Establishing a lineage tracing system for studying thymus-derived innate lymphoid cells

建立研究胸腺源性先天淋巴细胞谱系追踪系统

• 批准号: 10644626
• 负责人: Xiao-Hong Sun
• 金额: $ 8.53万
• 依托单位: OKLAHOMA MEDICAL RESEARCH FOUNDATION
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-03-01 至 2025-02-28
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10644626
• 关键词: Biological; Biological Assay; Blood; Bone Marrow; Bone Marrow Cells; Bone Marrow Transplantation; Brain; CD3 Antigens; Cell Lineage; Cell Ontogeny; Cell Separation; Cell physiology; Cells; Cellular biology; Common Lymphoid Progenitor; Complex; Data; Dependence; Enterobacteria phage P1 Cre recombinase; Exhibits; Future; Generations; Genes; Growth; Heterozygote; IL7R gene; Internal Ribosome Entry Site; Knock-in Mouse; Label; Laboratories; Lung; Lymphoid; Lymphoid Cell; Monitor; Mouse Strains; Mus; Nude Mice; Organ; PTPRC gene; Pattern; Peripheral; Population; Production; Proteins; Reagent; Reporter; Reporter Genes; Research; Series; Small Intestines; Source; Specificity; Surface; System; T-Cell Development; T-Lymphocyte; Testing; Thymus Gland; Tissues; Transcript; Transgenes; Translating; Transplantation; Validation; cell type; detection platform; experimental study; field study; lymphoid organ; novel; polypeptide; response; single-cell RNA sequencing; stem cells; tool; transcriptome

ABSTRACT Innate lymphoid cells (ILCs) include three major groups, ILC1, ILC2 and ILC3. The ontogeny of ILCs is complex. ILCs are thought to differentiate from common lymphoid progenitors in the bone marrow. Tissue- resident ILCs are also believed to replenish the ILC pools. We and others have accumulated evidence suggesting that ILCs can be made in the thymus and exported to peripheral tissues. Recently, by using single cell RNA sequencing (scRNAseq), we compared the transcriptomes of ILCs in the blood of wild type (WT) and athymic nude mice and found that a substantial fraction of Lineage negative (Lin-) Thy1+ population was dramatically reduced in nude mice, suggesting that these ILC precursors likely arise in the thymus. However, this conclusion has not been verified using the lineage tracing experiment due to the lack of a suitable Cre transgene that is specifically and efficiently expressed at early stages of T cell development. Our scRNAseq data showed that these thymus-dependent cells express genes encoding CD3 chains, Cd3d, Cd3e and Cd3g. Flow cytometric analyses detected CD3 intracellularly (icCD3ε) but not on the surface of Lin-Thy1+ cells of WT but not nude mice. In peripheral tissues such as the lung and small intestine, icCD3ε+ cells were also found but they are mostly immature ILC2s or other ILC subsets. We have evidence to suggest that CD3 expression is down-regulated as the precursors differentiate into ILC2s. If thymus-derived cells can be permanently labeled in the thymus using a lineage tracing system, we would be able to recognize them in peripheral tissues even if they down-regulate Cd3 expression in tissues. The purpose of the R03 application is to establish a lineage tracing system for detecting thymus-derived ILCs by creating Cd3-iCre knock-in mice and testing the specificity and efficiency of iCre-induced tdTomato expression. There are two aims: (1) To generate and characterize Cd3-iCre knock-in mice. We choose to make Cd3e-iCre and Cd3g-iCre mice because of the different specificities and efficiencies of expression of the two Cd3 gene. The mice will be generated by Applied StemCell Inc. Founder mice will be crossed with ROSA26stop-tdTomato mice. tdTomato expression in lymphoid and non-lymphoid organs will be examined. (2) To further validate the specificity and efficiency of Cd3-iCre/ROSA26stop-tdTomato mice. We will perform bone marrow transplant using bone marrow of Cd3-iCre/ROSA26stop-tdTomato mice as donors. The recipients will be the athymic nude mice and their heterozygous controls. tdTomato expression in the recipients will be examined. In addition, we will validate our scRNAseq data of WT and nude small intestine ILCs by scRNA sequencing the same cells from the reporter mice to test if thymus-dependent clusters express tdTomato and the efficiency of expression. In short, establishing this system will provide a powerful tool for studying the function of thymus-derived ILCs and solidifying a new paradigm of ILC ontogeny. This system will also be useful to others in the field of early T cell development.

抽象的 先天淋巴样细胞（ILC）包括三个主要组ILC1，ILC2和ILC3。 ILC的个体发育是 复杂的。 ILC被认为与骨髓中常见的淋巴祖细胞区分开。组织- 居民ILC还被认为复制ILC池。我们和其他人积累了证据 表明可以在胸腺中制作ILC并导出到外围组织。最近，使用单个 细胞RNA测序（SCRNASEQ），我们比较了野生型（WT）和 无胸腺裸鼠，发现谱系负（lin-）thy1+种群的大部分是 裸鼠动态减少，表明这些ILC前体可能在胸腺中出现。然而， 由于缺乏合适的Cre 转基因在T细胞发育的早期阶段特别有效地表达。我们的scrnaseq 数据表明，这些依赖性的细胞表达编码CD3链，CD3D，CD3E和CD3G的基因。 流式细胞仪分析检测到CD3细胞内（ICCD3ε），但在WT的Lin-Thy1+细胞表面未检测到。 但不是裸鼠。在肺和小肠等周围组织中，还发现了ICCD3ε+细胞 但是它们主要是未成熟的ILC2或其他ILC子集。我们有证据表明CD3表达 当前体分化为ILC2时，被下调。如果胸腺衍生的细胞可以永久 使用谱系示踪系统在胸腺中标记，我们将能够在外围组织中识别它们 即使它们下调组织中的CD3表达。 R03应用程序的目的是建立一个 通过创建CD3-ICRE敲入小鼠和 测试ICRE诱导的TDTOMATO表达的特异性和效率。有两个目标：（1） 生成并表征CD3-ICRE敲入小鼠。我们选择制作CD3E-ICRE和CD3G-ICRE小鼠 由于两个CD3基因的表达的规格和效率不同。老鼠会 由Applied Stemcell Inc.生成的创始人小鼠将与Rosa26stop-Tdtomato小鼠交叉。 tdtomato 将检查淋巴机器人和非淋巴器官中的表达。 （2）进一步验证特异性和 CD3-ICRE/ROSA26STOP-TDTOMATO小鼠的效率。我们将使用骨髓移植 CD3-ICRE/ROSA26STOP-TDTOMATO小鼠作为供体。接收者将是无胸腺裸鼠及其 杂合对照。将检查接收者中的TDTOMATO表达。此外，我们将验证 我们的scrnaseq数据和裸体小肠ILC通过SCRNA测序从相同的细胞中 记者小鼠测试胸腺依赖性簇是否表达tdtomato和表达效率。简而言之， 建立该系统将为研究胸腺来源的ILC和 巩固ILC个体发育的新范式。该系统也对早期T细胞领域的其他人有用 发展。