Specification of Treg cells: learning from FoxP3 deficiencies

在许多情况下，T调节细胞(Treg)有助于维持免疫耐受和控制炎症。他们的 功能障碍导致FoxP3缺陷小鼠和人IPEX患者的多器官自身免疫，变量 临床表现。Treg功能和动态平衡依赖于转录因子FoxP3， 编码在chrx上，这决定了他们特定转录组的很大一部分。我们之前的工作是 FOXP3结构/功能探讨FOXP3‘S如何与多分子内不同辅因子相互作用 驻留在不同核区的复合体，并将其反式激活潜力限制在不同的 转录靶标。此外，FOXP3缺陷型T细胞的单细胞流式细胞术和转录组学研究 患者和小鼠揭示了这种缺陷的一个狭窄的细胞固有特征，直接由FoxP3控制，并且 一种较大的疾病标志，以细胞外源性方式授予，同时影响Treg和传统的CD4+ T细胞。我们假设，这些成分根据实际的FOXP3损伤，突变在 不同的相互作用导致不同的病理，环境和激活触发 促进完整的细胞-外部IPEX签名的展开。在Aim1中，为了更好地了解 IPEX患者Treg功能障碍，我们将分析FOXP3错义突变对单细胞的影响 来自配对的IPEX患者和携带者女性亲属(通常是母亲)的CD4+T细胞中的RNAseq，其中我们 随机chRX失活和单核苷酸多态性对表达野生型和突变型FOXP3的Treg样细胞的区分 基于小区标识。对染色质的影响也将被绘制(ATACseq)，并与 这些患者的症状学。为了在基因和环境控制的环境中进行进一步分析 环境，其中样本访问不受限制，AIM2使用CRISPR编辑来介绍八个选定的 来自IPEX患者的FOXP3错义突变进入B6小鼠，在那里淋巴和组织驻留Tregs将 进行了检查和分析。超分辨率显微镜和HiChIPseq将评估FOXP3的效果 突变蛋白在细胞核内的定位，从核结构和增强子- 启动子循环。我们还将分析获得主导的Treg-Extrative的时机和决定因素 签名。Aim3将使用这个FoxP3突变小鼠小组来询问突变特异的转录特征 与IPEX相关的小鼠临床特征有关：出现自发性自身免疫(Scrfy- 易患诱导自身免疫(结肠炎、皮炎)。挑战定义的微生物和 微生物分子将测试这一假设，即IPEX的临床变异性至少部分来自 环境诱因和传染性诱因。这些相互关联的目标，以及对小鼠的互补探索 以及人类，将带来关于转录因子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。

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.