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

CD4 and CD8 coreceptor double-negative TCRαβ+ 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. Single-cell RNA sequencing on five cell types of TCRαβ+ 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