Integrated Single Cell Analysis Reveals the Transcriptional Heterogeneity of Mouse Double Negative T Cells

Double negative T cells (DNT cells) are a rare T cell population involved in immune regulation, inflammation, autoimmunity, transplantation, and tumor immunity. However, their low abundance and dispersed distribution across tissues have limited a systematic understanding of their cellular organization and transcriptional diversity. To address this problem, we developed an integrative single cell framework to reconstruct the mouse DNT cell landscape across tissues using multiple public single cell RNA sequencing datasets. Using transcriptomic criteria, we identified and integrated 7984 mouse RNA defined DNT cells. The integrated population was resolved into multiple transcriptionally distinct clusters. These clusters were organized into naive like, proinflammatory, cytotoxic, proliferative, and myeloid associated states, revealing that mouse RNA defined DNT cells constitute a highly heterogeneous yet structured transcriptional compartment. Integrated downstream analyses indicated transcriptional relationships among these subsets. Naive like populations showed inferred transcriptional relationships with inflammatory and cytotoxic programs, while virtual knockout and intercellular communication analyses identified distinct predicted regulatory and signaling features across states. To place these mouse DNT associated genes in a human disease context, selected genes were mapped to their corresponding human homologs and examined using TCGA pan cancer transcriptomic data. Together, these findings define a cross tissue transcriptional framework for mouse RNA defined DNT cells and provide a basis for further evaluating the conservation and relevance of these transcriptional programs in human DNT biology. More broadly, this study provides a generalizable integrative strategy for reconstructing and characterizing rare immune cell populations that are insufficiently represented in individual single cell datasets.