*Purpose: Older organs represent an underutilized potential. We dissected alloimmune responses when transplanting older organs and tested if the depletion of senescent cells in older organs ameliorates alloimmunity and improves transplant outcomes.

*Methods: Ischemia reperfusion Injury (IRI) was induced in young and old mice (2 and 18 months) through bilateral clamping of the renal pedicle. Systemic and local mt-DNA levels were measured by qPCR; the accumulation of senescence cells was assessed; T cell and Dendritic cell (DC) frequency and activation were tested by FACS and co-culturing experiments. Senolytics (Dasitinib and Quercetin, D&Q) were applied to donor animals and cardiac transplantation was performed in a model of strong histoincompatibility (C57BL/6 to DBA/2).

*Results: Old animals showed higher systemic mitochondrial DNA (mt-DNA) levels (n=5/group; p=0.05) that increased dramatically after IRI (n=5/group after 48h; p=0.019); elevated systemic mt-DNA levels were linked to highly increased IFN-γ expression on splenic CD8+ T cells (p=0.0001). Old DCs co-cultured with mt-DNA showed an upregulation of costimulatory molecules (p<0.05 compared to young DCs). Inhibition of TLR9, in turn, prevented the activation of DCs through mt-DNA. Old but not young DCs promoted an enhanced IFN-y and IL-17 response of allogeneic T cells in vitro. Next, we tested if the accumulation of senescent cells was the source of enhanced mt-DNA release upon IRI. Old donor animals were treated with senolytics (D&Q) that have been shown to clear senescent cells through disabling senescence-associated anti-apoptotic pathways (SCAPs). Indeed, senolytics reduced mt-DNA levels in old animals (p=0.01 compared to young controls). Furthermore, senolytics decreased the expression of IFN-γ on CD8+ T cells (p=0.01). Finally, we confirmed the therapeutic potential of senolytics: A single application of D&Q prolonged the survival of old cardiac allografts significantly (p=0,028 compared to untreated controls).

*Conclusions: The accumulation of senescent cells is linked to augmented mt-DNA levels that activate old DCs causing an amplified alloimmune response. Senolytics applied to donor animals decrease mt-DNA levels and the activation of DCs. Treating donor animals with senolytics improved graft survival of older organs. Those experimental data provide a rationale to apply senolytics to donors or organs during preservation.

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