Transcriptome landscape of double negative T cells by single-cell RNA sequencing

CD4 and CD8 coreceptor double-negative TCRaß+ T (DNT) cells are increasingly being recognized for their critical and diverse roles in the immune system. However, their molecular and functional signatures remain poorly understood and controversial. We performed single-cell RNA sequencing on 28,835 single immune cells isolated from mixed splenocytes of mice using strict fluorescence-activated cell sorting. In this study, our analysis revealed five transcriptionally distinct naïve DNT clusters, which expressed unique sets of genes and mainly performed T helper, cytotoxic and innate immune functions. Anti-CD3/CD28 activation enhanced their T helper and cytotoxic functions. Moreover, by comparing with CD4+, CD8+ T cells and NK cells, we found Ly6c2 and Ikzf2 were highly expressed by naïve DNT cells. Upon activation, cytotoxic DNT subpopulation, had significantly higher Ikzf2 expression and protein expression of Ly-6C. Our results presented a comprehensive view of the transcriptional states of individual functional subset of DNT cells, highlighting the importance of DNT cells in immune surveillance, defense, and homeostasis. Overall design: Single-cell RNA sequencing on five cell types of TCRaß+ T cells using 10x Genomics. Naive DNT and activated DNT cells have two biological replicates. Grant Information: Funding Agency: National Natural Science Foundation of China Grant ID: No. 81570510, 81870399 and 82001694

CD4与CD8共受体双阴性TCRαβ+ T细胞（double-negative TCRαβ+ T cells，以下简称DNT细胞）在免疫系统中发挥的关键且多样的作用正日益受到学界认可。然而，其分子与功能特征仍未得到充分阐明，且相关研究尚存争议。本研究通过严格的荧光激活细胞分选术（fluorescence-activated cell sorting, FACS），从小鼠混合脾细胞中分离得到28835个单个免疫细胞，并对其开展单细胞RNA测序（single-cell RNA sequencing）。本研究的分析结果显示，存在5个转录特征各异的初始型DNT细胞簇，它们各自表达独特的基因集，且主要行使T辅助、细胞毒性及天然免疫功能。经抗CD3/CD28激活后，这类细胞的T辅助与细胞毒性功能得到增强。此外，通过与CD4+、CD8+ T细胞及自然杀伤细胞（Natural Killer, NK细胞）进行对比，本研究发现初始型DNT细胞高表达Ly6c2与Ikzf2基因。激活后，细胞毒性DNT细胞亚群的Ikzf2表达水平及Ly-6C的蛋白表达量均显著升高。本研究结果全面解析了不同功能亚群DNT细胞的转录状态，凸显了DNT细胞在免疫监视、免疫防御及免疫稳态维持中的重要作用。
实验设计：采用10×基因组学（10x Genomics）平台，对5类TCRαβ+ T细胞开展单细胞RNA测序。其中，初始型DNT细胞与激活型DNT细胞均设置2个生物学重复。
资助信息：资助机构为中国国家自然科学基金（National Natural Science Foundation of China），项目编号为No. 81570510、81870399及82001694。