Abstract Heart transplantation (Tx) remains the therapy of choice for patients with end-stage heart failure. However, long- term graft and patient survival remains suboptimal due to chronic rejection and high rates of morbidity and mortality associated with life-long immunosuppression (IS). IS-resistant memory T cells (Tmem) play a major role in chronic rejection and are considered a major barrier to graft survival. To date, no immunotherapy has been clinically proven to be effective in the prevention or treatment of Tmem-mediated rejection. Post-Tx inflammatory milieux promote cytokine-mediated pathogenic donor-reactive Tmem (darTmem) function and are equally detrimental to donor-reactive regulatory T cells (darTreg). Reactive oxygen species (ROS) promote interleukin (IL)-6 mediated graft injury and dysfunction. Concomitantly, IL-6 prevents T cell regulation, promotes IS-resistant T cells, and abrogates long-term graft acceptance. Our preliminary data suggest that ROS and IL-6 promote graft endothelial cell dysfunction in recipient monkeys. It is known that IL-7 and IL-15 jointly orchestrate Tmem generation and persistence. IL-7 prevents T cell regulation, while IL-15 augments clonal expansion of IS-resistant darTmem. Our preliminary studies demonstrate increased IL-7 and IL-15 mediated pathogenic darTmem percentages and function at the time of rejection in recipient monkeys. Adoptive Treg therapy remains at the forefront of innovative cell-based therapy in Tx. Treg engineered with chimeric antigen receptors (CARTreg) that recognize specific donor MHC molecules home and reside in target tissues of humanized mouse models, with superior suppressive function compared to conventional Treg. In this application, we propose to test the unifying hypothesis that early post-Tx cytokine modulation and donor- specific T cell regulation will induce durable high darTreg-to-darTmem ratios, promote long-term graft protection, and maintain protective immunity. We propose to test this hypothesis, through combined (i) cytokine modulation using newly developed rhesus-specific (rh) mAbs and (ii) administration of novel monkey Bw6- specific CARTreg, in heart allograft recipient monkeys. We have 3 specific aims: Aim 1: To investigate the impact of IL-6 neutralization on early post-Tx ROS-mediated graft injury and darTmem development, using rh anti-IL-6 neutralizing mAb. Aim 2: To Attenuate the persistence of IS-resistant darTmem through dual targeting of IL-7 and IL15 receptors in allograft recipients, using rh blocking mAbs. Aim 3: To induce durable T cell regulation through combined administration of Bw6-specific CARTreg and cytokine modulation conducive of prolonged graft acceptance. The proposed research will define inciting molecular and cellular mechanisms that promote IS-resistant rejection and provide the foundation for the development of novel and clinically relevant therapeutic strategies amenable to rapid clinical translation.