Allograft TLR9 is required for endogenous donor-reactive memory CD8 T cell activation to mediate CTLA-4Ig resistant rejection of high-risk cardiac allografts

Abstract Organ cold ischemic storage (CIS) is inherent to deceased-donor solid organ transplantation and CIS time correlates with poorer transplant outcomes. Prolonged CIS enhances ischemia-reperfusion injury (IRI) following graft revascularization, generating an inflammatory environment that promotes endogenous donor-reactive memory T cell activation leading to CTLA-4Ig costimulatory blockade-resistant rejection. Using a murine heterotopic heart transplant model, we tested the impact of prolonged (8hr) versus minimal (0.5hr) CIS on cell-free mitochondrial DNA (cf-mtDNA) release and activation of the cf-mtDNA sensor TLR9 to understand their contributions to early post-transplant donor-reactive endogenous memory T cell activation and CTLA-4Ig resistant rejection. Plasma cf-mtDNA was significantly elevated in 8hr versus 0.5hr CIS allograft recipients as early as 12hr post-transplant. Flow cytometry analysis of graft infiltrating leukocytes showed reduced memory CD4 and CD8 T cell proliferation on D2 following 8hr CIS in TLR9KO compared to WT allografts. In contrast, graft TLR9KO did not impact the de novo donor-reactive T cell response. 8hr CIS TLR9KO and WT allografts had similar survival in untreated recipients (MST 7.5 vs 7 days) and there was no difference in IFN-γ producing donor-reactive T cells in recipient spleens on D7. When recipients of allografts subjected to 8hr CIS were conditioned with CTLA-4Ig to inhibit de novo T cell priming, graft TLR9KO significantly prolonged graft survival compared to WT (MST 59 vs 28 days). These findings indicate that IRI-induced cf-mtDNA release and TLR9 activation are novel targets for abrogating CTLA-4Ig resistant donor-reactive memory T cell activation within high-risk allografts. This work was supported by NIH grants R01AI40459, T32AI089474, and T32GM007250.