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展。 认可，但对自我组织保持容忍。这种免疫耐受性的崩溃可能会导致 自身免疫性疾病，因此有多种机制来确保免疫自我耐受。胸腺 是T细胞发展和教育的关键部位，通过表达 组织特异性抗原(TSA)由特化的胸腺髓质上皮细胞(MTECs)产生。转录本 已知调节因子Aire和Fezf2在mTEC内作用，促进数千个TSA自我表达 抗原的目的是去除发育中的自我反应性T细胞，这一过程称为负选择。 研究表明，在mTECs中，部分TSA基因的表达需要AIRE和Fezf2，但不是全部，提示 在mTEC中观察到的TSA表达的完整谱系需要额外的转录调控因子。 此外，最近的工作突出了至少有四个mTEC种群的mTEC的复杂性：AIRE+ MTECs、Ccl21a mTECs、晚期/后期mTECs和簇状细胞。然而，人们对此的了解有限。 这些异质mTEC亚群的发育调控和祖细胞。我们已经确定了 转录因子Ikaros(IKZF1)作为一种新的mTEC组成和功能调节因子，Ikaros缺失 在mTECs中，导致Aire+mTECs减少和簇状细胞扩张。此外，缺乏伊卡洛斯的mTECs 有TSA基因表达缺陷，导致自身免疫的特定迹象。有趣的是，基因突变 IKZF1与人类自身免疫性疾病有关。因此，我们假设伊卡洛斯在 MTEC谱系发育、TSA表达和中枢耐受，mTEC功能缺陷可能 有助于在携带IKZF1突变的人类中看到的自身免疫。我们建议检验我们的假设，并 通过以下具体目标增加我们对这种新型mTEC转录调控因子的了解：(1) 确定mTEC发展的阶段(S)，在这个阶段，伊卡洛斯调节mTEC功能，(2)询问 Ikaros在mTECs中作用的分子机制，以及(3)确定TEC特异性的Ikaros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 基因工具和老鼠报告系，允许我们扰乱mTEC中的Ikaros功能。我们将使用Flow 流式细胞术研究mTEC和胸腺免疫细胞表型，联合scRNA-seq/scATAC-seq 分析基因表达和染色质可及性，并补充体外细胞机制研究 台词。我们的长期目标是增加我们对mTEC发展和功能调控的了解。 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.