ABSTRACT Transplantation is the most effective treatment for end-stage organ diseases. Clinically, two important risk factors for poor transplant outcomes are the pre-transplant presence of donor-reactive T cells in the recipient and the length of graft cold ischemic storage (CIS) time prior to transplant. In kidney transplant patients, the pre- transplant presence of circulating donor-reactive memory T cells is associated with an increased risk of acute rejection episodes, delayed and decreased graft function and worse long-term graft survival. In unsensitized recipients, donor-reactivity of memory T cells is due to heterologous (cross-reactive) immunity. However, the mechanisms activating donor-reactive endogenous memory T cells within allografts to mediate acute graft injury remain poorly understood. Prolonged CIS increases ischemia-reperfusion injury (IRI), which is characterized by production of reactive oxygen species and proinflammatory cytokines that direct infiltration of recipient leukocytes into the graft and cause activation of these cells within the graft. IRI also induces the graft to release damage associated molecular patterns (DAMPs) from injured and dying cells that exacerbate inflammation and contribute to worse outcomes in higher risk allografts. We previously showed that these two risk factors for poor transplant outcomes are linked. The sustained high-inflammatory environment seen following transplant of cardiac allografts subjected to prolonged CIS is necessary for sufficient activation of donor-reactive memory T cells to mediate costimulatory blockade resistant acute rejection in unsensitized recipients. Multiple clinical transplant studies have shown that one DAMP, cell-free DNA is elevated in the circulation during allograft injury, representing a promising non-invasive biomarker for early detection of acute rejection. Additionally, recent work from our collaborators has found new, pharmacologically targetable signaling partners required for activation of innate immune sensors of cell free DNA, such as TLR9 which can recognize mitochondrial DNA (mtDNA). These findings and our current preliminary data have led us to hypothesize that prolonged CIS and subsequent increased IRI enhance the release of mtDNA, leading to greater TLR9 activation and downstream type I IFN production, triggering a pro-inflammatory cycle that is sustained by endogenous donor-reactive memory T cell mediated acute graft injury. This hypothesis will be tested in two specific aims using our vascularized mouse heterotopic heart transplant model: first, we will test modulation of TLR9 signaling in vivo to assess impact on donor-reactive memory T cell mediated acute graft injury; and second, we will test the mechanism(s) by which type I interferon contributes to early post-transplant inflammation and activation of donor-reactive T cells to mediate rejection. We anticipate that these studies will identify new targets for therapeutic strategies to improve s...