Project Summary The aging population is on the rise. By 2030, for the first time, both the population older than 65 and those younger than 18 years will contribute equally with 21% to the overall US population. Organ transplantation is hampered by a limited supply of organs with many patients waiting for numerous years and numerous patients dying before getting a transplant. Organs from older donors are available, however, frequently not considered or discarded with concerns of compromised function and augmented immunogenicity. We have previously documented the impact of aging in clinical transplantation and have dissected some of the mechanisms that drive the augmented immunogenicity of older organs. We have identified old intragraft dendritic cells (DC) as drivers of an IL-17-driven immune response. Mechanisms that activate old DCs, however, remain to be determined. In additional preliminary data, we have now documented a systemic increase of cell- free mitochondrial DNA (cf-mt-DNA), a damage-associated molecular pattern (DAMP) that accumulates in aging. Notably, blocking TLR-9, the ligand for mt-DNA, reduced the immunogenicity of old DCs and prolonged the survival of old transplants. Additional preliminary data documented a compromised clearance of senescent cells in older mice. Senolytics, agents that deplete senescence cells reduced inflammatory responses associated with ischemia-reperfusion injury (IRI) in an age-dependent fashion. In specific aim 1, we will therefore define the impact of aging on DAMP release and delineate whether the transplantation of old organs can promote inflammation via the dissemination of senescent cells. Our preliminary experiments have also shown that the communication between DCs and T cells is impacted by aging. In specific aim 2, we will therefore elucidate the underlying mechanisms that promote mtDNA release and inflamm-aging. Here, we will test the role of nitrogen permease regulator-like 3 (NLRP3) and will assess whether elevated DAMP levels mediate inflammation via this signaling pathway in an age dependent manner. We have also accumulated preliminary data showing that senolytics reduced mtDNA levels while ameliorating DC and T-cell activation in an age-specific fashion. Additional preliminary data have also shown that a single application of senolytics in older donor animals prolonged transplant survival. We thus submit that senolytics will have the capacity to improve organ quality and reduce the augmented immunogenicity in aging. In specific aim 3, we will therefore dissect the translational capacity of senolytics administered to donors, recipients, or as an addition to the preservation solution. We believe that our proposal is built on a strong collection of preliminary data, the availability of unique experimental models and the support of a distinctive group of researchers. We are thus confident that our proposal will contribute substantially to an improved understanding in an area of unmet need.