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 ont...