Innate-like functions of natural killer T cell subsets result from highly divergent gene programs [single_cell_RNA-seq]

Natural killer T (NKT) cells have immune stimulatory or inhibitory effects on the immune response that are context-dependent. This may be attributed in part to the existence of functional NKT cell subsets; however, these functional subsets have only been characterized on the basis of differential expression of a few transcription factors and cell surface molecules. Here we have analyzed purified populations of thymic NKT cell subsets at both the transcriptomic and epigenomic levels, and by single-cell RNA sequencing. Our data indicate that despite their similar antigen specificity, the functional NKT cell subsets are highly divergent populations characterized by many gene expression and epigenetic differences. Therefore the thymus imprints innate-like NKT cells with novel combinations of properties, including differences in proliferative capacity, homing, and effector functions that were not previously anticipated. Analysis of single cell transcriptomic heterogeneity in mouse Valpha14 iNKT thymocyte subsets (NKT1, NKT2, NKT17 and NKT0). Samples were generated from individual experiment using a pool of thymocytes prepared from five five-week old C57BL/6J females. NKT cells subtypes were isolated from thymuses and directly sorted by flow cytometry into lysis buffer (96 well plate single cell sort). The preparation of samples occurred in 2 different batches (both having a equal representation of the different cell populations).自然杀伤T（Natural killer T, NKT）细胞对免疫应答的影响具有情境依赖性，既可发挥免疫激活作用，亦可介导免疫抑制。这种功能多样性一定程度上可归因于功能性NKT细胞亚群的存在，但目前仅通过少数转录因子与细胞表面分子的差异表达对这些功能性亚群进行了鉴定。本研究通过单细胞RNA测序技术，从转录组学与表观基因组学层面分析了纯化的胸腺NKT细胞亚群。研究数据显示，尽管不同功能性NKT细胞亚群的抗原特异性相似，但其基因表达与表观遗传特征存在大量差异，属于高度异质性的细胞群体。因此，胸腺可通过全新的特性组合对先天样NKT细胞进行印记，包括此前未被发现的增殖能力、归巢特性与效应功能差异。本研究针对小鼠Valpha14不变自然杀伤T（invariant NKT, iNKT）胸腺细胞亚群（NKT1、NKT2、NKT17及NKT0）的单细胞转录组异质性展开分析。实验样本来源于独立实验，以5只5周龄C57BL/6J雌性小鼠的混合胸腺细胞制备。从胸腺中分离NKT细胞亚型后，通过流式细胞术直接将其分选至裂解缓冲液中（采用96孔板单细胞分选方式）。样本制备分为2个独立批次，两个批次的各细胞群体占比均保持一致。