Andrew Eric Gelman, Ph.D. - US grants

PROJECT SUMMARY Survival rates after lung transplantation continue to lag substantially behind those observed after transplantation of other solid organs. A major barrier to success of lung transplantation is primary graft dysfunction, a form of acute lung injury that is mediated by ischemia reperfusion injury. Primary graft dysfunction not only results in an increase in early morbidity and mortality, but it also predisposes to chronic lung allograft dysfunction. We have developed new clinically relevant mouse models of lung transplantation and novel intravital imaging approaches to examine mechanistic links between primary graft dysfunction and rejection. In our previous funding period we have discovered that monocytes play a central role in mediating primary graft dysfunction by regulating neutrophil entry into the reperfused lung graft. Furthermore, we have described mechanisms how neutrophilic uptake of endogenous ligands that are released from injured lungs propagates primary graft dysfunction. In this application we propose to examine pathways that result in the activation of monocytes and neutrophils in lung grafts and to uncover mechanisms how these cells promote alloimmunity and graft rejection. In Aim 1 we will define mechanisms that activate neutrophils upon graft infiltration. In Aim 2 we will analyze pathways that mediate the activation and differentiation of monocytes in lung grafts. In Aim 3 we will examine interactions between monocytes and T cells that can trigger graft rejection.

DESCRIPTION (provided by applicant): Outcomes for lung transplant recipients remain significantly worse when compared to other organ recipients. Lung allograft injury in the first 72 hours post-transplant, also termed Primary Graft Dysfunction, is the leading cause for early mortality following lung transplantation and has also been shown to be a risk factor for chronic lung rejection. Ischemia-reperfusion injury is the leading cause of Primary Graft Dysfunction but the mechanisms that control this type of injury remain unclear. To address this problem we developed an orthotopic vascularized aerated lung transplant method that models Primary Graft Dysfunction in humans. New data from this model shows that a subset of IL-10+ TNF-¿- neutrophils accumulate in graft tissue shortly after transplantation and may protect against lung graft ischemia-reperfusion injury. We observed that IL-10+ TNF-¿ - neutrophils are the predominant IL-10+ cell type following lung transplantation and that their development is dependent on stress-induced or 'emergency granulopiesis'. In the absence of IL-10 expression in the recipient lung graft injury was severe indicating IL-10 is important to prevent lung graft ischemia reperfusion injury. We hypothesize that emergency granulopoiesis is required to generate IL-10+ neutrophils to prevent lung graft ischemia reperfusion injury. We have three specific aims. Our first aim is to examine factors that regulate the production of IL-10+ neutrophils in lung recipients. Our second aim is to develop IL-10+ neutrophil-based strategies to prevent or treat lung graft ischemia reperfusion injury. Our third aim is to assess patterns of IL-10 and TNF-¿ expression in the peripheral blood neutrophils of human lung recipients with varying degrees of primary graft dysfunction.

Lung transplantation is the treatment of choice for end-stage lung disease, but survival after lung transplant remains disappointingly low. Acute rejection episodes put lung grafts at greater risk of failing. However, acute rejection is often clinically silent and difficult to detect by any means other than biopsy, which requires an invasive procedure. Therefore, noninvasive methods for detecting and quantifying the presence of acute rejection could potentially improve outcomes for lung transplant recipients by improving our ability to detect acute rejection that requires treatment. Because T cells must proliferate in the lungs to cause acute rejection, we propose using the novel positron emission tomography (PET) tracer [18F]ISO-1, which targets the proliferation marker sigma-2 receptor, also recently characterized as the progesterone receptor membrane component-1 (PGRMC1), to image T cell activation in lung grafts. We have shown that PET imaging with [18F]fluorodeoxyglucose ([18F]FDG) also detects acute rejection and can measure its response to treatment; therefore, we will evaluate the ability of [18F]ISO-1 and [18F]FDG to detect acute rejection in a novel mouse model of left lung transplantation and in human lung transplant recipients.

PROJECT SUMMARY/ABSTRACT Lung recipients suffer from shorter life spans and higher rejection rates when compared to other solid organ recipients. To better advance a program project focused on the mechanisms of lung transplant rejection an administrative core is proposed with three specific aims: (1) to organize and facilitate communication between three projects and the microsurgical core (2) to maximize the use of fiscal resources and (3) to assemble a scientific advisory committee. This administrative core will consist of a core leader, an administrative lead and a biostatistician, who will provide statistical support for each of the projects. The overall goal of the administrative core is to leverage the intellectual and financial resources of the individual projects to maximize overall scientific productivity.

PROJECT SUMMARY/ABSTRACT Lung recipients suffer from shorter life spans and higher rejection rates when compared to other solid organ recipients. Neutrophilia is associated with graft injury but it remains unclear how neutrophils regulate adaptive immune response that control lung transplant tolerance. Recent reports from our laboratory show neutrophils trigger acute rejection by direct interactions between CD11c+ antigen presenting cells and T lymphocytes. However, how neutrophils control chronic rejection, the major cause of death of lung recipients who have had their grafts for more than one year, remains largely unknown. Using a new model of mouse orthotopic lung transplantation and novel intravital imaging techniques new data from our lab indicates that both high neutrophilia and the complete lack of neutrophils promotes chronic rejection in response to airway epithelial injury. Moreover, neutrophils are required to induce immunosuppression-mediated tolerance. In this proposal we will define mechanisms by which neutrophils differentially regulate rejection and tolerance. We have 3 specific aims. In Aim 1 we will define neutrophil activity that promotes tolerance through analyzing trafficking patterns that promote the removal of injured cells, which we hypothesize prevents chronic rejection. We also determine if the homing of central memory CD8+ T cells to lung allografts, which we have recently shown promotes the induction of immunosuppression-mediated tolerance, is dependent on neutrophils. In Aim 2 we hypothesize that high neutrophilia triggers chronic rejection through altering the stability of bronchus associated lymphoid tissue, an ectopic lymphoid aggregates that we have shown is associated with tolerance. In Aim 3, we determine if neutrophils extracellular trap (NET) generation is associated with worse primary graft dysfunction in clinical lung transplant recipients and chronic rejection in the mouse lung transplant model.

? DESCRIPTION (provided by applicant): Median graft survival for recipients of lung transplants remains approximately 5 years, the worst of all solid organ transplants. The major cause of death after the first year post-transplantation is chronic rejection, manifest primarily as bronchiolitis obliterans syndrome (BOS). Therefore, there is a critical unmet need for new treatment strategies that prevent or limit the progression of BOS and will thereby effectively prolong graft survival. Extracorporeal photopheresis has shown promise in the treatment of BOS, however it is typically used as a salvage therapy, instituted only after conventional approaches have failed. Initially developed for the treatment of cutaneous T cell lymphoma, ECP has also been successfully used to treat a number of diseases, including cardiac allograft rejection and chronic graft versus host disease following allogeneic stem cell transplantation. However, our ability optimally utilize this treatment is severely hampered by a lack of mechanistic understanding. The major goals of this proposal are to develop a murine model of ECP in lung transplantation, and to determine the mechanism by which ECP modulates the immune response. To accomplish this, we have developed in vitro and in vivo model systems which recapitulate the key effects of ECP, as well as the essential features of bronchiolitis obliterans. In specific aim 1, we will use an established murine orthotopic lung transplant model, pioneered by Washington University researchers, to determine the effectiveness of ECP to prevent the development of obliterative airways disease. This system will be the first, animal model to investigate ECP in lung transplantation. In specific aim 2, we will use both in vitro and in vivo approaches to dissect the mechanism by which ECP modulates effector T cell function. If successful, we will have established the first in vivo murine model system of the treatment of chronic lung allograft rejection with ECP, allowing us to explore mechanism, and most importantly, to determine how best to use ECP in the prevention and treatment of BOS in lung transplant recipients. The data obtained from this pilot project will provide essential information that will guide future studies in both mice and humans, and may provide critical supportive evidence for the early use of ECP to prevent bronchiolitis obliterans.

PROJECT SUMMARY/ABSTRACT Lung recipients suffer from shorter life spans and higher rejection rates when compared to other solid organ recipients. Bronchiolitis obliterans syndrome (BOS) is the most common cause of chronic lung transplant rejection. Based on clinical outcome studies that have identified many known non-alloimmune mediators of epithelial injury as risk factors for BOS, we have developed a new mouse orthotopic lung transplant model that recapitulates this relationship. This innovation has allowed us to a identify a key airway epithelial cell progenitor, the club cell, as vital for the maintenance of allograft tolerance and the prevention of obliterative bronchiolitis (OB) - the major pathological indicator of BOS. We have also observed that OB is driven by graft-infiltrating effector CD8+ T cells that inhibit club cell-mediated repair of airway epithelium. New data from our laboratory suggests that follicular cytotoxic CD8+ T cells, a poorly defined CD8+ T cell subset with no reported role in solid organ transplantation, may facilitate responses against pulmonary-self antigens. Our group has previously reported that CCR2+ monocytes (CM) and neutrophil extracellular traps (NETs) play critical roles in both lung transplant ischemia-reperfusion injury and the induction of lung allograft tolerance, but their role in the maintenance of tolerance remains unclear. We have now uncovered evidence implicating graft-infiltrating CM and NETs in OB pathogenesis. Here we propose to dissect the mechanisms by which CM (Aim 1), effector CD8+ T cells (Aim 2) and NETs (Aim 3) promote OB pathogenesis. We will also examine how CM and NETs shape effector CD8+ T cell responses that promote OB pathogenesis. Through these studies, we expect to reveal targetable pathways that promote BOS.