Specification of Treg cells: FOXP3 functional facets

Treg 细胞的规格：FOXP3 功能方面

• 批准号: 9461146
• 负责人: CHRISTOPHE O. BENOIST
• 金额: $ 9.75万
• 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/9461146
• 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; Gene Targeting; Generic Drugs; Genetic Transcription; Genomics; Homeostasis; Human; Immune Tolerance; Immunoblotting; Immunologics; In Vitro; Individual; Inflammation; Laboratories; Link; Location; Mass Spectrum Analysis; Measures; Mediator of activation protein; Missense Mutation; Mus; Mutagenesis; Mutant Strains Mice; Mutation; Nuclear Extract; 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; immunoregulation; in vivo; mutant; public health relevance; 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（由申请人提供）：FoxP3+ Treg 细胞在许多情况下有助于维持免疫耐受并控制炎症，如 FoxP3 缺陷小鼠或人类“IPEX”患者的破坏性多器官炎症以及发现不同类型的 Treg 细胞所示。 Treg 亚表型发挥着不同的抗炎作用，转录因子 FoxP3 指定了 Treg 分化和功能的许多方面，但其他几种转录辅助因子和表观遗传修饰因子也参与指定了 Treg 的分化和功能。 Treg 谱系及其不同的效应子功能。为了解决 FoxP3 如何协调这些活动，我们构建了一组涵盖整个 FoxP3 蛋白的突变体，对其转录结果的评估揭示了突变效应的多样化拼凑，从而提供了详细的线索。 FoxP3 的功能方面，识别具有相关影响的突变类别，并将 FoxP3 目标解析为依赖于相同相互作用者的簇。我们将基于这些数据来绘制 FoxP3 的物理相互作用。通过突变的影响，测量 DNA 序列基序特异性的变化，并使用多重质谱法来识别 FoxP3 相关蛋白的变化。 FoxP3 突变体还将与已知的辅助因子共转染，以观察哪些突变消除了协同效应。该框架将用于分析 IPEX 患者的错义 FoxP3 突变的转录效应，将这些突变引入相同的转染系统中，以将其转录和物理效应与已经确定的突变体类别进行比较。波士顿儿童医院和巴黎内克尔医院的同事们将通过高通量单细胞 RNA 测序对来自具有相同突变的患者的离体 CD4+FOXP3+ Treg 样细胞进行分析来补充这些结果。将分析 FoxP3 突变体类别在体内的影响，利用基于 CRISPR 的小鼠种系诱变的速度和效率，在体内 Treg 细胞的环境中测试六到八个突变，这些突变被选择来代表分析突变小鼠的免疫稳态，将显示扰乱 FoxP3 的相互作用如何影响 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。这些结果将提供对 Foxp3 参与控制 Treg 细胞不同功能和功能的途径的机制理解。对 IPEX 患者突变的新认识。