M-CSFR⁺ precursors can become NK cells but have differential developmental requirements. (A) The CD56⁻CD117⁺ cell fraction was isolated from day 18 NK-cell differentiation culture of CD34⁺ cells and labeled for M-CSFR (CD115) expression. FACS gates for the M-CSFR⁺ and M-CSFR⁻ subsets are shown. On further culture for 2 weeks with cytokines alone (IL-15, IL-7, SCF, FLT-3L) the M-CSFR⁺ subset gives rise to CD56⁺CD94⁻ NK cells (top plot), whereas M-CSFR⁻ subset generates predominantly CD56⁺CD94⁺ NK cells (bottom plot). (B) The addition of HDC, stroma, or both to CD56⁻CD117⁺M-CSFR⁺ myeloid precursors induces increasing acquisition of CD94 (top) and KIR (bottom). (C) The CD56⁻CD117⁺M-CSFR⁺ subset generates increasing numbers of CD14⁺ cells on the addition of M-CSF (□, no M-CSF; ▩, 10 ng/mL; ■, 50 ng/mL). The number of CD14⁺ cells is shown after 1 week of culture with NK-supporting cytokines (IL-15, IL-7, SCF, FLT-3L) and stroma (left) or cytokines and stroma and HDC (right). (D) M-CSF at high doses abolishes NK-cell development of CD56⁻CD117⁺M-CSFR⁺ precursors. Shown are the number of CD56⁺ cells when CD56⁻CD117⁺M-CSFR⁺ precursors are cultured in NK-supporting cytokines (IL-15, IL-7, SCF, FLT-3L) and in stroma (left) or cytokines, stroma, and HDC (right) and cultured with/without M-CSF (□ indicates no M-CSF; ▩, 10 ng/mL; ■, 50 ng/mL). The number of NK cells at 2 weeks is shown. (E) The CD56⁻CD117⁺M-CSFR⁺ subset cultured with low concentrations (0, 1, 10, and 20 ng/mL) of M-CSF induces CD56⁺ NK cells with coexpression of CD14 (percentages of CD14⁺ in CD56⁺ fraction indicated). Cells were present in the lymphocyte gate by SSC versus FSC characteristics (not shown).Groups showing significant differences are indicated by asterisks (***P < .001, **P = .001-.01, and *P = .01-.05), 2-way analysis of variance on log-transformed values with Bonferroni posttest.