*Purpose: Longer cold ischemic storage (CIS) time prior to transplant results in increased proliferation of endogenous donor-reactive memory CD8 T cells within complete MHC-mismatched cardiac allografts at 24 hrs after transplant and in enhanced effector functions to mediate CTLA-4Ig-resistant rejection of the high-risk allografts. This increased memory CD8 T cell proliferation within high-risk allografts requires CD4 T cell help via CD40-CD154 interactions with graft dendritic cell (DC)s to produce IL-12p40 homodimer (p40 HD) that is a key factor for driving the memory CD8 T cell proliferation.

*Methods: Here, we investigated how p40HD stimulate endogenous memory CD8 T cell proliferation within high-risk allografts.

*Results: When testing the impact of p40HD on cytokine production in the allografts, p40 HD injection into recipients of low-risk allografts induced marked increases in graft IL-15. Consistent with this, the longer CIS time increased mRNA expression of IL2Rα (CD25), IL2Rβ(CD122) and IL15Rα in purified CD8 T cells infiltrating the allografts on day 2 post-transplant and markedly increased IL-15 protein in the allografts. Blocking IL-15 signaling with anti-CD122 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 low-risk allografts. Moreover, anti-CD122 mAb inhibited p40HD induced memory CD8 T cell proliferation within low-risk allografts. To test the source of IL-15, we transplanted diphtheria toxin (DT) receptor-CD11c transgenic mice subjected to 8hr CIS into A/J recipients. Depletion of CD11c+ cells abrogated the p40HD-induced endogenous memory CD8 T cell proliferation and IL-15 production within low-risk allograft. To further test the role of allograft IL-15 signaling, we used B6.IL15Rα^-/- as donors. 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.

*Conclusions: These results indicate that p40 HD produced by graft DCs stimulate graft DCs 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 attenuate CTLA-4Ig resistant rejection mediated by these donor reactive T cells.

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