*Purpose: We have reported that longer duration of cold ischemic storage (CIS) prior to transplant resulted in increased proliferation of endogenous donor-reactive CD8 T cells within complete MHC-mismatched cardiac allografts at 24 hrs after transplant and in increased expression of their effector functions to mediate CTLA-4Ig-resistant rejection of the high-risk allografts. This increased memory CD8 T cell activation within high-risk allografts requires CD4 T cell help via CD40-CD154 interactions with graft dendritic cells to induce IL-12p40 homodimer (p40 HD) production that is required to drive the memory CD8 T cell proliferation.

*Methods: In this study, we investigated how p40HD stimulate endogenous memory CD8 T cell proliferation within allografts subjected to 8hrs CIS.

*Results: The longer CIS time prior to transplant increased mRNA expression of IL2Rα (CD25), IL2Rβ (CD122) and IL15Rα in isolated CD8 T cells infiltrating the allografts on day 2 post-transplant and markedly increased IL-15 protein in lysate of the allografts. Blocking IL-15 signaling with anti-CD122 mAb, but not IL-2 signaling with anti-CD25 mAb, inhibited endogenous memory CD8 T cell proliferation within high-risk allografts at 48 hrs after transplant and prolonged allograft survival in CTLA-4Ig conditioned recipients to that observed in CTLA-4Ig conditioned recipients of heart allografts subjected to minimal (30 min) CIS (Mean Survival Time 53 days vs. 18 days in CTLA-4Ig conditioned recipients treated with control IgG). When testing the impact of p40HD on cytokine production in the allografts, p40 HD administration increased IL-15 mRNA expression and protein within allograft subjected to 30 min CIS. To test the source of IL-15/IL-15Rα transpresentation on CD8 T cell proliferation within high-risk allografts, we transplanted B6.IL15Rα^-/- allografts subjected to 8 hr CIS into Balb/c recipients. Graft deficiency of IL-15Rα decreased the endogenous memory CD8 T cell proliferation within allografts at 48 hrs post-transplant and extended survival of grafts in CTLA-4Ig conditioned recipients to MST of 69 days.

*Conclusions: These results indicate that p40 HD produced by graft DCs stimulate graft cells to produce IL-15 that directly drives endogenous donor-reactive memory CD8 T cell proliferation to mediate CTLA-4 resistant rejection. The IL-15 mediated regulation of endogenous memory CD8 T cell may provide a new strategy to prevent or attenuate CTLA-4Ig resistant rejection.

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