A2 antigens via transgenes. The homing of these cells into transplanted islets and lymph nodes will be studied. The responses of healthy donors (HD) will be compared with T1DM patients. Two well characterized islet autoantigens are insulin and GAD. Since, regulation and amino acid sequence differences exist between human and mouse autoantigens. Th character of the immune response could be affected. Thus, a human preproinsulin transgene with and without a EGFP reporter gene transgene will be constructed and an existing GAD transgene will be bred onto the immunodeficient mice. These mice, expressing human antigens will be used as cell recipients. In the second aim, antigen specific CD4, CD8 and Treg cells for insulin and GAD will be expanded and immortalized with a human telomerase retrovirus in order to provide adequate numbers of cloned antigen-specific cells to study in vivo homing and functional characteristics. Finally, in aim 3, the T cell clones will be studied in HLA and insulin and GAD transgenic murine models and human islet transplanted models to characterize the immune responses. This will include extensive immunohistochemical analysis as well as real time imaging analysis with 2-photon microscopy.

Anticipated Outcome:

The strategies that will be employed to generate islet specific T cell lines are the best available and the generation of the optimal mouse strain for development of a humanized mouse is in this Center. We anticipate making substantial progress in the development of an autoimmune disease model for T1DM.

Relevance to Type I Diabetes:

These studies should permit mechanistic studies of human autoreactive lymphocytes responsible for T1DM. There is significant clinical relevance to these studies since cells from patients in clinical trials in project 5 will be studied.

Applicant: Herold, Kevan

PROJECT 5: Mechanistic studies of antigen/anti-CD3 therapy in humanized mice

SCIENTIFIC ABSTRACT

Objective:

Our studies of anti-CD3 mAb in patients with Type 1 diabetes (T1DM) indicate that a single course of treatment with the mAb can attenuate the loss of insulin production over the first years of the disease. The goal of this Project is to use humanized mice to understand the mechanisms of action of the anti- CD3 mAb, to improve the safety of its use, and to test the effects of a combination therapy with anti-CD3 mAb and antigen.

Background/Rationale:

The major limitation to the use of anti-CD3 mAb is the development of cytokine release syndrome with the initial doses of the mAb. We do not know, however, who will develop this adverse event, but the safety of drug administration could be significantly improved if a means of identifying patients at risk were available. In addition, we have found that the anti-CD3 mAb induces CD8+ T cells that we believe have regulatory function. In a proposed clinical trial, we are planning to test whether the combination of anti- CD3 mAb with antigen will improve the duration of the clinical effects of the mAb by inducing regulatory T cells.

Description of Project:

In the proposed studies, we plan to use mice that have been been reconstituted with PBMC from patients enrolled in clinical trials to test whether the responses to anti-CD3 in these humanized mice will predict the clinical responses when patients are treated with the drug. We will also isolate cells from the treated patients and test their ability to regulate immune responses in the reconstituted mice. These cells will be immortalized and studied in collaboration with Drs. Bothwell and Ruddle in Project 4. Finally, we will also test the effects of the anti-CD3 mAb in mice with fully humanized immune systems (HHL mice).

Anticipated Outcome:

We predict that the development of cytokine release syndrome will be a feature of the PBMC from patients, and therefore, that we will be able to identify individuals who are likely to develop this adverse event by studying the responses to the mAb in mice that receive cells from the subject. We also anticipate that the cells that we isolate from subjects in the clinical trials will exhibit regulatory function. An important question that we will be able to answer is the duration of the regulatory effect and the correlation of the findings in the reconstituted mice compared to patients. Finally, we predict that we will see changes in PBMC in the HHL mice that are similar to what has been found in patients treated with the anti-CD3 mAb. Studies of the peripheral lymphoid compartments will shed light on the usefulness of studies of human PBMC to identify the mechanisms of the drug action in patients.

Relevance to Type I Diabetes:

Anti-CD3 mAb has been shown to attenuate the loss of insulin production in 3 randomized controlled trials with two different molecules. These studies should improve the safety of the use of these drugs and will help to understand the mechanisms of action of the drugs. This information may allow the development of more specific application of these agents so that systemic toxicity can be avoided. In addition, the 3rd aim will be an initial test of the utility of the HHL mouse as a screen for human drugs. If this model proves useful, it will greatly improve the safety of the development of new therapies and accelerate their development.

Applicant: FLAVELL, Richard A

Core A : Administrative Core

SCIENTIFIC ABSTRACT

Objective:

This Core will manage the administrative tasks of the Center. These include providing secretarial support, maintaining a website, arranging regular meetings of the investigators, organizing meetings with the Scientific Advisory Board and the annual review, and facilitating interactions with other JDRF Centers and Regeneron.

Description of Project:

This core will:

1. Provide secretarial support

2. Maintain a website

3. Arrange regular meetings of the investigators and Scientific Advisory Board

4. Arrange annual review

5. Facilitate interactions with other JDRF Centers and Regeneron

Applicant: FLAVELL, Richard A

Core B: Humanized mice

SCIENTIFIC ABSTRACT

Objective:

The objective of this Core is to generate novel humanized mouse models that allow study of type 1 diabetes process and testing of therapies.

Background/Rationale:

Although the NOD mouse is one of the best animal models for the study of autoimmune insulin- dependent type 1 diabetes (T1D), and has facilitated many important discoveries, it does not allow for study of specific interactions of human lymphocytes with human MHC molecules. We have previously generated a number of humanized mice that lack mouse MHC class II molecules (mII-) and express either HLA DQ8 or DR4 or both. These mice are useful for studying peptide binding to the HLA molecules and provide information about generation of immune responses to particular autoantigens in vivo and the binding of those autoantigenic peptides in vitro. However, Our existing mice are not suitable recipients for reconstitution with human cells as they are not immunodeficient, they also lack some of important human genes to facilitate human cells engraftment. Development of mice that are suitable for reconstitution with human cells is of vital importance. This Core will develop new mouse lines that are immunodeficient and suitable as models of Type 1 diabetes.

Description of Project:

This core will 1. Maintain and distribute diabetes prone humanized mice carrying HLA DR and DQ transgenes. 2. Generate constructs for new humanized transgene and knockin replacement mice. 3. Generate and maintain other mice as needed for the members of the Center.

Anticipated Outcome:

We anticipate to expanding the existing “humanized” mice and providing to all the investigators in the center. We also anticipate to generating new “fully humanized” transgene and knockin mice for the center investigators in their projects.

Relevance to Type I Diabetes:

The development of humanized animal models that allow study of disease process and testing of therapy is of major importance in developing strategies to translate knowledge in preclinical models to the ultimate prevention and cure of type 1 diabetes in humans.

Applicant: Bothwell, Alfred

Core C: Human Tissue Repository

SCIENTIFIC ABSTRACT

Objective:

All of the Projects in the Center will use human cells for study. Obtaining and processing clinical material for study is a time consuming task. This core will provide clinical material for studies in the Center Projects.

Background/Rationale:

In order to generate humanized mice, we will need human stem and peripheral blood cells. One of the goals of studies in the Center is to reconstitute mice with cells from patients with Type 1 diabetes so that mechanistic studies regarding disease pathogenesis and actions of immune interventions can be addressed. There are not any existing model systems in which human immune cells that cause diabetes can be studied but the studies in mice may not completely model human immune responses. Therefore, to develop mice that can be used to study human immune responses and even disease specific responses, we plan to establish a core facility that will collect the samples needed for these studies. The projects in this Center require the characterization of clinical samples with regard to HLA type, effective utilization of clinical material by appropriate preparation and sample aliquoting and the integration of clinical information.

Description of Project:

The materials to be collected include peripheral blood mononuclear cells (PBMC), bone marrow aspirates, serum, and DNA from patients and normal control subjects, and human islets from organ donors. The Core functions will include fulfilling the regulatory requirements for obtaining this material, isolating and storing the cells, serum, and DNA, and arranging the procurement of human pancreata that will be used for isolation of islets at the islet isolation laboratory at Massachusetts General Hospital. In addition, the Core will prepare lines of EBV-transformed B lymphocytes that can be used in the projects as antigen presenting cells or other purposes. The donors of the cells will be HLA typed for HLA-A2 and HLA-DR3, 4, and DQ8 by real-time PCR. The cells and other materials will be stored in the Yale University Repository that will be part of the Yale Clinical and Translational Science Award.

Anticipated Outcome:

The clinical samples stored and characterized will be an invaluable resource for the Center projects. Storage in liquid nitrogen will be used for all samples containing cells for subsequent functional studies. During the first year we will build up a stock of cells as described below totaling about 1500 vials. Samples will be consumed and replaced in subsequent years such that up to a total of 3,000 vials would accrue in subsequent years. From our functional studies in vivo samples are good for up to 2 years but show reduced function at later times. Therefore we will gauge the use and not harvest more than would be used within a one year period.

Relevance to Type I Diabetes:

Support for the development of the in vivo model proposed in this Center grant would provide a system to further identify and study basic mechanisms in the induction of type 1 diabetes and provide criteria for assessing the effectiveness of clinical treatments.

The BDC JDRF Autoimmunity Prevention Center

John C. Hutton

of

University of Colorado at Denver

and Health Sciences Center

Applicant: Hutton, John C.

Composite: The BDC JDRF Autoimmunity Prevention Center

SCIENTIFIC ABSTRACT

The Barbara Davis Center for Childhood Diabetes (BDC) is a leading international research institution affiliated with the University of Colorado Health Sciences Center with a focus on type 1 diabetes treatment and prevention. It has a long tradition of collaborative research, (ITN, DPT, TRIALNET, SEARCH, TEDDY, Brehm Coalition), generation and sharing of novel research reagents (T-cell clones BDC 2.5, 6.9 and 10.1, Phogrin P2/P7; autoantigens (IGRP, Phogrin and ZnT8), transgenic mice (insulin B16AlaNOD and IGRP-/-NOD), and human genes (PTPN22 and DQB1 0602 recombinants). Its website hosts the Genespeed bioinformatics resource genespeed.uchsc.edu, and seminal publications including Peter Chase’s “Understanding Diabetes” and the “Immunology of Diabetes” edited by George Eisenbarth www.barbaradaviscenter.org. The BDC recently expanded 4 fold and occupies a 125,000 sq ft. building that accommodates T1D clinical care, clinical research, translational research and basic research under the same roof alongside Computational Biology (Larry Hunter), Autoimmunity Research (Mike Holers) a Research Infusion unit and an Islet Transplantation Immunobiology unit incorporating a GMP facility (Alex Wiseman). The establishment of a JDRF Autoimmunity Center Grant at this time will build upon the achievements of the BDC, will facilitate recruitment of researchers, the development of new technologies, and accelerate the design of safe interventions to prevent or slow the progression of type 1 diabetes. Importantly, the latter will be based upon a mechanistic understanding of immune-based therapies. The JDRF Center proposal focuses on two ß cell-specific autoantigens (insulin and ZnT8) that hold promise as components of therapeutic agents with minimal off-target drug effects. Three research projects and 2 clinical trials are outlined: the research component to gain insight into the role of the molecules in the pathogenesis of type 1 diabetes defined at the cellular and molecular level; the trials to implement antigen- specific therapies in human subjects aimed at restoration of immune tolerance. Besides an Administrative Core, a biomedical resource core is proposed that will service the basic and clinical research components and at the same time develop procedures to monitor the effectiveness of therapies interventions and disease recurrence.

Specific program components are as follows:

Project 1: Evaluation of a conserved alpha chain T-cell receptor clonotype (P.I. Eisenbarth) Project 5: Humoral autoreactivity to the novel T1D autoantigen Slc30A8 (P.I. Hutton) Project 6: T-cell immunity to the novel T1D autoantigen Slc30A8 (P.I. Davidson) Project 8: Pre-Point Immune Response Monitoring (P.I. Gottlieb) Core A: Lymphocyte Analysis Core (P.I Zipris) Core B: BDC JDRF Autoimmunity Prevention Center Administrative Core (Director Hutton)

A 5 year plan is proposed with a budget of $ 6.5m direct costs. Each project will span for 4 to 5 years and provision is made for additional studies funded by annually awarded pilot and feasibility grants of up to $100K. Operation of the Center will be managed by an Executive committee headed by Dr. Hutton and comprised of the PI’s with governance from an Advisory committee of national scientific experts and JDRF scientific and lay committee members. Applicant: Eisenbarth, George

Project 1 - Clonotype

SCIENTIFIC ABSTRACT

Objective:

Given data that insulin B:9-23 peptide may be a primary target essential for development of spontaneous diabetes of NOD mice and that T cells recognizing this peptide have a shared “non- stringent” T cell receptor (TCR) with only conserved Valpha and Jalpha, targeting the conserved T cell receptor has the potential to achieve a robust antigen-based immunotherapy.

Background/Rationale:

We will directly test the hypothesis that such genomically encoded T cell receptor chains are crucial for disease in the NOD mouse and recognition by the T cell repertoire of the B:9-23 insulin peptide.

Description of Project:

We propose to create NOD mice having single Jalpha 56, which has different sequence from the conserved Jalpha (53/42) and to evaluate them for development of anti-islet autoimmunity. The remainder of the T cell receptor repertoire will be available (use of different Valpha, N alpha, Vbeta, nDnbeta, Jbeta). C57BL/6 mice bearing only Jalpha 56 were established where a single Jalpha is used to create immune repertoire, and we will create congenic Jalpha56 NOD mice using speed congenic techniques. We predict if the Jalpha 53/42 conserved sequences are critical, anti-insulin/islet autoimmunity will not develop for NOD mice having only the “control” Jalpha 56 sequence. If prevention occurs in mice with the single Jalpha 56, we will molecularly create mice with the single Jalpha 56 sequence modified to be a Jalpha 53 sequence, and we predict restoring autoimmunity.

Anticipated Outcome:

In addition, we will define sequences of the conserved alpha chain that confer anti-insulin autoimmunity. Producing retrogenic mice with the original and modified alpha chain T cell receptors will allow us to define crucial sequences. We will molecularly transfer amino acid cassettes from the conserved Valpha and Jalpha of anti-B:9-23 diabetogenic clones into a “control” alpha chain to assess whether we can create anti-insulin autoimmunity (allowing beta chains to be provided endogenously and thus vary), and transfer amino acid cassettes from control alpha chain into anti-B:9-23 diabetogenic alpha chains to suppress or abrogate autoimmunity.

Relevance to Type I Diabetes:

We believe that these studies has the clear potential to disprove our hypothesis of critical germ- line T cell receptor sequences, but more important if such a pathway is critical in the NOD mouse will stimulate a search for an analogous human critical pathway and clinically relevant immunotherapy for disease prevention based on targeting specific TCR germ line sequences. Applicant: Hutton, John C.

Project 5 - Autoantigen

SCIENTIFIC ABSTRACT

Humoral Autoreactivity of Novel Human Diabetes Autoantigen ZnT8

ZnT8 (Slc30A8) is a recently discovered multispanning transmembrane protein localized to the ß- cell insulin granule that is the target of circulating autoantibodies in humans. It is the basis of a novel and sensitive radioimmunoprecipitation assay that can be applied to predict disease often in the absence of classical autoimmune markers such as anti-insulin, GAD65 and IA2 or islet cell antibodies. Unlike GAD65 and IA2, the autoantigen shows high ß-cell specificity and can potentially be used to monitor changes in residual ß-cell mass post disease onset. It has superior diagnostic value in older subjects than insulin and unlike insulin can be used as an autoimmune marker following initiation of hormone replacement therapy. The proposed research aims at the further development of this marker in terms of:

1) Defining changes in autoantibody titer and prevalence leading up to and following disease and cataloging the relationship of autoantibody persistence and functional beta cell mass.

2) Mapping of conformational epitopes in ZnT8 bound by diabetic autoantibodies.

3) Evaluation of epitope-specific antibodies and B-cell assays as disease specific predictive markers

The primary objectives are to validate and optimize the assay as a diagnostic tool that can be applied to screening of at-risk populations and to explore its utility as a potential therapeutic agent aimed at halting or reversing diabetic autoimmunity prior to clinical disease. Antibody-expressing B-cells play an important role as antigen presenting cells in the pathogenesis of type 1 diabetes through interaction with antigen-specific T-cells (Project 6) and by exploring both aspects of the immune response to ZnT8 we aim to define molecular components of ZnT8 and immunological mechanisms that can be exploited therapeutically (Project 8). The project utilizes clinical sample archives from ongoing studies in the natural history of type 1 diabetes in man (e.g. DAISY and TEDDY at the BDC) and national and international consortia (e.g. ITN and TRIALNET).

Applicant: Davidson, Howard W

Project 6 – T1D

SCIENTIFIC ABSTRACT

Type 1A insulin dependent diabetes mellitus (T1D) is an autoimmune disease characterized by the selective destruction of pancreatic ß-cells, likely due to an imbalance in the immune system of susceptible individuals that allows pathogenic T-cells specific for ß-cell proteins to overcome the regulatory mechanisms that would normally prevent them from attacking self-tissues. Recently we demonstrated that the ß-cell specific zinc transporter hZnT8 (Slc30A8) is a major target of humoral immunity in human T1D (see project 5). As the generation of high affinity class switched antibodies is a T-dependent process we hypothesized that hZnT8 is also a target of potentially diabetogenic T-cells, and our preliminary data support this idea. The goals of this study are to confirm and expand our preliminary observations by completing a comprehensive analysis of T1D associated T-cell responses to hZnT8 in humans. We propose 3 specific aims.

1. To map CD4+ T-cell epitopes in hZnT8 that elicit IFN-? responses in PBMCs from pre- diabetic and diabetic subjects, determine their restriction elements, and investigate whether the same epitopes are recognized by regulatory cells in control subjects.

2. To map CD8+ T-cell epitopes in hZnT8 that elicit IFN-? responses in PBMCs from pre- diabetic and diabetic subjects and determine whether they show disease association.

3. To use the data generated in aims 1 and 2 to create oligomeric peptide-MHC complexes that can be used for flow cytometry based analyses of PBMCs, the expansion of antigen-specific regulatory cells in vitro, and the creation of microarrays for improved monitoring of antigen- specific cytokine responses.

The data thus obtained should identify a panel of peptides that are prime candidates for future phase I clinical trials, and also represent key biomarkers for monitoring progression or remission of T1D.

Applicant: Gottlieb, Peter

Project 8 – PrePoint

SCIENTIFIC ABSTRACT

Objective:

Specific Aim 1: To delineate the effect of oral/intranasal insulin therapy on T cell responses to autoantigens with ELISPOT analysis and CD45RO T cell CSFE assay.

Specific Aim 2: To determine the effect of oral/intranasal insulin therapy on the frequency of T regulatory populations including CD4+CD25+ T cells.

Background/Rationale:

We now have the ability to identify a rare group of genetically susceptible young children with a 50% or greater risk for the development of B-cell autoantibodies (BCA) and type 1 diabetes (T1D). Mucosal (oral and intranasal) insulin has been safely used in subjects positive for BCA to delay or prevent T1D. Pre-POINT (Primary Oral/intranasal INsulin Trial), an international JDRF study headed by Ezio Bonifacio, with Jennifer Barker at the BDC as US coordinator and BDC core laboratories, has been developed with the objective to determine the feasibility, safety and bioavailability of oral and/or intranasal insulin in children with high genetic risk for T1D in a dose escalation primary intervention pilot study.

Hypothesis:

Insulin-specific therapy can prevent the development of autoantibodies in individuals who are at high genetic risk for the development of type 1diabetes (T1D). B and T cell studies can detect responses to insulin and other autoantigens that will correlate with the prevention of autoimmunity and overt hyperglycemia.

Description of Project:

The Pre-POINT Study will recruit young children who are first degree relatives of type 1 patients with high risk HLA genes that are inherited in a haploidentical way to their affected proband. The study will administer oral or nasal insulin in a dose escalation schedule to determine both safety and efficacy regarding preventing the development of autoantibodies and changes in T cell responses and phenotype. The CD4 indirect ELISPOT assay will be performed with the assistance of Core A. The mechanistic studies will examine both B and T cell responses to insulin. IgG and IgA antibodies to insulin in serum, and IgA antibodies to insulin in saliva in samples measured before commencing treatment, and after 3 months of treatment will be examined. BCA and T cell studies will be obtained at baseline, 3, 9 and 15 months after initiating therapy. In subjects positive for insulin autoantibodies, IgG subtyping and affinity will be performed. For participants who participate 9 months or longer, samples will also be taken at 9 months and 15 months after commencing treatment. Cell mediated responses to insulin and proinsulin peptides will be performed in consultation with Dr Gottlieb core laboratory, Denver and Dr Peakman, London and Dr. Ezio Bonifacio in Germany. The ELISPOT assay to proinsulin peptides as described by Arif (46) is already established at the University of London in the UK, in Munich, and in Denver and will also be used in TrialNet mechanistic studies. Antigens will include the study drug (whole insulin), four overlapping insulin peptides, a proinsulin peptide, and diluent control, a recall peptide control, and GAD65. IFNg and IL-10 responses will be measured. Dr. Bonifacio will also use his CD45RO CFSE recall assay to examine T cell reactivity in a subset of patients as well.

Anticipated Outcome:

We expect to be able to detect responses to proinsulin peptides which will be stimulated by insulin since this is the antigen being used in the Pre-POINT study. Further we expect to see that antigen specific therapy given by oral or intranasal routes should generate protective immune responses that would be reflected in positivity in the IL-10 ELISPOT assay and in the production of Th2 associated cytokines such as IL-4, IL-13 and TGF . We hope to detect native responses to autoantigenic peptides that are defined as responses in the IFN wells and hopefully modulation of those responses over time. This could reflect decreases in the number of IFN spots and/or the generation of IL-10 response to the same peptides or new peptides of insulin and perhaps other autoantigens such as GAD or IA-2 (infectious tolerance). Alternatively, this type of therapy may lead to deletion of the response rather than deviation and we may observe decreased spot numbers without a generation of the IL-10 response or other Th2 or Treg cytokines.

Relevance to Type I Diabetes:

Understanding the mechanism of treatment will be critically important in determine the optimal dose and timing of therapy as the Pre-POINT study moves forward into a larger clinical trial. Treatment with insulin by the mucosal route should allow us to see how this route of therapy influences the development of T cell reactivity and phenotype. Using immunologic endpoints to better understand how the character of the T cell response is changed by therapy will be vitally important in allowing us to define the best dose and route of administration of antigen-specific therapy. This will be critical in making this approach as effective as possible in preventing the development of T1D

Applicant: Zipris, Danny

Core A - Lymphocyte

SCIENTIFIC ABSTRACT

Objective:

High-quality facilities providing services for lymphocyte subset purification and phenotypic and functional analyses of lymphocytes are a fundamental requirement for a wide variety of studies addressing the role of T and B cells in the pathogenesis of type 1 diabetes (T1D). The main goal of the proposed Lymphocyte Analysis Core is to provide Project Investigators with critical experimental resources that are described in the following four specific aims:

Specific Aim 1 is to perform ELISPOT assay analyses to monitor the function of rare antigen- specific T- and B-cell responses at the single-cell level. Core personnel will be responsible for quantitating T and B cell responses using the ELISPOT Bioreader.

Specific Aim 2 is to purify T-cell subsets and B lymphocytes using the AutoMacs system and Miltyenyi beads.

Specific Aim 3 is to perform phenotypic analyses of T and B lymphocytes by means of flow cytometry. For this purpose, the Core will use the FACS Calibur and BD LSR II systems available at the Barbara Davis Center.

Specific Aim 4 is to perform multiplex cytokine analysis using the Luminex 200 system. The core will evaluate the level of multiple cytokines produced in vitro by autoreactive T and B cells and in sera from humans or animal models of diabetes.

Background/Rationale:

The proposed Autoimmunity and Prevention Center will identify the role of humoral and cellular responses in the mechanism of autoimmune diabetes in the following experimental systems, each of which will be supported by this Core: Project 1: Evaluation of a conserved alpha chain T cell Receptor clonotype as a critical determinant for type 1 diabetes in the NOD mouse.

Project 5: Humoral autoreactivity of novel human diabetes autoantigen ZnT8

Project 6: Cell mediated autoimmunity to a novel human diabetes autoantigen ZnT8

Project 8: Pre-Point: Immune response monitoring and application of novel assays post mucosal insulin in autoantibody negative children

Description of Project:

The Lymphocyte Analysis Core intends to collaborate with the investigators and contribute to all aspects of the research process. This includes consultation in experimental design, technical assistance in the operation of instruments, trouble shooting, data analysis and storage, and data interpretation. The Core facility will also communicate with investigators at other institutions in order to introduce and develop new applications. The Core will also adhere to a rigorous set of quality control guidelines for turn-around of data, trouble-shooting and other parameters for daily monitoring of all assays.

Anticipated Outcome:

The primary benefit of the Lymphocyte Analysis Core is the establishment of a high-quality, cost- effective facility for addressing T and B cell phenotype and functions in T1D. The proposed core will provide critical experimental tools to the Project Investigators required for performing their studies in the most efficient manner. The majority of the instruments that will be used by the Core for providing the proposed services in this application are available and up and running at the Barbara Davis Center. We do not currently have access to the Luminex system. We will need to purchase this instrument and we are requesting funds to cover a portion of its cost.

Relevance to Type I Diabetes:

It has been demonstrated that genes in the Class II region play a key role in disease susceptibility (8,9). There is a strong link between susceptibility to diabetes and specific alleles of MHC II genes, in particular HLA-DR and HLA-DQ (10-13). Susceptibility to diabetes is thought to be mediated by the HLA-DRB1*0401-DQA1*0301-DQB1*0302 (DR4/DQ8) haplotype, and protection by DRB1*1501-DQA1*0102-DQB1*0602 (DR2/DQ6) (12). Three major islet autoantibody specificities against insulin (14), glutamic acid decarboxylase (GAD) (15), and islet tyrosine phosphatase, IA-2 (16) were identified and most of the studies analyzing T cell responses against autoantigens in T1D have focused on these three autoantigens. It has been demonstrated that class-switched IgG autoantibodies against these islet proteins are detectable in the peripheral blood of patients with T1D and individuals at high risk for disease development, implying T cells in the ongoing anti-islet immune response (17,18). This hypothesis is further supported by recent studies demonstrating that T cells directed against insulin, GAD and IA-2 can be derived from patients with T1D (19-21).

Applicant: Hutton, John C.

Core B - Administration

SCIENTIFIC ABSTRACT

The Administrative Core will be established that will be headed by the Principal Investigator Dr. Hutton as Core Center Director and Dr. Eisenbarth as Associate Director. The Core will be staffed by a part-time administrator (50%), a part-time finance officer (25%), a part-time (10%) IT / website manager who will also act as the HIPPA compliance office, and a part time (25%) professional research assistant whose principal role will be as a coordinator of human subjects recruited for clinical trials. An executive committee will be established which includes the Center Director, the principal investigators of each project, the head of the Immunoassay Core. The Administrative Core will interact with staff employed by the Center, BDC staff, University administrators, JDRF staff, and the lay community to enhance the visibility of the JDRF Autoimmunity Prevention Center and to promote it as an instrument for world-class diabetes research. External advisors will be drafted as appropriate to conduct a site visit of the Center in its second year. The principal activity of the Administrative Core is to support the Research Base of the Center and its research efforts as formulated in the projects 1, 5,6, and 8 and the immunoassay core. Its staff will also interact with directors of the Diabetes and Endocrine Research Center (DERC) and which operate core facilities in the areas of Clinical Research, Histology, Flow Cytometry, Animal resources, Molecular Biology, Bioinformatics and viral vector construction.

JDRF Center on Immunological Tolerance in Type-1 Diabetes at Harvard Medical School

Diane Mathis

of

Harvard Medical School

Applicant: Mathis, Diane

Composite: JDRF Center on Immunological Tolerance in Type-1 Diabetes at Harvard Medical School

SCIENTIFIC ABSTRACT

Objective:

The major goals of the JDRF Center on Immunological Tolerance in Type-1 Diabetes at Harvard Medical School are to identify mechanisms responsible for the defective T cell tolerance at the root of type-1 diabetes, and to exploit this knowledge to develop novel strategies for preventing or reversing the disease. The essence of the renewed Center will be a JDRF-funded confederation of 8 research Projects, 2 technological Cores and an Administrative Core. A cluster of three Projects will develop novel murine:human chimeric systems, and then exploit them to study the breaking and remaking of T cell tolerance in T1D patients. A second cluster of four Projects will focus on new molecular and cellular immonoregulatory agents, how they translate to the human context, and how they can be harnessed to therapeutic ends. The eighth Project will explore the relationship between autoimmunity and inflammation in NOD diabetes, and will test the therapeutic efficacy of anti-inflammatory reagents in halting disease. The technological Cores will provide access to state-of-the-art mouse and murine:human models, and to clinically, immunologically and genetically characterized T1D patient and control blood samples. The Center can also draw on a number of allied resources at the Joslin Diabetes Center and within the Harvard Community more generally, which greatly enriches its scientific base and potential.

Relevance to Type I Diabetes:

The Center is focused on learning to re-establish immunological tolerance, which is central to prevention and therapy of T1D.

Applicant: Mathis, Diane

Project 1: Defective central tolerance induction in humans?

SCIENTIFIC ABSTRACT Objective: To determine whether human T1D patients, like NOD mice, have a T cell-intrinsic defect in immunological tolerance at the “central” (i.e. thymic) level.

Background/Rationale: The central role of -cell-reactive T cells in T1D implies that individuals with this disease harbor some kind of defect in the induction of T cell tolerance to self-antigens (Ags). Indeed, it has recently become clear that NOD mice have a genetic deficiency in the clonal deletion of self- reactive thymocytes. Our studies during the last funding cycle of the JDRF Tolerance Center at HMS established that the NOD genetic background is much less effective than the C57Bl/6 (B6) background at supporting clonal deletion of BDC2.5 T cell receptor (TCR) transgenic (tg) thymocytes in fetal thymic organ cultures (FTOCs), in response to either endogenous autoAg or exogenously added mimotope peptide. This abnormality is thymocyte-intrinsic. Parallel observations have been made with other TCR specificities (eg the LCMV gp peptide-reactive P14) and in the in vivo context. During the Center’s next funding cycle, we propose to translate these observations to the human arena: do T cell precursors derived from T1D patients have an inherent defect in clonal deletion in response to cognate Ag in the thymus?

Description of Project: To render the above question tractable, we will develop a novel murine:human chimeric system that exploits mimotope engagement of the BDC2.5 TCR to read out intrinsic human T cell signaling pathways. More specifically, we will:

1) Optimize a procedure for routine differentiation and expansion of human T cell precursors from hematopoetic cell progenitors (HCPs). This procedure will involve culture of HCPs on OP9 stromal cells expressing the Notch ligand Delta-like-1 (OP9-DL1), followed by cell sorting of the desired prothymocyte population.

2) Develop a method to assay clonal deletion of human T cell precursors when confronted with cognate antigen in the thymus. This method will entail retrovirus-mediated transduction of the prothymocytes with the BDC2.5 (or P14) TCR genes, seeding of NOD reconstituted thymic organ cultures (RTOCs) (or eventually NOD mouse thymi in vivo) with the transduced precursors, addition of varying amounts of BDC2.5mi (or LCMVgp) peptide, and flow cytometric analysis of the resulting thymocyte subsets.

3) Compare the inherent ability of T cell precursors generated from T1D patients and controls to undergo thymic deletion.

Anticipated Outcome: Data from these studies should allow us to answer an important question about the pathophysiology of human T1D: do patients harbor inherent defects in T cell central tolerance induction, as do NOD mice? The novel murine:human chimeric system that we will develop should provide an excellent framework for downstream experiments aimed at molecularly and genetically defining any abnormalities identified, and can also be used in a number of allied applications.

Relevance to Type I Diabetes: It is important to understand the pathogenesis of T1D. Pinpointing abnormalities in immunological tolerance would be an important insight. Applicant: Greiner, Dale

Project 2: Autoreactive humanized TCR transgenic mice in T1D

SCIENTIFIC ABSTRACT

Objective: We propose to use a new humanized mouse model of type 1 diabetes to study the development and function of human islet autoreactive T cells. This model is based on three technological breakthroughs: 1) Generation of human islet autoreactive T-cell clones. 2) Development of retroviral technology enabling efficient transduction of human hematopoietic stem cells (HSCs). 3) Availability of new generation immunodeficient HLA-transgenic NOD-scid IL2rγnull mice. NOD-scid IL2rγnull mice engrafted with human HSC generate a complete functional human immune system.

Background/Rationale: We have the next generations of genetically modified NOD-scid IL2rγnull mice available for our studies, including HLA transgenic mice and mice expressing the human costimulatory molecule B7.1 on their beta cells that will provide the appropriate intrathymic environment for the proper selection of the HLA-A2-expressing transgenic T cells as well as providing optimal HLA-restricted islet targets for the autoreactive T cells. We have also established lentiviral technology in our laboratory, and we have human HLA-A2-restricted T cell clones that are specific for human IGRP265-273 (islet-specific glucose-6-phosphatase catalytic subunit-related protein) and for human insulin B10-18 that have been provided by Dr. Bart Roep.

Description of Project: Aim 1 will construct the lentivirus vector containing the human IGRP-autoreactive TCR and validate its ability to transduce human HSC that generate human TCR Tg T cells. Aim 2 will generate human IGRP TCR Tg retrogenic humanized mice. Aim 3 will investigate mechanisms by which therapeutics in clinical trials ameliorate autoimmune type 1 diabetes. Humanized mice provide important pre-clinical model systems without putting individuals at risk.

Anticipated Outcome: We will use these new technologies to develop “retrogenic” NOD-scid IL2rγnull (Tg) HLA-A2 RIP- B7.1 mice engrafted with a human immune system expressing a transgenic autoreactive TCR directed against IGRP265-273. These mice will be used to investigate human autoreactive T cell development and function at various stages of the disease process to study the efficacy of currently available therapeutic interventions. Aim 1 will construct the lentivirus vector containing the human IGRP-autoreactive TCR and validate its ability to transduce human HSC that generate human TCR Tg T cells. Aim 2 will generate human IGRP TCR Tg retrogenic humanized mice.

Relevance to Type I Diabetes: The generation, validation, and use of these new animal models to investigate underlying mechanisms of currently available therapeutic interventions will facilitate the investigation of mechanisms important in the development, prevention, and reversal of autoimmune type 1 diabetes.

Applicant: Sykes, Megan

Project 3: Regulation and homeostasis of diabetic human T cells

SCIENTIFIC ABSTRACT

Objective:

To identify hematopoietic stem cell-intrinsic defects in T cell homeostasis and regulation in Type I diabetic patients.

Background/Rationale:

Studies in NOD mice, the best known model for T1DM immunopathogenesis, indicate that the autoimmunity associated with Type 1 diabetes mellitus (T1DM) is driven in large part by genetically-determined abnormalities in hematopoietic cells. Many of these abnormalities have been mapped to loci containing immunoregulatory genes. Studies in NOD mice and in patients with T1DM implicate abnormalities in peripheral T cell survival and homeostasis, and in regulatory T cells and NKT cells, in disease pathogenesis. However, models in which these abnormalities in human T cells can be dissected and studied prospectively are lacking.

Description of Project:

This proposal builds on a unique humanized mouse model we have developed in which robust peripheral reconstitution with human antigen-presenting cells (APCs) in combination with a human thymus graft allows the optimal development, peripheral survival and function of human T cells, including Treg. We will build on these data to: 1) Develop a reaggregate human thymus transplant model allowing human thymopoiesis in HLA-defined human thymus grafts. These studies will extend our humanized mouse model; instead of implanting intact fetal thymus tissue, we will implant CD45-negative thymic stromal cells cryopreserved from HLA-typed human fetal thymic tissue under the kidney capsule of NOD-SCID mice. Thymi bearing common diabetes- susceptibility HLA alleles will support the thymopoiesis of T cells from i.v. injected CD34+ cells from normal control and Type 1 diabetic subjects sharing these alleles; 2) Use the model in Aim 1 to compare peripheral survival, homeostatic expansion, phenotypic conversion and self tolerance of conventional T cells derived from CD34 cells of T1DM versus normal controls; 3) Use the model in Aim 1 to compare numbers, function and peripheral phenotypic conversion of regulatory T cells derived from CD34 cells of T1DM versus normal controls.

Anticipated Outcome:

We hypothesize that T cells from T1DM patients will show increased lymphopenia-driven expansion in association with loss of tolerance to “self” antigens of the thymus donor. We hypothesize that Tregs and NKT cells derived from stem cells of T1DM patients will show defects. These studies will identify intrinsic abnormalities in regulatory cells from T1DM patients and their thymic versus post-thymic origin.

Relevance to Type I Diabetes:

Together, our studies in this unique model should provide novel insights into the role of HSC- determined immunoregulatory abnormalities in driving autoimmune pathogenesis in T1DM. They will pave the way for more detailed genetic and genomic analyses. Applicant: Benoist, Christophe

Project 4: Genomics of FoxP3+ Treg cells in mice and humans

SCIENTIFIC ABSTRACT

Objective: 1) To identify the molecular basis of the functional abnormality of Treg cells in NOD mice. 2) To determine whether Treg cells in human T1D patients have functional abnormalities and, if so, to identify them.

Background/Rationale: Peripheral mechanisms of immunological tolerance and immunoregulation are essential to prevent the damage that may be caused by autoreactive T cells that escape the filters of thymic tolerance induction. The best studied of these mechanisms is a subpopulation of CD4+ regulatory T (Treg) cells, characterized by high-level expression of the transcription factor FoxP3 and a distinct gene-expression signature. These cells have the ability to suppress immune and autoimmune responses in several experimental settings. We have recently found that, although the proportion of Treg cells is normal or even high in NOD mice, they show subtle functional defects accompanied by clear differences in a small set of genes. Some of these candidate genes are quite suggestive of a role in Treg function, although this remains speculative. Second, we and others have found that the number of FoxP3+ cells is normal in the blood of T1D patients. Whether Treg function is fully normal in diabetic individuals is an issue that remains open, and is difficult to address given the obvious limitations to experimentation in humans. This project will explore Treg defects in parallel in mice and humans.

Description of Project: In NOD mice, we will establish the root of the perturbation of Treg function, testing how the functional and gene expression differences evolve over the course of diabetes progression, and whether the differences observed result from variation in Treg activity or from differential susceptibility of Tconv cells to Treg action. We will test the candidate genes we have identified by constructing lentigenic mice with RNAi knockdowns, or by tetracycline-controlled transgenesis with T cell-specific drivers. In T1D patients, we will search for similar perturbations, by investigating whether the candidate genes/pathways identified in mice might also have perturbed expression. We will also perform a full genomic analysis of Treg cells in human T1D patients, searching for specific alterations of the Treg signature that correlate with disease status.

Anticipated Outcome: We expect to understand why Treg cells are abnormal in NOD mice, and whether (and why) they are also functionally deficient in T1D patients.

Relevance to Type I Diabetes: We stand to learn about T1D pathogenesis in both mice and humans. This knowledge may be applied to the currently attractive proposition of harnessing Treg cells therapeutically or preventatively.

Applicant: Anjana Rao

Project 5: Dissecting TGF- signaling during differentiation of effector and regulatory T cells

SCIENTIFIC ABSTRACT

Objective:

To determine how the cytokine TGF- influences the course of type I diabetes.

Background/ Rationale:

The pathogenesis of autoimmune diseases, such as type I diabetes, is strongly influenced by whether naive T cells differentiate into effector or regulatory T cells (Tregs). Whereas effector T cells, such as Th1 and Th17 cells, mediate tissue inflammation and damage, Tregs blunt effector T cell responses and so have a central role in establishing peripheral immune tolerance. A variety of Treg lineages have been reported, including inducible Tregs (iTregs) and IL-10 producing Tr1 cells. The transforming growth factor (TGF)- family of cytokines plays an essential role in differentiation of effector Th17 cells as well as two separate regulatory lineages, iTreg and Tr1. Briefly, iTreg differentiation is promoted by TGF- alone, Th17 differentiation by the combination of TGF- and IL-6, and Tr1 differentiation by the combination of TGF- and IL-27. However, the signalling pathways used by TGF- to promote differentiation in these various directions have not been sorted out.

Description of project:

Aim 1 of this proposal is to delineate the signalling pathways downstream of TGF- that promote the in vitro differentiation of cultured murine Th17 and iTreg cells from naïve precursor T cells.

Aim 2 is to define the signalling pathways downstream of TGF- that promote the differentiation of cultured human T cells.

Aim 3 is to extend the in vitro studies to investigate in vivo responses in the NOD mouse model of autoimmune diabetes. The experiments take advantage of a potent pharmacological inhibitor of TGF- signalling, SB431542, and the existence of a mutant TGF- RI receptor (ALK-5 S275M) that is resistant to this inhibitor but is otherwise entirely functional.

Anticipated outcome:

In Aims 1 and 2, signalling pathways downstream of the TGF- R1 receptor will be dissected by retroviral delivery of the inhibitor-resistant (ALK-5 S275M) TGF- RI receptor into primary mouse and human T cells respectively, under conditions where signalling through the endogenous receptors is blocked with the pharmacological inhibitor SB431542. We will then use mutated versions of the inhibitor-resistant TGF- R1 receptor to define the specific contributions of SMAD and non-SMAD signalling pathways to Th17, iTreg and Tr1 differentiation (by culturing the transduced cells with the TGF- R1 inhibitor and TGF- alone or with the appropriate cytokine). If R-Smad signaling is required, we will define the transcriptional interactions among the Smads and Foxp3 and ROR-gammat, transcription factors characteristic of the iTreg and Th17 lineages respectively. If R-Smad signaling is not required, we will ask which non-SMAD pathways are important in each case.

For Aim 3, naïve T cells from BDC2.5/ NOD mice will be retrovirally-transduced with wildtype and mutant versions of the inhibitor-resistant ALK-5 receptor, and cultured under conditions that promote differentiation into Th17, iTreg and Tr1 cells as described in Aim 1. The cells will then be injected in place of either “effector” or “regulatory” populations into newborn mice, and diabetes development will be assessed. The experiments will establish whether the same TGF- signals that are needed for in vitro differentiation of Th17, iTreg and Tr1 cells are required for functional responses in vivo, in the context of autoimmune diabetes.

Relevance to Type I diabetes:

The balance between autoimmunity and tolerance is regulated by whether naïve T cells differentiate into effector or regulatory T cells (Tregs). Effector T cells, such as Th1 and Th17 cells, mediate tissue inflammation and damage, whereas Tregs blunt effector T cell responses and so have a central role in establishing peripheral immune tolerance. This balance strongly influences the course of autoimmune diseases such as type I diabetes. If distinct TGF- signalling pathways are found to underlie effector and regulatory T cell differentiation, selective inhibitors of these pathways could potentially be used in the clinic to manipulate the course of autoimmune diabetes by promoting the development of tolerance-inducing iTreg and Tr1 cells while suppressing Th17 differentiation and associated inflammatory responses.

Applicant: KUCHROO, Vijay K.

Project 6: Pathogenic and Regulatory T Cells in Type 1 Diabetes

SCIENTIFIC ABSTRACT

Objective: Type I diabetes is an autoimmune disease in which proinflammatory Th1 cells that produce IFN- have been shown to induce disease. However, recently another effector T cell subset that produces IL-17 (named Th17) has been identified and implicated to be the effector T cell involved in the induction of many autoimmune diseases. Whether Th17 cells also induce type 1 diabetes has not been addressed. Induction of type 1 diabetes by the pathogenic T cells is regulated by another class of T cells called regulatory T cells, which includes CD4+ CD25+, Fox-P3+ regulatory T cells (T-reg) and IL-10-producing Tr1 cells. While the role of CD4+ CD25+ Treg cells has been studied, the role of Tr1 cells in the regulation of diabetes has not been studied. The main objective of this grant proposal is to determine how the balance between pathogenic and regulatory T cells can be regulated so as to affect the course of autoimmune diabetes.

Background/Rationale: The NOD mice serves as a good model for human type 1 diabetes, in which proinflammatory CD4+ and CD8+ T cells have been shown to induce diabetes. Development of diabetes is dictated by a balance between pathogenic and regulatory T cells. Whereas TGF- induces Fox- P3 and generates T-reg cells, we have found that presence of IL-6 inhibits the generation of T-reg cells and induces development of pathogenic Th17 cells suggesting that there is a reciprocal relationship in the induction of pathogenic Th17 and protective T-reg cells. IL-6 a cytokine produced by the activated innate immune system plays a pivotal role in regulating this balance. In addition to IL-6, we have found that another cytokine produced by activated innate immune system called IL-27 also suppresses the induction of Fox-P3 but induces IL-10-producing Tr1 cells. We hypothesize that IL-6 and IL-27 produced by the activated innate immune system plays a crucial role in regulating the balance between the pathogenic and regulatory T cells.

Description of Project: This grant proposal will focus on studying i) Whether Th17 cells with specificity for islet antigens are pathogenic in inducing type 1 diabetes; ii) Whether Tr1 cells generated in vitro in the presence of TGF plus IL-27 can transfer protection against diabetes when transferred in vivo and III) whether loss of IL-6 will result in the predominant generation of T-reg cells in NOD mice and thus protect them from developing diabetes (on the other hand whether IL-27R deficiency on the NOD background will accelerate development of diabetes due to loss of protective Tr1 cells).

Anticipated Outcome: We anticipate that islet specific Th17 cells will be highly pathogenic in inducing diabetes, and that Tr1 cells with specificity for islet antigens will be protective. Furthermore, we predict that IL-6 will be a good target for regulating autoimmune diabetes in that the loss of IL-6 would shift the balance in the favor of T-reg cells and thus make NOD mice highly resistant to type 1 diabetes.

Relevance to Type I Diabetes: By analyzing the cytokine requirements for the generation of pathogenic and regulatory T cells, we will be able to readily regulate the balance between these two subsets of T cells. The analysis will be useful in identifying the role of two innate cytokines (like IL-6 and IL-27) in regulating the balance and the development of type 1 diabetes. This information can be gainfully employed to design therapeutic tools to target cytokines of the innate immune system for the development of treatments for type 1 diabetes in humans. Applicant: Wucherpfennig, Kai W.

Project 7: Induction of beta cell specific regulatory T cells

SCIENTIFIC ABSTRACT

Objective:

The major goal of the project is to develop a novel approach for the prevention of T1D.

Background/Rationale:

A series of studies have demonstrated that regulatory CD4 T cells have the potential to prevent and even reverse type 1 diabetes in NOD mice. Small numbers of cells can be sufficient, but the regulatory T cells need to be specific for beta cell antigens. However, the induction of regulatory CD4 T cells with defined specificity is challenging due to the diversity of antigen presenting cell populations that induce differentiation of CD4 T cells into a variety of different functional subsets.

Description of Project:

Recent work has shown that some cytokine receptors such as the IL-2 receptor localize to the immunological synapse, and our approach is based on this physiological mechanism. We propose to generate bifunctional molecules comprised of a MHC class II/peptide complex and a regulatory cytokine in order to induce differentiation of regulatory T cells with defined specificity. We hypothesize that bifunctional complexes containing a single peptide/MHC (pMHC) and a cytokine will lead to the activation and expansion of T cells with defined specificity (based on the pMHC component) and cytokine profile (determined by the differentiation pathway induced by the linked cytokine). Our approach circumvents the requirement for antigen presenting cells by direct targeting of the relevant receptors on T cells via the pMHC and cytokine arms of these bifunctional molecules.

Anticipated Outcome:

In Aim 1, we will generate cytokine-pMHC complexes using the NOD MHC class II molecule and examine the ability of the complexes to induce T cell activation and differentiation into Foxp3+ Treg or Tr1 cells with defined pMHC specificity. In

Aim 2, we will examine if injection of such regulatory CD4 T cells provides protection from T1D and whether administration of cytokine-pMHC complexes can induce such T cells populations in vivo.

In Aim 3, we will generate the corresponding human complexes using a diabetes-associated DR molecule that we have already expressed and study the biological properties of the complexes with human T cells.

Relevance to Type I Diabetes:

This project thus has significant translational potential by aiming to develop a novel approach for the induction of regulatory T cells with defined specificity and function for the prevention of T1D.

Applicant: Strom, Terry

Project 8: The autoimmunity/inflammation connection in type-1 diabetes

SCIENTIFIC ABSTRACT

Objective:

To obtain the best possible therapeutic effect in patients with new onset T1D, the treatment should restore euglycemia, ablate invasive insulitis, restore immune tolerance to islets, ablate insulin resistance/ restore insulin signaling and enable restoration of the diminished beta cell mass.

Aim 1: We will determine whether short-term treatment with a neutralizing anti-TNF- mAb serves to (1A) permanently restore euglycemia; (1B) ablate invasive insulitis and selectively restore tolerance to syngeneic islets; (1C) ablate inflammation within critical insulin responsive tissues and thereby restore insulin signaling and responsiveness; (1D) enable expansion of the beta cell mass in new onset T1D NOD mice rendered euglycemic by therapy

Aim 2: We will determine whether treatment with IL-1Ra, the soluble IL-1 R antagonist, short- term serves to (2A) permanently restore euglycemia; short-term serves to (2B) ablate invasive insulitis and selectively restore tolerance to syngeneic islets; (2C) ablate inflammation within critical insulin responsive tissues and thereby restore insulin signaling and responsiveness; and (2D) enable expansion of the beta cell mass in new onset T1D NOD mice rendered euglycemic by therapy.

Aim 3: We will determine whether treatment with a neutralizing anti-IL-6 mAb serves to (3A) permanently restore euglycemia; We will employ a 6 day course of neutralizing anti-IL-6(3B) ablate invasive insulitis and selectively restore tolerance to syngeneic islets; (3C) ablate inflammation within critical insulin responsive tissues and thereby restore insulin signaling and responsiveness; and (3D) enable expansion of the beta cell mass in new onset T1D NOD mice rendered euglycemic by therapy.

Aim 4: As anti-CD3 mAb therapy ablates invasive insulitis and restores tolerance but does not ablate insulin resistance and enable restoration of the beta cell mass, we will test combined anti- CD3 mAb therapy with treatments that target TNF- IL-1 Ra or IL-6

Background/Rationale:

Short term treatment of new onset T1D NODs with either anti-CD3 mAb, power mix or alpha1 antitrypsin (AAT) ablates invasive insulitis and permanently restores euglycemia and tolerance to islets in the vast majority of treated mice. To obtain the best possible therapeutic effect in patients with new onset T1D, the treatment should restore euglycemia, ablate invasive insulitis, restore immune tolerance to islets, and also restore insulin signaling and thereby ablate insulin resistance as well as enable restoration of the beta cell mass. In the clinically informative new onset NOD T1D model, anti-CD3 mAb, the most effective T cell directed strategy tested to date, ablates invasive insulitis and restores tolerance but does not ablate insulin resistance and enable restoration of the beta cell mass We now want to test the hypothesis that new onset diabetes is associated with a highly detrimental form of inflammation that plays, in addition to its role in initiating T-cell dependent autoimmunity, an important role in hastening the onset of T1DM by creating insulin resistance. Hence, the insulin resistance compounds the difficulties arising from immune mediated loss of islets.

Description of Project:

Through the specific therapeutic targeting of pro-inflammatory proteins we believe that we can restore normal blood glucose levels, thereby setting the stage for immune tolerance therapies.

Anticipated Outcome:

We believe that immune tolerance will be more easily induced in non-inflamed islets than in inflamed islets. As a consequence we believe that T-cell directed tolerizing regimens will prove more successful if given after a course of select anti-inflammatory therapy.

Relevance to Type I Diabetes:

The data suggest that a cure for T1DM will require silencing of inflammation and creation of immune tolerance.

Applicant: Benoist, Christophe

Core A: Manipulated NOD Mouse Core

SCIENTIFIC ABSTRACT

Objective:

1) To provide Center investigators with NOD-related mouse strains

2) To construct novel mouse lines for Center investigators (transgenesis, shRNA, etc)

Background/Rationale:

The NOD mouse has made important contributions to our understanding of the pathogenesis of T1D; studies on this model are a shared focus of the Center’s activities. Many variants of the NOD mouse that allow one to probe the role of particular molecules, pathways or cells have been established. Ready access to established NOD variants, and the ability to further manipulate the NOD genome, are essential tools for basic diabetes research.

Description of Project:

The Core will support the Center projects in two complementary ways. First, it will provide Center investigators with a panel of NOD-related mouse strains, encompassing the main transgenes or mutations used regularly in diabetes experimentation. The lines are maintained live, in numbers that allow for immediate delivery of breeding pairs or small experimental cohorts for exploratory experimentation. They are listed and described on a detailed website. Second, the core produced novel mouse lines by several modes of transgenesis, all performed directly on the NOD genetic background: i) conventional transgenesis by DNA microinjection; ii) construction of BAC transgenic mice by microinjection of large DNA fragments; iii) generation of lentigenic lines by microinfection of NOD zygotes with lentivirus vectors – used for gene knockdown by RNA interference (RNAi) or targeted cDNA expression. The success rates have been quite high (on average 15% for the former, 60% for the latter), making these effective tools for further probing of diabetes pathogenesis.

Anticipated Outcome:

Following in the tradition of the last funding cycle, the Core will serve the needs of the Center, but will also play an important role by distributing its strains and particular competence to diabetes investigators elsewhere, thus leveraging the investment in this technology beyond the Center.

Relevance to Type I Diabetes:

Provides a key resource for studies in the pathogenesis and treatment of T1D.

Applicant: Svoren, Britta

Core B: Human Sample Procurement Core

SCIENTIFIC ABSTRACT

Objective:

The Human Sample Procurement Core will support translational research endeavors within the JDRF Center for Immunological Tolerance in Type 1 Diabetes (T1D) at Harvard Medical School by providing the laboratories access to well-characterized blood samples from patients with diabetes at different stages of the disease.

Background/Rationale:

The goals of the JDRF Center are to identify the mechanisms underlying the defective T cell tolerance leading to T1D and to use this knowledge to develop novel strategies for preventing or reversing the disease. Every one of the projects has a clear path to the human arena with almost all of them having one or more Aims involving human material. The addition of a translational Human Sample Procurement Core, which did not exist in the Center’s previous organization, will allow the leads identified by the projects in mouse models to be more rapidly and comprehensively evaluated in human patients.

Description of Project:

Individuals with T1D (recent onset, long-standing) and matched controls (healthy or T2D) will be recruited from the patient populations at the Joslin Diabetes Center and neighboring institutions. The Core will perform and record a basic characterization of patients and their samples. The analysis will include a thorough evaluation of clinical characteristics from a diabetes and autoimmune standpoint, and an immunogenetic workup (outsourced to Joslin or other cores): autoantibody determination, HLA typing and genotyping for the best recognized susceptibility loci (INS, PTPN22, CTLA4). A relational database will be adapted to record all patient information, copies of which will be provided in a de-identified manner to the investigators.

Anticipated Outcome:

The availability of well-characterized blood samples from patients with diabetes will greatly facilitate the translational exploration of concepts and targets emerging from the Center’s basic research projects

Relevance to Type 1 Diabetes:

The Core will provide well-characterized samples from T1D patients for experimental evaluation by the Center’s laboratories thereby facilitating the translation of leads obtained in mouse systems to applications in patients with diabetes. The hope is to develop novel strategies for preventing or reversing the disease.

Applicant: Mathis, Diane

Core C: Administrative Core

SCIENTIFIC ABSTRACT

Objective:

The overall goal of the Administrative Core (Core C) is to administer the JDRF Center on Immunological Tolerance in Type-1 Diabetes at HMS, maximizing its effectiveness. As such, its major activities will fall under 3 areas:

1) Administration of the JDRF Center.

The Administrative Core will decide the Center’s overall scientific direction, integrating input from the Directors, an Internal Advisory Board, the other Project and Core PIs, additional Center participants, an External Advisory Board, and the JDRF scientific and lay leadership and reviewers. The Core will also administer the Center grant, requiring liaison between the Center, the JDRF, HMS, Joslin Diabetes Center and the other six affiliate institutions. It will assure the timely submission of all applications and reports, and the ascertainment of needed “permissions”. The Core will pay particular attention to integrating the Center’s activities with those of Dr. Strom’s Catalyst Center “A Pipeline to Induce Tolerance”.

2) Promoting synergy within the Center.

The Core will organize monthly scientific meetings and a yearly retreat. It will maintain, and enrich, the Center’s website, which serves as an amalgamating force for synergy between the Projects and Cores. It will keep a database of “live” contact information for all PIs, as well as for participating fellows and students from their labs.

3) Fostering outreach from the Center.

The Core will assure the Center’s participation in all JDRF-organized all-centers face-to-face, telephone and “e” meetings. It will advertise the Center’s activities and accomplishments to external scientists and to the public via email announcements, through the Center website and by providing sporadic “news briefs” to the JDRF, Harvard and Joslin for potential inclusion on their websites.

Relevance to Type I Diabetes:

The Center is focused on learning to re-establish immunological tolerance, which is central to prevention and therapy of T1D.

JDRF-BRI Center for Translational Research

Gerald Nepom

of

Benaroya Research Institute

Composite: JDRF-BRI Center for Translational Research

SCIENTIFIC ABSTRACT

Objective: The Benaroya Research Institute at Virginia Mason (BRI) is dedicated to research on the causes and cures of human autoimmune diseases, with a major institutional focus on type 1 diabetes (T1D). This alignment of institutional mission with the JDRF mission has created a very productive partnership, called the JDRF-BRI Center for Translational Research. Scientific goals for the Center include:

1. Identify the consequences of genotypic and phenotypic variation within T and B cell populations associated with progression toward or protection from type 1 diabetes (T1D);

2. Monitor and modulate this variation in the context of clinical trials designed to prevent or intervene in the progression of T1D;

3. Link concepts from the T1D research community to the clinical and immunologic outcomes in our well characterized and accessible human subject populations and serve as a resource center.

Background/Rationale: In our JDRF-BRI Center for Translational Research, data and samples have been obtained on nearly 1,300 unique subjects with diabetes or relatives of individuals with diabetes, and information is maintained in our diabetes translational research database (Core A). We also have data on several hundred healthy control subjects with neither personal nor family history of any autoimmune disease, which has proven essential, e.g., in understanding the role of PTPN22 (Projects 1 and 2). Our external research base, which improved during the previous granting period, consists of human T1D research projects and clinical trials that are conducted and funded elsewhere but use our center’s core resources.

Description of Project: The four major projects are designed as translational research initiatives, focused on effector memory T cells (Project 1), B cells (Project 2), regulatory T cells (Project 3), and DQ tetramers (Project 4). In each case, the objective is to identify biomarkers and targets for the different elements of the adaptive immune system that together shape autoimmune activation and progression in T1D. Our experimental approach in each case links genotypic variation with specific phenotypic traits of disease toward interpreting the immunologic mechanism in terms of cell population, signaling, and lineage maturation. Study subjects represent a broad range of experimental immunotherapy trials and clinical outcomes. This tight linkage between basic immunobiology and in vivo clinical immunology focuses our studies on cellular mechanisms with translational and therapeutic potential.

Anticipated Outcomes: Better understanding of the roles of T and B cells in T1D pathogenesis and the linkage of genotype and phenotype in autoimmunity Development of candidate biomarkers for disease- relevant phenotypes in T1D

Relevance to Type I Diabetes: All studies in this funded Center will focus on type 1 diabetes. The raison d’être of the JDRF-BRI Center for Translational Research is to optimize the bench-to-clinic effectiveness and application of research knowledge for patients with type 1 diabetes. Project 1: Modulation of autoreactive CD4+ T cells in T1 D

SCIENTIFIC ABSTRACT

Objective:

In this project we will analyze the generation and activation of human self-reactive CD4 T cells. Previous studies have focused on several key elements, which characterize the autoreactive CD4+ T cell profile in type 1 diabetes (T1D) and suggest potential strategies to alter the balance between pathogenic and regulatory pathways. These results will now be extended to specifically translate genetic and phenotypic properties of diabetes-associated T cells into tools for understanding and modulating islet-specific immunity.

Background/Rationale:

Many of the therapeutic interventions for T1D in our Center (and elsewhere) include either modulation of the effector T cell response or enhancement of the regulatory T cell response, or both. In our studies to date, we have identified properties associated with the effector and regulatory compartments, which hold promise for improved targeted therapy, and which can assist in development of better clinical biomarkers. Project 1 focuses on insights into effector T cells, which derive from recent studies of genotype-phenotype relationships in T1D (Project 3 addresses the regulatory T cell compartment).

Description of Project:

We will test three compelling hypotheses: 1. genetic predisposition to attenuated TCR signal transduction is associated with the development and persistence of a distinct population of CD4+ memory T cells in T1D; 2. islet antigen-specific effector memory T cells in T1D subjects express phenotypic properties attributed to altered TCR signaling, with therapeutic implications; and 3. CD4+ T cells specific for an immunodominant proinsulin epitope present in T1D and atrisk subjects, and have both effector and regulatory potential. This project utilizes Core A for sample collection and clinical information, Project 4 for tetramer technology, and Core C for genotyping, HLA, and arrays. Drawing from the same patient base, information in this project will be linked with the B cell studies in Project 2, particularly for the PTPN22 genotypic evaluations. Additionally, we will use a specialized mouse model developed at BRI.

Anticipated Outcome:

The properties we have identified that are associated with the effector and regulatory compartments of T cells hold promise for improved targeted therapy and can assist in development of better clinical biomarkers.

Relevance to Type I Diabetes:

In all studies we strive to connect the genotypic analysis with phenotypic characterization of T cells from samples of T1D subjects and turn the information around to effect therapeutic solutions for patients with diabetes. Proiect 2: Defects in the function and requlation of the B cell compartment in T1 D

SCIENTIFIC ABSTRACT

Objective:

In work resulting from the current JDRF Translational Center, we have identified alterations in B cell profiles and B cell receptor (BCR) signal transduction in subjects with type 1 diabetes (T1D). These findings suggest that the balance between long term memory and antibody production may be altered in T1D as a result of dampened B cell signaling. We hypothesize that this defect in the B cell compartment contributes to pathogenesis of disease through the production of high affinity islet specific antibodies themselves or the interaction of auto reactive B cells with other immune cells. These studies will extend our understanding of the roles B cells and BCR signaling play in the development of T1D, which will direct our future use of B cellspecific therapies.

Background/Rationale:

Although the destruction of pancreatic islets is thought to be due to T cell-mediated processes, several lines of evidence indicate a role for B cells in the pathogenesis of T1D. Islet-specific antibodies are seen in murine models of diabetes, and islet-specific antibodies precede disease in humans. These antibodies are currently the most effective marker for the prediction of T1D. Whether they contribute to the pathogenesis of disease, or are merely a product of that response, is still unclear. We have identified alterations in B cell profiles of subjects with T1D, compared with controls, raising the possibility that alterations in B cell selection and maturation checkpoints may contribute to the pathogenesis of T1D.

Description of Project:

We propose to more fully characterize the B cell compartment in individuals with T1D to better understand at which point in selection and maturation these alterations occur. Using a BCR repertoire analysis, we will determine the stage(s) of removal of auto reactive B cells is defective in subjects with T1D. We will utilize a technique that allows direct examination of BCR auto reactivity, based on cloning and expression of individual BCR from newly formed transitional and naïve mature B cells, respectively. The combination of characterizing the number and distribution of B cell subpopulations and evaluation of their relative auto reactivity should allow us to localize the point in B cell development at which selection, survival, and fate decisions are altered in T1D.

Anticipated Outcome:

We expect in this aim to fully define the alterations in the character of the circulating B cell pool associated with the PTPN22 1858T variant. We will then investigate in a more direct manner the functional impact of theLyp620W on B cell function, specifically, on its impact on BCR signal transduction. The combination of both the functional findings with the peripheral phenotype, will allow us to understand how this one variant can impact tolerance, maturation and fate decisions in the B cell compartment. These studies will then be used as a model to extend our understanding of how alterations within the BCR signaling pathway may impact autoimmunity specifically the development of T1D.

Relevance to Type I Diabetes:

All studies relate directly to better understanding of the mechanisms of T1D and determining new therapeutic options. Proiect 3: Maturation, persistence. and function of requlatorv T cells in T1 0

SCIENTIFIC ABSTRACT

Objective: The goal of this project is to explore the dynamic process in which regulatory T cell (TR) maturation, persistence, and function respond to contextual cues and the mediators that regulate it. Further, it is important to understand whether these processes are altered in type 1 diabetes (T1D). The analysis of cell population dynamics in patients by use of in vivo labeling, to be developed in this project, may in the future be applied to questions resulting from both Project 1 (effector memory T cells) and Project 2 (B cells) of the Center.

Background/Rationale: CD4+CD25+ TR are a subset of regulatory cells known to play a role in protecting the body from autoimmunity. TR represent 4-8% of all CD4+ T cells in humans and have been shown to respond in an antigen-specific manner to alloantigens and self peptides. A lack of TR produces a spectrum of spontaneous organ-specific autoimmune manifestations in mice and is also implicated in the pathogenesis of diabetes in both animals depleted of TR and in humans. The mechanism by which TR function works is not yet known; however, the transcription factor FoxP3 has been shown to be critical for the generation and function of TR in mice and humans.

Description of Project: We will perform in vivo and vitro studies to address the hypothesis that the peripheral induction, expansion, or survival of TR is altered in individuals with T1D. We will then utilize these methods to examine the impact of immunotherapeutic interventions on TR induction, expansion, and persistence. We will test the hypothesis that hyaluronic acid and its breakdown products can regulate TR function through modulation of FOXP3 expression directly in human TR cells, evaluate additional mediators that augment and inhibit TR function and persistence, and investigate the mechanisms involved. We hypothesize that the milieu present at sites of autoimmune inflammation results in the abrogation of TR function due to signaling through innate receptors. We will use a mouse model of type 1 diabetes to test this hypothesis and evaluate the role of innate signaling in TR cell function.

Anticipated Outcome: We expect to find that any therapy that depletes T cells, upon recovery, will result in transiently increased numbers of TR because of their increased proliferation rate compared with nonregulatory TR. However, this effect will diminish over time as the system restores homeostasis, reflecting no direct effect of alteration in TR survival or homing. In contrast, we expect that therapies, for which in vitro work suggests a preferential induction of TR (i.e., rapamycin and IL2), will produce an increased number of TR with prolonged survival. We expect to validate candidates suggested by the flow and transcript analysis by using similar specific siRNA constructs. In all cases, the FOXP3 persistence assay will be the primary outcome measured. Animal experiments are designed to uncover the mechanism that underlies the development of diabetes in DO/RIPmOVA mice. Based on what is known from other systems, it seems likely that innate immune signals are involved. If the genes chosen are not involved, the system is versatile enough to allow us to interrogate other possible players, including additional molecules involved in TLR signaling (e.g., Trif and Irak-3).

Relevance to Type I Diabetes: All studies directly relate to the study of mechanisms of T1D and avenues for potential therapies. Proiect 4: Generation of DQ tetramers

SCIENTIFIC ABSTRACT

Objective:

The major objectives of this project are to generate and evaluate DQ tetramers that can monitor the presence and phenotype of autoreactive T cells in T1D.

Background/Rationale:

T1D is an HLA-linked, T cell-mediated autoimmune disease. In Caucasian populations, the DR0401- DQ0302/DR0301/DQ0201 genotype confers the highest risk of disease susceptibility. Multiple T1D-associated autoantigens have also been identified. The ability to track islet-specific, autoreactive T cells in blood samples from T1D subjects will improve our understanding of the pathogenesis of T1D. However, the development of a reliable assay to track these autoreactive T cells has been elusive, in spite of significant effort in the immunology community for the past 15 years. One of the stumbling blocks is the lack of HLA-DQ-specific tetramers for studies of the CD4 T cells in T1D.

Description of Project:

We will produce HLA-DQ class II tetramers, extending our success with DQ0602/GAD65, by developing a reliable mammalian cell culture expression system for the production of DQ0302 and DQ0201 tetramers, and use these tetramers to identify novel DQ epitopes relevant to autoreactive CD4+ T cells ex vivo in T1D. This work represents a significant technical challenge and will require innovative and concerted effort in this project.

Anticipated Outcome:

We plan to develop at least one class II tetramer reagent for each of the major HLA-DQ genotypes associated with susceptibility and protection in T1D, which will allow for study of the specificity and phenotype of autoreactive DQrestricted CD4+ T cells. Following successful tetramer production, we plan to evaluate the relevance to T1D disease susceptibility and progression by flow cytometry analysis of peripheral CD4+ T cells from at-risk subjects, T1D subjects, and controls.

Relevance to Type I Diabetes:

We have successfully developed class II tetramers for the detection of DR-restricted T cells specific for T1D-associated autoantigens, which are now in various studies as potential biomarkers of disease and response to therapy. It is important to do the same for DQ-restricted T cells, as these cells may be closer in temporal and functional origin to the initial stages of T1D. Core A: Clinical and translational core

SCIENTIFIC ABSTRACT

Objective: The goal of the clinical core is to identify and characterize research subjects; develop and maintain a comprehensive database; establish and maintain a sample storage facility; support type 1 diabetes translational research projects within and external to our center; and provide a population for clinical trials

Background/Rationale: An underlying theme of our JDRF Center is that there are multiple metabolic, genetic, and immunologic pathways that lead to development of type 1 diabetes and that unraveling the etiopathology of disease requires detailed characterization of each individual. Beta cell survival is determined in part by local immune system activity, such as cellular immune response and cytokine responses. As now increasingly recognized even among youth with diabetes, autoimmunity and beta cell dysfunction co-exist with clinically significant insulin resistance. Thus, metabolic pathways to onset of diabetes include the interplay of both insulin secretion and utilization. Parallel with this concept is a modification of the classic understanding of the natural history of T1DM with respect to beta cell function, in terms of factors contributing to the duration of endogenous insulin secretion detectable or clinically relevant post diagnosis. These combined notions suggest that extension of the “honeymoon” period may occur via modifications to both the insulin resistance and immunity side of the equation. Yet, testing these hypotheses in clinical trials ideally requires development of simple, yet robust measures of islet directed immunity, insulin secretion, beta cell mass, and insulin sensitivity. Our JDRF Center for Translational Research has pursued development of each of these measures.

Description of Project: To support T1D translational research projects within and external to our center, the Clinical Core will perform metabolic tests exploring changes in beta cell function over time, including studies involving subjects with long-standing diabetes, as well as evaluating the role of insulin resistance in T1D in the context of residual endogenous insulin production. We will continue to expand our pool of research subjects and samples to maintain a ready supply for internal and external projects during the grant period. Projects 1 and 2 will require whole blood and PBMC samples from individuals genotyped for HLA and PTPN 22 in conjunction with autoantibody and clinical information. These will be individuals with T1DM, relatives of individuals with T1DM, and healthy control subjects. Project 3 will study in vivo T cell kinetics using deuterated labeling. We expect that only adult subjects will be used for these studies (healthy controls, individuals with T1DM, individuals with T1DM with pancreatic transplant, and others treated with immunomodulatory agents. The clinical core will recruit subjects and handle samples for this project, testing responses to GAD65/alum immunization.

Anticipated Outcome: Our infrastructure and database system allow for baseline and sequential samples for individuals on study to investigate potential surrogate markers of treatment effects with the interventions. We will continue to support type 1 diabetes translational research projects within and external to our center. Investigators can query our translational database from their workstations with download of data into programs for analysis in conjunction with genetic information. In addition to producing results data, the database encompasses a sample tracking system to pinpoint the location of samples.

Relevance to Type I Diabetes: All studies involve subjects with T1D to determine best possible treatments for each individual. Core C: Genotypinq core

SCIENTIFIC ABSTRACT

Objective: The primary objective of the genotyping core is to provide comprehensive and dedicated genetic support to JDRF Center projects and cores. The genetics component of the JDRF-BRI Center (formerly Project 2) was realigned as a Genetics Core laboratory, specializing in techniques established during the previous grant cycle and incorporated as core functions. Core C directly interacts with virtually all components of this Center grant.

Background/Rationale: Type 1 diabetes arises from the actions and interactions of multiple genetic loci and environmental factors. Epidemiological analysis further demonstrates that the most likely model for inheritance incorporates contributions from a single, major locus and several polygenes, which could interact with each other and/or with various environmental risk factors. Four genomic regions exhibiting association with T1D have been repeatedly identified in multiple studies examining independent populations. Linkage analysis indicates that the HLA complex accounts for up to 50% of the familial risk for T1D, and this is likely due to the actions of multiple loci within the complex. There is significant evidence for association between alleles at DQB1 and T1D, with different alleles having effects ranging from strongly predisposing (DQB1*0302 and 0201) to strongly protective (DQB1*0602). When considering evidence for genetic susceptibility to type 1 diabetes and other autoimmune disorders, a pattern is emerging of variants that confer susceptibility to multiple autoimmune disorders and have clear immunological significance.

Description of Project: We will characterize newly collected case and control populations for known T1D susceptibility loci, HLA (DQB1 and DRB1 typing), INS, CTLA4, and PTPN22; evaluate candidate susceptibility variants for T1D within phenotyped case and control populations, specifically targeting candidates involved with lymphocyte activation and signaling, insulin resistance, and other metabolic pathways relevant to type 1 diabetes; and provide routine genetic and molecular support for ongoing JDRF center projects.

Anticipated Outcome: Continued statistical and epidemiological analysis for effective integration of genotype data with physical measurements and clinical information Rapid genotyping of newly reported susceptibility genes from ongoing whole genome association studies Routine typed SNPs of variants associated with a phenotype and/or metabolic parameter of interest (measured by other cores or projects) Ongoing collaboration with Dr. Patrick Concannon, University of Virginia, to analyze these SNPS in larger cohorts of type 1 diabetes cases and controls Laser capture microdissection for more precise expression analysis of particular cell populations within tissues, analysis of exon usage with “all exon” expression arrays, and analysis of copy number variation using tiling or SNP microarrays

Relevance to Type I Diabetes: All work in the genotyping core relates to autoimmune disease, especially type 1 diabetes. Core D: Administrative and enrichment core

SCIENTIFIC ABSTRACT

Objective: The goals of this core are to coordinate organizational activities that facilitate scientific collaboration and productivity within the Center and report in a timely manner on scientific and financial information. The PI of Core D will be responsible for timely reporting to JDRF on scientific, financial, and review matters.

Background/Rationale: The JDRF-BRI Center for Translational Research has a successful track record of efficient and timely administrative performance over the last five years. Our flexible administrative model is responsive to changes as needed, and close interactions under one roof at BRI among our basic laboratory, translational, and clinical investigators provide a good opportunity to enrich the diabetes trainee environment. Center programs will build upon the BRI organization-wide research work-in-progress and visiting scientist seminars, which emphasize the interdisciplinary and translational aspects of immunology, immune-mediated diseases, and inflammatory pathways.

Description of Project: An executive committee, composed of the Center’s project and core PIs and chaired by Dr. Nepom, will meet bimonthly and be charged with administration of Center activities. Dr. Carla Greenbaum will serve as Vice Chair in Dr. Nepom’s absence and will step in as Center PI if necessary. We will overlay the scientific/clinical programs with Center-focused research meetings and seminars, which specifically target innovative approaches to the main goals of our Center. For the most part, we will address candidate genotype-phenotype questions, from the point of view of genetics, clinical phenotypes, other autoimmune diseases, response to therapy, and the like. We will also conduct an annual retreat sponsored by the Center in which we review progress and conduct scientific focus groups oriented around proposed new interactions. We intend to invite outside investigators each year, asking them to participate actively and present their own data. Because Seattle has several research groups interested in T1D, in addition to the BRI, including Institute for Systems Biology, Children’s Hospital and Regional Medical Center, Pacific Northwest Research Institute, and the University of Washington, we will have an opportunity to cement inter-institutional relationships on a regular basis. Finally, Core D plans to establish and maintain a JDRF-BRI website, which advertises our program and services to the diabetes research community and provides links to our tetramer and database resources. In addition to the web pages themselves, representing each project and core, we anticipate installing/upgrading an important set of hotlinks and search bars, which will guide the websurfer to some of our existing BRI website pages, including a searchable index of ongoing diabetes clinical trials (with contact information) and a listing of PDF files with a variety of tetramer protocols and helpful applications.

Anticipated Outcome: Continued timely reporting to JDRF of scientific progress and accurate grant accounting High quality seminar series and scientific retreat Inspiring milieu for young investigators of T1D translational research Greater collaboration among diabetes research groups in our community and among other JDRF Centers A renovated website for BRI programs, services, scientific milestones, and clinical opportunities

Relevance to Type I Diabetes: All activities and programs emanating from this core relate directly to diabetes research and translation to the clinic.

JDRF UK Center for Diabetes Genes, Autoimmunity and Prevention (D-GAP)

Mark Peakman

Of

King's College London

Applicant: Peakman, Mark Composite: JDRF UK Centre for Diabetes Genes, Autoimmunity and Prevention (D-GAP)

D-GAP, UK Scientific Abstract

The goal of this Center is to establish direct causal relationships between T1D-associated gene polymorphisms and immunological phenotypes.

The genetic profile of an individual determines whether he or she runs the risk of developing T1D. Of the T1D susceptibility genes identified, many by investigators in this Center, most, if not all, operate within the immune system, making it likely that T1D results from a failure of the regulation of immunity to self. Likewise, measurements of islet-specific immunity in the blood, again identified or elaborated by key personnel in our Center, are becoming recognizable as defining features of T1D. At least some of the blood immunophenotypes are likely, at some level, to be causative in T1D, although this is more challenging to prove directly because of the unknown effects of disease on their manifestation. As an a priori hypothesis, we propose that some of the T1D genes directly affect some of the immune phenotypes we study. By confirming this link we would identify causative immune phenotypes in T1D, which could become the legitimate targets of drug development and surrogate markers for measurement in prevention and intervention trials. We believe that robust gene-phenotype aetiological links of this type have not been made to date as a result of several factors; very few studies have attempted direct genotype- phenotype linking experiments; they have been carried out on small numbers; they have used T1D patients in whom the phenotypes may be unreliable or exaggerated as a result of disease. We propose that this field needs a dedicated programme of work, in which adequately powered studies are carried out on carefully genotyped populations, selected for the extremes of protection versus susceptibility for single gene polymorphisms. We further propose that these studies should be performed in populations without overt disease, because of its unknown modifying effects. Study groups should include first-degree relatives (FDRs) of T1D patients, themselves at defined low and high risk for disease, as well as healthy non-diabetic subjects with the same genetic background. This approach is the nearest one can aspire to in terms of mirroring the analysis of in-bred rodent strains, the classical, direct means for analyzing genotype-phenotype interactions. To undertake such a body of work requires the bringing together of the constellation of efforts and talents that are currently active in UK T1D research, with expertise in population- based studies; unrivalled genotype analysis; and internationally recognized expertise in human islet autoimmunity. Between 2008-2013 the UK will be an even more fertile environment for such studies, as a result of the Diabetes Research Network, created by government and covering a population of over 20 million. As we undergo transition to translation, we propose a research programme that will coalesce with ongoing UK T1D clinical networks and the new government-sponsored healthcare framework in the UK as part of an essential “next step” towards T1D prevention.

Applicant: Peakman, Mark

Project 1: T1D genes and CD4 T cell islet autoreactivity

Technical Abstract

The aim of this study is to establish whether INS and PTPN22 gene polymorphisms exert their effects on disease risk through direct pathways that affect the generation of islet-autoreactive T cells. The relationship between T1D susceptibility genotype and the development of the phenotype of islet-specific T cell reactivity is not known. Project 1 will test the hypothesis that insulin (INS) gene polymorphisms influence the frequency, and/or functional phenotype and/or avidity of proinsulin (PI)-reactive CD4 T cells; and that disease-related PTPN22 gene polymorphisms influence similar parameters in relation to PI- and other islet autoantigen-reactive T cells.

The proposal will utilize clinical groups selected to reflect extremes of susceptibility and protection in the relevant gene polymorphisms. These subjects will be recent onset T1D patients and first degree relatives (FDRs) with high risk for T1D progression, in which we expect to be studying predominantly the influence of genes on pro-inflammatory responses; and first degree relatives with low risk of T1D progression and non-diabetic control subjects in which we expect to be studying predominantly the influence of genes on regulatory responses.

Using these subjects, our specific aims are to examine whether the frequency or functional phenotype (IFN-g, IL-17, IL-10) of PI-reactive or islet-reactive CD4 T cells is influenced by INS or PTPN22 genotype, respectively; and to generate, from selected patients in these cohorts, PI- reactive and islet-reactive CD4 T cell clones, in order to examine whether there are gene-related differences in sensitivity to ligand, avidity or activation threshold. As a direct result of these studies, important additional sub-aims will be achieved, in that this will be the first large scale systematic study to examine the nature (epitopes, function) of islet-specific CD4 T cell autoreactivity in well defined cohorts of high risk and low risk FDRs; as well as the first to examine the presence of a novel pro-inflammatory T cell subset producing IL-17 in response to islet autoantigens.

These studies address critical scientific gaps, namely in our understanding of how T1D susceptibility genes impact upon the disease process and the nature of CD4 T cell autoreactivity in preclinical disease. The insights the results offer into pathogenesis could undergo translation in the arenas of prediction as well as in the deployment of antigen-specific and non-specific immune prevention strategies for T1D, which could, on the basis of genotype-phenotype, be tailored and targeted to subjects with defects in antigen-specific Tregs, or excessive expansions of T effectors.

Applicant: Peakman, Mark

Project 2: Novel HLA class I risk genes in T1D

Technical Abstract

New research from the JDRF/Wellcome Trust Diabetes Inflammation Laboratory in Cambridge, in collaboration with the Type 1 diabetes Genetic Consortium, has identified HLA class I genes associated with increased risk for T1D. Indeed, these genes confer a relative risk comparable to, or greater than, the more classically associated T1D genes, such as INS and PTPN22, and two alleles, HLA-B*39 and HLA-A*24, are associated with younger disease onset.

In this proposal we will examine the structure-function relationship of the gene products, HLA class I molecules, using novel technologies recently developed in our laboratory to identify naturally processed and presented HLA class I epitopes of preproinsulin (PPI). In recent studies, this approach has revealed a highly distinctive and unusual PPI processing pathway for one of the HLA molecules, HLA-A2.1 (A*0201). We have found that a major A2.1-restricted cytotoxic epitope arises from preproinsulin signal peptide, and that its presentation by human beta cells appears to be directly glucose-regulated.

We wish to test the hypothesis that HLA class I molecules associated with T1D risk have distinctive PPI processing and presentation characteristics and direct cytotoxic reactivity against beta cells, which could account for their disease association. Our specific aims are to identify PPI peptides naturally processed and presented by HLA-B*39, -B*18 and –A*24, recently identified as new T1D susceptibility genes; to validate these as disease-relevant epitopes using T1D patients and at-risk subjects from Core A; and to generate epitope-specific CD8 T cell clones restricted by these elements for analysis of cytotoxic potential against human beta cells.

These studies address a critical scientific gap in our understanding of how HLA class I susceptibility genes impact upon the disease process. These will be the first studies to specifically address functional aspects of these risk genes. The insights the results offer could undergo translation in the arenas of prediction and monitoring (eg through tetramer development), as well as in the development of novel antigen-specific and non-specific immune prevention strategies for T1D aimed at CD8 T cells, particularly in view of the fact that >75% of T1D patients will bear at least one of the four HLA class I susceptibility alleles.

Applicant: Wicker, Linda Susan

Project 4: Immune phenotypes and T1D genes

Technical Abstract

Type 1 diabetes (T1D) is a common disease of the immune system in genetically-susceptible individuals leading to autoimmune destruction of the insulin-producing cells of the pancreas, a process that begins early in life, often many years before diagnosis. Our hypothesis is that inherited dysregulation of the immune system precedes and causes T1D.

Our aim is to identify immunophenotypes associated with T1D susceptibility genotypes. Using blood samples from healthy volunteers, and from unaffected siblings of T1D cases, we will correlate T1D genetic risk with immunophenotypes. These will bedetermined by multicolour flow cytometry to detect and quantitate subpopulations of circulating immune cells, including T lymphocytes and dendritic cells, likely to be involved in T1D pathogenesis.

We will also correlate in collaboration with Projects 1 and 2 (Peakman) and 3 (Tree), immunophenotypes and T1D susceptibility genotypes with insulin-specific autoreactive T cells and the functionality of regulatory T cells.

The main aim is to identify disease precursors in the immune system of genetically-susceptible individuals that, in the future, could be targeted by therapeutics to prevent T1D.

Applicant: Dunger, David B.

Core A: Clinical Cohorts

Technical Abstract

Type 1 diabetes (T1D) is a common disease of the immune system in genetically-susceptible individuals leading to autoimmune destruction of the insulin-producing cells of the pancreas, a process that begins early in life, often many years before diagnosis. Now that the genetic basis of type 1 diabetes is being unravelled, the next step is to link T1D susceptibility genes with characteristics of the immune system.

Our Centre aims to identify these characteristics that are genetically determined by the T1D susceptibility genes, in order to pinpoint precursors or biomarkers of the disease that could be used in the future as targets for potential preventative therapies. To achieve, this we will recruit 4,000 unaffected siblings of T1D cases, 400 newly-diagnosed T1D patients and 1,000 healthy volunteers, and assess their T1D genetic susceptibility and analyse blood samples in detail for early markers of disease that are under the control of the T1D genes.

Our clinical core will be based on the unique, ongoing clinical networks in the UK: the UK Diabetes Research Network, the Bristol University Network and the JDRF/Wellcome Trust paediatric UK cohort (GRID).

Applicant: Todd, John Andrew

Core B: Genotyping, databasing and statistics

Technical Abstract

Genotyping, databasing and statistics is responsible for providing DNA extraction, genotyping, collation and storage of data and samples and analyses for the Centre. Data and sample management is based on the JDRF/Wellcome Trust Diabetes and Inflammation Laboratory sample and data management systems that have been established over the last seven years.

A laboratory database, based on T1DBase (www.t1dbase.org), will be developed to provide internal and external research groups access to data correlating genetic susceptibility to type 1 diabetes (T1D) with inherited characteristics, or immunophenotypes, of the peripheral immune system.

The main aim is to identify disease precursors in the immune system of genetically-susceptible individuals that, in the future, could be targeted by therapeutics to prevent T1D.

Applicant: Bingley, Polly

Core C: Autoantibodies

Technical Abstract

The goal of this Center is to establish causal relationships between type 1 diabetes associated gene polymorphisms and immunological phenotypes. The autoantibody core will be responsible for autoantibody determination in samples collected from clinical cohorts identified in Core A in order support the immunophenotyping of individuals in Projects 1-5. Autoantibody testing will form the basis for identification of unaffected first degree relatives (FDR) with evidence of ongoing islet autoimmunity defined by established markers of high risk of future diabetes, and of relatives at low risk, as well as characterization of newly diagnosed cases.

Presence of islet autoimmunity will be defined in terms of an autoantibody profile that is strongly associated with later development of type 1 diabetes, and absence by a profile strongly associated with remaining diabetes-free. Antibody-based risk assessment strategies using multiple markers can provide both sensitive and specific prediction and long term follow-up of relatives with multiple antibodies shows no diminution of risk over time and suggests that all of them may ultimately develop diabetes. At the same time we have shown in a family study population very comparable to that which will be recruited to this Center that, using a low threshold to define antibody positivity and a panel of four antibodies, the risk of progression to diabetes within 20 years in antibody negative siblings is less than 0.5%. We therefore plan to use the finding of two or more antibodies to define the ‘high risk’ category to be used in the Center, and negativity for four antibodies to define the ‘low risk’ group.

Autoantibodies to islet autoantigens (GAD, IA-2, and insulin if appropriate) will be measured by radiobinding assay in sera collected from all individuals recruited via Core A (520 individuals with newly onset type 1 diabetes, 4000 unaffected siblings and 1000 healthy volunteers). Additional testing for autoantibodies to recently identified novel islet antigens is planned subject to confirmation of their prognostic significance in existing study populations. IA-2 beta antibody measurement will be used to further stratify risk within the antibody positive unaffected siblings. Markers of other organ specific autoimmune disease (tissue transglutaminase, thyroid peroxidase and gastric parietal cell antibodies) will be measured in the cohort of healthy volunteers.

The proposed Core laboratory has an excellent track record in islet autoantibody testing as measured in international comparisons such as the Diabetes Antibody Standardization Program, and plays a major role in the development, evaluation and standardization of new assays and markers. It also has extensive experience in measuring autoantibodies for large studies of this type and is fully equipped to handle the number of samples that will be collected for the Center.

JDRF / WT Diabetes and Inflammation

Laboratory

John Todd

of

University of Cambridge

Applicant: Todd, J.A., Clayton, D.G., Wicker, L.S.

Title: JDRF / WT Diabetes and Inflammation Laboratory

SCIENTIFIC ABSTRACT

Specific aims:

(1) Carry out a genome-wide association study (GWAS) with the Wellcome Trust Case Control Consortium (wtccc.org.uk) and complete a GWAS of nonsynonymous single nucleotide polymorphisms (SNPs) to identify novel type 1 diabetes (T1D) susceptibility regions using the JDRF/WT cases.

(2) Complete the analysis of the MHC HLA region in T1D using a high density SNP map and classical typing data.

(3) Continue major collaborations with the WTCCC and the Type 1 Diabetes Genetics Consortium (t1dgc.org) in the genetic analysis of T1D, including GWAS results access and bioinformatics tools in T1DBase.

(4) Ship DNA samples and cell lines to the NIDDK repository.

(5) In vivo modelling of human T1D susceptibility genes in the NOD mouse model.

(6) Correlate T1D susceptibility genotypes with phenotypes, such as gene expression, biomarkers and surface and induced phenotype in immune cells, using plasma, serum and blood samples from people with T1D and their relatives, and healthy volunteers from the Cambridge BioResource (cambridgebioresource.org.uk) in order to identify disease pathways and mechanisms, with a particular focus on the interleukin-2 and vitamin D pathways.

Accomplishments:

(1) 38 manuscripts and publications since December 2006.

(2) Identification and analysis of at least four new T1D susceptibility regions (WTCCC, 2007, Nature 447, 661-678; Todd JA et al, 2007 Nat. Genet. 39, 857-864) (http://t1dbase.org/page/MarkerOverview/display/?marker_id=rs1000112).

(3) Identification of the HLA-B and HLA-A genes in the MHC as additional determinant of T1D susceptibility over and above the HLA class II genes (Nejentsev S et al, 2007, Nature 450, 887- 892).

(4) Launched a major new GWAS of JDRF/WT cases and controls with the T1DGC.

(5) Detailed genetic mapping of the IL2RA region and correlation of IL2RA genotype with plasma concentration of the immune activation biomarker soluble IL-2RA/CD25 (Lowe C et al, 2007, Nat. Genet. 39, 1074-1082).

(6) Cell lines shipped to NIDDK repository, with DNA samples to be sent by Q3, 2008.

(7) Evidence that NOD mouse PTPN22 gene is altered genetically and that increased expression, and presumably, activity, correlates with T1D susceptibility, as found in humans.

Publications/Presentations:

The Wellcome Trust Case Control Consortium. (2007) Genome-wide association study of 14,000 cases of seven common diseases and 3,000 shared controls. Nature 447, 661-678. Todd, J.A., Walker, N.M., Cooper, J.D., Smyth, D.J., Downes, K., Plagnol, V., Bailey, R., Nejentsev, S., Field, S.F., Payne, F., Lowe, C.E., Szeszko, J.S., Hafler, J.P., Zeitels, L., Yang, J.H.M., Vella, A., Nutland, S., Stevens, H.E., Schuilenburg, H., Coleman, G., Maisuria, M., Meadows, W., Smink, L.J., Healy, B., Burren, O.S., Lam, A.H., Ovington, N.R., Allen, J., Adlem, E., Leung, H.-T., Wallace, C., Howson, J.M.M., Guja, C., Ionescu-Tirgoviste, C., Genetics of Type 1 Diabetes in Finland, Simmonds, M.J., Heward, J.M., Gough, S.C.G., The Wellcome Trust Case Control Consortium, Dunger, D.B., Wicker, L.S. and Clayton,

D.G. (2007) Robust associations of four new chromosome regions from genome-wide analyses of type 1 diabetes. Nat. Genet. 39, 857-864. Lowe, C.E., Cooper, J.D., Brusko, T., Walker, N.M., Smyth, D.J., Bailey, R., Bourget, K., Plagnol, V., Field, S., Atkinson, M., Clayton, D.G., Wicker,

L.S. and Todd, J.A. (2007) Large-scale genetic fine mapping and genotype-phenotype associations implicate IL2RA region polymorphism in type 1 diabetes. Nat. Genet. 39, 1074-1082.

Who benefits from this research:

Identification and characterization of the genetic basis of T1D reveals the major pathways and mechanisms of disease. This information can help inform in the design of clinical studies and prevention trials, and their outcomes, including in the selection of participants in clinical studies. T1D susceptibility genes can help identify disease biomarkers. The JDRF/WT DIL provides samples, expertise, genetic information, informatics and statistical tools and methods to the research community.

Future plans:

(1) With the Type 1 Diabetes Genetics Consortium (t1dgc.org) undertake a genome-wide association study (GWAS) of 2,500 controls and 4,000 cases and combine the data with the GWAS results from the Wellcome Trust Case Control Consortium (wtccc.org.uk) from 3,000 controls and 2,000 and other available T1D and control data (e.g. GAIN data for 1,800 T1D cases from the GoKinD study) in order to locate novel susceptibility regions and genes.

(2) Analyse the data from the WTCCC available in 2008 from resequencing and genotyping of known T1D regions to design a “T1D SNP chip” to genotype the JDRF/WT T1D cases (7,000) and controls (7,000) to define and localise haplotype associations and candidate variants, as a prerequisite for functional studies.

(3) Correlate T1D susceptibility genotypes with phenotypes, such as gene expression, biomarkers and surface and induced phenotype in immune cells, using plasma, serum and blood samples from people with T1D and their relatives, and healthy volunteers from the Cambridge BioResource (cambridgebioresource.org.uk), including in vivo mouse modelling, in order to identify disease pathways and mechanisms, with a particular focus on the interleukin-2 and vitamin D