Identification of aceNKPs, a committed common progenitor of the ILC1 and NK cell continuum. 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 0.1; Sirpa, Cd63, Csf1r, Csf2ra, Trpm2, Clec9a expression < 0.002.

先天淋巴细胞（innate lymphoid cell, ILC）转录因子报告小鼠的研发，揭示了此前未被认知的ILC造血系统复杂性。本研究借助新型多色荧光小鼠实现更高分辨率的表型分型，对定向分化为1组ILC的骨髓祖细胞开展了特征鉴定。这类被我们命名为"aceNKPs"的共同ILC1/自然杀伤（natural killer, NK）祖细胞，其表型定义为谱系标记阴性、Id2+、IL-7Ra+、CD25–、α4β7–、NKG2A/C/E+、Bcl11b–。

体外实验中，aceNKPs可分化为1组ILC，包括无需白细胞介素-15（interleukin-15, IL-15）即可表达Eomes的NK样细胞，且在IL-15刺激后可分泌干扰素-γ（interferon-γ, IFN-γ）与穿孔素。将aceNKPs移植至Rag2^–/–Il2rg^–/–受体小鼠后，其可产生一系列成熟ILC1/NK细胞（无论其组织定位如何），且无法被明确划分为传统的ILC1与NK细胞亚群，这提示1组ILC构成了由共同祖细胞发育而来的动态连续谱系。此外，在肺转移瘤模型中，aceNKP衍生的ILC1/NK细胞可获得细胞毒性NK细胞表型，有效减轻肿瘤负荷。本研究结果明确了一类核心的ILC1/NK祖细胞，该祖细胞不具备分化为ILC2或ILC3的潜能，且无论组织归巢特性如何，均严格定向分化为ILC1/NK细胞。

实验整体设计：通过流式细胞术从骨髓细胞中纯化得到小鼠aceNKPs，将其置于热灭活胎牛血清中，用磷酸盐缓冲液（phosphate-buffered saline, PBS）稀释至50%浓度后重悬洗涤。随后用注射器吸取细胞悬液，重悬于无内毒素无菌PBS中，调整浓度至1000 aceNKPs/mL，经尾静脉注射至接受过450拉德亚致死剂量辐射的CD45.1 Rag2^–/–Il2rg^–/–受体小鼠（每只小鼠移植100-200个细胞）。细胞移植7周后，对供体细胞子代进行分析。

处死小鼠后摘取肺组织，将组织切碎后，用750 U/mL胶原酶I（GIBCO）与0.3 mg/mL脱氧核糖核酸酶I（DNaseI，Sigma-Aldrich）进行预消化，随后通过70mm细胞滤器（实际应为70 μm细胞滤器，系原文笔误）过滤制备单细胞悬液。洗涤细胞后，使用红细胞裂解液（140 mM NH4Cl、17 mM Tris，pH 7.2）去除血细胞。通过850×g离心10分钟，采用30% Percoll（GE Healthcare）富集肺淋巴细胞，最后用含3%胎牛血清的PBS洗涤细胞。

将样本用抗CD45.1抗体、Live/Dead可固定染色染料eF780以及抗NK1.1抗体进行染色。通过流式细胞术分离得到活细胞、CD45.2+Id2+的aceNKP子代细胞，离心收集后送至10x Genomics Chromium平台进行单细胞RNA测序。

采用10x Genomics技术完成单细胞文库制备，通过10x Genomics Chromium单细胞3'端v3试剂盒构建3'端文库并进行后续测序。将测序reads比对至小鼠转录组（GRCm38），利用10x Genomics Cell Ranger（版本6.0.1）完成表达量分析与细胞聚类。进一步分析与统计计算采用10x Genomics Loupe Browser（版本5.1）（https://support.10xgenomics.com/single-cell-gene-expression/software/visualization/latest/what-is-loupe-cell-browser）。

通过以下参数剔除分析中的异常值（如污染的髓系细胞与死细胞）：唯一分子标识符（unique molecular identifier, UMI）计数区间500-20000；每个条形码对应的基因数>500；线粒体UMI占比<0.1；Sirpa、Cd63、Csf1r、Csf2ra、Trpm2、Clec9a的表达量<0.002。