Evidence for an autocrine loop in the Axl/Gas6 pathway. CD34⁺ cells isolated from blood of 2 different donors were cultured for 3 days in the presence of IL-15. Harvested cells were fixed, permeabilized, and stained with antibodies against Gas6, Axl, and phospho-STAT5. Gating was on CD34⁺ cells, and numbers in the upper right quadrant indicate percentage of double-positive cell populations (Gas6⁺Axl⁺ or Gas6⁺ phospho-STAT5⁺).

Expression of Axl family members and their ligands in human CD34⁺ HPCs. Isolated CD34⁺cells from human peripheral blood were cultured in the presence of IL-15 for 0, 5, 10, or 15 days. Then nonquantitative assessment of Gas6, Axl, Sky (Tyro3), Mer, protein S, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA expression was performed by RT-PCR. These results are representative of 2 experiments.

The Axl/Gas6 pathway is required for normal IL-15 signaling in human CD34⁺ HPCs. (A) CD34⁺ HPCs were isolated from human peripheral blood and treated with KL and FL for 5 days. Cells were washed, rested, and then treated with either irrelevant Fc or Axl-Fc in the presence of IL-15 for 30 minutes. Harvested cells were subject to Western blot to detect phospho-STAT5, and actin was used as a loading control. The figure is the representative of 2 similar experiments. (B) In a separate experiment, CD34⁺ cells were prepared and treated as described in panel A. Assessment of intracellular STAT5 phosphorylation using flow cytometry was performed while gating on CD34⁺ cells. Shown is 1 of 2 similar experiments. Dotted line represent media only; filled histogram, IL-15 + irrelevant Fc; open histogram, IL-15 + Axl-Fc. Dotted line and open histogram were indistinguishably overlapped.

Blockade of Axl/Gas6 pathway inhibits IL-15–induced in vitro differentiation of human CD34⁺ HPCs into NK cells. (A) Representative flow cytometry plot showing differentiation of NK cells (CD3⁻CD56⁺) from blood CD34⁺ HPCs. Value in parentheses indicates a percentage of CD3⁻CD56⁺ NK cells (upper left quadrant) detected among the total cells after a 3-week culture under the experimental conditions indicated. (B) Top graph shows a percentage of differentiated CD3⁻CD56⁺ NK cells among the total cells after culture for 3 weeks (triplicate wells). Bottom graph shows absolute numbers of NK cells (calculated by multiplying total number of cells counted in each well times the percentage of NK cells noted by flow cytometric analysis). Results for panel B are summarized from at least 3 experiments; error bar represents SD. *P < .03; **P < .001. (C) CD34⁺ cells isolated from human bone marrow were cultured with IL-15 in the presence of irrelevant Fc or Axl-Fc for 3 weeks, and the resultant percentage of CD3⁻CD56⁺ NK cells among the total cells is shown. The results show the mean plus or minus SD and are from 3 separate donors, each in triplicate wells. *P < .02.

Assessment of the Axl/Gas6 pathway in purified mature NK cells. (A) After total RNA was isolated from sorted fresh human CD34⁺ HPCs (34⁺), CD56^bright (bright), or CD56^dim (dim) NK cells, RT-PCR was performed to detect Gas6, Axl, Sky, and Mer mRNA. GAPDH was used as a loading control. The figure represents 1 of 2 experiments performed with separate donors. (B) Purified CD56^bright and CD56^dim NK cells were sorted from human peripheral blood, plated with the same cell numbers, and stimulated with IL-12 and IL-15 in the presence of irrelevant Fc or Axl-Fc for 24 hours. Supernatants were collected and IFN-γ production was measured by ELISA. Results illustrate the mean plus or minus SD from triplicate wells and represent 1 of 3 similar experiments showing no significant difference in the presence or absence of Axl-Fc for CD56^bright or CD56^dim NK cells. (C) CD56^bright and CD56^dim NK cells were prepared as described in panel B and cultured with IL-15 in the presence of irrelevant Fc or Axl-Fc for 24 hours and then mixed with ⁵¹Cr-labeled K-562 target cells at an effector:target ratio of 3:1, again without significant difference. These results are representative of 3 similar experiments.

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.

The Axl/Gas6 pathway is important for generation of functionally competent NK cells from human CD34⁺ HPCs. (A) CD34⁺ HPCs from human peripheral blood were cultured for 21 days under different experimental conditions as indicated. On day 21, equal numbers of CD3⁻CD56⁺ NK cells were plated into each well, followed by stimulation with IL-12 and IL-15 for 48 hours. Supernatants were harvested, and the concentration of IFN-γ was measured by ELISA. Results shown are the compilation of data from 2 donors, and each experiment was performed in triplicate wells. Error bar represents SD. *P < .05; **P < .01; ***P < .001. (B) On day 21, NK cells that were derived from CD34⁺ HPCs under different experimental conditions as described in panel A were plated in equal numbers into a 96-well culture plate. After being treated with IL-12 and IL-15 for 6 hours, cells were lysed, total RNA was isolated, and IFN-γ or T-BET mRNA was quantified by Taqman real-time RT-PCR. 18S rRNA was used for normalization. Error bar represents SD. These results are representative of 3 experiments. *P < .05; **P < .01. (C) After a 21-day culture of CD34⁺ cells under the indicated experimental conditions, the same number of differentiated CD3⁻CD56⁺ NK cells were plated in each well and mixed with ⁵¹Cr-labeled NK-sensitive K-562 target cells at a 2:1 ratio of effector to target cells (triplicate wells). Results are representative of 3 experiments and show mean plus or minus SD. No significant differences in cytotoxicity were noted.