Generation of Lytic Natural Killer 1.1⁺, Ly-49⁻ Cells from Multipotential Murine Bone Marrow Progenitors in a Stroma-free Culture: Definition of Cytokine Requirements and Developmental Intermediates

We have developed a stroma-free culture system in which mouse marrow or thymus cells, known to be enriched for lymphoid progenitors, can be driven to generate natural killer (NK) cells. Culture of lineage marker (Lin)⁻, c-kit⁺, Sca2⁺, interleukin (IL)-2/15Rβ (CD122)⁻ marrow cells in IL-6, IL-7, stem cell factor (SCF), and flt3 ligand (flt3-L) for 5–6 d followed by IL-15 alone for an additional 4–5 d expanded the starting population 30–40-fold and gave rise to a virtually pure population of NK1.1⁺, CD3⁻ cells. Preculture in IL-6, IL-7, SCF, and flt3-L was necessary for inducing IL-15 responsiveness in the progenitors because the cells failed to significantly expand when cultured in IL-15 alone from the outset. Although culture of the sorted progenitors in IL-6, IL-7, SCF, and flt3-L for the entire 9–11-d culture period caused significant expansion, no lytic NK1.1⁺ cells were generated if IL-15 was not added, demonstrating a critical role for IL-15 in NK differentiation. Thus, two distinct populations of NK progenitors, IL-15 unresponsive and IL-15 responsive, have been defined. Similar results were obtained with Lin⁻, CD44⁺, CD25⁻, c-kit⁺ lymphoid progenitors obtained from adult thymus. The NK cells generated by this protocol lysed the NK-sensitive target YAC-1 and expressed markers of mature NK cells with the notable absence of Ly-49 major histocompatibility complex (MHC) receptors. However, despite the apparent lack of these inhibitory MHC receptors, the NK cells generated could distinguish MHC class I⁺ from class I⁻ syngeneic targets, suggesting the existence of novel class I receptors.