Division of Hepato-Biliary-Pancreatic Surgery and Transplantation, Department of Surgery, Graduate School of Medicine, Kyoto University, Kyoto, Japan.
Department of Surgery , Division of Abdominal Transplantation, Stanford University School of Medicine, Stanford, CA, United States.
Front Immunol. 2023 Jun 16;14:1186653. doi: 10.3389/fimmu.2023.1186653. eCollection 2023.
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 (), while sham procedure was performed in non-sensitized controls (). 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. received Anti-C5 intravenously on PTD-0 and -3. showed increased anti-donor (DA) antibody-titers (0.001) and more C4d deposition in transplanted livers than in (0.001). Alanine aminotransferase (ALT), alkaline phosphatase (ALP), total bile acid (TBA), and total bilirubin (T-Bil) were all significantly higher in than in (all 0.01). Thrombocytopenia (0.01), coagulopathies (PT-INR, =0.04), and histopathological deterioration (C4d+h-score, 0.001) were also confirmed in . Anti-C5 administration significantly lowered anti-DA IgG (0.05), resulting in decreased ALP, TBA, and T-Bil on PTD-7 than in Group-PS (all 0.01). Histopathological improvement was also confirmed on PTD-1, -3, and -7 (all 0.001). Of the 9,543 genes analyzed by RNA sequencing, 575 genes were upregulated in LT-AMR (). Of these, 6 were directly associated with the complement cascades. In particular, were specific to the classical pathway. Volcano plot analysis identified 22 genes that were downregulated by Anti-C5 treatment (). Of these, Anti-C5 significantly down-regulated , and , 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 ( =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.
抗体介导的排斥反应（AMR）在供体特异性抗体（DSA）阳性或血型不相容肝移植（LT）后仍然是一种难治性排斥反应，即使在利妥昔单抗预移植脱敏时代也是如此。这是因为不仅缺乏有效的移植后治疗方法，而且缺乏强大的动物模型来开发/验证新的干预措施。我们使用正交 LT 从雄性 Dark Agouti（DA）到雄性 Lewis（LEW）大鼠来开发大鼠 LT-AMR 模型。在 LT 前 4-6 周，LEW 通过先前的来自 DA 的皮肤移植进行预致敏（），而非致敏对照（）中进行假手术。在 LT 后第 7 天（PTD）或处死之前，每天给予他克莫司以抑制细胞排斥反应。使用该模型，我们验证了抗 C5 抗体（Anti-C5）治疗 LT-AMR 的疗效。在 PTD-0 和 -3 时，给予 静脉内注射 Anti-C5。与对照组（）相比，显示出更高的抗供体（DA）抗体滴度（0.001）和移植肝脏中更多的 C4d 沉积（0.001）。丙氨酸氨基转移酶（ALT）、碱性磷酸酶（ALP）、总胆汁酸（TBA）和总胆红素（T-Bil）在 中均显著高于对照组（均为 0.01）。血小板减少症（0.01）、凝血障碍（PT-INR，0.04）和组织病理学恶化（C4d+h-score，0.001）也在 中得到证实。Anti-C5 给药显著降低抗-DA IgG（0.05），导致 PTD-7 时的 ALP、TBA 和 T-Bil 低于 PS 组（均为 0.01）。在 PTD-1、-3 和 -7 时也证实了组织病理学改善（均为 0.001）。在通过 RNA 测序分析的 9543 个基因中，575 个基因在 LT-AMR 中上调（）。其中，有 6 个基因与补体级联直接相关。特别是，有 3 个基因与经典途径有关。火山图分析确定了 22 个被 Anti-C5 治疗下调的基因（）。其中，Anti-C5 显著下调了和，这两个基因在 LT-AMR 中被放大。值得注意的是，仅在 PTD-0 和 -3 时给予两次 Anti-C5 抗体治疗即可显著改善胆管损伤和肝纤维化，直至 PTD-100，从而提高长期动物生存率（0.02）。我们新开发了一种符合所有 Banff 诊断标准的 LT-AMR 大鼠模型，并证明了 Anti-C5 抗体治疗 LT-AMR 的疗效。
