Robert R. Redfield III, MD
University of Wisconsin

Defining the Role and Mechanism of APRIL/BLyS Inhibition For the Prevention and Reduction of Alloantibody to Improve Long Term Kidney Allograft Survival

Synopsis: My research goal at the University of Wisconsin is to develop novel strategies that are safe and effective in preventing and reversing the production of detrimental MHC antibody in transplant patients. Alloantibody directed against graft MHC antigens is a significant barrier to kidney transplantation for sensitized patients, and a significant barrier to improving long-term kidney allograft survival. It is estimated that greater than 50% of kidney allograft failures can be attributed to alloantibody (3). A major source of alloantibody production is the plasma cell. APRIL (a proliferation inducing ligand) and BLyS (B lymphocyte stimulator), are B cell survival factors that are required for plasma cell activity. This research plan, if successful, will show that blockade of APRIL and BLyS, with the novel immunotherapeutic TACI-Ig, in a well-defined and established sensitized rodent model of functional kidney transplantation, will be an effective agent for the prevention and elimination of alloantibody. Substantial support for this hypothesis exists in the autoimmune literature, however its effect on MHC-directed alloantibody is currently unknown (1, 2).

APRIL and BLyS blockade is a novel avenue of investigation in the transplant field and could lead to a paradigm shift in the way we treat patients with alloantibody. These therapies are currently being tested clinically in autoimmune patients, and if these preclinical studies demonstrate positive results in the context of solid organ transplantation, we will be poised to rapidly test this hypothesis clinically.

Jaimie D. Nathan, MD
Cincinnati Children's Hospital Medical Center

The Role of Intestinal Microbiota in Acute Rejection in Small Bowel Transplant Recipients

Synopsis: Early detection and prompt treatment of acute rejection (AR) is one of the most important determinants for long-term patient and allograft survival after small bowel transplantation (SBT). However, the timely diagnosis of AR remains challenging due to the paucity of clinical signs and biopsy findings during very early stages. Recently, shifts in microbial communities were identified at the onset of AR in SBT patients, suggesting the potential of utilizing intestinal microbiota as a non-invasive diagnostic biomarker of SBT rejection. Moreover, regulatory T cells (Tregs) have been suggested to have a direct interaction with intestinal microbiota to maintain immune tolerance in the intestine. The present study is designed to test the overall hypothesis that changes in intestinal microbiota play a role in AR episodes after SBT via interactions with Tregs and may be used as new non-invasive biomarkers to predict the development of AR. In Aim 1, we will investigate potential changes in intestinal microbiota associated with AR after SBT. In Aim 2, we will evaluate local Treg responses in allograft associated with changes in microbiota. In Aim 3, we will characterize systemic Treg responses associated with changes in microbiota.

Data from the present study will provide novel evidence on the potential of using microbiota as noninvasive biomarkers. Specific bacteria identified in this study will be further examined as therapeutic targets for AR management after SBT. Data generated from the study will provide novel evidence and generate new hypotheses on mechanisms by which microbiota may affect AR development and SBT outcomes.

Qizhi Tang, PhD
University of California, San Francisco

The NIH-funded multisite trial HIVTR found surprisingly that HIV+ patients have three times higher rate of acute rejection than HIV- recipients. Rejections mostly occurred in the first 6 months of transplant and frequently recurred despite intensification of immunosuppression. These observations suggest that HIV+ patients have preformed memory immune responses against alloantigens.

We have performed phenotypic and functional analysis of the PBMC samples banked from the HIVTR study. Our results thus far show that HIVTR patients have similar leukocyte compositions and phenotypes except a moderate reduction of CD4 counts. TCR sequencing shows that HIVTR patients have significant T cell clonal expansion before transplant. Preliminary analysis has found higher frequency of donor-reactive CD8 T cells among rejecters when compared to non-rejecters. These results are consistent with our hypothesis that HIV+ patients have preformed memory alloimmune responses.

We further hypothesize that cytotoxic CD8 T cells are the drivers of rejection in HIVTR patients and the donorreactive CD8 arise as a result of cross-reactivity between HIV-reactive and alloreactive T cells. In this study, we propose to compare CD4 and CD8 T cells from rejecters and non-rejecters using global gene expression profiling and cross-reactivity assay to determine correlates of rejection (Aim 1). We also plan to analyze biopsy using gene expression and multiplex immunofluorescence analyses to determine the nature of the rejection (Aim2). We have samples archived from the HIVTR trial ready to be used in this study.

Mariusz Bral, MD
University of Alberta

Additive and Protective Strategies in an Experimental DCD Porcine Model of Ex vivo
Liver Perfusion

Synopsis: Remarkable progress has occurred in the development of portable technologies to support ex vivo normothermic liver perfusion (EVNLP). Clinical trials have been initiated, and preliminary outcomes suggest potent protection of marginal liver grafts. These findings will have huge bearing in expanding the limited liver donor pool, and will save lives where up to 1:5 die waiting on lists.

We have developed a stable DCD donor model of liver transplantation in the pig using EVNLP, modeled closely to the OrganOx Metra system that we are currently piloting in pilot clinical trial in Edmonton. Based on our extensive previous work of cellular protection in experimental and clinical islet transplantation, we herein propose to explore the potential of a series of potent anti-inflammatory and pan-caspase cytoprotective strategies. We hypothesize that the rational addition of selective protectant compounds to the ex vivo circuit will improve the safe transplantation of more marginal DCD liver grafts. Furthermore, administration within the ex vivo circuit will circumvent early hurdles commonly associated with early phase clinical trial testing, as the liver graft will be treated rather than the recipient. The compounds that have demonstrated strong potency in our laboratory include 1) pan-caspase inhibitors 2) inhibitors of regulated necrosis and 3) anti-freeze glycoproteins, all of which would serve well in additive strategies.

We are excited about the potential combination of these cytoprotective additives to EVNLP, and are confident that this work will yield translatable findings that will have major impact in the future practice of clinical liver transplantation.

James R. Butler, MD
Indiana University School of Medicine

Defining the co-stimulatory properties of vascular endothelium in a porcine model: a strategy to promote anergy in clinical xenografts

Synopsis: Cellular-mediated rejection (CMR) remains a topic of great importance to the allograft community. CMR operates through a donor T-cell intermediary; subsequently, recent success in mediating this process has been achieved by pharmacologically disrupting the costimulatory pathway. At present, inhibitors to CD28 and CD40L exist but their use is limited by untoward side-effects. Our preliminary findings document an ability to create porcine models of xenotransplant that reduce human antibody-mediated response (AMR) to levels comparable with current allograft standards. Furthermore, we have documented a reduced-but-capable T-cell response to porcine vs. human stimulation. Better understanding the xenoreactive CMR pathway is now critical; this could provide an avenue to create pig organs that illicit an anergic response from human lymphocytes, thereby limiting the chance of chronic rejection in the setting of clinical xenotransplantaion.

The aim of this project is to evaluate the individual role of co-stimulatory molecules on the vascular endothelium in the direct pig-to-human CMR pathway. Specifically, I propose to validate the hypothesis that silencing co-stimulatory genes on the porcine endothelium will eliminate the direct human antiporcine T cell response -towards anergy- without pharmacologic intervention; furthermore I will discover if the reduced stimulation between porcine and human stimulator populations can be salvaged by introducing human co-stimulatory transgenes into porcine endothelial cells. Through these geneticallymodified models, we will gain important knowledge of both allo- and xeno-CMR pathways. Moving from model to treatment, genetically modified porcine organs for clinical xenotransplantation may ultimately circumvent CMR with less pharmacologic reliance.

Steven C. Kim, MD
Emory University

Costimulatory Blockade and Mesenchymal Stromal Cell Therapy to Induce Transplantation Tolerance

Synopsis: Transplantation tolerance represents a theoretically desirable state of long-term graft acceptance. In recent years, mesenchymal stromal cells (MSCs) have demonstrated immunoregulatory potential through both their suppression of T-cell function and proliferation as well as their effect to preferentially differentiate T cells toward a regulatory phenotype. While the regenerative applications of MSCs have been a focus of research for some time, their clinical applications for both autoimmune disease and transplantation have gained traction as well. With our significant experience in non-human primate (NHP) transplantation models, clinical models of transplantation and cellular therapeutics we propose a series of rigorous NHP studies designed to translate MSC-based tolerance-induction strategies to the clinic. We hypothesize that MSC therapy can be utilized as an adoptive cellular therapy to induce immune tolerance to renal allografts in a non-human primate model. We aim to characterize MSCs in vitro to look at this cellular therapy's immunosuppressive effects in combination with costimulation blockade and also evaluate the effects of gamma IFN-y licensing prior to administration. We will then assess the impact of combined MSC therapy with costimulation blockade in vivo by assessing renal allograft survival in rhesus macaques. With a better understanding of the complex mechanisms by which MSCs exert their protective effects against T cell recognition and proliferation in the inflammatory environment of allograft transplantation, we hope to further characterize MSCs' effects on T cell function and cytokine production as well as their effects on allograft survival and their tolerogenic potential when used in concert with costimulation blockade.

Steven A. Wisel, MD
University of California, San Francisco

Optimizing Embryonic Stem Cell-Derived Islet Survival in the Encapsulated Setting

Synopsis: Although islet transplantation has shown great progress in the treatment of diabetes mellitus, broader application of the technology is limited by the scarcity of human donor tissue and the massive islet loss in the peri-transplant period. Human embryonic stem cell (h-ESC)-derived islets hold great promise as a robust option for clinical application, providing an unlimited source of beta cells with the ability to recover cell mass over time and more durable response to hypoxia.1-4 However, these h-ESC-derived islets remain susceptible to primary graft failure (PGF), with death of 60-80% of islets in the first 3-5 days prior to revascularization.5-7 Early clinical trials with h-ESC islets will take place within an encapsulation device in a subcutaneous location, providing immunoprotective barrier function and protection against malignant transformation. The Tang lab at UCSF has observed that the high density packing required for encapsulation further exacerbates PGF. Optimizing graft survival remains the central challenge to establishing islet transplantation as a feasible therapeutic option. As PGF mainly occurs in the early transplant period prior to revascularization, I hypothesize that nutrient deprivation and hypoxia are two independent primary triggers of PGF. I will test this hypothesis with h-ESC-derived islets in vitro and in a humanized mouse model. Successful completion of this study will help to optimize early h-ESC islet survival, decrease encapsulation device size, and make h-ESC islet transplantation a viable clinical therapeutic option on the human scale.

Nicolas Goldaracena, MD
University of Toronto

Liver Graft Modification Using Subnormothermic Ex Vivo Liver Perfusion—A Novel Strategy Against Hepatitis C Infection During Transplantation

Synopsis: Subnormothermic Ex Vivo Liver Perfusion (SNEVLP) is a novel preservation strategy for liver transplantation. Because SNEVLP liver grafts are preserved at close to physiologic temperatures with an active metabolism, this new preservation technique offers an opportunity to modify grafts during the storage period. Therapeutic agents are currently available that could potentially treat the graft to induce resistance to viral infection, modulate immune responses to the graft, or prevent cancer recurrence. However, while these ideas offer great promise, to date there are no data in large or small animal studies, or human models confirming proof of the concept.

Endogenous miRNA-122 (miR-122) is a critical factor for HCV replication and stability; Hepatitis C replication cannot occur in the absence of miRA-122 in hepatocytes. Therefore, sequestration and inactivation of miR-122 in liver cells should make livers resistant against Hepatitis C infection. Treatment of liver grafts with antisense miRNA122 sequesters and blocks miRNA-122. We hypothesize that using a novel subnormothermic ex vivo liver perfusion system to deliver anti miR-122 to liver grafts during perfused organ preservation will prevent HCV infection after transplantation.

We will evaluate drug uptake, and inhibition of miRNA-122 during the preservation phase in a pig model of liver transplantation. We will then perform a pilot study investigating the effects of miRNA-122 inhibition during organ preservation on HCV recurrence during human transplantation.

We anticipate this study will confirm that graft modification is possible with SNEVLP, opening exciting new avenues for novel therapies to improve the outcomes of liver transplantation.

Andrew Atia, BS
Duke University School of Medicine

CD57+CD4+ T cells in Belatacept-resistant Renal Allograft Rejection

Synopsis: The risk of T cell-mediated transplant rejection remains ubiquitous. Thus, clinical transplantation is predicated on the use of drugs to impair T cell function. Unfortunately, these drugs are not allospecific, but rather target broad aspects of T cell function including help, activation, proliferation, and effector functions. Thus, all transplant patients incur some degree of immune incompetence with regard to protective, particularly viral-specific, immunity. Currently, calcineurin inhibitor (CNI) based regimens are by far the most common in clinical transplantation, however, belatacept, a B7- specific fusion protein that mediates CD28-B7 costimulation blockade, was recently approved as a CNI alternative. Belatacept has been shown to prevent rejection without many off-target side effects. However, belatacept appears less able to prevent rejection than CNIs, particularly when rejection is mediated by previously activated memory T cells. We surmise that belatacept is a suitably effective agent for many, but not all, transplant recipients. Thus, a key clinical question is how to distinguish patients well served by belatacept from those better served by CNIs. Previous studies suggest that

CD57+CD4+ T cells play a role in belatacept-resistant renal allograft rejection. Our study is designed to investigate the in-vitro functionality of CD57+CD4+ T cells as it relates to the role these cells play in belatacept-resistant rejection, and initiate studies surveying these cells in renal allograft recipients treated with conventional tacrolimus-based immunosuppression. This investigation will allow us to tailor post-transplant immunosuppressive therapies to individuals to optimize clinical results.

Hillary J. Braun, BA
University of California, San Francisco

MELD/PELD Exceptions for Pediatric Liver Transplant Candidates: A Review of UNOS Proposal Narratives from 2009-2014

Synopsis: Preliminary data from UNOS/OPTN suggests that as many as 53% of pediatric liver transplant recipients are granted “exceptions” to their MELD/PELD scores based on narratives submitted to regional review boards. Recent investigations in this area have also demonstrated significant variation in exception requests by region and demographics. However, the content of the narratives submitted by transplant centers has not been analyzed or compared across regions. The purpose of this study is to review patient-level exception narratives to characterize medical conditions for which exceptions are applied for and granted and differences in exception applications and awards among regions.

David C. Cron, BS
University of Michigan Medical School

Frailty and Healthcare Utilization in Liver Transplant Recipients

Synopsis: Frailty, a biologic syndrome of decreased physiologic reserve, has recently been shown to be associated with decreased survival, low quality of life, and depression in patients with end-stage liver disease (ESLD). The outcomes of frail liver transplant recipients are currently unknown. To this end, we aim to study the relationship between pre-transplant frailty and post-transplant healthcare utilization, morbidity, and mortality. We hypothesize that frail patients will consume more hospital resources and have inferior post-transplant outcomes.

Since 2009, we have prospectively administered frailty assessments to over 600 ESLD patients referred for transplantation at a single center. 150 of these patients have received liver transplants. Frailty is assessed with a validated instrument of 5 components (unintentional weight loss, self-reported exhaustion, grip strength, physical activity, and gait speed). Our primary outcomes of healthcare utilization will include 90-day hospital costs, length of stay, readmissions, and discharge disposition. Secondary outcomes will include post-transplant morbidity and survival.

We have recently developed a multidimensional prehabilitation program at our institution targeting frailty in patients with ESLD referred for transplant. If this proposed study shows that frail patients indeed have inferior post-transplant outcomes, then such an intervention may have positive implications for post-transplant outcomes. Further, an analysis of the healthcare utilization of frail transplant recipients may help argue a case for cost-effectiveness of such a program.

Anna Yegiants, BA
Columbia University Medical Center

Investigation of the Barriers to Wider Utilization of Living Donor Liver Transplantation (LDLT)

Synopsis: Living donor liver transplantation (LDLT) reduces waitlist mortality significantly and markedly improves access to liver transplantation [1]. LDLT has post-transplant outcomes that are equivalent or better to deceased donor transplant and has shown to have excellent outcomes for patients with decompensated cirrhosis and end-stage liver disease [2]. Despite these clear benefits, LDLT continues to be a small fraction of the liver transplants performed in the United States. Even at centers with significant LDLT volumes, the majority of recipients continue to receive deceased donor liver transplants (DDLT).

This study seeks to gain understanding of the barriers to wider utilization of LDLT in the United States. Potential barriers include recipient concerns and education, as well as donor concerns and education, about the possible benefits and risks of LDLT. We plan to survey potential recipients and donors at our center to gain their perspectives on various donor recovery times, potential health risks to the donor, possible financial strain to the donor, and recipients’ willingness and concerns about approaching potential donors and accepting a transplant from a living donor. With better characterization of the barriers to LDLT, transplant centers may be able to provide appropriate education to patients who might be currently underutilizing LDLT.

Benjamin S. Parker, BS
Duke University School of Medicine

Role of Mitochondria in Allospecific T Cell Activation

Synopsis: It is well recognized that the innate immune response to pathogen-associated molecular patterns (PAMPs), such as bacterial DNA and lipopolysaccharide, is critical in orchestrating an effective adaptive immune response against microbial pathogens. Increasing evidence now shows that the innate immune system also senses tissue injury and cellular necrosis through the recognition of endogenously derived damage-associated molecular patterns (DAMPs).1,2 In the setting of sterile tissue injury, DAMPs may have a similar role in shaping the adaptive immune response and play a critical part in pathogenic inflammation well as physiologic wound healing. A diverse array of DAMPs derived from cellular products and the extracellular matrix has already been described.3-5 Recent research has highlighted mitochondria as being a major source of endogenous DAMPs and as playing a central role in the immune response to tissue injury.6-8

This study seeks to test the hypothesis that mitochondria released from injured tissue are key contributors to the immune response that leads to allograft rejection (alloimmune response or alloresponse) through activation of the innate immune system, and further, aims to define the relative contributions of mitochondrial components towards eliciting the alloresponse. Such information will improve our understanding of allograft rejection, while illuminating potential high-yield therapeutic targets and establishing a strong foundation for further research.