The Notch ligands Jagged2, Delta1, and Delta4 induce differentiation and expansion of functional human NK cells from CD34+ cord blood hematopoietic progenitor cells

Abstract

Notch receptor signaling is required for T cell development, but its role in natural killer (NK) cell development is poorly understood. We compared the ability of the 5 mammalian Notch ligands (Jagged1, Jagged2, Delta1, Delta3, or Delta4) to induce NK cell development from human hematopoietic progenitor cells (HPCs). CD34(+) HPCs were cultured with OP9 stromal cell lines transduced with 1 of the Notch ligands or with OP9 stromal cells alone, in the presence of IL-7, Flt3L, and IL-15. Differentiation and expansion of CD56(+)CD3(-) cells were greatly accelerated in the presence of Jagged2, Delta-1, or Delta-4, versus culture in the absence of ligand or in the presence of Jagged1 or Delta3. At 4 weeks, cultures containing Jagged2, Delta1, or Delta4 contained 80% to 90% NK cells, with the remaining cells being CD33(+) myelogenous cells. Notch-induced NK (N-NK) cells resembled CD56(bright) NK cells in that they were CD16(-), CD94(-), CD117(+), and killer immunoglobulin-like receptors (KIR(-)). They also expressed NKp30, NKp44, NKp46, 2B4, and DNAM-1, with partial expression of NKG2D. The N-NK cells displayed cytotoxic activity against the K562 and RPMI-8226 cell lines, at levels similar to activated peripheral blood (PB) NK cells, although killing of Daudi cells was not present. N-NK cells were also capable of interferon (IFN)-gamma secretion. Thus, Notch ligands have differential ability to induce and expand immature, but functional, NK cells from CD34(+) HPCs. The use of Notch ligands to generate functional NK cells in vitro may be significant for cellular therapy purposes.

Fig. 1

The Notch ligands Jagged2, Delta1, and Delta4 accelerate NK cell differentiation from human CD34+ HPCs. (A) The development of NK cells (defined as CD56+CD3−) from CD34+ HPCs is enhanced by co-culture with OP9 cells expressing Jagged2, Delta1, or Delta4, versus OP9 cells expressing Jagged1 or Delta3 or containing vector (Ret-10) alone. All cultures contain IL-7 and Flt3-L plus IL-15 (black bars) or IL-2 (gray bars). Data shown is from day 21 of co-culture and represents the mean +/− SD of at least 3 experiments. (B) The percentage of Notch-induced NK (N-NK) cells in IL-15+ cultures increases over time and peaks after 4 weeks. Data shown represent the mean +/− SD of at least 3 experiments. (C) NK cell differentiation is inhibited by the presence of a gamma-secretase inhibitor (GSI), L-685,458, at day 21 of culture. Data shown represents the mean of 2 experiments.

Fig. 2

Culture of CD34+ HPCs in the presence of IL-15 and Notch ligand drives NK cell differentiation, without T or B cell maturation. Flow cytometric analysis of 4 week-old cultures containing the Notch ligand Delta4 and IL-15 demonstrate abundance of CD7+CD56+ NK cells and no T cell precursors (A), mature T cells (CD3+; B), or B cells (CD19+; C), while culture without IL-15 in the presence of Delta4 demonstrates predicted T cell precursors (CD1a+CD7+; A). Culture without Notch ligand (RET-10) in the presence of IL-15 demonstrates few CD7+ NK cell precursors and CD56+ NK cells (A, B), with mostly CD33+ myeloid cells and no development of T or B cells (A–C). All cultures contained IL-7 and FltL. Data from cultures containing the Notch ligands Jagged2 or Delta1 showed similar results to the Delta4 culture. Data shown is representative of at least 4 experiments.

Fig. 3

The Notch ligands Jagged2, Delta1, and Delta4 induce NK cell expansion from human CD34+ HPCs. The number of NK cells (defined as CD56+CD3−) derived per single CD34+ input cell at day 28 of culture in the presence of IL-15 is shown; each symbol represents an individual experiment with the horizontal bar indicating the mean (n = 5–7 independent experiments). P values generated by student’s t-test are shown.

Fig. 4

N-NK cells have a predominantly immature NK cell surface phenotype but do express NKG2D and the natural cytotoxicity receptors, NKp30, NKp44, and NKp46. Expression of NK cell markers was examined on gated CD56+CD3− cells from 4 week-old co-cultures containing Delta4 or no Notch ligand (RET-10). KIR expression was examined by an antibody cocktail (DX9 + CD158a + CD158b). Data from cultures containing the Notch ligands Jagged2 or Delta1 showed similar results to the Delta4 culture. Data shown is representative of at least 4 independent experiments.

Fig. 5

N-NK cells have perforin-dependent cytotoxic activity. (A) Cytotoxicity assays demonstrate activity against the human leukemia cell lines K562 and RPMI-8226 but not Daudi. N-NK cells from week 3 or 4 of co-culture were used as effector cells. (B) N-NK cell cytotoxicity assay against K562 cells is abrogated in the presence of the perforin inhibitor concanamycin-A (CMA). The Effector:Target ratio is 10:1. (C) N-NK cells from week 4 of Delta4 co-culture express perforin by flow cytometry; N-NK cells from Jagged2 and Delta1 cultures have similar levels of perforin expression (not shown). (D) The level of cytotoxic activity of Delta4-derived N-NK cells is similar, but not identical, to activated PB NK cells (PB Stim), and is greater than unstimulated PB NK cells (PB). PB NK cells were activated by overnight incubation with IL-15 (10 ng/ml). Averages of 3 independent experiments are shown.

Fig. 6

Both N-NK cells and NK cells developed in the absence of Notch ligand can secrete IFN-γ only after overnight incubation with IL-12 and IL-18 (100 ng/ml each). Levels of IFN-γ secreted by PB NK cells from three different healthy donors are shown for comparison. PB NK cells were cultured for two days in the presence of IL-15, with or without additional IL-12 and IL-18 on day 2.