*Purpose: Increasing the duration of cold ischemic storage (CIS) from 0.5 to 8 hrs prior to transplant induces the increased proliferation of endogenous donor-reactive CD4 and CD8 T cells in mice cardiac allografts within 24 hours of reperfusion and the effector functions of the activated memory CD8 T cells to directly mediate CTLA-4Ig-resistant rejection of the higher risk allografts. Our previous studies indicated this increased memory CD8 T cell proliferation 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. The goal of the current study was to test if p40HD directly or indirectly stimulate this CD8 T cell proliferation within allografts subjected to 8hrs CIS and to investigate the mechanisms of p40 HD driven CD8 T cell proliferation.

*Methods: To test this, we use a heterotopic cardiac model which transplanted complete MHC mismatched heart allograft subjected to various time of CIS prior to transplant.

*Results: Endogenous memory CD8 T cells within highly ischemic allografts expressed both IL12Rβ1 and β2. However, most proliferating CD8 T cells did not express IL12Rβ1 needed for p40 HD mediated signaling. Moreover, endogenous memory CD8 T cells isolated from allografts did not proliferate ex vivo when cultured with p40 HD. These results suggested p40 HD may provoke memory CD8 T cell proliferation within the higher risk allografts by indirectly binding to IL-12Rβ1 expressing cells leading us to investigate other candidate factors to drive this memory CD8 T cell proliferation. qPCR analysis indicated increased mRNA expression of IL2Rα (CD25), β(CD122) and IL15Rα in infiltrating CD8 T cells purified from allografts subjected to prolonged vs. minimal CIS on day 2 post-transplant. ELISA indicated longer CIS time markedly increased IL-12p40 and IL-15 protein in the allografts and these increases were dependent on recipient CD4 T cells. Blocking of p40 or IL-15 signaling with anti-p40 or anti-CD122 mAb inhibited endogenous memory CD8 T cell proliferation within high-risk cardiac allografts in CTLA-4Ig conditioned recipients and extended the survival of the high-risk allografts from days 16-23 to days 40-72 (anti-p40 mAb) and days 24-97 (anti-CD122 mAb).

*Conclusions: These results suggest that p40 HD produced by graft DCs stimulated the production of proliferative cytokines such as IL-15, which directdly stimulates endogenous donor-reactive memory CD8 T cell proliferation within allografts subjected to 8 hours CIS.

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