SUMMARY Induction of graft acceptance in the absence of chronic immunosuppressive therapy remains an elusive goal in organ transplantation. Transplant immunologists have historically attempted to prevent organ rejection by developing novel therapeutic approaches that target antigen-presentation (signal 1), co-stimulation (signal 2) and/or cytokine production (signal 3). While promising results have been obtained using these novel methodologies that target the adaptive immune response, long-term graft survival rates remain suboptimal. Recent data demonstrates that the innate immune system (macrophages) initiate transplant rejection. In line with these observations, our laboratory has recently discovered a novel pathway that contributes to allograft rejection, which is mediated by epigenetic reprograming of macrophages. This recently discovered macrophage functional state has been defined as “trained immunity” and is positively regulated by the mammalian target of rapamycin (mTOR). While regulation of macrophage differentiation and function represents a compelling therapeutic approach, its application to induce immunological tolerance remains unexplored clinically. We hypothesize that failure to induce long-term allograft survival may be due, in part, to the lack of therapeutic protocols that target myeloid cells in vivo. Therefore, the development of an immunotherapy that targets innate immune cells in vivo and prevents trained immunity represents a promising approach to facilitate long-term allograft survival. To this aim, we developed high-density lipoprotein (HDL) nanobiologics that target myeloid cells in vivo and regulate trained immunity by blunting the mTOR pathway. We observed that these mTORi- HDL nanobiologics prevented aerobic glycolysis and epigenetic modifications underlying inflammatory cytokine production (signal 3) associated with trained immunity. To enhance therapeutic efficacy, we developed a second inhibitory CD40-TRAF6 specific nanobiologic CD40i-HDL that prevents co-stimulation (signal 2). Remarkably, a short-term combined mTORi-HDL and CD40i-HDL nanoimmunotherapy regimen resulted in indefinite allograft survival with no signs of toxicity or chronic allograft vasculopathy. Having described that trained immunity can be negatively regulated by HDL nanoparticles to promote tolerance for transplantation in "naïve" mice, we propose to evaluate the robustness of our combined nanoimmunotherapy in two settings that compromise the long-term function of the transplanted organ: infection and allosenstization.