CORE SUMMARY / ABSTRACT Tolerance of kidney allografts has been achieved in monkeys and patients via transient mixed hematopoietic chimerism. However, until now, our current mixed chimerism protocols have consistently failed to induce tolerance of other more immunogenic transplants, including hearts. Enhancing our mixed chimerism strategy to achieve tolerance of heart allografts is the main objective of our overall Program. We will accomplish this objective by testing the overall hypothesis that enhancing current mixed chimerism protocols using novel strategies that achieve durable, high level donor chimerism will lead to an effective and safe tolerance protocol that can be rapidly translated to human recipients of heart allografts. The specific goal of Core A is to test mechanistic hypotheses generated by the in vivo treatments described in Projects 1-3. Improving our mechanistic understanding of tolerance inducing procedures that work and those that do not will inform the design of future protocols. We will test these mechanistic hypotheses by 1) deciphering the mechanisms by which donor hematopoietic mixed chimerism, leukocyte recovery, and alloimmunity by memory T cells affect tolerance induction, 2) determining if successful tolerance protocols are associated with extracellular vesicle generation and donor antigen cross-dressing, and 3) evaluating the role of T cell deletion and regulation in rejection vs. tolerance. Understanding the mechanisms driving the immune response towards rejection or tolerance in monkeys treated with the mixed chimerism protocols described in Projects 1-3 will contribute greatly to the refinement of those protocols and to the design of future tolerance strategies that can be tested in Projects 1-3 in anticipation of rapid translation to human heart transplant recipients.