PROJECT SUMMARY / ABSTRACT The unifying goal of this Program is to design new and innovative strategies that achieve clinically-relevant heart allograft tolerance in 2-month-delayed tolerance induction protocols which induce either transient or durable mixed chimerism. The PI of this Program application has recently developed novel protocols that have, for the first time, achieved long-term, stable tolerance of fully MHC mismatched hearts in cynomolgus monkeys. This remarkable result was attained in heart recipients by combining a transient mixed chimerism protocol with donor kidney co-transplantation. Recent pilot studies have demonstrated the feasibility of replacing the previous requirement for donor kidney co-transplantation with approaches that both inhibit CD8 effector/memory T cells and enhance host regulatory T cells. Project 3 will build on the clinically-applicable heart tolerance induction platform developed by the Madsen team (2-month D-Protocol; presented in the Overview and in detail in Project 1) and the Pierson/Azimzadeh lab's extensive experience with dual costimulation pathway blockade in NHP heart allograft models. We will test whether enhanced costimulation pathway blockade (αCD2 with αCD154 and/or αCD28) can replace the requirement for renal co- transplantation while evaluating αCD2 (Aim 1) or inhibition of JAK 1/3 (Aim 2) or Bcl-2 (Aim 3) as alternative and potentially complementary approaches to deplete or inhibit tolerance-resistent effector memory T cells (TEM) cells and reduce toxicity (eliminate the need for αCD8 and reduce or eliminate host total body irradiation (TBI)) in the 2-month D-Protocol. Guided by initial results, we will also deploy TReg-supportive treatments (from Project 1) and radiation-free conditioning using αCD117-Amanitin antibody drug conjugate (from Project 2) with αCD2-based enhanced costimulation blockade, aiming to reduce or eliminate the need for radiation and thereby reduce toxicity. Core A will elucidate the cellular and molecular mechanisms associated with success or failure to consistently induce tolerance in the three coordinated but distinct Project 3 Aims, and enable mechanistically informative comparisons between results from distinct but closely aligned Aims in Projects 1 and 2. We anticipate that one or more Aims in Project 3 will generate innovative, effective, and safe tolerance protocols that will be ready for clinical trials in heart recipients by the end of the funding period, and enhance our understanding of tolerance induction in a clinically relevant context.