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，FoxP3缺陷小鼠或人类“IPEX”患者的破坏性多器官炎症以及不同Treg亚型Treg细胞的发现发挥不同的抗炎作用表明了这一点。转录因子FoxP3规定了Treg分化和功能的许多方面，但其他几个转录辅助因子和表观遗传修饰物也参与了Treg谱系及其不同效应功能的确定。为了解决FoxP3如何协调这些活动，我们构建了一组广泛的突变体，这些突变体跨越了FoxP3蛋白的整个。对其转录结果的评估揭示了突变效应的精细拼凑，提供了FoxP3 S功能面的详细线索，识别了具有相关影响的突变类别，并将FoxP3靶标解析为依赖相同交互作用的簇。我们将在这些数据的基础上，通过突变的影响绘制FOXP3与S的物理相互作用图，测量DNA序列基序的特异性变化，并使用多重质谱学来确定FOXP3相关蛋白质的变化。Foxp3突变体也将与已知的辅助因子共转染，看看哪些突变消除了协同效应。这一框架将被用来分析来自IPEX患者的错义FoxP3突变的转录效应，将这些突变引入同一转染系统，以比较它们的转录和物理影响与已经确定的突变类别。与儿童医院(波士顿)和Necker医院(巴黎)的同事合作，这些结果将得到补充，通过高通量单细胞RNA测序，来自具有相同突变的患者的体外CD4+FOXP3+Treg样细胞。最后，我们将分析FoxP3突变类别在体内的影响，利用基于CRISPR的小鼠生殖系突变的速度和效率来测试，在体内Treg细胞的设置下，选择6到8个突变来代表主要的突变类别和IPEX突变。分析突变小鼠的免疫稳态将显示干扰FoxP3‘S相互作用如何影响Treg细胞的分化和功能，以及它们控制耐受和自身免疫性疾病的能力。这将在一个易于处理和不混乱的系统中揭示IPEX患者突变的后果。这些结果将提供对Foxp3参与控制不同口味和功能的Treg细胞的途径的机械理解，并为IPEX患者的突变提供新的理解。

 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.