Specification of Treg cells: learning from FoxP3 deficiencies

Treg 细胞的规范：从 FoxP3 缺陷中学习

基本信息

批准号：
10521755
负责人：
CHRISTOPHE O. BENOIST
金额：
$ 55.59万
依托单位：
HARVARD MEDICAL SCHOOL
依托单位国家：
美国
项目类别：
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/10521755
关键词：
3-Dimensional ATAC-seq Affect Alanine Appearance Architecture Australia Autoimmune Autoimmune Diseases Autoimmunity Back Boston CD4 Positive T Lymphocytes Cell Nucleus Cells Characteristics Chromatin Chromatin Structure Clinical Clustered Regularly Interspaced Short Palindromic Repeats Colitis Collaborations Complex Controlled Environment Cytometry Data Defect Dermatitis Disease Disease susceptibility Engineering Enhancers FOXP3 gene Family Female Foundations Functional disorder Gene Expression Gene Expression Profile Genes Genetic Genetic Transcription Genomics Grant Homeostasis Human Immune Tolerance Immunologics Inflammation Internal Ribosome Entry Site Italy Learning Lesion Link Location Logistics Lymphoid Tissue Maps Mediator of activation protein Microbe Microscopy Missense Mutation Modeling Molecular Mothers Mus Mutant Strains Mice Mutation Nuclear Nuclear Structure Organ Organism Paris, France Pathology Patients Phenotype Play Proteins Recording of previous events Regulation Regulatory T-Lymphocyte Reporter Resolution Sampling Scanning Severities Structure Symptoms T-Lymphocyte Testing United States National Institutes of Health Variant Work base 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 symptomatology transcription factor transcriptome transcriptome sequencing transcriptomics

项目摘要

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 (Treg) 细胞在许多情况下有助于维持免疫耐受并控制炎症。功能障碍导致 FoxP3 缺陷小鼠和人类 IPEX 患者的多器官自身免疫，具有可变的临床表现 Treg 功能和稳态依赖于转录因子 FoxP3，编码在 ChrX 上，它决定了我们之前的工作的特定转录组的很大一部分。 FoxP3 结构/功能探讨了 FoxP3 如何与多分子内不同辅因子相互作用复合物驻留在不同的核区室中，并根据不同的条件调节其反式激活潜力此外，FOXP3 缺陷型 T 细胞的单细胞细胞计数和转录组学。患者和小鼠揭示了缺陷的狭窄细胞固有特征，由 FoxP3 直接控制，并且以细胞外源性方式赋予的更大的疾病特征，影响 Treg 和传统 CD4+ 我们发现这些成分会根据实际的 FOXP3 病变、突变而变化。不同的相互作用方面导致不同的病理，以及环境和激活的触发促进完整的细胞外源性 IPEX 特征的展开，以更好地理解其组成部分。 IPEX患者Treg功能障碍，我们将通过单细胞分析FOXP3错义突变的影响来自配对 IPEX 患者和携带者女性亲属（典型母亲）的 CD4+ T 细胞中的 RNAseq，其中我们通过随机 ChrX 失活和 SNP- 区分表达野生型或突变型 FOXP3 的 Treg 样细胞基于细胞识别的对染色质的影响也将被映射（ATACseq），并关联回这些患者的症状学，以便在遗传和环境控制下进行进一步分析。在样本访问不受限制的环境中，Aim2 使用 CRISPR 编辑引入了八个选定的 IPEX 患者的 FOXP3 错义突变转化为 B6 小鼠，其中淋巴和组织驻留性 Tregs 将被超分辨率显微镜和 HiChIPseq 将评估 FOXP3 的效果。就核结构和增强子而言，突变蛋白在细胞核中的定位发生突变- 我们还将分析获得显性 Treg 外源性的时间和决定因素。 Aim3 将使用该 FoxP3 突变小鼠面板来询问突变特异性转录特征如何。小鼠中与 IPEX 相关的临床特征：自发性自身免疫的出现（scurfy- 疾病）、对诱导性自身免疫的易感性（结肠炎、皮炎）和特定微生物的挑战。微生物分子将检验这样的假设：IPEX 的临床变异至少部分是由这些相互关联的目标，以及对小鼠的互补探索。和人类，将带来关于转录因子的变异如何调节其能力的独特信息通过染色质和基因组 3D 结构影响基因表达，以及它们如何与人类患者的单基因耐受性丧失。