Specification of Treg cells: FOXP3 functional facets

Treg 细胞的规格：FOXP3 功能方面

• 批准号: 9038990
• 负责人: CHRISTOPHE O. BENOIST
• 金额: $ 42.38万
• 依托单位: HARVARD MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-04-01 至 2020-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9038990
• 关键词: Affect; Alanine; Anti-Inflammatory Agents; Anti-inflammatory; Autoimmune Diseases; Binding; Biochemical; Biological Assay; Boston; CD4 Positive T Lymphocytes; Cells; Child; Clustered Regularly Interspaced Short Palindromic Repeats; Collaborations; Complement; Consent; DNA Binding; DNA Sequence; Data; Electrophoretic Mobility Shift Assay; Epigenetic Process; FOXP3 gene; Family; Flavoring; Gene Targeting; Generic Drugs; Genomics; Health; Homeostasis; Human; Immune Tolerance; Immunoblotting; In Vitro; Individual; Inflammation; Laboratories; Link; Location; Mass Spectrum Analysis; Measures; Mediator of activation protein; Mus; Mutagenesis; Mutant Strains Mice; Mutation; Nuclear Extract; Organ; Paris, France; Pathway interactions; Patients; Pediatric Hospitals; Play; Proteins; Regulation; Regulatory T-Lymphocyte; Role; Scanning; Series; Severity of illness; Specific qualifier value; Specificity; Speed; Syndrome; System; Testing; Transcriptional Activation; Transfection; Wit; base; clinically relevant; cofactor; ds-DNA; forkhead protein; gene repression; hypoxia inducible factor 1; immunopathology; in vivo; mutant; transcription factor; transcriptome sequencing; transcriptomics; vector

 DESCRIPTION (provided by applicant): FoxP3+ Treg cells help maintain immunologic tolerance and control inflammation in many contexts, as shown by the devastating multi-organ inflammation of FoxP3-deficient mice or human "IPEX" patients, and the discovery of Treg cells of different Treg subphenotypes playing diverse anti-inflammatory roles. The transcription factor FoxP3 specifies many aspects of Treg differentiation and function, but several other transcriptional cofactors and epigenetic modifiers partake in specifying the Treg lineage and its different effector functions. To tackle how FoxP3 orchestrates these activities, we constructed a broad set of mutants that span the entirety of the FoxP3 protein. Assessment of their transcriptional consequences revealed a finely variegated patchwork of mutation effects, providing detailed leads on FoxP3's functional facets, identifying classes of mutations with related impact, and parsing FoxP3 targets into clusters that depend on the same interactors. We will build on these data to chart FoxP3's physical interactions through the impact of the mutations, measuring changes in specificity to DNA sequence motifs, and using multiplexed mass spectrometry to identify changes in FoxP3-associated proteins. FoxP3 mutants will also be co-transfected with known co-factors to see which mutations abolish synergistic effects. This framework will be used to analyze the transcriptional effect of missense FoxP3 mutations from IPEX patients, introducing these mutations into the same transfection system to compare their transcriptional and physical effects to the mutant classes already identified. In collaboration wit colleagues at Children's (Boston) and Necker (Paris), these results will be complemented by profiling, with high-throughput single-cell RNA sequencing, ex vivo CD4+FOXP3+ Treg-like cells from patients with the same mutations. Finally, we will analyze the impact of the FoxP3 mutant classes in vivo, harnessing the speed and efficiency of CRISPR-based germline mutagenesis in mice to test, in the setting of Treg cells in vivo, six to eight mutations chosen to represent the main mutants classes and IPEX mutations. Analyzing immunological homeostasis in the mutant mice will show how perturbing FoxP3's interactions affects the differentiation and function of Treg cells, and their ability to control tolerance and autoimmune disease. This will reveal, in a tractable and non-confounded system, the consequences of the mutations from IPEX patients. These results will provide a mechanistic understanding of the pathways through which Foxp3 partakes in the control of different flavors and functions of Treg cells, and provide a new understanding of mutations in IPEX patients.

 描述（由适用提供）：FOXP3+ Treg细胞在许多情况下有助于维持免疫学耐受性和控制注射，如FOXP3缺乏小鼠或人类“ IPEX”患者的毁灭性多器官炎症，以及发现不同Treg treg亚表格的Treg细胞的发现，发挥了不同的抗激素抗激素疾病。转录因子FOXP3指定了Treg分化和功能的许多方面，但是其他几个转录辅助因子和表观遗传修饰符都参与指定Treg谱系及其不同效应子功能。为了解决FOXP3如何编排这些活动，我们构建了一组跨越整个Foxp3蛋白质的突变体。对其转录后果的评估表明，突变效应的各种拼布，为Foxp3功能方面提供了详细的潜在线索，识别出具有相关影响的突变类别，并将FOXP3目标解析为依赖相同相互作用者的群集。我们将通过这些数据来构建通过突变的影响，测量特异性对DNA序列基序的变化以及使用多重质谱法以识别FOXP3相关蛋白的变化，从而构建了FOXP3的物理相互作用。 FOXP3突变体还将与已知的共同因子共转染，以查看哪些突变消除了协同作用。该框架将用于分析来自IPEX患者的失误FOXP3突变的转录效应，将这些突变引入相同的翻译系统中，以将其转录和物理效应与已经确定的突变类别进行比较。在儿童（波士顿）和内克（Baris）（巴黎）的合作同事中，这些结果将通过分析完成，并通过同一突变患者的高通量单细胞RNA测序，离体CD4+ Foxp3+ Treg样细胞来完成。最后，我们将在体内分析FOXP3突变类别的影响，利用小鼠基于CRISPR的生殖线诱变的速度和效率，以测试，在体内Treg细胞的情况下，被选择代表主要突变体类别和IPEX突变的六到八个突变。分析突变小鼠中的免疫稳态会表明Foxp3相互作用的扰动如何影响Treg细胞的分化和功能，以及它们控制耐受性和自身免疫性疾病的能力。这将揭示在可探讨且非共鸣的系统中，这是IPEX患者突变的后果。这些结果将提供对途径的机械理解，因为Foxp3部分控制了Treg细胞的不同口味和功能，并提供了对IPEX患者突变的新理解。