*Purpose: Increased ischemia-reperfusion graft inflammation provokes activation of endogenous donor-reactive memory CD8 T cells within complete MHC-mismatched cardiac allografts at 24 hrs after graft reperfusion to express effector functions that mediate CTLA-4Ig-resistant graft rejection.

*Methods: The current study tested the role of TLR signaling for the innate immune induction of this endogenous donor-reactive memory CD8 T cell response in “high-risk” cardiac allografts subjected to prolonged cold-ischemic storage (CIS) prior to transplant.

*Results: High-risk TLR4^-/- allografts did not compromise memory CD8 T cell activation or CTLA-4Ig resistant rejection observed in wild type (WT) allografts. When RNA from WT allografts subjected to 0.5 vs. 8 hrs CIS was interrogated 48 hrs after transplant, expression of TLR9, but not TLR4 or TLR7, was increased >3-fold in the high-risk grafts. In contrast to WT C57BL/6 allografts, unsensitized BALB/c recipient endogenous memory CD8 T cell activation within high-risk B6.TLR9^-/- allografts was virtually absent and recipient conditioning with CTLA-4Ig prolonged allograft survival to a median of 44 days vs. 27 days for high-risk WT allografts. High-risk WT B6 and B6.TLR9-deficient allografts were rejected at the same time (day 8-9) in untreated BALB/c recipients and numbers of donor-reactive T cells producing IFN-γ were the same in the spleens of both recipients, indicating that the presence or absence of TLR9 did not impact the activation of the de novo donor-reactive response. Reciprocal wild type and TLR9^-/- radiation-induced/bone marrow reconstituted chimeras were generated for use as donors of cardiac allografts subjected to 8 hrs CIS prior to transplant. High risk cardiac allografts from TLR9 bone marrow to WT chimeras had a median survival time of 28.5 days in CTLA-4Ig conditioned BALB/c recipients (CTLA-4Ig resistant rejection is maintained when TLR9-deficiency is restricted to graft bone marrow-derived cells) whereas allografts from WT bone marrow reconstituted TLR9^-/- recipient chimeras survived with a median of 110 days (CTLA-4Ig resistant rejection is abrogated when TLR9-deficiency is restricted to graft non-bone marrow-derived cells).

*Conclusions: These results indicate that TLR9 signaling on parenchymal cells in allografts subjected to prolonged CIS prior to transplant is required for endogenous donor-reactive memory CD8 T cell activation to mediate CTLA-4 resistant rejection.

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