I. Raul Badell, MD
Emory University

Circulating T follicular helper cells as a biomarker for donor-specific alloantibodies in transplantation

Synopsis: As an early career surgeon-scientist I am applying for this grant to support my interest in developing circulating T follicular helper (Tfh) cells as a biomarker to aid in the diagnosis and management of deleterious donor-specific antibodies (DSA) in transplantation. With the now well recognized role anti-donor antibodies play in shortening kidney allograft survival, there is great need for the discovery of biomarkers of humoral alloimmunity in kidney transplant recipients to facilitate the clinical management of antibody-mediated allograft injury and the initiation of effective therapeutic interventions to prevent premature allograft loss. In this application, I first propose to define the circulating Tfh (cTfh) cell repertoire and examine its kinetics in non-transplant and transplant patient cohorts without DSA. I will then use the data acquired from these control cohorts to guide the subsequent analysis and interpretation of cTfh cell quality and kinetics in a unique cohort of transplant recipients with de novo DSA formation. My goal is to identify the kinetics and characteristics of cTfh cells during the development of DSA to support their use as a cellular biomarker for the diagnosis and prediction of alloantibody-mediated processes in transplantation that could also help identify new therapeutic targets against humoral alloimmunity. The data obtained from this proposal will inform the design of prospective human immunology studies aimed at further development and validation of cTfh cells and their subsets for diagnostic and predictive purposes in the field of transplantation.

Adam D. Griesemer, MD
Columbia University Medical Center

Mechanisms of Liver Transplant Tolerance and Alloreactivity in a Translational Model

Synopsis: The liver is the second-most transplanted organ in the US. Outcomes are limited due to the need for the vast majority of patients to take immunosuppressive drugs for life. We therefore established the only existing nonhuman primate (NHP) liver transplant model in the US to develop protocols for early tolerance induction. Our preliminary data show that memory T-cell responses (particularly effector/memory CD8) and enhanced levels of inflammatory cytokines represent significant barriers to liver tolerance. Control of the memory CD8 T-cell response led to long-term survival off immunosuppression. Interestingly, some animals had a rejection crisis with a spontaneous resolution of the inflammation and normalization of LFTs. We now aim to develop a protocol that eliminates the rejection crisis. We will characterize the mechanisms that led to the rejection crisis and resolution of rejection in the new and previous protocols. In Aim 1, we will eliminate late allograft dysfunction by additional memory CD8 depletion after systemic immunosuppression withdrawal. In Aim 2, we will use a novel T-cell receptor deep sequencing platform to track the expansion or contraction of alloreactive CD4, CD8 and Tregs in the peripheral blood and liver. In Aim 3, we will use the VECTRA multiplex immunohistochemistry platform to characterize the changes in liver-resident APC and graft-infiltrating CD8 phenotype to identify markers of the tolerogenic liver environment that can guide immunosuppression weaning. These studies will enhance our understanding of the mechanisms of liver tolerance and lead to clinical trials of rapid immunosuppression withdrawal following liver transplantation.

Ari Cohen, MD
Ochsner Health System

Srikanta Dash, PhD
Tulane University

Tumor-derived exosomes in early stage HCC: the role of exosome shedding in HCC patients undergoing locoregional therapy, assessment of tumor biological aggressiveness, and tumor induced-tolerance

Synopsis: Tumor-derived exosomes (TDE) are released in the circulation bearing tumor antigens and carrying lipid, protein, RNA and microRNA (miRNA) cargo capable of altering target cell function. With research efforts focused on TDE contents and target-cell function, changes in shedding frequency following treatment and correlations between shedding behavior and outcomes are unclear. Treatment-induced shedding may provide an important indication of tumor stress-adaptation and relapse, despite a positive radiographic treatment effect. We propose a two-year prospective study containing two primary aims. We will utilize and expand upon an existing prospective cohort of treatment-naïve very early - intermediate stage hepatocellular carcinoma (HCC) patients undergoing de novolocoregional therapy as a downstage or bridge to liver transplantation. In the first aim, the HCC-associated surface biomarker glypican-3 (GPC3) will be utilized to identify and quantify baseline and 90-day post-treatment changes in TDE levels in the plasma and correlated with treatment response and waitlist progression-free survival. In the second year of the study, we will quantify TDE levels pre- and post-transplantation and monitor 1 year tumor-free survival outcomes. In the second aim, we will test the hypothesis that the TDE cargo promotes tumor-induced tolerance. Tumor-induced tolerance is observed in ~ 1/3 of early stage HCC patients and is closely associated with tumor resistance to embolization and waitlist dropout. The results of this proposal will establish the utility of a TDE based approach to assess tumor biological aggressiveness and provide a mechanism uniting a shedding phenotype with poor prognosis in transplant-relevant HCC patients.

Robert L. Gottlieb, MD, PhD, FACC
Baylor University Medical Center

Antiviral-Facilitated Transplantation of Hepatitis C Hearts to Expand the Donor Pool

Synopsis: The ongoing, critical shortage of available donor organs for cardiac transplantation is aggravated by a low acceptance rate of the hearts offered. Otherwise acceptable hearts from hepatitis C virus (HCV)-positive donors are routinely rejected due to risk of transplant-associated transmission. However, recently developed hepatitis C therapies warrant a reassessment of the risks involved.

The proposed institution-sponsored, consortium study will establish the safety of an expanded transplant donor pool that includes donors infected with hepatitis C. We will monitor HCV transmission in 20 patients across 3 sites in Texas, North Carolina, and California, and assess the response to direct-acting antiviral therapy (sofosbuvir-velpatasvir) in immunosuppressed heart transplant patients. Following transplantation, subjects will be closely surveilled while inpatient using serial HCV polymerase chain reaction and, if no HCV viremia is detected by hospital discharge, weekly for 8 weeks. If/when recipients develop confirmed viremia, sofosbuvir-velpatasvir therapy will be initiated and administered for 12 weeks. The primary endpoint is the sustained virologic response 12 weeks after discontinuation of therapy. The secondary objective is to evaluate 1-year post-transplant survival. Complete safety assessments will occur at 6 weeks and 1 year.

The results will provide insight in the kinetics of hepatitis C viral transmission and response to therapy in immunosuppressed heart transplant patients. If safe and feasible, this approach may lead to a potential 20%-increase in the number of acceptable donor hearts. While the study drug will be provided generously by Gilead Sciences, we presently seek funding for the operating costs of this 2-year study. 

Kazunari Sasaki, MD
Cleveland Clinic Foundation

Developing new organ allocation scheme using continuous risk score in liver transplantation for hepatocellular carcinoma

Synopsis: The current organ allocation schema in liver transplantation (LT) for hepatocellular carcinoma (HCC) has major drawback. With increasing penetration of bridging and down-staging locoregional therapy (LRT) comes increasing heterogeneity of oncological risk among certain tumor morphologic group. The Milan criteria (MC) which is the main trunk of allocation scheme might be no more suitable for current LTx practice. MC and subsequent reactive frameworks enacted in a piece-wise fashion in allocation metric would waste certain number of precious organs by allocating. To mitigate this problem, we developed preoperatively assessable continuous risk score in liver transplant for HCC which outperformed current major preoperatively assessable selection metrics (Sasaki Kand Firl D et al. Lancet Gastroenterol Hepatol 2017; 2: 595–603). The biggest advantage of continuous risk score comparing to dichotomous selection criteria with exception point were; 1) able to precisely follow longitudinally oncological risk increase/decrease during courses of LRT, 2) able to stratify the candidate according to the postoperative prognostic risk.

We aim to develop liver graft allocation metric using continuous risk score which potentially decrease inappropriate organ waste and feasible to adjust organ allocation number between liver transplant candidate with HCC and non-HCC. 

Kevin Janek, MD
University of Wisconsin

Targeting the AHR with Nanoparticles in Models of Organ Transplantation

Synopsis: Current immunosuppressive strategies after transplantation rely on non-specific global suppression of the immune system. Patients are subjected to ongoing treatment with extensive side-effects, including severe nephrotoxicity. While one-year survival is excellent, patients continue to struggle with chronic rejection and graft loss. There is a need for a novel, targeted approach to extend graft survival, reduce side-effects, and avoid global immunosuppression. The aryl hydrocarbon receptor (AHR) has been shown to have a central role in the ability of DCs to direct differentiation of Tregs from naïve T cells, and have been utilized to prolong murine skin grafts with intraperitoneal, oral or inhaled AHR ligands. Nanoparticles (NPs) have been extensively investigated for targeted drug delivery to treat various major diseases, and can significantly enhance the stability and solubility of the payloads and deliver the payloads more specifically to the targeted tissues/cells, thereby maximizing their therapeutic indexes and minimizing their potential side-effects. We have designed NPs that can be loaded with both AHR ligand and alloantigen to assess their ability to generate toleragenic anti-donor immune response in transplant models. We aim to characterize these NPs for their ability to target DCs and reduce an allogeneic immune response, and test the efficacy of treatment on recipient mice in both splenocyte and skin graft models.

Brendan P. Lovasik, MD
Emory University

Improving Xenograft Outcomes: Exploring Novel Molecular Targets and Developing Translational Therapies

Synopsis: Kidney transplantation offers a life-saving and life-improving surgical therapy to patients with end-stage renal disease. Xenotransplantation, using genetically-modified porcine organs, has the potential to dramatically increase the organ supply to provide more patients with a new kidney. The proposed study seeks to explore two major barriers to applications of xenotransplantation: hyperacute rejection due to pre-formed antibodies against nonhuman donor cell markers, and acute rejection due to recipient lymphocytic cell-mediated response.

First, we will examine the effect of a genetically optimized porcine donor on rejection, by performing renal transplants using the newly-developed double-knockdown GAL -/-, β4GAL-NT2 -/-, hCD55 pig on as donors into rhesus macaques with the resulting elimination of targets for xeno-directed antibody. Next, using this optimized double-knockout pig, we can then examine impact of “next generation” costimulatory blockade gents (anti-CD28 dAb and anti-CD154 dAb) in a pig-to-NHP model using these genetically optimized donor pigs.

Second, we will explore the T-cell derived lymphocytic response to xenotransplanted donor organs through analysis of the CD4+/CD8+ cells that mediate rejection. Here, we will explore the impact of CD4+ and CD8+ T cell-directed depletion immunosuppressive regimens, as well the impact of swine leukocyte antigen (SLA) class I or II deletion on T-cell mediated cytotoxicity by generating porcine donors with SLA class I or class II deletions on the Gal-/-, β4GAL-NT2 -/-, hCD55 tg background pigs.

These novel and highly efficacious new strategies in to the field of xenotransplantation has the potential to bring direct clinical and therapeutic applications to the >100,000 patients on the waiting list for a new kidney.

Benjamin Smood, BS
University of Alabama at Birmingham School of Medicine

Development of pig composite islet-kidney grafts (for future xenotransplantation)

Synopsis: Diabetes is a global problem and major cause of end-stage renal disease. Treatment often requires pancreatic and/or kidney transplant from deceased human donors. Due to limited donor supply, cross-species transplantation (xenotransplantation) is considered a potential solution, if hurdles of immunogenicity and rejection can be overcome.

Anna Zhang, BS
Tufts University School of Medicine

Prevention of Ischemia-Reperfusion Injury in Liver Transplantation: Vasoprotection by Nitric Oxide during Ex Vivo Normothermic Machine Perfusion in a Steatotic Human Split-Liver Model

Synopsis: A severe shortage of viable donor livers prevents half the patients on the waiting list from undergoing potentially lifesaving transplantations each year. Worse, over 40% of the livers are macrosteatotic. Large fat droplets impair sinusoidal perfusion, lowering the tolerance to ischemia and ultimately leading to decreased graft survival. Thus, there is a need to induce vasoprotective recovery of sinusoidal flow in steatotic livers. If successful, such an approach could greatly expand the donor pool to include steatotic, human livers once deemed unacceptable for transplantation. Past research has demonstrated the vasoprotective effects of simvastatin in nitric oxide (NO) upregulation via the KLF2 pathway in rat livers. However, no studies have evaluated simvastatin, or other anti-inflammatory molecules like alpha-1 antitrypsin (AAT), administered during machine perfusion of human, steatotic livers. Therefore, this study seeks to study the individual effects of AAT and simvastatin on NO production in steatotic human livers during ex vivo normothermic (37°C) machine perfusion (NMP) using a novel split-liver model. We hypothesize that such treatments can restore vasoprotective pathways via NO upregulation in steatotic, human livers for the prevention of hepatic IRI during organ preservation prior to transplantation.