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

尽管肠道干细胞（ISCs）对胃肠道（GI）上皮维持的重要性， 以及炎症性肠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区室接合的潜力仍然知之甚少。GI 移植物抗宿主病（GVHD）是同种异体造血/骨髓移植的最大挑战之一 移植（allo-BMT），引起严重毒性，需要深度免疫抑制干预，和 限制了allo-BMT在潜在可固化条件下的更广泛使用。我们发现急性GVHD导致 Lgr 5 + ISCs和提供其上皮小生境的潘氏细胞的显著减少。虽然有限 了解免疫应答诱导ISC区室损伤的确切机制， 再生，这是不可能完全了解在胃肠道或如何免疫相关的并发症 细胞可能会影响特定的上皮成分，而无需检查三维（3-D）组织 肠的环境和免疫应答对ISC区室的接近性。我们寻求 了解免疫系统和ISC区室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 生物学可以被治疗性地操纵以用于临床干预。为了实现这些目标，我们 提出了一项研究建议，强调胃肠道内免疫效应物的三维成像， 小鼠和人免疫细胞募集至ISC区室的体内建模，以及在 实验移植模型我们还组建了一个多学科的合作者团队， 基础和转化免疫学，ISC生物学和先进的显微成像的前沿。我们 初步研究结果表明，ISC区室是同种异体T细胞入侵的第一个位点， BMT后的肠粘膜和因此GVHD的初始靶点。此外，我们还发现， 免疫细胞和肠道类器官的生物学特性可以用于模拟和机械地剖析这些过程。 使用三维共焦和双光子活体成像，我们已经开发出准确量化损失的能力 ISC的特异性侵袭，以及T细胞归巢配体的定位。 体内平衡和主动免疫期间。我们还开发了用于分析体内谱系的策略- ISC区室内的特异性基因表达。因此，本研究将检验淋巴细胞 ISC区室的募集和调节是移植后GI损伤的中心特征， 影响生理和病理粘膜反应，我们将评估调节 淋巴细胞归巢并促进受损上皮的恢复。这个项目将导致一个机械的 了解ISC区室和免疫系统之间的基本相互作用， 通过保护ISCs和增强再生，为GVHD和其他GI疾病的治疗提供了新的方向。

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.