Tissue-Resident Natural Killer (NK) Cells Are Cell Lineages Distinct From Thymic and Conventional Splenic NK Cells (part 1)

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 cell specification 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 NK 1.1+CD49+, liver NK 1.1+CD49-, spleen NK 1.1+ 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（Natural killer, NK）细胞隶属于先天免疫系统，可通过细胞毒性作用与炎性细胞因子的分泌，调控病毒感染及肿瘤发生发展。然而，当前针对小鼠NK细胞的研究大多聚焦于脾脏中的常规NK（conventional NK, cNK）细胞。近期本团队报道了肝脏内的两类NK细胞群：组织驻留NK（tissue-resident NK, trNK）细胞，以及与脾脏cNK细胞表型相似的细胞群。 但由于trNK细胞呈现出未成熟发育态cNK细胞的表型，其与cNK细胞的谱系关联始终不明。此外，肝脏trNK细胞可能与胸腺NK细胞存在关联，亦或是独立于cNK与胸腺NK细胞的独特谱系。 本研究通过对多种转录因子缺陷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细胞是独立于cNK与胸腺NK细胞的独特谱系——尤其体现在其无需此前报道的NK细胞特化因子NFIL3（E4BP4）。 对其他组织的分析显示，皮肤与子宫中亦存在trNK细胞群，且其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（Fluorescence-Activated Cell Sorting, FACS）从单只小鼠体内分离汇集相关NK细胞群，最终每份样本获得约10万个细胞，所分选的细胞群包括肝脏NK1.1+CD49+、肝脏NK1.1+CD49-，以及脾脏NK1.1+CD49- NK细胞。 随后利用Invitrogen寡聚dT磁珠从细胞裂解液中提取mRNA。