DOT1L controls NK cell maturation and lineage integrity [RNA-seq D5]. DOT1L controls NK cell maturation and lineage integrity [RNA-seq D5]

In the innate immune system, natural killer (NK) cells represent a highly important subset of cells with utmost importance in the control of transformed cell and tumour inflammation. Previous studies showed that NK cells can convert into ILC1-like cells in a TGF-β-rich tumour microenvironment (TME). In addition, cancer patients with acute myeloid leukemia (AML) or chronic lymphocytic leukemia (CLL) have increased frequencies of ILC1 cells in peripheral blood mononuclear cells (PBMCs), which show reduced production of the proinflammatory cytokines IFN-γ and TNF in addition to decreased granzyme B production. Here, we identify the histone methyltransferase DOT1L as a critical regulator of NK cell lineage integrity in vitro and in vivo. NK cells from NKp46-conditional DOT1L knockout mice (DOT1L.Ncr1) show increased frequencies of ILC1-like cells (CD49b+ CD49a+), and also express more of the ILC1 markers CD200R, TRAIL, while showing reduced expression of Ly49H. We further identify that the increased ILC1-like phenotype is largely independent of TGFβ. Assessment of transcription factor (TF) availability in the absence of DOT1L shows that the Myocyte-specific enhancer factor (MEF)2C is the highest downregulated TF in the absence of DOT1L. CRISPRCas9-mediated deletion of MEF2C ultimately provides evidence that this TF is limiting NK cell plasticity in vivo. Our findings provide evidence for a previously unknown role of DOT1L and MEF2C in NK cell biology for maintaining NK cell lineage integrity and may ultimately result in improved therapies for patients with leukemia or other NK cell-lineage dependent malignancies. Overall design: RNA-seq of enriched splenic NK cells after culturing for 5 days in the presence of 50 ng/ml IL-15; Three replicates each for WT (Ncr1-Cre- DOT1L fl/fl) and DOT1L knockout (DOT1L.Ncr1; Ncr1-Cre+ DOT1L fl/fl).

先天免疫系统中，自然杀伤（natural killer, NK）细胞是一类至关重要的细胞亚群，在调控转化细胞与肿瘤炎症过程中发挥核心作用。既往研究表明，NK细胞在富含转化生长因子β（transforming growth factor-β, TGF-β）的肿瘤微环境（tumour microenvironment, TME）中可转化为类固有淋巴细胞1（innate lymphoid cell 1-like, ILC1-like）细胞。此外，急性髓系白血病（acute myeloid leukemia, AML）或慢性淋巴细胞白血病（chronic lymphocytic leukemia, CLL）患者的外周血单个核细胞（peripheral blood mononuclear cells, PBMCs）中ILC1细胞频率升高，此类细胞的促炎细胞因子干扰素γ（interferon-γ, IFN-γ）与肿瘤坏死因子（tumour necrosis factor, TNF）生成量减少，同时颗粒酶B（granzyme B）的产生也出现下调。

本研究鉴定出组蛋白甲基转移酶DOT1L是体内外调控NK细胞谱系完整性的关键调节因子。对NKp46条件性敲除DOT1L的小鼠（DOT1L.Ncr1）的NK细胞进行分析发现，其体内类ILC1细胞（CD49b+ CD49a+）的频率升高，同时高表达类ILC1细胞标志物CD200R、TRAIL，而Ly49H的表达水平显著降低。进一步研究表明，类ILC1表型的升高在很大程度上不依赖于TGFβ信号通路。

对缺失DOT1L的细胞进行转录因子（transcription factor, TF）可及性评估发现，肌细胞特异性增强因子2C（myocyte-specific enhancer factor 2C, MEF2C）是缺失DOT1L后下调幅度最大的转录因子。通过CRISPR-Cas9介导的MEF2C敲除实验最终证实，该转录因子可在体内限制NK细胞的可塑性。本研究结果揭示了DOT1L与MEF2C在NK细胞生物学中维持谱系完整性的全新功能，有望为白血病或其他NK细胞谱系相关恶性肿瘤患者的治疗提供改进方向。

整体实验设计：将富集获得的脾脏NK细胞在含50 ng/mL IL-15的培养基中培养5天后进行RNA测序（RNA-seq）；野生型对照组（WT，Ncr1-Cre- DOT1L fl/fl）与DOT1L敲除组（DOT1L.Ncr1; Ncr1-Cre+ DOT1L fl/fl）各设置3个生物学重复。