Identification of aceNKPs, a committed common progenitor of the ILC1 and NK cell continuum

The development of innate lymphoid cell (ILC) transcription factor reporter mice has shown a previously unexpected complexity in ILC haematopoiesis. Using novel polychromic mice to achieve higher phenotypic resolution we have characterised bone marrow progenitors that are committed to the group 1 ILC lineage. These common ILC1/NK progenitors, which we call 'aceNKPs', are defined as lineage–Id2+IL-7Ra+CD25–a4b7–NKG2A/C/E+Bcl11b–. In vitro, aceNKPs differentiate into group 1 ILCs, including NK-like cells that express Eomes without the requirement for IL-15, and produce IFN-g and perforin upon IL-15 stimulation. Following reconstitution of Rag2–/–Il2rg–/– hosts, aceNKPs give rise to a spectrum of mature ILC1/NK cells (regardless of their tissue location) that cannot be clearly segregated into the traditional ILC1 and NK subsets, suggesting that group 1 ILCs constitute a dynamic continuum of ILCs that can develop from a common progenitor. In addition, aceNKP-derived ILC1/NK cells effectively ameliorate tumour burden in a model of lung metastasis where they acquired a cytotoxic NK cell phenotype. Our results identify the primary ILC1/NK progenitor that lacks ILC2 or ILC3 potential and is strictly committed to ILC1/NK cell production irrespective of tissue homing. Overall design: Mouse aceNKPs from the bone marrow cells were purified by flow cytometry into heat-inactivated FCS, diluted to 50% with PBS and washed with PBS before injection. Cell suspensions were aspirated with a syringe, resuspended in endotoxin-free sterile PBS to a concentration of 1000 aceNKPs/mL, and implanted via tail vein injection into sublethally-irradiated (450 rad) CD45.1 Rag2–/–Il2rg–/– recipients (100-200 cells per mouse). Analysis of donor cell progeny was performed 7 weeks after cell transfer. Lungs were harvested and pre-digested by finely chopping the tissue and incubating it with 750 U/mL collagenase I (GIBCO) and 0.3 mg/mL DNaseI (Sigma-Aldrich) prior to obtaining a single cell suspension by straining the tissue through a 70mm cell strainer. After washing, blood cells were removed by incubation with RBC lysis solution (140 mM NH4Cl, 17 mM Tris; pH 7.2). Lung lymphocytes were further enriched by centrifugation in 30% Percoll at 850 x g for 10 min (GE Healthcare), followed by a final wash with PBS with 3% FCS. Samples were stained with anti-CD45.1, Life/Dead fixable dye eF780, anti NK1.1. aceNKP-derived progeny was isolated by flow cytometry as Live CD45.2+Id2+, pelleted and submitted for 10x Chromium single cell RNA sequencing. Single-cell library preparation was performed using the 10x Genomics technology, and the 3' libraries were obtained through the 10x Genomics Chromium Single-Cell 3' v3 protocol and underwent subsequent sequencing. The reads were aligned to the mouse transcriptome (GRCm38), and expression and clusters were determined using 10x Genomics Cell Ranger (version 6.0.1). Further analysis and statistical calculations were performed using the 10x Genomics Loupe Browser (version 5.1) (https://support.10xgenomics.com/single-cell-gene-expression/software/visualization/latest/what-is-loupe-cell-browser). Data outliers such as contaminating myeloid cells and dead cells were removed from the analysis by applying the following parameters: UMI threshold: 500-20000; genes per barcode > 500; mitochondrial UMI < 5%; Ptprc expression > 0.1; Sirpa, Cd63, Csf1r, Csf2ra, Trpm2, Clec9a expression < 0.002.