Hedgehog-Induced Activation of Alloimmune T Cells During Ischemia Reperfusion Injury

缺血再灌注损伤（IRI）是影响实体器官移植物的病理过程， 当移植的器官经受长时间的血流中断时， 外科植入IRI经常发生在已故的捐赠者中，并易患慢性抗体- 介导的排斥反应（CABMR），降低移植物存活率和恶化的患者结果。这种情况没有 有效的医学治疗，特别是与经历更高风险的资深移植接受者相关。 死亡供体移植的发生率很高，因此有发生IRI及其相关并发症的风险。 认识到IRI对退伍军人患者造成的重大终身健康负担和成本， 最近的联邦授权已经建立了机制， 系统地改善对实体器官移植，特别是肾移植退伍军人接受者的护理。这 任务将增加移植VA中心的数量，研究所系统为基础的变化，以改善 手术前和手术后获得护理，并促进基础研究，以提高同种异体移植物的存活率。 在这种情况下，我们提交了这个移植免疫学应用程序，以探索刺猬（Hh）的作用。 CABMR信号，实体器官移植中的一种IRI相关并发症。补体（C '）是 参与宿主防御的免疫蛋白，在免疫过程中在内皮细胞（EC）上被病理激活， IRI是我们发现的一种选择性扩增外周辅助性T细胞（TPH细胞）的过程， 专门提供B细胞帮助的细胞亚群。Hh信号是一种广泛研究的创伤调节途径 通过引发血管细胞增殖来愈合。我们推断Hh配体是由C '-损伤的EC释放的， 作为自体伤口修复的信号，但同时这些相同的配体可以扩增， 同种免疫TPH细胞，继续促进同种抗体反应和CABMR相关病理。 为了验证这一观点，我们使用了体外和体内的人源化模型，并使用了前瞻性收集的 患者样本，以增加我们发现的临床相关性。我们的数据显示，IRI处理的EC 以C '依赖的方式产生Hh配体，选择性激活T外周辅助细胞（TPH）， 新描述的专门提供B细胞帮助的T细胞亚群。在机制上，Hh诱导ZFYVE 21， 我们发现了一种新的Rab 5效应物，在TPH细胞中引发Akt介导的NRLP 3炎性小体。这导致 IL-18释放和IL-18介导的IL-18 R1 +TPH细胞的扩增促进CABMR样病理 包括血管炎症、alloAb产生和血管病变。这些令人兴奋的初步数据地址 这是一个重要和普遍的临床问题，是最近退伍军人事务部联邦倡议的重点领域。 根据这些数据，我们假设在IRI期间由C '-损伤的EC释放的Hedgehog配体激活 CD 4 + TPH细胞以引发CABMR相关的病理。我们将在2个非- 相互依存的目标。在目标1中，我们将采用生物化学和功能测定来确定机制， 该Hh激动激活ZFYVE 21-Akt-Casp 1轴以引起TPH的炎性小体依赖性扩增 细胞我们提出了包括磷酸化蛋白质组学分析在内的尖端方法，以促进 的发现在目标2中，我们将使用2个人源化细胞在体内测试ZFYVE 21-Akt-Casp 1信号传导的相关性 CABMR小鼠模型，我们将前瞻性地使用 收集的患者样本。我们的研究探索刺猬信号作为一种新的途径，可能是可行的 在临床环境中使用最近FDA批准的药物进行调节，以阻断与虹膜相关的 如实体器官移植中的CABMR等并发症。因此，这些研究的结果可能是相关的， 用于提高有IRI及其相关并发症风险的退伍军人患者的同种异体移植物存活率。

Ischemia reperfusion injury (IRI) is a pathological process affecting solid organ allografts and occurs when a transplanted organ subjected to prolonged disruption of blood flow undergoes tissue damage following surgical implantation. IRI frequently occurs among deceased donors and predisposes to chronic antibody- mediated rejection (CABMR), decreased graft survival, and worsened patient outcomes. This condition has no effective medical treatments and is especially relevant to veteran transplant recipients who undergo higher rates of deceased donor transplants and are thus at risk for developing IRI and its related complications. In recognition of the significant lifetime health burden and costs incurred by IRI to veteran patients and to the VA Healthcare System, respectively, recent federal mandates have established mechanisms to systemically improve care for veteran recipients of solid organ transplants, in particular renal transplants. This mandate will increase the number of transplanting VA centers, institute systems-based changes to improve pre- and post-operative access to care, and promote basic research to improve allograft survival. In this context, we submit this transplant immunology application to explore a role for Hedgehog (Hh) signaling in CABMR, an IRI-associated complication in solid organ transplantation. Complement (C') are immune proteins involved in host defense that are pathologically activated on endothelial cells (ECs) during IRI, a process we have found selectively expands T peripheral helper (TPH cells), a recently discovered CD4+ T cell subset specialized in provision of B cell help. Hh signaling is a widely studied pathway regulating wound healing by eliciting vascular cell proliferation. We reasoned that Hh ligands are released by C'-injured ECs during IRI as a signal for autologous wound repair, but concurrently these same ligands could expand alloimmune TPH cells that go on to promote alloantibody responses and CABMR-related pathologies. To test this notion, we used humanized models in vitro and in vivo, and we used prospectively collected patient samples to increase the clinical relevance of our findings. Our data showed that IRI-treated ECs produced Hh ligands in a C'-dependent manner that selectively activated T peripheral helper (TPH) cells, a newly described T cell subset specialized in provision of B cell help. Mechanistically, Hh induced ZFYVE21, a novel Rab5 effector we discovered, to elicit Akt-mediated NRLP3 inflammasomes in TPH cells. This resulted in IL-18 release and IL-18-mediated expansion of IL-18R1+TPH cells that promoted CABMR-like pathologies including vascular inflammation, alloAb production, and vasculopathy. These exciting preliminary data address an important and prevalent clinical problem that is a focus area of recent federal initiatives at the VA. From these data, we hypothesize that Hedgehog ligands released by C'-injured EC during IRI activate CD4+ TPH cells to elicit CABMR-associated pathologies. We will explore this hypothesis in 2 non- interdependent Aims. In Aim 1, we will employ biochemical and functional assays to identify mechanisms(s) by which Hh agonism activates a ZFYVE21-Akt-Casp1 axis to elicit inflammasome-dependent expansion of TPH cells. We propose cutting edge approaches including phosphoproteomic analyses to facilitate mechanistic discovery. In Aim 2, we will test the relevance of ZFYVE21-Akt-Casp1 signaling in vivo using 2 humanized mouse models of CABMR, and we will test the clinical relevance of lead molecule(s) using prospectively collected patient specimens. Our studies explore Hedgehog signaling as a new pathway that may be feasibly modulated in clinical settings with recently FDA-approved agents to putatively block IRI-associated complications like CABMR in solid organ transplantation. As such, findings from these studies may be relevant for improving allograft survival in veteran patients who are at risk for IRI and its associated complications.