Intestinal surveillance by intraepithelial lymphocytes

项目摘要 肠上皮内淋巴细胞(IEL)是构成粘膜免疫的重要分支之一的T细胞 系统，提供了针对病原体的第一道免疫防御系统，可能还提供了针对上皮癌的免疫防御系统 它们位于肠腔和身体核心之间的关键界面上。始终如一， IELs调节失调导致粘膜屏障完整性丧失，对肠道感染和 炎症性肠病(IBD)和癌症。近几年来，一些机制控制着 针对肠道病原体的IEL群体的发展和功能已被阐明，包括工作 在本提案的第一个供资周期内制定。除了它们在免疫监测中的作用外 肠道感染，最近的数据表明γδT细胞相关基因标志是最有利的预后 所有癌症类型的因素，包括结直肠癌(CRC)。结直肠癌是世界上第二致命的癌症。 美国，每年影响超过14万人，在美国造成大约5万人死亡。高达20%的 IBD患者发生结直肠癌，尽管大多数结直肠癌发生在没有潜在炎症的患者身上。 在常见的结直肠癌和IBD诱导的结直肠癌中，肿瘤引发的炎症都会触发EC损伤 导致微生物入侵，从而维持炎症，进而推动癌症的进展。因此， IEL对粘膜屏障的监测在结直肠癌中可能起到双重作用：(I)防止结直肠癌进展和早期 通过免疫细胞介导的杀伤或额外的抗肿瘤反应进行传播；(Ii)促进结直肠癌 通过炎性细胞因子或免疫调节分子的进展和转移。基于现有的 关于小鼠和人类结直肠癌的文献，我们最近的工作，以及这里提供的大量初步数据，我们 假设γδ上皮细胞监测对肿瘤形成的调控至关重要。我们在这里展示了这一点。 在稳定状态下，大多数肠道γδIEL表达Vγ7或Vγ1 TCR和IEL标志，包括细胞毒性 机械设备。然而，在结肠炎相关(AOM+DSS)和突变相关(CDX2-APC)模型中， 结直肠癌进展与Vγ7或Vγ1+相对减少和表达γδIEL的积聚有关 Vγ6或Vγ4 TCR，产生IL-17并表达PD-1。在目标1中，我们将解决组织驻留的Vγ7+ 或Vγ1+γδIEL亚群在免疫监视上皮细胞中发挥作用，防止肿瘤的形成。在AIM 2，我们将表征在结直肠癌进展过程中积累并可能促进肿瘤生长的γδIEL亚群。 本文提出的研究将描述γδ开发早期和后期阶段的行为。 一种创新成像方法的组合。我们还将跟踪互动的EC和周围的IELs CRC使用一种新的小鼠模型来识别细胞伙伴和单细胞转录产物。可感应的 交叉遗传学将被用于靶向γδIEL的分化或功能，而γδIEL亚群将得到丰富 在结直肠癌的不同阶段，将使用缺乏特定V-伽马的新菌株进行靶向；这些小鼠品系将 使用补充性儿童权利公约办法，接受诺维菌素和感染模型。

Project Summary Intestinal intraepithelial lymphocytes (IELs) are T cells that form one of the key branches of the mucosal immune system, providing a first line of immune defense against pathogens and possibly against epithelial cancers due to their location at the critical interface between the intestinal lumen and the core of the body. Consistently, dysregulation of IELs leads to loss of mucosal barrier integrity, susceptibility to enteric infections and inflammatory bowel diseases (IBD) and cancer. In recent years some of the mechanisms controlling the development and function of IEL populations against enteric pathogens have been elucidated, including work developed during the first funding cycle of this proposal. In addition to their role in immune surveillance against enteric infections, recent data suggest a γδ T cell-associated gene signature as the most favorable prognostic factor across cancer types, including colorectal cancer (CRC). CRC is the second most deadly cancer in the United States, affecting over 140,000 people each year, killing approximately 50,000 in the US. Up to 20% of IBD patients develop CRC, although the majority of CRCs develop in patients without underlying inflammation. In both the common forms of CRC and IBD-induced CRC tumor-elicited inflammation triggers EC damage resulting in microbial invasion, which sustains inflammation that in turn drives cancer progression. Therefore, IEL surveillance of the mucosal barrier may play dual roles in CRC: (i) prevention of CRC progression and early dissemination by immune cell-mediated killing or additional anti-tumor responses; (ii) promotion of CRC progression and metastasis through inflammatory cytokines or immune-regulatory molecules. Based on existing literature in murine and human CRC, our recent work, and extensive preliminary data presented here, we hypothesize that γδ IEL epithelial surveillance is crucial for the regulation of tumor formation. We show that at steady state, the majority of intestinal γδ IELs express Vγ7 or Vγ1 TCRs and IEL hallmarks including a cytotoxic machinery. However, in both colitis-associated (AOM+DSS) and mutation-associated (CDX2-APC) models, CRC progression was associated with relative reduction of Vγ7 or Vγ1+ and accumulation of γδ IELs expressing Vγ6 or Vγ4 TCRs, which produce IL-17 and express PD-1. In Aim 1, we will address whether tissue-resident Vγ7+ or Vγ1+ γδ IEL subsets play a role in immune surveillance of the epithelium, preventing tumor formation. In Aim 2, we will characterize γδ IEL subsets that accumulate during CRC progression and may facilitate tumor growth. Studies proposed here will characterize γδ IEL behavior during early and late stages of CRC development using a combination of innovative imaging approaches. We will also track interacting ECs and surrounding IELs during CRC using a novel mouse model to identify cellular partners and single cell transcriptomics. Inducible intersectional genetics will be used to target differentiation or function of γδ IELs, while γδ IEL subsets enriched in different stages of CRC will be targeted using novel strains lacking specific V-gamma; these murine lines will be subjected to gnotobiotic and infection models using complementary CRC approaches.