Identification and localization of bone marrow ILC2s using Id2/Cxcr6 dual-reporter mice

Type 2 innate lymphoid cells (ILC2s) are present in the bone marrow, where their identification and spatial localization remain challenging because of their low abundance and the expression of shared markers by other lymphoid populations. Here, we evaluated the use of Id2YFP/+ Cxcr6GFP/+ dual-reporter mice to identify and visualize bone marrow ILC2s. Using spectral flow cytometry, we showed that both ST2- and ST2 ILC2s expressed high levels of ID2, whereas CXCR6 expression differed between these subsets. However, the ID2 expressing compartments also contained NK cells, NKT cells, conventional T cells and ILC progenitors. Surface marker profiling identified CD25 and ICOS as useful markers for enriching ILC2 populations, with ST2 enabling the segregation of the ST2- and ST2 subsets. Single-cell RNA sequencing further characterized the diversity of Id2 and Cxcr6 expressing lymphoid populations and supported the use of CD5 and NKp46 as exclusion markers. Combining these markers with the dual-reporter system, confocal microscopy revealed YFPGFPCD5-NKp46- cells compatible with an ILC2 phenotype, distributed throughout the bone marrow as isolated cells or within multicellular clusters. Complementary flow cytometric analyses demonstrated that ILC2s accounted for more than 90% of a phenotypically related reporter-positive population after exclusion of major T and NK cell populations, providing additional support for the identification of the cells visualized in situ as putative ILC2s. Together, these findings establish a strategy combining dual-reporter mice, flow cytometry, transcriptomic profiling and confocal imaging to investigate the localization and spatial organization of putative bone marrow ILC2s.