Specification of Treg cells: learning from FoxP3 deficiencies

Treg 细胞的规范：从 FoxP3 缺陷中学习

• 批准号: 10652618
• 负责人: CHRISTOPHE O. BENOIST
• 金额: $ 55.6万
• 依托单位: HARVARD MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-06-27 至 2027-05-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10652618
• 关键词: 3-Dimensional; ATAC-seq; Affect; Alanine; Appearance; Architecture; Australia; Autoimmune; Autoimmune Diseases; Autoimmunity; Back; Boston; CD4 Positive T Lymphocytes; Cell Nucleus; Cell physiology; Cells; Characteristics; Chromatin; Chromatin Structure; Clinical; Clustered Regularly Interspaced Short Palindromic Repeats; Colitis; Collaborations; Complex; Controlled Environment; Cytometry; Data; Defect; Dermatitis; Disease; Engineering; Enhancers; Environment; FOXP3 gene; Family; Female; Foundations; Functional disorder; Gene Expression; Gene Expression Profile; Genes; Genetic; Genetic Transcription; Genomics; Grant; Heterozygote; Homeostasis; Human; Immune Tolerance; Immunologics; Inflammation; Internal Ribosome Entry Site; Italy; Learning; Lesion; Link; Location; Lymphoid; Maps; Mediator; Microbe; Missense Mutation; Modeling; Molecular; Mothers; Mus; Mutant Strains Mice; Mutation; Nuclear; Nuclear Structure; Organ; Organism; Paris, France; Pathology; Patients; Phenotype; Play; Predisposition; Proteins; Recording of previous events; Regulation; Regulatory T-Lymphocyte; Reporter; Repression; Resolution; Sampling; Scanning; Severities; Specific qualifier value; Structure; Symptoms; T-Lymphocyte; Testing; Tissues; United States National Institutes of Health; Variant; Work; clinically relevant; cofactor; conditioning; forkhead protein; immunopathology; immunoregulation; in vivo; insight; male; microbial; multiple omics; mutant; novel; pathobiont; programs; promoter; response; single-cell RNA sequencing; superresolution microscopy; symptomatology; transcription factor; transcriptome; transcriptome sequencing; transcriptomics

T regulatory (Treg) cells help maintain immunologic tolerance and control inflammation in many contexts. Their dysfunction leads to multi-organ autoimmunity in FoxP3-deficient mice and human IPEX patients, with variable clinical manifestations. Treg function and homeostasis are dependent on the transcription factor FoxP3, encoded on ChrX, which determines a substantial portion of their specific transcriptome. Our prior work on FoxP3 structure/function explored how FoxP3’s interactions with different cofactors within multimolecular complexes that reside in different nuclear compartments, and condition its transactivating potential on different transcriptional targets. Further, single-cell cytometry and transcriptomics of T cells from FOXP3-deficient patients and mice revealed a narrow cell-intrinsic signature of the deficiency, directly controlled by FoxP3, and a larger disease signature, conferred in a cell-extrinsic manner, that affects both Treg and conventional CD4+ T cells. We hypothesize that these components vary according to the actual FOXP3 lesion, mutations in different interaction facets leading to different pathology, and that environmental and activation triggers promote the unfolding of the full cell-extrinsic IPEX signature. In Aim1, to better understand the components of Treg dysfunction in IPEX patients, we will analyze the impact of missense FOXP3 mutations by single-cell RNAseq in CD4+ T cells from paired IPEX patients and carrier female relatives (typically mothers), in which we distinguish Treg-like cells expressing wild-type or mutant FOXP3 through random ChrX-inactivation and SNP- based cell identification. Effects on chromatin will also be mapped (ATACseq), and related back to symptomatology in these patients. To enable further analysis in a genetically and environmentally controlled environment, where sample access is not limiting, Aim2 uses CRISPR editing to introduce eight selected missense FOXP3 mutations from IPEX patients into B6 mice, where lymphoid and tissue-resident Tregs will be examined and profiled. Super-resolution microscopy and HiChIPseq will assess the effect of the FOXP3 mutations on localization of the mutant proteins in the nucleus, in terms of nuclear structure and enhancer- promoter loops. We will also analyze the timing and determinants of acquisition of the dominant Treg-extrinsic signature. Aim3 will use this FoxP3 mutant mouse panel to ask how mutation-specific transcriptional features relate to the IPEX-relevant clinical characteristics in mice: appearance of spontaneous autoimmunity (scurfy- like disease), susceptibility to induced autoimmunity (colitis, dermatitis). Challenges with defined microbes and microbial molecules will test the hypothesis that clinical variability in IPEX results, at least in part, from environmental and infectious triggers. These interconnected Aims, with complementary explorations on mice and humans, will bring unique information on how variation in a transcription factor modulates its ability to influence gene expression through chromatin and genomic 3D architecture, and how these relate to a monogenic loss of tolerance in human patients.

调节性T细胞（Treg）在许多情况下有助于维持免疫耐受和控制炎症。他们的 功能障碍导致FoxP 3缺陷小鼠和人IPEX患者的多器官自身免疫， 临床表现Treg的功能和稳态依赖于转录因子FoxP 3， 在ChrX上编码，这决定了它们特定转录组的大部分。我们之前的工作 FoxP 3的结构/功能探讨了FoxP 3如何与多分子内的不同辅因子相互作用。 复合物驻留在不同的核隔室，并在不同的条件下，其反式激活潜力 转录靶点。此外，来自FOXP 3缺陷型的T细胞的单细胞细胞计量术和转录组学研究显示， 患者和小鼠显示出由FoxP 3直接控制的狭窄的细胞内在缺陷特征， 以细胞外在方式赋予的更大的疾病特征，其影响Treg和常规CD 4 + T细胞。我们假设这些成分根据实际FOXP 3病变而变化， 不同的相互作用方面导致不同的病理，环境和激活触发 促进全细胞外在IPEX签名的解折叠。在目标1中，为了更好地理解 IPEX患者的Treg功能障碍，我们将通过单细胞免疫组化分析错义FOXP 3突变的影响。 来自配对IPEX患者和携带者女性亲属（通常是母亲）的CD 4 + T细胞中的RNAseq，其中我们 通过随机ChrX失活和SNP-1区分表达野生型或突变型FOXP 3的Treg样细胞。 基于细胞识别。对染色质的影响也将被映射（ATACseq），并与 在这些患者中，为了能够在基因和环境控制下进行进一步分析， 环境中，在样本访问不受限制的情况下，Aim 2使用CRISPR编辑来引入八个选定的 将IPEX患者的错义FOXP 3突变导入B6小鼠，其中淋巴和组织驻留型THBG将被 检查和分析。超分辨率显微镜和HiChIPseq将评估FOXP 3 突变对突变蛋白在细胞核中的定位，就细胞核结构和增强子而言， 启动子环我们还将分析获得显性Treg-外源性的时机和决定因素， 签名. Aim 3将使用这个FoxP 3突变小鼠小组来询问突变特异性转录特征如何影响基因表达。 与小鼠中IPEX相关的临床特征有关：自发性自身免疫的出现（皮屑- 类似疾病），对诱导的自身免疫的易感性（结肠炎、皮炎）。确定的微生物的挑战， 微生物分子将测试IPEX结果的临床变异性的假设，至少部分来自 环境和传染诱因这些相互关联的目标，以及对小鼠的互补探索 和人类，将带来独特的信息，如何在转录因子的变化调节其能力， 通过染色质和基因组3D结构影响基因表达，以及这些如何与 人类患者的单基因耐受性丧失。