Specification of Treg cells: FOXP3 functional facets

 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性和控制炎症，如FoxP3缺陷小鼠或人“IPEX”患者的破坏性多器官炎症以及发现不同Treg亚表型的Treg细胞发挥不同的抗炎作用所示。转录因子FoxP3指定Treg分化和功能的许多方面，但其他几种转录辅因子和表观遗传修饰剂参与指定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，我们构建了一系列跨越整个FoxP3蛋白的突变体。对它们的转录结果的评估揭示了突变效应的精细拼凑，提供了FoxP3功能方面的详细线索，确定了具有相关影响的突变类别，并将FoxP3靶点解析为依赖于相同相互作用的簇。我们将在这些数据的基础上，通过突变的影响来绘制FoxP3的物理相互作用，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突变体也将与已知的辅因子共转染，以观察哪些突变消除协同效应。该框架将用于分析来自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鉴定的突变类型进行比较。与Children's（波士顿）和Necker（巴黎）的同事合作，这些结果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。最后，我们将分析FoxP3突变体类别在体内的影响，利用小鼠中基于CRISPR的种系诱变的速度和效率，在体内Treg细胞的环境中测试6到8个突变，选择这些突变来代表主要的突变体类别和IPEX突变。分析突变小鼠的免疫稳态将显示干扰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。这将揭示，在一个易于处理和非混淆的系统，从IPEX患者的突变的后果。这些结果将提供Foxp3参与控制Treg细胞不同风味和功能的途径的机制理解，并提供对IPEX患者突变的新理解。

 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.