Differential Ability of the Notch Ligands Jagged2, Delta1, and Delta4 To Accelerate Development of Human CD34+ Hematopoietic Progenitor Cells (HPCs) into Killer Inhibitory Receptor (KIR)-Negative NK Cells with Cytotoxic Activity.

The evolutionarily conserved Notch receptors play important roles in cell fate decisions. Ligation of Notch-1 causes differentiation of T/NK cell precursors from HPCs and is critical for development of T cells in the thymic microenvironment. Five known Notch ligands exist in mammals (Delta1, Delta3, Delta4, Jagged1, and Jagged2), and Delta4 in particular has a greater capacity to support T cell development than Delta1. Notch ligation through Delta1 has also been shown to potentiate CD56+ NK cell differentiation from human HPCs in the presence of IL-15, although the phenotype and functionality of these cells has not been extensively described. We compared the ability of the 5 mammalian Notch ligands to induce the development of functional NK cells from human CD34+ HPCs derived from umbilical cord blood (CB). CD34+ cells isolated from CB were cultured in RPMI + 10% FBS on a murine stromal cell line, OP-9, expressing one of the five mammalian Notch receptors (Jagged1, Jagged2, Delta1, Delta3, or Delta4) or OP-9 cells transfected with vector alone, in the presence of IL-7, Flt3 ligand (FL), and IL-15. After three weeks of culture, development of CD56+CD3− cells was greatly accelerated by the ligands Jagged2 (53.4 +/− 5.5% CD56+CD3− cells), Delta-1 (38.6 +/− 5.7%), and Delta-4 (65.0 +/− 3.9%) versus culture in the absence of ligand (17.6 +/−10.3%, p = < 0.02) or in the presence of Jagged1 or Delta3. By 5 weeks, the percentage of NK cells seen in cultures containing Jagged2, Delta1, or Delta4 reached 80–90%. These NK cells expressed CD117 but only partially expressed CD94, with positivity ranging from 12.1 to 34.1% of NK cells derived from these 3 ligands after 5 weeks in culture; similarly, few CD16+ NK cells were seen in these cultures (0 to 12.1% of NK cells after 5 weeks). KIR expression in more than 1% of NK cells was not identified under any culture condition. In preliminary experiments, the addition of IL-2 or IL-21, both of which have been shown to induce KIR expression in non-Notch mediated models of NK cell development, did not significantly alter the percentages of NK cells expressing CD94, CD16, or KIR. Because the ligands Jagged2 and Delta4 induced the highest percentages of NK cells in culture, we examined the cytotoxic activity of these cells. NK cells derived from Jagged2 or Delta4 ligation expressed perforin and displayed in vitro cytotoxic activity against the human leukemia cell lines K562 (34.1% or 40.8% target cell lysis, respectively, at an E:T of 10:1) and HL-60 (14.1% or 31.6%, respectively). These cells also produced IFN-gamma, with Delta4 cultures producing higher levels of IFN-gamma versus Jagged2 cultures (1112 vs. 163.9 pg/ml, respectively). These data demonstrate that the Notch ligands vary in their ability to induce differentiation of NK cells from human CD34+ HPCs. Jagged2 and Delta4 in particular have greater capacity to generate functional NK cells which have cytolytic activity and can secrete IFN-gamma, while at the same time lacking a majority of inhibitory NK receptors (KIR and the NKG family of receptors which dimerize with CD94). The generation of cytolytic KIR-negative NK cells is of interest for cellular therapy against malignancies that are susceptible to NK cell killing.