Andrew S. Barbas, MD
Duke University Medical Center

Enhancing Mitochondrial Quality Control Programs to Improve Organ Preservation by Normothermic Ex vivo Liver Perfusion

Synopsis:The primary limitation of liver transplantation is the severe shortage of donor organs. To ameliorate this shortage, transplant centers have increased utilization of extended criteria donor (ECD) livers. Experience with ECD liver transplantation has demonstrated that these organs poorly tolerate standard organ preservation (cold static storage). This has prompted a paradigm shift, with normothermic ex-vivo liver perfusion (NEVLP) emerging as a promising alternative. Preclinical studies have demonstrated improved graft function with NEVLP, but the cellular mechanisms underlying this benefit are not understood. Mitochondrial dysfunction from ischemia-reperfusion injury leads to energy failure and cell death. Cellular recovery occurs through activation of mitochondrial quality control (QC) programs. These programs are likely to be relevant during NEVLP and have not been characterized in transplantation. Improved understanding of mitochondrial QC will foster development of novel therapeutic strategies to improve transplantation of ECD livers. The overall hypothesis of this project is that NEVLP enhances activation of mitochondrial QC programs during organ preservation, leading to restoration of mitochondrial integrity and improved graft function.

James Gardner, MD, PhD
University of California, San Francisco

Donor-Specific Tolerance Induction by Extrathymic Aire-Expressing Cells

Synopsis:The purpose of our research is to understand the fundamental mechanisms regulating self-tolerance in the adaptive immune system, and to translate these findings into relevant therapeutics for inducing and maintaining donor-specific tolerance. In mentored collaboration with the Mark Anderson lab at the UCSF Diabetes Center, our lab studies a gene called the Autoimmune Regulator, or Aire, which was found to be a key regulator of central tolerance, and is required by educator cells in the thymus to expose the developing T-cell pool to a diverse array of one’s own antigenic diversity. In 2008 we discovered of a novel population of Aire-expressing cells outside the thymus—distributed throughout the body’s secondary lymphoid organs—which we named extrathymic Aire-expressing cells (eTACs), and which we demonstrated could be modified to induce robust self-tolerance and prevent autoimmunity (Gardner et al. Science 2008, Immunity 2013).

Our current research has three aims. First, to better understand the basic biology and immunology of eTACs. To this end, we have defined and are currently publishing work describing the family of antigen-presenting cells to which eTACs belong, and the mechanisms by which they induce tolerance. This basic biology and immunology is essential to any further therapeutic efforts. Second, to define the developmental biology of this cell population, and specifically how eTACs can be differentiated in vitro for diagnostic and therapeutic purposes. This work will greatly facilitate the ability to both study these cells and to develop translational cellular therapies. Third, to pioneer a therapeutic role for eTACs in inducing and maintaining donor-specific tolerance in a murine allogeneic transplant model. Together this research program aims to define a novel branch of adaptive immunity, and to apply these findings directly to transplant tolerance.

Nicole Conkling, MD
University of California, San Francisco

Chimeric Antigen Reception T-regulatory Cells for Targeted Rejection Therapy in Murine Hindlimb Composite Tissue Transplant

Synopsis: Vascularized composite tissue allotransplantation (VCTA) still faces many challenges regarding the unique immunologic demands of composite grafts and their propensity toward rejection. Acute rejection is extremely common in clinical VCTA, and chronic rejection remains poorly described. Interest has emerged in cellular therapies as an alternative to immunosuppressive drugs to treat rejection. T-regulatory cells (T-regs), which induce a more tolerogenic state, are known to be instrumental in long-term graft survival, attenuating the long-term sequelae of chronic rejection. Engineered chimeric antigen receptor (CAR) T-regs activated by donor antigens may allow for efficient, targeted therapy for VCTA rejection.

This study employs a murine hindlimb transplant model with two specific aims. First, transplanted mice will be observed clinically and serially biopsied to study the kinetics and cellular aspects of VCTA rejection, examining the architecture of the tissues and populations of graft cellular infiltrates. Second, recipients with rejection will be treated with CAR T-regs as salvage therapy. We hypothesize that CAR T-reg infusion will halt or reverse the rejection process. This project seeks to elucidate what is known about rejection in VCTA, as well as apply a targeted cellular intervention for salvage of rejected grafts. CAR T-cells have shown clinical promise in other fields, and they could feasibly be translated to the treatment of rejection in human VCTA. CAR T-regs represent a powerful tool for treating composite allografts threatened by rejection, as well as progress toward achieving the goal of tolerance without the life-long use of immunosuppressive drugs.

Ashley N. Suah, MD
University of Chicago

Induction of Sensitization and Resistance to Transplant Tolerance by Allogenic Pregnancy

Synopsis: Pregnancy is the second most common cause of immune sensitization and significantly contributes to gender disparities in access to transplantation. Pregnancy-induced sensitization has been reported using clinical registry data, with 54 – 84% of mothers developing antibodies specific for paternal antigens. Gestation may also induce a cellular response against paternal HLA antigens, resulting in T cell sensitization. In contrast to the clinical observations, Rowe and colleagues used a mouse model to demonstrate a sustained expansion of maternal FoxP3+ regulatory T cells with fetal specificity throughout pregnancy and post-gestation, suggesting that regulatory T cells play a role in maternal tolerance to the fetus. In our study, we used Rowe’s mouse model to investigate the apparent paradox of sensitization to F1 allografts based on clinical data, and acquired regulatory tolerance to the F1 fetus. We show that a single successful allogeneic pregnancy results in sensitization and an acquired resistance to costimulatory blockade-induced tolerance.

The aim of this study is to define the immunological consequences of pregnancy for transplantation, and identify therapies that can reverse sensitization and allow for the successful induction of tolerance in individuals sensitized by pregnancy. We hypothesize that 1) humoral and T cell sensitization to fetal antigen occurs after pregnancy, when the percentage of Tregs have declined; 2) that fetus/donor-specific antibodies are the mediators of failed tolerance in post-partum recipients; and 3) that desensitization to diminish the levels of circulating DSA will be sufficient to restore susceptibility to CoB-induced tolerance.

Imad Aljabban, MMSc, BS
Massachusetts General Hospital, Center for Transplantation Sciences

Analyzing the Role of Inducible Nitric Oxide Synthase as a Regulator of Renal Allograft Tolerance

Synopsis: Nitric oxide synthase (NOS) is associated with the innate axis of the immune system, and is responsible for antimicrobial and tumoricidal activity. The development of new technologies, such as NOS directed antibodies and knock-out mice, reveal a broader function of this enzyme in regulating lymphocyte activity¹. This is most likely a result of nitric oxide’s (NO) ability to react with a myriad of organic molecules^2,3,4. There are three isoforms of NOS that produce NO using L-arginine as a substrate. However, they differ in their regulation at the transcriptional level. Both endothelial NOS (eNOS) and neuronal NOS (nNOS) are expressed constitutively, but inducible NOS (iNOS) is upregulated in leukocytes during proinflammatory settings⁵. Although the function of NOS is known, the role of iNOS expressing cells in transplantation is unclear.

iNOS+ myeloid-derived suppressor cells (MDSCs) have been shown to prevent intratumoral infiltration of antigen-specific T cells through the nitration of chemokines and adhesion receptors^6,7. Additionally, iNOS deficiency in donor tissue accelerates allograft destruction⁸. Collectively, this data indicates iNOS as a potential regulator of T cell infiltration and possibly cytotoxic function. Our preliminary results show that spontaneously accepted allografts are heavily nitrosylated, while rejecting allografts lack this characteristic, and that iNOS+ cells infiltrate and organize around regulatory T cell lymphoid sheaths. The aim of our study is to examine the role of iNOS+ infiltrating MDSCs in promoting long-term survival of renal allografts.

Steven M. Elzein, BS
The Ohio State University College of Medicine

Investigating the Susceptibility of Specific CD8+ Tab-supp Cell-Mediated Mechanisms of Calcineurin Inhibition

Synopsis: Alloantibodies produced after transplant contribute to rejection and poor long-term graft survival. Recent published findings from our laboratory indicate that mammalian target of rapamycin inhibitors (mTORi) suppress in vivo alloantibody production by alloprimed IgG1+ B cells and prolong allograft survival compared with non-treated controls. While many report that mTORi has direct inhibitory effects on B cells, we are the first to report that combination of mTORi with calcineurin inhibitors (CNi) results in a reversal of the beneficial effects of mTORi on antibody production. Our lab is also the first to report that production of alloantibody is downregulated by a novel CD8+ T cell subset (CD8+ TAbsupp cells) that kills alloprimed B cells. Since we have shown that CNi inhibits CD8+ TAbsupp cell-mediated cytotoxicity to IgG1+ B cells, the CNi-mediated suppression of CD8+ TAb-supp cells is a likely reason why mTORi/CNi dual treatment does not significantly inhibit alloantibody production posttransplant. The proposed studies will investigate the precise mechanisms by which CD8+ TAb-supp cells are suppressed by CNi. I hypothesize that CNi critically impairs CD8+ TAb-supp cell acquisition of cytolytic effector molecules or release of perforin/granzyme. In order to address this hypothesis, I will investigate the phenotype and effector function of CD8+ TAb-supp cells in vivo following adoptive transfer of naïve CD8+ T cells into CD8 KO transplant recipients treated with mTORi, CNi, or both. Understanding these pathways will significantly enhance our knowledge of alloantibody regulation by the CD8+ TAb-supp cell subset and may lead to novel immunotherapy regimens for transplant patients.

Richard E. Seeber II
University of Alabama at Birmingham School of Medicine

Humanizing Pig CD59 Loci and Control by Endogenous Promoter

Synopsis: Despite nationwide efforts to increase organ donation, the United States continues to suffer from widespread organ scarcity. Recent data provided by the Organ Procurement and Transplantation Network reveal 119,996 patients in need of life-saving organ transplantation in America and that only 24,892 organ transplants were performed between January and October 2016. Because of such organ scarcity, researchers have investigated xenotransplantation, the transplantation of tissues or organs from non-human animals into humans, in hopes of providing patients on the wait with an otherwise unavailable transplant.

Rapid progress in xenotransplantation has been stymied by challenges in breeding organisms with specific mutations of xenoantigen-encoding loci, but the recent development of the CRISPR/Cas9 system allows facile, rapid, and reliable editing of organisms’ genes of interest.

Ultimately, our laboratory seeks to ameliorate the problem of organ scarcity by taking advantage of CRISPR/Cas9-mediated genome editing to generate porcine kidneys compatible with transplant into humans. For example, after deleting two xenoantigen-encoding genes, GGTA1 and B4GALNT2, we have been successful in transplanting a genetically-engineered pig kidney into a rhesus macaque, which survived for 15 months – the longest-surviving kidney xenograft to date. While we continue to diminish graft antigenicity by eliminating specific xenoantigens, we are interested in “humanizing” pig tissue by swapping porcine genes with human homologs using CRISPR/Cas9. Specifically, we will humanize the porcine CD59 locus, which encodes a complement regulatory protein inhibiting formation of the membrane attack complex. We hope to thereby simultaneously delete a potential xenoantigen, more appropriately regulate human complement, and ultimately increase xenograft survival.

Dor Yoeli, BA
Baylor College of Medicine

Percutaneous Spleno-Portal Bypass as a Minimally Invasive Alterative to Meso-Rex Bypass

Synopsis: My research focuses on developing a minimally invasive technique to bypass an extra-hepatic portal vein occlusion (EHPVO), a condition in which a thrombosis in the main portal vein blocks splanchnic blood flow in an otherwise healthy liver and causes potentially life-threatening portal hypertension. The meso-Rex bypass offers a surgical cure for EHPVO by anastomosing a venous graft between the superior mesenteric vein (SMV) and left portal vein to restore splanchnic blood flow through the liver. Although effective at resolving the portal hypertension caused by EHPVO, this operation represents a major abdominal surgery that most commonly requires an extensive neck dissection to harvest an autologous internal jugular vein graft to use as the venous conduit. My research aims to develop a technique for creating a percutaneous spleno-portal bypass. This minimally invasive procedure will effectively cure EHPVO without the associated morbidity of major abdominal surgery and neck dissection. With support from this grant, I will study the feasibility, efficacy, and safety of this experimental technique, which I designed under the mentorship of transplant surgery and interventional radiology faculty, in a swine model. This procedure utilized innovative magnetic technology to facilitate percutaneous through-and-through access between the left portal and splenic veins. In addition to providing a minimally invasive alternative to the meso-Rex bypass for patients with EHPVO, this study, if successful, will be the first to demonstrate a percutaneous vascular bypass, potentially ushering in a new wave of minimally invasive vascular and interventional procedures.