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

缺血再灌注损伤(IRI)是影响同种异体器官移植的一种病理过程。 当移植器官遭受长期血流中断时， 外科植入术。IRI经常发生在已故捐赠者中，并易患慢性抗体- 介导性排斥反应(CABMR)，移植物存活率降低，患者预后恶化。这种情况没有 有效的医疗治疗，尤其适用于经历了更高 死亡的供者移植的比率，因此有发生IRI及其相关并发症的风险。 认识到IRI给退伍军人患者带来的巨大终身健康负担和费用 分别到退伍军人管理局医疗保健系统，最近的联邦任务已经建立了机制，以 系统地改善对实体器官移植，特别是肾移植退伍军人的护理。这 任务规定将增加退伍军人管理局移植中心的数量，以制度变革为基础加以改进 术前和术后获得护理，并促进基础研究，以提高同种异体移植物的存活率。 在此背景下，我们提交这项移植免疫学申请，以探索Hedgehog(HH)的作用 CABMR中的信号传递，实体器官移植中IRI相关的并发症。补语(C‘)是 参与宿主防御的免疫蛋白在内皮细胞(ECs)上被病理激活 IRI，我们发现的一种过程，选择性地扩大T外周辅助细胞(TPH细胞)，这是最近发现的一种CD4T 专门提供B细胞帮助的细胞亚群。HH信号是一种被广泛研究的调节创伤的途径 通过诱导血管细胞增殖来愈合。我们推测HH配体是由C‘损伤的内皮细胞释放的 在IRI期间，作为自体伤口修复的信号，但同时这些相同的配体可以扩大 同种异体免疫的TPH细胞继续促进同种异体抗体反应和CABMR相关的病理。 为了验证这一概念，我们使用了体外和体内的人性化模型，并使用了前瞻性收集的 患者样本，以增加我们的发现的临床相关性。我们的数据显示，接受IRI治疗的内皮细胞 以C‘依赖的方式产生HH配体，选择性激活T外周辅助(TPH)细胞，a 新描述的T细胞亚群专门提供B细胞帮助。从机理上讲，HH诱导的ZFYVE21，a 我们发现了新的Rab5效应器，可以在TPH细胞中诱导Akt介导的NRLP3炎症体。这导致了 IL-18释放及IL-18介导的IL-18R1TPH细胞的扩增促进CABMR样病变 包括血管炎症、同种异体抗体的产生和血管病变。这些令人兴奋的初步数据地址 一个重要而普遍的临床问题，是退伍军人管理局最近联邦倡议的重点领域。 根据这些数据，我们推测，在IRI激活过程中，C‘损伤的EC释放了Hedgehog配体 CD4TPH细胞诱导CABMR相关病理改变。我们将在两个非 相互依存的目标。在目标1中，我们将使用生化和功能分析来确定机制(S)，方法是 哪种HH激动剂激活ZFYVE21-Akt-CASP1轴以诱导炎症依赖的TPH扩张 细胞。我们提出了尖端的方法，包括磷酸蛋白质组分析，以促进机械 发现号。在目标2中，我们将使用2人源化的方法来测试ZFYVE21-Akt-CASP1信号在体内的相关性 小鼠CABMR模型，我们将前瞻性地检验铅分子(S)的临床相关性 收集病人样本。我们的研究将Hedgehog信号作为一种可能可行的新途径。 在临床环境中使用最近FDA批准的药物进行调节，以推测阻止IRI相关的药物 实体器官移植中的CABMR等并发症。因此，这些研究的发现可能是相关的 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.