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. Moreover, the majority of studies are descriptive because of the relative low frequency of cells and non-standardized definition of this lineage. In this study, we performed single-cell RNA sequencing on 28,835 single immune cells isolated from mixed splenocytes of male C57BL/6 mice using strict fluorescence-activated cell sorting. The data was replicated in a subsequent study. Our analysis revealed five transcriptionally distinct naïve DNT cell clusters, which expressed unique sets of genes and primarily performed T helper, cytotoxic and innate immune functions. Anti-CD3/CD28 activation enhanced their T helper and cytotoxic functions. Moreover, in comparison with CD4⁺, CD8⁺ T cells and NK cells, Ikzf2 was highly expressed by both naïve and activated cytotoxic DNT cells. In conclusion, we provide a map of the heterogeneity in naïve and active DNT cells, addresses the controversy about DNT cells, and provides potential transcription signatures of DNT cells. The landscape approach herein will eventually become more feasible through newer high throughput methods and will enable clustering data to be fed into a systems analysis approach. Thus the approach should become the “backdrop” of similar studies in the myriad murine models of autoimmunity, potentially highlighting the importance of DNT cells and other minor lineage of cells in immune homeostasis. The clear characterization of functional DNT subsets into helper DNT, cytotoxic DNT and innate DNT will help to better understand the intrinsic roles of different functional DNT subsets in the development and progression of autoimmune diseases and transplant rejection, and thereby may facilitate diagnosis and therapy.

CD4 和 CD8 辅助受体双阴性 TCRαβ ⁺T (DNT) 细胞因其在免疫系统中的关键和多样化作用而越来越受到认可。然而，它们的分子和功能特征仍然知之甚少且存在争议。此外，由于细胞频率相对较低且该谱系的定义不规范，因此大多数研究都是描述性的。在这项研究中，我们使用严格的荧光激活细胞分选技术对从雄性 C57BL/6 小鼠的混合脾细胞中分离出的 28,835 个单免疫细胞进行了单细胞 RNA 测序。在随后的研究中复制了这些数据。我们的分析揭示了五个转录不同的幼稚 DNT 细胞簇，它们表达独特的基因组，主要执行 T 辅助、细胞毒性和先天免疫功能。抗 CD3/CD28 激活增强了它们的 T 辅助和细胞毒性功能。而且，⁺、CD8 ⁺ T 细胞和 NK 细胞、Ikzf2由幼稚和活化的细胞毒性 DNT 细胞高度表达。总之，我们提供了幼稚和活跃 DNT 细胞的异质性图谱，解决了关于 DNT 细胞的争议，并提供了 DNT 细胞的潜在转录特征。本文中的景观方法最终将通过更新的高通量方法变得更加可行，并将使聚类数据能够输入到系统分析方法中。因此，该方法应该成为在无数小鼠自身免疫模型中进行类似研究的“背景”，可能突出 DNT 细胞和其他次要细胞谱系在免疫稳态中的重要性。将功能 DNT 子集明确表征为辅助 DNT，