Sustained Notch1 signaling instructs the earliest human intrathymic precursors to adopt a γδ T-cell fate in fetal thymus organ culture

Notch1 activity is essential for the specification of T-lineage fate in hematopoietic progenitors. Once the T-cell lineage is specified, T-cell precursors in the thymus must choose between alphabeta and gammadelta lineages. However, the impact of Notch1 signaling on intrathymic pro-T cells has not been addressed directly. To approach this issue, we used retroviral vectors to express constitutively active Notch1 in human thymocyte progenitors positioned at successive developmental stages, and we followed their differentiation in fetal thymus organ culture (FTOC). Here we show that sustained Notch1 signaling impairs progression to the double-positive (DP) stage and efficiently diverts the earliest thymic progenitors from the main alphabeta T-cell pathway toward development of gammadelta T cells. The impact of Notch1 signaling on skewed gammadelta production decreases progressively along intrathymic maturation and is restricted to precursor stages upstream of the pre-T-cell receptor checkpoint. Close to and beyond that point, Notch1 is not further able to instruct gammadelta cell fate, but promotes an abnormal expansion of alphabeta-committed thymocytes. These results stress the stage-specific impact of Notch1 signaling in intrathymic differentiation and suggest that regulation of Notch1 activity at defined developmental windows is essential to control alphabeta versus gammadelta T-cell development and to avoid deregulated expansion of alphabeta-lineage cells.