Notch signaling induces cytoplasmic CD3ϵ expression in human differentiating NK cells

It has been proposed that heterogeneity in natural killer (NK)–cell phenotype and function can be achieved through distinct thymic and bone marrow pathways of NK-cell development. Here, we show a link between Notch signaling and the generation of intracellular CD3ϵ (cyCD3)–expressing NK cells, a cell population that can be detected in vivo. Differentiation of human CD34+ cord blood progenitors in IL-15–supplemented fetal thymus organ culture or OP9-Delta-like 1 (DL1) coculture resulted in a high percentage of cyCD3+ NK cells that was blocked by the γ-secretase inhibitor DAPT. The requirement for Notch signaling to generate cyCD3+ NK cells was further illustrated by transduction of CD34+ cord blood (CB) cells with either the active intracellular part of Notch or the dominant-negative mutant of mastermind-like protein 1 that resulted in the generation of NK cells with respectively high or low frequencies of cyCD3. Human thymic CD34+ progenitor cells displayed the potential to generate cyCD3+ NK cells, even in the absence of Notch/DL1 signaling. Peripheral blood NK cells were unable to induce cyCD3 expression after DL1 exposure, indicating that Notch-dependent cyCD3 expression can only be achieved during the early phase of NK-cell differentiation.