Tissue-Resident Natural Killer (NK) Cells Are Cell Lineages Distinct From Thymic and Conventional Splenic NK Cells (part 2)

Natural killer (NK) cells belong to the innate immune system where they can control virus infections and developing tumors by cytotoxicity and production of inflammatory cytokines. Most studies of mouse NK cells, however, have focused on conventional NK (cNK) cells found in the spleen. Recently, we described two populations of NK cells within the liver, tissue-resident NK (trNK) cells and those resembling splenic cNK cells. However, the lineage relationship of trNK to cNK cells was unclear because trNK cells display a phenotype associated with immature, developing cNK cells. Moreover, liver trNK cells could be related to thymic NK cells or alternatively, a lineage distinct from both cNK and thymic NK cells. Herein we used detailed transcriptomic, flow cytometric, and functional analysis of mice deficient in several transcription factors to determine that liver trNK cells form a distinct lineage from cNK and thymic NK cells, especially because they do not require NFIL3 (E4BP4), the previously described NK cellspecification factor. Analysis of other tissues indicate the presence of trNK cells in skin and uterus with different transcription factor requirements. Thus, there are at least four distinct lineages of NK cells: cNK, thymic, liver (and skin) trNK, and uterine trNK cells. Liver DX5-CD49+, liver DX5+CD49-, spleen DX5+ CD49- populations of NK cells were sorted with FACS pooling cells from individual mice to end up with ~100k cells for each samples. mRNA was derived from lysates using Invitrogen oligo-dT beads

自然杀伤细胞（Natural Killer，NK）隶属于固有免疫系统，可通过细胞毒性作用与炎性细胞因子的分泌，调控病毒感染与肿瘤发生发展。然而，现有针对小鼠NK细胞的研究大多聚焦于脾脏中存在的经典NK细胞（conventional NK，cNK）。近期，我们在肝脏中发现了两类NK细胞亚群：组织驻留NK细胞（tissue-resident NK，trNK），以及一类与脾脏经典NK细胞表型相似的亚群。但trNK细胞与cNK细胞的谱系发育关系尚未明确，因trNK细胞的表型与未成熟的发育中cNK细胞相似。此外，肝脏trNK细胞可能与胸腺NK细胞存在关联，亦或是一类独立于cNK细胞与胸腺NK细胞的独特谱系。本研究通过对多种转录因子敲除小鼠开展细致的转录组学、流式细胞术与功能学分析，证实肝脏trNK细胞是一类独立于cNK细胞与胸腺NK细胞的独特谱系，尤其是其并不依赖此前被鉴定为NK细胞定向分化因子的NFIL3（E4BP4）。对其他组织的分析显示，皮肤与子宫中亦存在trNK细胞，且这类细胞对转录因子的需求存在差异。综上，NK细胞至少存在四类独立谱系：cNK细胞、胸腺NK细胞、肝脏（及皮肤）trNK细胞，以及子宫trNK细胞。本研究通过荧光激活细胞分选术（FACS），从单只小鼠体内分离并混合对应NK细胞亚群：肝脏DX5-CD49+ NK细胞、肝脏DX5+CD49- NK细胞与脾脏DX5+CD49- NK细胞，最终每份样本获得约10万个细胞。随后通过Invitrogen公司的寡聚dT磁珠从细胞裂解液中分离提取mRNA。