Blocking the Axl/Gas6 pathway reduces NK precursor frequency and inhibits c-Kit phosphorylation. (A) Isolated CD34⁺ HPCs from human peripheral blood were plated using serial dilution (from 6000 to 94 cells/well × 12 wells/condition). CD34⁺ HPCs were next cultured in 1 of 3 conditions and then directly stained with anti-CD3 and anti-CD56 antibodies, and analyzed by flow cytometry to quantify CD3⁻CD56⁺ NK cells: (1) IL-15 for 2 weeks (IL-15); (2) KL plus irrelevant Fc for 7 days followed by culture in IL-15 alone for 2 weeks (KL + irrelevant Fc → IL-15); or (3) KL plus Axl-Fc for 7 days, followed by culture in IL-15 alone for 2 weeks (KL + Axl-Fc → IL-15). CD56 expression was analyzed by flow cytometry, and the NK precursor frequency was determined as described in “Limiting dilution assay.” Results are from 3 donors. Error bar represents SEM. *P < .05. (B) CD34⁺ cells from human peripheral blood were plated, rested overnight, and treated with media only, KL plus irrelevant Fc, or KL + Axl-Fc for the indicated times. Cells were then harvested, and the level of phospho–c-Kit was measured by Western blot (top). In a separate experiment, intracellular staining for phospho–c-Kit that was gated on CD34⁺ cells was performed (bottom). Dotted line represents media only; thick line, KL + irrelevant Fc; thin line, KL + Axl-Fc. Results are representative of 4 separate experiments with similar results.