Aging lymphocytes: senescence programs in lymphocytes relevant to human inflammatory bowel disease

项目摘要/摘要 Foxp3+CD4+细胞在克罗恩病(CD)患者发炎的粘膜内扩张；然而 持续的肠道炎症表明这些CD相关的CD4+FOXP3+细胞缺乏典型的调节 T细胞(Treg)抑制功能。FOXP3+Treg衰老的功能和治疗意义 细胞是未知的。这个应用程序的长期目标是剖析驱动 肠道炎症时CD4+FOXP3+细胞的分化和功能因此， 这项建议的总体目标是确定导致FOXP3+细胞衰老的机制 胃肠道炎症和测试治疗机会，以恢复调节功能。中心假设是 CD相关的FOXP3+细胞代表衰老状态，并有助于肠道炎症。此外， 这种新的细胞表型(衰老的FOXP3+细胞，SnFOXP3)代表了一个潜在的治疗靶点。这 假设是通过对椎板中的CD4+FOXP3+细胞进行单细胞转录分析而形成的 由申请人的实验室制作的CD患者的专有材料。其基本原理是定义这些机制 在炎症环境中对SnFOXP3细胞的发育和功能至关重要的将发现靶点 Treg指导的治疗，包括用于过继细胞治疗试验的人类Treg的先进工程。 在强劲的初步数据的指导下，这一假设将通过追求三个具体目标来检验：1)为什么 Foxp3+细胞在没有BMI1的情况下衰老；2)为什么在克罗恩病中FOXP3+细胞衰老 疾病？；以及3)为什么SnFOXP3细胞是促炎细胞？在第一个目标下，申请者将利用 在申请人手中已被确定为可行的表观遗传学分析，以建立BMI1 在FOXP3+细胞中抑制ETS1调节的衰老程序。抗体和检测试剂 已经在手中的都将被使用。在第二个目标下，申请者将利用生物化学方法 培养树的处理以确定肿瘤坏死因子α对Bmi-1介导的抑制的破坏作用 衰老。最后，在第三个目标下，申请者将使用一种新颖的体外共培养系统来建立 SnFOXP3细胞的促炎能力，并建立治疗靶点。这种方法是创新的 因为它脱离了利用单个细胞对CD4+FOXP3+细胞进行功能表征的现状 方法，以及定义导致致病性SnFOXP3的表观遗传事件的机制方法 手机。在结论中，申请人将了解导致发展的表观遗传机制 SnFOXP3细胞在肠道炎症中的设置。这一贡献意义重大，因为SnFOXP3 在克罗恩病变中扩张，是促炎的，代表着一个重要的表观遗传药物靶点。这 该项目首次阐明了调控衰老激活的标志性和精确的表观遗传事件 在肠道炎症方面，因此推进了工程细胞疗法的治疗领域 自身免疫性疾病的背景。

PROJECT SUMMARY/ABSTRACT FOXP3+ CD4+ cells are expanded within the inflamed mucosa of Crohn’s disease (CD) patients; yet the persistence of intestinal inflammation suggests these CD associated CD4+FOXP3+ cells lack typical regulatory T cell (TREG) suppressive function. The functional and therapeutic implications of senescence in FOXP3+ TREG cells is unknown. The long-term goal of this application is to dissect the epigenetic mechanisms driving the differentiation and function of CD4+FOXP3+ cells in the setting of intestinal inflammation. Consequently, the overall objective of this proposal is to identify the mechanisms leading to senescent FOXP3+ cells in the setting of GI inflammation and test therapeutic opportunities to restore regulatory function. The central hypothesis is that CD associated FOXP3+ cells represent a senescent state and contribute to intestinal inflammation. Furthermore, this new cellular phenotype (senescent FOXP3+ cell, snFOXP3) represents a potential therapeutic target. This hypothesis was formulated through single cell transcriptomic analysis of CD4+FOXP3+ cells from the lamina propria of CD patients, produced by this applicant’s laboratory. The rationale is that defining the mechanisms critical to the development and function of snFOXP3 cells in the setting of inflammation will uncover targets for TREG-directed therapies, including the advanced engineering of human TREGS for adoptive cell therapy trials. Guided by strong preliminary data, this hypothesis will be tested by pursuing three specific aims: 1) Why do FOXP3+ cells become senescent in the absence of BMI1?; 2) Why are FOXP3+ cells senescent in Crohn’s disease?; and 3) Why are snFOXP3 cells proinflammatory? Under the first aim, the applicant will utilize epigenetic assays, which have been established as feasible in the applicant’s hands, to establish BMI1 repression on an ETS1-regulated senescence program in FOXP3+ cells. Antibodies and assay reagents that are already on hand will be used. Under the second aim, the applicant will utilize a biochemistry approach with treatment of cultured TREGS to determine a TNFα-induced disruption of BMI1-mediated repression of senescence. Finally, under the third aim, the applicant will use a novel in vitro co-culture system to establish the proinflammatory capacity of snFOXP3 cells and establish therapeutic targets. The approach is innovative because it departs from the status quo of functional characterization of CD4+FOXP3+ cells utilizing single cell approaches, and a mechanistic approach to define the epigenetic events that lead to a pathogenic snFOXP3 cell. Upon conclusion, the applicant will understand the epigenetic mechanisms leading to the development of snFOXP3 cells in the setting of intestinal inflammation. This contribution is significant as the snFOXP3 is expanded in the Crohn’s lesion, is proinflammatory, and represents an important epigenetic drug target. This project is the first to articulate both signature and precise epigenetic events that regulate activation of senescence in TREGS in intestinal inflammation, therefore advancing the field of therapeutics in engineered cell therapies in the setting of autoimmune diseases.