Notch Signaling in Alloimmunity

摘要 同种异体T细胞对外来宿主抗原的应答介导了移植物抗宿主病， 异基因造血细胞移植（allo-HCT）的严重并发症。在此过程中， 我们的提案定义了Notch信号在调节致病性同种异体反应性T细胞中的关键作用 其在多种同种异体HCT小鼠模型中介导移植物抗宿主病（GVHD）。Notch抑制 供体T细胞导致了对GVHD发病率和死亡率的长期保护。使用单克隆抗体， 我们确定了T细胞中Notch 1/2受体和Delta-like 1/4（Dll 1/4）Notch配体在T细胞中的关键作用。 以Notch 1和Dll 4为优势效应。出现了具有短疗程抗体的Dll1/4阻断 作为靶向Notch信号传导的最有前途的策略，同时避免泛Notch的全身副作用 抑制作用我们最近发现了T细胞同种异体免疫中Notch调控的几个显著特征 需要进一步调查首先，我们鉴定了特化的抗辐射基质细胞， 用Ccl19-Cre转基因作为Notch配体在次级淋巴器官中的关键来源， GVHD的发生。这些发现揭示了成纤维基质细胞亚群在GVHD中的核心作用。第二、 在allo-HCT后数天内短期抑制Delta-like Notch配体是赋予长期 在多个小鼠模型中对GVHD的长期保护。在这个早期的时间窗口内，Notch诱导了独特的 在同种异体抗原特异性T细胞活化过程中的转录效应， 他们的区别。第三，我们研究了非人灵长类动物allo-HCT模型中的Notch配体抑制， 模仿人体移植单剂抗DLL4抗体具有显着的单药活性， 预防GVHD，显示出从小鼠到非人灵长类的Notch信号传导的高度保守效应。在 在这两种模型中，我们观察到肠道中调节性T细胞与常规T细胞的比例增加， 保护免受肠道GVHD，急性GVHD的最危险成分。我们假设 同种异体抗原特异性T细胞参与与成纤维细胞基质细胞特化亚群的早期相互作用 表达Delta样Notch配体，诱导T细胞中Notch驱动的致病和肠道归巢程序 促进GVHD为了探讨这一假说，我们将鉴定成纤维细胞间质细胞的单个亚群， 在allo-HCT后早期将Dll 1和/或Dll 4 Notch配体呈递给供体来源的T细胞的细胞，绘制了 支持同种异体反应性T细胞中Notch激活的解剖部位，并定义免疫- 介导对次级淋巴器官中基质网络随后完整性的损伤。此外，本发明还提供了一种方法， 我们将确定抑制Notch缺陷型T细胞在肠道中积累的机制，从而防止 肠GVHD，并研究同种异体反应性T细胞中Notch信号传导的早期转录作用。 这些研究将为同种免疫的调节带来新的见解，并可能导致发展 新的方法来限制同种异体移植后T细胞反应性的破坏性后果。

ABSTRACT Allogeneic T cell responses against foreign host antigens mediate graft-versus-host disease, the most serious complication of allogeneic hematopoietic cell transplantation (allo-HCT). During the course of this proposal we defined a critical role for Notch signaling in the regulation of pathogenic alloreactive T cells that mediate graft-versus-host disease (GVHD) in multiple mouse models of allo-HCT. Notch inhibition in donor T cells led to long-term protection from GVHD morbidity and mortality. Using monoclonal antibodies, we identified a critical role for Notch1/2 receptors in T cells and Delta-like1/4 (Dll1/4) Notch ligands in the host, with dominant effects of Notch1 and Dll4. Dll1/4 blockade with a short course of antibodies emerged as the most promising strategy to target Notch signaling while avoiding systemic side effects of pan-Notch inhibition. We recently uncovered several remarkable features of Notch regulation in T cell alloimmunity that warrant further investigation. First, we identified specialized radioresistant stromal cells lineage-traced with a Ccl19-Cre transgene as the critical source of Notch ligands in secondary lymphoid organs at the onset of GVHD. These findings uncover a central role for fibroblastic stromal cell subsets in GVHD. Second, short-term inhibition of Delta-like Notch ligands within days after allo-HCT was essential to confer long- term protection from GVHD in multiple mouse models. Within this early time window, Notch induced unique transcriptional effects during the activation of alloantigen-specific T cells that impacted selected aspects of their differentiation. Third, we studied Notch ligand inhibition in a non-human primate allo-HCT model that mimics human transplantation. A single dose of anti-DLL4 antibodies had marked single agent activity to prevent GVHD, showing highly conserved effects of Notch signaling from mice to non-human primates. In both models, we observed an increased ratio of regulatory to conventional T cells in the gut and striking protection from intestinal GVHD, the most dangerous component of acute GVHD. We hypothesize that alloantigen-specific T cells engage in early interactions with specialized subsets of fibroblastic stromal cells expressing Delta-like Notch ligands, inducing a Notch-driven pathogenic and gut-homing program in T cells that promotes GVHD. To explore this hypothesis, we will identify individual subsets of fibroblastic stromal cells that present Dll1 and/or Dll4 Notch ligands to donor-derived T cells early after allo-HCT, map the anatomical sites that support Notch activation in alloreactive T cells, and define the impact of immune- mediate injury on the subsequent integrity of stromal networks in secondary lymphoid organs. In addition, we will identify mechanisms that blunt the accumulation of Notch-deficient T cells in the gut, thus preventing intestinal GVHD, and investigate the early transcriptional effects of Notch signaling in alloreactive T cells. These studies will bring novel insights into the regulation of alloimmunity and might lead to the development of new approaches to limit damaging consequences of T cell reactivity after allogeneic transplantation.