Constitutive Notch signalling promotes CD4^- CD8 ^- thymocyte differentiation in the absence of the pre-TCR complex, by mimicking pre-TCR signals

Notch1 signalling is essential for the commitment of multipotent lymphocyte precursors towards the αβ T-cell lineage and plays an important role in regulating β-selection in CD4^- CD8^- double-negative (DN) thymocytes. However, the role played by Notch in promoting the development of CD4⁺ CD8⁺ double-positive (DP) thymocytes is poorly characterized. Here, we demonstrate that the introduction of a constitutively active Notch1 (ICN1) construct into RAG^-/- lymphocyte precursors resulted in the generation of DP thymocytes in in vitro T-cell culture systems. Notably, developmental rescue was dependent not only on the presence of an intact Notch1 RAM domain but also on Delta-like signals, as ICN1-induced DP development in RAG^-/- thymocytes occurred within an intact thymus or in OP9-DL1 co-cultures, but not in OP9-control co-cultures. Interestingly, ICN1 expression in SLP-76^-/- precursors resulted in only a minimal developmental rescue to the immature CD8^- single-positive stage, suggesting that Notch is utilizing the same signalling pathway as the pre-TCR complex. In support of this, ICN1 introduction resulted in the activation of the ERK-MAPK-signalling cascade in RAG^-/- thymocytes. Taken together, these studies demonstrate that constitutive Notch signalling can bypass β-selection during early T-cell development by inducing pre-TCR-like signals within a T-cell-promoting environment.