Regulation of medullary thymic epithelial cells and thymic central tolerance by Ikaros

项目摘要 一个正常和强大的免疫系统依赖于具有不同抗原库的细胞的发育 但仍能容忍自身组织。这种免疫耐受性的破坏可以引起 自身免疫性疾病，并因此多种机制到位，以确保免疫自身耐受性。胸腺 是T细胞发育和教育的关键部位，通过表达 组织特异性抗原（TSA）通过特化的胸腺髓质上皮细胞（mTEC）。转录 已知调节剂Aire和Fezf 2在mTEC内起作用，以促进数千个TSA自身的表达， 抗原，目的是在称为阴性选择的过程中去除正在发育的自身反应性T细胞。 Aire和Fezf 2被证明是mTEC中表达的一些但不是所有TSA基因所必需的，这表明 对于在mTEC中观察到的TSA表达的全部库，需要额外的转录调节因子。 此外，最近的工作突出了mTEC的复杂性，至少有四个mTEC群体： mTEC、Ccl 21 a mTEC、晚期/后Aire mTEC和簇细胞。然而，对这一问题的认识有限。 这些异质mTEC亚群的发育调节和祖细胞。我们已经确定了 转录因子Ikaros（Ikzf 1）作为mTEC组成和功能的新调节因子，缺失Ikaros 导致Aire+ mTEC减少和簇细胞扩增。此外，Ikaros缺陷型mTEC TSA基因表达缺陷导致自身免疫的特异性体征。有趣的是， IKZF 1与人类自身免疫性疾病有关。因此，我们假设Ikaros在以下方面的新角色： mTEC谱系发育、TSA表达和中枢耐受，mTEC功能缺陷可导致 导致IKZF 1突变的人类出现自身免疫。我们建议测试我们的假设， 通过以下具体目标增加我们对这种新型mTEC转录调节因子的了解：（1） 确定Ikaros调节mTEC功能的mTEC发育阶段，（2）询问 Ikaros在mTEC中功能的分子机制，以及（3）确定Ikaros的TEC特异性缺失是否影响 T细胞耐受和自身免疫。为了实现这些目标，我们开发了一套独特而强大的 遗传工具和小鼠报告基因系，使我们能够干扰Ikaros在mTEC中的功能。我们将使用Flow 流式细胞术以研究mTEC和胸腺免疫细胞表型，组合scRNA-seq/scATAC-seq以 分析基因表达和染色质可及性，并补充体外细胞中的机制研究 线我们的长期目标是增加我们对mTEC发育和功能调节的理解 中枢耐受性，以及改变如何导致耐受性和自身免疫的破坏。

Project Summary A normal and robust immune system relies on the development of cells with diverse repertoire of antigen recognition but that remain tolerant of self-tissues. Breakdown of this immune tolerance can give rise to autoimmune disease, and hence multiple mechanisms are in place to ensure immune self-tolerance. The thymus is a critical site for the development and education of T cells to promote tolerance to self through expression of tissue specific antigens (TSAs) by specialized medullary thymic epithelial cells (mTECs). The transcriptional regulators Aire and Fezf2 are known to act within mTECs to promote the expression of thousands of TSA self- antigens for the purpose of removing developing self-reactive T cells in a process known as negative selection. Aire and Fezf2 are shown to be required for some, but not all TSA genes expressed in mTECs, suggesting additional transcriptional regulators are required for the full repertoire of the observed TSA expression in mTECs. Furthermore, recent work has highlighted the complexity of mTECs with at least four mTEC populations: Aire+ mTECs, Ccl21a mTECs, Late/Post Aire mTECs, and tuft cells. However, there is limited knowledge about the developmental regulation and progenitors of these heterogeneous mTEC subsets. We have identified the transcription factor Ikaros (Ikzf1) as a novel regulator of mTEC composition and function, with deletion of Ikaros in mTECs causing a reduction of Aire+ mTECs and an expansion of tuft cells. Moreover, Ikaros-deficient mTECs have a defect in TSA gene expression resulting in specific signs of autoimmunity. Interestingly, mutations in IKZF1 have been linked to human autoimmune diseases. Thus, we hypothesize a novel role for Ikaros in mTEC lineage development, TSA expression and central tolerance, and that defects in mTEC function could contribute to the autoimmunity seen in humans with IKZF1 mutations. We propose to test our hypothesis and increase our knowledge of this novel mTEC transcriptional regulator through the following specific aims: (1) Identify the stage(s) of mTEC development at which Ikaros modulates mTEC function, (2) Interrogate the molecular mechanism of Ikaros function in mTECs, and (3) Determine if TEC specific deletion of Ikaros affects T cell tolerance and autoimmunity. To complete these aims, we have developed a unique and powerful set of genetic tools and mouse reporter lines that allow us to perturb Ikaros function in mTECs. We will use flow cytometry to investigate mTEC and thymic immune cell phenotypes, combined scRNA-seq/scATAC-seq to analyze gene expression and chromatin accessibility, and supplement with mechanistic studies in vitro in cell lines. Our long-term goal is to increase our understanding of the regulation of mTEC development and function in central tolerance, and how alterations can lead to a break of tolerance and autoimmunity.