Qizhi Tang - US grants

Dr. Bluestone's laboratory has developed a novel class of anti-CD3-based immunosuppressant that was effective at suppressing immune response without the severe side effects associated with the use of conventional anti-CD3 mAb therapy. The novel anti-CD3 mAbs appear to induce immune tolerance by anergizing the pathogenic Th1 cells, and promote the opposing Th2 response. Biochemical analysis showed identical early TCR signaling patterns in both subsets of T cells similar to that observed in T cells treated with altered peptide ligands. It is hypothesized that imbalanced signal is responsible for the differential activity and may be a common mechanism to regulate T cell activation and differentiation in vivo. The goal of this proposed study is to is to define the signaling mechanisms that regulate these processes. The first aim of this study is to further map signaling abnormality in T cells treated with the novel anti-CD3 mAb using conventional biochemical approaches. The molecules to be analyzed include Ick, fyn, TCR zetu, JNK, and p38 kinase. The second aim is focused on defining the minimal signaling requirements for anergy induction and Th differentiation using genetic approaches. Altered forms of the above signaling molecules will be introduced into T cells in vitro to either block or enhance individual signaling pathways and effects of such manipulation on anergy induction or Th differentiation will be analyzed. A newly developed retroviral gene transduction system will be used to introduce these genes into either naive or cloned T cells. The results from these experiments will provide information on the functional outcome of differential TCR signaling, which cannot be obtained using transformed T cell lines. As this novel class of anti-CD-3 mAb moves into clinical trials, there is an urgent need to understand the molecular basis of its in vivo effect. The information will be invaluable in designing new safer and more specific therapeutic avenues for preventing transplant rejection and treating autoimmune disorders.

Accumulating evidence suggests that CD4+CD25+ regulatory T cells (Tregs) are an essential component of immune regulation, and loss of Treg functions may be one of the underlying causes of autoimmunity. However, how Tregs suppress immune responses in vivo remains unclear. This is likely due to the low frequency of Tregs and the difficulty in analyzing in vivo responses. We have recently developed a robust system to restore Tregs and control autoimmune diabetes in non-obese diabetic mice by transferring in vitro expanded islet antigen-specific Tregs purified from the BDC2.5 TCR transgenic mice. We hypothesize that Tregs both control immune homeostasis in steady state and respond to inflammatory stimuli to suppress immune responses at local tissue sites. New imaging technologies offer the most direct and unmanipulated ways to analyze in vivo cellular activities. However the field is facing the challenges of moving into clinically relevant disease models and establishing causal relationships between various imaging observations with functional outcomes. We will directly analyze both the steady-state and inflammatory responses of Tregs in vivo in our model system using novel in vivo imaging technologies. Specifically, we will use bioluminescence to monitor the lymph node homing and tissue trafficking of luciferase-tagged diabetogenic CD4 and CDS T cells in the presence and absence of co-transferred BDC Tregs. We will analyze the priming and effector function development of the diabetogenic T cells using two-photon laser scanning microscopy and determine the effect of BDC Tregs on these activities in both LN and islet tissues. We will directly visualize Treg interaction with antigen presenting cells and effector T cells in vivo with two-photon microscopy to determine the direct cellular target of the Tregs. Finally, we will examine the roles of immunosuppressive molecules IL-10, TGF- beta, and CTLA-4 using this novel technology and determine the effect of these molecules on the homing, trafficking, priming, and effector function development of the diabetogenic T cells. We will combine the imaging analysis with conventional cellular immunological approaches, multicolor immunofluorescence and immunohistochemistry techniques to pinpoint the time and location of Treg activation and function. The results of these studies will provide new insights into our understanding of autoimmune diseases by specifically studying the balance between immune regulation and auto-aggression

DESCRIPTION (provided by applicant): Type 1 diabetes (T1D) is one of the most prevalent chronic childhood diseases worldwide. In addition to its negative impact on quality of life for patients and their families, the disease also poses a significant financial burden on society. Thus, a curative solution, instead of the current maintenance therapy, is urgently needed. One such curative treatment shown to be very successful in mouse models is the infusion of autologous regulatory T cells (Tregs). Tregs are a small subset of CD4+ T lymphocytes that are primarily responsible for controlling pathogenic autoimmune responses in the periphery. Mounting evidence in animal models and patients demonstrates that T1D is associated with an imbalance between pathogenic T cells and Tregs. Treg therapy restores the balance and enables the immune system to regain self-control. With these encouraging results, a clinical trial of Treg-based therapy is being actively developed and is scheduled to start in 2009. At this juncture, it is important to understand the cellular and molecular basis of Treg function in controlling T1D. Our previous experiments demonstrate that a single infusion of islet-antigen-specific Tregs isolated from BDC2.5 T cell receptor transgenic mice (BDC Tregs) can prevent and reverse diabetes in the NOD mice. The BDC Tregs migrate to pancreatic lymph nodes and islets. In the pancreatic LN, they engage dendritic cells and effectively block further activation of pathogenic T cells by dendritic cells. How BDC Tregs halt 2 cell destruction in the inflamed islets has not been studied. In this grant application, we propose to systematically investigate the mechanism of T1D control in the NOD mice by BDC Tregs. We will determine the direct cellular target of BDC Tregs in vivo, and identify their impact on the ongoing inflammatory response in the islets at cellular and molecular levels. We will further determine the molecular profile of the therapeutic Tregs and identify molecule(s) responsible for their protective effect in vivo. Through the studies proposed in this grant application, we expect to gain better understanding of the pathogenic events critical for T1D progression and how therapeutic Tregs control these processes. The insight gained from these mechanistic studies will help to improve the design of Treg- based cellular therapy and to identify new targets for therapeutic intervention of T1D. PUBLIC HEALTH RELEVANCE: Type 1 diabetes is a chronic childhood disease that results from an immune-mediated destruction of the insulin-producing 2 cells. Regulatory T cells constitute a small population of immune cells that is mainly responsible for preventing unwanted immune response in healthy people. In animal models, regulatory T cell therapy can effectively prevent and reverse type 1 diabetes. Studies proposed herein are designed to understand the cellular and molecular basis for regulatory T cell control the disease. The insight gained from these mechanistic studies will help to improve the design of regulatory T cell-based therapy for patients and to identify new targets for therapeutic intervention of type 1 diabetes.

DESCRIPTION (provided by applicant): This Shared Instrument Grant application is for the purchase of a high-speed fluorescence-activated cell sorter, FACSAria II by Becton Dickinson. This instrument is peerless in its user friendly design and offers maximal flexibility to adjust to various experimental needs;thus it is an ideal choice for user-operated shared equipment. We have custom configured the FACSAria II to have four lasers at 405 nm, 488 nm, 561 nm, and 640 nm and twelve photomultiplier tubes for simultaneous analysis of ten fluorescent parameters. The custom FACSAria II also comes with a fully enclosed fluidic system and an advanced aerosol management option that meets the safety standard for processing samples contaminated with human pathogens. The impetus for this application is insufficient cell sorting capacity currently available, increased cell sorting demand, and the lack of appropriately configured devices for new research developments at the University of California, San Francisco (UCSF). The new instrument has a projected usage of over 2500 hours per year by fifteen defined investigators, demonstrating a real need for this instrument. Six of the investigators are new and early-career investigators, two are new to UCSF Parnassus campus, and three are new users of cell sorters due to new project development;together these new activities represent over 70% of the projected use of this instrument. These new activities cannot be fully accommodated by existing, already oversubscribed sorters at UCSF. Furthermore, clinical and human sample processing represent over 40% of the estimated usage of this machine. Lastly, in addition to the general shortage of the sorters, there is very limited capacity to sort cells based on red fluorescence due to the lack of green lasers on existing sorters. In response to these needs, we have configured the new sorter with improved aerosol containment for human sample processing and an additional yellow-green laser for sorting cells with red fluorescent tags. Thus, the new sorter is tailored to maximally meet new research demands at UCSF. Researchers directly benefiting from this instrument are from five departments in diverse fields ranging from molecular and cellular research in animal models to translational and clinical projects. This instrument will directly support research projects by 9 investigators with active NIH funding and 6 new early career investigators. The new sorter will be jointly managed by the Laboratory of Cell Analysis (LCA) and the Transplantation Research Laboratory (TRL). The LCA is a campus-wide core program that has over sixty years of experience in flow cytometry support. It will be responsible for technical and administrative support of the instrument. The TRL will house the instrument and provide backup onsite technical support. The new instrument will be integrated into the existing network of sorters managed by LCA to ensure expert maintenance of the instrument. Coordinated management of similar equipment will also ensure consistency in sorter recharge rate and access across the campus, which will likely benefit the UCSF research community at large. PUBLIC HEALTH RELEVANCE: We are requesting a high-speed Sorter to be shared by more than 15 researchers at UCSF Parnassus campus. This instrument will support research in a wide range of research fields such as Transplantation, Diabetes, Asthma, Cancer, and Immunology.

Purpose: The DERC Cytometry &Cell Sorting Core assists investigators whose research requires the characterization of molecular markers in dispersed cells and/or the isolation of cells based on those markers Components: 1. Flow Cytometers. The LSRII enables simultaneous detection of 9 fluorescent colors on a single cell for thorough, quantitative characterization of large populations of cells. 2. Cell Sorters. Up to 25,000 cells per minute can be collected on the MoFlo and FACSAria cell sorters within the Core. The MoFlo is exclusively service-oriented whereas users can be trained to operate the FACSAria independently. 3. Assistance and Training. Core personnel are experts who can assist novice and more routine investigators either through direct, hands-on assistance or through proper training in equipment use and data interpretation. Benefits to DERC Community: The highly specialized flow cytometry and cell sorting equipment requires dedicated scientists to maintain and operate. Those same Core personnel are experts who can assist novice and more routine investigators. The availability of this expertise ensures sound experimental designs, expert advice and proper analysis. Technology Development: The instrumentations are highly sophisticated. In order to reach their full potential, they must be accurately and quantitatively calibrated. Improved capabilities will be implemented within the next DERC cycle in order to provide improved service that individual laboratories would struggle to achieve and maintain. Plans to expand service also include plans to increase capacity through the upgrade of existing instruments and the purchase of new ones.

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

DESCRIPTION (provided by applicant): Type 1 diabetes (T1D) is one of the most prevalent chronic childhood diseases worldwide. In addition to its negative impact on quality of life for patients and their families, the disease also poses a significant financial burden on society. Thus, a curative solution, instead of the current maintenance therapy, is urgently needed. One such curative treatment shown to be very successful in mouse models is the infusion of autologous regulatory T cells (Tregs). Tregs are a small subset of CD4+ T lymphocytes that are primarily responsible for controlling pathogenic autoimmune responses in the periphery. Mounting evidence in animal models and patients demonstrates that T1D is associated with an imbalance between pathogenic T cells and Tregs. Treg therapy restores the balance and enables the immune system to regain self-control. With these encouraging results, a clinical trial of Treg-based therapy is being actively developed and is scheduled to start in 2009. At this juncture, it is important to understand the cellular and molecular basis of Treg function in controlling T1D. Our previous experiments demonstrate that a single infusion of islet-antigen-specific Tregs isolated from BDC2.5 T cell receptor transgenic mice (BDC Tregs) can prevent and reverse diabetes in the NOD mice. The BDC Tregs migrate to pancreatic lymph nodes and islets. In the pancreatic LN, they engage dendritic cells and effectively block further activation of pathogenic T cells by dendritic cells. How BDC Tregs halt 2 cell destruction in the inflamed islets has not been studied. In this grant application, we propose to systematically investigate the mechanism of T1D control in the NOD mice by BDC Tregs. We will determine the direct cellular target of BDC Tregs in vivo, and identify their impact on the ongoing inflammatory response in the islets at cellular and molecular levels. We will further determine the molecular profile of the therapeutic Tregs and identify molecule(s) responsible for their protective effect in vivo. Through the studies proposed in this grant application, we expect to gain better understanding of the pathogenic events critical for T1D progression and how therapeutic Tregs control these processes. The insight gained from these mechanistic studies will help to improve the design of Treg- based cellular therapy and to identify new targets for therapeutic intervention of T1D. PUBLIC HEALTH RELEVANCE: Type 1 diabetes is a chronic childhood disease that results from an immune-mediated destruction of the insulin-producing 2 cells. Regulatory T cells constitute a small population of immune cells that is mainly responsible for preventing unwanted immune response in healthy people. In animal models, regulatory T cell therapy can effectively prevent and reverse type 1 diabetes. Studies proposed herein are designed to understand the cellular and molecular basis for regulatory T cell control the disease. The insight gained from these mechanistic studies will help to improve the design of regulatory T cell-based therapy for patients and to identify new targets for therapeutic intervention of type 1 diabetes.

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

Abstract/Summary Core B: Flow Cytometry & Cell Sorting Core. The initiation and progression of events leading to type 1 and type 2 diabetes is accompanied by alterations in multiple cell types within the subject. Characterization, tracking and isolation of those cell populations by DRC investigators are enabled by the instruments and expertise available in the Flow Cytometry & Cell Sorting Core. Purpose: The Flow Cytometry & Cell Sorting Core assists investigators whose research requires the characterization of molecular markers in dispersed cells and/or the isolation of cells based on those markers. The Core provides the following services that is currently averaging 5501 accesses and 9419 cytometry/sorting hours by 143 researchers in 20 DRC laboratories: 1. Flow Cytometry. Five high-speed, multi-laser flow cytometers and analysis programs with highly complementary capabilities are available to meet DRC demand for cell characterization. 2. Cell Sorting. Six high-speed, multi-laser fluorescence activated cell sorters enable DRC investigators to isolate cell populations that can then be extensively characterized molecularly or that may be re-introduced to host animals to examine the effect of those cells on disease progression. 3. Information and Training. The Core trains investigators on the proper use of equipment and provides advice on the application of the technology. Benefits to DRC Community: The Core accelerates diabetes research by providing DRC investigators with access to advanced technologies and operational expertise that are beyond the abilities of individual laboratories to maintain. 39 NIH-funded and 31 other diabetes-related projects totaling $13,640,397 annual direct costs have benefited from the practical and economic access to this highly needed technology. Technology Development: Few laboratories have the resources to develop the most advanced capabilities to realize the full potential of the instruments. Support from the DRC enables one Core staff member to spend up to one day a week developing advanced capabilities that the DRC Executive Committee finds to be required by large numbers of DRC investigators. For example, new spectral analysis capabilities on advanced instruments not yet publicly available are now being developed in the Core to increase the accuracy and number of channels available for the analysis of specific cellular populations during disease progression. In the past three years, this Core has been successfully consolidated with other UCSF cytometry and cell sorting facilities to coordinate equipment procurement, expand instrument access and enhance staff availability. A robust co-pay mechanism ensures that DRC support is channeled to diabetes research in an institution-wide Core that provides access to a much greater degree of complementary instrumentation at lowered costs through the defraying of Core maintenance and personnel costs over a larger user base..

? DESCRIPTION (provided by applicant): Current gold standard tissue diagnosis for renal allograft rejection is based on light microscopic evaluation of biopsies aided by a semi-quantitative scoring system combined with limited immunophenotypical and electron microscopic analysis. Major weaknesses of this system include poor inter-observer reproducibility, over- simplification of the pathogenetic processes, and applying arbitrary histologic criteria to define disease categories. Furthermore, in spite of the fact that a significnt proportion of the pathologic changes in the transplanted kidneys are linked to cellular and/or antibody-mediated anti-graft immunologic activity, no comprehensive tissue based assays are available to assess the cellular and molecular underpinnings of these processes. The emerging molecular diagnostic approaches such as gene expression profiling have their own shortcomings particularly in their lack of cellular and structural context and limited mechanistic insights of the disease process can be gleaned. Therefore, the potential diagnostic power of allograft kidney biopsies is currently under-utilized. In this proposal, we aim to 1) develop multiplex immunofluorescence and in situ hybridization (miFish) assays to characterize inflammation and structural alterations in the kidney allograft biopsies and 2) and validate the miFish assays against the current gold standard of Banff scoring and molecular profiling to better define disease categories. Overall, the miFish assays will aim to develop in this proposal will help to identify prognostic markers and therapeutic targets for kidney allograft pathologies. t may also be used to re-define the current diagnostic criteria for some of the problematic categories in Banff, such as borderline changes. Although the miFish assays we propose to develop will be optimized to a transplant environment in the kidneys, the very same assays should also be applicable (with some modifications) to assess inflammation in other settings, such as various inflammatory conditions in multiple organs, including autoimmune diseases, and also in neoplastic lesions.