*Purpose: Prolonged allograft cold ischemic storage (CIS) and subsequent increased ischemia-reperfusion injury (IRI) following solid organ transplant act through innate immune pathways that promote early activation of endogenous donor-reactive memory T cells to express effector functions to mediate CTLA-4Ig-resistant allograft rejection. Here, we investigated the role of innate immune ligands and receptors in IRI and their impact on this memory T cell activation and CTLA-4Ig resistant rejection.

*Methods: Mouse complete MHC-mismatched heart allografts were subjected to long (8hr) or short (0.5hr) CIS before heterotopic transplant. Infiltration and proliferation of endogenous donor-reactive memory T cells in allografts was assessed by flow cytometry. Plasma mitochondrial DNA was measured using a custom TaqMan qPCR assay. IFN-γ producing donor-reactive T cells in the spleen were quantified by ELISPOT.

*Results: Recipients of WT allografts subjected to long CIS had significantly elevated plasma mitochondrial DNA (mtDNA) as early as 12hr post-transplant (2.6-fold) compared to recipients of allografts subjected to short CIS. Because mtDNA is a TLR9 ligand, we then explored how long CIS affects expression of TLR9 versus other TLRs. On D2 post-transplant, WT allografts subjected to long CIS had significantly increased TLR9 (2.3-fold), but not TLR4 or TLR7, expression compared to WT allografts subjected to short CIS. Endogenous donor-reactive memory T cell activation on D2 in allografts subjected to long CIS was markedly reduced in TLR9 KO allografts compared to WT allografts. Allografts subjected to long CIS from TLR9 KO donors and transplanted into untreated recipients showed no survival benefit compared to allografts from WT donors (MST 7.5 vs 7 days). Consistent with this, numbers of splenic donor-reactive T cells producing IFN-γ on D7 did not differ between recipients of TLR9 KO and WT allografts, further indicating that graft TLR9 deficiency does not alter the generation of de novo donor-reactive T cell responses. In contrast, allografts from TLR9 KO donors subjected to long CIS had significantly prolonged survival in recipients conditioned with CTLA-4Ig on D0 and +1 compared to high-risk WT allografts (MST 59 vs 28 days).

*Conclusions: IRI induced release of mtDNA and activation of TLR9 signaling represent novel targets for abrogating CTLA-4Ig resistant endogenous donor-reactive memory T cell activation within high-risk allografts.

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