Figure 2. Hypoxia enriches pNK cell cultures in CD56^BrightCD16^-cells and turns pNKs into pro-angiogenic cells.

a) CD56 and CD16 expression profile of pNK cells cultured for one week under hypoxia (1% O₂) or normal tissue culture conditions (21% O₂) in the presence of IL-15. Squares delineate CD56^Bright CD16⁻ NK and CD56^Dim CD16⁺ NK cell populations. Numbers indicate percentage of cells. b) CD56^Bright CD16⁻ NK cell to CD56^Dim CD16⁺ NK cell ratio in hypoxia and 21% O₂ cultures in experiments similar to the one shown in A. Each line represents an independent experiment. ** p-value=6×10⁻⁵ by paired t-test. c) VEGF-A levels in the supernatants of pNK cells, FACS sorted CD56^Bright CD16⁻ pNK cells and FACS sorted CD56^Dim CD16⁺ pNK cells cultured under 21% or 1% O₂ in the presence of IL-15 for one week. Cells were seeded at a density of 4×10⁴ cells in 300 μl of media. Error bars represent standard deviation. Average of 3 experiments. * p-value=0.029, ** p-value=0.009 d) Tube formation by HUVECs in the presence of supernatants of pNK cells seeded at 1×10⁶ cells per ml and cultured one week under 21% O₂ or 1% O₂. Representative fields (left) and tube formation quantitative evaluation (right) by the average percentage area per field covered by HUVECs. Results are average of 6 experiments. Error bars represent standard deviation. ** p-value=0.002. e) Cytotoxic activity of freshly isolated pNK and of pNK cells cultured in the presence of IL-15 under 21% O₂ or 1% O₂ on K562 target cells. Average of 2 ⁵¹Chromium release assays. f) CD9 (top panel) and KIR (bottom panel) expression by CD3⁻CD56^Bright CD16⁻ NK and CD3^-CD56^Dim CD16⁺ NK cells from pNK cell cultures maintained for a week under 1% O2 (black histogram) or 21% O₂ (gray histogram) in the presence of 10 ng/mL IL-15. In the top panels histograms are not visualized due to overlap with isotyope control..Filled histograms, isotype controls. Histograms are representative of 3 experiments.