Increased posttransplant ischemia reperfusion injury (IRI) prevents costimulatory blockade induced heart graft survival but mechanisms linking innate and adaptive immunity are poorly understood. Based on previous work indicating that complement participates in IRI and amplifies adaptive alloimmunity we tested the impact of C3 on CTLA4Ig induced allograft survival in recipients of heart grafts that underwent 8 h cold ischemia (CI). Whereas a single dose of CTLA4Ig prolonged survival of BALB/C hearts subjected to standard CI (30 min) in B6 recipients to >60 d (n=4), median survival time of BALB/c hearts subjected to 8h CI was 35 d (n=6, p<0.05) with histology showing mononuclear cell infiltrates consistent with acute cellular rejection. Remarkably, WT BALB/c hearts subjected to 8h CI survived >60 d upon transplantation into B6 C3KO recipients treated with CTLA4Ig (n=6, p<0.05). BALB/c C3KO grafts subjected to 8h CI rejected with similar kinetics to WT (n=6, p=NS). To assess mechanisms we quantified intragraft proinflammatory gene expression by RT-PCR 24h after transplant. Grafts exposed to 8h CI implanted in WT hosts showed 2-6x higher expression of TNFα, IL6, IL1β, and the chemokine CXCL1 compared to controls (n=3/grp, p<0.05). The absence of recipient C3 prevented the upregulation of all genes induced by the 8h CI (expression levels same as in WT recipients of standard CI grafts). To assess effects of prolonged CI on adaptive alloimmunity in the presence or absence of recipient C3, we quantified alloreactive T cells by ELISPOT and serum donor specific alloantibodies (DSA) by flow cytometry. Prolonged CI led to a 2 fold increase in allospecific IFN-g producing splenocytes (170/500k vs 68/500k, n=6, p<0.05). In contrast, anti-donor T cell immunity in C3KO recipients of 8 h CI grafts (71/500k) was similar to those in WT recipients of standard CI grafts. Whereas WT recipients of prolonged CI allografts developed high DSA titers by 28d posttransplant (Mean 1:10,000), the DSA titers in C3KO recipients were 10-100 fold lower (n=6, p<0.05). Together the data demonstrate that the effects of IRI on amplifying early inflammation, adaptive alloimmunity and late allograft failure are dependent upon recipient C3, and support the need for testing effects of early posttransplant complement blockade on graft survival in human recipients of allografts at risk for IRI.

CITATION INFORMATION: Chun N, Zhang M, Fairchild R, Heeger P. Ischemia Reperfusion Induced Murine Heart Transplant Rejection Is Complement Dependent. Am J Transplant. 2016;16 (suppl 3).

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