T cell invasion of the stem cell compartment during immune-mediated GI damage

尽管肠干细胞(ISCs)对胃肠道(GI)上皮的维持很重要 以及炎症性肠道疾病和免疫介导的胃肠道损伤的临床意义， 免疫系统直接与ISC隔室接触的潜力仍然知之甚少。美国退伍军人协会 移植物抗宿主病(GVHD)是异基因造血/骨髓面临的最大挑战之一 移植(allo-BMT)，造成严重毒性，需要深度免疫抑制干预，以及 限制allo-BMT在潜在可治愈条件下的更广泛使用。我们发现急性移植物抗宿主病先导 导致Lgr5+ISCs和Paneth细胞显著减少，为其提供上皮龛。在有限的时间里 了解免疫反应导致ISC间隔室损伤和 再生，不可能完全了解胃肠道的骨髓移植相关并发症或如何免疫 细胞可能会影响特定的上皮成分，而不检查三维(3-D)组织 肠道环境和免疫反应对ISC隔间的接近程度。我们寻求 了解免疫系统和ISC之间发生的基本相互作用， 这些相互作用是如何在造血移植的背景下进行的，以及潜在的 生物学可以在治疗上被操纵，用于临床干预。为了实现这些目标，我们有 开发了一项研究方案，强调胃肠道内免疫效应器的3D成像，机械性试验 小鼠和人类免疫细胞募集到ISC隔室的活体模型，并在体内验证 实验性移植模型。我们还组建了一个由多学科合作伙伴组成的团队， 基础免疫学和翻译免疫学、ISC生物学和高级显微成像的前沿。我们的 初步研究结果表明，ISC室是同种异体T细胞入侵的第一个部位 骨髓移植后的肠粘膜，因此是移植物抗宿主病的最初靶点。此外，我们还发现，共同文化 免疫细胞和肠道类器官可以用来模拟和机械地剖析这些过程。 使用三维共聚焦和双光子活体成像，我们已经开发了准确量化损失的能力 间充质干细胞的特异性侵袭，以及T细胞归巢配体的定位 动态平衡和主动免疫期间。我们还开发了分析体内血统的策略- 在ISC间隔内的特定基因表达。因此，这项研究将检验淋巴细胞 移植后胃肠道损伤的主要特征是ISC的募集和调节。 影响生理和病理黏膜反应，我们将评估调节方法 淋巴细胞归巢，促进受损上皮的修复。这个项目将导致一个机械化的 了解ISC间隔和免疫系统之间的基本相互作用，打开了一个 通过保护ISCs和增强再生来治疗GVHD和其他胃肠道疾病的新方向。

Despite the importance of intestinal stem cells (ISCs) for epithelial maintenance in the gastrointestinal (GI) tract and the clinical significance of inflammatory intestinal disorders and immune-mediated GI damage, the potential of the immune system to directly engage with the ISC compartment remains poorly understood. GI graft vs. host disease (GVHD) is one of the greatest challenges for allogeneic hematopoietic/bone marrow transplantation (allo-BMT), causing severe toxicity, necessitating deeply immunosuppressive interventions, and limiting the wider usage of allo-BMT for potentially curable conditions. We have found that acute GVHD leads to substantial reduction of Lgr5+ ISCs and the Paneth cells providing their epithelial niche. While there is limited understanding of the precise mechanisms by which immune responses induce ISC compartment damage and regeneration, it is not possible to fully understand BMT-related complications in the GI tract or how immune cells may impact specific epithelial components without examining the three-dimensional (3-D) tissue environment of the intestines and the proximity of immune responses to the ISC compartment. We seek to understand the fundamental interactions occurring between the immune system and the ISC compartment, how these interactions are engaged in the context of hematopoietic transplantation, and how the underlying biology may be manipulated therapeutically for clinical intervention. To address these goals, we have developed a research proposal emphasizing 3-D imaging of immune effectors within GI tract, mechanistic ex vivo modeling of murine and human immune cell recruitment to the ISC compartment, and in vivo validation in experimental transplant models. We have also assembled a multi-disciplinary team of collaborators at the forefront of basic and translational immunology, ISC biology, and advanced microscopic imaging. Our preliminary findings indicate that the ISC compartment is the first site of allogeneic T cell invasion within the intestinal mucosa after BMT and thus the initial target of GVHD. Furthermore, we have found that co-cultures of immune cells and intestinal organoids can be used to model and mechanistically dissect these processes. Using 3-D confocal and 2-photon intravital imaging, we have developed the capacity to accurately quantify loss of ISCs, specific invasion of the ISC compartment, and localization of T cell homing ligands during homeostasis and during active immunity. We have also developed strategies for analysis of in vivo lineage- specific gene expression within the ISC compartment. This study will thus test the hypothesis that lymphocyte recruitment to and regulation of the ISC compartment are central features of GI damage post-transplant, impacting both physiologic and pathologic mucosal responses, and we will evaluate approaches to modulate lymphocyte homing and promote recovery of injured epithelium. This project will lead to a mechanistic understanding of fundamental interactions between the ISC compartment and the immune system, opening a new direction for treatment of GVHD and other GI disorders by protecting ISCs and augmenting regeneration.