Anti-complement 5 antibody ameliorates antibody-mediated rejection after liver transplantation in rats

Abstract

Antibody-mediated rejection (AMR) remains a refractory rejection after donor-specific antibody (DSA)-positive or blood-type incompatible liver transplantation (LT), even in the era of pre-transplant rituximab desensitization. This is due to the lack of not only effective post-transplant treatments but also robust animal models to develop/validate new interventions. Orthotopic LT from male Dark Agouti (DA) to male Lewis (LEW) rats was used to develop a rat LT-AMR model. LEW were pre-sensitized by a preceding skin transplantation from DA 4-6 weeks before LT (<i>Group-PS</i>), while sham procedure was performed in non-sensitized controls (<i>Group-NS</i>). Tacrolimus was daily administered until post-transplant day (PTD)-7 or sacrifice to suppress cellular rejections. Using this model, we validated the efficacy of anti-C5 antibody (Anti-C5) for LT-AMR. <i>Group-PS+Anti-C5</i> received Anti-C5 intravenously on PTD-0 and -3. <i>Group-PS</i> showed increased anti-donor (DA) antibody-titers (<i>P <</i>0.001) and more C4d deposition in transplanted livers than in <i>Group-NS</i> (<i>P <</i>0.001). Alanine aminotransferase (ALT), alkaline phosphatase (ALP), total bile acid (TBA), and total bilirubin (T-Bil) were all significantly higher in <i>Group-PS</i> than in <i>Group-NS</i> (all <i>P <</i>0.01). Thrombocytopenia (<i>P <</i>0.01), coagulopathies (PT-INR, <i>P</i> =0.04), and histopathological deterioration (C4d+h-score, <i>P <</i>0.001) were also confirmed in <i>Group-PS</i>. Anti-C5 administration significantly lowered anti-DA IgG (<i>P <</i>0.05), resulting in decreased ALP, TBA, and T-Bil on PTD-7 than in Group-PS (all <i>P <</i>0.01). Histopathological improvement was also confirmed on PTD-1, -3, and -7 (all <i>P <</i>0.001). Of the 9,543 genes analyzed by RNA sequencing, 575 genes were upregulated in LT-AMR (<i>Group-PS vs. Group-NS</i>). Of these, 6 were directly associated with the complement cascades. In particular, <i>Ptx3, Tfpi2, and C1qtnf6</i> were specific to the classical pathway. Volcano plot analysis identified 22 genes that were downregulated by Anti-C5 treatment (<i>Group-PS+Anti-C5 vs. Group-PS</i>). Of these, Anti-C5 significantly down-regulated <i>Nfkb2, Ripk2, Birc3</i>, and <i>Map3k1</i>, the key genes that were amplified in LT-AMR. Notably, just two doses of Anti-C5 only on PTD-0 and -3 significantly improved biliary injury and liver fibrosis up to PTD-100, leading to better long-term animal survival (<i>P</i> =0.02). We newly developed a rat model of LT-AMR that meets all the Banff diagnostic criteria and demonstrated the efficacy of Anti-C5 antibody for LT-AMR.