Introduction: Memory T cells provide protection against recurrent pathogens, but memory T cells with donor-reactivity pose a major barrier to successful allograft transplant and tolerance induction. We have previously shown that increasing the duration of cold ischemic storage (CIS) from 0.5 to 8 hrs prior to transplant induces early infiltration of innate and adaptive leukocytes, the increased proliferation of endogenous donor-reactive CD4 and CD8 T cells in cardiac allografts within 48 hours of reperfusion and their effector functions to directly mediate CTLA-4Ig-resistant rejection of the higher risk allografts. In addition, this increased proliferation of endogenous memory CD8 T cells within the higher risk allografts requires memory CD4 T cell help via CD40-CD154 interactions with graft dendritic cells. However, whether recipient derived monocytes play in a role in early inflammatory responses and affect the endogenous memory CD8 T cell activation within the high risk graft have not been investigated.
Methods: To test the role of recipient monocytes in endogenous memory T cell activation in allografts, we transplanted complete MHC mismatched A/J allografts subjected to 8hrs CIS into chimeric B6 CD11b (diphtheria toxin receptor)DTR recipients and administered DT to deplete bone marrow derived CD11b+ cells prior to the transplant. The infiltrating cell proliferation was assessed on day 2 post-transplant following recipient pulsing with BrdU.
Results: Depletion of recipient CD11b+ cells inhibited the infiltration and proliferation of early graft infiltrating memory CD4 and CD8 T cell on day2 post-transplant and markedly extended survival of highly ischemic allografts in CTLA-4Ig conditioned recipients (MST 49 days vs. 24 days in CTLA-4Ig conditioned recipients treated without DT).While the expression of TLR9 but not TLR4 or TLR7 mRNA was increased in the highly ischemic allografts, its expression was decreased in the absence of recipient CD11b+ cells to the levels observed in allografts subjected to 30 min CIS. Furthermore, prolonged CIS induced increases in IL-1β and TNFα mRNA expression were also downregulated in the high-risk grafts from recipients depleted of CD11b+ cells. To further analyze the infiltrating recipient CD11b+ cell population, we transplanted A/J allograft into B6 CX3CR1^GFP/+:CCR2^RFP/+ reporter transgenic mice. The frequency of recipient derived CX3CR1-expressing inflammatory monocytes (Ly6C+ CD11b+) were increased within the allografts subjected to 8hrs vs 0.5hrs CIS on days 2 post-transplant.
Conclusion: These results suggest that graft infiltrating recipient inflammatory monocytes sustains early post-transplant inflammation induced by prolonged CIS and activate endogenous donor reactive memory CD8 T cells to mediate CTLA-4Ig resistant rejection.