ABSTRACT This proposal presents a five-year research career development program focused on targeting donor liver- resident cells with regulatory properties to decrease rejection after transplantation. I am an Assistant Professor of Surgery at the University of Wisconsin-Madison, with previous research and clinical experience in transplant immunology and transplant surgery involving normothermic ex vivo machine perfusion (NEVLP), whereby an organ is housed under physiologic conditions. The present project will advance the field of transplant immunology by using NEVLP technology to modify the immune cells within the liver prior to transplantation. I have assembled an outstanding mentorship team of investigators with expertise in transplant immunology, dendritic cell biology, and extracellular vesicle biology. The proposed training will guide and enhance my development in core competencies, including transplant immunology, communication, biostatistics, and ethical research design that will enable me to transition to research independence as a surgeon-scientist dedicated to reducing organ rejection in the field of transplant surgery. Liver transplantation is the only treatment option for patients with end-stage liver disease; however, rejection of the transplant can decrease liver and patient survival. In addition, patients still require lifelong use of anti-rejection medications that suppress the immune system. Modification of the donor liver, and the immune cells within it, has the potential to promote acceptance of the liver and minimize the need for anti-rejection drugs. Advances in an innovative technique called normothermic ex vivo liver perfusion (NEVLP) offer a unique opportunity to benefit significantly the 25% of liver transplant recipients that develop acute rejection, as well as many more transplant recipients who would benefit from using fewer anti-rejection drugs. Recent studies have demonstrated the importance of regulatory dendritic cells (DCregs) for prolonging transplant survival. My central hypothesis is that expansion of the number of liver-resident DCregs during NEVLP will promote a regulatory environment for the organ after transplant. Using a rat model of NEVLP and liver transplantation that my research group has optimized, I expect NEVLP to expand DCregs potently, leading to an increase in immune checkpoint molecule expression and production of anti-inflammatory extracellular vesicles and cytokines that can reduce immune-mediated rejection. This innovative approach of expanding graft-resident DCregs to decrease rejection could be used in deceased donor liver transplantation as well as translated to other types of solid organ transplants. To achieve these objectives, I propose the following scientific aims: 1) Determine the dominant regulatory function of liver-resident DCregs after NEVLP, and 2) Measure the impact of expanded liver-resident DCregs generated by combination cytokine therapy during NEVLP on liver graft rejection in vitro and in vivo.

PUBLIC HEALTH RELEVANCE STATEMENT Transplant recipients must take lifelong anti-rejection medications that cause side effects and decrease their ability to defend against infections and cancers. We will use a novel method of liver storage in a rat model of liver transplantation to examine the impact of interventions aimed at increasing regulatory cells within the liver, thereby making the entire organ less likely to cause an immune reaction in the recipient. This exploration of strategies to decrease patient reliance on harmful drugs holds tremendous promise not only for liver transplantation, but all solid organ transplants.