Intestinal surveillance by intraepithelial lymphocytes

项目摘要 肠上皮内淋巴细胞（IEL）是形成粘膜免疫应答的关键分支之一的T细胞， 系统，提供针对病原体和可能针对上皮癌的第一道免疫防御， 到它们在肠腔和身体核心之间的关键界面的位置。我们一贯认为， IEL的失调导致粘膜屏障完整性的丧失，对肠道感染的易感性， 炎症性肠病（IBD）和癌症。近年来，一些机制控制 已经阐明了IEL群体对肠道病原体的发展和功能，包括工作 在本提案的第一个供资周期制定的。除了它们在免疫监视中的作用外， 肠道感染，最近的数据表明γδ T细胞相关的基因特征是最有利的预后 包括结直肠癌（CRC）在内的癌症类型的因素。CRC是世界上第二大致命癌症。 美国，每年影响超过14万人，在美国造成约5万人死亡。高达20%的 IBD患者发展为CRC，尽管大多数CRC在没有潜在炎症的患者中发展。 在两种常见形式的CRC和IBD诱导的CRC中，肿瘤引起的炎症触发EC损伤 导致微生物入侵，从而维持炎症，进而推动癌症进展。因此，我们认为， 粘膜屏障的IEL监测可能在CRC中发挥双重作用：（i）预防CRC进展和早期 通过免疫细胞介导的杀伤或额外的抗肿瘤应答传播;（ii）促进CRC 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 在鼠和人CRC的文献中，我们最近的工作，以及这里提出的广泛的初步数据，我们 假设γδ IEL上皮监视对于肿瘤形成的调节是至关重要的。我们表明，在 稳态时，大多数肠道γδ IEL表达Vγ7或Vγ1 TCR和IEL标志，包括细胞毒性T细胞受体， 机械.然而，在结肠炎相关（AOM+DSS）和突变相关（CDX 2-APC）模型中， CRC进展与Vγ7或Vγ1+的相对减少和γδ IEL表达的积累相关。 Vγ6或Vγ4 TCR，其产生IL-17并表达PD-1。在目标1中，我们将解决组织驻留Vγ7+ 或Vγ1+ γδ IEL亚群在上皮的免疫监视中起作用，防止肿瘤形成。在Aim中 2，我们将描述在CRC进展过程中积累并可能促进肿瘤生长的γδ IEL亚群。 本文提出的研究将使用以下方法表征CRC发展早期和晚期阶段的γδ IEL行为： 创新成像方法的组合。我们还将跟踪相互作用的EC和周围的IEL， 使用新型小鼠模型鉴定细胞伴侣和单细胞转录组学的CRC。诱导型 交叉遗传学将用于靶向γδ IEL的分化或功能，而γδ IEL亚群富集 将使用缺乏特异性V-γ的新菌株靶向CRC的不同阶段;这些鼠系将 使用互补的CRC方法进行gnotobiotic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.