Figure 4. Aza, hypoxia and TGFβ1 combined induce CD9⁺KIR⁺dNK-like cells that secrete VEGF-A and have reduced cytotoxicity.

a) KIR (top panels) and CD9 (bottom panels) expression by CD56^Bright CD16⁻ (left) and CD56^Dim CD16⁺ (right) pNK cells from one week-long pNK cell cultures under 1% O₂ in the presence of 2ng/mL TGFβ1 with (dark gray histograms) or without (light gray histogram) the addition of 1μM Aza, evaluated by flow cytometry. Tinted histograms, isotype control. Bars and numbers indicate the percentage of KIR⁺ cells in the corresponding NK cell subset in cultures performed under 1% O₂ in the presence of Aza and TGFβ1. One experiment representative of 3. b) Percentage of KIR+ cells among CD56^Bright CD16⁻ pNK cells from a one week long culture under 1% O₂ (circles) or 21% O₂ (squares), in the presence (white symbols) or absence (black symbols) of 2ng/mL TGFβ1, at different concentrations of Aza. One experiment representative of two. c) VEGF-A content in the supernatants of one week long cultures of pNK cells, seeded at 1×10⁶ cells /mL, in the presence of TGFβ1 under hypoxia (circles) or 21% O₂ (squares) at different concentrations of Aza. Average of 2 experiments d) Cytotoxic activity of effector pNK cells cultured for a week under hypoxia in the presence (triangles) or absence (circles) of 1μM Aza, in the presence (white symbols) or absence (black symbols) of 2 ng/mL TGFβ1, on K562 target cells. Results are average of 2 independent experiments.