The T-box transcription factor plays an important role in regulating the immunoregulatory function of double-negative...

Double-negative T (DNT) cells are important immunoregulatory cells that play a key role in maintaining immune homeostasis. However, the specific immune molecular mechanisms regulating DNT cell function have yet to be studied in depth. This study revealed that compared with conventional T cells, natural DNT cells and CD4⁺ T cell-converted DNT (cDNT) cells can secrete high levels of IFN-γ. Further analysis revealed that DNT cells highly expressed Th1-related genes and the T-box transcription factor (T-bet). Knocking out T-bet significantly reduced the level of IFN-γ secretion by DNT cells, indicating that T-bet is involved in the regulation of IFN-γ. Knocking out T-bet did not affect the conversion, survival or proliferation of cDNT cells but weakened the ability of cDNT cells to kill monocytes and inhibit monocytes TNF-α secretion. Transcriptome sequencing analysis confirmed that T-bet knockout in cDNT cells significantly reduced the immune-killing ability and activation level of cDNT cells. The ChIP-seq analysis revealed that T-bet directly transcriptionally regulated genes associated with cytotoxicity and activation, such as Gzma, Gzmb, Prf1, and Cd28. After T-bet knockout, the expression levels of these genes in cDNT cells were significantly reduced. In summary, this study revealed the key role of T-bet in regulating the immunoregulatory function of DNT cells, expanded knowledge on the mechanisms of action by which DNT cells exert immunoregulatory effects and provided a theoretical basis for the application of DNT cells in immune cell therapy.