Abstract Immunosuppression management follows standard protocol that is aimed at predetermined drug levels, with minimal adjustments over the lifetime of the recipient. While successful in preventing rejection, the high burden of suppression is associated with an increased incidence of drug-related side effects impacting the well-being of the recipient. The absence of objective assays to evaluate immune activation poses a challenge in determining the optimal dosage of immunosuppressive drugs and the ability to personalize therapy. Our NIH Immune Tolerance Network trial (ITN030ST) demonstrated that many of the recipients can maintain good allograft function with less immunosuppression, starting at a relatively early stage after transplantation. However, tapering immunosuppression based on clinical observation of allograft function risks the development of acute rejection, and the need for additional treatment to reverse rejection and rescue allograft function. Mechanistic studies using ITN030ST serum samples retrieved prior to, during, and after tapering of immunosuppression demonstrated that the trajectories of serum miRNAs are sensitive, specific, prognostic, and diagnostic of acute rejection. Taken together we hypothesize that highly sensitive and specific miRNA profiles can detect early molecular activation of immune response and allograft injury and will greatly improve safe management and allow personalizing immunosuppression while reducing the risk of rejection. The R34 application outlines a design for a prospective randomized study to assess whether serum miRNA profiling can assist in safe tapering of immunosuppression when compared to clinically guided management. The 3-phase approach to the design involve establishing trial procedures, followed by protocol design towards implementation, and concludes with an outcome assessment plan. A subsequent clinical study will assess whether miRNA profiles can be utilized as a predictor of rejection and aid in personalizing immunosuppression. We also propose the design of mechanistic studies that will leverage clinical samples and data to improve prediction of rejection. A novel technology developed by our investigators for methylation-based determination the cell/tissue origins of cfDNA. Using this method, it will be possible to interrogate both liver pathology and the activation of specific immune and inflammatory cell types, to identify early allograft injury and expose the underlying causes of dysfunction and immune activation when tapering immunosuppression. In the long run, this technology can get incorporated into protocols for personalized management of immunosuppression. The proposed application takes us one step closer towards developing a clinical study aimed at implementing biomarker-guided personalized immunosuppression in the transplantation setting. The study will also offer valuable insights into the molecular mechanisms underlying immune-mediated allograft injury associated with th...