Mouse fetal and embryonic liver cells differentiate human umbilical cord blood progenitors into CD56-negative natural killer cell precursors in the absence of interleukin-15

Abstract

Objective-Human NK cell maturation involves the orderly acquisition of NK cell receptors. Our aim was to understand how stromal interactions and cytokines are important in this developmental process.Methods-Human UCB CD34 + /Lin − /CD38 − cells were cultured on two murine stromal cell lines (AFT024 and EL08-1D2) in a switch culture to study NK cell development.Results-When human progenitors were cultured on AFT024 with IL-3 and Flt3-L in the absence of IL-15, NK cell differentiation occurred, albeit at low frequency. These conditions favored the accumulation of CD56 − NK cell precursors (CD34 + CD7 − , CD34 + CD7 + and CD34 − CD7 + cells), which are populations rare in adult blood but abundant in fresh UCB. In secondary culture, addition of IL-3 or IL-3+Flt3L to IL-15 increased the absolute number of CD56 + NK cells from precursors and the acquisition of CD94 and KIR. To further explore the microenvironment in early NK cell maturation, a cell line derived from murine embryonic liver (EL08-1D2) was studied. NK cell development and KIR acquisition was superior with EL08-1D2 which supported the differentiation of NK cell precursors, NK cell commitment, and proliferation.Conclusion-Although the earliest events in NK cell maturation do not require exogenous human IL-15, it is required at a later stage of NK cell commitment. At a minimum, murine stroma, IL-3, and Flt3-L are required to recapitulate early NK cell development and differentiation into distinct NK cell precursors. EL08-1D2 induces KIR acquisition suggesting that extrinsic signals in NK cell development are conserved between mouse and man.

Notch Activation Induces the Generation of Functional NK Cells from Human Cord Blood CD34-Positive Cells Devoid of IL-15

“…IL-15 has been considered to have a more physiologic role than IL-2 in NK development (30). Notably, IL-15-independent NK cell differentiation has recently been published (6). This culture system, however, has been reported to be stromal cell dependent while the potential molecules and signaling pathways are unknown and, thus, the conclusion whether IL-15 is indispensable is yet to be determined.…”

“…In humans, IL-15 is considered to be required for in vitro NK cell development and virtually most current protocols for human NK cell differentiation culture depend on IL-15. IL-15-independent NK cell differentiation has been reported in which human cord blood (CB) 9 cells are cocultured with murine stromal cell lines (6). Signaling, however, substituting IL-15 signaling that is responsible for the NK cell differentiation in this culture system was not described.…”

Generation of natural killer cells from hematopoietic stem cells in vitro for immunotherapy

“…Their results show that NK cell receptor fate is acquired after NK cell commitment and does not require stromal presentation of MHC-1 by the stromal cells. 87 Later in 2008, McCullar et al 88 investigated AFT024 and EL08-1D2 potential to generate NK cells, and found that EL08-1D2 is significantly better at recapitulating NK cell development. EL08-1D2 was cloned from embryonic liver at day 11, and was proven in this study to support the generation of NK cells from human hematopoietic precursors, and used also by other groups.…”

Lineage relationships of human interleukin-22–producing CD56+ RORγt+ innate lymphoid cells and conventional natural killer cells

“…1 cells cocultured with stroma and recombinant cytokines can differentiate into both cNK cells and ILC22 cells, 7,19,20,22 but the lineage relationship between these two cell types in humans has been debated. 10,15,23,24 To investigate this, CD34 1 cells were cultured with IL-3 (for the first week), SCF, FLT3L, IL-7, and IL-15 for 3 weeks.…”