Ali Zarrinpar, MD, PhD
University of California, Los Angeles

Role of Neutrophil Activation in Ischemia Reperfusion Injury

Synopsis: Reperfusion of ischemic tissues leads to damage in the absence of exogenous antigens. The resulting ischemia/reperfusion injury (IRI) initiates a cascade of innate immunity dominated responses leading to local inflammation, cell death, and even organ failure. Neutrophil activation is a major effector mechanism in liver IRI. Recently, we showed that Bruton’s tyrosine kinase (Btk) signaling is essential in neutrophil activation and that its selective inhibition blocks IR-induced hepatocellular damage and protects livers from severe inflammation. This has led us to propose that identifying and modulating pre-transplant neutrophil activity will permit us to select and monitor patients, and modulate IRI, thus improving liver transplant outcomes. We hypothesize that 1) Btk regulates innate immunity in hepatic IRI by controlling neutrophil activation; and 2) characterizing pre-transplant neutrophil activity and cytokine profiles in human recipients will permit us to predict post-transplant IRI.

Aim 1: To define the role of Btk in innate immune function. Hypothesis: Btk activation potentiates neutrophil activation and promotes innate immune activity. We will examine the role of Btk-activation in immune regulation in a warm hepatic ischemia reperfusion model.

Aim 2: To evaluate the role of neutrophil activation in human liver transplantation. Hypothesis: Heightened neutrophil activity before transplantation leads to increased IRI and subsequent activation of resident Kupffer cells and recruitment/activation of monocytes/macrophages and circulating T cells in human liver allografts, and that this pathological cascade perpetuates damage to the graft. We will examine how neutrophil activity in recipients prior to transplantation can affecting graft IRI and subsequent function.

Juan Echeverri, MD
Toronto General Hospital – Multi-Organ Transplant Program

Assessment of Graft Metabolism and Function During Normothermic Ex Vivo Liver Perfusion - Objective Assessment Prior to Transplantation

an increased risk for postoperative graft failure. Many of these marginal grafts would have provided good function after transplantation but our current inability to assess grafts prior to transplantation results in the wasting of this important resource.

To overcome this obstacle we have developed a novel Normothermic Ex Vivo Liver Perfusion (NEVLP) circuit as an alternative preservation technique for Liver Transplantation (LT). During NEVLP the liver is metabolically active with bile production, oxygen consumption, ATP synthesis, and lactate metabolism. Livers can be preserved on the NEVLP for several hours, which for the first time, offers the unique opportunity to study graft function prior to the transplant procedure. The aim of our study is to identify biomarkers during NEVLP that predict outcome after liver transplantation in a porcine model. We will investigate in heart beating and DCD pig liver transplant experiments NEVLP characteristics that will determine outcome of the graft after reperfusion. In addition, we will evaluate the metabolism of portal field zone 1 and 3 during NEVLP by administrating rocuronium and midazolam.

In Toronto, we have already initiated a human clinical trial using NEVLP as the preservation technique prior to liver transplantation. So far, 11 human liver transplants have been performed successfully in the Toronto Multi Organ Transplant Program. Our research team is fully involved in the experimental porcine model of ex vivo liver perfusion and in the NEVLP human clinical trial. We will apply the results of the animal experiments to the clinical trial and validate our findings in humans.

Identifying markers of graft function during NEVLP that predict outcome after transplantation will allow us to safely extend the donor pool and reduce the mortality on the waiting list.

Stephen Chiu, MD
Northwestern University

The Role Of Nonclassical Monocytes In Primary Lung Allograft Dysfunction

Synopsis: Primary graft dysfunction (PGD) occurs in up to 50% of lung recipients and is the principal risk factor for acute graft loss and chronic lung allograft rejection. Neutrophil infiltration into the allograft has been shown to mediate PGD. However, mechanisms of neutrophil infiltration remain incompletely characterized. In a murine lung transplant model, we found that CX₃CR1^hi Ly6C^low non-classical monocytes remain attached to the pulmonary endothelium despite flushing with standard preservatives and are, therefore, transplanted with the donor lung. Elimination of these monocytes in the donor resulted significant protection from neutrophil infiltration of the allograft and abrogated physiologic signs of primary dysfunction. CD14^lowCD16⁺ monocytes in humans are analogous to murine Ly6C^low monocytes, making these findings applicable to human lung transplantation. In additional preliminary experiments, we have found that knockout of CX₃CR1 and Toll-like receptor (TLR) signaling in donor Ly6Clow monocytes also protected against infiltration of the allograft by neutrophils. Therefore, we hypothesize that CX₃CR1 is necessary for attachment of Ly6C^low monocytes to the pulmonary endothelium and that TLR signaling is responsible for their activation during transplantation. In order to investigate this hypothesis, I propose to utilize core and innovative experiments in immunology, including adoptive transfer, flow cytometry, intravital multiphoton microscopy, cell culture, and live cell imaging techniques in a multidisciplinary collaborative training environment. Through the experiments outlined here, we hope to gain insight into the role of Ly6C^low monocytes as endothelial gatekeepers and mediators of neutrophil extravasation during PGD.

Juan S. Danobeitia, MD
University of Wisconsin-Madison

The Dual Role Of Macrophages In Transplant Immunity: Macrophage Differentiation In The Regulation Of Innate, Adaptive Immunity And Fibrosis After Renal Transplantation

Synopsis: Brain-death (BD) induces an inflammatory response associated to shortened graft life, acute rejection and maladaptive tissue repair. Innate immunity is at the forefront of this process and the complement system plays a determinant role in regulating inflammation and modulating adaptive immune responses. We developed a unique and clinically relevant model of delayed graft function (DGF) in non-human primates (NHP) and our preliminary data suggests that donor treatment with a protease inhibitor that regulates both complement and contact system activation (rC1INH), leads to protection from DGF by limiting recruitment of activated neutrophils and macrophages, decreasing inflammatory cytokine/chemokine expression and attenuating the progression to fibrosis. The mechanisms underlying these protective effects remain poorly understood. Here, we hypothesize that complement blockade interfered with monocyte recruitment into the graft and more importantly, modulated activation favoring alternative and regulatory-type macrophage differentiation. We propose to use our DGF model to characterize the recruitment, differentiation kinetics and function of monocytes in the setting of BD and their role in the pathogenesis of DGF, acute rejection and repair after transplant. In addition, this system will help uncover mechanistic insights surrounding the interplay between complement and monocytes in the regulation of antigen presenting cell activation, differentiation and function in allo-transplantation.

Victoria Ann Bendersky
Duke University School of Medicine

The Role of Rapamycin in Altering Graft Endothelial Cell Alloimmunogenicity

Synopsis: Transplant is the treatment of choice for irreversible organ disease. However, its benefit is associated with significant risks and the need for immunosuppression. Costimulation blockade (CoB) is emerging as a promising frontier in immunosuppression. This applicant’s mentor, Dr. Allan Kirk, has demonstrated that selected kidney transplant patients can be immunologically managed to prevent rejection using the CoB agent belatacept. However, patients for whom CoB is insufficient remain. Preliminary experimental and clinical data suggest that CoB-resistant rejection (CoBRR) is mediated by allospecific effector memory T cells no longer reliant on costimulation. Pilot human studies have shown that CoB with belatacept is more effective in mitigating the phenotype of CoBRR when combined with the mechanistic target of rapamycin (mTOR) inhibitor, rapamycin. However, the mechanism of rapamycin’s contribution to CoB by belatacept is unknown.

The purpose of this project is to define the role of rapamycin in controlling CoBRR, specifically examining extra-lymphocytic pathways for mollifying alloimmune memory T cell responses. While much attention has been directed toward the effects of rapamycin on T cell function, our hypothesis is that rapamycin is altering the alloimmunogenicity of the graft endothelium such that its propensity to activate memory T cell clones is reduced. To examine this hypothesis we developed two specific aims:

1. Characterize the changes in endothelial cells (ECs) with potential immunomodulatory effects induced by rapamycin.
2. Determine the mechanistic link between mTOR inhibition and phenotypic changes in ECs and the nature of the memory T cell response to rapamycin-treated ECs in vitro.

Catherine Dong, BS
Medical University of South Carolina

The Impact of Cellular Immunometabolism in Solid Organ transplantation

Synopsis: Transplantation is currently on the precipice of the next generation of significant advances including organ engineering, machine perfusion of livers, normothermic preservation, and nanotherapy-driven targeted drug delivery. The Transplant Immunobiology Laboratory (TIBL) at the Medical University of South Carolina (MUSC) has led the way in the development of nanotherapeutics as an immunosuppressive delivery mechanism to allografts both in vivo and as a pretreatment mechanism. Interesting preliminary data utilizing novel Targeted Rapamycin Micelle (TRaM) nanoparticles, developed in the TIBL, have shown that transplant vasculopathy (TV) can be prevented with the pretreatment of murine aortic allografts by TRaM therapy when used as an additive to University of Wisconsin (UW) solution. Further data suggests that early endothelial cell (EC) injury may incite a cascade of events leading to TV. Established and current data supports that inappropriate antigen presentation by endothelial cells exacerbates T cell priming promoting a pro-inflammatory milieu. Studies in the oncologic literature suggest that changes in cellular immunometabolism alter antigen presentation capacity and expression of inflammatory biomarkers; however, these concepts have yet to be applied to transplantation wherein organs are subjected to non-physiologic hypothermic and hypoxic environments. Here, we test the hypothesis that cold storage alters the immunometabolism of ECs as measured by mitochondrial spare capacity affecting their immunogenicity. The immunometablism of ECs is the focus of our proposal and we will investigate the ability of rapamycin alone or encapsulated in our novel TRaM nanotherapy to alter the immunogenicity of EC through changes in cellular immunometabolism in the setting of transplantation.

Samuel J. Gavzy, MS
Columbia Center for Translational Immunology

Impact of IL-17-Producing, Memory T Lymphocytes on Tolerance Induction in Nonhuman Primate Model of Liver Transplantation

Synopsis: While there have been many advances in immunosuppressive therapies to combat rejection in patients undergoing solid organ allotransplantion, these agents still demonstrate significant and debilitating side effects. Global immune dampening in transplant patients can result in organ toxicity as well as increased risk of infection and malignancy. Immunological tolerance induction toward the donor allograft is an attractive alternative to immunosuppression. Generating mixed chimerism, allowing hematopoietic progenitors of both donor and recipient to coexist in the immune compartment of the recipient, has shown great promise in yielding central and peripheral tolerance.

With the goal of establishing life-long, donor-specific tolerance in a preclinical transplant setting, we are investigating mixed chimerism induction through combined liver and bone marrow transplantation (CL-BMT) using a nonhuman primate model. While we have had success with previous models of combined kidney and bone marrow transplants, initial CL-BMT recipients displayed mixed chimerism, but developed rejection or Graft Versus Host Disease. We found that both host versus graft (HvG) and graft versus host (GvH) responses were mediated predominantly by IL-17 producing, graft-infiltrating T cells. In particular, the GvH response appears rooted in liver passenger memory T cells that produce IL-17. We will study and compare the provenance, costimulation blockade resistance, and in situ inflammatory homing of GvH passenger cells within the donor liver and infiltrating HvG lymphocytes in post-transplant livers. Understanding the properties and therapeutic susceptibilities of these pathogenic lymphocytes will allow our team to effectively modulate induction therapy for CL-BMT, bringing successful allograft tolerance closer to clinical implementation.

Joseph M. Ladowski
Indiana University School of Medicine

Evaluating the Role of Class I MHC in Pig-to-Human Xenotransplantation

Synopsis: The widening disparity between patients requiring transplant and the number of available organs requires a supply side solution. Xenotransplantation using pig organs could eliminate transplant waiting lists. Genetic engineering of donor pigs could make it easier to prevent rejection in clinical xenotransplantation. The Major Histocompatibiity Complex (MHC) is one potential barrier to xenotransplantation, interacting with recipient T cell receptors to trigger rejection. The role of swine class I MHC proteins (SLA) in human CD8+ T cell mediated xenograft injury is unclear. This proposal aims to determine whether human CD8+ T cells bind SLA class I to initiate rejection of pig xenografts. The proposal will also evaluate if swapping the recipient’s class I HLA for class I SLA in the donor pig cell will reduce or eliminate the ability of the CD8+ T cell to reject pig cells. This MHC “swap” would represent a personalized approach to medicine. If successful, the HLA class I knock-in (KI) cells developed under this protocol could eventually be used in somatic cell nuclear transfer (SCNT) to clone a HLA class I KI pig.