Murine Embryonic Liver Differentiates Human Stem Cells into a Spectrum of NK Precursors and Polyclonal KIR Expressing NK Cells.

We have shown that a murine fetal liver cell line (AFT024) and human cytokines (IL-15, IL-7, IL-3, Flt3-ligand and c-kit ligand) are needed to induce NK cell differentiation and KIR acquisition. To understand the level of maturation where these factors orchestrate NK cell development, a switch culture was designed to separate early and late events. Cord blood CD34⁺/Lin⁻/CD38⁻ stem cells were cultured on AFT024 for 28 days. Use of IL-3 or Flt3-L alone resulted in minimal growth. In contrast, we show that NK cell differentiation can occur, albeit at low frequency, with a combination of IL-3 and Flt3-L, in the absence of IL-15. These early NK cells were negative for both CD94 and KIR. These conditions also allowed accumulation of CD56⁻ NK cell precursors. CD34⁺CD7⁻, CD34⁺CD7⁺ and CD34⁻CD7⁺ cells were detected in cultures lacking IL-15. Each precursor was tested in secondary cultures containing AFT024 with IL-15 alone, IL-15+IL-3, or IL15+IL-3+Flt3-L. After an additional 2–4 weeks, NK cells differentiated from each distinct cell population. A few predominantly KIR negative NK cells resulted from IL-15 alone. Addition of IL-3 or IL-3+Flt3-L significantly increased the absolute number of NK cells as well as the acquisition of CD94 heterodimers and KIR. We next explored other stromal cell lines in attempt to identify novel factors important in early NK cell maturation. A novel cell line derived from murine embryonic liver (EL08-1D2), identified for its ability to support expansion of mouse stem cells, was compared to AFT024. To test the differential capacity of these microenvironments, single cord blood stem cells were plated on the two feeders supplemented with all cytokines. After 4 weeks, EL08-1D2 induced 125,852±1400 NK cells from a single stem cell, significantly more than with AFT024 (23,143±8117). KIR⁺ NK cells were also significantly more frequent with EL08-1D2 (3689±801 vs. 799±491), always in a polyclonal pattern. NK cell development and KIR acquisition were dependent on direct contact with EL08-1D2. Increased development could be from greater differentiation, proliferation or both. Cord blood stem cells were cultured in direct contact with EL08-1D2 under primary culture conditions with IL-3 and Flt3-L but in the absence of IL-15. All CD56⁻ NK cell precursors developed with greater frequency on EL08-1D2 than AFT024. In conclusion, EL08-1D2, derived from a primitive microenvironment during mouse ontogeny, efficiently recapitulates NK cell development by inducing NK cell differentiation and proliferation. IL-3 and Flt3-L, but not IL-15, facilitate the isolation and study of distinct NK cell precursors. Direct contact with EL08-1D2 induces KIR acquisition, suggesting that unique environmental factors conserved between mouse and man contribute to the extrinsic signals which lead to KIR acquisition.