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

摘要 先天性淋巴样细胞（ILC）包括三大类，ILC 1、ILC 2和ILC 3。ILC的个体发育是 复杂. ILC被认为是从骨髓中的普通淋巴祖细胞分化而来。组织- 居民ILC也被认为补充ILC池。我们和其他人已经积累了证据 提示ILC可以在胸腺中产生并输出到外周组织。最近，通过使用单 细胞RNA测序（scRNAseq），我们比较了野生型（WT）和 无胸腺裸鼠，发现大部分谱系阴性（Lin-）Thy 1+群体是 在裸鼠中显著减少，表明这些ILC前体可能在胸腺中产生。然而，在这方面， 由于缺乏合适的Cre，这一结论尚未得到谱系追踪实验的验证 在T细胞发育的早期阶段特异性和有效表达的转基因。我们的scRNAseq 数据显示，这些胸腺依赖性细胞表达编码CD 3链、CD 3d、CD 3e和CD 3g的基因。 流式细胞术分析检测到WT的Lin-Thy 1+细胞的细胞内CD 3（icCD 3 ε），但未检测到WT的Lin-Thy 1+细胞表面CD 3 但不是裸鼠。在外周组织如肺和小肠中也发现icCD 3 ε+细胞 但它们大多是不成熟的ILC 2或其他ILC子集。我们有证据表明CD 3表达 随着前体分化为ILC 2，其表达下调。如果胸腺衍生的细胞可以永久地 在胸腺中使用谱系追踪系统标记，我们将能够在外周组织中识别它们。 即使它们下调了组织中Cd 3的表达。R 03应用程序的目的是建立 通过建立Cd 3-iCre基因敲入小鼠检测胸腺来源的ILC的谱系追踪系统， 测试iCre诱导的tdTomato表达的特异性和效率。有两个目的：（1） 产生并表征Cd 3-iCre基因敲入小鼠。我们选择了Cd 3e-iCre和Cd 3g-iCre小鼠 这是由于两种Cd 3基因表达的特异性和效率不同。将小鼠 由Applied StemCell Inc.将建立者小鼠与ROSA 26 stop-tdTomato小鼠杂交。tdTomato 将检查淋巴和非淋巴器官中的表达。(2)为了进一步验证专属性， Cd 3-iCre/ROSA 26 stop-tdTomato小鼠的效率。我们将使用骨髓进行骨髓移植， Cd 3-iCre/ROSA 26 stop-tdTomato小鼠作为供体。受体将是无胸腺裸鼠及其 杂合子对照。将检查接受者中的tdTomato表达。此外，我们将验证 我们通过scRNA测序获得的WT和裸小肠ILC的scRNAseq数据， 报告小鼠以测试胸腺依赖性簇是否表达tdTomato和表达效率。简而言之， 该系统的建立将为研究胸腺ILC的功能提供有力的工具， 巩固了ILC个体发育的新范式。该系统也可用于早期T细胞免疫领域的研究。 发展

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.