Differentiation of Nk1.1⁺, Ly49⁺ NK cells from flt3⁺ multipotent marrow progenitor cells

To delineate factors involved in NK cell development, we established an in vitro system in which lineage marker (Lin)^-, c.kit⁺, Sca2⁺ bone marrow cells differentiate into lyric NK1.1⁺ but Ly49^- cells upon culture in IL-7, stem cell factor (SCF), and flt3 ligand (flt3L), followed by IL-15 alone. A comparison of the ability of IL-7, SCF, and flt3L to generate IL-15- responsive precursors suggested that NK progenitors express the receptor for flt3L. In support of this, when Lin^-, c-kit⁺, flt3⁺ or Lin^-, c-kit⁺, flt3^- progenitors were utilized, 3-fold more NK cells arose from the flt3⁺ than from the flt3^- progenitors. Furthermore, NK cells that arose from flt3^- progenitors showed an immature NK1.1(dim), CD2^-, c-kit⁺ phenotype as compared with the more mature NK1.1(bright), CD2(+/-), c-kit^- phenotype displayed by NK cells derived from flt3⁺ progenitors. Both progenitors, however, gave rise to NK cells that were Ly49 negative. To test the hypothesis that additional marrow-derived signals are necessary for Ly49 expression on developing NK cells, flt3⁺ progenitors were grown in IL-7, SCF, and flt3L followed by culture with IL-15 and a marrow-derived stromal cell line. Expression of Ly49 molecules, including those of which the MHC class I ligands were expressed on the stromal or progenitor cells, as well as others of which the known ligands were absent, was induced within 6-13 days. Thus, we have established an in vitro system in which Ly49 expression on developing NK cells can be analyzed and possibly experimentally manipulated.